Sanjiv Sam Gambhir, MD, PhD

All Publications

[(18)F]FPRGD2 PET/CT imaging of integrin avß3 levels in patients with locally advanced rectal carcinoma. European journal of nuclear medicine and molecular imaging Withofs, N., Martinive, P., Vanderick, J., Bletard, N., Scagnol, I., Mievis, F., Giacomelli, F., Coucke, P., Delvenne, P., Cataldo, D., Gambhir, S. S., Hustinx, R. 2016; 43 (4): 654-662
Abstract

Our primary objective was to determine if [(18)F]FPRGD2 PET/CT performed at baseline and/or after chemoradiotherapy (CRT) could predict tumour regression grade (TRG) in locally advanced rectal cancer (LARC). Secondary objectives were to compare baseline [(18)F]FPRGD2 and [(18)F]FDG uptake, to evaluate the correlation between posttreatment [(18)F]FPRGD2 uptake and tumour microvessel density (MVD) and to determine if [(18)F]FPRGD2 and FDG PET/CT could predict disease-free survival.Baseline [(18)F]FPRGD2 and FDG PET/CT were performed in 32 consecutive patients (23 men, 9 women; mean age 63 ± 8 years) with LARC before starting any therapy. A posttreatment [(18)F]FPRGD2 PET/CT scan was performed in 24 patients after the end of CRT (median interval 7 weeks, range 3 - 15 weeks) and before surgery (median interval 4 days, range 1 - 15 days).All LARC showed uptake of both [(18)F]FPRGD2 (SUVmax 5.4 ± 1.5, range 2.7 - 9) and FDG (SUVmax 16.5 ± 8, range 7.1 - 36.5). There was a moderate positive correlation between [(18)F]FPRGD2 and FDG SUVmax (Pearson's r = 0.49, p = 0.0026). There was a moderate negative correlation between baseline [(18)F]FPRGD2 SUVmax and the TRG (Spearman's r = -0.37, p = 0.037), and a [(18)F]FPRGD2 SUVmax of >5.6 identified all patients with a complete response (TRG 0; AUC 0.84, 95 % CI 0.68 - 1, p = 0.029). In the 24 patients who underwent a posttreatment [(18)F]FPRGD2 PET/CT scan the response index, calculated as [(SUVmax1 - SUVmax2)/SUVmax1] × 100 %, was not associated with TRG. Post-treatment [(18)F]FPRGD2 uptake was not correlated with tumour MVD. Neither [(18)F]FPRGD2 nor FDG uptake predicted disease-free survival.Baseline [(18)F]FPRGD2 uptake was correlated with the pathological response in patients with LARC treated with CRT. However, the specificity was too low to consider its clinical routine use.

View details for DOI 10.1007/s00259-015-3219-y

View details for PubMedID 26490751
AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models JOURNAL OF NEURO-ONCOLOGY Chang, E., Pohling, C., Natarajan, A., Witney, T. H., Kaur, J., Xu, L., Gowrishankar, G., D'Souza, A. L., Murty, S., Schick, S., Chen, L., Wu, N., Khaw, P., Mischel, P., Abbasi, T., Usmani, S., Mallick, P., Gambhir, S. S. 2016; 126 (2): 253-264
Abstract

Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O'Neill (J Neurooncol 107: 359-364, 2012). An extract from the winter cherry plant (Withania somnifera ), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985-1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3-5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (U87-MG, GBM2 and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 µM respectively. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM.

View details for DOI 10.1007/s11060-015-1972-1

View details for Web of Science ID 000368728300005
F-18-FPRGD(2) PET/CT imaging of musculoskeletal disorders ANNALS OF NUCLEAR MEDICINE Withofs, N., Charlier, E., Simoni, P., Alvarez-Miezentseva, V., Mievis, F., Giacomelli, F., Mella, C., Gambhir, S. S., Malaise, O., de Seny, D., Malaise, M., Hustinx, R. 2015; 29 (10): 839-847
Abstract

This work reports on musculoskeletal uptake of (18)F-FPRGD2, targeting the integrin αvβ3, in patients who had undergone (18)F-FPRGD2 positron emission tomography combined with computed tomography (PET/CT) for oncologic purposes.Whole-body (18)F-FPRGD2 PET/CT images of 62 cancer patients were retrospectively reviewed to detect foci of musculoskeletal (18)F-FPRGD2 uptake. For 37 patients, a FDG PET/CT performed in clinical settings was available. In each joint with an abnormal uptake, the maximum standardized uptake value (SUVmax) was estimated.A total of 260 musculoskeletal foci of (18)F-FPRGD2 uptake were detected. Most common sites of uptake were joints and discs (n = 160; 61.5 %), entheses (osteotendinous and osteoligamentous junctions; n = 55; 21.2 %) and recent fractures (n = 18; 6.9 %). In addition, 27 (10.4 %) miscellaneous foci were detected. Out of the 146 lesions for which a FDG PET was available, 63 % showed both (18)F-FPRGD2 and FDG uptake, 33.6 % did not show FDG avidity and 3.4 % showed only FDG uptake. The uptake intensity of the 92 lesions positive with (18)F-FPRGD2 and FDG was similar with both radiopharmaceuticals, but the target-to-background (blood pool or muscle) ratios were significantly higher with (18)F-FPRGD2 than with FDG (p < 0.0001).The (18)F-FPRGD2 uptake in joints, spine degenerative diseases and tendons was highly prevalent in our population. Up to one-third of (18)F-FPRGD2 foci showed no FDG uptake suggesting that (18)F-FPRGD2 signal may not be related to inflammatory angiogenesis only.

View details for DOI 10.1007/s12149-015-1011-5

View details for Web of Science ID 000365723900001

View details for PubMedID 26254227
Multiscale Framework for Imaging Radio labeled Therapeutics MOLECULAR PHARMACEUTICS Natarajan, A., Tuerkcan, S., Gambhir, S. S., Pratx, G. 2015; 12 (12): 4554-4560
View details for DOI 10.1021/acs.molpharmaceur.5b00392

View details for Web of Science ID 000366151500036
Prospective Comparison of Tc-99m-MDP Scintigraphy, Combined F-18-NaF and F-18-FDG PET/CT, and Whole-Body MRI in Patients with Breast and Prostate Cancer JOURNAL OF NUCLEAR MEDICINE Minamimoto, R., Loening, A., Jamali, M., Barkhodari, A., Mosci, C., Jackson, T., Obara, P., Taviani, V., Gambhir, S. S., Vasanawala, S., Iagaru, A. 2015; 56 (12): 1862-1868
Abstract

We prospectively evaluated the combined (18)F-NaF/(18)F-FDG PET/CT in patients with breast and prostate cancers, and compared the results to (99m)Tc MDP bone scintigraphy (BS) and whole-body MRI (WBMRI).30 patients (15 women with breast cancer and 15 men with prostate cancer) referred for standard of care BS were prospectively enrolled in this study. (18)F-NaF/(18)F-FDG PET/CT and WBMRI were performed following BS. WBMRI protocol consisted of both non-contrast enhanced and contrast enhanced sequences. Lesions detected with each test were tabulated and the results were compared.For extra skeletal lesions, (18)F-/(18)F-FDG PET/CT and WBMRI had no statistically significant differences in sensitivity (92.9% vs 92.9%, P = 1.00), PPV (81.3% vs 86.7%, P = 0.68) and accuracy (76.5% vs 82.4%, P = 0.56). However, (18)F-/(18)F-FDG PET/CT showed significantly higher sensitivity and accuracy than WBMRI (96.2% vs 81.4%, P<0.001, 89.8% vs 74.7%, P = 0.01) and BS (96.2% vs 64.6%, P<0.001, 89.8% vs 65.9%, P<0.001) for the detection of skeletal lesions. Overall, (18)F-/(18)F-FDG PET/CT showed higher sensitivity and accuracy than WBMRI (95.7% vs 83.3%, P<0.002, 87.6% vs 76.0%, P< 0.02), but not statistically significant when compared to a combination of WBMRI and BS (95.7% vs 91.6%, P = 0.17, 87.6% vs 83.0%, P = 0.53). (18)F-/(18)F-FDG PET/CT showed no significant difference with a combination of (18)F-/(18)F-FDG PET/CT and WBMRI. No statistically significant differences in PPV were noted among the 3 examinations.The (18)F NaF/(18)F FDG PET/CT is superior to WBMRI and (99m)Tc-MDP scintigraphy for evaluation of skeletal disease extent. Further, (18)F NaF/(18)F FDG PET/CT and WBMRI detected extra-skeletal disease that may change the management of these patients. The (18)F NaF/(18)F FDG PET/CT provide similar diagnostic ability with combination of WBMRI and BS in patients with breast and prostate cancers. Larger cohorts are needed in order to confirm these preliminary findings, ideally using the newly introduced simultaneous PET/MRI scanners.

View details for DOI 10.2967/jnumed.115.162610

View details for Web of Science ID 000365724800011

View details for PubMedID 26405167
Photoacoustic Tomography Detects Early Vessel Regression and Normalization During Ovarian Tumor Response to the Antiangiogenic Therapy Trebananib JOURNAL OF NUCLEAR MEDICINE Bohndiek, S. E., Sasportas, L. S., Machtaler, S., Jokerst, J. V., Hori, S., Gambhir, S. S. 2015; 56 (12): 1942-1947
Abstract

The primary aim of this study was to assess the potential of in vivo photoacoustic tomography (PAT) for direct functional measurement of ovarian tumor response to anti-angiogenic therapy.In vivo studies were performed with institutional animal care and use committee approval. We used an orthotopic mouse model of ovarian cancer treated with Trebananib (n = 9) or vehicle (n = 9). Tumor-bearing mice were randomized into Trebananib or vehicle groups at day 10 and dosed on days 12, 15 and 18 post implantation. PAT and blood draws were performed at day 10, then 24 hours after each drug dose. Tumors were excised for histopathology following the final studies on day 19. Data analysis to test for statistical significance was performed blinded.Blockade of angiopoietin signaling using Trebananib resulted in reduced total hemoglobin-weighted PA signal (n = 9, P = 0.01) and increased oxyhemoglobin-weighted PA signal (n = 9, P<0.01). The latter observation indicated normalization of the residual tumor vessels, which was also implied by low levels of angiopoietin 1 in serum biomarker profiling (0.76±0.12ng/mL). These non-invasive measures reflected a 30% reduction in microvessel density and increased vessel maturation in ex vivo sections.PAT is able to evaluate both vessel regression and normalization in response to Trebananib. Non-invasive imaging data was supported by modulation of serum markers in vitro and ex vivo histopathology.

View details for DOI 10.2967/jnumed.115.160002

View details for Web of Science ID 000365724800023

View details for PubMedID 26315834
Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging. Proceedings of the National Academy of Sciences of the United States of America Maute, R. L., Gordon, S. R., Mayer, A. T., McCracken, M. N., Natarajan, A., Ring, N. G., Kimura, R., Tsai, J. M., Manglik, A., Kruse, A. C., Gambhir, S. S., Weissman, I. L., Ring, A. M. 2015; 112 (47): E6506-14
Abstract

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

View details for DOI 10.1073/pnas.1519623112

View details for PubMedID 26604307
Glioblastoma Multiforme Recurrence: An Exploratory Study of F-18 FPPRGD(2) PET/CT1 RADIOLOGY Iagaru, A., Mosci, C., Mittra, E., Zaharchuk, G., Fischbein, N., Harsh, G., Li, G., Nagpal, S., Recht, L., Gambhir, S. S. 2015; 277 (2): 497-506
Abstract

Purpose To prospectively evaluate fluorine 18 ((18)F) 2-fluoropropionyl-labeled PEGylated dimeric arginine-glycine-aspartic acid (RGD) peptide (PEG3-E[c{RGDyk}]2) (FPPRGD2) positron emission tomography (PET) in patients with glioblastoma multiforme (GBM). Materials and Methods The institutional review board approved this HIPAA-compliant protocol. Written informed consent was obtained from each patient. (18)F FPPRGD2 uptake was measured semiquantitatively in the form of maximum standardized uptake values (SUVmax) and uptake volumes before and after treatment with bevacizumab. Vital signs and laboratory results were collected before, during, and after the examinations. A nonparametric version of multivariate analysis of variance was used to assess safety outcome measures simultaneously across time points. A paired two-sample t test was performed to compare SUVmax. Results A total of 17 participants (eight men, nine women; age range, 25-65 years) were enrolled prospectively. (18)F FPPRGD2 PET/computed tomography (CT), (18)F fluorodeoxyglucose (FDG) PET/CT, and brain magnetic resonance (MR) imaging were performed within 3 weeks, prior to the start of bevacizumab therapy. In eight of the 17 patients (47%), (18)F FPPRGD2 PET/CT was repeated 1 week after the start of bevacizumab therapy; six patients (35%) underwent (18)F FPPRGD2 PET/CT a third time 6 weeks after starting bevacizumab therapy. There were no changes in vital signs, electrocardiographic findings, or laboratory values that qualified as adverse events. One patient (6%) had recurrent GBM identified only on (18)F FPPRGD2 PET images, and subsequent MR images enabled confirmation of recurrence. Of the 17 patients, 14 (82%) had recurrent GBM identified on (18)F FPPRGD2 PET and brain MR images, while (18)F FDG PET enabled identification of recurrence in 13 (76%) patients. Two patients (12%) had no recurrent GBM. Conclusion (18)F FPPRGD2 is a safe PET radiopharmaceutical that has increased uptake in GBM lesions. Larger cohorts are required to confirm these preliminary findings. (©) RSNA, 2015 Online supplemental material is available for this article.

View details for DOI 10.1148/radiol.2015141550

View details for Web of Science ID 000368435100026

View details for PubMedID 25965900
Biodistribution of the F-18-FPPRGD(2) PET radiopharmaceutical in cancer patients: an atlas of SUV measurements EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Minamimoto, R., Jamali, M., Barkhodari, A., Mosci, C., Mittra, E., Shen, B., Chin, F., Gambhir, S. S., Iagaru, A. 2015; 42 (12): 1850-1858
Abstract

The aim of this study was to investigate the biodistribution of 2-fluoropropionyl-labeled PEGylated dimeric arginine-glycine-aspartic acid (RGD) peptide (PEG3-E[c{RGDyk}]2) ((18)F-FPPRGD2) in cancer patients and to compare its uptake in malignant lesions with (18)F-FDG uptake.A total of 35 patients (11 men, 24 women, mean age 52.1 ± 10.8 years) were enrolled prospectively and had (18)F-FPPRGD2 PET/CT prior to treatment. Maximum standardized uptake values (SUVmax) and mean SUV (SUVmean) were measured in 23 normal tissues in each patient, as well as in known or suspected cancer lesions. Differences between (18)F-FPPRGD2 uptake and (18)F-FDG uptake were also evaluated in 28 of the 35 patients.Areas of high (18)F-FPPRGD2 accumulation (SUVmax range 8.9 - 94.4, SUVmean range 7.1 - 64.4) included the bladder and kidneys. Moderate uptake (SUVmax range 2.1 - 6.3, SUVmean range 1.1 - 4.5) was found in the choroid plexus, salivary glands, thyroid, liver, spleen, pancreas, small bowel and skeleton. Compared with (18)F-FDG, (18)F-FPPRGD2 showed higher tumor-to-background ratio in brain lesions (13.4 ± 8.5 vs. 1.1 ± 0.5, P < 0.001), but no significant difference in body lesions (3.2 ± 1.9 vs. 4.4 ± 4.2, P = 0.10). There was no significant correlation between the uptake values (SUVmax and SUVmean) for (18)F FPPRGD2 and those for (18)F-FDG.The biodistribution of (18)F-FPPRGD2 in cancer patients is similar to that of other RGD dimer peptides and it is suitable for clinical use. The lack of significant correlation between (18)F-FPPRGD2 and (18)F-FDG uptake confirms that the information provided by each PET tracer is different.

View details for DOI 10.1007/s00259-015-3096-4

View details for Web of Science ID 000361997600010
PET imaging of tumor glycolysis downstream of hexokinase through noninvasive measurement of pyruvate kinase M2. Science translational medicine Witney, T. H., James, M. L., Shen, B., Chang, E., Pohling, C., Arksey, N., Hoehne, A., Shuhendler, A., Park, J., Bodapati, D., Weber, J., Gowrishankar, G., Rao, J., Chin, F. T., Gambhir, S. S. 2015; 7 (310): 310ra169-?
Abstract

Cancer cells reprogram their metabolism to meet increased biosynthetic demands, commensurate with elevated rates of replication. Pyruvate kinase M2 (PKM2) catalyzes the final and rate-limiting step in tumor glycolysis, controlling the balance between energy production and the synthesis of metabolic precursors. We report here the synthesis and evaluation of a positron emission tomography (PET) radiotracer, [(11)C]DASA-23, that provides a direct noninvasive measure of PKM2 expression in preclinical models of glioblastoma multiforme (GBM). In vivo, orthotopic U87 and GBM39 patient-derived tumors were clearly delineated from the surrounding normal brain tissue by PET imaging, corresponding to exclusive tumor-associated PKM2 expression. In addition, systemic treatment of mice with the PKM2 activator TEPP-46 resulted in complete abrogation of the PET signal in intracranial GBM39 tumors. Together, these data provide the basis for the clinical evaluation of imaging agents that target this important gatekeeper of tumor glycolysis.

View details for DOI 10.1126/scitranslmed.aac6117

View details for PubMedID 26491079
Novel Radiotracer for ImmunoPET Imaging of PD-1 Checkpoint Expression on Tumor Infiltrating Lymphocytes BIOCONJUGATE CHEMISTRY Natarajan, A., Mayer, A. T., Xu, L., Reeves, R. E., Gano, J., Gambhir, S. S. 2015; 26 (10): 2062-2069
Abstract

Immune checkpoint signaling through the programmed death 1 (PD-1) axis to its ligand (PD-L1) significantly dampens anti-tumor immune responses. Cancer patients treated with checkpoint inhibitors that block this suppressive signaling have exhibited objective response rates of 20-40% for advanced solid tumors, lymphomas, and malignant melanomas. This represents a tremendous advance in cancer treatment. Unfortunately, all patients do not respond to immune checkpoint blockade. Recent findings suggest that patients with tumor infiltrating lymphocytes (TILs) expressing PD-1 may be most likely to respond to αPD-1/PD-L1 checkpoint inhibitors. There is a compelling need for diagnostic and prognostic imaging tools to assess the PD-1 status of TILs in vivo. Here we have developed a novel immunoPET tracer to image PD-1 expressing TILs in a transgenic mouse model bearing melanoma. A (64)Cu labeled anti-mouse antibody (IgG) PD-1 immuno positron emission tomography (PET) tracer was developed to detect PD-1 expressing murine TILs. Quality control of the tracer showed >95% purity by HPLC and >70% immunoreactivity in an in vitro cell binding assay. ImmunoPET scans were performed over 1-48 h on Foxp3+.LuciDTR4 mice bearing B16-F10 melanoma tumors. Mice receiving anti-PD-1 tracer (200 ± 10 μCi/10-12 μg/200 μL) revealed high tracer uptake in lymphoid organs and tumors. BLI images of FoxP3(+) CD4(+) Tregs known to express PD-1 confirmed lymphocyte infiltration of tumors at the time of PET imaging. Biodistribution measurements performed at 48 h revealed a high (11×) tumor to muscle uptake ratio of the PET tracer (p < 0.05). PD-1 tumors exhibited 7.4 ± 0.7%ID/g tracer uptake and showed a 2× fold signal decrease when binding was blocked by unlabeled antibody. To the best of our knowledge this data is the first report to image PD-1 expression in living subjects with PET. This radiotracer has the potential to assess the prognostic value of PD-1 in preclinical models of immunotherapy and may ultimately aid in predicting response to therapies targeting immune checkpoints.

View details for DOI 10.1021/acs.bioconjchem.5b00318

View details for Web of Science ID 000363438100010

View details for PubMedID 26307602
A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers PLOS ONE Jokerst, J. V., Chen, Z., Xu, L., Nolley, R., Chang, E., Mitchell, B., Brooks, J. D., Gambhir, S. S. 2015; 10 (9)
View details for DOI 10.1371/journal.pone.0139484

View details for Web of Science ID 000362175700122
Further validation to support clinical translation of [F-18]FTC-146 for imaging sigma-1 receptors EJNMMI RESEARCH Shen, B., James, M. L., Andrews, L., Lau, C., Chen, S., Palner, M., Miao, Z., Arksey, N. C., Shuhendler, A. J., Scatliffe, S., Kaneshige, K., Parsons, S. M., McCurdy, C. R., Salehi, A., Gambhir, S. S., Chin, F. T. 2015; 5
View details for DOI 10.1186/s13550-015-0122-2

View details for Web of Science ID 000363020600001
Diketopyrrolopyrrole-Based Semiconducting Polymer Nanoparticles for In Vivo Photoacoustic Imaging ADVANCED MATERIALS Pu, K., Mei, J., Jokerst, J. V., Hong, G., Antaris, A. L., Chattopadhyay, N., Shuhendler, A. J., Kurosawa, T., Zhou, Y., Gambhir, S. S., Bao, Z., Rao, J. 2015; 27 (35): 5184-5190
Abstract

Diketopyrrolopyrrole-based semiconducting polymer nanoparticles with high photostability and strong photoacoustic brightness are designed and synthesized, which results in 5.3-fold photoacoustic signal enhancement in tumor xenografts after systemic administration.

View details for DOI 10.1002/adma.201502285

View details for Web of Science ID 000361205700011
Combined F-18-NaF and F-18-FDG PET/CT in the Evaluation of Sarcoma Patients CLINICAL NUCLEAR MEDICINE Jackson, T., Mosci, C., von Eyben, R., Mittra, E., Ganjoo, K., Biswal, S., Gambhir, S. S., Iagaru, A. 2015; 40 (9): 720-724
Abstract

The combined administration of F-NaF and F-FDG in a single PET/CT scan has the potential to improve patient convenience and cancer detection. Here we report the use of this approach for patients with sarcomas.This is a retrospective review of 21 patients (12 men, 9 women; age, 19-66 years) with biopsy-proven sarcomas who had separate F-NaF PET/CT, F-FDG PET/CT, and combined F-NaF/F-FDG PET/CT scans for evaluation of malignancy. Two board-certified nuclear medicine physicians and 1 board-certified musculoskeletal radiologist were randomly assigned to review the scans. Results were analyzed for sensitivity and specificity, using linear regression and receiver operating characteristics.A total of 13 patients had metastatic disease on F-NaF PET/CT, F-FDG PET/CT, and combined F-NaF/F-FDG PET/CT. Skeletal disease was more extensive on the F-NaF PET/CT scan than on the F-FDG PET/CT in 3 patients, whereas in 1 patient, F-FDG PET/CT showed skeletal disease and the F-NaF PET/CT was negative. Extraskeletal lesions were detected on both F-FDG and combined F-NaF/F-FDG PET/CT in 20 patients, with 1 discordant finding in the lung.The combined F-NaF/F-FDG PET/CT scan allows for accurate evaluation of sarcoma patients. Further evaluation of this proposed imaging modality is warranted to identify the most suitable clinical scenarios, including initial treatment strategy and evaluation of response to therapy.

View details for DOI 10.1097/RLU.0000000000000845

View details for Web of Science ID 000359668600005

View details for PubMedID 26053718
Androgen Receptor Splice Variants Dimerize to Transactivate Target Genes CANCER RESEARCH Xu, D., Zhan, Y., Qi, Y., Cao, B., Bai, S., Xu, W., Gambhir, S. S., Lee, P., Sartor, O., Flemington, E. K., Zhang, H., Hu, C., Dong, Y. 2015; 75 (17): 3663-3671
Abstract

Constitutively active androgen receptor splice variants (AR-V) lacking the ligand-binding domain have been implicated in the pathogenesis of castration-resistant prostate cancer and in mediating resistance to newer drugs that target the androgen axis. AR-V regulates expression of both canonical AR targets and a unique set of cancer-specific targets that are enriched for cell-cycle functions. However, little is known about how AR-V controls gene expression. Here, we report that two major AR-Vs, termed AR-V7 and AR(v567es), not only homodimerize and heterodimerize with each other but also heterodimerize with full-length androgen receptor (AR-FL) in an androgen-independent manner. We found that heterodimerization of AR-V and AR-FL was mediated by N- and C-terminal interactions and by the DNA-binding domain of each molecule, whereas AR-V homodimerization was mediated only by DNA-binding domain interactions. Notably, AR-V dimerization was required to transactivate target genes and to confer castration-resistant cell growth. Our results clarify the mechanism by which AR-Vs mediate gene regulation and provide a pivotal pathway for rational drug design to disrupt AR-V signaling as a rational strategy for the effective treatment of advanced prostate cancer.

View details for DOI 10.1158/0008-5472.CAN-15-0381

View details for Web of Science ID 000361917100021

View details for PubMedID 26060018
A Systematic Comparison of F-18-C-SNAT to Established Radiotracer Imaging Agents for the Detection of Tumor Response to Treatment CLINICAL CANCER RESEARCH Witney, T. H., Hoehne, A., Reeves, R. E., Ilovich, O., Namavari, M., Shen, B., Chin, F. T., Rao, J., Gambhir, S. S. 2015; 21 (17): 3896-3905
Abstract

An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of (18)F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3-triggered nanoaggregation.Here, we compared the preclinical utility of (18)F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo.In drug-treated lymphoma cells, (18)F-FDG, (99m)Tc-Annexin V, and (18)F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R(2) > 0.8; P < 0.001), with no correlation measured for (18)F-ML-10 (R(2) = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of (99m)Tc-Annexin V and (18)F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although (99m)Tc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either (18)F-FDG or (18)F-ML-10.We have demonstrated here that (18)F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with (18)F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility. Clin Cancer Res; 21(17); 3896-905. ©2015 AACR.

View details for DOI 10.1158/1078-0432.CCR-14-3176

View details for Web of Science ID 000361910000013

View details for PubMedID 25972517
Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics. Proceedings of the National Academy of Sciences of the United States of America Inci, F., Filippini, C., Baday, M., Ozen, M. O., Calamak, S., Durmus, N. G., Wang, S., Hanhauser, E., Hobbs, K. S., Juillard, F., Kuang, P. P., Vetter, M. L., Carocci, M., Yamamoto, H. S., Takagi, Y., Yildiz, U. H., Akin, D., Wesemann, D. R., Singhal, A., Yang, P. L., Nibert, M. L., Fichorova, R. N., Lau, D. T., Henrich, T. J., Kaye, K. M., Schachter, S. C., Kuritzkes, D. R., Steinmetz, L. M., Gambhir, S. S., Davis, R. W., Demirci, U. 2015; 112 (32): E4354-63
Abstract

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

View details for DOI 10.1073/pnas.1510824112

View details for PubMedID 26195743
Radiation Dosimetry Study of [Zr-89]rituximab Tracer for Clinical Translation of B cell NHL Imaging using Positron Emission Tomography MOLECULAR IMAGING AND BIOLOGY Natarajan, A., Gambhir, S. S. 2015; 17 (4): 539-547
Abstract

We evaluated the dosimetry of [(89)Zr]rituximab, an anti-CD20 immunoPET tracer to image B cell non-Hodgkin's lymphoma (NHL) using a humanized transgenic mouse model that expresses human CD20 transgenic mice (huCD20TM).Rituximab was conjugated to desferrioxamine (Df) for radiolabeling of Zirconium-89. [(89)Zr]rituximab (2.8 ± 0.2 MBq) was tail vein-injected into huCD20T mice. Positron emission tomography (PET)/CT imaging was performed on the two groups of mice (blocking = 2 mg/kg pre-dose of rituximab and non-blocking; n = 5) at eight time points (1, 4, 24, 48, 72, 96, 120, and 168 h) post injection.The novel [(89)Zr]rituximab PET tracer had good immunoreactivity, was stable in human serum, and was able to specifically target human CD20 in mice. The human equivalents of highest dose (mean ± SD) organs with and without pre-dose are liver (345 ± 284 μSv/MBq) and spleen (1165 ± 149 μSv/MBq), respectively.Dosimetry of the human patient whole-body dose was found to be 145 MBq per annum, and the patient dose-limiting organ will be the liver (with rituximab pre-dose blocking) and spleen for non-blocking. The [(89)Zr]rituximab (t½ = 78.4 h) imaging of B cell NHL patients could permit the observation of targeting lesions in NHL patients over an extended period due to longer half-life as compared to the [(64)Cu] rituximab (t½ = 12.7 h).

View details for DOI 10.1007/s11307-014-0810-8

View details for Web of Science ID 000357504300013

View details for PubMedID 25500766
Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2 PLOS ONE Park, S., Park, S., Kim, D., Pyo, A., Kimura, R. H., Sathirachinda, A., Choy, H. E., Min, J., Gambhir, S. S., Hong, Y. 2015; 10 (7)
View details for DOI 10.1371/journal.pone.0132976

View details for Web of Science ID 000358197600194
Early detection of sporadic pancreatic cancer: summative review. Pancreas Chari, S. T., Kelly, K., Hollingsworth, M. A., Thayer, S. P., Ahlquist, D. A., Andersen, D. K., Batra, S. K., Brentnall, T. A., Canto, M., Cleeter, D. F., Firpo, M. A., Gambhir, S. S., Go, V. L., Hines, O. J., Kenner, B. J., Klimstra, D. S., Lerch, M. M., Levy, M. J., Maitra, A., Mulvihill, S. J., Petersen, G. M., Rhim, A. D., Simeone, D. M., Srivastava, S., Tanaka, M., Vinik, A. I., Wong, D. 2015; 44 (5): 693-712
Abstract

Pancreatic cancer (PC) is estimated to become the second leading cause of cancer death in the United States by 2020. Early detection is the key to improving survival in PC. Addressing this urgent need, the Kenner Family Research Fund conducted the inaugural Early Detection of Sporadic Pancreatic Cancer Summit Conference in 2014 in conjunction with the 45th Anniversary Meeting of the American Pancreatic Association and Japan Pancreas Society. This seminal convening of international representatives from science, practice, and clinical research was designed to facilitate challenging interdisciplinary conversations to generate innovative ideas leading to the creation of a defined collaborative strategic pathway for the future of the field. An in-depth summary of current efforts in the field, analysis of gaps in specific areas of expertise, and challenges that exist in early detection is presented within distinct areas of inquiry: Case for Early Detection: Definitions, Detection, Survival, and Challenges; Biomarkers for Early Detection; Imaging; and Collaborative Studies. In addition, an overview of efforts in familial PC is presented in an addendum to this article. It is clear from the summit deliberations that only strategically designed collaboration among investigators, institutions, and funders will lead to significant progress in early detection of sporadic PC.

View details for DOI 10.1097/MPA.0000000000000368

View details for PubMedID 25931254
Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope RADIOLOGY Ilovich, O., Natarajan, A., Hori, S., Sathirachinda, A., Kimura, R., Srinivasan, A., Gebauer, M., Kruip, J., Focken, I., Lange, C., Carrez, C., Sassoon, I., Blanc, V., Sarkar, S. K., Gambhir, S. S. 2015; 276 (1): 191-198
Abstract

Purpose To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. Materials and Methods Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. Results The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. Conclusion Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo. (©) RSNA, 2015 Online supplemental material is available for this article.

View details for DOI 10.1148/radiol.15140058

View details for Web of Science ID 000359708400021
Predictive Modeling of Drug Response in Non-Hodgkin's Lymphoma PLOS ONE Frieboes, H. B., Smith, B. R., Wang, Z., Kotsuma, M., Ito, K., Day, A., Cahill, B., Flinders, C., Mumenthaler, S. M., Mallick, P., Simbawa, E., Al-Fhaid, A. S., Mahmoud, S. R., Gambhir, S. S., Cristini, V. 2015; 10 (6)
Abstract

We combine mathematical modeling with experiments in living mice to quantify the relative roles of intrinsic cellular vs. tissue-scale physiological contributors to chemotherapy drug resistance, which are difficult to understand solely through experimentation. Experiments in cell culture and in mice with drug-sensitive (Eµ-myc/Arf-/-) and drug-resistant (Eµ-myc/p53-/-) lymphoma cell lines were conducted to calibrate and validate a mechanistic mathematical model. Inputs to inform the model include tumor drug transport characteristics, such as blood volume fraction, average geometric mean blood vessel radius, drug diffusion penetration distance, and drug response in cell culture. Model results show that the drug response in mice, represented by the fraction of dead tumor volume, can be reliably predicted from these inputs. Hence, a proof-of-principle for predictive quantification of lymphoma drug therapy was established based on both cellular and tissue-scale physiological contributions. We further demonstrate that, if the in vitro cytotoxic response of a specific cancer cell line under chemotherapy is known, the model is then able to predict the treatment efficacy in vivo. Lastly, tissue blood volume fraction was determined to be the most sensitive model parameter and a primary contributor to drug resistance.

View details for DOI 10.1371/journal.pone.0129433

View details for Web of Science ID 000355979500143

View details for PubMedID 26061425
Cu-64-Labeled Divalent Cystine Knot Peptide for Imaging Carotid Atherosclerotic Plaques JOURNAL OF NUCLEAR MEDICINE Jiang, L., Tu, Y., Kimura, R. H., Habte, F., Chen, H., Cheng, K., Shi, H., Gambhir, S. S., Cheng, Z. 2015; 56 (6): 939-944
Abstract

The rupture of vulnerable atherosclerotic plaques that lead to stroke and myocardial infarction may be induced by macrophage infiltration and augmented by the expression of integrin αvβ3. Indeed, atherosclerotic angiogenesis may be a promising marker of inflammation. In this study, an engineered integrin αvβ3-targeting PET probe, (64)Cu-NOTA-3-4A, derived from a divalent knottin miniprotein was evaluated in a mouse model for carotid atherosclerotic plaques.Atherosclerotic plaques in BALB/C mice, maintained on a high-fat diet, were induced with streptozotocin injection and carotid artery ligation and verified by MR imaging. Knottin 3-4A was synthesized by solid-phase peptide synthesis chemistry and coupled to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) before radiolabeling with (64)Cu. PET probe stability in mouse serum was evaluated. Mice with carotid atherosclerotic plaques were injected via the tail vein with (64)Cu-NOTA-3-4A or (18)F-FDG, followed by small-animal PET/CT imaging at different time points. Receptor targeting specificity of the probe was verified by coinjection of c(RGDyK) administered in molar excess. Subsequently, carotid artery dissection and immunofluorescence staining were performed to evaluate target expression.(64)Cu-NOTA-3-4A was synthesized in high radiochemical purity and yield and demonstrated molecular stability in both phosphate-buffered saline and mouse serum at 4 h. Small-animal PET/CT showed that (64)Cu-NOTA-3-4A accumulated at significantly higher levels in the neovasculature of carotid atherosclerotic plaques (7.41 ± 1.44 vs. 0.67 ± 0.23 percentage injected dose/gram, P < 0.05) than healthy or normal vessels at 1 h after injection. (18)F-FDG also accumulated in atherosclerotic lesions at 0.5 and 1 h after injection but at lower plaque-to-normal tissue ratios than (64)Cu-NOTA-3-4A. For example, plaque-to-normal carotid artery ratios for (18)F-FDG and (64)Cu-NOTA-3-4A at 1 h after injection were 3.75 and 14.71 (P < 0.05), respectively. Furthermore, uptake of (64)Cu-NOTA-3-4A in atherosclerotic plaques was effectively blocked (∼90% at 1 h after injection) by coinjection of c(RGDyK). Immunostaining confirmed integrin αvβ3 expression in both the infiltrating macrophages and the neovasculature of atherosclerotic plaques.(64)Cu-NOTA-3-4A demonstrates specific accumulation in carotid atherosclerotic plaques in which macrophage infiltration and angiogenesis are responsible for elevated integrin αvβ3 levels. Therefore, (64)Cu-NOTA-3-4A may demonstrate clinical utility as a PET probe for atherosclerosis imaging or for the evaluation of therapies used to treat atherosclerosis.

View details for DOI 10.2967/jnumed.115.155176

View details for Web of Science ID 000355570300026
Development of a High-Throughput Molecular Imaging-Based Orthotopic Hepatocellular Carcinoma Model. Cureus Hwang, G. L., van den Bosch, M. A., Kim, Y. I., Katzenberg, R., Willmann, J. K., Paulmurugan, R., Gambhir, S. S., Hofmann, L. 2015; 7 (6)
Abstract

We have developed a novel orthotopic rat hepatocellular (HCC) model and have assessed the ability to use bioluminescence imaging (BLI), positron emission tomography (PET), and ultrasound for early tumor detection and monitoring of disease progression. Briefly, rat HCC cells were stably transfected with click beetle red as a reporter gene for BLI. Tumor cells were injected under direct visualization into the left or middle lobe of the liver in 37 rats. In six animals, serial PET, BLI, and ultrasound imaging were performed at 10-time points in 28 days. The remainder of the animals underwent PET imaging at 14 days. Tumor implantation was successful in 34 of 37 animals (91.9%). In the six animals that underwent serial imaging, tumor formation was first detected with BLI on Day 4 with continued increase through Day 21, and hypermetabolic activity on PET was first noted on Days 14-15 with continued increase through Day 28. PET activity was seen on Day 14 in the 28 other animals that demonstrated tumor development. Anatomic tumor formation was detected with ultrasound at Days 10-12 with continued growth through Day 28. The first metastases were detected by PET after Day 24. We have successfully developed and validated a novel orthotopic HCC small animal model that permits longitudinal assessment of change in tumor size using molecular imaging techniques. BLI is the most sensitive imaging method for detection of early tumor formation and growth. This model permits high-throughput in vivo evaluation of image-guided therapies.

View details for DOI 10.7759/cureus.281

View details for PubMedID 26180705
Semiquantitative Analysis of the Biodistribution of the Combined F-18-NaF and F-18-FDG Administration for PET/CT Imaging JOURNAL OF NUCLEAR MEDICINE Minamimoto, R., Mosci, C., Jamali, M., Barkhodari, A., Habte, F., Jackson, T., Mittra, E., Gambhir, S. S., Iagaru, A. 2015; 56 (5): 688-694
Abstract

In this study we evaluated the biodistribution of the (18)F-/(18)F-FDG administration compared to separate (18)F-NaF and (18)F-FDG. We also estimated the interaction of (18)F-NaF and (18)F-FDG in the (18)F-/(18)F-FDG administration by semiquantitative analysis.We retrospectively analyzed data of 49 patients (male 39, female 10; mean ± SD age: 59.3 ± 15.2 years) who had separate (18)F-FDG PET/CT and (18)F-NaF PET/CT, as well as the (18)F-/(18)F-FDG PET/CT sequentially. The most common primary diagnosis was prostate cancer (n = 28), followed by sarcoma (n = 9) and breast cancer (n = 6). The mean standardized uptake values (SUVmean) were recorded for 18 organs in all patients, while maximum SUV (SUVmax) and SUVmean were recorded for all the identified malignant lesions. We also estimated the (18)F-/(18)F-FDG uptake by sum of (18)F-FDG uptake and adjusted (18)F-NaF uptake based on the ratio of (18)F-NaF injected dose in (18)F-/(18)F-FDG PET/CT. Lastly, we compared the results in order to explore the interaction of (18)F-FDG and (18)F-NaF uptake in the (18)F-/(18)F-FDG scan.The (18)F-/(18)F-FDG uptake in the cerebral cortex, cerebellum, parotid grand, myocardium and bowel mostly reflect the (18)F-FDG uptake, while the uptake in the other analyzed structures is influenced by both the (18)F-FDG and the (18)F-NaF uptake. The (18)F-/(18)F-FDG uptake in extra skeletal lesions shows no significant difference when compared to the uptake from the separate (18)F-FDG scan. The (18)F-/(18)F-FDG uptake in skeletal lesions reflected mostly the (18)F-NaF uptake. Tumor to background (T/B) ratio of (18)F-/(18)F-FDG in extra skeletal lesions showed no significant difference when compared with that from (18)F-FDG alone (P = 0.73). For skeletal lesions, T/B ratio of (18)F-/(18)F-FDG was lower than that from (18)F-NaF alone (P <0.001); however, this difference did not result in missed skeletal lesions on the (18)F-/(18)F-FDG scan.The understanding of the biodistribution of radiopharmaceuticals and the lesions uptake of the (18)F-/(18)F-FDG scan, as well as the variations compared to the uptake on the separate (18)F-FDG PET/CT and (18)F-NaF PET/CT are valuable for more in depth evaluation of the combined scanning technique.

View details for DOI 10.2967/jnumed.115.153767

View details for Web of Science ID 000353831000013
Optical coherence contrast imaging using gold nanorods in living mice eyes CLINICAL AND EXPERIMENTAL OPHTHALMOLOGY de la Zerda, A., Prabhulkar, S., Perez, V. L., Ruggeri, M., Paranjape, A. S., Habte, F., Gambhir, S. S., Awdeh, R. M. 2015; 43 (4): 358-366
Abstract

Optical coherence tomography (OCT) is a powerful imaging modality to visualize tissue structures, with axial image pixel resolution as high as 1.6 μm in tissue. However, OCT is intrinsically limited to providing structural information as the OCT contrast is produced by optically scattering tissues.Gold nanorods (GNRs) were injected into the anterior chamber (AC) and cornea of mice eyes which could create a significant OCT signal and hence could be used as a contrast agent for in vivo OCT imaging.A dose of 30 nM of GNRs (13 nm in diameter and 45 nm in length) were injected to the AC of mice eyes and produced an OCT contrast nearly 50-fold higher than control mice injected with saline. Furthermore, the lowest detectable concentration of GNRs in living mice AC was experimentally estimated to be as low as 120 pM.The high sensitivity and low toxicity of GNRs brings great promise for OCT to uniquely become a high-resolution molecular imaging modality.

View details for DOI 10.1111/ceo.12299

View details for Web of Science ID 000356810200009
A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles PLOS ONE Garai, E., Sensarn, S., Zavaleta, C. L., Loewke, N. O., Rogalla, S., Mandella, M. J., Felt, S. A., Friedland, S., Liu, J. T., Gambhir, S. S., Contag, C. H. 2015; 10 (4)
View details for DOI 10.1371/journal.pone.0123185

View details for Web of Science ID 000353711600032
Synthesis of [F-18]-labelled Maltose Derivatives as PET Tracers for Imaging Bacterial Infection MOLECULAR IMAGING AND BIOLOGY Namavari, M., Gowrishankar, G., Hoehne, A., Jouannot, E., Gambhir, S. S. 2015; 17 (2): 168-176
Abstract

To develop novel positron emission tomography (PET) agents for visualization and therapy monitoring of bacterial infections.It is known that maltose and maltodextrins are energy sources for bacteria. Hence, (18)F-labelled maltose derivatives could be a valuable tool for imaging bacterial infections. We have developed methods to synthesize 4-O-(α-D-glucopyranosyl)-6-deoxy-6-[(18)F]fluoro-D-glucopyranoside (6-[(18)F]fluoromaltose) and 4-O-(α-D-glucopyranosyl)-1-deoxy-1-[(18)F]fluoro-D-glucopyranoside (1-[(18)F]fluoromaltose) as bacterial infection PET imaging agents. 6-[(18)F]fluoromaltose was prepared from precursor 1,2,3-tri-O-acetyl-4-O-(2',3',-di-O-acetyl-4',6'-benzylidene-α-D-glucopyranosyl)-6-deoxy-6-nosyl-D-glucopranoside (5). The synthesis involved the radio-fluorination of 5 followed by acidic and basic hydrolysis to give 6-[(18)F]fluoromaltose. In an analogous procedure, 1-[(18)F]fluoromaltose was synthesized from 2,3, 6-tri-O-acetyl-4-O-(2',3',4',6-tetra-O-acetyl-α-D-glucopyranosyl)-1-deoxy-1-O-triflyl-D-glucopranoside (9). Stability of 6-[(18)F]fluoromaltose in phosphate-buffered saline (PBS) and human and mouse serum at 37 °C was determined. Escherichia coli uptake of 6-[(18)F]fluoromaltose was examined.A reliable synthesis of 1- and 6-[(18)F]fluoromaltose has been accomplished with 4-6 and 5-8 % radiochemical yields, respectively (decay-corrected with 95 % radiochemical purity). 6-[(18)F]fluoromaltose was sufficiently stable over the time span needed for PET studies (∼96 % intact compound after 1-h and ∼65 % after 2-h incubation in serum). Bacterial uptake experiments indicated that E. coli transports 6-[(18)F]fluoromaltose. Competition assays showed that the uptake of 6-[(18)F]fluoromaltose was completely blocked by co-incubation with 1 mM of the natural substrate maltose.We have successfully synthesized 1- and 6-[(18)F]fluoromaltose via direct fluorination of appropriate protected maltose precursors. Bacterial uptake experiments in E. coli and stability studies suggest a possible application of 6-[(18)F]fluoromaltose as a new PET imaging agent for visualization and monitoring of bacterial infections.

View details for DOI 10.1007/s11307-014-0793-5

View details for Web of Science ID 000351092900003
Validation of Cu-64-DOTA-Rituximab Injection Preparation Under Good Manufacturing Practices: A PET Tracer for Imaging of B-Cell Non-Hodgkin Lymphoma MOLECULAR IMAGING Natarajan, A., Arksey, N., Iagaru, A., Chin, F. T., Gambhir, S. S. 2015; 14
View details for DOI 10.2310/7290.2014.00055

View details for Web of Science ID 000365595700005
Detecting cancers through tumor-activatable minicircles that lead to a detectable blood biomarker PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ronald, J. A., Chuang, H., Dragulescu-Andrasi, A., Hori, S. S., Gambhir, S. S. 2015; 112 (10): 3068-3073
Abstract

Earlier detection of cancers can dramatically improve the efficacy of available treatment strategies. However, despite decades of effort on blood-based biomarker cancer detection, many promising endogenous biomarkers have failed clinically because of intractable problems such as highly variable background expression from nonmalignant tissues and tumor heterogeneity. In this work we present a tumor-detection strategy based on systemic administration of tumor-activatable minicircles that use the pan-tumor-specific Survivin promoter to drive expression of a secretable reporter that is detectable in the blood nearly exclusively in tumor-bearing subjects. After systemic administration we demonstrate a robust ability to differentiate mice bearing human melanoma metastases from tumor-free subjects for up to 2 wk simply by measuring blood reporter levels. Cumulative change in reporter levels also identified tumor-bearing subjects, and a receiver operator-characteristic curve analysis highlighted this test's performance with an area of 0.918 ± 0.084. Lung tumor burden additionally correlated (r(2) = 0.714; P < 0.05) with cumulative reporter levels, indicating that determination of disease extent was possible. Continued development of our system could improve tumor detectability dramatically because of the temporally controlled, high reporter expression in tumors and nearly zero background from healthy tissues. Our strategy's highly modular nature also allows it to be iteratively optimized over time to improve the test's sensitivity and specificity. We envision this system could be used first in patients at high risk for tumor recurrence, followed by screening high-risk populations before tumor diagnosis, and, if proven safe and effective, eventually may have potential as a powerful cancer-screening tool for the general population.

View details for DOI 10.1073/pnas.1414156112

View details for Web of Science ID 000350646500049
18F-FPRGD2 PET/CT imaging of integrin avß3 in renal carcinomas: correlation with histopathology. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Withofs, N., Signolle, N., Somja, J., Lovinfosse, P., Nzaramba, E. M., Mievis, F., Giacomelli, F., Waltregny, D., Cataldo, D., Gambhir, S. S., Hustinx, R. 2015; 56 (3): 361-364
Abstract

This study aimed to correlate (18)F-FB-mini-PEG-E[c(RGDyK)](2) ((18)F-FPRGD2) uptake to integrin αvβ3 expression and angiogenesis in renal tumors.(18)F-FPRGD2 PET/CT was performed on 27 patients before surgical resection (median 4 d) of a renal mass. The (18)F-FPRGD2 uptake was compared with integrin αvβ3, CD31, CD105, and Ki-67 using immunohistochemistry; with placental growth factor and vascular endothelial growth factor receptors 1 and 2 using reverse transcription polymerase chain reaction; and with vascular endothelial growth factor A isoforms using enzyme-linked immunosorbent assay.Overall, (18)F-FPRGD2 uptake significantly correlated (P < 0.0001) with integrin αvβ3 expression in renal masses. However, it correlated only with integrin αvβ3-positive vessels in the group of papillary carcinomas whereas it correlated with integrin αvβ3 expression by tumor cells in the clear cell carcinoma group.(18)F-FPRGD2 uptake reflects the expression of integrin αvβ3 in renal tumors but represents angiogenesis only when tumor cells do not express the integrin.

View details for DOI 10.2967/jnumed.114.149021

View details for PubMedID 25655629
Detection of Osseous Metastasis by F-18-NaF/F-18-FDG PET/CT Versus CT Alone CLINICAL NUCLEAR MEDICINE Sampath, S. C., Sampath, S. C., Mosci, C., Lutz, A. M., Willmann, J. K., Mittra, E. S., Gambhir, S. S., Iagaru, A. 2015; 40 (3): E173-E177
View details for Web of Science ID 000349471100001
PET Imaging of Translocator Protein (18 kDa) in a Mouse Model of Alzheimer's Disease Using N-(2,5-Dimethoxybenzyl)-2-F-18-Fluoro-N-(2-Phenoxyphenyl)Acetamide JOURNAL OF NUCLEAR MEDICINE James, M. L., Belichenko, N. P., Nguyen, T. V., Andrews, L. E., Ding, Z., Liu, H., Bodapati, D., Arksey, N., Shen, B., Cheng, Z., Wyss-Coray, T., Gambhir, S. S., Longo, F. M., Chin, F. T. 2015; 56 (2): 311-316
Abstract

Herein we aimed to evaluate the utility of N-(2,5-dimethoxybenzyl)-2-(18)F-fluoro-N-(2-phenoxyphenyl)acetamide ((18)F-PBR06) for detecting alterations in translocator protein (TSPO) (18 kDa), a biomarker of microglial activation, in a mouse model of Alzheimer's disease (AD).Wild-type (wt) and AD mice (i.e., APP(L/S)) underwent (18)F-PBR06 PET imaging at predetermined time points between the ages of 5-6 and 15-16 mo. MR images were fused with PET/CT data to quantify (18)F-PBR06 uptake in the hippocampus and cortex. Ex vivo autoradiography and TSPO/CD68 immunostaining were also performed using brain tissue from these mice.PET images showed significantly higher accumulation of (18)F-PBR06 in the cortex and hippocampus of 15- to 16-mo-old APP(L/S) mice than age-matched wts (cortex/muscle: 2.43 ± 0.19 vs. 1.55 ± 0.15, P < 0.005; hippocampus/muscle: 2.41 ± 0.13 vs. 1.55 ± 0.12, P < 0.005). And although no significant difference was found between wt and APP(L/S) mice aged 9-10 mo or less using PET (P = 0.64), we were able to visualize and quantify a significant difference in (18)F-PBR06 uptake in these mice using autoradiography (cortex/striatum: 1.13 ± 0.04 vs. 0.96 ± 0.01, P < 0.05; hippocampus/striatum: 1.266 ± 0.003 vs. 1.096 ± 0.017, P < 0.001). PET results for 15- to 16-mo-old mice correlated well with autoradiography and immunostaining (i.e., increased (18)F-PBR06 uptake in brain regions containing elevated CD68 and TSPO staining in APP(L/S) mice, compared with wts).(18)F-PBR06 shows great potential as a tool for visualizing TSPO/microglia in the progression and treatment of AD.

View details for DOI 10.2967/jnumed.114.141648

View details for Web of Science ID 000349324300035

View details for PubMedID 25613536
Sol-Gel Synthesis and Electrospraying of Biodegradable (P2O5)(55)-(CaO)(30)-(Na2O)(15) Glass Nanospheres as a Transient Contrast Agent for Ultrasound Stem Cell Imaging ACS NANO Foroutan, F., Jokerst, J. V., Gambhir, S. S., Vermesh, O., Kim, H., Knowles, J. C. 2015; 9 (2): 1868-1877
Abstract

Ultrasound imaging is a powerful tool in medicine because of the millisecond temporal resolution and submillimeter spatial resolution of acoustic imaging. However, the current generation of acoustic contrast agents is primarily limited to vascular targets due to their large size. Nanosize particles have the potential to be used as a contrast agent for ultrasound molecular imaging. Silica-based nanoparticles have shown promise here; however, their slow degradation rate may limit their applications as a contrast agent. Phosphate-based glasses are an attractive alternative with controllable degradation rate and easily metabolized degradation components in the body. In this study, biodegradable P2O5-CaO-Na2O phosphate-based glass nanospheres (PGNs) were synthesized and characterized as contrast agents for ultrasound imaging. The structure of the PGNs was characterized using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), (31)P magic angle spinning nuclear magnetic resonance ((31)P MAS NMR), and Fourier transform infrared (FTIR) spectroscopy. The SEM images indicated a spherical shape with a diameter size range of 200-500 nm. The XRD, (31)P NMR, and FTIR results revealed the amorphous and glassy nature of PGNs that consisted of mainly Q(1) and Q(2) phosphate units. We used this contrast to label mesenchymal stem cells and determined in vitro and in vivo detection limits of 5 and 9 μg/mL, respectively. Cell counts down to 4000 could be measured with ultrasound imaging with no cytoxicity at doses needed for imaging. Importantly, ion-release studies confirmed these PGNs biodegrade into aqueous media with degradation products that can be easily metabolized in the body.

View details for DOI 10.1021/nn506789y

View details for Web of Science ID 000349940500083
F-18-FAZA PET Imaging Response Tracks the Reoxygenation of Tumors in Mice upon Treatment with the Mitochondrial Complex I Inhibitor BAY 87-2243 CLINICAL CANCER RESEARCH Chang, E., Liu, H., Unterschemmann, K., Ellinghaus, P., Liu, S., Gekeler, V., Cheng, Z., Berndorff, D., Gambhir, S. S. 2015; 21 (2): 335-346
Abstract

We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity.Four PET tracers [(18)F-FDG, (18)F-Fpp(RGD)2, (18)F-FLT, and (18)F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes.Significant changes in uptake of (18)F-FAZA, (18)F-FLT, and (18)F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with (18)F-FAZA being the most prominent. (18)F-FDG uptake was unaffected. (18)F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. (18)F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting (18)F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced (18)F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed.Our studies suggest suitability of (18)F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243). Clin Cancer Res; 21(2); 335-46. ©2014 AACR.

View details for Web of Science ID 000347938200015
Validation of 64Cu-DOTA-rituximab injection preparation under good manufacturing practices: a PET tracer for imaging of B-cell non-Hodgkin lymphoma. Molecular imaging Natarajan, A., Arksey, N., Iagaru, A., Chin, F. T., Gambhir, S. S. 2015; 14
Abstract

AbstractManufacturing of 64Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-rituximab injection under good manufacturing practices (GMP) was validated for imaging of patients with CD20+ B-cell non-Hodgkin lymphoma. Rituximab was purified by size exclusion high performance liquid chromatography (HPLC) and conjugated to DOTA-mono-(N-hydroxysuccinimidyl) ester. 64CuCl2, buffers, reagents, and other raw materials were obtained as high-grade quality. Following a semi-automated synthesis of 64Cu-DOTA-rituximab, a series of quality control tests was performed. The product was further tested in vivo using micro-positron emission tomography/computed tomography (PET/CT) to assess targeting ability towards human CD20 in transgenic mice. Three batches of 64Cu-DOTA-rituximab final product were prepared as per GMP specifications. The radiolabeling yield from these batches was 93.1 ± 5.8%; these provided final product with radiopharmaceutical yield, purity, and specific activity of 59.2 ± 5.1% (0.9 ± 0.1 GBq of 64Cu), > 95% (by HPLC and radio-thin layer chromatography), and 229.4 ± 43.3 GBq/µmol (or 1.5 ± 0.3 MBq/µg), respectively. The doses passed apyrogenicity and human serum stability specifications, were sterile up to 14 days, and retained > 60% immunoreactivity. In vivo micro-PET/CT mouse images at 24 hours postinjection showed that the tracer targeted the intended sites of human CD20 expression. Thus, we have validated the manufacturing of GMP grade 64Cu-DOTA-rituximab for injection in the clinical setting.

View details for DOI 10.2310/7290.2014.00055

View details for PubMedID 25762106
A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers. PloS one Jokerst, J. V., Chen, Z., Xu, L., Nolley, R., Chang, E., Mitchell, B., Brooks, J. D., Gambhir, S. S. 2015; 10 (9)
Abstract

Novel biomarker assays and upgraded analytical tools are urgently needed to accurately discriminate benign prostatic hypertrophy (BPH) from prostate cancer (CaP). To address this unmet clinical need, we report a piezeoelectric/magnetic bead-based assay to quantitate prostate specific antigen (PSA; free and total), prostatic acid phosphatase, carbonic anhydrase 1 (CA1), osteonectin, IL-6 soluble receptor (IL-6sr), and spondin-2. We used the sensor to measure these seven proteins in serum samples from 120 benign prostate hypertrophy patients and 100 Gleason score 6 and 7 CaP using serum samples previously collected and banked. The results were analyzed with receiver operator characteristic curve analysis. There were significant differences between BPH and CaP patients in the PSA, CA1, and spondin-2 assays. The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation-the area under the curve was 0.84 with a p value below 10-6. Some of these data seem to contradict previous reports and highlight the importance of sample selection and proper assay building in the development of biomarker measurement schemes. This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair.

View details for DOI 10.1371/journal.pone.0139484

View details for PubMedID 26421725
Parts per billion detection of uranium with a porphyrinoid-containing nanoparticle and in vivo photoacoustic imaging ANALYST Ho, I., Sessler, J. L., Gambhir, S. S., Jokerst, J. V. 2015; 140 (11): 3731-3737
Abstract

Chemical tools that can report radioactive isotopes would be of interest to the defense community. Here we report ∼250 nm polymeric nanoparticles containing porphyrinoid macrocycles with and without pre-complexed depleted uranium and demonstrate that the latter species may be detected easily and with high sensitivity via photoacoustic imaging. The porphyrinoid macrocycles used in the present study are non-aromatic in the absence of the uranyl cation, but aromatic after cation complexation. We solubilized both the freebase and metalated forms of the macrocycles in poly(lactic-co-glycolic acid) and found a peak in the photoacoustic spectrum at 910 nm excitation in the case of the uranyl complex. The signal was stable for at least 15 minutes and allowed detection of uranium concentrations down to 6.2 ppb (5.7 nM) in vitro and 0.57 ppm (19 fCi; 0.52 μM) in vivo. To the best of our knowledge, this is the first report of a nanoparticle that detects an actinide cation via photoacoustic imaging.

View details for DOI 10.1039/c5an00207a

View details for Web of Science ID 000354650300006

View details for PubMedID 25854506
A multimodal imaging agent for intrinsic surface enhanced Raman scattering of biological tissue PLASMONICS IN BIOLOGY AND MEDICINE XII Pohling, C. B., Campbell, J. L., Larson, T. A., Gambhir, S. S. 2015; 9340
View details for DOI 10.1117/12.2077890

View details for Web of Science ID 000353615600006
Theranostic mesoporous silica nanoparticles biodegrade after pro-survival drug delivery and ultrasound/magnetic resonance imaging of stem cells. Theranostics Kempen, P. J., Greasley, S., Parker, K. A., Campbell, J. L., Chang, H., Jones, J. R., Sinclair, R., Gambhir, S. S., Jokerst, J. V. 2015; 5 (6): 631-642
Abstract

Increasing cell survival in stem cell therapy is an important challenge for the field of regenerative medicine. Here, we report theranostic mesoporous silica nanoparticles that can increase cell survival through both diagnostic and therapeutic approaches. First, the nanoparticle offers ultrasound and MRI signal to guide implantation into the peri-infarct zone and away from the most necrotic tissue. Second, the nanoparticle serves as a slow release reservoir of insulin-like growth factor (IGF)-a protein shown to increase cell survival. Mesenchymal stem cells labeled with these nanoparticles had detection limits near 9000 cells with no cytotoxicity at the 250 µg/mL concentration required for labeling. We also studied the degradation of the nanoparticles and showed that they clear from cells in approximately 3 weeks. The presence of IGF increased cell survival up to 40% (p<0.05) versus unlabeled cells under in vitro serum-free culture conditions.

View details for DOI 10.7150/thno.11389

View details for PubMedID 25825602
A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors MICRON Kempen, P. J., Kircher, M. F., de la Zerda, A., Zavaleta, C. L., Jokerst, J. V., Mellinghoff, I. K., Gambhir, S. S., Sinclair, R. 2015; 68: 70-76
Abstract

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy.

View details for DOI 10.1016/j.micron.2014.09.004

View details for Web of Science ID 000348016500011

View details for PubMedID 25464144
Simultaneous Whole-Body Time-of-Flight F-18-FDG PET/MRI A Pilot Study Comparing SUVmax With PET/CT and Assessment of MR Image Quality CLINICAL NUCLEAR MEDICINE Iagaru, A., Mittra, E., Minamimoto, R., Jamali, M., Levin, C., Quon, A., Gold, G., Herfkens, R., Vasanawala, S., Gambhir, S. S., Zaharchuk, G. 2015; 14 (1): 1-8
View details for Web of Science ID 000346633400023
Syntheses and Discovery of a Novel Class of Cinnamic Hydroxamates as Histone Deacetylase Inhibitors by Multimodality Molecular Imaging in Living Subjects CANCER RESEARCH CHAN, C. T., Qi, J., Smith, W., Paranol, R., Mazitschek, R., West, N., Reeves, R., Chiosis, G., Schreiber, S. L., Bradner, J. E., Paulmurugan, R., Gambhir, S. S. 2014; 74 (24): 7475-7486
Abstract

Histone deacetylases (HDAC) that regulate gene expression are being explored as cancer therapeutic targets. In this study, we focused on HDAC6 based on its ability to inhibit cancerous Hsp90 chaperone activities by disrupting Hsp90/p23 interactions. To identify novel HDAC6 inhibitors, we used a dual-luciferase reporter system in cell culture and living mice by bioluminescence imaging (BLI). On the basis of existing knowledge, a library of hydrazone compounds was generated for screening by coupling cinnamic hydroxamates with aldehydes and ketones. Potency and selectivity were determined by in vitro HDAC profiling assays, with further evaluation to inhibit Hsp90(α/β)/p23 interactions by BLI. In this manner, we identified compound 1A12 as a dose-dependent inhibitor of Hsp90(α/β)/p23 interactions, UKE-1 myeloid cell proliferation, p21(waf1) upregulation, and acetylated histone H3 levels. 1A12 was efficacious in tumor xenografts expressing Hsp90(α)/p23 reporters relative to carrier control-treated mice as determined by BLI. Small animal (18)F-FDG PET/CT imaging on the same cohort showed that 1A12 also inhibited glucose metabolism relative to control subjects. Ex vivo analyses of tumor lysates showed that 1A12 administration upregulated acetylated-H3 by approximately 3.5-fold. Taken together, our results describe the discovery and initial preclinical validation of a novel selective HDAC inhibitor.

View details for DOI 10.1158/0008-5472.CAN-14-0197

View details for Web of Science ID 000346363900031

View details for PubMedID 25320008
Noninvasive Reporter Gene Imaging of Human Oct4 (Pluripotency) Dynamics During the Differentiation of Embryonic Stem Cells in Living Subjects MOLECULAR IMAGING AND BIOLOGY Ahn, B., Parashurama, N., Patel, M., Ziv, K., Bhaumik, S., Yaghoubi, S. S., Paulmurugan, R., Gambhir, S. S. 2014; 16 (6): 865-876
View details for DOI 10.1007/s11307-014-0744-1

View details for Web of Science ID 000345281600014
A Radiofluorinated Divalent Cystine Knot Peptide for Tumor PET Imaging MOLECULAR PHARMACEUTICS Jiang, L., Kimura, R. H., Ma, X., Tu, Y., Miao, Z., Shen, B., Chin, F. T., Shi, H., Gambhir, S. S., Cheng, Z. 2014; 11 (11): 3885-3892
View details for DOI 10.1021/mp500018s

View details for Web of Science ID 000344307700012
Cerenkov luminescence endoscopy: improved molecular sensitivity with ß--emitting radiotracers. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Carpenter, C. M., Ma, X., Liu, H., Sun, C., Pratx, G., Wang, J., Gambhir, S. S., Xing, L., Cheng, Z. 2014; 55 (11): 1905-1909
Abstract

Cerenkov luminescence endoscopy (CLE) is an optical technique that captures the Cerenkov photons emitted from highly energetic moving charged particles (β(+) or β(-)) and can be used to monitor the distribution of many clinically available radioactive probes. A main limitation of CLE is its limited sensitivity to small concentrations of radiotracer, especially when used with a light guide. We investigated the improvement in the sensitivity of CLE brought about by using a β(-) radiotracer that improved Cerenkov signal due to both higher β-particle energy and lower γ noise in the imaging optics because of the lack of positron annihilation.The signal-to-noise ratio (SNR) of (90)Y was compared with that of (18)F in both phantoms and small-animal tumor models. Sensitivity and noise characteristics were demonstrated using vials of activity both at the surface and beneath 1 cm of tissue. Rodent U87MG glioma xenograft models were imaged with radiotracers bound to arginine-glycine-aspartate (RGD) peptides to determine the SNR.γ noise from (18)F was demonstrated by both an observed blurring across the field of view and a more pronounced fall-off with distance. A decreased γ background and increased energy of the β particles resulted in a 207-fold improvement in the sensitivity of (90)Y compared with (18)F in phantoms. (90)Y-bound RGD peptide produced a higher tumor-to-background SNR than (18)F in a mouse model.The use of (90)Y for Cerenkov endoscopic imaging enabled superior results compared with an (18)F radiotracer.

View details for DOI 10.2967/jnumed.114.139105

View details for PubMedID 25300598
Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications JOURNAL OF CLINICAL INVESTIGATION Fan, X., Rai, A., Kambham, N., Sung, J. F., Singh, N., Petitt, M., Dhal, S., Agrawal, R., Sutton, R. E., Druzin, M. L., Gambhir, S. S., Ambati, B. K., Cross, J. C., Nayak, N. R. 2014; 124 (11): 4941-4952
View details for DOI 10.1172/JCI76864

View details for Web of Science ID 000344203300029
F-18-FPPRGD2 PET/CT: Pilot Phase Evaluation of Breast Cancer Patients RADIOLOGY Lagaru, A., Mosci, C., Shen, B., Chin, F. T., Mittra, E., Telli, M. L., Gambhir, S. S. 2014; 273 (2): 549-559
View details for DOI 10.1148/radiol.14140028

View details for Web of Science ID 000345069800028
Endoscopic molecular imaging of human bladder cancer using a CD47 antibody SCIENCE TRANSLATIONAL MEDICINE Pan, Y., Volkmer, J., Mach, K. E., Rouse, R. V., Liu, J., Sahoo, D., Chang, T. C., Metzner, T. J., Kang, L., van de Rijn, M., Skinner, E. C., Gambhir, S. S., Weissman, I. L., Liao, J. C. 2014; 6 (260)
View details for DOI 10.1126/scitranslmed.3009457

View details for Web of Science ID 000343920500006
Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging. Journal of the American Chemical Society Fan, Q., Cheng, K., Hu, X., Ma, X., Zhang, R., Yang, M., Lu, X., Xing, L., Huang, W., Gambhir, S. S., Cheng, Z. 2014; 136 (43): 15185-15194
Abstract

Developing multifunctional and easily prepared nanoplatforms with integrated different modalities is highly challenging for molecular imaging. Here, we report the successful transferring an important molecular target, melanin, into a novel mul-timodality imaging nanoplatform. Melanin is abundantly expressed in melanotic melanomas and thus has been actively studied as a target for melanoma imaging. In our work, the multifunctional biopolymer nanoplatform based on ultrasmall (< 10 nm) water-soluble melanin nanoparticle (MNP) was developed and showed unique photoacoustic property and natural binding ability with metal ions (for example, 64Cu2+, Fe3+). Therefore MNP can serve not only as a photoacoustic contrast agent, but also as a nanoplatform for positron emission tomography (PET) and magnetic resonance imaging (MRI). Traditional passive nanoplatforms require complicated and time-consuming processes for pre-building reporting moieties or chemical modifications using active groups to integrate different contrast properties into one entity. In comparison, utilizing functional biomarker melanin can greatly simplify the building process. We further conjugated αvβ3 integrins targeting peptide, cyclic c(RGDfC) peptide, to MNPs and this allowed targeting of these nanoparticles to allow for greater U87MG tumor accumulation than that simply possible due to the enhanced permeability and retention (EPR) effect. The multimodal properties of MNPs demonstrate the high potential of endogenous materials with multifunctions as nanoplatforms for molecular theranostics and clinical translation.

View details for DOI 10.1021/ja505412p

View details for PubMedID 25292385
Investigation of 6-[F-18]-Fluoromaltose as a Novel PET Tracer for Imaging Bacterial Infection PLOS ONE Gowrishankar, G., Namavari, M., Jouannot, E. B., Hoehne, A., Reeves, R., Hardy, J., Gambhir, S. S. 2014; 9 (9)
View details for DOI 10.1371/journal.pone.0107951

View details for Web of Science ID 000343679800058
Detection and Quantitation of Circulating Tumor Cell Dynamics by Bioluminescence Imaging in an Orthotopic Mammary Carcinoma Model PLOS ONE Sasportas, L. S., Hori, S. S., Pratx, G., Gambhir, S. S. 2014; 9 (9)
View details for DOI 10.1371/journal.pone.0105079

View details for Web of Science ID 000341271500005
Circulating Tumor Microemboli Diagnostics for Patients with Non-Small-Cell Lung Cancer JOURNAL OF THORACIC ONCOLOGY Carlsson, A., Nair, V. S., Luttgen, M. S., Keu, K. V., Horng, G., Vasanawala, M., Kolatkar, A., Jamali, M., Iagaru, A. H., Kuschner, W., Loo, B. W., Shrager, J. B., Bethel, K., Hoh, C. K., Bazhenova, L., Nieva, J., Kuhn, P., Gambhir, S. S. 2014; 9 (8): 1111-1119
Abstract

Circulating tumor microemboli (CTM) are potentially important cancer biomarkers, but using them for cancer detection in early-stage disease has been assay limited. We examined CTM test performance using a sensitive detection platform to identify stage I non-small-cell lung cancer (NSCLC) patients undergoing imaging evaluation.First, we prospectively enrolled patients during 18F-FDG PET-CT imaging evaluation for lung cancer that underwent routine phlebotomy where CTM and circulating tumor cells (CTCs) were identified in blood using nuclear (DAPI), cytokeratin (CK), and CD45 immune-fluorescent antibodies followed by morphologic identification. Second, CTM and CTC data were integrated with patient (age, gender, smoking, and cancer history) and imaging (tumor diameter, location in lung, and maximum standard uptake value [SUVmax]) data to develop and test multiple logistic regression models using a case-control design in a training and test cohort followed by cross-validation in the entire group.We examined 104 patients with NSCLC, and the subgroup of 80 with stage I disease, and compared them to 25 patients with benign disease. Clinical and imaging data alone were moderately discriminating for all comers (Area under the Curve [AUC] = 0.77) and by stage I disease only (AUC = 0.77). However, the presence of CTM combined with clinical and imaging data was significantly discriminating for diagnostic accuracy in all NSCLC patients (AUC = 0.88, p value = 0.001) and for stage I patients alone (AUC = 0.87, p value = 0.002).CTM may add utility for lung cancer diagnosis during imaging evaluation using a sensitive detection platform.

View details for Web of Science ID 000340138700012
Imaging of hepatocellular carcinoma patient-derived xenografts using Zr-89-labeled anti-glypican-3 monoclonal antibody BIOMATERIALS Yang, X., Liu, H., Sun, C. K., Natarajan, A., Hu, X., Wang, X., Allegretta, M., Guttmann, R. D., Gambhir, S. S., Chua, M., Cheng, Z., So, S. K. 2014; 35 (25): 6964-6971
Abstract

Imaging probes for early detection of hepatocellular carcinoma (HCC) are highly desired to overcome current diagnostic limitations which lead to poor prognosis. The membrane protein glypican-3 (GPC3) is a potential molecular target for early HCC detection as it is over-expressed in >50% of HCCs, and is associated with early hepatocarcinogenesis. We synthesized the positron emission tomography (PET) probe (89)Zr-DFO-1G12 by bioconjugating and radiolabeling the anti-GPC3 monoclonal antibody (clone 1G12) with (89)Zr, and evaluated its tumor-targeting capacity. In vitro, (89)Zr-DFO-1G12 was specifically taken up into GPC3-positive HCC cells only, but not in the GPC3-negative prostate cancer cell line (PC3). In vivo, (89)Zr-DFO-1G12 specifically accumulated in subcutaneous GPC3-positive HCC xenografts only, but not in PC3 xenografts. Importantly, (89)Zr-DFO-1G12 delineated orthotopic HCC xenografts from surrounding normal liver, with tumor/liver (T/L) ratios of 6.65 ± 1.33 for HepG2, and 4.29 ± 0.52 for Hep3B xenografts. It also delineated orthotopic xenografts derived from three GPC3-positive HCC patient specimens, with T/L ratios of 4.21 ± 0.64, 2.78 ± 0.26, and 2.31 ± 0.38 at 168 h p.i. Thus, (89)Zr-DFO-1G12 is a highly translatable probe for the specific and high contrast imaging of GPC3-positive HCCs, which may aid early detection of HCC to allow timely intervention.

View details for DOI 10.1016/j.biomaterials.2014.04.089

View details for Web of Science ID 000338386800028

View details for PubMedID 24836949
A High-Affinity, High-Stability Photoacoustic Agent for Imaging Gastrin-Releasing Peptide Receptor in Prostate Cancer CLINICAL CANCER RESEARCH Levi, J., Sathirachinda, A., Gambhir, S. S. 2014; 20 (14): 3721-3729
Abstract

To evaluate the utility of targeted photoacoustic imaging (PAI) in providing molecular information to complement intrinsic functional and anatomical details of the vasculature within prostate lesion.We developed a PAI agent, AA3G-740, that targets gastrin-releasing peptide receptor (GRPR), found to be highly overexpressed in prostate cancer. The binding specificity of the agent was evaluated in human prostate cancer cell lines, PC3 and LNCaP, and antagonist properties determined by cell internalization and intracellular calcium mobilization studies. The imaging sensitivity was assessed for the agent itself and for the PC3 cells labeled with agent. The in vivo stability of the agent was determined in human plasma and in the blood of living mice. The in vivo binding of the agent was evaluated in PC3 prostate tumor models in mice, and was validated ex vivo by optical imaging.AA3G-740 demonstrated strong and specific binding to GRPR. The sensitivity of detection in vitro indicated suitability of the agent to image very small lesions. In mice, the agent was able to bind to GRPR even in poorly vascularized tumors leading to nearly 2-fold difference in photoacoustic signal relative to the control agent.The ability to image both vasculature and molecular profile outside the blood vessels gives molecular PAI a unique advantage over currently used imaging techniques. The imaging method presented here can find application both in diagnosis and in image-guided biopsy.

View details for DOI 10.1158/1078-0432.CCR-13-3405

View details for Web of Science ID 000339611500013

View details for PubMedID 24850845
Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery. Nature nanotechnology Smith, B. R., Ghosn, E. E., Rallapalli, H., Prescher, J. A., Larson, T., Herzenberg, L. A., Gambhir, S. S. 2014; 9 (6): 481-487
Abstract

In cancer imaging, nanoparticle biodistribution is typically visualized in living subjects using 'bulk' imaging modalities such as magnetic resonance imaging, computerized tomography and whole-body fluorescence. Accordingly, nanoparticle influx is observed only macroscopically, and the mechanisms by which they target cancer remain elusive. Nanoparticles are assumed to accumulate via several targeting mechanisms, particularly extravasation (leakage into tumour). Here, we show that, in addition to conventional nanoparticle-uptake mechanisms, single-walled carbon nanotubes are almost exclusively taken up by a single immune cell subset, Ly-6C(hi) monocytes (almost 100% uptake in Ly-6C(hi) monocytes, below 3% in all other circulating cells), and delivered to the tumour in mice. We also demonstrate that a targeting ligand (RGD) conjugated to nanotubes significantly enhances the number of single-walled carbon nanotube-loaded monocytes reaching the tumour (P < 0.001, day 7 post-injection). The remarkable selectivity of this tumour-targeting mechanism demonstrates an advanced immune-based delivery strategy for enhancing specific tumour delivery with substantial penetration.

View details for DOI 10.1038/nnano.2014.62

View details for PubMedID 24727688
99mTc-labeled cystine knot peptide targeting integrin avß6 for tumor SPECT imaging. Molecular pharmaceutics Zhu, X., Li, J., Hong, Y., Kimura, R. H., Ma, X., Liu, H., Qin, C., Hu, X., Hayes, T. R., Benny, P., Gambhir, S. S., Cheng, Z. 2014; 11 (4): 1208-1217
Abstract

Integrin αvβ6 is overexpressed in a variety of cancers, and its expression is often associated with poor prognosis. Therefore, there is a need to develop affinity reagents for noninvasive imaging of integrin αvβ6 expression since it may provide early cancer diagnosis, more accurate prognosis, and better treatment planning. We recently engineered and validated highly stable cystine knot peptides that selectively bind integrin αvβ6 with no cross-reactivity to integrins αvβ5, α5β1, or αvβ3, also known to be overexpressed in many cancers. Here, we developed a single photon emission computed tomography (SPECT) probe for imaging integrin αvβ6 positive tumors. Cystine knot peptide, S02, was first conjugated with a single amino acid chelate (SAAC) and labeled with [(99m)Tc(H2O)3(CO)3](+). The resulting probe, (99m)Tc-SAAC-S02, was then evaluated by in vitro cell uptake studies using two αvβ6 positive cell lines (human lung adenocarcinoma cell line HCC4006 and pancreatic cancer cell line BxPC-3) and two αvβ6 negative cell lines (human lung adenocarcinoma cell line H838 and human embryonic kidney cell line 293T). Next, SPECT/CT and biodistribution studies were performed in nude mice bearing HCC4006 and H838 tumor xenografts to evaluate the in vivo performance of (99m)Tc-SAAC-S02. Significant differences in the uptake of (99m)Tc-SAAC-S02 were observed in αvβ6 positive vs negative cells (P < 0.05). Biodistribution and small animal SPECT/CT studies revealed that (99m)Tc-SAAC-S02 accumulated to moderate levels in antigen positive tumors (∼2% ID/g at 1 and 6 h postinjection, n = 3 or 4/group). Moreover, the probe demonstrated tumor-to-background tissue ratios of 6.81 ± 2.32 (tumor-to-muscle) and 1.63 ± 0.18 (tumor-to-blood) at 6 h postinjection in αvβ6 positive tumor xenografts. Co-incubation of the probe with excess amount of unlabeled S02 as a blocking agent demonstrated significantly reduced tumor uptake, which is consistent with specific binding to the target. Renal filtration was the main route of clearance. In conclusion, knottin peptides are excellent scaffolds for which to develop highly stable imaging probes for a variety of oncological targets. (99m)Tc-SAAC-S02 demonstrates promise for use as a SPECT agent to image integrin αvβ6 expression in living systems.

View details for DOI 10.1021/mp400683q

View details for PubMedID 24524409
A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation BIOMATERIALS Ziv, K., Nuhn, H., Ben-Haim, Y., Sasportas, L. S., Kempen, P. J., Niedringhaus, T. P., Hrynyk, M., Sinclair, R., Barron, A. E., Gambhir, S. S. 2014; 35 (12): 3736-3743
Abstract

One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk-alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk-alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk-alginate ratio and the concentration of crosslinker - a characteristic that is very hard to accomplish in current hydrogels. The hydrogel and the presented results represents key steps on the way of creating artificial stem cell niche, opening up new paths in regenerative medicine.

View details for DOI 10.1016/j.biomaterials.2014.01.029

View details for Web of Science ID 000332431700003

View details for PubMedID 24484675
A Titratable Two-Step Transcriptional Amplification Strategy for Targeted Gene Therapy Based on Ligand-Induced Intramolecular Folding of a Mutant Human Estrogen Receptor MOLECULAR IMAGING AND BIOLOGY Chen, I. Y., Paulmurugan, R., Nielsen, C. H., Wang, D. S., Chow, V., Robbins, R. C., Gambhir, S. S. 2014; 16 (2): 224-234
Abstract

The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium.We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ERG521T) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression.Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given.The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.

View details for DOI 10.1007/s11307-013-0673-4

View details for Web of Science ID 000332828200010

View details for PubMedID 23955099
Construction and Validation of Nano Gold Tripods for Molecular Imaging of Living Subjects JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Cheng, K., Kothapalli, S., Liu, H., Koh, A. L., Jokerst, J. V., Jiang, H., Yang, M., Li, J., Levi, J., Wu, J. C., Gambhir, S. S., Cheng, Z. 2014; 136 (9): 3560-3571
Abstract

Anisotropic colloidal hybrid nanoparticles exhibit superior optical and physical properties compared to their counterparts with regular architectures. We herein developed a controlled, stepwise strategy to build novel, anisotropic, branched, gold nanoarchitectures (Au-tripods) with predetermined composition and morphology for bioimaging. The resultant Au-tripods with size less than 20 nm showed great promise as contrast agents for in vivo photoacoustic imaging (PAI). We further identified Au-tripods with two possible configurations as high-absorbance nanomaterials from various gold multipods using a numerical simulation analysis. The PAI signals were linearly correlated with their concentrations after subcutaneous injection. The in vivo biodistribution of Au-tripods favorable for molecular imaging was confirmed using small animal positron emission tomography (PET). Intravenous administration of cyclic Arg-Gly-Asp-d-Phe-Cys (RGDfC) peptide conjugated Au-tripods (RGD-Au-tripods) to U87MG tumor-bearing mice showed PAI contrasts in tumors almost 3-fold higher than for the blocking group. PAI results correlated well with the corresponding PET images. Quantitative biodistribution data revealed that 7.9% ID/g of RGD-Au-tripods had accumulated in the U87MG tumor after 24 h post-injection. A pilot mouse toxicology study confirmed that no evidence of significant acute or systemic toxicity was observed in histopathological examination. Our study suggests that Au-tripods can be reliably synthesized through stringently controlled chemical synthesis and could serve as a new generation of platform with high selectivity and sensitivity for multimodality molecular imaging.

View details for DOI 10.1021/ja412001e

View details for Web of Science ID 000332684700036

View details for PubMedID 24495038
Ultrasound Molecular Imaging in a Human CD276 Expression-Modulated Murine Ovarian Cancer Model CLINICAL CANCER RESEARCH Lutz, A. M., Bachawal, S. V., Drescher, C. W., Pysz, M. A., Willmann, J. K., Gambhir, S. S. 2014; 20 (5): 1313-1322
Abstract

To develop a mouse ovarian cancer model that allows modulating the expression levels of human vascular targets in mouse xenograft tumors and to test whether expression of CD276 during tumor angiogenesis can be visualized by molecularly targeted ultrasound in vivo.CD276-expressing MILE SVEN 1 (MS1) mouse endothelial cells were engineered and used for coinjection with 2008 human ovarian cancer cells for subcutaneous xenograft tumor induction in 15 nude mice. Fourteen control mice were injected with 2008 cells only. After confirming their binding specificity in flow chamber cell attachment studies, anti-CD276 antibody-functionalized contrast microbubbles were used for in vivo CD276-targeted contrast-enhanced ultrasound imaging.CD276-targeted ultrasound imaging signal was significantly higher (P = 0.006) in mixed MS1/2008 tumors than in control tumors. Compared with control microbubbles, the ultrasound signal using CD276-targeted microbubbles was significantly higher (P = 0.002), and blocking with purified anti-CD276 antibody significantly decreased (P = 0.0096) the signal in mixed MS1/2008 tumors. Immunofluorescence analysis of the tumor tissue confirmed higher quantitative immunofluorescence signal in mixed MS1/2008 tumors than in control 2008 only tumors, but showed not significantly different (P = 0.54) microvessel density.Our novel small animal model allows for modulating the expression of human tumor-associated vascular endothelial imaging targets in a mouse host and these expression differences can be visualized noninvasively by ultrasound molecular imaging. The animal model can be applied to other human vascular targets and may facilitate the preclinical development of new imaging probes such as microbubbles targeted at human vascular markers not expressed in mice. Clin Cancer Res; 20(5); 1313-22. ©2014 AACR.

View details for DOI 10.1158/1078-0432.CCR-13-1642

View details for Web of Science ID 000332403100027
Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice NATURE NANOTECHNOLOGY Pu, K., Shuhendler, A. J., Jokerst, J. V., Mei, J., Gambhir, S. S., Bao, Z., Rao, J. 2014; 9 (3): 233-239
Abstract

Photoacoustic imaging holds great promise for the visualization of physiology and pathology at the molecular level with deep tissue penetration and fine spatial resolution. To fully utilize this potential, photoacoustic molecular imaging probes have to be developed. Here, we introduce near-infrared light absorbing semiconducting polymer nanoparticles as a new class of contrast agents for photoacoustic molecular imaging. These nanoparticles can produce a stronger signal than the commonly used single-walled carbon nanotubes and gold nanorods on a per mass basis, permitting whole-body lymph-node photoacoustic mapping in living mice at a low systemic injection mass. Furthermore, the semiconducting polymer nanoparticles possess high structural flexibility, narrow photoacoustic spectral profiles and strong resistance to photodegradation and oxidation, enabling the development of the first near-infrared ratiometric photoacoustic probe for in vivo real-time imaging of reactive oxygen species-vital chemical mediators of many diseases. These results demonstrate semiconducting polymer nanoparticles to be an ideal nanoplatform for developing photoacoustic molecular probes.

View details for DOI 10.1038/NNANO.2013.302

View details for Web of Science ID 000332637200018
Light In and Sound Out: Emerging Translational Strategies for Photoacoustic Imaging CANCER RESEARCH Zackrisson, S., van de Ven, S. M., Gambhir, S. S. 2014; 74 (4): 979-1004
Abstract

Photoacoustic imaging (PAI) has the potential for real-time molecular imaging at high resolution and deep inside the tissue, using nonionizing radiation and not necessarily depending on exogenous imaging agents, making this technique very promising for a range of clinical applications. The fact that PAI systems can be made portable and compatible with existing imaging technologies favors clinical translation even more. The breadth of clinical applications in which photoacoustics could play a valuable role include: noninvasive imaging of the breast, sentinel lymph nodes, skin, thyroid, eye, prostate (transrectal), and ovaries (transvaginal); minimally invasive endoscopic imaging of gastrointestinal tract, bladder, and circulating tumor cells (in vivo flow cytometry); and intraoperative imaging for assessment of tumor margins and (lymph node) metastases. In this review, we describe the basics of PAI and its recent advances in biomedical research, followed by a discussion of strategies for clinical translation of the technique. Cancer Res; 74(4); 979-1004. ©2014 AACR.

View details for DOI 10.1158/0008-5472.CAN-13-2387

View details for Web of Science ID 000331876600001
Antiviral drug ganciclovir is a potent inhibitor of microglial proliferation and neuroinflammation JOURNAL OF EXPERIMENTAL MEDICINE Ding, Z., Mathur, V., Ho, P. P., James, M. L., Lucin, K. M., Hoehne, A., Alabsi, H., Gambhir, S. S., Steinman, L., Luo, J., Wyss-Coray, T. 2014; 211 (2): 189-198
Abstract

Aberrant microglial responses contribute to neuroinflammation in many neurodegenerative diseases, but no current therapies target pathogenic microglia. We discovered unexpectedly that the antiviral drug ganciclovir (GCV) inhibits the proliferation of microglia in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis (MS), as well as in kainic acid-induced excitotoxicity. In EAE, GCV largely prevented infiltration of T lymphocytes into the central nervous system (CNS) and drastically reduced disease incidence and severity when delivered before the onset of disease. In contrast, GCV treatment had minimal effects on peripheral leukocyte distribution in EAE and did not inhibit generation of antibodies after immunization with ovalbumin. Additionally, a radiolabeled analogue of penciclovir, [(18)F]FHBG, which is similar in structure to GCV, was retained in areas of CNS inflammation in EAE, but not in naive control mice, consistent with the observed therapeutic effects. Our experiments suggest GCV may have beneficial effects in the CNS beyond its antiviral properties.

View details for DOI 10.1084/jem.20120696

View details for Web of Science ID 000331281600002

View details for PubMedID 24493798
Imaging Circulating Tumor Cells in Freely Moving Awake Small Animals Using a Miniaturized Intravital Microscope PLOS ONE Sasportas, L. S., Gambhir, S. S. 2014; 9 (1)
Abstract

Metastasis, the cause for 90% of cancer mortality, is a complex and poorly understood process involving the invasion of circulating tumor cells (CTCs) into blood vessels. These cells have potential prognostic value as biomarkers for early metastatic risk. But their rarity and the lack of specificity and sensitivity in measuring them render their interrogation by current techniques very challenging. How and when these cells are circulating in the blood, on their way to potentially give rise to metastasis, is a question that remains largely unanswered. In order to provide an insight into this "black box" using non-invasive imaging, we developed a novel miniature intravital microscopy (mIVM) strategy capable of real-time long-term monitoring of CTCs in awake small animals. We established an experimental 4T1-GL mouse model of metastatic breast cancer, in which tumor cells express both fluorescent and bioluminescent reporter genes to enable both single cell and whole body tumor imaging. Using mIVM, we monitored blood vessels of different diameters in awake mice in an experimental model of metastasis. Using an in-house software algorithm we developed, we demonstrated in vivo CTC enumeration and computation of CTC trajectory and speed. These data represent the first reported use we know of for a miniature mountable intravital microscopy setup for in vivo imaging of CTCs in awake animals.

View details for DOI 10.1371/journal.pone.0086759

View details for Web of Science ID 000330621900038

View details for PubMedID 24497977
Detection and quantitation of circulating tumor cell dynamics by bioluminescence imaging in an orthotopic mammary carcinoma model. PloS one Sasportas, L. S., Hori, S. S., Pratx, G., Gambhir, S. S. 2014; 9 (9)
Abstract

Circulating tumor cells (CTCs) have been detected in the bloodstream of both early-stage and advanced cancer patients. However, very little is know about the dynamics of CTCs during cancer progression and the clinical relevance of longitudinal CTC enumeration. To address this, we developed a simple bioluminescence imaging assay to detect CTCs in mouse models of metastasis. In a 4T1 orthotopic metastatic mammary carcinoma mouse model, we demonstrated that this quantitative method offers sensitivity down to 2 CTCs in 0.1-1mL blood samples and high specificity for CTCs originating from the primary tumor, independently of their epithelial status. In this model, we simultaneously monitored blood CTC dynamics, primary tumor growth, and lung metastasis progression over the course of 24 days. Early in tumor development, we observed low numbers of CTCs in blood samples (10-15 cells/100 µL) and demonstrated that CTC dynamics correlate with viable primary tumor growth. To our knowledge, these data represent the first reported use of bioluminescence imaging to detect CTCs and quantify their dynamics in any cancer mouse model. This new assay is opening the door to the study of CTC dynamics in a variety of animal models. These studies may inform clinical decision on the appropriate timing of blood sampling and value of longitudinal CTC enumeration in cancer patients.

View details for DOI 10.1371/journal.pone.0105079

View details for PubMedID 25188396
Cellulose Nanoparticles are a Biodegradable Photoacoustic Contrast Agent for Use in Living Mice. Photoacoustics Jokerst, J. V., Van de Sompel, D., Bohndiek, S. E., Gambhir, S. S. 2014; 2 (3): 119-127
Abstract

Molecular imaging with photoacoustic ultrasound is an emerging field that combines the spatial and temporal resolution of ultrasound with the contrast of optical imaging. However, there are few imaging agents that offer both high signal intensity and biodegradation into small molecules. Here we describe a cellulose-based nanoparticle with peak photoacoustic signal at 700 nm and an in vitro limit of detection of 6 pM (0.02 mg/mL). Doses down to 0.35 nM (1.2 mg/mL) were used to image mouse models of ovarian cancer. Most importantly, the nanoparticles were shown to biodegrade in the presence of cellulase both through a glucose assay and electron microscopy.

View details for DOI 10.1016/j.pacs.2014.07.001

View details for PubMedID 25225633
Evaluation of s-1 receptor radioligand 18F-FTC-146 in rats and squirrel monkeys using PET. Journal of nuclear medicine : official publication, Society of Nuclear Medicine James, M. L., Shen, B., Nielsen, C. H., Behera, D., Buckmaster, C. L., Mesangeau, C., Zavaleta, C., Vuppala, P. K., Jamalapuram, S., Avery, B. A., Lyons, D. M., McCurdy, C. R., Biswal, S., Gambhir, S. S., Chin, F. T. 2014; 55 (1): 147-153
Abstract

The noninvasive imaging of σ-1 receptors (S1Rs) could provide insight into their role in different diseases and lead to novel diagnostic/treatment strategies. The main objective of this study was to assess the S1R radiotracer (18)F-FTC-146 in rats. Preliminary squirrel monkey imaging and human serum/liver microsome studies were performed to gain information about the potential of (18)F-FTC-146 for eventual clinical translation.The distribution and stability of (18)F-FTC-146 in rats were assessed via PET/CT, autoradiography, γ counting, and high-performance liquid chromatography (HPLC). Preliminary PET/MRI of squirrel monkey brain was conducted along with HPLC assessment of (18)F-FTC-146 stability in monkey plasma and human serum.Biodistribution studies showed that (18)F-FTC-146 accumulated in S1R-rich rat organs, including the lungs, pancreas, spleen, and brain. Pretreatment with known S1R compounds, haloperidol, or BD1047, before radioligand administration, significantly attenuated (18)F-FTC-146 accumulation in all rat brain regions by approximately 85% (P < 0.001), suggesting radiotracer specificity for S1Rs. Similarly, PET/CT and autoradiography results demonstrated accumulation of (18)F-FTC-146 in rat brain regions known to contain S1Rs and that this uptake could be blocked by BD1047 pretreatment. Ex vivo analysis of (18)F-FTC-146 in the brain showed that only intact radiotracer was present at 15, 30, and 60 min, whereas rapid metabolism of residual (18)F-FTC-146 was observed in rat plasma. Preliminary monkey PET/MRI studies demonstrated specific accumulation of (18)F-FTC-146 in the brain (mainly in cortical structures, cerebellum, and vermis) that could be attenuated by pretreatment with haloperidol. HPLC of monkey plasma suggested radioligand metabolism, whereas (18)F-FTC-146 appeared to be stable in human serum. Finally, liver microsome studies revealed that (18)F-FTC-146 has a longer half-life in human microsomes, compared with rodents.Together, these results indicate that (18)F-FTC-146 is a promising tool for visualizing S1Rs in preclinical studies and that it has potential for mapping these sites in the human brain.

View details for DOI 10.2967/jnumed.113.120261

View details for PubMedID 24337599
Cellulose nanoparticles: Photoacoustic contrast agents that biodegrade to simple sugars PHOTONS PLUS ULTRASOUND: IMAGING AND SENSING 2014 Jokerst, J. V., Bohndiek, S. E., Gambhir, S. S. 2014; 8943
View details for DOI 10.1117/12.2036256

View details for Web of Science ID 000338768500011
Photoacoustic Imaging of Mesenchymal Stem Cells in Living Mice via Silica-Coated Gold Nanorods PHOTONS PLUS ULTRASOUND: IMAGING AND SENSING 2014 Jokerst, J. V., Thangaraj, M., Gambhir, S. S. 2014; 8943
View details for DOI 10.1117/12.2036786

View details for Web of Science ID 000338768500035
Tracking Cellular and Immune Therapies in Cancer EMERGING APPLICATIONS OF MOLECULAR IMAGING TO ONCOLOGY Kurtz, D. M., Gambhir, S. S. 2014; 124: 257-296
View details for DOI 10.1016/B978-0-12-411638-2.00008-2

View details for Web of Science ID 000344511500008
A simple model for deep tissue attenuation correction and large organ analysis of Cerenkov luminescence imaging MEDICAL IMAGING 2014: PHYSICS OF MEDICAL IMAGING Habte, F., Natarajan, A., Paik, D. S., Gambhir, S. S. 2014; 9033
View details for DOI 10.1117/12.2043879

View details for Web of Science ID 000338775800154
Gold nanorods combine photoacoustic and Raman imaging for detection and treatment of ovarian cancer PHOTONS PLUS ULTRASOUND: IMAGING AND SENSING 2014 Jokerst, J. V., Cole, A. J., Bohndiek, S. E., Gambhir, S. S. 2014; 8943
View details for DOI 10.1117/12.2036776

View details for Web of Science ID 000338768500134
A Novel Engineered Anti-CD20 Tracer Enables Early Time PET Imaging in a Humanized Transgenic Mouse Model of B-cell Non-Hodgkins Lymphoma CLINICAL CANCER RESEARCH Natarajan, A., Hackel, B. J., Gambhir, S. S. 2013; 19 (24): 6820-6829
Abstract

The aim of this article was to evaluate the use of a novel engineered anti-CD20 protein based on the 10 kDa human fibronectin type 3 domain (FN3) and subsequently compare with (64)Cu-rituximab for positron emission tomography (PET) imaging of CD20.The engineered FN3(CD20) and FN3(WT) were produced in Escherichia coli cells at 2 to 5 mg/L, conjugated to DOTA, labeled with (64)Cu, and used for PET imaging of huCD20 expression in B cells. Humanized transgenic mice and subcutaneously xenografted mice each received intravenous (64)Cu-FN3(CD20) or FN3(WT) (3.7 MBq/4 μg Do-FN3 in 200 μL PBS). Control group received a blocking dose (50-fold excess) of unconjugated FN3(CD20) two hours before radiotracer injection. PET imaging was carried out at 1 to 24 hours postinjections.In vitro assay demonstrated FN3 binds CD20 with 20 nmol/L affinity on CD20-expressing cells. (64)Cu-FN3(CD20) showed clear, high-contrast visualization of huCD20-expressing B cells in the spleen of transgenic mice as early as 1 hour postinjection [38 ± 3% injected dose (ID)/g] and exhibited a spleen-to-blood ratio of 13 by 4 hours. This is higher uptake (P = 0.04) and 10-fold greater signal-to-background (P = 0.04) than the (64)Cu-rituximab antibody radiotracer. Tumor uptake (16.8 ± 1.6 vs. 5.6 ± 1.4%ID/g) and tumor:background ratios were superior for FN3CD20 relative to rituximab in xenograft studies as well.The (64)Cu-Do-FN3(CD20) radiotracer represents a novel small, high-affinity binder for imaging human CD20, which may be well suited for B-cell non-Hodgkin's lymphoma imaging in patients at early time points.

View details for DOI 10.1158/1078-0432.CCR-13-0626

View details for Web of Science ID 000328938700019
A F-18-Labeled Saxitoxin Derivative for in Vivo PET-MR Imaging of Voltage-Gated Sodium Channel Expression Following Nerve Injury JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hoehne, A., Behera, D., Parsons, W. H., James, M. L., Shen, B., Borgohain, P., Bodapati, D., Prabhakar, A., Gambhir, S. S., Yeomans, D. C., Biswal, S., Chin, F. T., Du Bois, J. 2013; 135 (48): 18012-18015
Abstract

Both chronic and neuropathic pain conditions are associated with increased expression of certain voltage-gated sodium ion channel (NaV) isoforms in peripheral sensory neurons. A method for noninvasive imaging of these channels could represent a powerful tool for investigating aberrant expression of NaV and its role in pain pathogenesis. Herein, we describe the synthesis and evaluation of a positron emission tomography (PET) radiotracer targeting NaVs, the design of which is based on the potent, NaV-selective inhibitor saxitoxin. Both autoradiography analysis of sciatic nerves excised from injured rats as well as whole animal PET-MR imaging demonstrate that a systemically administered [(18)F]-labeled saxitoxin derivative concentrates at the site of nerve injury, consistent with upregulated sodium channel expression following axotomy. This type of PET agent has potential use for serial monitoring of channel expression levels at injured nerves throughout wound healing and/or following drug treatment. Such information may be correlated with pain behavioral analyses to help shed light on the complex molecular processes that underlie pain sensation.

View details for DOI 10.1021/ja408300e

View details for Web of Science ID 000328100000002
Combined 18F-fluoride and 18F-FDG PET/CT: a response based on actual data from prospective studies. European journal of nuclear medicine and molecular imaging Iagaru, A., Mosci, C., Dick, D. W., Sathekge, M., Lapa, P., de Lima, J. M., Gambhir, S. S. 2013; 40 (12): 1922-1924
View details for DOI 10.1007/s00259-013-2556-y

View details for PubMedID 24057457
Single-Cell Photonic Nanocavity Probes NANO LETTERS Shambat, G., Kothapalli, S., Provine, J., Sarmiento, T., Harris, J., Gambhir, S. S., Vuckovic, J. 2013; 13 (11): 4999-5005
Abstract

In this report, we demonstrate for the first time photonic nanocavities operating inside single biological cells. Here we develop a nanobeam photonic crystal (PC) cavity as an advanced cellular nanoprobe, active in nature, and configurable to provide a multitude of actions for both intracellular sensing and control. Our semiconductor nanocavity probes emit photoluminescence (PL) from embedded quantum dots (QD) and sustain high quality resonant photonic modes inside cells. The probes are shown to be minimally cytotoxic to cells from viability studies, and the beams can be loaded in cells and tracked for days at a time, with cells undergoing regular division with the beams. We present in vitro label-free protein sensing with our probes to detect streptavidin as a path towards real-time biomarker and biomolecule detection inside single cells. The results of this work will enable new areas of research merging the strengths of photonic nanocavities with fundamental cell biology.

View details for DOI 10.1021/nl304602d

View details for Web of Science ID 000327111700001
Integrin-Targeted Molecular Imaging of Experimental Abdominal Aortic Aneurysms by 18F-labeled Arg-Gly-Asp Positron-Emission Tomography. Circulation. Cardiovascular imaging Kitagawa, T., Kosuge, H., Chang, E., James, M. L., Yamamoto, T., Shen, B., Chin, F. T., Gambhir, S. S., Dalman, R. L., McConnell, M. V. 2013; 6 (6): 950-956
Abstract

Background- Both inflammation and neoangiogenesis contribute to abdominal aortic aneurysm (AAA) disease. Arg-Gly-Asp-based molecular imaging has been shown to detect the integrin αvβ3. We studied a clinical dimeric (18)F-labeled Arg-Gly-Asp positron-emission tomography (PET) agent ((18)F-FPPRGD2) for molecular imaging of experimental AAAs. Methods and Results- Murine AAAs were induced in Apo-E-deficient mice by angiotensin II infusion, with monitoring of aortic diameter on ultrasound. AAA (n=10) and saline-infused control mice (n=7) were injected intravenously with (18)F-FPPRGD2, as well as an intravascular computed tomography contrast agent, then scanned using a small-animal PET/computed tomography scanner. Aortic uptake of (18)F-FPPRGD2 was quantified by percentage-injected dose per gram and target-to-=0.003; median target-to-=0.0008). Ex vivo autoradiography demonstrated high uptake of (18)F-FPPRGD2 into the AAA wall, with immunohistochemistry showing substantial cluster of differentiation (CD)-11b(+) macrophages and CD-31(+) neovessels. Target-to-=-0.29, P=0.41) but did strongly correlate with both mural macrophage density (r=0.79, P=0.007) and neovessel counts (r=0.87, P=0.001) on immunohistochemistry. Conclusions- PET imaging of experimental AAAs using (18)F-FPPRGD2 detects biologically active disease, correlating to the degree of vascular inflammation and neoangiogenesis. This may provide a clinically translatable molecular imaging approach to characterize AAA biology to predict risk beyond size alone.

View details for DOI 10.1161/CIRCIMAGING.113.000234

View details for PubMedID 23995363
Nanooncology: The Future of Cancer Diagnosis and Therapy CA-A CANCER JOURNAL FOR CLINICIANS Thakor, A. S., Gambhir, S. S. 2013; 63 (6): 395-418
Abstract

In recent years, there has been an unprecedented expansion in the field of nanomedicine with the development of new nanoparticles for the diagnosis and treatment of cancer. Nanoparticles have unique biological properties given their small size and large surface area-to-volume ratio, which allows them to bind, absorb, and carry compounds such as small molecule drugs, DNA, RNA, proteins, and probes with high efficiency. Their tunable size, shape, and surface characteristics also enable them to have high stability, high carrier capacity, the ability to incorporate both hydrophilic and hydrophobic substances and compatibility with different administration routes, thereby making them highly attractive in many aspects of oncology. This review article will discuss how nanoparticles are able to function as carriers for chemotherapeutic drugs to increase their therapeutic index; how they can function as therapeutic agents in photodynamic, gene, and thermal therapy; and how nanoparticles can be used as molecular imaging agents to detect and monitor cancer progression.

View details for DOI 10.3322/caac.21199

View details for Web of Science ID 000326887000004

View details for PubMedID 24114523
Preclinical Efficacy of the Anti-Hepatocyte Growth Factor Antibody Ficlatuzumab in a Mouse Brain Orthotopic Glioma Model Evaluated by Bioluminescence, PET, and MRI CLINICAL CANCER RESEARCH Mittra, E. S., Fan-Minogue, H., Lin, F. I., Karamchandani, J., Sriram, V., Han, M., Gambhir, S. S. 2013; 19 (20): 5711-5721
Abstract

Ficlatuzumab is a novel therapeutic agent targeting the hepatocyte growth factor (HGF)/c-MET pathway. We summarize extensive preclinical work using this agent in a mouse brain orthotopic model of glioblastoma.Sequential experiments were done using eight- to nine-week-old nude mice injected with 3 × 10(5) U87 MG (glioblastoma) cells into the brain. Evaluation of ficlatuzumab dose response for this brain tumor model and comparison of its response to ficlatuzumab and to temozolamide were conducted first. Subsequently, various small-animal imaging modalities, including bioluminescence imaging (BLI), positron emission tomography (PET), and MRI, were used with a U87 MG-Luc 2 stable cell line, with and without the use of ficlatuzumab, to evaluate the ability to noninvasively assess tumor growth and response to therapy. ANOVA was conducted to evaluate for significant differences in the response.There was a survival benefit with ficlatuzumab alone or in combination with temozolamide. BLI was more sensitive than PET in detecting tumor cells. Fluoro-D-thymidine (FLT) PET provided a better signal-to-background ratio than 2[(18)F]fluoro-2-deoxy-d-glucose (FDG) PET. In addition, both BLI and FLT PET showed significant changes over time in the control group as well as with response to therapy. MRI does not disclose any time-dependent change. Also, the MRI results showed a temporal delay in comparison to the BLI and FLT PET findings, showing similar results one drug cycle later.Targeting the HGF/c-MET pathway with the novel agent ficlatuzumab appears promising for the treatment of glioblastoma. Various clinically applicable imaging modalities including FLT, PET, and MRI provide reliable ways of assessing tumor growth and response to therapy. Given the clinical applicability of these findings, future studies on patients with glioblastoma may be appropriate.

View details for DOI 10.1158/1078-0432.CCR-12-1015

View details for Web of Science ID 000325797600019
A Scanning Transmission Electron Microscopy Approach to Analyzing Large Volumes of Tissue to Detect Nanoparticles MICROSCOPY AND MICROANALYSIS Kempen, P. J., Thakor, A. S., Zavaleta, C., Gambhir, S. S., Sinclair, R. 2013; 19 (5): 1290-1297
Abstract

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue, but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work, we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol-coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time-consuming analytical characterization. We utilized this technique to analyze 243,000 μm3 of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail vein accumulated in the liver, whereas those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation.

View details for DOI 10.1017/S143192761300192X

View details for Web of Science ID 000324550900022

View details for PubMedID 23803218
Noninvasive Imaging of Hypoxia-Inducible Factor-1 alpha Gene Therapy for Myocardial Ischemia HUMAN GENE THERAPY METHODS Chen, I. Y., Gheysens, O., Li, Z., Rasooly, J. A., Wang, Q., Paulmurugan, R., Rosenberg, J., Rodriguez-Porcel, M., Willmann, J. K., Wang, D. S., Contag, C. H., Robbins, R. C., Wu, J. C., Gambhir, S. S. 2013; 24 (5): 279-288
View details for DOI 10.1089/hgtb.2013.028

View details for Web of Science ID 000336911000001
MicroRNA-regulated non-viral vectors with improved tumor specificity in an orthotopic rat model of hepatocellular carcinoma GENE THERAPY Ronald, J. A., Katzenberg, R., Nielsen, C. H., Jae, H. J., Hofmann, L. V., Gambhir, S. S. 2013; 20 (10): 1006-1013
Abstract

In hepatocellular carcinoma (HCC), tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs (miRNAs) are powerful negative regulators of gene expression and many are downregulated in human HCC. We identified seven miRNAs that are also downregulated in tumors in a rat hepatoma model (P<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these miRNAs. Attenuation of luciferase expression by the corresponding miRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two miRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared with the control vector (6.40 and 0.26%, respectively; P<0.05). In tumor, miR-122 had a nonsignificant trend towards decreased (∼50%) expression, whereas miR-26 had no significant effect on tumor expression. To our knowledge, this is the first work using differentially expressed miRNAs to de-target transgene expression in an orthotopic hepatoma model and to identify miR-26a, in addition to miR-122, for de-targeting liver. Considering the heterogeneity of miRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease.Gene Therapy advance online publication, 30 May 2013; doi:10.1038/gt.2013.24.

View details for DOI 10.1038/gt.2013.24

View details for Web of Science ID 000325633500006

View details for PubMedID 23719066
Development and Application of Stable Phantoms for the Evaluation of Photoacoustic Imaging Instruments PLOS ONE Bohndiek, S. E., Bodapati, S., Van de Sompel, D., Kothapalli, S., Gambhir, S. S. 2013; 8 (9)
Abstract

Photoacoustic imaging combines the high contrast of optical imaging with the spatial resolution and penetration depth of ultrasound. This technique holds tremendous potential for imaging in small animals and importantly, is clinically translatable. At present, there is no accepted standard physical phantom that can be used to provide routine quality control and performance evaluation of photoacoustic imaging instruments. With the growing popularity of the technique and the advent of several commercial small animal imaging systems, it is important to develop a strategy for assessment of such instruments. Here, we developed a protocol for fabrication of physical phantoms for photoacoustic imaging from polyvinyl chloride plastisol (PVCP). Using this material, we designed and constructed a range of phantoms by tuning the optical properties of the background matrix and embedding spherical absorbing targets of the same material at different depths. We created specific designs to enable: routine quality control; the testing of robustness of photoacoustic signals as a function of background; and the evaluation of the maximum imaging depth available. Furthermore, we demonstrated that we could, for the first time, evaluate two small animal photoacoustic imaging systems with distinctly different light delivery, ultrasound imaging geometries and center frequencies, using stable physical phantoms and directly compare the results from both systems.

View details for DOI 10.1371/journal.pone.0075533

View details for Web of Science ID 000325218700097

View details for PubMedID 24086557
A c-Myc Activation Sensor-Based High-Throughput Drug Screening Identifies an Antineoplastic Effect of Nitazoxanide MOLECULAR CANCER THERAPEUTICS Fan-Minogue, H., Bodapati, S., Solow-Cordero, D., Fan, A., Paulmurugan, R., Massoud, T. F., Felsher, D. W., Gambhir, S. S. 2013; 12 (9): 1896-1905
Abstract

Deregulation of c-Myc plays a central role in the tumorigenesis of many human cancers. Yet, the development of drugs regulating c-Myc activity has been challenging. To facilitate the identification of c-Myc inhibitors, we developed a molecular imaging sensor based high throughput-screening (HTS) system. This system uses a cell-based assay to detect c-Myc activation in a HTS format, which is established from a pure clone of a stable breast cancer cell line that constitutively expresses a c-Myc activation sensor. Optimization of the assay performance in the HTS format resulted in uniform and robust signals at the baseline. Using this system, we performed a quantitative HTS against approximately 5,000 existing bioactive compounds from five different libraries. Thirty-nine potential hits were identified, including currently known c-Myc inhibitors. There are a few among the top potent hits that are not known for anti-c-Myc activity. One of these hits is nitazoxanide (NTZ), a thiazolide for treating human protozoal infections. Validation of NTZ in different cancer cell lines revealed a high potency for c-Myc inhibition with IC50 ranging between 10 - 500nM. Oral administration of NTZ in breast cancer xenograft mouse models significantly suppressed tumor growth by inhibition of c-Myc and induction of apoptosis. These findings suggest a potential of NTZ to be repurposed as a new anti-tumor agent for inhibition of c-Myc associated neoplasia. Our work also demonstrated the unique advantage of molecular imaging in accelerating discovery of drugs for c-Myc targeted cancer therapy.

View details for DOI 10.1158/1535-7163.MCT-12-1243

View details for Web of Science ID 000324174600019

View details for PubMedID 23825064
High-sensitivity, real-time, ratiometric imaging of surface-enhanced Raman scattering nanoparticles with a clinically translatable Raman endoscope device. Journal of biomedical optics Garai, E., Sensarn, S., Zavaleta, C. L., Van de Sompel, D., Loewke, N. O., Mandella, M. J., Gambhir, S. S., Contag, C. H. 2013; 18 (9): 096008-?
Abstract

ABSTRACT. Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.

View details for DOI 10.1117/1.JBO.18.9.096008

View details for PubMedID 24008818
Development and Validation of Non-Integrative, Self-Limited, and Replicating Minicircles for Safe Reporter Gene Imaging of Cell-Based Therapies PLOS ONE Ronald, J. A., Cusso, L., Chuang, H., Yan, X., Dragulescu-Andrasi, A., Gambhir, S. S. 2013; 8 (8)
Abstract

Reporter gene (RG) imaging of cell-based therapies provides a direct readout of therapeutic efficacy by assessing the fate of implanted cells. To permit long-term cellular imaging, RGs are traditionally required to be integrated into the cellular genome. This poses a potential safety risk and regulatory bottleneck for clinical translation as integration can lead to cellular transformation. To address this issue, we have developed non-integrative, replicating minicircles (MCs) as an alternative platform for safer monitoring of cells in living subjects. We developed both plasmids and minicircles containing the scaffold/matrix attachment regions (S/MAR) of the human interferon-beta gene, driven by the CMV promoter, and expressing the bioluminescence RG firefly luciferase. Constructs were transfected into breast cancer cells, and expanded S/MAR minicircle clones showed luciferase signal for greater than 3 months in culture and minicircles remained as episomes. Importantly, luciferase activity in clonal populations was slowly lost over time and this corresponded to a loss of episome, providing a way to reversibly label cells. To monitor cell proliferation in vivo, 1.5×10(6) cells carrying the S/MAR minicircle were implanted subcutaneously into mice (n = 5) and as tumors developed significantly more bioluminescence signal was noted at day 35 and 43 compared to day 7 post-implant (p<0.05). To our knowledge, this is the first work examining the use of episomal, self-limited, replicating minicircles to track the proliferation of cells using non-invasive imaging in living subjects. Continued development of S/MAR minicircles will provide a broadly applicable vector platform amenable with any of the numerous RG technologies available to allow therapeutic cell fate to be assessed in individual patients, and to achieve this without the need to manipulate the cell's genome so that safety concerns are minimized. This will lead to safe tools to assess treatment response at earlier time points and improve the precision of cell-based therapies.

View details for DOI 10.1371/journal.pone.0073138

View details for Web of Science ID 000323733800093

View details for PubMedID 24015294
A Transgenic Tri-Modality Reporter Mouse PLOS ONE Yan, X., Ray, P., Paulmurugan, R., Tong, R., Gong, Y., Sathirachinda, A., Wu, J. C., Gambhir, S. S. 2013; 8 (8)
Abstract

Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent (tdTomato), and positron emission tomography (PET) (ttk) modalities. Transgenic colonies with different levels of tri-fusion reporter gene expression showed a linear correlation between all three-reporter proteins (R(2)=0.89 for TdTomato vs Fluc, R(2)=0.94 for Fluc vs TTK, R(2)=0.89 for TdTomato vs TTK) in vitro from tissue lysates and in vivo by optical and PET imaging. Mesenchymal stem cells (MSCs) isolated from this transgenics showed high level of reporter gene expression, which linearly correlated with the cell numbers (R(2)=0.99 for bioluminescence imaging (BLI)). Both BLI (R(2)=0.93) and micro-PET (R(2)=0.94) imaging of the subcutaneous implants of Tri-Modality Reporter Mouse derived MSCs in nude mice showed linear correlation with the cell numbers and across different imaging modalities (R(2)=0.97). Serial imaging of MSCs transplanted to mice with acute myocardial infarction (MI) by intramyocardial injection exhibited significantly higher signals in MI heart at days 2, 3, 4, and 7 (p<0.01). MSCs transplanted to the ischemic hindlimb of nude mice showed significantly higher BLI and PET signals in the first 2 weeks that dropped by 4(th) week due to poor cell survival. However, laser Doppler perfusion imaging revealed that blood circulation in the ischemic limb was significantly improved in the MSCs transplantation group compared with the control group. In summary, this mouse can be used as a source of donor cells and organs in various research areas such as stem cell research, tissue engineering research, and organ transplantation.

View details for DOI 10.1371/journal.pone.0073580

View details for Web of Science ID 000326473200094

View details for PubMedID 23951359
Evaluation of Zr-89-rituximab Tracer by Cerenkov Luminescence Imaging and Correlation with PET in a Humanized Transgenic Mouse Model to Image NHL MOLECULAR IMAGING AND BIOLOGY Natarajan, A., Habte, F., Liu, H., Sathirachinda, A., Hu, X., Cheng, Z., Nagamine, C. M., Gambhir, S. S. 2013; 15 (4): 468-475
Abstract

PURPOSE: This research aimed to study the use of Cerenkov luminescence imaging (CLI) for non-Hodgkin's lymphoma (NHL) using (89)Zr-rituximab positron emission tomography (PET) tracer with a humanized transgenic mouse model that expresses human CD20 and the correlation of CLI with PET. PROCEDURES: Zr-rituximab (2.6 MBq) was tail vein-injected into transgenic mice that express the human CD20 on their B cells (huCD20TM). One group (n = 3) received 2 mg/kg pre-dose (blocking) of cold rituximab 2 h prior to tracer; a second group (n = 3) had no pre-dose (non-blocking). CLI was performed using a cooled charge-coupled device optical imager. We also performed PET imaging and ex vivo studies in order to confirm the in vivo CLI results. At each time point (4, 24, 48, 72, and 96 h), two groups of mice were imaged in vivo and ex vivo with CLI and PET, and at 96 h, organs were measured by gamma counter. RESULTS: huCD20 transgenic mice injected with (89)Zr-rituximab demonstrated a high-contrast CLI image compared to mice blocked with a cold dose. At various time points of 4-96 h post-radiotracer injection, the in vivo CLI signal intensity showed specific uptake in the spleen where B cells reside and, hence, the huCD20 biomarker is present at very high levels. The time-activity curve of dose decay-corrected CLI intensity and percent injected dose per gram of tissue of PET uptake in the spleen were increased over the time period (4-96 h). At 96 h, the (89)Zr-rituximab uptake ratio (non-blocking vs blocking) counted (mean ± standard deviation) for the spleen was 1.5 ± 0.6 for CLI and 1.9 ± 0.3 for PET. Furthermore, spleen uptake measurements (non-blocking and blocking of all time points) of CLI vs PET showed good correlation (R (2) = 0.85 and slope = 0.576), which also confirmed the corresponding correlations parameter value (R (2) = 0.834 and slope = 0.47) obtained for ex vivo measurements. CONCLUSIONS: CLI and PET of huCD20 transgenic mice injected with (89)Zr-rituximab demonstrated that the tracer was able to target huCD20-expressing B cells. The in vivo and ex vivo tracer uptake corresponding to the CLI radiance intensity from the spleen is in good agreement with PET. In this report, we have validated the use of CLI with PET for NHL imaging in huCD20TM.

View details for DOI 10.1007/s11307-013-0624-0

View details for Web of Science ID 000321972500014

View details for PubMedID 23471750
Molecular imaging with surface-enhanced Raman spectroscopy nanoparticle reporters MRS BULLETIN Jokerst, J. V., Pohling, C., Gambhir, S. S. 2013; 38 (8): 625-630
View details for DOI 10.1557/mrs.2013.157

View details for Web of Science ID 000322918100014
Real-time, continuous, fluorescence sensing in a freely-moving subject with an implanted hybrid VCSEL/CMOS biosensor BIOMEDICAL OPTICS EXPRESS O'Sullivan, T. D., Heitz, R. T., Parashurama, N., Barkin, D. B., Wooley, B. A., Gambhir, S. S., Harris, J. S., Levi, O. 2013; 4 (8): 1332-1341
View details for DOI 10.1364/BOE.4.001332

View details for Web of Science ID 000322618900008
Imaging Tumor Angiogenesis: The Road to Clinical Utility AMERICAN JOURNAL OF ROENTGENOLOGY Iagaru, A., Gambhir, S. S. 2013; 201 (2): W183-W191
Abstract

OBJECTIVE. Tumor growth and progression require the formation of new blood vessels from preexisting vasculature, a process called angiogenesis. The ability to noninvasively visualize angiogenesis may provide new opportunities to more appropriately select patients for antiangiogenesis treatment and to monitor treatment efficacy. CONCLUSION. The superior molecular sensitivity of PET and the lack of radiation from MRI and contrast-enhanced ultrasound put these techniques at the forefront of clinical translation.

View details for DOI 10.2214/AJR.12.8568

View details for Web of Science ID 000322225400003

View details for PubMedID 23883233
Activatable Oligomerizable Imaging Agents for Photoacoustic Imaging of Furin-Like Activity in Living Subjects JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Dragulescu-Andrasi, A., Kothapalli, S., Tikhomirov, G. A., Rao, J., Gambhir, S. S. 2013; 135 (30): 11015-11022
Abstract

Photoacoustic (PA) imaging is continuing to be applied for physiological imaging and more recently for molecular imaging of living subjects. Owing to its high spatial resolution in deep tissues, PA imaging holds great potential for biomedical applications and molecular diagnostics. There is however a lack of probes for targeted PA imaging, especially in the area of enzyme-activatable probes. Here we introduce a molecular probe, which upon proteolytic processing is retained at the site of enzyme activity and provides PA contrast. The probe oligomerizes via a condensation reaction and accumulates in cells and tumors that express the protease. We demonstrate that this probe reports furin and furin-like activity in cells and tumor models by generating a significantly higher PA signal relative to furin-deficient and nontarget controls. This probe could report enzyme activity in living subjects at depths significantly greater than fluorescence imaging probes and has potential for molecular imaging in deep tumors.

View details for DOI 10.1021/ja4010078

View details for Web of Science ID 000322752900032

View details for PubMedID 23859847
A small animal Raman instrument for rapid, wide-area, spectroscopic imaging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bohndiek, S. E., Wagadarikar, A., Zavaleta, C. L., Van de Sompel, D., Garai, E., Jokerst, J. V., Yazdanfar, S., Gambhir, S. S. 2013; 110 (30): 12408-12413
Abstract

Raman spectroscopy, amplified by surface enhanced Raman scattering (SERS) nanoparticles, is a molecular imaging modality with ultra-high sensitivity and the unique ability to multiplex readouts from different molecular targets using a single wavelength of excitation. This approach holds exciting prospects for a range of applications in medicine, including identification and characterization of malignancy during endoscopy and intraoperative image guidance of surgical resection. The development of Raman molecular imaging with SERS nanoparticles is presently limited by long acquisition times, poor spatial resolution, small field of view, and difficulty in animal handling with existing Raman spectroscopy instruments. Our goal is to overcome these limitations by designing a bespoke instrument for Raman molecular imaging in small animals. Here, we present a unique and dedicated small-animal Raman imaging instrument that enables rapid, high-spatial resolution, spectroscopic imaging over a wide field of view (> 6 cm(2)), with simplified animal handling. Imaging of SERS nanoparticles in small animals demonstrated that this small animal Raman imaging system can detect multiplexed SERS signals in both superficial and deep tissue locations at least an order of magnitude faster than existing systems without compromising sensitivity.

View details for DOI 10.1073/pnas.1301379110

View details for Web of Science ID 000322112300062

View details for PubMedID 23821752
An Observational Study of Circulating Tumor Cells and F-18-FDG PET Uptake in Patients with Treatment-Naive Non-Small Cell Lung Cancer PLOS ONE Nair, V. S., Keu, K. V., Luttgen, M. S., Kolatkar, A., Vasanawala, M., Kuschner, W., Bethel, K., Iagaru, A. H., Hoh, C., Shrager, J. B., Loo, B. W., Bazhenova, L., Nieva, J., Gambhir, S. S., Kuhn, P. 2013; 8 (7)
View details for DOI 10.1371/journal.pone.0067733

View details for Web of Science ID 000321425300025
Noninvasive Monitoring of Oxidative Stress in Transplanted Mesenchymal Stromal Cells JACC-CARDIOVASCULAR IMAGING Psaltis, P. J., Peterson, K. M., Xu, R., Franchi, F., Witt, T., Chen, I. Y., Lerman, A., Simari, R. D., Gambhir, S. S., Rodriguez-Porcel, M. 2013; 6 (7): 795-802
Abstract

OBJECTIVES: The goal of this study was to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells. BACKGROUND: In cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function. METHODS: Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67(phox) subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67(phox)), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67(phox) promoter. MSCs cotransfected with NAD(P)H p67(phox)-Fluc and a cell viability reporter gene (cytomegalovirus-Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions. RESULTS: After in vitro validation of the sensor during low-serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 ± 0.003 for sham vs. 0.026 ± 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor. CONCLUSIONS: Pathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.

View details for DOI 10.1016/j.jcmg.2012.11.018

View details for Web of Science ID 000321677300006

View details for PubMedID 23643284
Pilot Prospective Evaluation of Tc-99m-MDP Scintigraphy, F-18 NaF PET/CT, F-18 FDG PET/CT and Whole-Body MRI for Detection of Skeletal Metastases CLINICAL NUCLEAR MEDICINE Iagaru, A., Young, P., Mittra, E., Dick, D. W., Herfkens, R., Gambhir, S. S. 2013; 38 (7): E290-E296
View details for Web of Science ID 000330354600003
F-18-Fluorobenzoate-Labeled Cystine Knot Peptides for PET Imaging of Integrin alpha(v)beta(6) JOURNAL OF NUCLEAR MEDICINE Hackel, B. J., Kimura, R. H., Miao, Z., Liu, H., Sathirachinda, A., Cheng, Z., Chin, F. T., Gambhir, S. S. 2013; 54 (7): 1101-1105
Abstract

Integrin αvβ6 is a cell surface receptor minimally expressed by healthy tissue but elevated in lung, colon, skin, ovarian, cervical, and pancreatic cancers. A molecular PET agent for integrin αvβ6 could provide significant clinical utility by facilitating both cancer staging and treatment monitoring to more rapidly identify an effective therapeutic approach. METHODS: Here, we evaluated 2 cystine knot peptides, R01 and S02, previously engineered with a 3-6 nM affinity for integrin αvβ6, for (18)F radiolabeling and PET imaging of BxPC3 pancreatic adenocarcinoma xenografts in mice. Cystine knot peptides were labeled with N-succinimidyl-4-(18)F-fluorobenzoate and evaluated for binding affinity and serum stability. Peptides conjugated with (18)F-fluorobenzoate (2-3 MBq) were injected via the tail vein into nude mice xenografted with BxPC3 (integrin αvβ6-positive) or 293 (integrin αvβ6-negative) tumors. Small-animal PET scans were acquired at 0.5, 1, and 2 h after injection. Ex vivo γ-counting of dissected tissues was performed at 0.5 and 2 h. RESULTS: (18)F-fluorobenzoate peptides were produced in 93% ((18)F-fluorobenzoate-R01) and 99% ((18)F-fluorobenzoate-S02) purity. (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02 had affinities of 1.1 ± 0.2 and 0.7 ± 0.4 nM, respectively, and were 87% and 94%, respectively, stable in human serum at 37°C for 2 h. (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02 exhibited 2.3 ± 0.6 and 1.3 ± 0.4 percentage injected dose per gram (%ID/g), respectively, in BxPC3 xenografted tumors at 0.5 h (n = 4-5). Target specificity was confirmed by low tumor uptake in integrin αvβ6-negative 293 tumors (1.4 ± 0.6 and 0.5 ± 0.2 %ID/g, respectively, for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02; both P < 0.05; n = 3-4) and low muscle uptake (3.1 ± 1.0 and 2.7 ± 0.4 tumor to muscle for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02, respectively). Small-animal PET data were corroborated by ex vivo γ-counting of dissected tissues, which demonstrated low uptake in nontarget tissues with only modest kidney uptake (9.2 ± 3.3 and 1.9 ± 1.2 %ID/g, respectively, at 2 h for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02; n = 8). Uptake in healthy pancreas was low (0.3% ± 0.1% for (18)F-fluorobenzoate-R01 and 0.03% ± 0.01% for (18)F-fluorobenzoate-S02; n = 8). CONCLUSION: These cystine knot peptide tracers, in particular (18)F-fluorobenzoate-R01, show translational promise for molecular imaging of integrin αvβ6 overexpression in pancreatic and other cancers.

View details for DOI 10.2967/jnumed.112.110759

View details for Web of Science ID 000321113500019
A Raman-based endoscopic strategy for multiplexed molecular imaging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zavaleta, C. L., Garai, E., Liu, J. T., Sensarn, S., Mandella, M. J., Van de Sompel, D., Friedland, S., Van Dam, J., Contag, C. H., Gambhir, S. S. 2013; 110 (25): E2288-E2297
View details for DOI 10.1073/pnas.1211309110

View details for Web of Science ID 000321500200010
Molecular Imaging of Inflammation in Inflammatory Bowel Disease with a Clinically Translatable Dual-Selectin-targeted US Contrast Agent: Comparison with FDG PET/CT in a Mouse Model RADIOLOGY Wang, H., Machtaler, S., Bettinger, T., Lutz, A. M., Luong, R., Bussat, P., Gambhir, S. S., Tranquart, F., Tian, L., Willmann, J. K. 2013; 267 (3): 818-829
Abstract

Purpose: To develop and test a molecular imaging approach that uses ultrasonography (US) and a clinically translatable dual-targeted (P- and E-selectin) contrast agent (MBSelectin) in the quantification of inflammation at the molecular level and to quantitatively correlate selectin-targeted US with fluorodeoxyglucose (FDG) combined positron emission tomography (PET) and computed tomography (CT) in terms of visualization and quantification of different levels of inflammation in a murine acute colitis model. Materials and Methods: Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. MBSelectin was developed by covalently binding an analog of the naturally occurring binding ligand P-selectin glycoprotein ligand 1 fused to a human fragment crystallizable(or Fc) domain onto the lipid shell of perfluorobutane and nitrogen-containing MBs. Binding specificity of MBSelectin was assessed in vitro with a flow chamber assay and in vivo with a chemically induced acute colitis murine model. US signal was quantitatively correlated with FDG uptake at PET/CT and histologic grade. Statistical analysis was performed with the Student t test, analysis of variance, and Pearson correlation analysis. Results: MBSelectin showed strong attachment to both human and mouse P- and E-selectin compared with MBControl in vitro (P ≤ .002). In vivo, US signal was significantly increased (P < .001) in mice with acute colitis (173.8 arbitrary units [au] ± 134.8 [standard deviation]) compared with control mice (5.0 au ± 4.5). US imaging signal strongly correlated with FDG uptake on PET/CT images (ρ = 0.89, P < .001). Ex vivo analysis enabled confirmation of inflammation in mice with acute colitis and high expression levels of P- and E-selectin in mucosal capillaries (P = .014). Conclusion: US with MBSelectin specifically enables detection and quantification of inflammation in a murine acute colitis model, leveraging the natural pathway of leukocyte recruitment in inflammatory tissue. US imaging with MBSelectin correlates well with FDG uptake at PET/CT imaging. © RSNA, 2013 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.13122509/-/DC1.

View details for DOI 10.1148/radiol.13122509

View details for Web of Science ID 000319445400018
A comparison of noise models in a hybrid reference spectrum and principal components analysis algorithm for Raman spectroscopy JOURNAL OF RAMAN SPECTROSCOPY Van de Sompel, D., Garai, E., Zavaleta, C., Gambhir, S. S. 2013; 44 (6): 841-856
View details for DOI 10.1002/jrs.4258

View details for Web of Science ID 000319935700009
[F-18]CAIP a smart PET tracer for imaging caspase-3 induced Apoptosis Shen Bin, B., Jeon, J., Palner, M., Tong Ling, L., Felsher, D., Gambhir, S. S., Chin, F. T., Rao Jianghong, J. H. WILEY-BLACKWELL. 2013: S6-S6
View details for Web of Science ID 000318694100007
Evaluation of the antitumor effects of rilotumumab by PET imaging in a U-87 MG mouse xenograft model. Nuclear medicine and biology Rex, K., Lewis, X. Z., Gobalakrishnan, S., Glaus, C., Silva, M. D., Radinsky, R., Burgess, T. L., Gambhir, S. S., Coxon, A. 2013; 40 (4): 458-463
Abstract

Dysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using (18)F-FDG and (18)F-FLT PET.U-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 μg) were administered, and mice were evaluated with (18)F-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 μg of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using (18)F-FDG and (18)F-FLT PET.In the dose response study, rilotumumab at doses of 300 and 500 μg was similarly effective against tumor growth. Treatment with 300 and 500 μg rilotumumab inhibited (18)F-FDG accumulation with significant decreases of -37% and -40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 μg rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation with a maximum %ID/g of -41% and -64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed.Rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate (18)F-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer.

View details for DOI 10.1016/j.nucmedbio.2013.01.004

View details for PubMedID 23454250
High-resolution, serial intravital microscopic imaging of nanoparticle delivery and targeting in a small animal tumor model NANO TODAY Smith, B. R., Zavaleta, C., Rosenberg, J., Tong, R., Ramunas, J., Liu, Z., Dai, H., Gambhir, S. S. 2013; 8 (2): 126-137
View details for DOI 10.1016/j.nantod.2013.02.004

View details for Web of Science ID 000319236400006
Intracellular Aggregation of Multimodal Silica Nanoparticles for Ultrasound-Guided Stem Cell Implantation SCIENCE TRANSLATIONAL MEDICINE Jokerst, J. V., Khademi, C., Gambhir, S. S. 2013; 5 (177)
View details for DOI 10.1126/scitranslmed.3005228

View details for Web of Science ID 000316454100004
Molecular Photoacoustic Imaging of Follicular Thyroid Carcinoma CLINICAL CANCER RESEARCH Levi, J., Kothapalli, S., Bohndiek, S., Yoon, J., Dragulescu-Andrasi, A., Nielsen, C., Tisma, A., Bodapati, S., Gowrishankar, G., Yan, X., Chan, C., Starcevic, D., Gambhir, S. S. 2013; 19 (6): 1494-1502
Abstract

To evaluate the potential of targeted photoacoustic imaging as a noninvasive method for detection of follicular thyroid carcinoma.We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP-activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP-activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual-wavelength imaging method.Active forms of both MMP-2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, nonactivatable, agent.With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas.

View details for DOI 10.1158/1078-0432.CCR-12-3061

View details for Web of Science ID 000316188900021

View details for PubMedID 23349314
Earlier Detection of Breast Cancer with Ultrasound Molecular Imaging in a Transgenic Mouse Model CANCER RESEARCH Bachawal, S. V., Jensen, K. C., Lutz, A. M., Gambhir, S. S., Tranquart, F., Tian, L., Willmann, J. K. 2013; 73 (6): 1689-1698
Abstract

While there is an increasing role of ultrasound for breast cancer screening in patients with dense breast, conventional anatomical ultrasound lacks sensitivity and specificity for early breast cancer detection. In this study, we assessed the potential of ultrasound molecular imaging using clinically translatable vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted microbubbles (MB(VEGFR2)) to improve the diagnostic accuracy of ultrasound in earlier detection of breast cancer and ductal carcinoma in situ (DCIS) in a transgenic mouse model [FVB/N-Tg(MMTV-PyMT)634Mul]. In vivo binding specificity studies (n = 26 tumors) showed that ultrasound imaging signal was significantly higher (P < 0.001) using MB(VEGFR2) than nontargeted microbubbles and imaging signal significantly decreased (P < 0.001) by blocking antibodies. Ultrasound molecular imaging signal significantly increased (P < 0.001) when breast tissue (n = 315 glands) progressed from normal [1.65 ± 0.17 arbitrary units (a.u.)] to hyperplasia (4.21 ± 1.16), DCIS (15.95 ± 1.31), and invasive cancer (78.1 ± 6.31) and highly correlated with ex vivo VEGFR2 expression [R(2) = 0.84; 95% confidence interval (CI), 0.72-0.91; P < 0.001]. At an imaging signal threshold of 4.6 a.u., ultrasound molecular imaging differentiated benign from malignant entities with a sensitivity of 84% (95% CI, 78-88) and specificity of 89% (95% CI, 81-94). In a prospective screening trail (n = 63 glands), diagnostic performance of detecting DCIS and breast cancer was assessed and two independent readers correctly diagnosed malignant disease in more than 95% of cases and highly agreed between each other [intraclass correlation coefficient (ICC) = 0.98; 95% CI, 97-99]. These results suggest that VEGFR2-targeted ultrasound molecular imaging allows highly accurate detection of DCIS and breast cancer in transgenic mice and may be a promising approach for early breast cancer detection in women.

View details for DOI 10.1158/0008-5472.CAN-12-3391

View details for Web of Science ID 000316187500006
An Integrated Computational/Experimental Model of Lymphoma Growth PLOS COMPUTATIONAL BIOLOGY Frieboes, H. B., Smith, B. R., Chuang, Y., Ito, K., Roettgers, A. M., Gambhir, S. S., Cristini, V. 2013; 9 (3)
Abstract

Non-Hodgkin's lymphoma is a disseminated, highly malignant cancer, with resistance to drug treatment based on molecular- and tissue-scale characteristics that are intricately linked. A critical element of molecular resistance has been traced to the loss of functionality in proteins such as the tumor suppressor p53. We investigate the tissue-scale physiologic effects of this loss by integrating in vivo and immunohistological data with computational modeling to study the spatiotemporal physical dynamics of lymphoma growth. We compare between drug-sensitive E?-myc Arf-/- and drug-resistant E?-myc p53-/- lymphoma cell tumors grown in live mice. Initial values for the model parameters are obtained in part by extracting values from the cellular-scale from whole-tumor histological staining of the tumor-infiltrated inguinal lymph node in vivo. We compare model-predicted tumor growth with that observed from intravital microscopy and macroscopic imaging in vivo, finding that the model is able to accurately predict lymphoma growth. A critical physical mechanism underlying drug-resistant phenotypes may be that the E?-myc p53-/- cells seem to pack more closely within the tumor than the E?-myc Arf-/- cells, thus possibly exacerbating diffusion gradients of oxygen, leading to cell quiescence and hence resistance to cell-cycle specific drugs. Tighter cell packing could also maintain steeper gradients of drug and lead to insufficient toxicity. The transport phenomena within the lymphoma may thus contribute in nontrivial, complex ways to the difference in drug sensitivity between E?-myc Arf-/- and E?-myc p53-/- tumors, beyond what might be solely expected from loss of functionality at the molecular scale. We conclude that computational modeling tightly integrated with experimental data gives insight into the dynamics of Non-Hodgkin's lymphoma and provides a platform to generate confirmable predictions of tumor growth.

View details for DOI 10.1371/journal.pcbi.1003008

View details for Web of Science ID 000316864200070

View details for PubMedID 23555235
BIOENGINEERING AND REGENERATIVE MEDICINE Keeping track NATURE MATERIALS Ziv, K., Gambhir, S. S. 2013; 12 (3): 180-181
View details for Web of Science ID 000315707200009

View details for PubMedID 23422714
Non-Invasive Imaging of Phosphoinositide-3-Kinase-Catalytic-Subunit-Alpha (PIK3CA) Promoter Modulation in Small Animal Models PLOS ONE Gaikwad, S. M., Gunjal, L., Junutula, A. R., Astanehe, A., Gambhir, S. S., Ray, P. 2013; 8 (2)
Abstract

Activation of the PI3K/Akt pathway, a critical step for survival in cancer cells is often associated with decreased sensitivity to several chemotherapeutic drugs. PIK3CA gene amplification is observed in 16-24% of epithelial ovarian cancer (EOC) patients in conjunction with p53 mutations. A 900 bp long PIK3CA promoter is shown to be negatively regulated by p53 in ovarian surface epithelial cells but the consequence of chemotherapeutic drug treatments on this promoter in ovarian cancer cells is largely unknown. We aim to study the modulation of this promoter by cisplatin using an improved fusion reporter in ovarian cancer cells and tumor xenografts by non-invasive imaging approach. A PIK3CA sensor was developed using a bi-fusion reporter from a newly constructed library of bi- and tri-fusion vectors comprising of two mutant far red fluorescent proteins (mcherry/mch and tdTomato/tdt), a mutant firefly luciferase (fluc2), and a PET reporter protein (ttk). In vivo imaging of mice implanted with 293T cells transiently expressing these bi- and tri-fusion reporters along with respective controls revealed comparable activity of each reporter in the fusion background and fluc2-tdt as the most sensitive one. Repression of the PIK3CA sensor by drugs was inversely proportional to cellular p53 level in a germline (PA1) and in an EOC (A2780) cell line but not in a p53 deficient EOC (SKOV3) cell line. Bioluminescence imaging of tumor xenografts stably expressing the PIK3CA sensor in PA1 and A2780 cells exhibited attenuating activity without any change in SKOV3 tumors expressing the PIK3CA sensor after cisplatin treatment. Sequential mutation at p53 binding sites showed gradual increase in promoter activity and decreased effects of the drugs. These newly developed PIK3CA-fluc2-tdt and the mutant reporter sensors thus would be extremely useful for screening new drugs and for functional assessment of PIK3CA expression from intact cells to living subjects.

View details for DOI 10.1371/journal.pone.0055971

View details for Web of Science ID 000314692800061

View details for PubMedID 23393606
Dissection of the role of the tumor microenvironment in oncogene addiction by ex vivo and in situ imaging Tong, L., Jeon, J., Shen, B., Jianghong, R., Chin, F., Gambhir, S., Felsher, D. SOC NUCLEAR MEDICINE INC. 2013: 25-25
View details for Web of Science ID 000314691400075
Combined F-18-Fluoride and F-18-FDG PET/CT Scanning for Evaluation of Malignancy: Results of an International Multicenter Trial JOURNAL OF NUCLEAR MEDICINE Iagaru, A., Mittra, E., Mosci, C., Dick, D. W., Sathekge, M., Prakash, V., Iyer, V., Lapa, P., Isidoro, J., de Lima, J. M., Gambhir, S. S. 2013; 54 (2): 176-183
Abstract

(18)F-FDG PET/CT is used in a variety of cancers, but because of variable rates of glucose metabolism, not all cancers are reliably identified. (18)F(-) PET/CT allows for the acquisition of highly sensitive and specific images of the skeleton. We prospectively evaluated combined (18)F(-)/(18)F-FDG as a single PET/CT examination for evaluation of cancer patients and compared it with separate (18)F(-) PET/CT and (18)F-FDG PET/CT scans.One hundred fifteen participants with cancer were prospectively enrolled in an international multicenter trial evaluating (18)F(-) PET/CT, (18)F-FDG PET/CT, and combined (18)F(-)/(18)F-FDG PET/CT. The 3 PET/CT scans were performed sequentially within 4 wk of one another for each patient.(18)F(-)/(18)F-FDG PET/CT allowed for accurate interpretation of radiotracer uptake outside the skeleton, with findings similar to those of (18)F-FDG PET/CT. In 19 participants, skeletal disease was more extensive on (18)F(-) PET/CT and (18)F(-)/(18)F-FDG PET/CT than on (18)F-FDG PET/CT. In another 29 participants, (18)F(-) PET/CT and (18)F(-)/(18)F-FDG PET/CT showed osseous metastases where (18)F-FDG PET/CT was negative. The extent of skeletal lesions was similar in 18 participants on all 3 scans.This trial demonstrated that combined (18)F(-)/(18)F-FDG PET/CT shows promising results when compared with separate (18)F(-) PET/CT and (18)F-FDG PET/CT for evaluation of cancer patients. This result opens the possibility for improved patient care and reduction in health-care costs, as will be further evaluated in future trials.

View details for DOI 10.2967/jnumed.112.108803

View details for Web of Science ID 000314691200016
Colony-stimulating factor 1 receptor (CSF1R) signaling in injured neurons facilitates protection and survival JOURNAL OF EXPERIMENTAL MEDICINE Luo, J., Elwood, F., Britschgi, M., Villeda, S., Zhang, H., Ding, Z., Zhu, L., Alabsi, H., Getachew, R., Narasimhan, R., Wabl, R., Fainberg, N., James, M. L., Wong, G., Relton, J., Gambhir, S. S., Pollard, J. W., Wyss-Coray, T. 2013; 210 (1): 157-172
Abstract

Colony-stimulating factor 1 (CSF1) and interleukin-34 (IL-34) are functional ligands of the CSF1 receptor (CSF1R) and thus are key regulators of the monocyte/macrophage lineage. We discovered that systemic administration of human recombinant CSF1 ameliorates memory deficits in a transgenic mouse model of Alzheimer's disease. CSF1 and IL-34 strongly reduced excitotoxin-induced neuronal cell loss and gliosis in wild-type mice when administered systemically before or up to 6 h after injury. These effects were accompanied by maintenance of cAMP responsive element-binding protein (CREB) signaling in neurons rather than in microglia. Using lineage-tracing experiments, we discovered that a small number of neurons in the hippocampus and cortex express CSF1R under physiological conditions and that kainic acid-induced excitotoxic injury results in a profound increase in neuronal receptor expression. Selective deletion of CSF1R in forebrain neurons in mice exacerbated excitotoxin-induced death and neurodegeneration. We conclude that CSF1 and IL-34 provide powerful neuroprotective and survival signals in brain injury and neurodegeneration involving CSF1R expression on neurons.

View details for DOI 10.1084/jem.20120412

View details for Web of Science ID 000313560900014
Synthesis of ligand-functionalized water-soluble [F-18]YF3 nanoparticles for PET imaging NANOSCALE Xiong, L., Shen, B., Behera, D., Gambhir, S. S., Chin, F. T., Rao, J. 2013; 5 (8): 3253-3256
Abstract

We report a simple, efficient synthesis of novel (18)F-labeled imaging agents based on YF3 nanoparticles. Targeting ligands and antitumor drug molecules can be introduced onto the YF3 nanoparticles in a one-pot synthesis. The (18)F-labeling reaction proceeds in aqueous solutions at room temperature with excellent radiolabeling yields (>80%) in a very short time (5-10 min). (18)F-labeled YF3 nanoparticles displayed high stability in mouse and human serum, and their application for mapping lymph nodes in live rats after local injection has also been demonstrated.

View details for DOI 10.1039/c3nr00335c

View details for Web of Science ID 000316959500019

View details for PubMedID 23508229
Evolution of BRET Biosensors from Live Cell to Tissue-Scale In vivo Imaging. Frontiers in endocrinology De, A., Jasani, A., Arora, R., Gambhir, S. S. 2013; 4: 131-?
Abstract

Development of bioluminescence resonance energy transfer (BRET) based genetic sensors for sensing biological functions such as protein-protein interactions (PPIs) in vivo has a special value in measuring such dynamic events at their native environment. Since its inception in the late nineties, BRET related research has gained significant momentum in terms of adding versatility to the assay format and wider applicability where it has been suitably used. Beyond the scope of quantitative measurement of PPIs and protein dimerization, molecular imaging applications based on BRET assays have broadened its scope for screening pharmacologically important compounds by in vivo imaging as well. In this mini-review we focus on an in-depth analysis of engineered BRET systems developed and their successful application to cell-based assays as well as in vivo non-invasive imaging in live subjects.

View details for DOI 10.3389/fendo.2013.00131

View details for PubMedID 24065957
Comparison of Gaussian and Poisson Noise Models in a Hybrid Reference Spectrum and Principal Component Analysis Algorithm for Raman Spectroscopy SINGLE MOLECULE SPECTROSCOPY AND SUPERRESOLUTION IMAGING VI Van de Sompel, D., Garai, E., Zavaleta, C., Gambhir, S. S. 2013; 8590
View details for DOI 10.1117/12.2005455

View details for Web of Science ID 000321741600011
A Brain Tumor Molecular Imaging Strategy using a New Triple-Modality MRI-Photoacoustic-Raman Nanoparticle PHOTONS PLUS ULTRASOUND: IMAGING AND SENSING 2013 de la Zerda, A., Kircher, M. F., Jokerst, J. V., Zavaleta, C. L., Kempen, P. J., Mittra, E., Pitter, K., Huang, R., Campos, C., Habte, F., Sinclair, R., Brennan, C. W., Mellinghoff, I. K., Holland, E. C., Gambhir, S. S. 2013; 8581
View details for DOI 10.1117/12.2001719

View details for Web of Science ID 000322832800007
Stable phantoms for characterization of photoacoustic tomography (PAT) systems DESIGN AND PERFORMANCE VALIDATION OF PHANTOMS USED IN CONJUNCTION WITH OPTICAL MEASUREMENT OF TISSUE V Bohndiek, S. E., Van de Sompel, D., Bodapati, S., Kothapalli, S. R., Gambhir, S. S. 2013; 8583
View details for DOI 10.1117/12.2005195

View details for Web of Science ID 000322903900005
Human Flexor Tendon Tissue Engineering: Revitalization of Biostatic Allograft Scaffolds TISSUE ENGINEERING PART A Woon, C. Y., Farnebo, S., Schmitt, T., Kraus, A., Megerle, K., Hung Pham, H., Yan, X., Gambhir, S. S., Chang, J. 2012; 18 (23-24): 2406-2417
Abstract

Cadaveric tendon allografts form a readily available and underutilized source of graft material. Because of their material properties, allografts are biomechanically and biologically superior to synthetic scaffolds. However, before clinical use, allografts must undergo decellularization to reduce immunogenicity and oxidation to increase porosity, leaving a nonvital biostatic scaffold. Ex vivo seeding, or revitalization, is thought to hasten graft incorporation and stimulate intrinsic tendon healing, permitting early mobilization and return to function. In this study, we examined physical and biochemical augmentation methods, including scaffold surface scoring (physical) and rehydration of lyophilized scaffolds in serum (biochemical). Scaffolds were divided into four groups: (1) scored scaffolds, (2) lyophilized scaffolds rehydrated in fetal calf serum (FCS), (3) scaffolds both scored and rehydrated in FCS, and (4) control scaffolds. Scaffolds were reseeded with adipose-derived stem cells (ADSCs). Reseeding efficacy was quantified by a live cell and total cell assays and qualified histologically with hematoxylin and eosin, live/dead and SYTO green nucleic acid stains, TUNEL apoptosis stains, procollagen stains, and transmission electron microscopy. Scaffold-seeded cell viability at up to 2 weeks in vitro and up to 4 weeks in vivo was demonstrated with bioluminescent imaging of scaffolds seeded with luciferase-positive ADSCs. The effect of seeding on scaffold biomechanical properties was demonstrated with evaluation of ultimate tensile stress (UTS) and an elastic modulus (EM). We found that scaffold surface scoring led to an increase in live and total cell attachment and penetration (MTS assay, p<0.001 and DNA assay, p=0.003, respectively). Histology confirmed greater total cell number in both construct core and surface in scored compared with unscored constructs. Cells reseeded on scored constructs displayed reduced apoptosis, persistent procollagen production, and had a similar ultrastructural relationship to the surrounding matrix as native tenocytes on transmission electron microscopy. Rehydration of lyophilized scaffolds in serum did not improve reseeding. Seeded constructs demonstrated greater UTS and EM than unseeded constructs. Scaffolds seeded with ADSC-luc2-eGFP demonstrated persistent viability for at least 2 weeks in vitro. In conclusion, tendon surface scoring increases surface and core reseeding in vitro and may be incorporated as a final step in allograft processing before clinical implantation.

View details for DOI 10.1089/ten.tea.2012.0152

View details for Web of Science ID 000311600800002
Exogenous MC3T3 Preosteoblasts Migrate Systemically and Mitigate the Adverse Effects of Wear Particles TISSUE ENGINEERING PART A Fritton, K., Ren, P., Gibon, E., Rao, A. J., Ma, T., Biswal, S., Gambhir, S. S., Goodman, S. B. 2012; 18 (23-24): 2559-2567
Abstract

Understanding how relevant cell types respond to wear particles will reveal new avenues for treating osteolysis following joint replacements. In this study, we investigate the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on preosteoblast migration and function. We infused UHMWPE particles or saline into the left femur of mice and injected luciferase-expressing preosteoblasts (MC3T3 cells) into each left ventricle. Bioluminescence imaging (BLI) confirmed systemic administration of MC3T3 cells. BLI throughout the 28-day experiment showed greater MC3T3 migration to the site of particle infusion than to the site of saline infusion, with significant differences on days 0, 4, and 6 (p?0.055). Immunostaining revealed a greater number of osteoblasts and osteoclasts in the particle-infused femora, indicating greater bone turnover. The bone mineralization of the particle-infused femora increased significantly when compared to saline-infused femora (an increase of 146.4±27.9 vs. 12.8±8.7?mg/mL, p=0.008). These results show that infused preosteoblasts can migrate to the site of wear particles. Additionally, as the migrated cells were associated with increased bone mineralization in spite of the presence of particles, increasing osteoblast recruitment is a potential strategy for combating bone loss due to increased osteoclast/macrophage number and decreased osteoblast function.

View details for DOI 10.1089/ten.tea.2012.0086

View details for Web of Science ID 000311600800016

View details for PubMedID 22741555
Unexpected Dissemination Patterns in Lymphoma Progression Revealed by Serial Imaging within a Murine Lymph Node CANCER RESEARCH Ito, K., Smith, B. R., Parashurama, N., Yoon, J., Song, S. Y., Miething, C., Mallick, P., Lowe, S., Gambhir, S. S. 2012; 72 (23): 6111-6118
Abstract

Non-Hodgkin lymphoma (NHL) is a heterogeneous and highly disseminated disease, but the mechanisms of its growth and dissemination are not well understood. Using a mouse model of this disease, we used multimodal imaging, including intravital microscopy (IVM) combined with bioluminescence, as a powerful tool to better elucidate NHL progression. We injected enhanced green fluorescent protein and luciferase-expressing E?-Myc/Arf(-/-) (Cdkn2a(-/-)) mouse lymphoma cells (EL-Arf(-/-)) into C57BL/6NCrl mice intravenously. Long-term observation inside a peripheral lymph node was enabled by a novel lymph node internal window chamber technique that allows chronic, sequential lymph node imaging under in vivo physiologic conditions. Interestingly, during early stages of tumor progression we found that few if any lymphoma cells homed initially to the inguinal lymph node (ILN), despite clear evidence of lymphoma cells in the bone marrow and spleen. Unexpectedly, we detected a reproducible efflux of lymphoma cells from spleen and bone marrow, concomitant with a massive and synchronous influx of lymphoma cells into the ILN, several days after injection. We confirmed a coordinated efflux/influx of tumor cells by injecting EL-Arf(-/-) lymphoma cells directly into the spleen and observing a burst of lymphoma cells, validating that the burst originated in organs remote from the lymph nodes. Our findings argue that in NHL an efflux of tumor cells from one disease site to another, distant site in which they become established occurs in discrete bursts.

View details for DOI 10.1158/0008-5472.CAN-12-2579

View details for Web of Science ID 000311893100005

View details for PubMedID 23033441
Continuous sensing of tumor-targeted molecular probes with a vertical cavity surface emitting laser-based biosensor JOURNAL OF BIOMEDICAL OPTICS Parashurama, N., O'Sullivan, T. D., de la Zerda, A., El Kalassi, P., Cho, S., Liu, H., Teed, R., Levy, H., Rosenberg, J., Cheng, Z., Levi, O., Harris, J. S., Gambhir, S. S. 2012; 17 (11)
Abstract

Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets ?V?3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications.

View details for DOI 10.1117/1.JBO.17.11.117004

View details for Web of Science ID 000314502700046

View details for PubMedID 23123976
Gold Nanorods for Ovarian Cancer Detection with Photoacoustic Imaging and Resection Guidance via Raman Imaging in Living Mice ACS NANO Jokerst, J. V., Cole, A. J., Van de Sompel, D., Gambhir, S. S. 2012; 6 (11): 10366-10377
Abstract

Improved imaging approaches are needed for ovarian cancer screening, diagnosis, staging, and resection guidance. Here, we propose a combined photoacoustic (PA)/Raman approach using gold nanorods (GNRs) as a passively targeted molecular imaging agent. GNRs with three different aspect ratios were studied. Those with an aspect ratio of 3.5 were selected for their highest ex vivo and in vivo PA signal and used to image subcutaneous xenografts of the 2008, HEY, and SKOV3 ovarian cancer cell lines in living mice. Maximum PA signal was observed within 3 h for all three lines tested and increased signal persisted for at least two days postadministration. There was a linear relationship (R(2) = 0.95) between the PA signal and the concentration of injected molecular imaging agent with a calculated limit of detection of 0.40 nM GNRs in the 2008 cell line. The same molecular imaging agent could be used for clear visualization of the margin between tumor and normal tissue and tumor debulking via surface-enhanced Raman spectroscopy (SERS) imaging. Finally, we validated the imaging findings with biodistribution data and elemental analysis. To the best of our knowledge, this is the first report of in vivo imaging of ovarian cancer tumors with a photoacoustic and Raman imaging agent.

View details for DOI 10.1021/nn304347g

View details for Web of Science ID 000311521700112

View details for PubMedID 23101432
New Positron Emission Tomography (PET) Radioligand for Imaging sigma-1 Receptors in Living Subjects JOURNAL OF MEDICINAL CHEMISTRY James, M. L., Shen, B., Zavaleta, C. L., Nielsen, C. H., Mesangeau, C., Vuppala, P. K., Chan, C., Avery, B. A., Fishback, J. A., Matsumoto, R. R., Gambhir, S. S., McCurdy, C. R., Chin, F. T. 2012; 55 (19): 8272-8282
Abstract

?-1 receptor (S1R) radioligands have the potential to detect and monitor various neurological diseases. Herein we report the synthesis, radiofluorination, and evaluation of a new S1R ligand 6-(3-fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one ([(18)F]FTC-146, [(18)F]13). [(18)F]13 was synthesized by nucleophilic fluorination, affording a product with >99% radiochemical purity (RCP) and specific activity (SA) of 2.6 ± 1.2 Ci/?mol (n = 13) at end of synthesis (EOS). Positron emission tomography (PET) and ex vivo autoradiography studies of [(18)F]13 in mice showed high uptake of the radioligand in S1R rich regions of the brain. Pretreatment with 1 mg/kg haloperidol (2), nonradioactive 13, or BD1047 (18) reduced the binding of [(18)F]13 in the brain at 60 min by 80%, 82%, and 81%, respectively, suggesting that [(18)F]13 accumulation in mouse brain represents specific binding to S1Rs. These results indicate that [(18)F]13 is a promising candidate radiotracer for further evaluation as a tool for studying S1Rs in living subjects.

View details for DOI 10.1021/jm300371c

View details for Web of Science ID 000309643500008

View details for PubMedID 22853801
Improving Image Quality by Accounting for Changes in Water Temperature during a Photoacoustic Tomography Scan PLOS ONE Van de Sompel, D., Sasportas, L. S., Dragulescu-Andrasi, A., Bohndiek, S., Gambhir, S. S. 2012; 7 (10)
Abstract

The emerging field of photoacoustic tomography is rapidly evolving with many new system designs and reconstruction algorithms being published. Many systems use water as a coupling medium between the scanned object and the ultrasound transducers. Prior to a scan, the water is heated to body temperature to enable small animal imaging. During the scan, the water heating system of some systems is switched off to minimize the risk of bubble formation, which leads to a gradual decrease in water temperature and hence the speed of sound. In this work, we use a commercially available scanner that follows this procedure, and show that a failure to model intra-scan temperature decreases as small as 1.5°C leads to image artifacts that may be difficult to distinguish from true structures, particularly in complex scenes. We then improve image quality by continuously monitoring the water temperature during the scan and applying variable speed of sound corrections in the image reconstruction algorithm. While upgrading to an air bubble-free heating pump and keeping it running during the scan could also solve the changing temperature problem, we show that a software correction for the temperature changes provides a cost-effective alternative to a hardware upgrade. The efficacy of the software corrections was shown to be consistent across objects of widely varying appearances, namely physical phantoms, ex vivo tissue, and in vivo mouse imaging. To the best of our knowledge, this is the first study to demonstrate the efficacy of modeling temporal variations in the speed of sound during photoacoustic scans, as opposed to spatial variations as focused on by previous studies. Since air bubbles pose a common problem in ultrasonic and photoacoustic imaging systems, our results will be useful to future small animal imaging studies that use scanners with similarly limited heating units.

View details for DOI 10.1371/journal.pone.0045337

View details for Web of Science ID 000309807700008

View details for PubMedID 23071512
Remodeling of Endogenous Mammary Epithelium by Breast Cancer Stem Cells STEM CELLS Parashurama, N., Lobo, N. A., Ito, K., Mosley, A. R., Habte, F. G., Zabala, M., Smith, B. R., Lam, J., Weissman, I. L., Clarke, M. F., Gambhir, S. S. 2012; 30 (10): 2114-2127
Abstract

Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC.

View details for DOI 10.1002/stem.1205

View details for Web of Science ID 000308928300005

View details for PubMedID 22899386
Intraoperative Imaging of Tumors Using Cerenkov Luminescence Endoscopy: A Feasibility Experimental Study JOURNAL OF NUCLEAR MEDICINE Liu, H., Carpenter, C. M., Jiang, H., Pratx, G., Sun, C., Buchin, M. P., Gambhir, S. S., Xing, L., Cheng, Z. 2012; 53 (10): 1579-1584
Abstract

Cerenkov luminescence imaging (CLI) is an emerging new molecular imaging modality that is relatively inexpensive, easy to use, and has high throughput. CLI can image clinically available PET and SPECT probes using optical instrumentation. Cerenkov luminescence endoscopy (CLE) is one of the most intriguing applications that promise potential clinical translation. We developed a prototype customized fiberscopic Cerenkov imaging system to investigate the potential in guiding minimally invasive surgical resection.All experiments were performed in a dark chamber. Cerenkov luminescence from (18)F-FDG samples containing decaying radioactivity was transmitted through an optical fiber bundle and imaged by an intensified charge-coupled device camera. Phantoms filled with (18)F-FDG were used to assess the imaging spatial resolution. Finally, mice bearing subcutaneous C6 glioma cells were injected intravenously with (18)F-FDG to determine the feasibility of in vivo imaging. The tumor tissues were exposed, and CLI was performed on the mouse before and after surgical removal of the tumor using the fiber-based imaging system and compared with a commercial optical imaging system.The sensitivity of this particular setup was approximately 45 kBq (1.21 ?Ci)/300 ?L. The 3 smallest sets of cylindric holes in a commercial SPECT phantom were identifiable via this system, demonstrating that the system has a resolution better than 1.2 mm. Finally, the in vivo tumor imaging study demonstrated the feasibility of using CLI to guide the resection of tumor tissues.This proof-of-concept study explored the feasibility of using fiber-based CLE for the detection of tumor tissue in vivo for guided surgery. With further improvements of the imaging sensitivity and spatial resolution of the current system, CLE may have a significant application in the clinical setting in the near future.

View details for DOI 10.2967/jnumed.111.098541

View details for Web of Science ID 000309432400017

View details for PubMedID 22904353
Exploratory Clinical Trial of (4S)-4-(3-[F-18]fluoropropyl)-L-glutamate for Imaging x(C) Transporter Using Positron Emission Tomography in Patients with Non-Small Cell Lung or Breast Cancer CLINICAL CANCER RESEARCH Baek, S., Choi, C., Ahn, S. H., Lee, J. W., Gong, G., Ryu, J., Oh, S. J., Bacher-Stier, C., Fels, L., Koglin, N., Hultsch, C., Schatz, C. A., Dinkelborg, L. M., Mittra, E. S., Gambhir, S. S., Moon, D. H. 2012; 18 (19): 5427-5437
Abstract

(4S)-4-(3-[(18)F]fluoropropyl)-l-glutamate (BAY 94-9392, alias [(18)F]FSPG) is a new tracer to image x(C)(-) transporter activity with positron emission tomography (PET). We aimed to explore the tumor detection rate of [(18)F]FSPG in patients relative to 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG). The correlation of [(18)F]FSPG uptake with immunohistochemical expression of x(C)(-) transporter and CD44, which stabilizes the xCT subunit of system x(C)(-), was also analyzed.Patients with non-small cell lung cancer (NSCLC, n = 10) or breast cancer (n = 5) who had a positive [(18)F]FDG uptake were included in this exploratory study. PET images were acquired following injection of approximately 300 MBq [(18)F]FSPG. Immunohistochemistry was done using xCT- and CD44-specific antibody.[(18)F]FSPG PET showed high uptake in the kidney and pancreas with rapid blood clearance. [(18)F]FSPG identified all 10 NSCLC and three of the five breast cancer lesions that were confirmed by pathology. [(18)F]FSPG detected 59 of 67 (88%) [(18)F]FDG lesions in NSCLC, and 30 of 73 (41%) in breast cancer. Seven lesions were additionally detected only on [(18)F]FSPG in NSCLC. The tumor-to-blood pool standardized uptake value (SUV) ratio was not significantly different from that of [(18)F]FDG in NSCLC; however, in breast cancer, it was significantly lower (P < 0.05). The maximum SUV of [(18)F]FSPG correlated significantly with the intensity of immunohistochemical staining of x(C)(-) transporter and CD44 (P < 0.01).[(18)F]FSPG seems to be a promising tracer with a relatively high cancer detection rate in patients with NSCLC. [(18)F]FSPG PET may assess x(C)(-) transporter activity in patients with cancer.

View details for DOI 10.1158/1078-0432.CCR-12-0214

View details for Web of Science ID 000311906600027

View details for PubMedID 22893629
Positron Emission Tomography of Cu-64-DOTA-Rituximab in a Transgenic Mouse Model Expressing Human CD20 for Clinical Translation to Image NHL MOLECULAR IMAGING AND BIOLOGY Natarajan, A., Gowrishankar, G., Nielsen, C. H., Wang, S., Iagaru, A., Goris, M. L., Gambhir, S. S. 2012; 14 (5): 608-616
Abstract

This study aims to evaluate (64)Cu-DOTA-rituximab (PETRIT) in a preclinical transgenic mouse model expressing human CD20 for potential clinical translation.(64)Cu was chelated to DOTA-rituximab. Multiple radiolabeling, quality assurance, and imaging experiments were performed. The human CD20 antigen was expressed in B cells of transgenic mice (CD20TM). The mice groups studied were: (a) control (nude mice, n?=?3) that received 7.4 MBq/dose, (b) with pre-dose (CD20TM, n?=?6) received 2 mg/kg pre-dose of cold rituximab prior to PETRIT of 7.4 MBq/dose, and (c) without pre-dose (CD20TM, n?=?6) PETRIT alone received 7.4 MBq/dose. Small animal PET was used to image mice at various time points (0, 1, 2, 4, 24, 48, and 72 h). The OLINDA/EXM software was used to determine the human equivalent dose for individual organs.PETRIT was obtained with a specific activity of 545?±?38.91 MBq/nmole, radiochemical purity >95%, and immunoreactivity >75%. At 24 h, spleenic uptake of PETRIT%ID/g (mean?±?STD) with and without pre-dose was 1.76?±?0.43% and 16.5?±?0.45%, respectively (P value?=?0.01). Liver uptake with and without pre-dose was 0.41?±?0.51% and 0.52?±?0.17% (P value?=?0.86), respectively. The human equivalents of highest dose organs with and without pre-dose are osteogenic cells at 30.8?±?0.4 ?Sv/MBq and the spleen at 99?±?4 ?Sv/MBq, respectively.PET imaging with PETRIT in huCD20 transgenic mice provided human dosimetry data for eventual applications in non-Hodgkins lymphoma patients.

View details for DOI 10.1007/s11307-011-0537-8

View details for Web of Science ID 000308819300011

View details for PubMedID 22231277
FEASIBILITY OF AN INTRAMOLECULAR COMPLEMENTATION STRATEGY FOR SPLIT-REPORTER GENE IMAGING OF DRUGGABLE PROTEIN MISFOLDING IN BRAIN CANCER Massoud, T. F., Paulmurugan, R., Gambhir, S. S. OXFORD UNIV PRESS INC. 2012: 11-11
View details for Web of Science ID 000310971300048
alpha v beta 3 Integrins as a Biomarker of Disease Recurrence in Glioblastoma Multiforme: Initial Clinical Results Using 18F FPPRGD2 PET/CT Iagaru, A., Mosci, C., Mittra, E. S., Shin, B., Chin, F., Gambhir, S. S. SPRINGER. 2012: S244-S245
View details for Web of Science ID 000309726600324
The Impact of Partial Volume Correction in the Evaluation of Solitary Pulmonary Nodules by FDG PET/CT in a Population at Intermediate Risk of Lung Cancer Keu, K., Nair, V. S., Mittra, E., Gambhir, S. S., Iagaru, A. SPRINGER. 2012: S455-S455
View details for Web of Science ID 000309726602293
Designed hydrophilic and charge mutations of the fibronectin domain: towards tailored protein biodistribution PROTEIN ENGINEERING DESIGN & SELECTION Hackel, B. J., Sathirachinda, A., Gambhir, S. S. 2012; 25 (10): 639-647
Abstract

Engineered proteins are attractive affinity scaffolds for molecular imaging and drug delivery. Although exquisite binding specificity and affinity can be engineered, many proteins exhibit off-target uptake, particularly in the kidneys and liver, from physiologic effects. We quantified the ability to alter renal and hepatic uptake via hydrophilic and charge mutations. As a model protein, we used the 10th type III domain of human fibronectin, which has been engineered to bind many targets and has been validated for molecular imaging. We screened rational mutants, identified by structural and phylogenetic analyses, to yield eight mutations that collectively substantially increase protein hydrophilicity. Mutation of two parental clones yielded four domains with a range of hydrophilicity. These proteins were labeled with (64)Cu, injected intravenously into nu/nu mice (n = 3-5 each) and evaluated by positron emission tomography. Renal uptake strongly correlated with hydrophilicity (Pearson's correlation coefficient = 0.97), ranging from 29 ± 11 to 100 ± 22% ID/g at 1 h. Hepatic uptake inversely correlated with hydrophilicity (Pearson's correlation coefficient = -0.92), ranging from 30 ± 7 to 3 ± 1% ID/g. Thus, renal and hepatic uptake are directly tunable through hydrophilic mutation, identifiable by structural and phylogenetic analyses. To investigate charge, we mutated acidic and basic residues in both parental clones and evaluated (64)Cu-labeled mutants in nu/nu mice (n = 5-7). Selected charge removal reduced kidney signal: 78 ± 13 to 51 ± 8%ID/g (P < 0.0001) for the hydrophilic clone and 32 ± 10 to 21 ± 3 (P = 0.0005) for the hydrophobic clone. Elucidation of hydrophilicity and charge enabled modulation of background signal thereby enhancing the utility of protein scaffolds as translatable targeting agents for molecular imaging and therapy.

View details for DOI 10.1093/protein/gzs036

View details for Web of Science ID 000309468100016

View details for PubMedID 22691700
Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation CIRCULATION RESEARCH Gu, M., Nguyen, P. K., Lee, A. S., Xu, D., Hu, S., Plews, J. R., Han, L., Huber, B. C., Lee, W. H., Gong, Y., de Almeida, P. E., Lyons, J., Ikeno, F., Pacharinsak, C., Connolly, A. J., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2012; 111 (7): 882-893
Abstract

Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

View details for DOI 10.1161/CIRCRESAHA.112.269001

View details for Web of Science ID 000308868800015

View details for PubMedID 22821929
Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C. T., Reeves, R. E., Geller, R., Yaghoubi, S. S., Hoehne, A., Solow-Cordero, D. E., Chiosis, G., Massoud, T. F., Paulmurugan, R., Gambhir, S. S. 2012; 109 (37): E2476-E2485
Abstract

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (? and ?) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(?/?)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(?/?) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(?/?)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90?/p23 as compared with Hsp90?/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(?/?)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.

View details for DOI 10.1073/pnas.1205459109

View details for Web of Science ID 000309208000012

View details for PubMedID 22895790
Circulating tumour cells in early breast cancer LANCET ONCOLOGY Nair, V. S., Keu, K. V., Kuhn, P., Gambhir, S. S. 2012; 13 (9): E370-E371
View details for Web of Science ID 000308425600024

View details for PubMedID 22935234
Cationic versus Neutral Microbubbles for Ultrasound-mediated Gene Delivery in Cancer RADIOLOGY Wang, D. S., Panje, C., Pysz, M. A., Paulmurugan, R., Rosenberg, J., Gambhir, S. S., Schneider, M., Willmann, J. K. 2012; 264 (3): 721-732
Abstract

To test whether plasmid-binding cationic microbubbles (MBs) enhance ultrasound-mediated gene delivery efficiency relative to control neutral MBs in cell culture and in vivo tumors in mice.Animal studies were approved by the institutional animal care committee. Cationic and neutral MBs were characterized in terms of size, charge, circulation time, and DNA binding. Click beetle luciferase (CBLuc) reporter plasmids were mixed with cationic or neutral MBs. The ability of cationic MBs to protect bound plasmids from nuclease degradation was tested by means of a deoxyribonuclease (DNase) protection assay. Relative efficiencies of ultrasound-mediated transfection (ultrasound parameters: 1 MHz, 1 W/cm(2), 20% duty cycle, 1 minute) of CBLuc to endothelial cells by using cationic, neutral, or no MBs were compared in cell culture. Ultrasound-mediated gene delivery to mouse hind limb tumors was performed in vivo (n = 24) with insonation (1 MHz, 2 W/cm(2), 50% duty cycle, 5 minutes) after intravenous administration of CBLuc with cationic, neutral, or no MBs. Tumor luciferase activity was assessed by means of serial in vivo bioluminescence imaging and ex vivo analysis. Results were compared by using analysis of variance.Cationic MBs (+15.8 mV; DNA binding capacity, 0.03 pg per MB) partially protected bound DNA from DNase degradation. Mean CBLuc expression of treated endothelial cells in culture was 20-fold higher with cationic than with neutral MBs (219.0 relative light units [RLUs]/µg protein ± 92.5 [standard deviation] vs 10.9 RLUs/µg protein ± 2.7, P = .001) and was significantly higher (P < .001) than that in the no MB and no ultrasound control groups. Serial in vivo bioluminescence of mouse tumors was significantly higher with cationic than with neutral MBs ([5.9 ± 2.2] to [9.3 ± 5.2] vs [2.4 ± 0.8] to [2.9 ± 1.1] × 10(4) photons/sec/cm(2)/steradian, P < .0001) and versus no MB and no ultrasound controls (P < .0001). Results of ex vivo analysis confirmed these results (? = 0.88, P < .0001).Plasmid-binding cationic MBs enhance ultrasound-mediated gene delivery efficiency relative to neutral MBs in both cell culture and mouse hind limb tumors.

View details for DOI 10.1148/radiol.12112368

View details for Web of Science ID 000308645500013

View details for PubMedID 22723497
Fluorescent Magnetic Nanoparticles for Magnetically Enhanced Cancer Imaging and Targeting in Living Subjects ACS NANO Fu, A., Wilson, R. J., Smith, B. R., Mullenix, J., Earhart, C., Akin, D., Guccione, S., Wang, S. X., Gambhir, S. S. 2012; 6 (8): 6862-6869
Abstract

Early detection and targeted therapy are two major challenges in the battle against cancer. Novel imaging contrast agents and targeting approaches are greatly needed to improve the sensitivity and specificity of cancer theranostic agents. Here, we implemented a novel approach using a magnetic micromesh and biocompatible fluorescent magnetic nanoparticles (FMN) to magnetically enhance cancer targeting in living subjects. This approach enables magnetic targeting of systemically administered individual FMN, containing a single 8 nm superparamagnetic iron oxide core. Using a human glioblastoma mouse model, we show that nanoparticles can be magnetically retained in both the tumor neovasculature and surrounding tumor tissues. Magnetic accumulation of nanoparticles within the neovasculature was observable by fluorescence intravital microscopy in real time. Finally, we demonstrate that such magnetically enhanced cancer targeting augments the biological functions of molecules linked to the nanoparticle surface.

View details for DOI 10.1021/nn301670a

View details for Web of Science ID 000307988900039

View details for PubMedID 22857784
Impact of Screening Test Performance and Cost on Mortality Reduction and Cost-effectiveness of Multimodal Ovarian Cancer Screening CANCER PREVENTION RESEARCH Drescher, C. W., Hawley, S., Thorpe, J. D., Marticke, S., McIntosh, M., Gambhir, S. S., Urban, N. 2012; 5 (8): 1015-1024
Abstract

Ongoing ovarian cancer screening trials are investigating the efficacy of a two-step screening strategy using currently available blood and imaging tests [CA125 and transvaginal sonography (TVS)]. Concurrently, efforts to develop new biomarkers and imaging tests seek to improve screening performance beyond its current limits. This study estimates the mortality reduction, years of life saved, and cost-effectiveness achievable by annual multimodal screening using increasing CA125 to select women for TVS, and predicts improvements achievable by replacing currently available screening tests with hypothetical counterparts with better performance characteristics. An existing stochastic microsimulation model is refined and used to screen a virtual cohort of 1 million women from ages 45 to 85 years. Each woman is assigned a detailed disease course and screening results timeline. The preclinical behavior of CA125 and TVS is simulated using empirical data derived from clinical trials. Simulations in which the disease incidence and performance characteristics of the screening tests are independently varied are conducted to evaluate the impact of these factors on overall screening performance and costs. Our results show that when applied to women at average risk, annual screening using increasing CA125 to select women for TVS achieves modest mortality reduction (~13%) and meets currently accepted cost-effectiveness guidelines. Screening outcomes are relatively insensitive to second-line test performance and costs. Identification of a first-line test that does substantially better than CA125 and has similar costs is required for screening to reduce ovarian mortality by at least 25% and be reasonably cost-effective.

View details for DOI 10.1158/1940-6207.CAPR-11-0468

View details for Web of Science ID 000308223500004

View details for PubMedID 22750949
Tissue-engineered collateral ligament composite allografts for scapholunate ligament reconstruction: an experimental study. journal of hand surgery Endress, R., Woon, C. Y., Farnebo, S. J., Behn, A., Bronstein, J., Pham, H., Yan, X., Gambhir, S. S., Chang, J. 2012; 37 (8): 1529-1537
Abstract

In patients with chronic scapholunate (SL) dissociation or dynamic instability, ligament repair is often not possible, and surgical reconstruction is indicated. The ideal graft ligament would recreate both anatomical and biomechanical properties of the dorsal scapholunate ligament (dorsal SLIL). The finger proximal interphalangeal joint (PIP joint) collateral ligament could possibly be a substitute ligament.We harvested human PIP joint collateral ligaments and SL ligaments from 15 cadaveric limbs. We recorded ligament length, width, and thickness, and measured the biomechanical properties (ultimate load, stiffness, and displacement to failure) of native dorsal SLIL, untreated collateral ligaments, decellularized collateral ligaments, and SL repairs with bone-collateral ligament-bone composite collateral ligament grafts. As proof of concept, we then reseeded decellularized bone-collateral ligament-bone composite grafts with green fluorescent protein-labeled adipo-derived mesenchymal stem cells and evaluated them histologically.There was no difference in ultimate load, stiffness, and displacement to failure among native dorsal SLIL, untreated and decellularized collateral ligaments, and SL repairs with tissue-engineered collateral ligament grafts. With pair-matched untreated and decellularized scaffolds, there was no difference in ultimate load or stiffness. However, decellularized ligaments revealed lower displacement to failure compared with untreated ligaments. There was no difference in displacement between decellularized ligaments and native dorsal SLIL. We successfully decellularized grafts with recently described techniques, and they could be similarly reseeded.Proximal interphalangeal joint collateral ligament-based bone-collateral ligament-bone composite allografts had biomechanical properties similar to those of native dorsal SLIL. Decellularization did not adversely affect material properties.These tissue-engineered grafts may offer surgeons another option for reconstruction of chronic SL instability.

View details for DOI 10.1016/j.jhsa.2012.05.020

View details for PubMedID 22835583
Shape Matters: Intravital Microscopy Reveals Surprising Geometrical Dependence for Nanoparticles in Tumor Models of Extravasation NANO LETTERS Smith, B. R., Kempen, P., Bouley, D., Xu, A., Liu, Z., Melosh, N., Dai, H., Sinclair, R., Gambhir, S. S. 2012; 12 (7): 3369-3377
Abstract

Delivery is one of the most critical obstacles confronting nanoparticle use in cancer diagnosis and therapy. For most oncological applications, nanoparticles must extravasate in order to reach tumor cells and perform their designated task. However, little understanding exists regarding the effect of nanoparticle shape on extravasation. Herein we use real-time intravital microscopic imaging to meticulously examine how two different nanoparticles behave across three different murine tumor models. The study quantitatively demonstrates that high-aspect ratio single-walled carbon nanotubes (SWNTs) display extravasational behavior surprisingly different from, and counterintuitive to, spherical nanoparticles although the nanoparticles have similar surface coatings, area, and charge. This work quantitatively indicates that nanoscale extravasational competence is highly dependent on nanoparticle geometry and is heterogeneous.

View details for DOI 10.1021/nl204175t

View details for Web of Science ID 000306296200004

View details for PubMedID 22650417
In vivo targeting of HER2-positive tumor using 2-helix affibody molecules AMINO ACIDS Ren, G., Webster, J. M., Liu, Z., Zhang, R., Miao, Z., Liu, H., Gambhir, S. S., Syud, F. A., Cheng, Z. 2012; 43 (1): 405-413
Abstract

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2<2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with 64Cu or 111In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I2 oxidation. DOTA-MUT-DS was then radiolabeled with 64Cu or 111In to prepare the HER2 imaging probe (64Cu/111In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with 64Cu or 111In. Biodistribution study showed that tumor uptake value of 64Cu or 111In-labeled DOTA-MUT-DS was 4.66±0.38 or 2.17±0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n=3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for 64Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for 111In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule ZHER2:342 with 64Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. 111In-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with 64Cu-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, 64Cu/111In DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.

View details for DOI 10.1007/s00726-011-1096-7

View details for Web of Science ID 000305210800041

View details for PubMedID 21984380
Photoacoustic Imaging of Mesenchymal Stem Cells in Living Mice via Silica-Coated Gold Nanorods ACS NANO Jokerst, J. V., Thangaraj, M., Kempen, P. J., Sinclair, R., Gambhir, S. S. 2012; 6 (7): 5920-5930
Abstract

Improved imaging modalities are critically needed for optimizing stem cell therapy. Techniques with real-time content to guide and quantitate cell implantation are especially important in applications such as musculoskeletal regenerative medicine. Here, we report the use of silica-coated gold nanorods as a contrast agent for photoacoustic imaging and quantitation of mesenchymal stem cells in rodent muscle tissue. The silica coating increased the uptake of gold into the cell more than 5-fold, yet no toxicity or proliferation changes were observed in cells loaded with this contrast agent. Pluripotency of the cells was retained, and secretome analysis indicated that only IL-6 was disregulated more than 2-fold from a pool of 26 cytokines. The low background of the technique allowed imaging of down to 100,000 cells in vivo. The spatial resolution is 340 ?m, and the temporal resolution is 0.2 s, which is at least an order of magnitude below existing cell imaging approaches. This approach has significant advantages over traditional cell imaging techniques like positron emission tomography and magnetic resonance imaging including real time monitoring of stem cell therapy.

View details for DOI 10.1021/nn302042y

View details for Web of Science ID 000306673800020

View details for PubMedID 22681633
A Hybrid Least Squares and Principal Component Analysis Algorithm for Raman Spectroscopy PLOS ONE Van de Sompel, D., Garai, E., Zavaleta, C., Gambhir, S. S. 2012; 7 (6)
Abstract

Raman spectroscopy is a powerful technique for detecting and quantifying analytes in chemical mixtures. A critical part of Raman spectroscopy is the use of a computer algorithm to analyze the measured Raman spectra. The most commonly used algorithm is the classical least squares method, which is popular due to its speed and ease of implementation. However, it is sensitive to inaccuracies or variations in the reference spectra of the analytes (compounds of interest) and the background. Many algorithms, primarily multivariate calibration methods, have been proposed that increase robustness to such variations. In this study, we propose a novel method that improves robustness even further by explicitly modeling variations in both the background and analyte signals. More specifically, it extends the classical least squares model by allowing the declared reference spectra to vary in accordance with the principal components obtained from training sets of spectra measured in prior characterization experiments. The amount of variation allowed is constrained by the eigenvalues of this principal component analysis. We compare the novel algorithm to the least squares method with a low-order polynomial residual model, as well as a state-of-the-art hybrid linear analysis method. The latter is a multivariate calibration method designed specifically to improve robustness to background variability in cases where training spectra of the background, as well as the mean spectrum of the analyte, are available. We demonstrate the novel algorithm's superior performance by comparing quantitative error metrics generated by each method. The experiments consider both simulated data and experimental data acquired from in vitro solutions of Raman-enhanced gold-silica nanoparticles.

View details for DOI 10.1371/journal.pone.0038850

View details for Web of Science ID 000305583300060

View details for PubMedID 22723895
Endoscopic imaging of Cerenkov luminescence BIOMEDICAL OPTICS EXPRESS Kothapalli, S., Liu, H., Liao, J. C., Cheng, Z., Gambhir, S. S. 2012; 3 (6): 1215-1225
Abstract

We demonstrate feasibility of endoscopic imaging of Cerenkov light originated when charged nuclear particles, emitted from radionuclides, travel through a biological tissue of living subjects at superluminal velocity. The endoscopy imaging system consists of conventional optical fiber bundle/ clinical endoscopes, an optical imaging lens system, and a sensitive low-noise charge coupled device (CCD) camera. Our systematic studies using phantom samples show that Cerenkov light from as low as 1 µCi of radioactivity emitted from (18)F-Fluorodeoxyglucose (FDG) can be coupled and transmitted through conventional optical fibers and endoscopes. In vivo imaging experiments with tumor bearing mice, intravenously administered with (18)F-FDG, further demonstrated that Cerenkov luminescence endoscopy is a promising new tool in the field of endoscopic molecular imaging.

View details for Web of Science ID 000304965700007

View details for PubMedID 22741069
Transatlantic Consensus Group on active surveillance and focal therapy for prostate cancer BJU INTERNATIONAL Ahmed, H. U., Akin, O., Coleman, J. A., Crane, S., Emberton, M., Goldenberg, L., Hricak, H., Kattan, M. W., Kurhanewicz, J., Moore, C. M., Parker, C., Polascik, T. J., Scardino, P., Van As, N., Villers, A. 2012; 109 (11): 1636-1647
Abstract

What's known on the subject? and What does the study add? Active surveillance for prostate cancer is gaining increasing acceptance for low risk prostate cancer. Focal therapy is an emerging tissue preservation strategy that aims for treat only areas of cancer. Early phase trials have shown that side-effects can be significantly reduced using focal therapy. There is significant uncertainty in both active surveillance and focal therapy. This consensus group paper provides a road-map for clinical practice and research for both tissue-preserving strategies in the areas of patient population, tools for risk stratification and cancer localisation, treatment interventions as well as comparators and outcome measures in future comparative trials.To reach consensus on key issues for clinical practice and future research in active surveillance and focal therapy in managing localized prostate cancer.A group of expert urologists, oncologists, radiologists, pathologists and computer scientists from North America and Europe met to discuss issues in patient population, interventions, comparators and outcome measures to use in both tissue-preserving strategies of active surveillance and focal therapy. Break-out sessions were formed to provide agreement or highlight areas of disagreement on individual topics which were then collated by a writing group into statements that formed the basis of this report and agreed upon by the whole Transatlantic Consensus Group.The Transatlantic group propose that emerging diagnostic tools such as precision imaging and transperineal prostate mapping biopsy can improve prostate cancer care. These tools should be integrated into prostate cancer management and research so that better risk stratification and more effective treatment allocation can be applied. The group envisaged a process of care in which active surveillance, focal therapy, and radical treatments lie on a continuum of complementary therapies for men with a range of disease grades and burdens, rather than being applied in the mutually exclusive and competitive way they are now.The changing landscape of prostate cancer epidemiology requires the medical community to re-evaluate the entire prostate cancer diagnostic and treatment pathway in order to minimize harms resulting from over-diagnosis and over-treatment. Precise risk stratification at every point in this pathway is required alongside paradigm shifts in our thinking about what constitutes cancer in the prostate.

View details for DOI 10.1111/j.1464-410X.2011.10633.x

View details for Web of Science ID 000303598400015

View details for PubMedID 22077593
Family of Enhanced Photoacoustic Imaging Agents for High-Sensitivity and Multiplexing Studies in Living Mice ACS NANO de la Zerda, A., Bodapati, S., Teed, R., May, S. Y., Tabakman, S. M., Liu, Z., Khuri-Yakub, B. T., Chen, X., Dai, H., Gambhir, S. S. 2012; 6 (6): 4694-4701
Abstract

Photoacoustic imaging is a unique modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a family of novel photoacoustic contrast agents that are based on the binding of small optical dyes to single-walled carbon nanotubes (SWNT-dye). We synthesized five different SWNT-dye contrast agents using different optical dyes, creating five "flavors" of SWNT-dye nanoparticles. In particular, SWNTs that were coated with either QSY(21) (SWNT-QSY) or indocyanine green (SWNT-ICG) exhibited over 100-times higher photoacoustic contrast in living animals compared to plain SWNTs, leading to subnanomolar sensitivities. We then conjugated the SWNT-dye conjugates with cyclic Arg-Gly-Asp peptides to molecularly target the ?(v)?(3) integrin, which is associated with tumor angiogenesis. Intravenous administration of these tumor-targeted imaging agents to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. Finally, we were able to spectrally separate the photoacoustic signals of SWNT-QSY and SWNT-ICG in living animals injected subcutaneously with both particles in the same location, opening the possibility for multiplexing in vivo studies.

View details for DOI 10.1021/nn204352r

View details for Web of Science ID 000305661300017

View details for PubMedID 22607191
Effect of a CCR1 receptor antagonist on systemic trafficking of MSCs and polyethylene particle-associated bone loss BIOMATERIALS Gibon, E., Yao, Z., Rao, A. J., Zwingenberger, S., Batke, B., Valladares, R., Smith, R. L., Biswal, S., Gambhir, S. S., Goodman, S. B. 2012; 33 (14): 3632-3638
Abstract

Particle-associated periprosthetic osteolysis remains a major issue in joint replacement. Ongoing bone loss resulting from wear particle-induced inflammation is accompanied by continued attempts at bone repair. Previously we showed that mesenchymal stem cells (MSCs) are recruited systemically to bone exposed to continuous infusion of ultra high molecular weight polyethylene (UHMWPE) particles. The chemokine-receptor axis that mediates this process is unknown. We tested two hypotheses: (1) the CCR1 receptor mediates the systemic recruitment of MSCs to UHMWPE particles and (2) recruited MSCs are able to differentiate into functional mature osteoblasts and decrease particle-associated bone loss. Nude mice were allocated randomly to four groups. UHMWPE particles were continuously infused into the femoral shaft using a micro-pump. Genetically modified murine wild type reporter MSCs were injected systemically via the left ventricle. Non-invasive imaging was used to assay MSC migration and bone mineral density. Bioluminescence and immunohistochemistry confirmed the chemotaxis of reporter cells and their differentiation into mature osteoblasts in the presence of infused particles. Injection of a CCR1 antagonist decreased reporter cell recruitment to the UHMWPE particle infusion site and increased osteolysis. CCR1 appears to be a critical receptor for chemotaxis of MSCs in the presence of UHMWPE particles. Interference with CCR1 exacerbates particle-induced bone loss.

View details for DOI 10.1016/j.biomaterials.2012.02.003

View details for Web of Science ID 000302425400003

View details for PubMedID 22364730
Intratumoral versus Intravenous Gene Therapy Using a Transcriptionally Targeted Viral Vector in an Orthotopic Hepatocellular Carcinoma Rat Model JOURNAL OF VASCULAR AND INTERVENTIONAL RADIOLOGY Kim, Y. I., Ahn, B., Ronald, J. A., Katzenberg, R., Singh, A., Paulmurugan, R., Ray, S., Gambhir, S. S., Hofmann, L. V. 2012; 23 (5): 704-711
Abstract

To evaluate the feasibility of intratumoral delivery of adenoviral vector carrying a bidirectional two-step transcriptional amplification (TSTA) system to amplify transcriptional strength of cancer-specific Survivin promoter in a hepatocellular carcinoma model.MCA-RH7777 cells were implanted in rat liver, and tumor formation was confirmed with [(18)F]fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). The adenoviral vector studied had Survivin promoter driving a therapeutic gene (tumor necrosis factor-?-related apoptosis-inducing ligand [TRAIL]) and a reporter gene (firefly luciferase [FL]; Ad-pSurvivin-TSTA-TRAIL-FL). Tumor-bearing rats were administered Ad-pSurvivin-TSTA-TRAIL-FL intravenously (n = 7) or intratumorally (n = 8). For control groups, adenovirus FL under cytomegalovirus (CMV) promoter (Ad-pCMV-FL) was administered intravenously (n = 3) or intratumorally (n = 3). One day after delivery, bioluminescence imaging was performed to evaluate transduction. At 4 and 7 days after delivery, 18F-FDG-PET was performed to evaluate therapeutic efficacy.With intravenous delivery, Ad-pSurvivin-TSTA-TRAIL-FL showed no measurable liver tumor FL signal on day 1 after delivery, but showed better therapeutic efficacy than Ad-pCMV-FL on day 7 (PET tumor/liver ratio, 3.5 ± 0.58 vs 6.0 ± 0.71; P = .02). With intratumoral delivery, Ad-pSurvivin-TSTA-TRAIL-FL showed positive FL signal from all tumors and better therapeutic efficacy than Ad-pCMV-FL on day 7 (2.4 ± 0.50 vs 5.4 ± 0.78; P = .01). In addition, intratumoral delivery of Ad-pSurvivin-TSTA-TRAIL-FL demonstrated significant decrease in tumoral viability compared with intravenous delivery (2.4 ± 0.50 vs 3.5 ± 0.58; P = .03).Intratumoral delivery of a transcriptionally targeted therapeutic vector for amplifying tumor-specific effect demonstrated better transduction efficiency and therapeutic efficacy for liver cancer than systemic delivery, and may lead to improved therapeutic outcome for future clinical practice.

View details for DOI 10.1016/j.jvir.2012.01.053

View details for Web of Science ID 000303557000020

View details for PubMedID 22387029
A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle NATURE MEDICINE Kircher, M. F., de la Zerda, A., Jokerst, J. V., Zavaleta, C. L., Kempen, P. J., Mittra, E., Pitter, K., Huang, R., Campos, C., Habte, F., Sinclair, R., Brennan, C. W., Mellinghoff, I. K., Holland, E. C., Gambhir, S. S. 2012; 18 (5): 829-U235
Abstract

The difficulty in delineating brain tumor margins is a major obstacle in the path toward better outcomes for patients with brain tumors. Current imaging methods are often limited by inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triple-modality magnetic resonance imaging-photoacoustic imaging-Raman imaging nanoparticle (termed here MPR nanoparticle) can accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively. The MPRs were detected by all three modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive tumor delineation using all three modalities through the intact skull. Raman imaging allowed for guidance of intraoperative tumor resection, and a histological correlation validated that Raman imaging was accurately delineating the brain tumor margins. This new triple-modality-nanoparticle approach has promise for enabling more accurate brain tumor imaging and resection.

View details for DOI 10.1038/nm.2721

View details for Web of Science ID 000303763500053

View details for PubMedID 22504484
Deep Tissue Photoacoustic Imaging Using a Miniaturized 2-D Capacitive Micromachined Ultrasonic Transducer Array IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Kothapalli, S., Ma, T., Vaithilingam, S., Oralkan, O., Khuri-Yakub, B. T., Gambhir, S. S. 2012; 59 (5): 1199-1204
Abstract

In this paper, we demonstrate 3-D photoacoustic imaging (PAI) of light absorbing objects embedded as deep as 5 cm inside strong optically scattering phantoms using a miniaturized (4 mm × 4 mm × 500 ?m), 2-D capacitive micromachined ultrasonic transducer (CMUT) array of 16 × 16 elements with a center frequency of 5.5 MHz. Two-dimensional tomographic images and 3-D volumetric images of the objects placed at different depths are presented. In addition, we studied the sensitivity of CMUT-based PAI to the concentration of indocyanine green dye at 5 cm depth inside the phantom. Under optimized experimental conditions, the objects at 5 cm depth can be imaged with SNR of about 35 dB and a spatial resolution of approximately 500 ?m. Results demonstrate that CMUTs with integrated front-end amplifier circuits are an attractive choice for achieving relatively high depth sensitivity for PAI.

View details for DOI 10.1109/TBME.2012.2183593

View details for Web of Science ID 000303201000001

View details for PubMedID 22249594
MC3T3-E1 Osteoprogenitor Cells Systemically Migrate to a Bone Defect and Enhance Bone Healing TISSUE ENGINEERING PART A Gibon, E., Batke, B., Jawad, M. U., Fritton, K., Rao, A., Yao, Z., Biswal, S., Gambhir, S. S., Goodman, S. B. 2012; 18 (9-10): 968-973
Abstract

Although iliac crest autologous bone graft remains the gold standard for treatment of bone defects, delayed- and nonunions, and arthrodeses, several alternative strategies have been attempted, including the use of mesenchymal stem cells. Whether cells from the osteoblast lineage demonstrate systemic recruitment to an acute bone defect or fracture, and whether these cells directly participate in bone healing is controversial. This study tests two hypotheses: (1) that exogenous murine MC3T3-E1 osteoprogenitor cells with a high propensity for osteoblast differentiation are able to systemically migrate to a bone defect and (2) that the migrated MC3T3-E1 cells enhance bone healing. Two groups of nude mice were used; a bone defect was drilled in the left femoral shaft in both groups. MC3T3-E1 were used as reporter cells and injected in the left ventricle of the heart, to avoid sequestration in the lungs. Injection of saline served as a control. We used bioluminescence and microCT to assay cell recruitment and bone mineral density (BMD). Immunohistochemical staining was used to confirm the migration of reporter cells. MC3T3-E1 cells were found to systemically migrate to the bone defect. Further, BMD at the defect was significantly increased when cells were injected. Systemic cell therapy using osteoprogenitor cells may be a potential strategy to enhance bone healing.

View details for DOI 10.1089/ten.tea.2011.0545

View details for Web of Science ID 000303540400008

View details for PubMedID 22129134
Twist1 Suppresses Senescence Programs and Thereby Accelerates and Maintains Mutant Kras-Induced Lung Tumorigenesis PLOS GENETICS Tran, P. T., Shroff, E. H., Burns, T. F., Thiyagarajan, S., Das, S. T., Zabuawala, T., Chen, J., Cho, Y., Luong, R., Tamayo, P., Salih, T., Aziz, K., Adam, S. J., Vicent, S., Nielsen, C. H., Withofs, N., Sweet-Cordero, A., Gambhir, S. S., Rudin, C. M., Felsher, D. W. 2012; 8 (5)
Abstract

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with Kras(G12D) to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

View details for DOI 10.1371/journal.pgen.1002650

View details for Web of Science ID 000304864000004

View details for PubMedID 22654667
Glioblastoma Therapy with Cytotoxic Mesenchymal Stromal Cells Optimized by Bioluminescence Imaging of Tumor and Therapeutic Cell Response PLOS ONE Alieva, M., Bago, J. R., Aguilar, E., Soler-Botija, C., Vila, O. F., Molet, J., Gambhir, S. S., Rubio, N., Blanco, J. 2012; 7 (4)
Abstract

Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 10(4) relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection.

View details for DOI 10.1371/journal.pone.0035148

View details for Web of Science ID 000305347400033

View details for PubMedID 22529983
A MOLECULAR IMAGING PRIMER: MODALITIES, IMAGING AGENTS, AND APPLICATIONS PHYSIOLOGICAL REVIEWS James, M. L., Gambhir, S. S. 2012; 92 (2): 897-965
Abstract

Molecular imaging is revolutionizing the way we study the inner workings of the human body, diagnose diseases, approach drug design, and assess therapies. The field as a whole is making possible the visualization of complex biochemical processes involved in normal physiology and disease states, in real time, in living cells, tissues, and intact subjects. In this review, we focus specifically on molecular imaging of intact living subjects. We provide a basic primer for those who are new to molecular imaging, and a resource for those involved in the field. We begin by describing classical molecular imaging techniques together with their key strengths and limitations, after which we introduce some of the latest emerging imaging modalities. We provide an overview of the main classes of molecular imaging agents (i.e., small molecules, peptides, aptamers, engineered proteins, and nanoparticles) and cite examples of how molecular imaging is being applied in oncology, neuroscience, cardiology, gene therapy, cell tracking, and theranostics (therapy combined with diagnostics). A step-by-step guide to answering biological and/or clinical questions using the tools of molecular imaging is also provided. We conclude by discussing the grand challenges of the field, its future directions, and enormous potential for further impacting how we approach research and medicine.

View details for DOI 10.1152/physrev.00049.2010

View details for Web of Science ID 000306562500009

View details for PubMedID 22535898
Prospective Evaluation of Tc-99m MDP Scintigraphy, F-18 NaF PET/CT, and F-18 FDG PET/CT for Detection of Skeletal Metastases MOLECULAR IMAGING AND BIOLOGY Iagaru, A., Mittra, E., Dick, D. W., Gambhir, S. S. 2012; 14 (2): 252-259
Abstract

Technetium (Tc) methylene diphosphonate (MDP) has been the standard method for bone scintigraphy for three decades. (18)F sodium fluoride ((18)F NaF) positron emission tomography (PET)/computed tomography (CT) has better resolution and is considered superior. The role of 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F FDG) PET/CT is proven in a variety of cancers, for which it has changed the practice of oncology. There are few prospective studies comparing these three methods of detection of skeletal metastases. Thus, we were prompted to initiate this prospective pilot trial.This is a prospective study (Sep 2007-Dec 2010) of 52 patients with proven malignancy referred for evaluation of skeletal metastases. There were 37 men and 15 women, 19-84 years old (average, 55.6?±?15.9). Technetium-99m ((99m)Tc) MDP bone scintigraphy, (18)F NaF PET/CT, and (18)F FDG PET/CT were subsequently performed within 1 month.Skeletal lesions were detected by (99m)Tc MDP bone scintigraphy in 22 of 52 patients, by (18)F NaF PET/CT in 24 of 52 patients, and by (18)F FDG PET/CT in 16 of 52 patients. The image quality and evaluation of extent of disease were superior by (18)F NaF PET/CT over (99m)Tc MDP scintigraphy in all 22 patients with skeletal lesions on both scans and over (18)F FDG PET/CT in 11 of 16 patients with skeletal metastases on (18)F FDG PET/CT. In two patients, (18)F NaF PET/CT showed skeletal metastases not seen on either of the other two scans. Extraskeletal lesions were identified by (18)F FDG PET/CT in 28 of 52 subjects.Our prospective pilot-phase trial demonstrates superior image quality and evaluation of skeletal disease extent with (18)F NaF PET/CT over (99m)Tc MDP scintigraphy and (18)F FDG PET/CT. At the same time, (18)F FDG PET detects extraskeletal disease that can significantly change disease management. As such, a combination of (18)F FDG PET/CT and (18)F NaF PET/CT may be necessary for cancer detection. Additional evaluation with larger cohorts is required to confirm these preliminary findings.

View details for DOI 10.1007/s11307-011-0486-2

View details for Web of Science ID 000301584100013

View details for PubMedID 21479710
Use of Cu-64-labeled Fibronectin Domain with EGFR-Overexpressing Tumor Xenograft: Molecular Imaging RADIOLOGY Hackel, B. J., Kimura, R. H., Gambhir, S. S. 2012; 263 (1): 179-188
Abstract

To assess the ability of an engineered epidermal growth factor receptor (EGFR)-binding fibronectin domain to serve as a positron emission tomographic (PET) probe for molecular imaging of EGFR in a xenograft mouse model.An EGFR-binding fibronectin domain (fibronectin abbreviated to Fn when bound) was site-specifically labeled with copper 64 ((64)Cu) (8 MBq/nmol). Copper 64-Fn binding was tested in cell cultures with varying EGFR expression. Stability in human and mouse serum was measured in vitro. Animal experiments were approved by the Stanford University Institutional Animal Care and Use Committee. Copper 64-Fn (approximately 2 MBq) was used for PET in mice (n = 5) bearing EGFR-overexpressing xenografted tumors (approximately 5-10 mm in diameter). Results of tomography were compared with those of ex vivo gamma counting of dissected tissues. Statistical analysis was performed with t tests and adjustment for multiple comparisons.Copper 64-Fn exhibited EGFR-dependent binding to multiple cell lines in culture. The tracer was stable for 24 hours in human and mouse serum at 37°C. The tracer exhibited good tumor localization (3.4% injected dose [ID]/g ± 1.0 [standard deviation] at 1 hour), retention (2.7% ID/g ± 0.6 at 24 hours), and specificity (8.6 ± 3.0 tumor-to-muscle ratio, 8.9 ± 4.7 tumor-to-blood ratio at 1 hour). Specific targeting was verified with low localization to low-expressing MDA-MB-435 tumors (0.7% ID/g ± 0.8 at 1 hour, P = .018); specificity was further demonstrated, as a nonbinding control fibronectin had low localization to EGFR-overexpressing xenografts (0.8% ID/g ± 0.2 at 1 hour, P = .013).The stability, low background, and target-specific tumor uptake and retention of the engineered fibronectin domain make it a promising EGFR molecular imaging agent. More broadly, it validates the fibronectin domain as a potential scaffold for a generation of various molecular imaging agents.

View details for DOI 10.1148/radiol.12111504

View details for Web of Science ID 000302642700018

View details for PubMedID 22344401
Immunomodulation of Curcumin on Adoptive Therapy with T Cell Functional Imaging in Mice CANCER PREVENTION RESEARCH Chang, Y., Chuang, H., Hsu, C., Liu, R., Gambhir, S. S., Hwang, J. 2012; 5 (3): 444-452
Abstract

Adoptive T-cell therapy involves the ex vivo expansion and subsequent transfusion of tumor-specific T lymphocytes to eliminate tumors. Using immune modulators to block immunosuppressive factors in the tumor microenvironment has emerged as a promising strategy to enhance T-cell-mediated tumor regression. Curcumin, a major component of turmeric, has been shown to possess antitumor and immunomodulatory effects by regulating a diverse range of molecular targets. Thus, we hypothesize that these beneficial effects of curcumin may improve the therapeutic efficacy of adoptive therapy. Here, we have shown that curcumin enhances cytotoxicity of CD8(+) T cells toward tumors via alteration of the tumor microenvironment when combined with adoptive therapy. We found that T-cell accumulation and function were increased in combined treatment due to the blockade of different immunosuppressors, including TGF-?, indoleamine 2,3-dioxygenase, and regulatory T cells. Furthermore, bioluminescent imaging with a granzyme B promoter-conjugated optical reporter also reflected improved cytotoxicity of antigen-specific CD8(+) T cells in tumor-bearing mice during treatment. These findings suggest that combination of multitargeting drugs, such as curcumin, with adoptive therapy may have potential for clinical application. In addition, using a granzyme B-specific imaging reporter to assess T-cell function may also be applied for the development and therapeutic evaluation of new immunotherapy in preclinical studies.

View details for DOI 10.1158/1940-6207.CAPR-11-0308

View details for Web of Science ID 000300987800011

View details for PubMedID 22135043
Optical Imaging with Her2-Targeted Affibody Molecules Can Monitor Hsp90 Treatment Response in a Breast Cancer Xenograft Mouse Model CLINICAL CANCER RESEARCH van de Ven, S. M., Elias, S. G., Chan, C. T., Miao, Z., Cheng, Z., De, A., Gambhir, S. S. 2012; 18 (4): 1073-1081
Abstract

To determine whether optical imaging can be used for in vivo therapy response monitoring as an alternative to radionuclide techniques. For this, we evaluated the known Her2 response to 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) treatment, an Hsp90 inhibitor.After in vitro 17-DMAG treatment response evaluation of MCF7 parental cells and 2 HER2-transfected clones (clone A medium, B high Her2 expression), we established human breast cancer xenografts in nude mice (only parental and clone B) for in vivo evaluation. Mice received 120 mg/kg of 17-DMAG in 4 doses at 12-hour intervals intraperitonially (n = 14) or PBS as carrier control (n = 9). Optical images were obtained both pretreatment (day 0) and posttreatment (day 3, 6, and 9), always 5 hours postinjection of 500 pmol of anti-Her2 Affibody-AlexaFluor680 via tail vein (with preinjection background subtraction). Days 3 and 9 in vivo optical imaging signal was further correlated with ex vivo Her2 levels by Western blot after sacrifice.Her2 expression decreased with 17-DMAG dose in vitro. In vivo optical imaging signal was reduced by 22.5% in clone B (P = 0.003) and by 9% in MCF7 parental tumors (P = 0.23) 3 days after 17-DMAG treatment; optical imaging signal recovered in both tumor types at days 6 to 9. In the carrier group, no signal reduction was observed. Pearson correlation of in vivo optical imaging signal with ex vivo Her2 levels ranged from 0.73 to 0.89.Optical imaging with an affibody can be used to noninvasively monitor changes in Her2 expression in vivo as a response to treatment with an Hsp90 inhibitor, with results similar to response measurements in positron emission tomography imaging studies.

View details for DOI 10.1158/1078-0432.CCR-10-3213

View details for Web of Science ID 000300628100017

View details for PubMedID 22235098
First Experience with Clinical-Grade [F-18]FPP(RGD)(2): An Automated Multi-step Radiosynthesis for Clinical PET Studies MOLECULAR IMAGING AND BIOLOGY Chin, F. T., Shen, B., Liu, S., Berganos, R. A., Chang, E., Mittra, E., Chen, X., Gambhir, S. S. 2012; 14 (1): 88-95
Abstract

A reliable and routine process to introduce a new ¹?F-labeled dimeric RGD-peptide tracer ([¹?F]FPP(RGD?) for noninvasive imaging of ?(v)?? expression in tumors needed to be developed so the tracer could be evaluated for the first time in man. Clinical-grade [¹?F]FPP(RGD)? was screened in mouse prior to our first pilot study in human.[¹?F]FPP(RGD)? was synthesized by coupling 4-nitrophenyl-2-[¹?F]fluoropropionate ([¹?F]NPE) with the dimeric RGD-peptide (PEG?-c(RGDyK)?). Imaging studies with [¹?F]FPP(RGD)? in normal mice and a healthy human volunteer were carried out using small animal and clinical PET scanners, respectively.Through optimization of each radiosynthetic step, [¹?F]FPP(RGD)? was obtained with RCYs of 16.9?±?2.7% (n?=?8, EOB) and specific radioactivity of 114?±?72 GBq/?mol (3.08?±?1.95 Ci/?mol; n?=?8, EOB) after 170 min of radiosynthesis. In our mouse studies, high radioactivity uptake was only observed in the kidneys and bladder with the clinical-grade tracer. Favorable [¹?F]FPP(RGD)? biodistribution in human studies, with low background signal in the head, neck, and thorax, showed the potential applications of this RGD-peptide tracer for detecting and monitoring tumor growth and metastasis.A reliable, routine, and automated radiosynthesis of clinical-grade [¹?F]FPP(RGD)? was established. PET imaging in a healthy human volunteer illustrates that [¹?F]FPP(RGD)? possesses desirable pharmacokinetic properties for clinical noninvasive imaging of ?(v)?? expression. Further imaging studies using [¹?F]FPP(RGD)? in patient volunteers are now under active investigation.

View details for DOI 10.1007/s11307-011-0477-3

View details for Web of Science ID 000301583900012

View details for PubMedID 21400112
Prospective comparison of combined F-18-FDG and F-18-NaF PET/CT vs. F-18-FDG PET/CT imaging for detection of malignancy EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Lin, F. I., Rao, J. E., Mittra, E. S., Nallapareddy, K., Chengapa, A., Dick, D. W., Gambhir, S. S., Iagaru, A. 2012; 39 (2): 262-270
Abstract

Typically, (18)F-FDG PET/CT and (18)F-NaF PET/CT scans are done as two separate studies on different days to allow sufficient time for the radiopharmaceutical from the first study to decay. This is inconvenient for the patients and exposes them to two doses of radiation from the CT component of the examinations. In the current study, we compared the clinical usefulness of a combined (18)F-FDG/(18)F-NaF PET/CT scan with that of a separate (18)F-FDG-only PET/CT scan.There were 62 patients enrolled in this prospective trial. All had both an (18)F-FDG-alone PET/CT scan and a combined (18)F-FDG/(18)F-NaF PET/CT scan. Of the 62 patients, 53 (85%) received simultaneous tracer injections, while 9 (15%) received (18)F-NaF subsequent to the initial (18)F-FDG dose (average delay 2.2 h). Images were independently reviewed for PET findings by two Board-Certified nuclear medicine physicians, with discrepancies resolved by a third reader. Interpreters were instructed to only report findings that were concerning for malignancy. Reading the (18)F-FDG-only scan first for half of the patients controlled for order bias.In 15 of the 62 patients (24%) neither the (18)F-FDG-only PET/CT scan nor the combined (18)F-FDG/(18)F-NaF PET/CT scan identified malignancy. In the remaining 47 patients who had PET findings of malignancy, a greater number of lesions were detected in 16 of 47 patients (34%) using the combined (18)F-FDG/(18)F-NaF PET/CT scan compared to the (18)F-FDG-only PET/CT scan. In 2 of these 47 patients (4%), the (18)F-FDG-only scan demonstrated soft tissue lesions that were not prospectively identified on the combined study. In 29 of these 47 patients (62%), the combined scan detected an equal number of lesions compared to the (18)F-FDG-only scan. Overall, 60 of all the 62 patients (97%) showed an equal or greater number of lesions on the combined scan than on the (18)F-FDG-only scan.The current study demonstrated that (18)F-FDG and (18)F-NaF can be combined in a single PET/CT scan by administering the two radiopharmaceuticals simultaneously or in sequence on the same day. In addition to patient convenience and reduced radiation exposure from the CT component, the combined (18)F-FDG/(18)F-NaF PET/CT scan appeared to increase the sensitivity for detection of osseous lesions compared to the (18)F-FDG-only PET/CT scan in the studied population.

View details for DOI 10.1007/s00259-011-1971-1

View details for Web of Science ID 000302286600009

View details for PubMedID 22065013
Proof-of-Concept Study of Monitoring Cancer Drug Therapy with Cerenkov Luminescence Imaging JOURNAL OF NUCLEAR MEDICINE Xu, Y., Chang, E., Liu, H., Jiang, H., Gambhir, S. S., Cheng, Z. 2012; 53 (2): 312-317
Abstract

Cerenkov luminescence imaging (CLI) has emerged as a less expensive, easier-to-use, and higher-throughput alternative to other nuclear imaging modalities such as PET. It is expected that CLI will find many applications in biomedical research such as cancer detection, probe development, drug screening, and therapy monitoring. In this study, we explored the possibility of using CLI to monitor drug efficacy by comparisons against PET. To assess the performance of both modalities in therapy monitoring, 2 murine tumor models (large cell lung cancer cell line H460 and prostate cancer cell line PC3) were given bevacizumab versus vehicle treatments. Two common radiotracers, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) and (18)F-FDG, were used to monitor bevacizumab treatment efficacy.One group of mice (n = 6) was implanted with H460 xenografts bilaterally in the shoulder region, divided into treatment and control groups (n = 3 each), injected with (18)F-FLT, and imaged with PET immediately followed by CLI. The other group of mice (n = 6) was implanted with PC3 xenografts in the same locations, divided into treatment and control groups (n = 3 each), injected with (18)F-FDG, and imaged by the same modalities. Bevacizumab treatment was performed by 2 injections of 20 mg/kg at days 0 and 2.On (18)F-FLT scans, both CLI and PET revealed significantly decreased signals from H460 xenografts in treated mice from pretreatment to day 3. Moderately increased to unchanged signals were observed in untreated mice. On (18)F-FDG scans, both CLI and PET showed relatively unchanged signals from PC3 tumors in both treated and control groups. Quantifications of tumor signals of Cerenkov luminescence and PET images showed that the 2 modalities had excellent correlations (R(2) > 0.88 across all study groups).CLI and PET exhibit excellent correlations across different tumor xenografts and radiotracers. This is the first study, to our knowledge, demonstrating the use of CLI for monitoring cancer treatment. The findings warrant further exploration and optimization of CLI as an alternative to PET in preclinical therapeutic monitoring and drug screening.

View details for DOI 10.2967/jnumed.111.094623

View details for Web of Science ID 000300032800024

View details for PubMedID 22241909
Pharmacokinetically Stabilized Cystine Knot Peptides That Bind Alpha-v-Beta-6 Integrin with Single-Digit Nanomolar Affinities for Detection of Pancreatic Cancer CLINICAL CANCER RESEARCH Kimura, R. H., Teed, R., Hackel, B. J., Pysz, M. A., Chuang, C. Z., Sathirachinda, A., Willmann, J. K., Gambhir, S. S. 2012; 18 (3): 839-849
Abstract

Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin ?(v)?(6), a cell surface receptor being evaluated as a novel clinical biomarker.To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3-6 nmol/L) to integrin ?(v)?(6). The binders do not cross-react with related integrin ?(v)?(5), integrin ?(5)?(1), or tumor-angiogenesis-associated integrin, ?(v)?(3).Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin ?(v)?(6). Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting ?(v)?(6)-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3).We have engineered highly stable cystine knot peptides with potent and specific integrin ?(v)?(6)-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging.

View details for DOI 10.1158/1078-0432.CCR-11-1116

View details for Web of Science ID 000300115000027

View details for PubMedID 22173551
Development of a Novel Long-Lived ImmunoPET Tracer for Monitoring Lymphoma Therapy in a Humanized Transgenic Mouse Model. Bioconjugate chemistry Natarajan, A., Habte, F., Gambhir, S. S. 2012
Abstract

Positron emission tomography (PET) is an attractive imaging tool to localize and quantify tracer biodistribution. ImmunoPET with an intact mAb typically requires two to four days to achieve optimized tumor-to-normal ratios. Thus, a positron emitter with a half-life of two to four days such as zirconium-89 [(89)Zr] (t(1/2): 78.4 h) is ideal. We have developed an antibody-based, long-lived immunoPET tracer (89)Zr-Desferrioxamine-p-SCN (Df-Bz-NCS)-rituximab (Zr-iPET) to image tumor for longer durations in a humanized CD20-expressing transgenic mouse model. To optimize the radiolabeling efficiency of (89)Zr with Df-Bz-rituximab, multiple radiolabelings were performed. Radiochemical yield, purity, immunoreactivity, and stability assays were carried out to characterize the Zr-iPET for chemical and biological integrity. This tracer was used to image transgenic mice that express the human CD20 on their B cells (huCD20TM). Each huCD20TM mouse received a 7.4 MBq/dose. One group (n = 3) received a 2 mg/kg predose (blocking) of cold rituximab 2 h prior to (89)Zr-iPET; the other group (n = 3) had no predose (nonblocking). Small animal PET/CT was used to image mice at 1, 4, 24, 48, 72, and 120 h. Quality assurance of the (89)Zr-iPET demonstrated NCS-Bz-Df: antibody ratio (c/a: 1.5 ± 0.31), specific activity (0.44-1.64 TBq/mol), radiochemical yield (>70%), and purity (>98%). The Zr-iPET immunoreactivity was >80%. At 120 h, Zr-iPET uptake (% ID/g) as mean ± STD for blocking and nonblocking groups in spleen was 3.2 ± 0.1% and 83.3 ± 2.0% (p value <0.0013.). Liver uptake was 1.32 ± 0.05% and 0.61 ± 0.001% (p value <0.0128) for blocking and nonblocking, respectively. The small animal PET/CT image shows the spleen specific uptake of Zr-iPET in mice at 120 h after tracer injection. Compared to the liver, the spleen specific uptake of Zr-iPET is very high due to the expression of huCD20. We optimized the radiolabeling efficiency of (89)Zr with Df-Bz-rituximab. These radioimmunoconjugate lots were stable up to 5 days in serum in vitro. The present study showed that (89)Zr is well-suited for mAbs to image cancer over an extended period of time (up to 5 days).

View details for PubMedID 22621257
Photoacoustic Imaging Using a 9F MicroLinear CMUT ICE Catheter 2012 IEEE INTERNATIONAL ULTRASONICS SYMPOSIUM (IUS) Nikoozadeh, A., Choe, J. W., Kothapalli, S., Moini, A., Sanjani, S. S., Kamaya, A., Oralkan, O., Gambhir, S. S., Khuri-Yakub, P. T. 2012: 24-27
View details for DOI 10.1109/ULTSYM.2012.0007

View details for Web of Science ID 000326960200006
Response to Intra-Arterial Oncolytic Virotherapy with the Herpes Virus NV1020 Evaluated by [F-18]Fluorodeoxyglucose Positron Emission Tomography and Computed Tomography HUMAN GENE THERAPY Sze, D. Y., Iagaru, A. H., Gambhir, S. S., de Haan, H. A., Reid, T. R. 2012; 23 (1): 91-97
Abstract

Oncolytic virotherapy poses unique challenges to the evaluation of tumor response. We hypothesized that the addition of [(18)F]fluorodeoxyglucose (FDG) positron emission tomography (PET) to standard computed tomography (CT) evaluation would improve diagnostic and prognostic power of the measurement of tumor response to oncolytic virotherapy. A phase I/II trial was conducted to investigate treatment of hepatic metastases from colorectal carcinoma using intra-arterial administration of the oncolytic herpes virus NV1020. Both contrast-enhanced CT and FDG PET were obtained on each patient at each time point. Quantitative FDG PET and CT responses were correlated with each other and with clinical outcome metrics. A majority of patients showed initial post-viral infusion increases in tumor size (69%) or in standardized uptake value (SUV) (80%) large enough to qualify as progressive disease. Most showed subsequent decreases in tumor size (64%) or SUV (83%) enough to be reclassified as partial response or stable disease. Late PET and CT imaging results correlated well with each other and with clinical outcomes, but results from early in the treatment scheme did not correlate with each other, with later results, or with clinical outcomes. The addition of FDG PET to the evaluation of tumor response to the oncolytic virus NV1020 did not provide useful diagnostic or prognostic data. More sophisticated molecular imaging will need to be developed to monitor the effects of this novel class of antineoplastic agents.

View details for DOI 10.1089/hum.2011.141

View details for Web of Science ID 000299604000011

View details for PubMedID 21895536
A Novel Clinically Translatable Fluorescent Nanoparticle for Targeted Molecular Imaging of Tumors in Living Subjects NANO LETTERS Gao, J., Chen, K., Luong, R., Bouley, D. M., Mao, H., Qiao, T., Gambhir, S. S., Cheng, Z. 2012; 12 (1): 281-286
Abstract

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance, and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding nontumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD(2) to integrin ?(v)?(3)-positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD-modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron-based nanoprobes in the clinical setting in the near future.

View details for DOI 10.1021/nl203526f

View details for Web of Science ID 000298943100049

View details for PubMedID 22172022
Raman's "Effect" on Molecular Imaging JOURNAL OF NUCLEAR MEDICINE Zavaleta, C. L., Kircher, M. F., Gambhir, S. S. 2011; 52 (12): 1839-1844
Abstract

Raman spectroscopy is an optical technique that offers unsurpassed sensitivity and multiplexing capabilities to the field of molecular imaging. In the past, Raman spectroscopy had predominantly been used as an analytic tool for routine chemical analysis, but more recently, researchers have been able to harness its unique properties for imaging and spectral analysis of molecular interactions in cell populations and preclinical animal models. Additionally, researchers have already begun to translate this optical technique into a novel clinical diagnostic tool using various endoscopic strategies.

View details for DOI 10.2967/jnumed.111.087775

View details for Web of Science ID 000298162500016

View details for PubMedID 21868625
In Vitro and in Vivo Molecular Imaging of Estrogen Receptor alpha and beta Homo- and Heterodimerization: Exploration of New Modes of Receptor Regulation MOLECULAR ENDOCRINOLOGY Paulmurugan, R., Tamrazi, A., Massoud, T. F., Katzenellenbogen, J. A., Gambhir, S. S. 2011; 25 (12): 2029-2040
Abstract

Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ER? and ER?, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ER? and ER? in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ER? and ER?, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ER?/ER? or ER?/ER? homodimerization, or ER?/ER? heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ER? and ER? form the strongest dimers, and subtype-preferential homodimerization was seen with ER?-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ER?-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ER?/ER? heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ER? and ER? dimers might be the mediators of the effects of different types of ER ligands given at different doses.

View details for DOI 10.1210/me.2011-1145

View details for Web of Science ID 000298055800005

View details for PubMedID 22052998
Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer MOLECULAR IMAGING AND BIOLOGY Gheysens, O., Chen, I. Y., Rodriguez-Porcel, M., Chan, C., Rasooly, J., Vaerenberg, C., Paulmurugan, R., Willmann, J. K., Deroose, C., Wu, J., Gambhir, S. S. 2011; 13 (6): 1124-1132
Abstract

We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1? (HIF-1?-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively.The plasmid pUbi-hrl-pUbi-HIF-1?-VP2, which expresses both hrl and HIF-1?-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1?-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI).Strong correlations existed between the expression of hRL and each of HIF-1?-VP2, VEGF, and PlGF (r(2) > 0.83, r(2) > 0.82, and r(2) > 0.97, respectively). In vivo, both transplanted cells and HIF-1?-VP2 transgene expression were successfully imaged using BLI.An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein.

View details for DOI 10.1007/s11307-011-0471-9

View details for Web of Science ID 000296794400009

View details for PubMedID 21267661
Non-Invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a Cu-64-Labeled Activity-Based Probe PLOS ONE Ren, G., Blum, G., Verdoes, M., Liu, H., Syed, S., Edgington, L. E., Gheysens, O., Miao, Z., Jiang, H., Gambhir, S. S., Bogyo, M., Cheng, Z. 2011; 6 (11)
Abstract

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.

View details for DOI 10.1371/journal.pone.0028029

View details for Web of Science ID 000297789900039

View details for PubMedID 22132198
Mathematical Model Identifies Blood Biomarker-Based Early Cancer Detection Strategies and Limitations SCIENCE TRANSLATIONAL MEDICINE Hori, S. S., Gambhir, S. S. 2011; 3 (109)
Abstract

Most clinical blood biomarkers lack the necessary sensitivity and specificity to reliably detect cancer at an early stage, when it is best treatable. It is not yet clear how early a clinical blood assay can be used to detect cancer or how biomarker-based strategies can be improved to enable earlier detection of smaller tumors. To address these issues, we developed a mathematical model describing dynamic plasma biomarker kinetics in relation to the growth of a tumor, beginning with a single cancer cell. To exemplify a realistic scenario in which biomarker is shed by both cancerous and noncancerous cells, we primed the model on ovarian tumor growth and CA125 shedding data, for which tumor growth parameters and shedding rates are readily available in published literature. We found that a tumor could grow unnoticed for more than 10.1 years and reach a volume of about ?/6(25.36 mm)(3), corresponding to a spherical diameter of about 25.36 mm, before becoming detectable by current clinical blood assays. Model parameters were perturbed over log orders of magnitude to quantify ideal shedding rates and identify other blood-based strategies required for early submillimeter tumor detectability. The detection times we estimated are consistent with recently published tumor progression time lines based on clinical genomic sequencing data for several cancers. Here, we rigorously showed that shedding rates of current clinical blood biomarkers are likely 10(4)-fold too low to enable detection of a developing tumor within the first decade of tumor growth. The model presented here can be extended to virtually any solid cancer and associated biomarkers.

View details for DOI 10.1126/scitranslmed.3003110

View details for Web of Science ID 000297218300004

View details for PubMedID 22089452
GLUT 5 Is Not Over-Expressed in Breast Cancer Cells and Patient Breast Cancer Tissues PLOS ONE Gowrishankar, G., Zitzmann-Kolbe, S., Junutula, A., Reeves, R., Levi, J., Srinivasan, A., Bruus-Jensen, K., Cyr, J., Dinkelborg, L., Gambhir, S. S. 2011; 6 (11)
Abstract

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.

View details for DOI 10.1371/journal.pone.0026902

View details for Web of Science ID 000297154900052

View details for PubMedID 22073218
A novel F-18-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Miao, Z., Ren, G., Jiang, L., Liu, H., Webster, J. M., Zhang, R., Namavari, M., Gambhir, S. S., Syud, F., Cheng, Z. 2011; 38 (11): 1977-1984
Abstract

Two-helix scaffold proteins (~ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an (18)F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors.An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I(2) oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-(18)F-fluorobenzyl aldehyde ((18)F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, (18)F-FBO-MUT-DS, was performed at 37°C. (18)F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 ?Ci/?g, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted.Cell uptake assays showed high and specific cell uptake (~12% applied activity at 1 h) by incubation of (18)F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K(D)) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of (18)F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 ?g of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p < 0.05).F-FBO-MUT-DS displays excellent HER2 targeting ability and tumor PET imaging quality. The two-helix scaffold proteins are suitable for development of (18)F-based PET probes.

View details for DOI 10.1007/s00259-011-1879-9

View details for Web of Science ID 000295680200004

View details for PubMedID 21761266
Noninvasive cell-tracking methods NATURE REVIEWS CLINICAL ONCOLOGY Kircher, M. F., Gambhir, S. S., Grimm, J. 2011; 8 (11): 677-688
Abstract

Cell-based therapies, such as adoptive immunotherapy and stem-cell therapy, have received considerable attention as novel therapeutics in oncological research and clinical practice. The development of effective therapeutic strategies using tumor-targeted cells requires the ability to determine in vivo the location, distribution, and long-term viability of the therapeutic cell populations as well as their biological fate with respect to cell activation and differentiation. In conjunction with various noninvasive imaging modalities, cell-labeling methods, such as exogenous labeling or transfection with a reporter gene, allow visualization of labeled cells in vivo in real time, as well as monitoring and quantifying cell accumulation and function. Such cell-tracking methods also have an important role in basic cancer research, where they serve to elucidate novel biological mechanisms. In this Review, we describe the basic principles of cell-tracking methods, explain various approaches to cell tracking, and highlight recent examples for the application of such methods in animals and humans.

View details for DOI 10.1038/nrclinonc.2011.141

View details for Web of Science ID 000296812500009

View details for PubMedID 21946842
Endothelial Cells Derived From Human iPSCS Increase Capillary Density and Improve Perfusion in a Mouse Model of Peripheral Arterial Disease ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Rufaihah, A. J., Huang, N. F., Jame, S., Lee, J. C., Nguyen, H. N., Byers, B., De, A., Okogbaa, J., Rollins, M., Reijo-Pera, R., Gambhir, S. S., Cooke, J. P. 2011; 31 (11): E72-U44
Abstract

Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease.Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and vascular endothelial growth factor. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144, and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging and green fluorescence protein for fluorescent detection. The hiPSC-ECs (5×10(5)) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). Bioluminescence imaging showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 versus 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284±155 versus 797±206 capillaries/mm(2)) (P<0.002).This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.

View details for DOI 10.1161/ATVBAHA.111.230938

View details for Web of Science ID 000296605400001

View details for PubMedID 21836062
Theranostic nanomedicine. Accounts of chemical research Chen, X., Gambhir, S. S., Cheon, J. 2011; 44 (10): 841-?
View details for DOI 10.1021/ar200231d

View details for PubMedID 22004477
Immobilizing Reporters for Molecular Imaging of the Extracellular Microenvironment in Living Animals ACS CHEMICAL BIOLOGY Xia, Z., Xing, Y., Jeon, J., Kim, Y., Gall, J., Dragulescu-Andrasi, A., Gambhir, S. S., Rao, J. 2011; 6 (10): 1117-1126
Abstract

We report here an immobilization strategy using a collagen binding protein to deliver and confine synthetic reporters to the extracellular microenvironment in vivo for noninvasively imaging the activity of targets in the microenvironment. We show that the immobilization of reporters on collagens in the local microenvironment is highly efficient and physiologically stable for repetitive, long-term imaging. By using this strategy we successfully developed an immobilized bioluminescent activatable reporter and a dual-modality reporter to map and quantitatively image the activity of extracellular matrix metalloproteinases (MMP) in tumor-bearing mice. The inhibition of MMP activity by chemical inhibitor was also demonstrated in living subjects. We further demonstrated the general applicability of this immobilization strategy by imaging MMP activity at the inflammation site in a mouse model. Our results show that the in vivo immobilization of reporters can be used as a general strategy for probing the local extracellular microenvironment.

View details for DOI 10.1021/cb200135e

View details for Web of Science ID 000296208100018

View details for PubMedID 21830814
Molecular Imaging with Theranostic Nanoparticles ACCOUNTS OF CHEMICAL RESEARCH Jokerst, J. V., Gambhir, S. S. 2011; 44 (10): 1050-1060
Abstract

Nanoparticles (NPs) offer diagnostic and therapeutic capabilities not available with small molecules or microscale tools. As the field of molecular imaging has emerged from the blending of molecular biology with medical imaging, NP imaging is increasingly common for both therapeutic and diagnostic applications. The term theranostic describes technology with concurrent and complementary diagnostic and therapeutic capabilities. Although NPs have been FDA-approved for clinical use as transport vehicles for nearly 15 years, full translation of their theranostic potential is incomplete. However, NPs have shown remarkable success in the areas of drug delivery and magnetic resonance imaging. Emerging applications include image-guided resection, optical/photoacoustic imaging in vivo, contrast-enhanced ultrasound, and thermoablative therapy. Diagnosis with NPs in molecular imaging involves the correlation of the signal with a phenotype. The location and intensity of NP signals emanating from a living subject indicate the disease area's size, stage, and biochemical signature. Therapy with NPs uses the image for resection or delivery of a small molecule or RNA therapeutic. Ablation of the affected area is also possible via heat or radioactivity. The ideal theranostic NP includes several features: (1) it selectively and rapidly accumulates in diseased tissue; (2) it reports biochemical and morphological characteristics of the area; (3) it delivers an effective therapeutic; and (4) it is safe and biodegrades with nontoxic byproducts. Such a system contains a central imaging core surrounded by small molecule therapeutics. The system targets via ligands such as IgG and is protected from immune scavengers by a cloak of protective polymer. Although no NP has achieved all of the above criteria, many NPs possess one or more of these features. While the most clinically translatable NPs have been used in the field of magnetic resonance imaging, other types in development are quickly becoming more biocompatible through methods that modify their toxicity and biodistribution profiles. In this Account, we describe diagnostic imaging and therapeutic uses of NPs. We propose and offer examples of five primary types of nanoparticles with concurrent diagnostic and therapeutic uses.

View details for DOI 10.1021/ar200106e

View details for Web of Science ID 000296682400022

View details for PubMedID 21919457
Synthesis of 2 '-Deoxy-2 '-[F-18]Fluoro-9-beta-D-Arabinofuranosylguanine: a Novel Agent for Imaging T-Cell Activation with PET MOLECULAR IMAGING AND BIOLOGY Namavari, M., Chang, Y., Kusler, B., Yaghoubi, S., Mitchell, B. S., Gambhir, S. S. 2011; 13 (5): 812-818
Abstract

9-(?-D-Arabinofuranosyl)guanine (AraG) is a guanosine analog that has a proven efficacy in the treatment of T-cell lymphoblastic disease. To test the possibility of using a radiofluorinated AraG as an imaging agent, we have synthesized 2'-deoxy-2'-[(18)F]fluoro-9-?-D-arabinofuranosylguanine ([(18)F]F-AraG) and investigated its uptake in T cells.We have synthesized [(18)F]F-AraG via a direct fluorination of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-trifyl-?-D-ribofuranosyl)guanine with [(18)F]KF/K.2.2.2 in DMSO at 85°C for 45 min. [(18)F]F-AraG uptake in both a CCRF-CEM leukemia cell line (unactivated) and activated primary thymocytes was evaluated.We have successfully prepared [(18)F]F-AraG in 7-10% radiochemical yield (decay corrected) with a specific activity of 0.8-1.3 Ci/?mol. Preliminary cell uptake experiments showed that both a CCRF-CEM leukemia cell line and activated primary thymocytes take up the [(18)F]F-AraG.For the first time to the best of our knowledge, [(18)F]F-AraG has been successfully synthesized by direct fluorination of an appropriate precursor of a guanosine nucleoside. This approach maybe also useful for the synthesis of other important positron emission tomography (PET) probes such as [(18)F]FEAU, [(18)F]FMAU, and [(18)F]FBAU which are currently synthesized by multiple steps and involve lengthy purification. The cell uptake studies support future studies to investigate the use of [(18)F]F-AraG as a PET imaging agent of T cells.

View details for DOI 10.1007/s11307-010-0414-x

View details for Web of Science ID 000295176200002

View details for PubMedID 20838911
Gold Nanoparticles: A Revival in Precious Metal Administration to Patients NANO LETTERS Thakor, A. S., Jokerst, J., Zavaleta, C., Massoud, T. F., Gambhir, S. S. 2011; 11 (10): 4029-4036
Abstract

Gold has been used as a therapeutic agent to treat a wide variety of rheumatic diseases including psoriatic arthritis, juvenile arthritis, and discoid lupus erythematosus. Although the use of gold has been largely superseded by newer drugs, gold nanoparticles are being used effectively in laboratory based clinical diagnostic methods while concurrently showing great promise in vivo either as a diagnostic imaging agent or a therapeutic agent. For these reasons, gold nanoparticles are therefore well placed to enter mainstream clinical practice in the near future. Hence, the present review summarizes the chemistry, pharmacokinetics, biodistribution, metabolism, and toxicity of bulk gold in humans based on decades of clinical observation and experiments in which gold was used to treat patients with rheumatoid arthritis. The beneficial attributes of gold nanoparticles, such as their ease of synthesis, functionalization, and shape control are also highlighted demonstrating why gold nanoparticles are an attractive target for further development and optimization. The importance of controlling the size and shape of gold nanoparticles to minimize any potential toxic side effects is also discussed.

View details for DOI 10.1021/nl202559p

View details for Web of Science ID 000295667000001

View details for PubMedID 21846107
Preclinical Derivation and Imaging of Autologously Transplanted Canine Induced Pluripotent Stem Cells JOURNAL OF BIOLOGICAL CHEMISTRY Lee, A. S., Xu, D., Plews, J. R., Nguyen, P. K., Nag, D., Lyons, J. K., Han, L., Hu, S., Lan, F., Liu, J., Huang, M., Narsinh, K. H., Long, C. T., de Almeida, P. E., Levi, B., Kooreman, N., Bangs, C., Pacharinsak, C., Ikeno, F., Yeung, A. C., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2011; 286 (37): 32697-32704
Abstract

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

View details for DOI 10.1074/jbc.M111.235739

View details for Web of Science ID 000294726800078

View details for PubMedID 21719696
Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds. Journal of biomedical materials research. Part A Neofytou, E. A., Chang, E., Patlola, B., Joubert, L., Rajadas, J., Gambhir, S. S., Cheng, Z., Robbins, R. C., Beygui, R. E. 2011; 98 (3): 383-393
Abstract

Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.

View details for DOI 10.1002/jbm.a.33113

View details for PubMedID 21630430
Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics CONTRAST MEDIA & MOLECULAR IMAGING de la Zerda, A., Kim, J., Galanzha, E. I., Gambhir, S. S., Zharov, V. P. 2011; 6 (5): 346-369
Abstract

Various nanoparticles have raised significant interest over the past decades for their unique physical and optical properties and biological utilities. Here we summarize the vast applications of advanced nanoparticles with a focus on carbon nanotube (CNT)-based or CNT-catalyzed contrast agents for photoacoustic (PA) imaging, cytometry and theranostics applications based on the photothermal (PT) effect. We briefly review the safety and potential toxicity of the PA/PT contrast nanoagents, while showing how the physical properties as well as multiple biological coatings change their toxicity profiles and contrasts. We provide general guidelines needed for the validation of a new molecular imaging agent in living subjects, and exemplify these guidelines with single-walled CNTs targeted to ?(v) ?(3) , an integrin associated with tumor angiogenesis, and golden carbon nanotubes targeted to LYVE-1, endothelial lymphatic receptors. An extensive review of the potential applications of advanced contrast agents is provided, including imaging of static targets such as tumor angiogenesis receptors, in vivo cytometry of dynamic targets such as circulating tumor cells and nanoparticles in blood, lymph, bones and plants, methods to enhance the PA and PT effects with transient and stationary bubble conjugates, PT/PA Raman imaging and multispectral histology. Finally, theranostic applications are reviewed, including the nanophotothermolysis of individual tumor cells and bacteria with clustered nanoparticles, nanothrombolysis of blood clots, detection and purging metastasis in sentinel lymph nodes, spectral hole burning and multiplex therapy with ultrasharp rainbow nanoparticles.

View details for DOI 10.1002/cmmi.455

View details for Web of Science ID 000300110400003

View details for PubMedID 22025336
Preclinical Evaluation of Raman Nanoparticle Biodistribution for their Potential Use in Clinical Endoscopy Imaging SMALL Zavaleta, C. L., Hartman, K. B., Miao, Z., James, M. L., Kempen, P., Thakor, A. S., Nielsen, C. H., Sinclair, R., Cheng, Z., Gambhir, S. S. 2011; 7 (15): 2232-2240
Abstract

Raman imaging offers unsurpassed sensitivity and multiplexing capabilities. However, its limited depth of light penetration makes direct clinical translation challenging. Therefore, a more suitable way to harness its attributes in a clinical setting would be to couple Raman spectroscopy with endoscopy. The use of an accessory Raman endoscope in conjunction with topically administered tumor-targeting Raman nanoparticles during a routine colonoscopy could offer a new way to sensitively detect dysplastic lesions while circumventing Raman's limited depth of penetration and avoiding systemic toxicity. In this study, the natural biodistribution of gold surface-enhanced Raman scattering (SERS) nanoparticles is evaluated by radiolabeling them with (64) Cu and imaging their localization over time using micropositron emission tomography (PET). Mice are injected either intravenously (IV) or intrarectally (IR) with approximately 100 microcuries (?Ci) (3.7 megabecquerel (MBq)) of (64) Cu-SERS nanoparticles and imaged with microPET at various time points post injection. Quantitative biodistribution data are obtained as % injected dose per gram (%ID g(-1)) from each organ, and the results correlate well with the corresponding microPET images, revealing that IV-injected mice have significantly higher uptake (p < 0.05) in the liver (5 h = 8.96% ID g(-1); 24 h = 8.27% ID g(-1)) than IR-injected mice (5 h = 0.09% ID g(-1); 24 h = 0.08% ID g(-1)). IR-injected mice show localized uptake in the large intestine (5 h = 10.37% ID g(-1); 24 h = 0.42% ID g(-1)) with minimal uptake in other organs. Raman imaging of excised tissues correlate well with biodistribution data. These results suggest that the topical application of SERS nanoparticles in the mouse colon appears to minimize their systemic distribution, thus avoiding potential toxicity and supporting the clinical translation of Raman spectroscopy as an endoscopic imaging tool.

View details for DOI 10.1002/smll.201002317

View details for Web of Science ID 000294361200015

View details for PubMedID 21608124
Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dragulescu-Andrasi, A., Chan, C. T., De, A., Massoud, T. F., Gambhir, S. S. 2011; 108 (29): 12060-12065
Abstract

Identifying protein-protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.

View details for DOI 10.1073/pnas.1100923108

View details for Web of Science ID 000292876900069

View details for PubMedID 21730157
Pilot Pharmacokinetic and Dosimetric Studies of F-18-FPPRGD2: A PET Radiopharmaceutical Agent for Imaging alpha(v)beta(3) Integrin Levels RADIOLOGY Mittra, E. S., Goris, M. L., Iagaru, A. H., Kardan, A., Burton, L., Berganos, R., Chang, E., Liu, S., Shen, B., Chin, F. T., Chen, X., Gambhir, S. S. 2011; 260 (1): 182-191
Abstract

To assess the safety, biodistribution, and dosimetric properties of the positron emission tomography (PET) radiopharmaceutical agent fluorine 18 ((18)F) FPPRGD2 (2-fluoropropionyl labeled PEGylated dimeric RGD peptide [PEG3-E{c(RGDyk)}2]), which is based on the dimeric arginine-glycine-aspartic acid (RGD) peptide sequence and targets ?(v)?(3) integrin, in the first volunteers imaged with this tracer.The protocol was approved by the institutional review board, and written informed consent was obtained from all participants. Five healthy volunteers underwent whole-body combined PET-computed tomography 0.5, 1.0, 2.0, and 3.0 hours after tracer injection (mean dose, 9.5 mCi ± 3.4 [standard deviation] [351.5 MBq ± 125.8]; mean specific radioactivity, 1200 mCi/mmol ± 714 [44.4 GBq/mmol ± 26.4]). During this time, standard vital signs, electrocardiographic (ECG) readings, and blood sample values (for chemistry, hematologic, and liver function tests) were checked at regular intervals and 1 and 7 days after the injection. These data were used to evaluate tracer biodistribution and dosimetric properties, time-activity curves, and the stability of laboratory values. Significant changes in vital signs and laboratory values were evaluated by using a combination of population-averaged generalized estimating equation regression and exact paired Wilcoxon tests.The administration of (18)F-FPPRGD2 was well tolerated, with no marked effects on vital signs, ECG readings, or laboratory values. The tracer showed the same pattern of biodistribution in all volunteers: primary clearance through the kidneys (0.360 rem/mCi ± 0.185 [0.098 mSv/MBq ± 0.050]) and bladder (0.862 rem/mCi ± 0.436 [0.233 mSv/MBq ± 0.118], voiding model) and uptake in the spleen (0.250 rem/mCi ± 0.168 [0.068 mSv/MBq ± 0.046]) and large intestine (0.529 rem/mCi ± 0.236 [0.143 mSv/MBq ± 0.064]). The mean effective dose of (18)F-FPPRGD2 was 0.1462 rem/mCi ± 0.0669 (0.0396 mSv/MBq ± 0.0181). With an injected dose of 10 mCi (370 MBq) and a 1-hour voiding interval, a patient would be exposed to an effective radiation dose of 1.5 rem (15 mSv). Above the diaphragm, there was minimal uptake in the brain ventricles, salivary glands, and thyroid gland. Time-activity curves showed rapid clearance from the vasculature, with a mean 26% ± 17 of the tracer remaining in the circulation at 30 minutes and most of the activity occurring in the plasma relative to cells (mean whole blood-plasma ratio, 0.799 ± 0.096).(18)F-FPPRGD2 has desirable pharmacokinetic and biodistribution properties. The primary application is likely to be PET evaluation of oncologic patients-especially those with brain, breast, or lung cancer. Specific indications may include tumor staging, identifying patients who would benefit from antiangiogenesis therapy, and separating treatment responders from nonresponders early.

View details for DOI 10.1148/radiol.11101139

View details for Web of Science ID 000291932300021

View details for PubMedID 21502381
Synthesis and Radioluminescence of PEGylated Eu3+-doped Nanophosphors as Bioimaging Probes ADVANCED MATERIALS Sun, C., Pratx, G., Carpenter, C. M., Liu, H., Cheng, Z., Gambhir, S. S., Xing, L. 2011; 23 (24): H195-H199
View details for DOI 10.1002/adma.201100919

View details for Web of Science ID 000293046600018

View details for PubMedID 21557339
Nanoparticle PEGylation for imaging and therapy NANOMEDICINE Jokerst, J. V., Lobovkina, T., Zare, R. N., Gambhir, S. S. 2011; 6 (4): 715-728
Abstract

Nanoparticles are an essential component in the emerging field of nanomedical imaging and therapy. When deployed in vivo, these materials are typically protected from the immune system by polyethylene glycol (PEG). A wide variety of strategies to coat and characterize nanoparticles with PEG has established important trends on PEG size, shape, density, loading level, molecular weight, charge and purification. Strategies to incorporate targeting ligands are also prevalent. This article presents a background to investigators new to stealth nanoparticles, and suggests some key considerations needed prior to designing a nanoparticle PEGylation protocol and characterizing the performance features of the product.

View details for DOI 10.2217/NNM.11.19

View details for Web of Science ID 000292994300019

View details for PubMedID 21718180
Potent, tumor-specific gene expression in an orthotopic hepatoma rat model using a Survivin-targeted, amplifiable adenoviral vector GENE THERAPY Ahn, B., Ronald, J. A., Kim, Y. I., Katzenberg, R., Singh, A., Paulmurugan, R., Ray, S., Hofmann, L. V., Gambhir, S. S. 2011; 18 (6): 606-612
Abstract

Ideal cancer gene therapies should have high tumor specificity and efficacy, and allow systemic administration to target metastases. We recently developed a bi-directional, two-step transcriptional amplification (TSTA) system driven by the tumor-specific Survivin promoter (pSurv) to amplify the correlated expression of both the reporter gene firefly luciferase (FL) and therapeutic gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we compare the specificity and potency of an adenovirus carrying this system (Ad-pSurv-TSTA-TRAIL-FL) to a nonspecific vector (Ad-pCMV-FL) in an orthotopic hepatocellular carcinoma (HCC) rat model after systemic administration. At 24?h after injection of Ad-pCMV-FL, bioluminescence imaging revealed a trend (P=0.30) towards greater FL expression in liver versus tumor. In striking contrast, Ad-pSurv-TSTA-TRAIL-FL showed increased FL activity within the tumor compared with the liver (P<0.01), a strong trend towards reduced liver expression compared with Ad-pCMV-FL (P=0.07), and importantly, similar FL levels within tumor compared with Ad-pCMV-FL (P=0.32). Hence, this vector shows potent, tumor-specific transgene expression even after extensive liver transduction and may be of significant value in avoiding hepatotoxicity in HCC patients. Future studies will explore the benefits of tumor-specific TRAIL expression in this model, the potential to target metastases and the extension of this vector for the treatment of other Survivin-positive tumors is warranted.

View details for DOI 10.1038/gt.2011.5

View details for Web of Science ID 000291438900010

View details for PubMedID 21307888
Therapeutic treatment of critical limb ischemia using human induced pluripotent stem cell-derived endothelial cells Huang, N. F., Jalil, R. A., Lee, J., Jame, S., Nguyen, H. N., De, A., Gambhir, S., Reijo-Pera, R., Cooke, J. P. SAGE PUBLICATIONS LTD. 2011: 221-221
View details for Web of Science ID 000291218600013
Engineering and Visualization of Bacteria for Targeting Infarcted Myocardium MOLECULAR THERAPY Le, U. N., Kim, H., Kwon, J., Kim, M. Y., Nguyen, V. H., Jiang, S. N., Lee, B., Hong, Y., Shin, M. G., Rhee, J. H., Bom, H., Ahn, Y., Gambhir, S. S., Choy, H. E., Min, J. 2011; 19 (5): 951-959
Abstract

Optimization of the specific affinity of cardiac delivery vector could significantly improve the efficiency of gene/protein delivery, yet no cardiac vectors to date have sufficient target specificity for myocardial infarction (MI). In this study, we explored bacterial tropism for infarcted myocardium based on our previous observations that certain bacteria are capable of targeting the hypoxic regions in solid tumors. Out of several Escherichia coli or Salmonella typhimurium strains, the S. typhimurium defective in the synthesis of ppGpp (?ppGpp S. typhimurium) revealed accumulation and selective proliferation in the infarcted myocardium without spillover to noncardiac tissue. The Salmonellae that were engineered to express a variant of Renilla luciferase gene (RLuc8), under the control of the E. coli arabinose operon promoter (P(BAD)), selectively targeted and delivered RLuc8 in the infarcted myocardium only upon injection of L-arabinose. An examination of the infarct size before and after infection, and estimations of C-reactive protein (CRP) and procalcitonin indicated that intravenous injection of ?ppGpp S. typhimurium did not induce serious local or systemic immune reactions. This current proof-of-principle study demonstrates for the first time the capacity of Salmonellae to target infarcted myocardium and to serve as a vehicle for the selective delivery of therapeutic agents in MI.

View details for DOI 10.1038/mt.2011.25

View details for Web of Science ID 000290146200021

View details for PubMedID 21364539
Multimodality Imaging of beta-Cells in Mouse Models of Type 1 and 2 Diabetes DIABETES Yong, J., Rasooly, J., Dang, H., Lu, Y., Middleton, B., Zhang, Z., Hon, L., Namavari, M., Stout, D. B., Atkinson, M. A., Tian, J., Gambhir, S. S., Kaufman, D. L. 2011; 60 (5): 1383-1392
Abstract

?-Cells that express an imaging reporter have provided powerful tools for studying ?-cell development, islet transplantation, and ?-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 "MIP-TF" mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their ?-cells. This should enable the noninvasive imaging of ?-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of ?-cells at the cellular level by fluorescent microscopy.MIP-TF mouse ?-cells were multimodality imaged in models of type 1 and type 2 diabetes.MIP-TF mouse ?-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF ?-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse ?-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve-intraperitoneal glucose tolerance test (AUC-IPGTT).MIP-TF mice provide a new tool for monitoring ?-cells from the single cell level to noninvasive assessments of ?-cells in models of type 1 diabetes and type 2 diabetes.

View details for DOI 10.2337/db10-0907

View details for Web of Science ID 000290349700004

View details for PubMedID 21441442
Early Diagnosis of Ovarian Carcinoma: Is a Solution in Sight? RADIOLOGY Lutz, A. M., Willmann, J. K., Drescher, C. W., Ray, P., Cochran, F. V., Urban, N., Gambhir, S. S. 2011; 259 (2): 329-345
Abstract

Ovarian cancer is the most lethal of the gynecologic malignancies. Because ovarian cancer symptoms are subtle and nonspecific, the diagnosis is often delayed until the disease is well advanced. Overall 5-year survival is a rather dismal 50% but can be improved to greater than 90% if the disease is confined to the ovary at the time of diagnosis (generally in fewer than 25% of patients). Effective screening tools are currently not available. Owing to the rather low incidence of the disease in the general population, potential screening tests must provide very high specificity to avoid unnecessary interventions in false-positive cases. This article reviews currently available serum biomarkers and imaging tests for the early detection of ovarian cancer and provides an outlook on the potential improvements in these noninvasive diagnostic tools that may lead to successful implementation in a screening program. Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11090563/-/DC1.

View details for DOI 10.1148/radiol.11090563

View details for Web of Science ID 000289667300006

View details for PubMedID 21502390
"Same Day" Ex-vivo Regional Gene Therapy: A Novel Strategy to Enhance Bone Repair MOLECULAR THERAPY Virk, M. S., Sugiyama, O., Park, S. H., Gambhir, S. S., Adams, D. J., Drissi, H., Lieberman, J. R. 2011; 19 (5): 960-968
Abstract

Ex-vivo regional gene therapy with bone marrow cells (BMCs) overexpressing bone morphogenetic protein-2 (BMP-2) has demonstrated efficacy in healing critical sized bone defects in preclinical studies. The purpose of this preclinical study was to compare the osteoinductive potential of a novel "same day" ex-vivo regional gene therapy versus a traditional two-step approach, which involves culture expansion of the donor cells before implantation. In the "same day" strategy buffy coat cells were harvested from the rat bone marrow, transduced with a lentiviral vector-expressing BMP-2 for 1 hour and implanted into a rat femoral defect in the same sitting. There was no significant difference (P = 0.22) with respect to the radiographic healing rates between the femoral defects treated with the "same day" strategy (13/13; 100%) versus the traditional two-step approach (11/14; 78%). However, the femoral defects treated with the "same day" strategy induced earlier radiographic bone healing (P = 0.004) and higher bone volume (BV) [micro-computed tomography (micro-CT); P < 0.001]. The "same day" strategy represents a significant advance in the field of ex-vivo regional gene therapy because it offers a solution to limitations associated with the culture expansion process required in the traditional ex vivo approach. This strategy should be cost-effective when adapted for human use.

View details for DOI 10.1038/mt.2011.2

View details for Web of Science ID 000290146200022

View details for PubMedID 21343916
The Fate and Toxicity of Raman-Active Silica-Gold Nanoparticles in Mice SCIENCE TRANSLATIONAL MEDICINE Thakor, A. S., Luong, R., Paulmurugan, R., Lin, F. I., Kempen, P., Zavaleta, C., Chu, P., Massoud, T. F., Sinclair, R., Gambhir, S. S. 2011; 3 (79)
Abstract

Raman spectroscopy is an optical imaging method that is based on the Raman effect, the inelastic scattering of a photon when energy is absorbed from light by a surface. Although Raman spectroscopy is widely used for chemical and molecular analysis, its clinical application has been hindered by the inherently weak nature of the Raman effect. Raman-silica-gold-nanoparticles (R-Si-Au-NPs) overcome this limitation by producing larger Raman signals through surface-enhanced Raman scattering. Because we are developing these particles for use as targeted molecular imaging agents, we examined the acute toxicity and biodistribution of core polyethylene glycol (PEG)-ylated R-Si-Au-NPs after different routes of administration in mice. After intravenous administration, PEG-R-Si-Au-NPs were removed from the circulation by macrophages in the liver and spleen (that is, the reticuloendothelial system). At 24 hours, PEG-R-Si-Au-NPs elicited a mild inflammatory response and an increase in oxidative stress in the liver, which subsided by 2 weeks after administration. No evidence of significant toxicity was observed by measuring clinical, histological, biochemical, or cardiovascular parameters for 2 weeks. Because we are designing targeted PEG-R-Si-Au-NPs (for example, PEG-R-Si-Au-NPs labeled with an affibody that binds specifically to the epidermal growth factor receptor) to detect colorectal cancer after administration into the bowel lumen, we tested the toxicity of the core nanoparticle after administration per rectum. We observed no significant bowel or systemic toxicity, and no PEG-R-Si-Au-NPs were detected systemically. Although additional studies are required to investigate the long-term effects of PEG-R-Si-Au-NPs and their toxicity when carrying the targeting moiety, the results presented here support the idea that PEG-R-Si-Au-NPs can be safely used in living subjects, especially when administered rectally.

View details for DOI 10.1126/scitranslmed.3001963

View details for Web of Science ID 000292976700004

View details for PubMedID 21508310
FDG-PET/CT in Cancers of the Head and Neck: What is the Definition of Whole Body Scanning? MOLECULAR IMAGING AND BIOLOGY Iagaru, A., Mittra, E. S., Gambhir, S. S. 2011; 13 (2): 362-367
Abstract

The role of 2-deoxy-2-[F-18]fluoro-D-glucose-positron emission tomography (FDG-PET) was studied in a variety of cancers, including head and neck squamous cell carcinomas (HNSCC) and nasopharyngeal carcinomas (NPC), with several presentations indicating that for these clinical entities a "whole-body" (i.e., eyes to thighs) may yield little additional information. Therefore, we were prompted to review our experience with PET/computed tomography (CT) in the management of patients with HNSCC and NPC.This is a retrospective study of 133 patients with HNSCC, 23-90 years old (average: 58.2?±?12.7) and 26 patients with NPC, ages 16-75 (average: 47.3?±?17.1), who had whole body PET/CT at our institution from Jan 2003 to Nov 2006. Reinterpretation of the imaging studies for accuracy and data analysis from medical records was performed. Lesions identified on PET/CT below the level of the adrenal glands were recorded and tabulated.Lesions were identified below the adrenal glands in seven patients (5.2%) with HNSCC. These included hepatic and osseous metastases from HNSCC in two patients (1.5%), a new renal cancer (0.75%), a new pancreatic cancer (0.75%), a new colon cancer (0.75%) and findings proven benign on follow-up (focal colon uptake in one patient and an inflammatory inguinal lymph node in another patient; 1.5%). Lesions were identified below the adrenal glands in three patients (11.5%) with NPC. These included osseous metastases from NPC in two patients (7.7%) and findings proven benign on follow-up (focal colon uptake in one patient; 3.84%).This study suggests that whole body PET/CT imaging in HNSCC has a relatively low yield (3%, 95% CI: 1.33-8.42) of significant findings below the level of the adrenal glands. Therefore, implementing a more limited protocol (through the level of adrenal glands), especially in low-risk cases of HNSCC, may be considered. However, whole body PET/CT imaging in NPC may have a significant yield (7.7%, 95% CI: 1.02-25.26) of medically relevant findings below the level of the adrenal glands. Thus, the whole body (i.e., vertex to thighs) PET/CT scan of NPC patients appears to be the appropriate imaging protocol for this population. This recommendation requires further evaluation and validation in larger prospective studies.

View details for DOI 10.1007/s11307-010-0343-8

View details for Web of Science ID 000288177700021

View details for PubMedID 20495879
Reproducibility study of [F-18]FPP(RGD)(2) uptake in murine models of human tumor xenografts EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Chang, E., Liu, S., Gowrishankar, G., Yaghoubi, S., Wedgeworth, J. P., Chin, F., Berndorff, D., Gekeler, V., Gambhir, S. S., Cheng, Z. 2011; 38 (4): 722-730
Abstract

An (18)F-labeled PEGylated arginine-glycine-aspartic acid (RGD) dimer {[(18)F]FPP(RGD)(2)} has been used to image tumor ?(v)?(3) integrin levels in preclinical and clinical studies. Serial positron emission tomography (PET) studies may be useful for monitoring antiangiogenic therapy response or for drug screening; however, the reproducibility of serial scans has not been determined for this PET probe. The purpose of this study was to determine the reproducibility of the integrin ?(v)?(3)-targeted PET probe, [(18)F]FPP(RGD)(2,) using small animal PET.Human HCT116 colon cancer xenografts were implanted into nude mice (n?=?12) in the breast and scapular region and grown to mean diameters of 5-15 mm for approximately 2.5 weeks. A 3-min acquisition was performed on a small animal PET scanner approximately 1 h after administration of [(18)F]FPP(RGD)(2) (1.9-3.8 MBq, 50-100 ?Ci) via the tail vein. A second small animal PET scan was performed approximately 6 h later after reinjection of the probe to assess for reproducibility. Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean or maximum activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility.The coefficient of variation (mean±SD) for %ID(mean)/g and %ID(max)/g values between [(18)F]FPP(RGD)(2) small animal PET scans performed 6 h apart on the same day were 11.1?±?7.6% and 10.4?±?9.3%, respectively. The corresponding differences in %ID(mean)/g and %ID(max)/g values between scans were -0.025?±?0.067 and -0.039?±?0.426. Immunofluorescence studies revealed a direct relationship between extent of ?(?)?(3) integrin expression in tumors and tumor vasculature with level of tracer uptake. Mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, noting tumor volume, as well as making uniform: the time of imaging after injection and the ROI size. Reanalysis of ROI placement displayed variability for %ID(mean)/g of 6.6?±?3.9% and 0.28?±?0.12% for %ID(max)/g.[(18)F]FPP(RGD)(2) small animal PET mouse tumor xenograft studies are reproducible with relatively low variability.

View details for DOI 10.1007/s00259-010-1672-1

View details for Web of Science ID 000288255500015

View details for PubMedID 21125268
Molecular Imaging Using Light-Absorbing Imaging Agents and a Clinical Optical Breast Imaging System-a Phantom Study MOLECULAR IMAGING AND BIOLOGY van de Ven, S. M., Mincu, N., Brunette, J., Ma, G., Khayat, M., Ikeda, D. M., Gambhir, S. S. 2011; 13 (2): 232-238
Abstract

The aim of the study was to determine the feasibility of using a clinical optical breast scanner with molecular imaging strategies based on modulating light transmission.Different concentrations of single-walled carbon nanotubes (SWNT; 0.8-20.0 nM) and black hole quencher-3 (BHQ-3; 2.0-32.0 µM) were studied in specifically designed phantoms (200-1,570 mm(3)) with a clinical optical breast scanner using four wavelengths. Each phantom was placed in the scanner tank filled with optical matching medium. Background scans were compared to absorption scans, and reproducibility was assessed.All SWNT phantoms were detected at four wavelengths, with best results at 684 nm. Higher concentrations (?8.0 µM) were needed for BHQ-3 detection, with the largest contrast at 684 nm. The optical absorption signal was dependent on phantom size and concentration. Reproducibility was excellent (intraclass correlation 0.93-0.98).Nanomolar concentrations of SWNT and micromolar concentrations of BHQ-3 in phantoms were reproducibly detected, showing the potential of light absorbers, with appropriate targeting ligands, as molecular imaging agents for clinical optical breast imaging.

View details for DOI 10.1007/s11307-010-0356-3

View details for Web of Science ID 000288177700006

View details for PubMedID 20532642
Multiparametric MRI reveals early response patterns to antiangiogenic therapy in primary breast cancer HUGHES, N. P., Mehta, S., Adams, R. F., Gambhir, S. S., Padhani, A. R., Harris, A. L. SOC NUCLEAR MEDICINE INC. 2011: 668-669
View details for Web of Science ID 000288804500051
Molecular imaging of the Epidermal Growth Factor Receptor in rodent colon via Affibody-functionalized surface enhanced Raman scattering (SERS) nanoparticles Jokerst, J. V., Miao, Z., Thakor, A. S., Cheng, Z., Gambhir, S. S. AMER CHEMICAL SOC. 2011
View details for Web of Science ID 000291982802272
MYC Phosphorylation, Activation, and Tumorigenic Potential in Hepatocellular Carcinoma Are Regulated by HMG-CoA Reductase CANCER RESEARCH Cao, Z., Fan-Minogue, H., Bellovin, D. I., Yevtodiyenko, A., Arzeno, J., Yang, Q., Gambhir, S. S., Felsher, D. W. 2011; 71 (6): 2286-2297
Abstract

MYC is a potential target for many cancers but is not amenable to existing pharmacologic approaches. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) by statins has shown potential efficacy against a number of cancers. Here, we show that inhibition of HMG-CoA reductase by atorvastatin (AT) blocks both MYC phosphorylation and activation, suppressing tumor initiation and growth in vivo in a transgenic model of MYC-induced hepatocellular carcinoma (HCC) as well as in human HCC-derived cell lines. To confirm specificity, we show that the antitumor effects of AT are blocked by cotreatment with the HMG-CoA reductase product mevalonate. Moreover, by using a novel molecular imaging sensor, we confirm that inhibition of HMG-CoA reductase blocks MYC phosphorylation in vivo. Importantly, the introduction of phosphorylation mutants of MYC at Ser62 or Thr58 into tumors blocks their sensitivity to inhibition of HMG-CoA reductase. Finally, we show that inhibition of HMG-CoA reductase suppresses MYC phosphorylation through Rac GTPase. Therefore, HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties. The inhibition of HMG-CoA reductase may be a useful target for the treatment of MYC-associated HCC as well as other tumors.

View details for DOI 10.1158/0008-5472.CAN-10-3367

View details for Web of Science ID 000288381300028

View details for PubMedID 21262914
Affibody-Functionalized Gold-Silica Nanoparticles for Raman Molecular Imaging of the Epidermal Growth Factor Receptor SMALL Jokerst, J. V., Miao, Z., Zavaleta, C., Cheng, Z., Gambhir, S. S. 2011; 7 (5): 625-633
Abstract

The affibody functionalization of fluorescent surface-enhanced Raman scattering gold-silica nanoparticles as multimodal contrast agents for molecular imaging specific to epidermal growth factor receptor (EGFR) is reported. This nanoparticle bioconjugate reports EGFR-positive A431 tumors with a signal nearly 35-fold higher than EGFR-negative MDA-435S tumors. The low-level EGFR expression in adjacent healthy tissue is 7-fold lower than in the positive tumors. Validation via competitive inhibition reduces the signal by a factor of six, and independent measurement of EGFR via flow cytometry correlates at R(2) = 0.92.

View details for DOI 10.1002/smll.201002291

View details for Web of Science ID 000288081900013

View details for PubMedID 21302357
Affibody-based nanoprobes for HER2-expressing cell and tumor imaging BIOMATERIALS Gao, J., Chen, K., Miao, Z., Ren, G., Chen, X., Gambhir, S. S., Cheng, Z. 2011; 32 (8): 2141-2148
Abstract

This article reports the affibody-based nanoprobes specifically target and image human epidermal growth factor receptor type 2 (HER2)-expressing cells and tumors. The affibody molecules are a promising class of targeting ligands with simple, robust, and precise structure and high affinity. Using near-infrared (NIR) quantum dots (QDs) and iron oxide (IO) nanoparticles as two representative nanomaterials, we designed anti-HER2 affibody molecules with a N-terminus cysteine residue (Cysteine-Z(HER2:342)) and precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates. The in vitro and in vivo study showed the conjugates are highly specific to target and image HER2-expressing cells and tumors. This work indicated the nanoparticle-affibody conjugates may be excellent candidates as targeting probes for molecular imaging and diagnosis.

View details for DOI 10.1016/j.biomaterials.2010.11.053

View details for Web of Science ID 000287061400015

View details for PubMedID 21147502
Use of DNA Microarray and Small Animal Positron Emission Tomography in Preclinical Drug Evaluation of RAF265, a Novel B-Raf/VEGFR-2 lInhibitor NEOPLASIA Tseng, J. R., Stuart, D., Aardalen, K., Kaplan, A., Aziz, N., Hughes, N. P., Gambhir, S. S. 2011; 13 (3): 266-U108
Abstract

Positron emission tomography (PET) imaging has become a useful tool for assessing early biologic response to cancer therapy and may be particularly useful in the development of new cancer therapeutics. RAF265, a novel B-Raf/vascular endothelial growth factor receptor-2 inhibitor, was evaluated in the preclinical setting for its ability to inhibit the uptake of PET tracers in the A375M(B-Raf(V600E)) human melanoma cell line. RAF265 inhibited 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) accumulation in cell culture at 28 hours in a dose-dependent manner. RAF265 also inhibited FDG accumulation in tumor xenografts after 1 day of drug treatment. This decrease persisted for the remaining 2 weeks of treatment. DNA microarray analysis of treated tumor xenografts revealed significantly decreased expression of genes regulating glucose and thymidine metabolism and revealed changes in apoptotic genes, suggesting that the imaging tracers FDG, 3-deoxy-3-[(18)F]fluorothymidine, and annexin V could serve as potential imaging biomarkers for RAF265 therapy monitoring. We concluded that RAF265 is highly efficacious in this xenograft model of human melanoma and decreases glucose metabolism as measured by DNA microarray analysis, cell culture assays, and small animal FDG PET scans as early as 1 day after treatment. Our results support the use of FDG PET in clinical trials with RAF265 to assess early tumor response. DNA microarray analysis and small animal PET studies may be used as complementary technologies in drug development. DNA microarray analysis allows for analysis of drug effects on multiple pathways linked to cancer and can suggest corresponding imaging tracers for further analysis as biomarkers of tumor response.

View details for DOI 10.1593/neo.101466

View details for Web of Science ID 000288905900008

View details for PubMedID 21390189
Noninvasive Monitoring of Placenta-Specific Transgene Expression by Bioluminescence Imaging PLOS ONE Fan, X., Ren, P., Dhal, S., Bejerano, G., Goodman, S. B., Druzin, M. L., Gambhir, S. S., Nayak, N. R. 2011; 6 (1)
Abstract

Placental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts.Zona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc) and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3). Animals were examined for Fluc expression by live bioluminescence imaging (BLI) at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2)/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages.These results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early detection and quantitative analysis of gene expression throughout pregnancy by live BLI. This method may be useful for a wide range of applications involving trophoblast-specific gene manipulations in utero.

View details for DOI 10.1371/journal.pone.0016348

View details for Web of Science ID 000286522300037

View details for PubMedID 21283713
Oxidative Stress Mediates the Effects of Raman-Active Gold Nanoparticles in Human Cells SMALL Thakor, A. S., Paulmurugan, R., Kempen, P., Zavaleta, C., Sinclair, R., Massoud, T. F., Gambhir, S. S. 2011; 7 (1): 126-136
Abstract

Polyethylene glycol (PEG)ylated Raman-active gold nanoparticles (PEG-R-AuNPs) consist of an interchangeable Raman organic molecule layer held onto a gold nanocore by a silica shell. PEG-R-AuNPs have been shown preclinically to increase the sensitivity and specificity of Raman spectroscopy, with picomolar sensitivity and multiplexing capabilities. Although clinical trials are being designed to use functionalized PEG-R-AuNPs in various applications (e.g., to target dysplastic bowel lesions during colonoscopy), the effects of these nanoparticles on human cells remain unknown. The occurrence and mechanisms underlying any potential cytotoxicity induced by these nanoparticles (0-1000 PEG-R-AuNPs/cell) are investigated in immortalized human HeLa and HepG2 cell lines at several time points (0-48 h) after exposure. Using fluorometric assays, cell viability (MTT), reactive oxygen species (ROS) generation (dichlorofluorescein diacetate), protein oxidation (protein carbonyl content), and total cellular antioxidant concentrations the concentrations (metmyoblobin-induced oxidation of ABTS) are assessed. Analysis of lipid oxidation using an enzyme immunoassay (8-isoprostane concentrations), gene expression of antioxidant enzymes using quantitative reverse transcription polymerase chain reactions, and the intracellular location of PEG-R-AuNPs using transmission electron microscopy is also undertaken. PEG-R-AuNPs cause no cytotoxicity in either HeLa or HepG2 cells in the acute setting as ROS generation is balanced by antioxidant enzyme upregulation. Following prolonged exposures (48 h) at relatively high concentrations (1000 PEG-R-AuNPs/cell), nanoparticles are found within vesicles inside cells. Under these conditions, a minimal amount of cytotoxicity is seen in both cell lines owing to increases in cellular oxidative stress, most likely due to ROS overwhelming the antioxidant defenses. Evidence of oxidative stress-induced damage includes increased lipid and protein oxidation. Although further in vivo toxicity studies are necessary, these initial encouraging results show that PEG-R-AuNPs cause minimal toxicity in human cells in the acute setting, which bodes well for potential future applications of these nanoparticles in living subjects.

View details for DOI 10.1002/smll.201001466

View details for Web of Science ID 000285794100015

View details for PubMedID 21104804
Radiolabeling of a Saxitoxin derivative for PET-MRI imaging of pain Hoehne, A., Parsons, W. H., Behera, D., Shen, B., Gambhir, S. S., Du Bois, J., Biswal, S., Chin, F. T. WILEY-BLACKWELL. 2011: S2-S2
View details for Web of Science ID 000295901600003
A Hybrid Least Squares and Principal Component Analysis Algorithm for Raman Spectroscopy Van de Sompel, D., Garai, E., Zavaleta, C., Gambhir, S. S. IEEE. 2011: 6971-6974
Abstract

The least squares fitting algorithm is the most commonly used algorithm in Raman spectroscopy. In this paper, however, we show that it is sensitive to variations in the background signal when the signal of interest is weak. To address this problem, we propose a novel algorithm to analyze measured spectra in Raman spectroscopy. The method is a hybrid least squares and principal component analysis algorithm. It explicitly accounts for any variations expected in the reference spectra used in the signal decomposition. We compare the novel algorithm to the least squares method with a low-order polynomial residual model, and demonstrate the novel algorithm's superior performance by comparing quantitative error metrics. Our experiments use both simulated data and data acquired from an in vitro solution of Raman-enhanced gold nanoparticles.

View details for Web of Science ID 000298810005123

View details for PubMedID 22255942
[F-18]YF3 nanoprobes: a novel synthetic strategy for F-18-labeled imaging agents Xiong Liqin, L. Q., Shen Bin, B., Gambhir, S. S., Chin, F. T., Rao Jianghong, J. H. WILEY-BLACKWELL. 2011: S77-S77
View details for Web of Science ID 000295901600078
[F-18]FTC-146 for imaging sigma-1 receptors in squirrel monkey brain using PET/MRI James, M. L., Shen, B., Nielsen, C. H., Buckmaster, C. L., Berganos, R. A., Zavaleta, C., Lyons, D., McCurdy, C. R., Gambhir, S. S., Chin, F. T. WILEY-BLACKWELL. 2011: S317-S317
View details for Web of Science ID 000295901600317
F-18-Cyanobenzolthiol ([F-18]CBT): A novel F-18-prosthetic group for labeling peptide or protein Shen Bin, B., Jeon, J., Gambhir, S. S., Rao, J., Chin, F. T. WILEY-BLACKWELL. 2011: S503-S503
View details for Web of Science ID 000295901600503
Longitudinal, Noninvasive Imaging of T-Cell Effector Function and Proliferation in Living Subjects CANCER RESEARCH Patel, M. R., Chang, Y., Chen, I. Y., Bachmann, M. H., Yan, X., Contag, C. H., Gambhir, S. S. 2010; 70 (24): 10141-10149
Abstract

Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB-CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models.

View details for DOI 10.1158/0008-5472.CAN-10-1843

View details for Web of Science ID 000285334200016

View details for PubMedID 21159636
Near-infrared fluorescent nanoprobes for cancer molecular imaging: status and challenges TRENDS IN MOLECULAR MEDICINE He, X., Gao, J., Gambhir, S. S., Cheng, Z. 2010; 16 (12): 574-583
Abstract

Near-infrared fluorescence (NIRF) imaging promises to improve cancer imaging and management; advances in nanomaterials allow scientists to combine new nanoparticles with NIRF imaging techniques, thereby fulfilling this promise. Here, we present a synopsis of current developments in NIRF nanoprobes, their use in imaging small living subjects, their pharmacokinetics and toxicity, and finally their integration into multimodal imaging strategies. We also discuss challenges impeding the clinical translation of NIRF nanoprobes for molecular imaging of cancer. Whereas utilization of most NIRF nanoprobes remains at a proof-of-principle stage, optimizing the impact of nanomedicine in cancer patient diagnosis and management will probably be realized through persistent interdisciplinary amalgamation of diverse research fields.

View details for DOI 10.1016/j.molmed.2010.08.006

View details for Web of Science ID 000285727200004

View details for PubMedID 20870460
PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model with a Novel Cu-64-DOTA-Knottin Peptide CANCER RESEARCH Nielsen, C. H., Kimura, R. H., Withofs, N., Tran, P. T., Miao, Z., Cochran, J. R., Cheng, Z., Felsher, D., Kjaer, A., Willmann, J. K., Gambhir, S. S. 2010; 70 (22): 9022-9030
Abstract

Due to the high mortality of lung cancer, there is a critical need to develop diagnostic procedures enabling early detection of the disease while at a curable stage. Targeted molecular imaging builds on the positive attributes of positron emission tomography/computed tomography (PET/CT) to allow for a noninvasive detection and characterization of smaller lung nodules, thus increasing the chances of positive treatment outcome. In this study, we investigate the ability to characterize lung tumors that spontaneously arise in a transgenic mouse model. The tumors are first identified with small animal CT followed by characterization with the use of small animal PET with a novel 64Cu-1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-knottin peptide that targets integrins upregulated during angiogenesis on the tumor associated neovasculature. The imaging results obtained with the knottin peptide are compared with standard 18F-fluorodeoxyglucose (FDG) PET small animal imaging. Lung nodules as small as 3 mm in diameter were successfully identified in the transgenic mice by small animal CT, and both 64Cu-DOTA-knottin 2.5F and FDG were able to differentiate lung nodules from the surrounding tissues. Uptake and retention of the 64Cu-DOTA-knottin 2.5F tracer in the lung tumors combined with a low background in the thorax resulted in a statistically higher tumor to background (normal lung) ratio compared with FDG (6.01±0.61 versus 4.36±0.68; P<0.05). Ex vivo biodistribution showed 64Cu-DOTA-knottin 2.5F to have a fast renal clearance combined with low nonspecific accumulation in the thorax. Collectively, these results show 64Cu-DOTA-knottin 2.5F to be a promising candidate for clinical translation for earlier detection and improved characterization of lung cancer.

View details for DOI 10.1158/0008-5472.CAN-10-1338

View details for Web of Science ID 000284213300008

View details for PubMedID 21062977
Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, H., Patel, M. R., Prescher, J. A., Patsialou, A., Qian, D., Lin, J., Wen, S., Chang, Y., Bachmann, M. H., Shimono, Y., Dalerba, P., Adorno, M., Lobo, N., Bueno, J., Dirbas, F. M., Goswami, S., Somlo, G., Condeelis, J., Contag, C. H., Gambhir, S. S., Clarke, M. F. 2010; 107 (42): 18115-18120
Abstract

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

View details for DOI 10.1073/pnas.1006732107

View details for Web of Science ID 000283184800050

View details for PubMedID 20921380
Dynamic Visualization of RGD-Quantum Dot Binding to Tumor Neovasculature and Extravasation in Multiple Living Mouse Models Using Intravital Microscopy SMALL Smith, B. R., Cheng, Z., De, A., Rosenberg, J., Gambhir, S. S. 2010; 6 (20): 2222-2229
View details for DOI 10.1002/smll.201001022

View details for Web of Science ID 000283890500003

View details for PubMedID 20862677
[F-18]FPPRGD2 PET/CT Imaging of Integrin Expression in Healthy Volunteers Mittra, E., Iagaru, A., Goris, M. L., Chin, F., Chen, X., Gambhir, S. S. SPRINGER. 2010: S287-S287
View details for Web of Science ID 000283023800400
Combined F-18 Fluoride and F-18 FDG PET/CT Scan for Evaluation of Malignancy: Beyond the Pilot Phase Study Iagaru, A., Mittra, E., Dick, D. W., Gambhir, S. S. SPRINGER. 2010: S200-S200
View details for Web of Science ID 000283023800010
Tumor Measurements by F-18-FDG PET: How Accurate are they? Mittra, E., Iagaru, A., Gambhir, S. S. SPRINGER. 2010: S330-S331
View details for Web of Science ID 000283023800598
A Comparison Between Time Domain and Spectral Imaging Systems for Imaging Quantum Dots in Small Living Animals MOLECULAR IMAGING AND BIOLOGY de la Zerda, A., Bodapati, S., Teed, R., Schipper, M. L., Keren, S., Smith, B. R., Ng, J. S., Gambhir, S. S. 2010; 12 (5): 500-508
Abstract

We quantified the performance of time-domain imaging (TDI) and spectral imaging (SI) for fluorescence imaging of quantum dots (QDs) in three distinct imaging instruments: eXplore Optix (TDI, Advanced Research Technologies Inc.), Maestro (SI, CRi Inc.), and IVIS-Spectrum (SI, Caliper Life Sciences Inc.).The instruments were compared for their sensitivity in phantoms and living mice, multiplexing capabilities (ability to resolve the signal of one QD type in the presence of another), and the dependence of contrast and spatial resolution as a function of depth.In phantoms, eXplore Optix had an order of magnitude better sensitivity compared to the SI systems, detecting QD concentrations of ~40 pM in vitro. Maestro was the best instrument for multiplexing QDs. Reduction of contrast and resolution as a function of depth was smallest with eXplore Optix for depth of 2-6 mm, while other depths gave comparable results in all systems. Sensitivity experiments in living mice showed that the eXplore Optix and Maestro systems outperformed the IVIS-Spectrum.TDI was found to be an order of magnitude more sensitive than SI at the expense of speed and very limited multiplexing capabilities. For deep tissue QD imaging, TDI is most applicable for depths between 2 and 6 mm, as its contrast and resolution degrade the least at these depths.

View details for DOI 10.1007/s11307-009-0290-4

View details for Web of Science ID 000282273200006

View details for PubMedID 20012220
Classical Hodgkin Lymphoma in First Complete Remission: Is There a Role for F-18 FDG PET/CT Surveillance? Lagaru, A., Maeda, L. S., Lin, F. I., Hoppe, R. T., Rosenberg, S. A., Gambhir, S. S., Advani, R. H. SPRINGER. 2010: S212-S213
View details for Web of Science ID 000283023800065
Noninvasive molecular imaging of c-Myc activation in living mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fan-Minogue, H., Cao, Z., Paulmurugan, R., Chan, C. T., Massoud, T. F., Felsher, D. W., Gambhir, S. S. 2010; 107 (36): 15892-15897
Abstract

The cytoplasmic Myc protein (c-Myc) regulates various human genes and is dysregulated in many human cancers. Phosphorylation mediates the protein activation of c-Myc and is essential for the function of this transcription factor in normal cell behavior and tumor growth. To date, however, the targeting of Myc as a therapeutic approach for cancer treatment has been achieved primarily at the nonprotein level. We have developed a molecular imaging sensor for noninvasive imaging of c-Myc activity in living subjects using a split Firefly luciferase (FL) complementation strategy to detect and quantify the phosphorylation-mediated interaction between glycogen synthase kinase 3beta (GSK3beta) and c-Myc. This sensor system consists of two fusion proteins, GSK 35-433-CFL and NFL-c-Myc, in which specific fragments of GSK3beta and c-Myc are fused with C-terminal and N-terminal fragments of the split FL, respectively. The sensor detects phosphorylation-specific GSK3beta-c-Myc interaction, the imaging signal of which correlates with the steady-state and temporal regulation of c-Myc phosphorylation in cell culture. The sensor also detects inhibition of c-Myc activity via differential pathways, allowing noninvasive monitoring of c-Myc-targeted drug efficacy in intact cells and living mice. Notably, this drug inhibition is detected before changes in tumor size are apparent in mouse xenograft and liver tumor models. This reporter system not only provides an innovative way to investigate the role of functional c-Myc in normal and cancer-related biological processes, but also facilitates c-Myc-targeted drug development by providing a rapid quantitative approach to assessing cancer response to therapy in living subjects.

View details for DOI 10.1073/pnas.1007443107

View details for Web of Science ID 000281637800049

View details for PubMedID 20713710
Biodistribution of Neural Stem Cells After Intravascular Therapy for Hypoxic-Ischemia STROKE Pendharkar, A. V., Chua, J. Y., Andres, R. H., Wang, N., Gaeta, X., Wang, H., De, A., Choi, R., Chen, S., Rutt, B. K., Gambhir, S. S., Guzman, R. 2010; 41 (9): 2064-2070
Abstract

Intravascular transplantation of neural stem cells represents a minimally invasive therapeutic approach for the treatment of central nervous system diseases. The cellular biodistribution after intravascular injection needs to be analyzed to determine the ideal delivery modality. We studied the biodistribution and efficiency of targeted central nervous system delivery comparing intravenous and intra-arterial (IA) administration of neural stem cells after brain ischemia.Mouse neural stem cells were transduced with a firefly luciferase reporter gene for bioluminescence imaging (BLI). Hypoxic-ischemia was induced in adult mice and reporter neural stem cells were transplanted IA or intravenous at 24 hours after brain ischemia. In vivo BLI was used to track transplanted cells up to 2 weeks after transplantation and ex vivo BLI was used to determine single organ biodistribution.Immediately after transplantation, BLI signal from the brain was 12 times higher in IA versus intravenous injected animals (P<0.0001). After IA injection, 69% of the total luciferase activity arose from the brain early after transplantation and 93% at 1 week. After intravenous injection, 94% of the BLI signal was detected in the lungs (P=0.004) followed by an overall 94% signal loss at 1 week, indicating lack of cell survival outside the brain. Ex vivo single organ analysis showed a significantly higher BLI signal in the brain than in the lungs, liver, and kidneys at 1 week (P<0.0001) and 2 weeks in IA (P=0.007).IA transplantation results in superior delivery and sustained presence of neural stem cells in the ischemic brain in comparison to intravenous infusion.

View details for DOI 10.1161/STROKEAHA.109.575993

View details for Web of Science ID 000281503000037

View details for PubMedID 20616329
DYNAMIC CONTRAST-ENHANCED MRI REVEALS CORE SIGNALLING PATHWAYS IN BREAST CANCER Mehta, S., HUGHES, N. P., Buffa, F. M., Adams, R. F., Gambhir, S. S., Harris, A. L. PERGAMON-ELSEVIER SCIENCE LTD. 2010: 13-13
View details for DOI 10.1016/j.ejcsup.2010.06.035

View details for Web of Science ID 000288598400035
Facile synthesis, silanization and biodistribution of biocompatible quantum dots Ma, N., Marshall, A. F., Gambhir, S., Rao, J. AMER CHEMICAL SOC. 2010
View details for Web of Science ID 000208164701771
Design, Synthesis, and Imaging of an Activatable Photoacoustic Probe JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Levi, J., Kothapalli, S. R., Ma, T., Hartman, K., Khuri-Yakub, B. T., Gambhir, S. S. 2010; 132 (32): 11264-11269
Abstract

Photoacoustic tomography is a rapidly growing imaging modality that can provide images of high spatial resolution and high contrast at depths up to 5 cm. We report here the design, synthesis, and evaluation of an activatable probe that shows great promise for enabling detection of the cleaved probe in the presence of high levels of nonactivated, uncleaved probe, a difficult task to attain in absorbance-based modality. Before the cleavage by its target, proteolytic enzyme MMP-2, the probe, an activatable cell-penetrating peptide, Ceeee[Ahx]PLGLAGrrrrrK, labeled with two chromophores, BHQ3 and Alexa750, shows photoacoustic signals of similar intensity at the two wavelengths corresponding to the absorption maxima of the chromophores, 675 and 750 nm. Subtraction of the images taken at these two wavelengths makes the probe effectively photoacoustically silent, as the signals at these two wavelengths essentially cancel out. After the cleavage, the dye associated with the cell-penetrating part of the probe, BHQ3, accumulates in the cells, while the other dye diffuses away, resulting in photoacoustic signal seen at only one of the wavelengths, 675 nm. Subtraction of the photoacoustic images at two wavelengths reveals the location of the cleaved (activated) probe. In the search for the chromophores that are best suited for photoacoustic imaging, we have investigated the photoacoustic signals of five chromophores absorbing in the near-infrared region. We have found that the photoacoustic signal did not correlate with the absorbance and fluorescence of the molecules, as the highest photoacoustic signal arose from the least absorbing quenchers, BHQ3 and QXL 680.

View details for DOI 10.1021/0104000a

View details for Web of Science ID 000280861300058

View details for PubMedID 20698693
[F-18]FTC-146: A novel and highly selective PET ligand for visualizing sigma-1 receptors in living subjects James, M. L., Shen, B., Zavaleta, C., Berganos, R. A., Mesangeau, C., Shaikh, J., Gambhir, S. S., Matsumoto, R. R., McCurdy, C. R., Chin, F. T. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S123-S124
View details for DOI 10.1016/j.neuroimage.2010.04.101

View details for Web of Science ID 000280181900103
Antiangiogenic Cancer Therapy: Monitoring with Molecular US and a Clinically Translatable Contrast Agent (BR55) RADIOLOGY Pysz, M. A., Foygel, K., Rosenberg, J., Gambhir, S. S., Schneider, M., Willmann, J. K. 2010; 256 (2): 519-527
Abstract

To develop and test human kinase insert domain receptor (KDR)-targeted microbubbles (MBs) (MB(KDR)) for imaging KDR at the molecular level and for monitoring antiangiogenic therapy in a human colon cancer xenograft tumor model in mice.Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. A heterodimeric peptide that binds to human KDR with low nanomolar affinity (K(D) = 0.5 nmol/L) was coupled onto the surface of perfluorobutane-containing lipid-shelled MBs (MB(KDR)). Binding specificity of MB(KDR) to human KDR and cross-reactivity with murine vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) were tested in cell culture under flow shear stress conditions (at 100 sec(-1)). In vivo binding specificity of MB(KDR) to VEGFR2 was tested in human LS174T colon cancer xenografts in mice with a 40-MHz ultrasonographic (US) transducer. Targeted contrast material-enhanced US imaging signal by using MB(KDR) was longitudinally measured during 6 days in tumors with (n = 6) and without (n = 6) antiangiogenic treatment (anti-VEGF antibody). Ex vivo VEGFR2 staining and microvessel density analysis were performed. Significant differences were evaluated (t, Mann-Whitney, or Wilcoxon test).Cell culture experiments showed four times greater binding specificity of MB(KDR) to human KDR and cross-reactivity to murine VEGFR2 (P < or = .01). In vivo imaging signal was more than three times higher (P = .01) with MB(KDR) compared with control MBs and decreased significantly (approximately fourfold lower, P = .03) following in vivo receptor blocking with anti-VEGFR2 antibody. One day after initiation of antiangiogenic therapy, imaging signal was significantly decreased (approximately 46% lower, P = .02) in treated versus untreated tumors; it remained significantly lower (range, 46%-84% decreased; P = .038) during the following 5 days. Microvessel density was significantly reduced (P = .04) in treated (mean, 7.3 microvessels per square millimeter +/- 4.7 [standard deviation]) versus untreated tumors (mean, 22.0 microvessels per square millimeter +/- 9.4); VEGFR2 expression was significantly decreased (>50% lower, P = .03) in treated tumors.Human MB(KDR) allow in vivo imaging and longitudinal monitoring of VEGFR2 expression in human colon cancer xenografts.

View details for DOI 10.1148/radiol.10091858

View details for Web of Science ID 000280272100023

View details for PubMedID 20515975
A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography NATURE MEDICINE Massoud, T. F., Paulmurugan, R., Gambhir, S. S. 2010; 16 (8): 921-U123
Abstract

Improved techniques to noninvasively image protein-protein interactions (PPIs) are essential. We molecularly engineered a positron emission tomography (PET)-based split reporter (herpes simplex virus type 1 thymidine kinase), cleaved between Thr265 and Ala266, and used this in a protein-fragment complementation assay (PCA) to quantify PPIs in mammalian cells and to microPET image them in living mice. An introduced point mutation (V119C) markedly enhanced thymidine kinase complementation in PCAs, on the basis of rapamycin modulation of FKBP12-rapamycin-binding domain (FRB) and FKBP12 (FK506 binding protein), the interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor, and in an estrogen receptor intramolecular protein folding assay. Applications of this unique split thymidine kinase are potentially far reaching, including, for example, considerably more accurate monitoring of immune and stem cell therapies, allowing for fully quantitative and tomographic PET localization of PPIs in preclinical small- and large-animal models of disease.

View details for DOI 10.1038/nm.2185

View details for Web of Science ID 000280649200033

View details for PubMedID 20639890
Facile Synthesis, Silanization, and Biodistribution of Biocompatible Quantum Dots SMALL Ma, N., Marshall, A. F., Gambhir, S. S., Rao, J. 2010; 6 (14): 1520-1528
Abstract

A facile strategy for the synthesis of silica-coated quantum dots (QDs) for in vivo imaging is reported. All the QD synthesis and silanization steps are conducted in water and methanol under mild conditions without involving any organometallic precursors or high-temperature, oxygen-free environments. The as-prepared silica-coated QDs possess high quantum yields and are extremely stable in mouse serum. In addition, the silanization method developed here produces nanoparticles with small sizes that are difficult to achieve via conventional silanization methods. The silica coating helps to prevent the exposure of the QD surface to the biological milieu and therefore increases the biocompatibility of QDs for in vivo applications. Interestingly, the silica-coated QDs exhibit a different biodistribution pattern from that of commercially available Invitrogen QD605 (carboxylate) with a similar size and emission wavelength. The Invitrogen QD605 exhibits predominant liver (57.2% injected dose (ID) g(-1)) and spleen (46.1% ID g(-1)) uptakes 30 min after intravenous injection, whereas the silica-coated QDs exhibit much lower liver (16.2% ID g(-1)) and spleen (3.67% ID g(-1)) uptakes but higher kidney uptake (8.82% ID g(-1)), blood retention (15.0% ID g(-1)), and partial renal clearance. Overall, this straightforward synthetic strategy paves the way for routine and customized synthesis of silica-coated QDs for biological use.

View details for DOI 10.1002/smll.200902409

View details for Web of Science ID 000280633900011

View details for PubMedID 20564726
Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy GENE THERAPY Chen, I. Y., Gheysens, O., Ray, S., Wang, Q., Padmanabhan, P., Paulmurugan, R., Loening, A. M., Rodriguez-Porcel, M., Willmann, J. K., Sheikh, A. Y., Nielsen, C. H., Hoyt, G., Contag, C. H., Robbins, R. C., Biswal, S., Wu, J. C., Gambhir, S. S. 2010; 17 (7): 827-838
Abstract

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

View details for DOI 10.1038/gt.2010.30

View details for Web of Science ID 000279614600002

View details for PubMedID 20237511
Molecular imaging: current status and emerging strategies CLINICAL RADIOLOGY Pysz, M. A., Gambhir, S. S., Willmann, J. K. 2010; 65 (7): 500-516
Abstract

In vivo molecular imaging has a great potential to impact medicine by detecting diseases in early stages (screening), identifying extent of disease, selecting disease- and patient-specific treatment (personalized medicine), applying a directed or targeted therapy, and measuring molecular-specific effects of treatment. Current clinical molecular imaging approaches primarily use positron-emission tomography (PET) or single photon-emission computed tomography (SPECT)-based techniques. In ongoing preclinical research, novel molecular targets of different diseases are identified and, sophisticated and multifunctional contrast agents for imaging these molecular targets are developed along with new technologies and instrumentation for multi-modality molecular imaging. Contrast-enhanced molecular ultrasound (US) with molecularly-targeted contrast microbubbles is explored as a clinically translatable molecular imaging strategy for screening, diagnosing, and monitoring diseases at the molecular level. Optical imaging with fluorescent molecular probes and US imaging with molecularly-targeted microbubbles are attractive strategies as they provide real-time imaging, are relatively inexpensive, produce images with high spatial resolution, and do not involve exposure to ionizing irradiation. Raman spectroscopy/microscopy has emerged as a molecular optical imaging strategy for ultrasensitive detection of multiple biomolecules/biochemicals with both in vivo and ex vivo versatility. Photoacoustic imaging is a hybrid of optical and US techniques involving optically-excitable molecularly-targeted contrast agents and quantitative detection of resulting oscillatory contrast agent movement with US. Current preclinical findings and advances in instrumentation, such as endoscopes and microcatheters, suggest that these molecular imaging methods have numerous potential clinical applications and will be translated into clinical use in the near future.

View details for DOI 10.1016/j.crad.2010.03.011

View details for Web of Science ID 000280379900002

View details for PubMedID 20541650
Reply to: The diagnostic accuracy of F-18-FDG PET in cutaneous malignant melanoma EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Delgado-Bolton, R. C., Jimenez-Requena, F., Fernandez-Perez, C., Gambhir, S. S., Schwimmer, J., Perez-Vazquez, J. M., Carreras-Delgado, J. L. 2010; 37 (7): 1436-1437
View details for DOI 10.1007/s00259-010-1476-3

View details for Web of Science ID 000278833100023
Implantable semiconductor biosensor for continuous in vivo sensing of far-red fluorescent molecules OPTICS EXPRESS O'Sullivan, T., Munro, E. A., Parashurama, N., Conca, C., Gambhir, S. S., Harris, J. S., Levi, O. 2010; 18 (12): 12513-12525
Abstract

We have fabricated miniature implantable fluorescence sensors for continuous fluorescence sensing applications in living subjects. These monolithically integrated GaAs-based sensors incorporate a 675 nm vertical-cavity surface-emitting laser (VCSEL), a GaAs PIN photodiode, and a fluorescence emission filter. We demonstrate high detection sensitivity for Cy5.5 far-red dye (50 nanoMolar) in living tissue, limited by the intrinsic background autofluorescence. These low cost, sensitive and scalable sensors are promising for long-term continuous monitoring of molecular dynamics for biomedical studies in freely moving animals.

View details for Web of Science ID 000278527700052

View details for PubMedID 20588377
Cu-64-Labeled Affibody Molecules for Imaging of HER2 Expressing Tumors MOLECULAR IMAGING AND BIOLOGY Cheng, Z., De Jesus, O. P., Kramer, D. J., De, A., Webster, J. M., Gheysens, O., Levi, J., Namavari, M., Wang, S., Park, J. M., Zhang, R., Liu, H., Lee, B., Syud, F. A., Gambhir, S. S. 2010; 12 (3): 316-324
Abstract

The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets.In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with (64)Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (Z(HER2:477)) and dimeric [(Z(HER2:477))(2)] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA). The resulting DOTA-Affibody conjugates were radiolabeled with (64)Cu and evaluated in nude mice bearing subcutaneous SKOV3 tumors. Biodistribution experiments showed that tumor uptake values of (64)Cu-DOTA-Z(HER2:477) and (64)Cu-DOTA-(Z(HER2:477))(2) were 6.12 +/- 1.44% and 1.46 +/- 0.50% ID/g, respectively, in nude mice (n = 3 each) at 4 h postinjection. Moreover, (64)Cu-labeled monomer exhibited significantly higher tumor/blood ratio than that of radiolabeled dimeric counterpart at all time points examined in this study. MicroPET imaging of (64)Cu-DOTA-Z(HER2:477) in SKOV3 tumor mice clearly showed good and specific tumor localization. This study demonstrates that (64)Cu-labeled Z(HER2:477) is a promising targeted molecular probe for imaging HER2 receptor expression in living mice. Further work is needed to improve the excretion properties, hence dosimetry and imaging efficacy, of the radiometal-based probe.

View details for DOI 10.1007/s11307-009-0256-6

View details for Web of Science ID 000277375300010

View details for PubMedID 19779897
Ultrahigh Sensitivity Carbon Nanotube Agents for Photoacoustic Molecular Imaging in Living Mice NANO LETTERS de la Zerda, A., Liu, Z., Bodapati, S., Teed, R., Vaithilingam, S., Khuri-Yakub, B. T., Chen, X., Dai, H., Gambhir, S. S. 2010; 10 (6): 2168-2172
Abstract

Photoacoustic imaging is an emerging modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high-contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a novel photoacoustic contrast agent, Indocyanine Green dye-enhanced single walled carbon nanotube (SWNT-ICG). We conjugated this contrast agent with cyclic Arg-Gly-Asp (RGD) peptides to molecularly target the alpha(v)beta(3) integrins, which are associated with tumor angiogenesis. Intravenous administration of this tumor-targeted contrast agent to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. The new contrast agent gave a markedly 300 times higher photoacoustic contrast in living tissues than previously reported SWNTs, leading to subnanomolar sensitivities. Finally, we show that the new contrast agent can detect approximately 20 times fewer cancer cells than previously reported SWNTs.

View details for DOI 10.1021/nl100890d

View details for Web of Science ID 000278449200033

View details for PubMedID 20499887
Antioxidants Improve Early Survival of Cardiomyoblasts After Transplantation to the Myocardium MOLECULAR IMAGING AND BIOLOGY Rodriguez-Porcel, M., Gheysens, O., Paulmurugan, R., Chen, I. Y., Peterson, K. M., Willmann, J. K., Wu, J. C., Zhu, X., Lerman, L. O., Gambhir, S. S. 2010; 12 (3): 325-334
Abstract

We tested the hypothesis that modulation of the microenvironment (using antioxidants) will increase stem cell survival in hypoxia and after transplantation to the myocardium.Rat cardiomyoblasts were stably transfected with a reporter gene (firefly luciferase) for bioluminescence imaging (BLI). First, we examined the role of oxidative stress in cells under hypoxic conditions. Subsequently, stem cells were transplanted to the myocardium of rats using high-resolution ultrasound, and their survival was monitored daily using BLI.Under hypoxia, oxidative stress was increased together with decreased cell survival compared to control cells, both of which were preserved by antioxidants. In living subjects, oxidative stress blockade increased early cell survival after transplantation to the myocardium, compared to untreated cells/animals.Modulation of the local microenvironment (with antioxidants) improves stem cell survival. Increased understanding of the interaction between stem cells and their microenvironment will be critical to advance the field of regenerative medicine.

View details for DOI 10.1007/s11307-009-0274-4

View details for Web of Science ID 000277375300011

View details for PubMedID 20013064
Radiation-Luminescence-Excited Quantum Dots for in vivo Multiplexed Optical Imaging SMALL Liu, H., Zhang, X., Xing, B., Han, P., Gambhir, S. S., Cheng, Z. 2010; 6 (10): 1087-1091
View details for DOI 10.1002/smll.200902408

View details for Web of Science ID 000278629300004

View details for PubMedID 20473988
Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope JOURNAL OF BIOMEDICAL OPTICS Ra, H., Gonzalez-Gonzalez, E., Smith, B. R., Gambhir, S. S., Kino, G. S., Solgaard, O., Kaspar, R. L., Contag, C. H. 2010; 15 (3)
Abstract

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

View details for DOI 10.1117/1.3432627

View details for Web of Science ID 000280642900042

View details for PubMedID 20615029
Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. 2010; 30 (5): 984-U224
Abstract

We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb.Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group.In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.

View details for DOI 10.1161/ATVBAHA.110.202796

View details for Web of Science ID 000276677700015

View details for PubMedID 20167654
Cetuximab-Based Immunotherapy and Radioimmunotherapy of Head and Neck Squamous Cell Carcinoma CLINICAL CANCER RESEARCH Niu, G., Sun, X., Cao, Q., Courter, D., Koong, A., Le, Q., Gambhir, S. S., Chen, X. 2010; 16 (7): 2095-2105
Abstract

To show the relationship between antibody delivery and therapeutic efficacy in head and neck cancers, in this study we evaluated the pharmacokinetics and pharmacodynamics of epidermal growth factor receptor (EGFR)-targeted immunotherapy and radioimmunotherapy by quantitative positron emission tomography (PET) imaging.EGFR expression on UM-SCC-22B and SCC1 human head and neck squamous cell cancer (HNSCC) cells were determined by flow cytometry and immunostaining. Tumor delivery and distribution of cetuximab in tumor-bearing nude mice were evaluated with small animal PET using (64)Cu-DOTA-cetuximab. The in vitro toxicity of cetuximab to HNSCC cells was evaluated by MTT assay. The tumor-bearing mice were then treated with four doses of cetuximab at 10 mg/kg per dose, and tumor growth was evaluated by caliper measurement. FDG PET was done after the third dose of antibody administration to evaluate tumor response. Apoptosis and tumor cell proliferation after cetuximab treatment were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Ki-67 staining. Radioimmunotherapy was done with (90)Y-DOTA-cetuximab.EGFR expression on UM-SCC-22B cells is lower than that on SCC1 cells. However, the UM-SCC-22B tumors showed much higher (64)Cu-DOTA-cetuximab accumulation than the SCC1 tumors. Cetuximab-induced apoptosis in SCC1 tumors and tumor growth was significantly inhibited, whereas an agonistic effect of cetuximab on UM-SCC-22B tumor growth was observed. After cetuximab treatment, the SCC1 tumors showed decreased FDG uptake, and the UM-SCC-22B tumors had increased FDG uptake. UM-SCC-22B tumors are more responsive to (90)Y-DOTA-cetuximab treatment than SCC1 tumors, partially due to the high tumor accumulation of the injected antibody.Cetuximab has an agonistic effect on the growth of UM-SCC-22B tumors, indicating that tumor response to cetuximab treatment is not necessarily related to EGFR expression and antibody delivery efficiency, as determined by PET imaging. Although PET imaging with antibodies as tracers has limited function in patient screening, it can provide guidance for targeted therapy using antibodies as delivery vehicles.

View details for DOI 10.1158/1078-0432.CCR-09-2495

View details for Web of Science ID 000278595800013

View details for PubMedID 20215534
Molecular imaging of biological gene delivery vehicles for targeted cancer therapy: beyond viral vectors. Nuclear medicine and molecular imaging Min, J., Nguyen, V. H., Gambhir, S. S. 2010; 44 (1): 15-24
Abstract

Cancer persists as one of the most devastating diseases in the world. Problems including metastasis and tumor resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of present clinical treatments. To overcome these limitations, cancer gene therapy has been developed over the last two decades for a broad spectrum of applications, from gene replacement and knockdown to vaccination, each with different requirements for gene delivery. So far, a number of genes and delivery vectors have been investigated, and significant progress has been made with several gene therapy modalities in clinical trials. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications. However, both have limitations and risks that restrict gene therapy applications, including the complexity of production, limited packaging capacity, and unfavorable immunological features. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents such as bacteria, bacteriophages, and bacteria-like particles. Recently, many molecular imaging techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been employed to assess vector-mediated gene expression in living subjects. In this review, molecular imaging techniques for monitoring biological gene delivery vehicles are described, and the specific use of these methods at different steps is illustrated. Linking molecular imaging to gene therapy will eventually help to develop novel gene delivery vehicles for preclinical study and support the development of future human applications.

View details for DOI 10.1007/s13139-009-0006-3

View details for PubMedID 24899933
Rosiglitazone Increases Myocardial Glucose Metabolism in Insulin-Resistant Cardiomyopathy JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Kao, D. P., Witteles, R. M., Quon, A., Wu, J. C., Gambhir, S. S., Fowler, M. B. 2010; 55 (9): 926-927
View details for DOI 10.1016/j.jacc.2009.08.085

View details for Web of Science ID 000274865100015

View details for PubMedID 20185047
Targeted Contrast-Enhanced Ultrasound Imaging of Tumor Angiogenesis with Contrast Microbubbles Conjugated to Integrin-Binding Knottin Peptides JOURNAL OF NUCLEAR MEDICINE Willmann, J. K., Kimura, R. H., Deshpande, N., Lutz, A. M., Cochran, J. R., Gambhir, S. S. 2010; 51 (3): 433-440
Abstract

Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quantification of tumor angiogenesis at the molecular level. The purpose of this study was to develop and test a new class of targeting ligands for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with small, conformationally constrained peptides that can be coupled to the surface of ultrasound contrast agents.Directed evolution was used to engineer a small, disulfide-constrained cystine knot (knottin) peptide that bound to alpha(v)beta(3) integrins with a low nanomolar affinity (Knottin(Integrin)). A targeted contrast-enhanced ultrasound imaging contrast agent was created by attaching Knottin(Integrin) to the shell of perfluorocarbon-filled microbubbles (MB-Knottin(Integrin)). A knottin peptide with a scrambled sequence was used to create control microbubbles (MB-Knottin(Scrambled)). The binding of MB-Knottin(Integrin) and MB-Knottin(Scrambled) to alpha(v)beta(3) integrin-positive cells and control cells was assessed in cell culture binding experiments and compared with that of microbubbles coupled to an anti-alpha(v)beta(3) integrin monoclonal antibody (MB(alphavbeta3)) and microbubbles coupled to the peptidomimetic agent c(RGDfK) (MB(cRGD)). The in vivo imaging signals of contrast-enhanced ultrasound with the different types of microbubbles were quantified in 42 mice bearing human ovarian adenocarcinoma xenograft tumors by use of a high-resolution 40-MHz ultrasound system.MB-Knottin(Integrin) attached significantly more to alpha(v)beta(3) integrin-positive cells (1.76 +/- 0.49 [mean +/- SD] microbubbles per cell) than to control cells (0.07 +/- 0.006). Control MB-Knottin(Scrambled) adhered less to alpha(v)beta(3) integrin-positive cells (0.15 +/- 0.12) than MB-Knottin(Integrin). After blocking of integrins, the attachment of MB-Knottin(Integrin) to alpha(v)beta(3) integrin-positive cells decreased significantly. The in vivo ultrasound imaging signal was significantly higher after the administration of MB-Knottin(Integrin) than after the administration of MB(alphavbeta3) or control MB-Knottin(Scrambled). After in vivo blocking of integrin receptors, the imaging signal after the administration of MB-Knottin(Integrin) decreased significantly (by 64%). The imaging signals after the administration of MB-Knottin(Integrin) were not significantly different in the groups of tumor-bearing mice imaged with MB-Knottin(Integrin) and with MB(cRGD). Ex vivo immunofluorescence confirmed integrin expression on endothelial cells of human ovarian adenocarcinoma xenograft tumors.Integrin-binding knottin peptides can be conjugated to the surface of microbubbles and used for in vivo targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Our results demonstrate that microbubbles conjugated to small peptide-targeting ligands provide imaging signals higher than those provided by a large antibody molecule.

View details for DOI 10.2967/jnumed.109.068007

View details for Web of Science ID 000275133100026

View details for PubMedID 20150258
Evaluation of a Cu-64-Labeled Cystine-Knot Peptide Based on Agouti-Related Protein for PET of Tumors Expressing alpha(v)beta(3) Integrin JOURNAL OF NUCLEAR MEDICINE Jiang, L., Kimura, R. H., Miao, Z., Silverman, A. P., Ren, G., Liu, H., Li, P., Gambhir, S. S., Cochran, J. R., Cheng, Z. 2010; 51 (2): 251-258
Abstract

Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to alpha(v)beta(3) integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects.Engineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetra-azacyclododecane-N,N',N''N'''-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with (64)Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of (64)Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine.AgRP-7C and DOTA-AgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 +/- 4 and 14 +/- 2 nM, respectively). DOTA-AgRP-7C was labeled with (64)Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, (64)Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 +/- 0.93 percentage injected dose per gram [%ID/g] and 2.37 +/- 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of (64)Cu-DOTA-AgRP-7C was confirmed in vitro and in vivo by showing that a large molar excess of the unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications.A radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the alpha(v)beta(3) integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.

View details for DOI 10.2967/jnumed.109.069831

View details for Web of Science ID 000274152800028

View details for PubMedID 20124048
Photoacoustic ocular imaging OPTICS LETTERS de la Zerda, A., Paulus, Y. M., Teed, R., Bodapati, S., Dollberg, Y., Khuri-Yakub, B. T., Blumenkranz, M. S., Moshfeghi, D. M., Gambhir, S. S. 2010; 35 (3): 270-272
Abstract

We developed a photoacoustic ocular imaging device and demonstrated its utility in imaging the deeper layers of the eye including the retina, choroid, and optic nerve. Using safe laser intensity, the photoacoustic system was able to visualize the blood distribution of an enucleated pig's eye and an eye of a living rabbit. Ultrasound images, which were simultaneously acquired, were overlaid on the photoacoustic images to visualize the eye's anatomy. Such a system may be used in the future for early detection and improved management of neovascular ocular diseases, including wet age-related macular degeneration and proliferative diabetic retinopathy.

View details for Web of Science ID 000274196100001

View details for PubMedID 20125691
A Dual-Labeled Knottin Peptide for PET and Near-Infrared Fluorescence Imaging of Integrin Expression in Living Subjects. Bioconjugate chemistry Kimura, R. H., Miao, Z., Cheng, Z., Gambhir, S. S., Cochran, J. R. 2010
Abstract

Previously, we used directed evolution to engineer mutants of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin that bind to alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity, and showed that Cy5.5- or (64)Cu-DOTA-labeled knottin peptides could be used to image integrin expression in mouse tumor models using near-infrared fluorescence (NIRF) imaging or positron emission tomography (PET). Here, we report the development of a dual-labeled knottin peptide conjugated to both NIRF and PET imaging agents for multimodality imaging in living subjects. We created an orthogonally protected peptide-based linker for stoichiometric coupling of (64)Cu-DOTA and Cy5.5 onto the knottin N-terminus and confirmed that conjugation did not affect binding to alpha(v)beta(3) and alpha(v)beta(5) integrins. NIRF and PET imaging studies in tumor xenograft models showed that Cy5.5 conjugation significantly increased kidney uptake and retention compared to the knottin peptide labeled with (64)Cu-DOTA alone. In the tumor, the dual-labeled (64)Cu-DOTA/Cy5.5 knottin peptide showed decreased wash-out leading to significantly better retention (p < 0.05) compared to the (64)Cu-DOTA-labeled knottin peptide. Tumor uptake was significantly reduced (p < 0.05) when the dual-labeled knottin peptide was coinjected with an excess of unlabeled competitor and when tested in a tumor model with lower levels of integrin expression. Finally, plots of tumor-to-background tissue ratios for Cy5.5 versus (64)Cu uptake were well-correlated over several time points post injection, demonstrating pharmacokinetic cross validation of imaging labels. This dual-modality NIRF/PET imaging agent is promising for further development in clinical applications where high sensitivity and high resolution are desired, such as detection of tumors located deep within the body and image-guided surgical resection.

View details for PubMedID 20131753
A red-shifted Renilla luciferase for transient reporter-gene expression NATURE METHODS Loening, A. M., Dragulescu-Andrasi, A., Gambhir, S. S. 2010; 7 (1): 5-6
View details for DOI 10.1038/nmeth0110-05

View details for Web of Science ID 000273128300003

View details for PubMedID 20038949
Optical Imaging with Radioactive Probes PLoS One Liu, H., Ren G, Miao Z, Zhang X, Tang X, Han P, Gambhir SS, Cheng Z 2010; 5 (3): E9470
Meta-analysis of the Performance of [18F] FDG-PET in Cutaneous Melanoma. European Journal of Nuclear Medicine and Molecular Imaging JImenez-Requena F, Delgado-Bolton R, Fernandez-Perez C, Gambhir SS, Schwimmer J, Perez-Vazquez J, Carreras-Delgado J. 2010; 37(2): 284-300
Combined F-18-FDG and Fluoride Approach in PET/CT Imaging: Is There a Clinical Future? REPLY JOURNAL OF NUCLEAR MEDICINE Iagaru, A., Mittra, E., Goris, M. L., Gambhir, S. S. 2010; 51 (1): 166-167
View details for DOI 10.2967/jnumed.109.067082

View details for Web of Science ID 000273263800030
A Novel Method of Monitoring Placenta-Specific Transgene Expression Throughout Pregnancy by Noninvasive Bioluminescence Imaging Fan, X., Ren, P., Dhal, S., Goodman, S. B., Gambhir, S. S., Druzin, M. L., Nayak, N. R. SOC STUDY REPRODUCTION. 2010: 144-145
View details for Web of Science ID 000284381300406
Functional and Transcriptional Characterization of Human Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Myocardial Infarction PLOS ONE Li, Z., Wilson, K. D., Smith, B., Kraft, D. L., Jia, F., Huang, M., Xie, X., Robbins, R. C., Gambhir, S. S., Weissman, I. L., Wu, J. C. 2009; 4 (12)
Abstract

Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time.In this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods.Taken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future.

View details for DOI 10.1371/journal.pone.0008443

View details for Web of Science ID 000273180200002

View details for PubMedID 20046878
Efficacy of F-18-FDG PET/CT in the evaluation of patients with recurrent cervical carcinoma EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Mittra, E., El-Maghraby, T., Rodriguez, C. A., Quon, A., McDougall, I. R., Gambhir, S. S., Iagaru, A. 2009; 36 (12): 1952-1959
Abstract

Only a limited number of studies have evaluated the efficacy of 18F-FDG PET/CT for recurrent cervical carcinoma, which this study seeks to expand upon.This is a retrospective study of 30 women with cervical carcinoma who had a surveillance PET/CT after initial therapy. Sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated using a 2 × 2 contingency table with pathology results (76%) or clinical follow-up (24%) as the gold standard. The Wilson score method was used to perform 95% confidence interval estimations.The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PET/CT for the detection of local recurrence at the primary site were 93, 93, 93, 86, and 96%, respectively. The same values for the detection of distant metastases were 96, 95, 95, 96, and 95%, respectively. Seventy-one percent of the scans performed in symptomatic patients showed true-positive findings. In comparison, 44% of scans performed in asymptomatic patients showed true-positive findings. But, all patients subsequently had a change in their management based on the PET/CT findings such that the effect was notable. The maximum standardized uptake value ranged from 5 to 28 (average: 13 ± 7) in the primary site and 3 to 23 (average: 8 ± 4) in metastases which were significantly different (p = 0.04).This study demonstrates favorable efficacy of 18F-FDG PET/CT for identification of residual/recurrent cervical cancer, as well as for localization of distant metastases.

View details for DOI 10.1007/s00259-009-1206-x

View details for Web of Science ID 000271979300004

View details for PubMedID 19585114
Development and intra-institutional and inter-institutional validation of a comprehensive new hepatobiliary software: part 1-liver and gallbladder function NUCLEAR MEDICINE COMMUNICATIONS Krishnamurthy, G. T., Krishnamurthy, S., Gambhir, S. S., Rodrigues, C., Rosenberg, J., Schiepers, C., Buxton-Thomas, M. 2009; 30 (12): 934-944
Abstract

To develop a software tool for quantification of liver and gallbladder function, and to assess the repeatability and reproducibility of measurements made with it.The software tool developed with the JAVA programming language uses the JAVA2 Standard Edition framework. After manual selection of the regions of interest on a 99mTc hepatic iminodiacetic acid study, the program calculates differential hepatic bile flow, basal duodeno-gastric bile reflux (B-DGBR), hepatic extraction fraction (HEF) of both the lobes with deconvolutional analysis and excretion half-time with nonlinear least squares fit. Gallbladder ejection fraction, ejection period (EP), ejection rate (ER), and postcholecystokinin (CCK) DGBR are calculated after stimulation with CCK-8. To assess intra-observer repeatability and intra-observer reproducibility, measurements from 10 normal participants were analyzed twice by three nuclear medicine technologists at the primary center. To assess inter-site reproducibility, measurements from a superset of 24 normal participants were also assessed once by three observers at the primary center and single observer at three other sites.For the 24 control participants, mean+/-SD of hepatic bile flow into gallbladder was 63.87+/-28.7%, HEF of the right lobe 100+/-0%, left lobe 99.43+2.63%, excretion half-time of the right lobe 21.50+6.98 min, left lobe 28.3+/-11.3 min. Basal DGBR was 1.2+/-1.0%. Gallbladder ejection fraction was 80+/-11%, EP 15.0+/-3.0 min, ER 5.8+/-1.6%/min, and DGBR-CCK 1.3+/-2.3%. Left and right lobe HEF was virtually identical across readers. All measures showed high repeatability except for gallbladder bile flow, basal DGBR, and EP, which exhibited marginal repeatability. Ejection fraction exhibited high reproducibility. There was high concordance among the three primary center observers except for basal DGBR, EP, and ER. Concordance between the primary site and one of the other sites was high, one was fair, and one was poor.New United States Food and Drug Administration-approved personal computer-based Krishnamurthy Hepato-Biliary Software for quantification of the liver and gallbladder function shows promise for consistently repeatable and reproducible results both within and between institutions, and may help to promote universal standardization of data acquisition and analysis in nuclear hepatology.

View details for DOI 10.1097/MNM.0b013e32832ed34a

View details for Web of Science ID 000272116100006

View details for PubMedID 19858769
Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion Huang, N. F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M. D., Wu, J. C., Gambhir, S. S., Cooke, J. P. LIPPINCOTT WILLIAMS & WILKINS. 2009: S1152-S1152
View details for Web of Science ID 000271831504251
A Novel Molecular Imaging Sensor of Cellular Oxidative Stress Peterson, K. M., Chen, I. Y., Simari, R. D., Gambhir, S. S., Lerman, A., Rodriguez-Porcel, M. LIPPINCOTT WILLIAMS & WILKINS. 2009: S1025-S1025
View details for Web of Science ID 000271831503510
F-18-FDG Uptake in Lung, Breast, and Colon Cancers: Molecular Biology Correlates and Disease Characterization JOURNAL OF NUCLEAR MEDICINE Jadvar, H., Alavi, A., Gambhir, S. S. 2009; 50 (11): 1820-1827
Abstract

It is hoped that in the not too distant future, noninvasive imaging-based molecular interrogation and characterization of tumors can improve our fundamental understanding of the dynamic biologic behavior of cancer. For example, the new dimension of diagnostic information that is provided by (18)F-FDG PET has led to improved clinical decision making and management changes in a substantial number of patients with cancer. In this context, the aim of this review is to bring together and summarize the current data on the correlation between the underlying molecular biology and the clinical observations of tumor (18)F-FDG accumulation in 3 major human cancers: lung, breast, and colon.

View details for DOI 10.2967/jnumed.108.054098

View details for Web of Science ID 000272554100015

View details for PubMedID 19837767
Three-Dimensional Photoacoustic Imaging Using a Two-Dimensional CMUT Array IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Vaithilingam, S., Ma, T., Furukawa, Y., Wygant, I. O., Zhuang, X., de la Zerda, A., Oralkan, O., Kamaya, A., Gambhir, S. S., Jeffrey, R. B., Khuri-Yakub, B. T. 2009; 56 (11): 2411-2419
Abstract

In this paper, we describe using a 2-D array of capacitive micromachined ultrasonic transducers (CMUTs) to perform 3-D photoacoustic and acoustic imaging. A tunable optical parametric oscillator laser system that generates nanosecond laser pulses was used to induce the photoacoustic signals. To demonstrate the feasibility of the system, 2 different phantoms were imaged. The first phantom consisted of alternating black and transparent fishing lines of 180 mum and 150 mum diameter, respectively. The second phantom comprised polyethylene tubes, embedded in chicken breast tissue, filled with liquids such as the dye indocyanine green, pig blood, and a mixture of the 2. The tubes were embedded at a depth of 0.8 cm inside the tissue and were at an overall distance of 1.8 cm from the CMUT array. Two-dimensional cross-sectional slices and 3-D volume rendered images of pulse-echo data as well as photoacoustic data are presented. The profile and beamwidths of the fishing line are analyzed and compared with a numerical simulation carried out using the Field II ultrasound simulation software. We investigated using a large aperture (64 x 64 element array) to perform photoacoustic and acoustic imaging by mechanically scanning a smaller CMUT array (16 x 16 elements). Two-dimensional transducer arrays overcome many of the limitations of a mechanically scanned system and enable volumetric imaging. Advantages of CMUT technology for photoacoustic imaging include the ease of integration with electronics, ability to fabricate large, fully populated 2-D arrays with arbitrary geometries, wide-bandwidth arrays and high-frequency arrays. A CMUT based photoacoustic system is proposed as a viable alternative to a piezoelectric transducer based photoacoustic systems.

View details for DOI 10.1109/TUFFC.2009.1329

View details for Web of Science ID 000271478600010

View details for PubMedID 19942528
Whole-body, real-time preclinical imaging of quantum dot fluorescence with time-gated detection JOURNAL OF BIOMEDICAL OPTICS May, A., Bhaumik, S., Gambhir, S. S., Zhan, C., Yazdanfar, S. 2009; 14 (6)
Abstract

We describe a wide-field preclinical imaging system optimized for time-gated detection of quantum dot fluorescence emission. As compared to continuous wave measurements, image contrast was substantially improved by suppression of short-lifetime background autofluorescence. Real-time (8 frames/s) biological imaging of subcutaneous quantum dot injections is demonstrated simultaneously in multiple living mice.

View details for DOI 10.1117/1.3269675

View details for Web of Science ID 000274267900004

View details for PubMedID 20059235
PET of Malignant Melanoma Using F-18-Labeled Metallopeptides JOURNAL OF NUCLEAR MEDICINE Ren, G., Liu, Z., Miao, Z., Liu, H., Subbarayan, M., Chin, F. T., Zhang, L., Gambhir, S. S., Cheng, Z. 2009; 50 (11): 1865-1872
Abstract

Melanocortin type 1 receptor (MC1R), also known as alpha-melanocyte-stimulating hormone (alpha-MSH) receptor, is an attractive molecular target for melanoma imaging and therapy. An (18)F-labeled linear alpha-MSH peptide ((18)F-FB-Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH(2) [NAPamide]) shows promising melanoma imaging properties but with only moderate tumor uptake and retention. A transition metal rhenium-cyclized alpha-MSH peptide, ReO[Cys(3,4,10),d-Phe(7),Arg(11)]alpha-MSH(3-13) (ReCCMSH(Arg(11))), has shown high in vitro binding affinity to MC1R and excellent in vivo melanoma-targeting profiles when labeled with radiometals. Therefore, we hypothesized that ReCCMSH(Arg(11)) could be a good platform for the further development of an (18)F-labeled probe for PET of MC1R-positive malignant melanoma.In this study, the metallopeptide Ac-d-Lys-ReCCMSH(Arg(11)) was synthesized using conventional solid-phase peptide synthesis chemistry and a rhenium cyclization reaction. The resulting peptides were then labeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The (18)F-labeled metallopeptides were further tested for their in vitro receptor binding affinities, in vivo biodistribution, and PET imaging properties.Both isomers of Ac-d-Lys-ReCCMSH(Arg(11)), named as RMSH-1 and RMSH-2, were purified and identified by high-performance liquid chromatography. The binding affinities of RMSH-1 and RMSH-2 and their respective (19)F-SFB-conjugated peptides ((19)F-FB-RMSH-1 and (19)F-FB-RMSH-2) were all determined to be within nanomolar range. Both (18)F-labeled metallopeptides showed good tumor uptake in the B16F10 murine model, with high MC1R expression, but much lower uptake in the A375M human melanoma xenografted in mice, indicating low MC1R expression. (18)F-FB-RMSH-1, when compared with (18)F-FB-RMSH-2, displayed more favorable in vivo performance in terms of slightly higher tumor uptakes and much lower accumulations in the kidney and liver at 2 h after injection. Small-animal PET of (18)F-FB-RMSH-1 and -2 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptakes and poorer tumor-to-normal organ contrasts were observed for the A375M model than for the B16F10 model. (18)F-FB-RMSH-1 and -2 showed higher tumor uptake and better tumor retention than did (18)F-FB-NAPamide.Specific in vivo targeting of (18)F-FB-RMSH-1 to malignant melanoma was successfully achieved in preclinical models with high MC1R expression. Thus, the radiofluorinated metallopeptide (18)F-FB-RMSH-1 is a promising molecular probe for PET of MC1R-positive tumors.

View details for DOI 10.2967/jnumed.109.062877

View details for Web of Science ID 000272554100021

View details for PubMedID 19837749
Matrix-insensitive protein assays push the limits of biosensors in medicine NATURE MEDICINE Gaster, R. S., Hall, D. A., Nielsen, C. H., Osterfeld, S. J., Yu, H., Mach, K. E., Wilson, R. J., Murmann, B., Liao, J. C., Gambhir, S. S., Wang, S. X. 2009; 15 (11): 1327-U130
Abstract

Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse clinical samples (for example, serum, urine, cell lysates or saliva) with high sensitivity and large linear dynamic range. A major limitation confounding other technologies is signal distortion that occurs in various matrices due to heterogeneity in ionic strength, pH, temperature and autofluorescence. Here we present a magnetic nanosensor technology that is matrix insensitive yet still capable of rapid, multiplex protein detection with resolution down to attomolar concentrations and extensive linear dynamic range. The matrix insensitivity of our platform to various media demonstrates that our magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense.

View details for DOI 10.1038/nm.2032

View details for Web of Science ID 000271543700023

View details for PubMedID 19820717
A strategy for blood biomarker amplification and localization using ultrasound PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA D'Souza, A. L., Tseng, J. R., Pauly, K. B., Guccione, S., Rosenberg, J., Gambhir, S. S., Glazer, G. M. 2009; 106 (40): 17152-17157
Abstract

Blood biomarkers have significant potential applications in early detection and management of various diseases, including cancer. Most biomarkers are present in low concentrations in blood and are difficult to discriminate from noise. Furthermore, blood measurements of a biomarker do not provide information about the location(s) where it is produced. We hypothesize a previously undescribed strategy to increase the concentration of biomarkers in blood as well as localize the source of biomarker signal using ultrasound energy directly applied to tumor cells. We test and validate our hypothesis in cell culture experiments and mouse tumor xenograft models using the human colon cancer cell line LS174T, while measuring the biomarker carcinoembryonic antigen (CEA) before and after the use of ultrasound to liberate the biomarker from the tumor cells. The results demonstrate that the application of low-frequency ultrasound to tumor cells causes a significant release of tumor biomarker, which can be measured in the blood. Furthermore, we establish that this release is specific to the direct application of the ultrasound to the tumor, enabling a method for localization of biomarker production. This work shows that it is possible to use ultrasound to amplify and localize the source of CEA levels in blood of tumor-bearing mice and will allow for a previously undescribed way to determine the presence and localization of disease more accurately using a relatively simple and noninvasive strategy.

View details for DOI 10.1073/pnas.0903437106

View details for Web of Science ID 000270537500053

View details for PubMedID 19805109
A novel multimodal approach to track neural progenitor cells in vivo Pendharkar, A., De, A., Wang, H., Gaeta, X., Wang, N., Chua, J. Y., Andres, R., Chen, X., Gambhir, S. S., Guzman, R. NATURE PUBLISHING GROUP. 2009: S472-S473
View details for Web of Science ID 000270329900575
Creatine modulates survival, migration and differentiation in neural stem cells Andres, R. H., Pendharkar, A., Guzman, R., De, A., Bliss, T. M., MacMillan, E., Svendsen, C., Gambhir, S. S., Widmer, H., Wallimann, T. NATURE PUBLISHING GROUP. 2009: S562-S562
View details for Web of Science ID 000270329900691
A Novel Estrogen Receptor Intramolecular Folding-based Titratable Transgene Expression System MOLECULAR THERAPY Paulmurugan, R., Padmanabhan, P., Ahn, B., Ray, S., Willmann, J. K., Massoud, T. F., Biswal, S., Gambhir, S. S. 2009; 17 (10): 1703-1711
Abstract

The use of regulated gene expression systems is important for successful gene therapy applications. In this study, ligand-induced structural change in the estrogen receptor (ER) was used to develop a novel ER intramolecular folding-based transcriptional activation system. The system was studied using ER-variants of different lengths, flanked on either side by the GAL4-DNA-binding domain and the VP16-transactivation domain (GAL4(DBD)-ER-VP16). The ER ligands of different types showed efficient ligand-regulated transactivation. We also characterized a bidirectional transactivation system based on the ER and demonstrated its utility in titrating both reporter and therapeutic gene expression. The ligand-regulated transactivation system developed by using a mutant form of the ER (G521T, lacking affinity for the endogenous ligand 17beta-estradiol, whereas maintaining affinity for other ligands) showed efficient activation by the ligand raloxifene in living mice without significant interference from the circulating endogenous ligand. The ligand-regulated transactivation system was used to test the therapeutic efficiency of the tumor suppressor protein p53 in HepG2 (p53(+/+)) and SKBr3 (p53(-/-)/mutant-p53(+/+)) cells in culture and tumor xenografts in living mice. The multifunctional capabilities of this system should be useful for gene therapy applications, to study ER biology, to evaluate gene regulation, ER ligand screening, and ER ligand biocharacterization in cells and living animals.

View details for DOI 10.1038/mt.2009.171

View details for Web of Science ID 000270851900008

View details for PubMedID 19654568
Melanin-Targeted Preclinical PET Imaging of Melanoma Metastasis JOURNAL OF NUCLEAR MEDICINE Ren, G., Miao, Z., Liu, H., Jiang, L., Limpa-Amara, N., Mahmood, A., Gambhir, S. S., Cheng, Z. 2009; 50 (10): 1692-1699
Abstract

Dialkylamino-alkyl-benzamides possess an affinity for melanin, suggesting that labeling of such benzamides with (18)F could potentially produce melanin-targeted PET probes able to identify melanotic melanoma metastases in vivo with high sensitivity and specificity.In this study, N-[2-(diethylamino)ethyl]-4-(18)F-fluorobenzamide ((18)F-FBZA) was synthesized via a 1-step conjugation reaction. The sigma-receptor binding affinity of (19)F-FBZA was determined along with the in vitro cellular uptake of radiofluorinated (18)F-FBZA in B16F10 cells. In vivo distribution and small-animal PET studies were conducted on mice bearing B16F10 melanoma, A375M amelanotic melanoma, and U87MG tumors, and comparative studies were performed with (18)F-FDG PET in the melanoma models.In vitro, uptake of (18)F-FBZA was significantly higher in B16F10 cells treated with l-tyrosine (P < 0.001). In vivo, (18)F-FBZA displayed significant tumor uptake; at 2 h, 5.94 +/- 1.83 percentage injected dose (%ID) per gram was observed in B16F10 tumors and only 0.75 +/- 0.09 %ID/g and 0.56 +/- 0.13 %ID/g was observed in amelanotic A375M and U87MG tumors, respectively. Lung uptake was significantly higher in murine lungs bearing melanotic B16F10 pulmonary metastases than in normal murine lungs (P < 0.01). Small-animal PET clearly identified melanotic lesions in both primary and pulmonary metastasis B16F10 tumor models. Coregistered micro-CT with small-animal PET along with biopsies further confirmed the presence of tumor lesions in the mouse lungs.(18)F-FBZA specifically targets primary and metastatic melanotic melanoma lesions with high tumor uptake and may have translational potential.

View details for DOI 10.2967/jnumed.109.066175

View details for Web of Science ID 000272553600023

View details for PubMedID 19759116
A 2-Helix Small Protein Labeled with Ga-68 for PET Imaging of HER2 Expression JOURNAL OF NUCLEAR MEDICINE Ren, G., Zhang, R., Liu, Z., Webster, J. M., Miao, Z., Gambhir, S. S., Syud, F. A., Cheng, Z. 2009; 50 (9): 1492-1499
Abstract

Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation.In this study, a 2-helix small protein, MUT-DS, was chemically modified with a metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA-MUT-DS was then site-specifically radiolabeled with an important PET radionuclide, (68)Ga. The resulting radiolabeled anti-HER2 2-helix molecule was further evaluated as a potential molecular probe for small-animal PET HER2 imaging in a SKOV3 tumor mouse model.The 2-helix DOTA-MUT-DS showed high HER2-binding affinity (dissociation constant, 4.76 nM). The radiolabeled probe displayed high stability in mouse serum and specificity toward HER2 in cell cultures. Biodistribution and small-animal PET studies further showed that (68)Ga-DOTA-MUT-DS had rapid and high SKOV3 tumor accumulation and quick clearance from normal organs. The specificity of (68)Ga-DOTA-MUT-DS for SKOV3 tumors was confirmed by monitoring modulation of HER2 protein on treatment of tumor mice with heat shock protein 90 inhibitor 17-N,N-dimethyl ethylene diamine-geldanamycin in vivo.This proof-of-concept research clearly demonstrated that synthetic 2-helix (68)Ga-DOTA-MUT-DS is a promising PET probe for imaging HER2 expression in vivo. The Affibody-derived small 2-helix protein scaffold has great potential for developing targeting agents for a variety of tumor-associated biomarkers.

View details for DOI 10.2967/jnumed.109.064287

View details for Web of Science ID 000272548900023

View details for PubMedID 19690041
Efficacy of F-18-FDG PET/CT for Breast Cancer Mittra, E., Quon, A., Gambhir, S. S., Iagaru, A. SPRINGER. 2009: S176-S176
View details for Web of Science ID 000208690800091
Combined F-18 Fluoride and F-18 FDG PET/CT Scan for Evaluation of Malignancy Lagaru, A., Mittra, E., Dick, D., Quon, A., Goris, M. L., Gambhir, S. S. SPRINGER. 2009: S214-S214
View details for Web of Science ID 000208690800268
Prospective Evaluation of Tc-99m-MDP Scintigraphy, F-18 NaF PET/CT and F-18 FDG PET/CT for Detection of Skeletal Metastases Iagaru, A., Mittra, E., Dick, D., Gambhir, S. S. SPRINGER. 2009: S187-S187
View details for Web of Science ID 000208690800141
Chemical tools for imaging glycosylation in vivo Chang, P. V., Prescher, J. A., Foss, C. A., Ray, P., Gambhir, S. S., Pomper, M. G., Bertozzi, C. R. AMER CHEMICAL SOC. 2009
View details for Web of Science ID 000207861907424
Visualizing Implanted Tumors in Mice with Magnetic Resonance Imaging Using Magnetotactic Bacteria CLINICAL CANCER RESEARCH Benoit, M. R., Mayer, D., Barak, Y., Chen, I. Y., Hu, W., Cheng, Z., Wang, S. X., Spielman, D. M., Gambhir, S. S., Matin, A. 2009; 15 (16): 5170-5177
Abstract

To determine if magnetotactic bacteria can target tumors in mice and provide positive contrast for visualization using magnetic resonance imaging.The ability of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1 (referred to from here as AMB-1), to confer positive magnetic resonance imaging contrast was determined in vitro and in vivo. For the latter studies, AMB-1 were injected either i.t. or i.v. Bacterial growth conditions were manipulated to produce small (approximately 25-nm diameter) magnetite particles, which were observed using transmission electron microscopy. Tumor targeting was confirmed using 64Cu-labeled bacteria and positron emission tomography and by determination of viable cell counts recovered from different organs and the tumor.We show that AMB-1 bacteria with small magnetite particles generate T1-weighted positive contrast, enhancing in vivo visualization by magnetic resonance imaging. Following i.v. injection of 64Cu-labeled AMB-1, positron emission tomography imaging revealed increasing colonization of tumors and decreasing infection of organs after 4 hours. Viable cell counts showed that, by day 6, the bacteria had colonized tumors but were cleared completely from other organs. Magnetic resonance imaging showed a 1.22-fold (P = 0.003) increased positive contrast in tumors on day 2 and a 1.39-fold increase (P = 0.0007) on day 6.Magnetotactic bacteria can produce positive magnetic resonance imaging contrast and colonize mouse tumor xenografts, providing a potential tool for improved magnetic resonance imaging visualization in preclinical and translational studies to track cancer.

View details for DOI 10.1158/1078-0432.CCR-08-3206

View details for Web of Science ID 000269024900019

View details for PubMedID 19671860
Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zavaleta, C. L., Smith, B. R., Walton, I., Doering, W., Davis, G., Shojaei, B., Natan, M. J., Gambhir, S. S. 2009; 106 (32): 13511-13516
Abstract

Raman spectroscopy is a newly developed, noninvasive preclinical imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of molecular imaging. In this study, we demonstrate the ability of Raman spectroscopy to separate the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s.c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i.v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally separated using our optimized noninvasive Raman imaging system. In addition, we were able to linearly correlate Raman signal with SERS concentration after injecting four spectrally unique SERS nanoparticles either s.c. (R(2) = 0.998) or i.v. (R(2) = 0.992). These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers associated with a specific disease.

View details for DOI 10.1073/pnas.0813327106

View details for Web of Science ID 000268877300066

View details for PubMedID 19666578
Cys-diabody Quantum Dot Conjugates (ImmunoQdots) for Cancer Marker Detection BIOCONJUGATE CHEMISTRY Barat, B., Sirk, S. J., McCabe, K. E., Li, J., Lepin, E. J., Remenyi, R., Koh, A. L., Olafsen, T., Gambhir, S. S., Weiss, S., Wu, A. M. 2009; 20 (8): 1474-1481
Abstract

The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-?-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655. Spectral characterization of the conjugate revealed that the spectrum was symmetrical and essentially identical to unconjugated Qdot. Specific receptor binding activity of anti-HER2 iQdot 655 was confirmed by flow cytometry on HER2 positive and negative cells. Immunofluorescence results showed homogeneous surface labeling of the cell membrane with Qdot 655 conjugate. In addition, cys-diabodies specific for HER2, as well as prostate stem cell antigen (PSCA), were conjugated successfully with amino PEG Qdot 800. All of these iQdots retain the photoluminescence properties of the unconjugated Qdot 800 as well as the antigen binding specificity of the cys-diabody as demonstrated by flow cytometry. Simultaneous detection of two tumor antigens on LNCaP/PSCA prostate cancer cells (which express PSCA and HER2) in culture was possible using two iQdots, anti-HER2 iQdot 655 and anti-PSCA iQdot 800. Thus, these iQdots are potentially useful as optical probes for sensitive, multiplexed detection of surface markers on tumor cells. The present thiol-specific conjugation method demonstrates a general approach for site-specific oriented coupling of cys-diabodies to a wide variety of nanoparticles without disturbing the antigen binding site and maintaining small size compared to intact antibody.

View details for DOI 10.1021/bc800421f

View details for Web of Science ID 000269042100006

View details for PubMedID 19642689
BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals FASEB JOURNAL De, A., Ray, P., Loening, A. M., Gambhir, S. S. 2009; 23 (8): 2702-2709
Abstract

Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon-efficient, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564-nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single-cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red-shifted light output, and good spectral resolution (approximately 85 nm), BRET3 shows improved spatial and temporal resolution for measuring intracellular events in single cells and in living small animal models. The development of further BRET3-based assays will allow imaging of protein-protein interactions using a single assay directly scalable from intact living cells to small living subjects, allowing accelerated drug discovery.

View details for DOI 10.1096/fj.08-118919

View details for Web of Science ID 000268836700038

View details for PubMedID 19351700
Role of Oxidative Stress in Stem Cell Survival Peterson, K. M., Abdelrhaman, A., Gambhir, S. S., Rodriguez-Porcel, M. G. LIPPINCOTT WILLIAMS & WILKINS. 2009: E88-E88
View details for Web of Science ID 000267102500416
Imaging Gene Expression in Human Mesenchymal Stem Cells: From Small to Large Animals RADIOLOGY Willmann, J. K., Paulmurugan, R., Rodriguez-Porcel, M., Stein, W., Brinton, T. J., Connolly, A. J., Nielsen, C. H., Lutz, A. M., Lyons, J., Ikeno, F., Suzuki, Y., Rosenberg, J., Chen, I. Y., Wu, J. C., Yeung, A. C., Yock, P., Robbins, R. C., Gambhir, S. S. 2009; 252 (1): 117-127
Abstract

To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 x 10(6) transduced (n = 5) and control nontransduced (n = 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphate-buffered saline (PBS) (n = 4) or matrigel (n = 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed.In cell culture, there was a viral dose-dependent increase of gene expression and FHBG accumulation in human MSCs. Human MSC viability was 96.7% (multiplicity of infection, 250). Cardiac FHBG uptake in rats was significantly elevated (P < .0001) after human MSC injection (0.0054% injected dose [ID]/g +/- 0.0007 [standard deviation]) compared with that in the remote myocardium (0.0003% ID/g +/- 0.0001). In swine, myocardial radiotracer uptake was not elevated after injection of up to 100 x 10(6) human MSCs (PBS group). In the matrigel group, signal-to-background ratio increased to 1.87 after injection of 100 x 10(6) human MSCs and positively correlated (R(2) = 0.97, P < .001) with the number of administered human MSCs.Reporter gene imaging in human MSCs can be translated to large animals. The study highlights the importance of co-administering a "scaffold" for increasing intramyocardial retention of human MSCs.

View details for DOI 10.1148/radiol.2513081616

View details for Web of Science ID 000268362900015

View details for PubMedID 19366903
Simulations of Virtual PET/CT 3-D Bronchoscopy Imaging Using a Physical Porcine Lung-Heart Phantom MOLECULAR IMAGING AND BIOLOGY Yerushalmi, D., Mullick, R., Quon, A., Fahrig, R., Pelc, N. J., Fann, J. I., Gambhir, S. S. 2009; 11 (4): 275-282
Abstract

We present a systematic approach for studying positron emission tomography-computed tomography (PET/CT) 3-D virtual fly-through endoscopy and for assessing the accuracy of this technology for visualizing and detecting endobronchial lesions as a function of focal lesion morphology and activity.Capsules designed to simulate endobronchial lesions were filled with activity and introduced into a porcine lung-heart phantom. PET/CT images were acquired, reconstructed, and volume rendered as 3-D fly-through and fly-around visualizations. Anatomical positioning of lesions seen on the 3-D-volume-rendered PET/CT images was compared to the actual position of the capsules.Lesion size was observed to be highly sensitive to PET threshold parameter settings and careful opacity and color transfer function parameter assignment.We have demonstrated a phantom model for studies of PET/CT 3-D virtual fly-through bronchoscopy and have applied this model for understanding the effect of PET thresholding on the visualization and detection of lesions.

View details for DOI 10.1007/s11307-009-0201-8

View details for Web of Science ID 000266830700010

View details for PubMedID 19434462
OXIDATIVE STRESS BLOCKADE IMPROVES STEM CELL SURVIVAL Peterson, K., Aly, A., Gambhir, S., Rodriguez-Porcel, M. ELSEVIER IRELAND LTD. 2009
View details for Web of Science ID 000207957101474
Tumor Metabolic Phenotypes on F-18 FDG PET REPLY JOURNAL OF NUCLEAR MEDICINE Iagaru, A. H., Gambhir, S. S., Goris, M. L. 2009; 50 (6): 1011-1012
View details for DOI 10.2967/jnumed.109.061945

View details for Web of Science ID 000272488000034
Engineered Two-Helix Small Proteins for Molecular Recognition CHEMBIOCHEM Webster, J. M., Zhang, R., Gambhir, S. S., Cheng, Z., Syud, F. A. 2009; 10 (8): 1293-1296
Abstract

Less is more: By starting with a high-affinity HER2-binding 3-helix affibody molecule, we successfully developed 2-helix small protein binders with 5 nM affinities by using a combination of several different strategies. Our efforts clearly suggest that 2-helix small proteins against important tumor targets can be obtained by rational protein design and engineering.

View details for DOI 10.1002/cbic.200900062

View details for Web of Science ID 000266561500003

View details for PubMedID 19422008
Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation MOLECULAR IMAGING AND BIOLOGY Chen, I. Y., Greve, J. M., Gheysens, O., Willmann, J. K., Rodriguez-Porcel, M., Chu, P., Sheikh, A. Y., Faranesh, A. Z., Paulmurugan, R., Yang, P. C., Wu, J. C., Gambhir, S. S. 2009; 11 (3): 178-187
Abstract

In this study, we compared firefly luciferase (Fluc) reporter gene and superparamagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively.Rats (n = 31) underwent an intramyocardial injection of cardiomyoblasts (2 x 10(6)) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 microg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis.BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (> or =16 days) and persistent MR signal (> or =42 days).Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.

View details for DOI 10.1007/s11307-008-0182-z

View details for Web of Science ID 000265686900005

View details for PubMedID 19034584
A Potent, Imaging Adenoviral Vector Driven by the Cancer-selective Mucin-1 Promoter That Targets Breast Cancer Metastasis CLINICAL CANCER RESEARCH Huyn, S. T., Burton, J. B., Sato, M., Carey, M., Gambhir, S. S., Wu, L. 2009; 15 (9): 3126-3134
Abstract

With breast cancer, early detection and proper staging are critical, and will often influence both the treatment regimen and the therapeutic outcome for those affected with this disease. Improvements in these areas will play a profound role in reducing mortality from breast cancer.In this work we developed a breast cancer-targeted serotype 5 adenoviral vector, utilizing the tumor-specific mucin-1 promoter in combination with the two-step transcriptional amplification system, a system used to augment the activity of weak tissue-specific promoters.We showed the strong specificity of this tumor-selective adenovirus to express the luciferase optical imaging gene, leading to diagnostic signals that enabled detection of sentinel lymph node metastasis of breast cancer. Furthermore, we were able to target hepatic metastases following systemic administration of this mucin-1 selective virus.Collectively, we showed that the amplified mucin-1 promoter-driven vector is able to deliver to and selectively express a desirable transgene in metastatic lesions of breast tumors. This work has strong clinical relevance to current diagnostic staging approaches, and could add to targeted therapeutic strategies to advance the fight against breast cancer.

View details for DOI 10.1158/1078-0432.CCR-08-2666

View details for Web of Science ID 000265712100022

View details for PubMedID 19366829
Molecular Imaging of Phosphorylation Events for Drug Development MOLECULAR IMAGING AND BIOLOGY CHAN, C. T., Paulmurugan, R., Reeves, R. E., Solow-Cordero, D., Gambhir, S. S. 2009; 11 (3): 144-158
Abstract

Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging.An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA).The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity.This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events.

View details for DOI 10.1007/s11307-008-0187-7

View details for Web of Science ID 000265686900002

View details for PubMedID 19048345
A NEAR-INFRARED FLUORESCENT DEOXYGLUCOSE DERIVATIVE FOR OPTICAL IMAGING OF EXPERIMENTAL ARTHRITIS JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES Liu, X., Xiong, Z., Lee, S., Levi, J., Yaghoubi, S., Biswal, S., Gambhir, S. S., Cheng, Z. 2009; 2 (2): 179-187
View details for DOI 10.1142/S1793545809000450

View details for Web of Science ID 000208647200008
Human adipose tissue-derived mesenchymal stromal cells as vehicles for tumor bystander effect: a model based on bioluminescence imaging GENE THERAPY Vilalta, M., Degano, I. R., Bago, J., Aguilar, E., Gambhir, S. S., Rubio, N., Blanco, J. 2009; 16 (4): 547-557
Abstract

Human adipose tissue mesenchymal stromal cells (AMSCs) share common traits, including similar differentiation potential and cell surface markers, with their bone marrow counterparts. Owing to their general availability, higher abundance and ease of isolation AMSCs may be convenient autologous delivery vehicles for localized tumor therapy. We demonstrate a model for tumor therapy development based on the use of AMSCs expressing renilla luciferase and thymidine kinase, as cellular vehicles for ganciclovir-mediated bystander killing of firefly luciferase expressing tumors, and noninvasive bioluminescence imaging to continuously monitor both, tumor cells and AMSCs. We show that the therapy delivering AMSCs survive long time within tumors, optimize the ratio of AMSCs to tumor cells for therapy, and asses the therapeutic effect in real time. Treatment of mice bearing prostate tumors plus therapeutic AMSCs with the prodrug ganciclovir induced bystander killing effect, reducing the number of tumor cells to 1.5 % that of control tumors. Thus, AMSCs could be useful vehicles to deliver localized therapy, with potential for clinical application in inoperable tumors and surgical borders after tumor resection. This approach, useful to evaluate efficiency of therapeutic models, should facilitate the selection of cell types, dosages, therapeutic agents and treatment protocols for cell-based therapies of specific tumors.

View details for DOI 10.1038/gt.2008.176

View details for Web of Science ID 000265021400012

View details for PubMedID 19092860
Novel Strategy for a Cocktail F-18-Fluoride and F-18-FDG PET/CT Scan for Evaluation of Malignancy: Results of the Pilot-Phase Study JOURNAL OF NUCLEAR MEDICINE Iagaru, A., Mittra, E., Yaghoubi, S. S., Dick, D. W., Quon, A., Goris, M. L., Gambhir, S. S. 2009; 50 (4): 501-505
Abstract

(18)F-FDG PET/CT is used for detecting cancer and monitoring cancer response to therapy. However, because of the variable rates of glucose metabolism, not all cancers are identified reliably. Sodium (18)F was previously used for bone imaging and can be used as a PET/CT skeletal tracer. The combined administration of (18)F and (18)F-FDG in a single PET/CT study for cancer detection has not been reported to date.This is a prospective pilot study (November 2007-November 2008) of 14 patients with proven malignancy (6 sarcoma, 3 prostate cancer, 2 breast cancer, 1 colon cancer, 1 lung cancer, and 1 malignant paraganglioma) who underwent separate (18)F PET/CT and (18)F-FDG PET/CT and combined (18)F/(18)F-FDG PET/CT scans for the evaluation of malignancy (a total of 3 scans each). There were 11 men and 3 women (age range, 19-75 y; average, 50.4 y).Interpretation of the combined (18)F/(18)F-FDG PET/CT scans compared favorably with that of the (18)F-FDG PET/CT (no lesions missed) and the (18)F PET/CT scans (only 1 skull lesion seen on an (18)F PET/CT scan was missed on the corresponding combined scan). Through image processing, the combined (18)F/(18)F-FDG scan yielded results for bone radiotracer uptake comparable to those of the (18)F PET/CT scan performed separately.Our pilot-phase prospective trial demonstrates that the combined (18)F/(18)F-FDG administration followed by a single PET/CT scan is feasible for cancer detection. This combined method opens the possibility for improved patient care and reduction in health care costs.

View details for DOI 10.2967/jnumed.108.058339

View details for Web of Science ID 000272487200003

View details for PubMedID 19289439
Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects CANCER RESEARCH Kimura, R. H., Cheng, Z., Gambhir, S. S., Cochran, J. R. 2009; 69 (6): 2435-2442
Abstract

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC(50), approximately 20 nmol/L) (64)Cu-DOTA-conjugated knottin peptides was 4.47% +/- 1.21% and 4.56% +/- 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC(50), approximately 0.4 mumol/L; 1.48 +/- 0.53%ID/g) and c(RGDyK) (IC(50), approximately 1 mumol/L; 2.32 +/- 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, (64)Cu-DOTA-conjugated knottin peptides generated lower levels of nonspecific liver uptake ( approximately 2%ID/g) compared with c(RGDyK) ( approximately 4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. (64)Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.

View details for DOI 10.1158/0008-5472.CAN-08-2495

View details for Web of Science ID 000264541300037

View details for PubMedID 19276378
Stem Cell-Mediated Accelerated Bone Healing Observed with in Vivo Molecular and Small Animal Imaging Technologies in a Model of Skeletal Injury JOURNAL OF ORTHOPAEDIC RESEARCH Lee, S., Padmanabhan, P., Ray, P., Gambhir, S. S., Doyle, T., Contag, C., Goodman, S. B., Biswal, S. 2009; 27 (3): 295-302
Abstract

Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [(18)F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [(18)F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC's traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p < 0.05). Systemically administered ADMC's traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route.

View details for DOI 10.1002/jor.20736

View details for Web of Science ID 000263307200003

View details for PubMedID 18752273
A Novel Method for Direct Site-Specific Radiolabeling of Peptides Using [F-18]FDG BIOCONJUGATE CHEMISTRY Namavari, M., Cheng, Z., Zhang, R., De, A., Levi, J., Hoerner, J. K., Yaghoubi, S. S., Syud, F. A., Gambhir, S. S. 2009; 20 (3): 432-436
Abstract

We have used the well-accepted and easily available 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET) tracer as a prosthetic group for synthesis of (18)F-labeled peptides. We herein report the synthesis of [(18)F]FDG-RGD ((18)F labeled linear RGD) and [(18)F]FDG-cyclo(RGD(D)YK) ((18)F labeled cyclic RGD) as examples of the use of [(18)F]FDG. We have successfully prepared [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) in 27.5% and 41% radiochemical yields (decay corrected) respectively. The receptor binding affinity study of FDG-cyclo(RGD(D)YK) for integrin alpha(v)beta(3), using alpha(v)beta(3) positive U87MG cells confirmed a competitive displacement with (125)I-echistatin as a radioligand. The IC(50) value for FDG-cyclo(RGD(D)YK) was determined to be 0.67 +/- 0.19 muM. High-contrast small animal PET images with relatively moderate tumor uptake were observed for [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) as PET probes in xenograft models expressing alpha(v)beta(3) integrin. In conclusion, we have successfully used [(18)F]FDG as a prosthetic group to prepare (18)F]FDG-RGD and [(18)F]FDG-cyclic[RGD(D)YK] based on a simple one-step radiosynthesis. The one-step radiosynthesis methodology consists of chemoselective oxime formation between an aminooxy-functionalized peptide and [(18)F]FDG. The results have implications for radiolabeling of other macromolecules and would lead to a very simple strategy for routine preclinical and clinical use.

View details for DOI 10.1021/bc800422b

View details for Web of Science ID 000264389800005

View details for PubMedID 19226160
Development of a breast cancer brain metastases model to study I-131 radioablative therapy. Renier, C., De, A., Hou, L., Dunkel, J., Sun, A., Prugpichailers, T., Gambhir, S. S., Tse, V., Wapnir, I. L. AMER ASSOC CANCER RESEARCH. 2009: 159S-160S
View details for Web of Science ID 000262583200304
Protein-Functionalized Synthetic Antiferromagnetic Nanoparticles for Biomolecule Detection and Magnetic Manipulation ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Fu, A., Hu, W., Xu, L., Wilson, R. J., Yu, H., Osterfeld, S. J., Gambhir, S. S., Wang, S. X. 2009; 48 (9): 1620-1624
Abstract

Direct protein functionalization provides synthetic antiferromagnetic nanoparticles with high chemical specificity and multifunctionality. These nanoparticle-protein conjugates function as improved magnetic labels for biological detection experiments, and exhibit tunable responses to a small external magnetic field gradient, thus allowing the observation of distinctive single nanoparticle motion.

View details for DOI 10.1002/anie.200803994

View details for Web of Science ID 000263642300018

View details for PubMedID 19156803
Human adipose tissue-derived menesenchymal stromal cells as vehicles for tumor bystander effect: A model based on bioluminescence imaging Gene Therapy Vilalta M, Degano I, Bago J, Aguilar E, Gambhir SS, Rubio N, Blanco J 2009; 16 (4): 547-557
Controlling the selection stringency of phage display using a microfluidic device LAB ON A CHIP Liu, Y., Adams, J. D., Turner, K., Cochran, F. V., Gambhir, S. S., Soh, H. T. 2009; 9 (8): 1033-1036
Abstract

We report the utilization of microfluidic technology to phage selection and demonstrate that accurate control of washing stringency in our microfluidic magnetic separator (MMS) directly impacts the diversity of isolated peptide sequences. Reproducible generation of magnetic and fluidic forces allows controlled washing conditions that enable rapid convergence of selected peptide sequences. These findings may provide a foundation for the development of automated microsystems for rapid in vitro directed evolution of affinity reagents.

View details for DOI 10.1039/b820985e

View details for Web of Science ID 000264978800001

View details for PubMedID 19350081
Endogenous NIS expression in triple negative breast cancers Annals of Surgical Oncology Renier C, Yao C, Goris M, Ghosh M, Katznelson L, Nowles K, Gambhir SS, Wapnir I 2009; 16 (4): 962-968
Lymphoid tissue-specific homing of bone marrow-derived dendritic cells Blood Creusot R, Yaghoubi S, Chang P, Chia J, Contag C, Gambhir SS, Fathman C 2009; 113 (26): 6638-47
Effects of Creatine on Survival, Migration, and Differentiation of Neural Stem Cells Andres, R. H., PENDHARKAR, A. V., Guzman, R., De, A., Bliss, T. M., McMillan, E., Svendsen, C. N., Gambhir, S. S., Widmer, H. R., Wallimann, T., Steinberg, G. K. COGNIZANT COMMUNICATION CORP. 2009: 208-208
View details for Web of Science ID 000266340800014
Embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia. Journal of visualized experiments : JoVE Huang, N. F., Niiyama, H., De, A., Gambhir, S. S., Cooke, J. P. 2009
Abstract

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD. In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.

View details for DOI 10.3791/1034

View details for PubMedID 19229180
A novel high-sensitivity rapid acquisition single photon cardiac imaging camera Journal of Nuclear Medicine Gambhir SS, Berman D, Ziffer J, Nagler M, Sandler M, Patton J, Hutton B, Sharir T, Haim S 2009; 50 (4): 635-643
AUTOMATED RADIOSYNTHESIS OF [F-18]EF-5 FOR IMAGING HYPOXIA IN HUMAN Chin, F. T., Subbarayan, M., Sorger, J., Gambhir, S. S., Graves, E. E. WILEY-BLACKWELL. 2009: S274-S274
View details for Web of Science ID 000268724900275
An Engineered Knottin Peptide Labeled with [18F] for PET Imaging of Integrin Expression. Bioconjugate Chemistry Miao Z, Ren G, Liu H, Kimura R, Jiang L, Cochran J, Gambhir SS, Cheng Z. 2009; 20(12): 2342-2347
Noninvasive detection of therapeutic cytolytic T cells with F-18-FHBG PET in a patient with glioma NATURE CLINICAL PRACTICE ONCOLOGY Yaghoubi, S. S., Jensen, M. C., Satyamurthy, N., Budhiraja, S., Paik, D., Czernin, J., Gambhir, S. S. 2009; 6 (1): 53-58
Abstract

A 57-year-old man had been diagnosed with grade IV glioblastoma multiforme and was enrolled in a trial of adoptive cellular immunotherapy. The trial involved infusion of ex vivo expanded autologous cytolytic CD8+ T cells (CTLs), genetically engineered to express the interleukin 13 zetakine gene (which encodes a receptor protein that targets these T cells to tumor cells) and the herpes simplex virus 1 thymidine kinase (HSV1 tk) suicide gene, and PET imaging reporter gene.MRI, whole-body and brain PET scan with (18)F-radiolabelled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) to detect CTLs that express HSV1 tk, and safety monitoring after injection of (18)F-FHBG.MRI detected grade III-IV glioblastoma multiforme plus two tumors recurrences that developed after resection of the initial tumor.Surgical resection of primary glioblastoma tumor, enrollment in CTL therapy trial, reresection of glioma recurrences, infusion of approximately 1 x 10(9) CTLs into the site of tumor reresection, and (18)F-FHBG PET scan to detect infused CTLs.

View details for DOI 10.1038/ncponc1278

View details for Web of Science ID 000261845300011

View details for PubMedID 19015650
Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice SMALL Schipper, M. L., Iyer, G., Koh, A. L., Cheng, Z., Ebenstein, Y., Aharoni, A., Keren, S., Bentolila, L. A., Li, J., Rao, J., Chen, X., Banin, U., Wu, A. M., Sinclair, R., Weiss, S., Gambhir, S. S. 2009; 5 (1): 126-134
Abstract

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

View details for DOI 10.1002/smll.200800003

View details for Web of Science ID 000262895300019

View details for PubMedID 19051182
Trafficking Mesenchymal Stem Cell Engraftment and Differentiation in Tumor-Bearing Mice by Bioluminescence Imaging STEM CELLS Wang, H., Cao, F., De, A., Cao, Y., Contag, C., Gambhir, S. S., Wu, J. C., Chen, X. 2009; 27 (7): 1548-1558
Abstract

The objective of the study was to track the distribution and differentiation of mesenchymal stem cells (MSCs) in tumor-bearing mice. The 4T1 murine breast cancer cells were labeled with renilla luciferase-monomeric red fluorescence protein (rLuc-mRFP) reporter gene. The MSCs labeled with firefly luciferase-enhanced green fluorescence protein (fLuc-eGFP) reporter gene (MSCs-R) were isolated from L2G85 transgenic mice that constitutively express fLuc-eGFP reporter gene. To study the tumor tropism of MSCs, we established both subcutaneous and lung metastasis models. In lung metastasis tumor mice, we injected MSCs-R intravenously either on the same day or 4 days after 4T1 tumor cell injection. In subcutaneous tumor mice, we injected MSCs-R intravenously 7 days after subcutaneous 4T1 tumor inoculation. The tumor growth was monitored by rLuc bioluminescence imaging (BLI). The fate of MSCs-R was monitored by fLuc BLI. The localization of MSCs-R in tumors was examined histologically. The osteogenic and adipogenic differentiation of MSCs-R was investigated by alizarin red S and oil red O staining, respectively. The mechanism of the dissimilar differentiation potential of MSCs-R under different tumor microenvironments was investigated. We found that the 4T1 cells were successfully labeled with rLuc-mRFP. The MSCs-R isolated from L2G85 transgenic mice constitutively express fLuc-eGFP reporter gene. When injected intravenously, MSCs-R survived, proliferated, and differentiated in tumor sites but not elsewhere. The localization of GFP(+) MSCs-R in tumor lesions was confirmed ex vivo. In conclusion, the MSCs-R can selectively localize, survive, and proliferate in both subcutaneous tumor and lung metastasis as evidenced by noninvasive bioluminescence imaging and ex vivo validation. The MSCs-R migrated to lung tumor differentiated into osteoblasts, whereas the MSCs-R targeting subcutaneous tumor differentiated into adipocytes.

View details for DOI 10.1002/stem.81

View details for Web of Science ID 000268257100011

View details for PubMedID 19544460
Multimodality Molecular Imaging of Transplanted Neural Progenitor Cells PENDHARKAR, A. V., De, A., Wang, H., Gaeta, X., Wang, N., Andres, R. H., Chen, X., Gambhir, S. S., Guzman, R. COGNIZANT COMMUNICATION CORP. 2009: 230-231
View details for Web of Science ID 000266340800077
F-18-FDG PET/CT evaluation of patients with ovarian carcinoma NUCLEAR MEDICINE COMMUNICATIONS Iagaru, A. H., Mittra, E. S., McDougall, I. R., Quon, A., Gambhir, S. S. 2008; 29 (12): 1046-1051
Abstract

The role of F-FDG PET has been studied in ovarian carcinoma, but its sensitivity and specificity calculations are based on dedicated PET acquisition, not PET/CT in the majority of the published studies. Therefore, we were prompted to review our experience with PET/CT in the management of patients with ovarian carcinoma.This is a retrospective study of 43 women with ovarian carcinoma, 27-80 years old (average: 53.9+/-7.8), who had whole-body PET/CT at our institution from 1 January 2003 to 31 August 2006. We reviewed the patients' outcomes from medical records and compared them to the interpretation of the PET/CT scans. Sensitivity and specificity were calculated using a 2 x 2 table with pathology results (79.1% of the patients) or clinical follow-up (20.9% of the cases) as the 'gold standard'. Confidence interval (CI) estimations were performed using the Wilson score method.All patients had advanced stage ovarian cancer and the study was requested for re-staging. A total of 60 scans were performed: 30 patients had one scan, nine patients had two scans and four patients had three scans. The administered doses of F-FDG ranged from 381.1 to 769.6 MBq (average: 569.8+/-73.3). PET/CT had a sensitivity of 88.4% (95% CI: 75.1-95.4) and a specificity of 88.2% (95% CI: 64.4-97.9) for detection of ovarian cancer. The SUV max of the detected lesions ranged from 3 to 27 (average: 9.4+/-5.9). The CA-125 tumor marker ranged from 3 to 935 kU/ml (average: 265.2) in patients with positive scans and 4-139 kU/ml (average: 17.1) in patients with negative scans. This difference was statistically significant (P value: 0.0242).This study confirms the good results of F-FDG PET/CT for identification of residual/recurrent ovarian cancer, as well as for distant metastases localization. PET/CT should be an integral part in evaluation of patients with high-risk ovarian cancer or rising values of tumor markers (CA-125), prior to selection of the most appropriate therapy.

View details for DOI 10.1097/MNM.0b013e32831089cb

View details for Web of Science ID 000261164200004

View details for PubMedID 18987524
Uptake kinetics and biodistribution of C-14-D-luciferin-a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: impact on bioluminescence based reporter gene imaging EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Berger, F., Paulmurugan, R., Bhaumik, S., Gambhir, S. S. 2008; 35 (12): 2275-2285
Abstract

Firefly luciferase catalyzes the oxidative decarboxylation of D: -luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated.Benzene ring (14)C(U)-labeled D-luciferin was utilized. Cell uptake and efflux assays, murine biodistribution, autoradiography and CCD-camera based optical bioluminescence imaging were carried out to examine the in vitro and in vivo characteristics of the tracer in cell culture and in living mice respectively.Radiolabeled and unlabeled D-luciferin revealed comparable levels of light emission when incubated with equivalent amounts of the firefly luciferase enzyme. Cell uptake assays in pCMV-luciferase-transfected cells showed slow trapping of the tracer and relatively low uptake values (up to 22.9-fold higher in firefly luciferase gene-transfected vs. nontransfected cells, p = 0.0002). Biodistribution studies in living mice after tail-vein injection of (14)C-D-luciferin demonstrated inhomogeneous tracer distribution with early predominant high radioactivity levels in kidneys (10.6% injected dose [ID]/g) and liver (11.9% ID/g), followed at later time points by the bladder (up to 81.3% ID/g) and small intestine (6.5% ID/g), reflecting the elimination routes of the tracer. Kinetics and uptake levels profoundly differed when using alternate injection routes (intravenous versus intraperitoneal). No clear trapping of (14)C-D-luciferin in firefly luciferase-expressing tissues could be observed in vivo.The data obtained with (14)C-D-luciferin provide insights into the dynamics of D: -luciferin cell uptake, intracellular accumulation, and efflux. Results of the biodistribution and autoradiographic studies should be useful for optimizing and adapting optical imaging protocols to specific experimental settings when utilizing the firefly luciferase and D: -luciferin system.

View details for DOI 10.1007/s00259-008-0870-6

View details for Web of Science ID 000261654000015

View details for PubMedID 18661130
Noninvasive Imaging of Therapeutic Gene Expression Using a Bidirectional Transcriptional Amplification Strategy MOLECULAR THERAPY Ray, S., Paulmurugan, R., Patel, M. R., Ahn, B. C., Wu, L., Carey, M., Gambhir, S. S. 2008; 16 (11): 1848-1856
Abstract

Promoters that limit transgene expression to tumors play a vital role in cancer gene therapy. Although tumor specific, the human Survivin promoter (pSurv) elicits low levels of transcription. A bidirectional two-step transcriptional amplification (TSTA) system was designed to enhance expression of the therapeutic gene (TG) tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL or TR) and the reporter gene firefly luciferase (FL) from pSurv. An adenoviral vector carrying the enhanced targeting apparatus (Ad-pSurv-TR-G8-FL) was tested for efficiency and specificity of gene expression in cells and in living animals. Compared to the one-step systems (Ad-pSurv-FL or Ad-pSurv-TR), the bidirectional TSTA system showed tenfold higher expression of both the therapeutic and the reporter gene and their expression correlated in cells (R(2) = 0.99) and in animals (R(2) = 0.67). Noninvasive quantitative monitoring of magnitude and time variation of TRAIL gene expression was feasible by bioluminescence imaging of the transcriptionally linked FL gene in xenograft tumors following intratumoral adenoviral injection. Moreover, the TSTA adenovirus maintained promoter specificity in nontarget tissues following tail vein administration. These studies demonstrate the potential of the bidirectional TSTA system to achieve high levels of gene expression from a weak promoter, while preserving specificity and the ability to image expression of the TG noninvasively.

View details for DOI 10.1038/mt.2008.180

View details for Web of Science ID 000260472600015

View details for PubMedID 18766175
Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes PLOS ONE Cao, F., Wagner, R. A., Wilson, K. D., Xie, X., Fu, J., Drukker, M., Lee, A., Li, R. A., Gambhir, S. S., Weissman, I. L., Robbins, R. C., Wu, J. C. 2008; 3 (10)
Abstract

Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

View details for DOI 10.1371/journal.pone.0003474

View details for Web of Science ID 000265126100005

View details for PubMedID 18941512
Noninvasive Raman spectroscopy in living mice for evaluation of tumor targeting with carbon nanotubes NANO LETTERS Zavaleta, C., de la Zerda, A., Liu, Z., Keren, S., Cheng, Z., Schipper, M., Chen, X., Dai, H., Gambhir, S. S. 2008; 8 (9): 2800-2805
Abstract

An optimized noninvasive Raman microscope was used to evaluate tumor targeting and localization of single walled carbon nanotubes (SWNTs) in mice. Raman images were acquired in two groups of tumor-bearing mice. The control group received plain-SWNTs, whereas the experimental group received tumor targeting RGD-SWNTs intravenously. Raman imaging commenced over the next 72 h and revealed increased accumulation of RGD-SWNTs in tumor ( p < 0.05) as opposed to plain-SWNTs. These results support the development of a new preclinical Raman imager.

View details for DOI 10.1021/nl801362a

View details for Web of Science ID 000259140200034

View details for PubMedID 18683988
F-18-FDG-PET/CT evaluation of response to treatment in lymphoma: when is the optimal time for the first re-evaluation scan? HELLENIC JOURNAL OF NUCLEAR MEDICINE Iagaru, A., Wang, Y., Mari, C., Quon, A., Goris, M. L., Horning, S., Gambhir, S. S. 2008; 11 (3): 153-156
Abstract

Assessing the response to treatment as soon after treatment initiation is one of the key reasons for imaging lymphoma patients. The optimal time after initiating treatment for assessing response to treatment has yet to be determined. Therefore, we were prompted to review our experience with serial (18)F-FDG PET/CT in patients undergoing treatment for Hodgkin's disease (HD) and non Hodgkin's lymphoma (NHL). This is a retrospective study (Feb 2003 - Oct 2004) of 20 patients, 11 men and 9 women, with age range of 7-75 years with diagnosis of HD (10) and NHL (10), who had PET/CT at our institution prior, during and at the completion of therapy. Restaging PET/CT was done after 2 cycles of chemotherapy in 10 patients (group A) and after 4 cycles of chemotherapy in 10 pts (group B). A total of 60 scans were reviewed. The DeltaSUV from baseline to first PET/CT was on average 67.6% in group A and 75.1% in group B. This had no statistical significance (P value: 0.31). The DeltaSUV from baseline to post-therapy PET/CT was on average 72.9% in group A and 79.8% in group B. This difference also had no statistical significance (P value: 0.24). The correlation coefficient was 0.98 in group A and 0.80 in group B. Results of PET/CT after 2 cycles of chemotherapy did not statistically differ from the results of PET/CT after 4 cycles of chemotherapy. These results need to be confirmed in larger, prospective, randomized trials.

View details for Web of Science ID 000262093600003

View details for PubMedID 19081857
Carbon nanotubes as photoacoustic molecular imaging agents in living mice NATURE NANOTECHNOLOGY de la Zerda, A., Zavaleta, C., Keren, S., Vaithilingam, S., Bodapati, S., Liu, Z., Levi, J., Smith, B. R., Ma, T., Oralkan, O., Cheng, Z., Chen, X., Dai, H., Khuri-Yakub, B. T., Gambhir, S. S. 2008; 3 (9): 557-562
Abstract

Photoacoustic imaging of living subjects offers higher spatial resolution and allows deeper tissues to be imaged compared with most optical imaging techniques. As many diseases do not exhibit a natural photoacoustic contrast, especially in their early stages, it is necessary to administer a photoacoustic contrast agent. A number of contrast agents for photoacoustic imaging have been suggested previously, but most were not shown to target a diseased site in living subjects. Here we show that single-walled carbon nanotubes conjugated with cyclic Arg-Gly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumours. Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes. These results were verified ex vivo using Raman microscopy. Photoacoustic imaging of targeted single-walled carbon nanotubes may contribute to non-invasive cancer imaging and monitoring of nanotherapeutics in living subjects.

View details for DOI 10.1038/nnano.2008.231

View details for Web of Science ID 000259013100014

View details for PubMedID 18772918
Cancer screening: A mathematical model relating secreted blood biomarker levels to tumor sizes PLOS MEDICINE Lutz, A. M., Willmann, J. K., Cochran, F. V., Ray, P., Gambhir, S. S. 2008; 5 (8): 1287-1297
Abstract

Increasing efforts and financial resources are being invested in early cancer detection research. Blood assays detecting tumor biomarkers promise noninvasive and financially reasonable screening for early cancer with high potential of positive impact on patients' survival and quality of life. For novel tumor biomarkers, the actual tumor detection limits are usually unknown and there have been no studies exploring the tumor burden detection limits of blood tumor biomarkers using mathematical models. Therefore, the purpose of this study was to develop a mathematical model relating blood biomarker levels to tumor burden.Using a linear one-compartment model, the steady state between tumor biomarker secretion into and removal out of the intravascular space was calculated. Two conditions were assumed: (1) the compartment (plasma) is well-mixed and kinetically homogenous; (2) the tumor biomarker consists of a protein that is secreted by tumor cells into the extracellular fluid compartment, and a certain percentage of the secreted protein enters the intravascular space at a continuous rate. The model was applied to two pathophysiologic conditions: tumor biomarker is secreted (1) exclusively by the tumor cells or (2) by both tumor cells and healthy normal cells. To test the model, a sensitivity analysis was performed assuming variable conditions of the model parameters. The model parameters were primed on the basis of literature data for two established and well-studied tumor biomarkers (CA125 and prostate-specific antigen [PSA]). Assuming biomarker secretion by tumor cells only and 10% of the secreted tumor biomarker reaching the plasma, the calculated minimally detectable tumor sizes ranged between 0.11 mm(3) and 3,610.14 mm(3) for CA125 and between 0.21 mm(3) and 131.51 mm(3) for PSA. When biomarker secretion by healthy cells and tumor cells was assumed, the calculated tumor sizes leading to positive test results ranged between 116.7 mm(3) and 1.52 x 10(6) mm(3) for CA125 and between 27 mm(3) and 3.45 x 10(5) mm(3) for PSA. One of the limitations of the study is the absence of quantitative data available in the literature on the secreted tumor biomarker amount per cancer cell in intact whole body animal tumor models or in cancer patients. Additionally, the fraction of secreted tumor biomarkers actually reaching the plasma is unknown. Therefore, we used data from published cell culture experiments to estimate tumor cell biomarker secretion rates and assumed a wide range of secretion rates to account for their potential changes due to field effects of the tumor environment.This study introduced a linear one-compartment mathematical model that allows estimation of minimal detectable tumor sizes based on blood tumor biomarker assays. Assuming physiological data on CA125 and PSA from the literature, the model predicted detection limits of tumors that were in qualitative agreement with the actual clinical performance of both biomarkers. The model may be helpful in future estimation of minimal detectable tumor sizes for novel proteomic biomarker assays if sufficient physiologic data for the biomarker are available. The model may address the potential and limitations of tumor biomarkers, help prioritize biomarkers, and guide investments into early cancer detection research efforts.

View details for DOI 10.1371/journal.pmed.0050170

View details for Web of Science ID 000258739200018

View details for PubMedID 18715113
A human estrogen receptor (ER)alpha mutation with differential responsiveness to nonsteroidal ligands: Novel approaches for studying mechanism of ER action MOLECULAR ENDOCRINOLOGY Paulmurugan, R., Tamrazi, A., Katzenellenbogen, J. A., Katzenellenbogen, B. S., Gambhir, S. S. 2008; 22 (7): 1552-1564
Abstract

Estrogens, acting through the estrogen receptors (ERs), play crucial roles in regulating the function of reproductive and other systems under physiological and pathological conditions. ER activity in regulating target genes is modulated by the binding of both steroidal and synthetic nonsteroidal ligands, with ligand binding inducing ERs to adopt various conformations that control their interactions with transcriptional coregulators. Previously, we developed an intramolecular folding sensor with a mutant form of ERalpha (ER(G521T)) that proved to be essentially unresponsive to the endogenous ligand 17beta-estradiol, yet responded very well to certain synthetic ligands. In this study, we have characterized this G521T-ER mutation in terms of the potency and efficacy of receptor response toward several steroidal and nonsteroidal ligands in two different ways: directly, by ligand effects on mutant ER conformation (by the split-luciferase complementation system), and indirectly, by ligand effects on mutant ER transactivation. Full-length G521T-ER shows no affinity for estradiol and does not activate an estrogen-responsive reporter gene. The synthetic pyrazole agonist ligand propyl-pyrazole-triol is approximately 100-fold more potent than estradiol in inducing intramolecular folding and reporter gene transactivation with the mutant ER, whereas both ligands have high potency on wild-type ER. This estradiol-unresponsive mutant ER can also specifically highlight the agonistic property of the selective ER modulator, 4-hydroxytamoxifen, by reporter gene transactivation, even in the presence of estradiol, and it can exert a dominant-negative effect on estrogen-stimulated wild-type ER. This system provides a model for ER-mutants that show differential ligand responsiveness to gene activation to gain insight into the phenomenon of hormone resistance observed in endocrine therapies of ER-positive breast cancers.

View details for DOI 10.1210/me.2007-0570

View details for Web of Science ID 000257144500004

View details for PubMedID 18451095
Direct site-specific radiolabeling of an affibody protein with 4-[F-18]fluorobenzaldehyde via oxime chemistry MOLECULAR IMAGING AND BIOLOGY Namavari, M., De Jesus, O. P., Cheng, Z., De, A., Kovacs, E., Levi, J., Zhang, R., Hoerner, J. K., Grade, H., Syud, F. A., Gambhir, S. S. 2008; 10 (4): 177-181
Abstract

In this study, we introduce a methodology for preparing 18F-labeled Affibody protein, specifically 18F-Anti-HER2 dimeric Affibody (14 kDa), for in vivo imaging of HER2neu with positron emission tomography (PET).We have used 4-[18F]fluorobenzaldehyde as a synthon to prepare 18F-Anti-HER2 Affibody. Aminooxy-functionalized Affibody (Anti-HER2-ONH2) was incubated with 4-[18F]fluorobenzaldehyde in ammonium acetate buffer at pH 4 in the presence of methanol at 70 degrees C for 15 min. The resulting 18F-labeled Affibody molecule was evaluated as a PET probe in xenograft models expressing HER2.We have successfully prepared 18F-Anti-HER2 dimeric Affibody (14 kDa), N-(4-[18F]fluorobenzylidine)oxime-Anti-HER2 Affibody, [18F]FBO-Anti-HER2, in 26-30% radiochemical yields (decay corrected). High-contrast small-animal PET images with relatively moderate tumor uptake (1.79 +/- 0.40% ID/g) were observed for the 18F-Anti-HER2 Affibody.Site-specific 18F-labeled Affibody against HER2 has been synthesized via chemoselective oxime formation between an aminooxy-functionalized Affibody and 18F-fluorobenzaldehyde. The results have implications for radiolabeling of other affibodies and macromolecules and should also be important for advancing Affibody imaging with PET.

View details for DOI 10.1007/s11307-008-0142-7

View details for Web of Science ID 000256929100001
A generalizable strategy for imaging pre-mRNA levels in living subjects using spliceosome-mediated RNA trans-splicing JOURNAL OF NUCLEAR MEDICINE Walls, Z. F., Puttaraju, M., Temple, G. F., Gambhir, S. S. 2008; 49 (7): 1146-1154
Abstract

Molecular imaging of gene expression is currently hindered by the lack of a generalizable platform for probe design. For any gene of interest, a probe that targets protein levels must often be generated empirically. Targeting gene expression at the level of mRNA, however, would allow probes to be built on the basis of sequence information alone. Presented here is a class of generalizable probes that can image pre-mRNA in a sequence-specific manner, using signal amplification and a facile method of delivery.Pre-trans-splicing molecules (PTMs) were engineered to capitalize on the phenomenon of spliceosome-mediated RNA trans-splicing. Using a modular binding domain that confers specificity by base-pair complementarity to the target pre-mRNA, PTMs were designed to target a chimeric target mini gene and trans-splice the Renilla luciferase gene onto the end of the target. PTMs and target genes were transfected in cell culture and assessed by luciferase assay, reverse-transcriptase polymerase chain reaction, Western blot, and rapid analysis of 5' cDNA ends. PTMs and target genes were also assessed in vivo by hydrodynamic delivery in mice.Efficiency and specificity of the trans-splicing reaction were found to vary depending on the binding domain length and structure. Specific trans-splicing was observed in living animals (P = 0.0862, Kruskal-Wallis test).Described here is a model system used to demonstrate the feasibility of spliceosome-mediated RNA trans-splicing for imaging gene expression at the level of pre-mRNA using optical imaging techniques in living animals. The experiments reported here show proof of principle for a generalizable imaging probe against RNA that can amplify signal on detection and be delivered using existing gene delivery methodology.

View details for DOI 10.2967/jnumed.107.047662

View details for Web of Science ID 000257599700021

View details for PubMedID 18552150
Molecular imaging of cancer: From molecules to humans - Introduction JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S. 2008; 49: 1S-4S
View details for DOI 10.2967/jnumed.108.053751

View details for Web of Science ID 000256491100001

View details for PubMedID 18523062
Molecular Imaging of Hypoxia-Inducible Factor 1 alpha and von Hippel-Lindau Interaction in Mice MOLECULAR IMAGING Choi, C. Y., Chau, D. A., Paulmurugan, R., Sutphin, P. D., Le, Q., Koong, A. C., Zundel, W., Gambhir, S. S., Giaccia, A. J. 2008; 7 (3): 139-146
Abstract

Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1 alpha is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1 alpha and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1 alpha and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1 alpha and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1 alpha are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.

View details for DOI 10.2310/7290.2008.00017

View details for Web of Science ID 000260954700004

View details for PubMedID 19123984
Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase METABOLIC ENGINEERING Goerke, A. R., Loening, A. M., Gambhir, S. S., Swartz, J. R. 2008; 10 (3-4): 187-200
Abstract

Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412mug/mL of purified GLuc, relative to 5mug/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2x10(24)photons/s/mol, the highest reported activity for any characterized luciferase.

View details for DOI 10.1016/j.ymben.2008.04.001

View details for Web of Science ID 000261595600006

View details for PubMedID 18555198
Imaging of VEGF receptor in a rat myocardial infarction model using PET JOURNAL OF NUCLEAR MEDICINE Rodriguez-Porcel, M., Cai, W., Gheysens, O., Willmann, J. K., Chen, K., Wang, H., Chen, I. Y., He, L., Wu, J. C., Li, Z., Mohamedali, K. A., Kim, S., Rosenblum, M. G., Chen, X., Gambhir, S. S. 2008; 49 (4): 667-673
Abstract

Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factor's receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject.In Sprague-Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3).Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 +/- 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 +/- 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels.We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.

View details for DOI 10.2967/jnumed.107.040576

View details for Web of Science ID 000254813600028

View details for PubMedID 18375924
Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging GENE THERAPY Sato, M., Figueiredo, M. L., Burton, J. B., Johnson, M., Chen, M., Powell, R., Gambhir, S. S., Carey, M., Wu, L. 2008; 15 (8): 583-593
Abstract

Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer.

View details for DOI 10.1038/gt.2008.19

View details for Web of Science ID 000254561200004

View details for PubMedID 18305574
Initial evaluation of F-18-fluorothymidine (FLT) PET/CT scanning for primary pancreatic cancer EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Quon, A., Chang, S. T., Chin, F., Kamaya, A., Dick, D. W., Loo, B. W., Gambhir, S. S., Koong, A. C. 2008; 35 (3): 527-531
Abstract

The aim of this study was to evaluate the potential of (18)F-fluorothymidine (FLT) PET/CT for imaging pancreatic adenocarcinoma.This was a pilot study of five patients (four males, one female) with newly diagnosed and previously untreated pancreatic adenocarcinoma. Patients underwent FLT PET/CT, (18)F-fluorodeoxyglucose (FDG) PET/CT, and contrast-enhanced CT scanning before treatment. The presence of cancer was confirmed by histopathological analysis at the time of scanning in all five patients. The degree of FLT and FDG uptake at the primary tumor site was assessed using visual interpretation and semi-quantitative SUV analyses.The primary tumor size ranged from 2.5 x 2.8 cm to 3.5 x 7.0 cm. The SUV of FLT uptake within the primary tumor ranged from 2.1 to 3.1. Using visual interpretation, the primary cancer could be detected from background activity in two of five patients (40%) on FLT PET/CT. By comparison, FDG uptake was higher in each patient with a SUV range of 3.4 to 10.8, and the primary cancer could be detected from background in all five patients (100%).In this pilot study of five patients with primary pancreatic adenocarcinoma, FLT PET/CT scanning showed poor lesion detectability and relatively low levels of radiotracer uptake in the primary tumor.

View details for DOI 10.1007/s00259-007-0630-z

View details for Web of Science ID 000254402800010

View details for PubMedID 17960376
Perspectives of molecular imaging and radioimmunotherapy in lymphoma RADIOLOGIC CLINICS OF NORTH AMERICA Iagaru, A., Goris, M. L., Gambhir, S. S. 2008; 46 (2): 243-252
Abstract

Successful treatment of Hodgkin lymphomas and non-Hodgkin lymphomas depends on accurate staging and prognostic estimations, as well as evaluation of response to therapy as early after initiation as possible. We focus on several aspects of molecular imaging and therapy that affect the management of patients who have lymphoma. First, we review prior use of gallium-67 citrate for evaluation of lymphoma patients, mainly from a historical perspective, since it was the mainstream lymphoma functional imaging tracer for decades. Next, we review current clinical uses of 18F Fluoro-2-Deoxyglucose (18F FDG) PET and PET/CT for evaluation of lymphoma patients and use of radioimmunotherapy in lymphoma. Finally, we discuss advances in molecular imaging that may herald the next generation of PET radiotracers after 18F FDG.

View details for DOI 10.1016/j.rcl.2008.03.007

View details for Web of Science ID 000258543500006

View details for PubMedID 18619379
Noninvasive molecular neuroimaging using reporter genes: Part II, experimental, current, and future applications AMERICAN JOURNAL OF NEURORADIOLOGY Massoud, T. F., Singh, A., Gambhir, S. S. 2008; 29 (3): 409-418
Abstract

In this second article, we review the various strategies and applications that make use of reporter genes for molecular imaging of the brain in living subjects. These approaches are emerging as valuable tools for monitoring gene expression in diverse applications in laboratory animals, including the study of gene-targeted and trafficking cells, gene therapies, transgenic animals, and more complex molecular interactions within the central nervous system. Further development of more sensitive and selective reporters, combined with improvements in detection technology, will consolidate the position of in vivo reporter gene imaging as a versatile technique for greater understanding of intracellular biologic processes and underlying molecular neuropathology and will potentially establish a future role in the clinical management of patients with neurologic diseases.

View details for DOI 10.3174/ajnr.A0863

View details for Web of Science ID 000254066700002

View details for PubMedID 18272565
Monitoring of the biological response to murine Hindlimb ischemia with Cu-64-labeled vascular endothelial growth factor-121 positron emission tomography CIRCULATION Willmann, J. K., Chen, K., Wang, H., Paulmurugan, R., Rollins, M., Cai, W., Wang, D. S., Chen, I. Y., Gheysens, O., Rodriguez-Porcel, M., Chen, X., Gambhir, S. S. 2008; 117 (7): 915-922
Abstract

Vascular endothelial growth factor-121 (VEGF121), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF121 positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training.64Cu-labeled VEGF121 and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF121 positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem gamma-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF121 showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF121 uptake in ischemic hindlimbs increased significantly (P < 0.001) from a control level of 0.61+/-0.17% ID/g (percentage of injected dose per gram) to 1.62+/-0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59+/-0.14% ID/g at day 29), and correlated with gamma-counting (R2 = 0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P < or = 0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2 = 0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice.64Cu-VEGF121 positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.

View details for DOI 10.1161/CIRCULATIONAHA.107.733220

View details for Web of Science ID 000253428100008

View details for PubMedID 18250264
Reporter gene imaging following percutaneous delivery in swine - Moving toward clinical applications JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Rodriguez-Porcel, M., Brinton, T. J., Chen, I. Y., Gheysens, O., Lyons, J., Ikeno, F., Willmann, J. K., Wu, L., Wu, J. C., Yeung, A. C., Yock, P., Gambhir, S. S. 2008; 51 (5): 595-597
View details for DOI 10.1016/j.jacc.2007.08.063

View details for Web of Science ID 000252908600013

View details for PubMedID 18237691
Noninvasive molecular neuroimaging using reporter genes: Part I, principles revisited AMERICAN JOURNAL OF NEURORADIOLOGY Massoud, T. F., Singh, A., Gambhir, S. S. 2008; 29 (2): 229-234
Abstract

In this first article, we review the basic principles of using reporter genes for molecular imaging of the brain in living subjects. This approach is emerging as a valuable tool for monitoring gene expression in diverse applications in laboratory animals, including the study of gene-targeted and trafficking cells, gene therapies, transgenic animals, and more complex molecular interactions within the central nervous system. Further development of more sensitive and selective reporters, combined with improvements in detection technology, will consolidate the position of in vivo reporter gene imaging as a versatile method for greater understanding of intracellular biologic processes and underlying molecular neuropathology and will potentially establish a future role in the clinical management of patients with neurologic diseases.

View details for DOI 10.3174/ajnr.A0864

View details for Web of Science ID 000253345200011

View details for PubMedID 18024575
US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice RADIOLOGY Willmann, J. K., Paulmurugan, R., Chen, K., Gheysens, O., Rodriguez-Porcel, M., Lutz, A. M., Chen, I. Y., Chen, X., Gambhir, S. S. 2008; 246 (2): 508-518
Abstract

To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance.In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types.US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.

View details for DOI 10.1148/radio1.2462070536

View details for Web of Science ID 000252796300021

View details for PubMedID 18180339
90Y Ibritumomal therapy in refractory non-Hodgkins lymphoma: Observations from 111 in-ibritumomab pre-treatment imaging Journal of Nuclear Medicine Iagaru A, Gambhir SS, Goris M 2008; 49 (11): 1809-1812
Molecular Imaging of Cancer: From Molecules to Humans. Introduction. Molecular Imaging of Cancer: From Molecules to Humans. Introduction. Gambhir, S. 2008; 49: 1S-4S
Use of Bioluminescent Imaging to Assay the Transplantation of Immortalized Human Fetal Hepatocytes Into Mice CELL TRANSPLANTATION Choi, M. S., Catana, A. M., Wu, J., Kim, Y. S., Yoon, S. J., Borowsky, A. D., Gambhir, S. S., Gupta, S., Zern, M. A. 2008; 17 (8): 899-909
Abstract

Noninvasive serial monitoring of the fate of transplanted cells would be invaluable to evaluate the potential therapeutic use of human hepatocyte transplantation. Therefore, we assessed the feasibility of bioluminescent imaging using double or triple fusion lentiviral vectors in a NOD-SCID mouse model transplanted with immortalized human fetal hepatocytes. Lentiviral vectors driven by the CMV promoter were constructed carrying reporter genes: firefly luciferase and green fluorescence protein with or without herpes simplex virus type 1 thymidine kinase. Human fetal hepatocytes immortalized by telomerase reconstitution (FH-hTERT) were successfully transduced with either of these fusion vectors. Two million stably transduced cells selected by fluorescence-activated cell sorting were injected into the spleens of NOD-SCID mice pretreated with methylcholanthrene and monocrotaline. The transplanted mice were serially imaged with a bioluminescence charged-coupled device camera after D-luciferin injection. Bioluminescence signal intensity was highest on day 3 (6.10 +/- 2.02 x 10(5) p/s/cm2/sr, mean +/- SEM), but decreased to 2.26 +/- 1.54 x 10(5) and 7.47 +/- 3.09 x 10(4) p/s/cm2/sr on day 7 and 10, respectively (p = 0.001). ELISA for human albumin in mice sera showed that levels were similar to those of control mice on day 2 (3.25 +/- 0.92 vs. 2.84 +/- 0.59 ng/ml, mean +/- SEM), peaked at 18.04 +/- 3.11 ng/ml on day 7, and decreased to 8.93 +/- 1.40 and 3.54 +/- 0.87 ng/ml on day 14 and 21, respectively (p = 0.02). Real-time quantitative RT-PCR showed gene expression levels of human albumin, alpha1-antitrypsin, and transferrin in mouse liver were 60.7 +/- 6.5%, 26.0 +/- 1.4%, and 156.8 +/- 62.4% of those of primary human adult hepatocytes, respectively, and immunohistochemistry revealed cells with human albumin and alpha1-antitrypsin expression in the mouse liver. In conclusion, our study demonstrated that bioluminescent imaging appears to be a sensitive, noninvasive modality for serial monitoring of transplanted hepatic stem cells.

View details for Web of Science ID 000261489600003

View details for PubMedID 19069633
Molecular imaging of PET reporter gene expression. Handbook of experimental pharmacology Min, J., Gambhir, S. S. 2008: 277-303
Abstract

Multimodality molecular imaging continues to rapidly expand and is impacting many areas of biomedical research as well as patient management. Reporter-gene assays have emerged as a very general strategy for indirectly monitoring various intracellular events. Furthermore, reporter genes are being used to monitor gene/cell therapies, including the location(s), time variation, and magnitude of gene expression. This chapter reviews reporter gene technology and its major pre-clinical and clinical applications to date. The future appears quite promising for the continued expansion of the use of reporter genes in many evolving biomedically related arenas.

View details for DOI 10.1007/978-3-540-77496-9_12

View details for PubMedID 18626607
Dual targeted contrast agent for US assessment of tumor angiogenesis in vivo Radiology Willmann J, Lutz A, Paulmurugan R, Patel M, Chu P, Rosenberg J, Gambhir SS 2008; 248 (3): 936-944
Preclinical efficacy of the c-met inhibitor CE-355621 in a U87 MG mouse xenograft model evaluated by F-18-FDG small-animal PET JOURNAL OF NUCLEAR MEDICINE Tseng, J. R., Kang, K. W., Dandekar, M., Yaghoubi, S., Lee, J. H., Christensen, J. G., Muir, S., Vincent, P. W., Michaud, N. R., Gambhir, S. S. 2008; 49 (1): 129-134
Abstract

The purpose of this study was to evaluate the efficacy of CE-355621, a novel antibody against c-Met, in a subcutaneous U87 MG xenograft mouse model using (18)F-FDG small-animal PET.CE-355621 or control vehicle was administered intraperitoneally into nude mice (drug-treated group, n = 12; control group, n = 14) with U87 MG subcutaneous tumor xenografts. Drug efficacy was evaluated over 2 wk using (18)F-FDG small-animal PET and compared with tumor volume growth curves.The maximum %ID/g (percentage injected dose per gram of tissue) of (18)F-FDG accumulation in mice treated with CE-355621 remained essentially unchanged over 2 wk, whereas the %ID/g of the control tumors increased 66% compared with the baseline. Significant inhibition of (18)F-FDG accumulation was seen 3 d after drug treatment, which was earlier than the inhibition of tumor volume growth seen at 7 d after drug treatment.CE-355621 is an efficacious novel antineoplastic chemotherapeutic agent that inhibits (18)F-FDG accumulation earlier than tumor volume changes in a mouse xenograft model. These results support the use of (18)F-FDG PET to assess early tumor response for CE-355621.

View details for DOI 10.2967/jnumed.106.038836

View details for Web of Science ID 000252391700026

View details for PubMedID 18077531
Molecular Imaging of Reporter Gene Expression in Prostate Cancer: An Overview. Seminars in Nuclear Medicine Singh A, Massoud T, Deroose C, Gambhir SS 2008; 39(1): 9-19
I-123 MIBG mapping with intraoperative gamma probe for recurrent neuroblastoma MOLECULAR IMAGING AND BIOLOGY Iagaru, A., Peterson, D., Quon, A., Dutta, S., Twist, C., Daghighian, F., Gambhir, S. S., Albanese, C. 2008; 10 (1): 19-23
Abstract

Intraoperative gamma probe guidance has become widely utilized for sentinel lymph node dissection in patients with breast cancer and melanoma, using (99m)Tc sulfur colloid. However, new indications are possible and need to continue to be investigated. We report the use during a wedge liver biopsy of a new hand-held gamma probe designed for (123)I intraoperative guidance. The patient studied is a 5-year-old boy with history of stage 4 high-risk neuroblastoma. Anatomic imaging (CT, MRI), (99m)Tc bone scintigraphy and 2-deoxy-2-[F-18]fluoro-d-glucose-positron emission tomography/computed tomography (FDG-PET/CT) were negative, but the (123)I MIBG scintigraphy suggested recurrent liver disease. A decision was made to biopsy these lesions to obtain histopathological confirmation. Intraoperative gamma probe mapping of the liver identified areas with signal above the background, but these were prove to be hemosiderin deposits on histo-pathology examination.

View details for DOI 10.1007/s11307-007-0116-1

View details for Web of Science ID 000252107800002

View details for PubMedID 17975716
Molecular imaging of the efficacy of heat shock protein 90 inhibitors in living subjects CANCER RESEARCH Chan, C. T., Paulmurugan, R., Gheysens, O. S., Kim, J., Chiosis, G., Gambhir, S. S. 2008; 68 (1): 216-226
Abstract

Heat shock protein 90 alpha (Hsp90 alpha)/p23 and Hsp90 beta/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90 alpha/p23 and Hsp90 beta/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purine-scaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90 alpha/p23 and Hsp90 beta/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90 alpha/p23 and Hsp90 beta/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoform-selective Hsp90 inhibitors.

View details for DOI 10.1158/0008-5472.CAN-07-2268

View details for Web of Science ID 000252072100029

View details for PubMedID 18172314
Applications of lentiviral vectors in non-invasive molecular imaging Methods in Molecular Biology De A, Yaghoubi S, Gambhir SS 2008; 433 (1): 177-202
Ovarian cancer early detection claims are biased Clinical Center Researh McIntosh M, Anderson G, Drescher C, Hanash S, Urban N, Brown P, Gambhir SS, Coukos G, Laird P, Nelson B, Palmer C 2008; 14 (22): 7574
Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects Clinical Cancer Research Ray P, De A, Patel M, Gambhir SS 2008; 14 (18): 5801-5809
Targeted microbubbles for imaging tumor angiogenesis: Assessment of whole body biodistribution with dynamic micro-PET in mice Radiology Willmann J, Cheng Z, Davis C, Lutz A, Schipper M, Nielsen C, Gambhir SS 2008; 249: 212-219
Uptake Kinetics and Biodistribution of (14)C-D:-Luciferin-a Radiolabeled Substrate for the Firefly Luciferase Catalyzed Bioluminescence Reaction: Impact on Bioluminescence Based Reporter Gene Imaging European Journal of Nuclear Medicine and Molecular Imaging Berger, F., Paulmurugan, R, Bhaumik, S, Gambhir, SS 2008
MOLECULAR IMAGING OF TUMOR VASCULATURE ANGIOGENESIS: IN VIVO SYSTEMS, PT B Cai, W., Gambhir, S. S., Chen, X. 2008; 445: 141-?
Abstract

Cancer, with more than 10 million new cases a year worldwide, is the third leading cause of death in developed countries. One critical requirement during cancer progression is angiogenesis, the formation of new blood vessels. Structural and functional imaging of tumor vasculature has been studied using various imaging modalities such as magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound. Molecular imaging, a key component of the 21st-century cancer-patient management strategy, takes advantage of these traditional imaging techniques and introduces molecular probes to determine the expression of indicative molecular markers at different stages of cancer development. In this chapter, we will focus on two tumor vasculature-related targets: integrin alpha(v)beta(3) and vascular endothelial growth factor receptor (VEGFR). For imaging of integrin alpha(v)beta(3) on the tumor vasculature, only nanoparticle-based probes will be discussed. VEGFR imaging will be discussed in depth, and we will give a detailed example of positron emission tomography (PET) imaging of VEGFR expression using radio-labeled VEGF(121) protein. Future clinical translation will be critical for maximum patient benefit from these agents. To achieve this goal, multidisciplinary approaches and cooperative efforts from many individuals, institutions, industries, and organizations are needed to quickly translate multimodality tumor vasculature imaging into multiple facets of cancer patient management.

View details for DOI 10.1016/S0076-6879(08)03007-3

View details for Web of Science ID 000260774900007

View details for PubMedID 19022059
A comparison between a time domain and continuous wave small animal optical imaging system IEEE TRANSACTIONS ON MEDICAL IMAGING Keren, S., Gheysens, O., Levin, C. S., Gambhir, S. S. 2008; 27 (1): 58-63
Abstract

We present a phantom study to evaluate the performance of the eXplore Optix (Advanced Research Technologies-GE Healthcare), the first commercially available time-domain tomography system for small animal fluorescence imaging, and compare its capabilities with the widely used IVIS 200 (Xenogen Corporation-Caliper) continuous wave planar imaging system. The eXplore Optix, based on point-wise illumination and collection scheme, is found to be a log order more sensitive with significantly higher detection depth and spatial resolution as compared with the wide-area illumination IVIS 200 under the conditions tested. A time-resolved detection system allows the eXplore Optix to measure the arrival time distribution of fluorescence photons. This enables fluorescence lifetime measurement, absorption mapping, and estimation of fluorescent inclusion depth, which in turn is used by a reconstruction algorithm to calculate the volumetric distribution of the fluorophore concentration. An increased acquisition time and lack of ability to image multiple animals simultaneously are the main drawbacks of the eXplore Optix as compared with the IVIS 200.

View details for DOI 10.1109/TMI.2007.902800

View details for Web of Science ID 000252098300007

View details for PubMedID 18270062
BRET-Based method for detection of specific RNA species BIOCONJUGATE CHEMISTRY Walls, Z. F., Gambhir, S. S. 2008; 19 (1): 178-184
Abstract

RNA detection and quantitation is a common necessity in modern molecular biology research. Most methods, however, are complex and/or time-intensive. Presented here is a BRET (bioluminescene resonance energy transfer)-based method that can accomplish the task of RNA identification quickly and easily. By conjugating BRET enzymes to two different oligonucleotides that are complementary to the same target sequence, probes were developed that could detect RNA using a solution-based assay. This assay was optimized for spacer length between the binding sites (found to be 10 nucleotides), and sensitivity was determined to be 1 microg for a specific species of RNA within a mixed population. Specificity of the assay was assessed using in vitro transcribed cRNA and found to be statistically siginificant ( p = 3.11 x 10 (-6), ANOVA, multiple range test). This assay represents a possibility for a less technically demanding, streamlined alternative to canonical RNA assays.

View details for DOI 10.1021/bc700278n

View details for Web of Science ID 000252520300024

View details for PubMedID 18072724
Comparison between adenoviral and retroviral vectors for the transduction of the thymidine kinase PET reporter gene in rat mesenchymal stem cells Journal of Nuclear Medicine Roelants V, Labar D, DeMeester C, Havaux X, Tabilio A, Gambhir SS, Dilanni M, Bertrand L, Vanoverschelde J 2008; 49 (11): 1836-1844
Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis JOURNAL OF MOLECULAR BIOLOGY Loening, A. M., Fenn, T. D., Gambhir, S. S. 2007; 374 (4): 1017-1028
Abstract

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 A and demonstrate a classic alpha/beta-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.

View details for DOI 10.1016/j.jmb.2007.09.078

View details for Web of Science ID 000251700900015

View details for PubMedID 17980388
Regulatory and reimbursement challenges for molecular imaging RADIOLOGY Hoffman, J. M., Gambhir, S. S., Kelloff, G. J. 2007; 245 (3): 645-660
Abstract

Molecular imaging is being hailed as the next great advance for imaging. Since molecular imaging typically involves the use of specific imaging probes that are treated like drugs, they will require regulatory approval. As with any drug, molecular imaging probes and techniques will also require thorough assessment in clinical trials to show safety and efficacy. The timeline for the regulatory approval will be long and potentially problematic because of the mounting costs of obtaining final regulatory approval. The current article is a detailed review of the regulatory and reimbursement process that will be required for molecular imaging probes and techniques to become a widespread clinical reality. The role of molecular imaging in the therapeutic drug discovery process will also be reviewed, as this is where these exciting new techniques have the potential to revolutionize the drug discovery and development process and, it is hoped, make it less costly. [(18)F]fluoro-2-deoxy-2-D-glucose positron emission tomography, one of the first molecular imaging techniques to be widely used, will be used as an example to illustrate the process of obtaining eventual reimbursement for widespread clinical use.

View details for DOI 10.1148/radiol.2453060737

View details for Web of Science ID 000251070700006

View details for PubMedID 18024447
Glia-dependent TGF-beta signaling, acting independently of the TH17 pathway, is critical for initiation of murine autoimmune encephalomyelitis JOURNAL OF CLINICAL INVESTIGATION Luo, J., Ho, P. P., Buckwalter, M. S., Hsu, T., Lee, L. Y., Zhang, H., Kim, D., Kim, S., Gambhir, S. S., Steinman, L., Wyss-Coray, T. 2007; 117 (11): 3306-3315
Abstract

Autoimmune encephalomyelitis, a mouse model for multiple sclerosis, is characterized by the activation of immune cells, demyelination of axons in the CNS, and paralysis. We found that TGF-beta1 synthesis in glial cells and TGF-beta-induced signaling in the CNS were activated several days before the onset of paralysis in mice with autoimmune encephalomyelitis. While early production of TGF-beta1 was observed in glial cells TGF-beta signaling was activated in neurons and later in infiltrating T cells in inflammatory lesions. Systemic treatment with a pharmacological inhibitor of TGF-beta signaling ameliorated the paralytic disease and reduced the accumulation of pathogenic T cells and expression of IL-6 in the CNS. Priming of peripheral T cells was not altered, nor was the generation of TH17 cells, indicating that this effect was directed within the brain, yet affected the immune system. These results suggest that early production of TGF-beta1 in the CNS creates a permissive and dangerous environment for the initiation of autoimmune inflammation, providing a rare example of the brain modulating the immune system. Importantly, inhibition of TGF-beta signaling may have benefits in the treatment of the acute phase of autoimmune CNS inflammation.

View details for DOI 10.1172/JCI31763

View details for Web of Science ID 000250676000023

View details for PubMedID 17965773
Molecular imaging techniques in body imaging RADIOLOGY Margolis, D. J., Hoffman, J. M., Herfkens, R. J., Jeffrey, R. B., Quon, A., Gambhir, S. S. 2007; 245 (2): 333-356
Abstract

Molecular imaging of the body involves new techniques to image cellular biochemical processes, which results in studies with high sensitivity, specificity, and signal-to-background. The most prevalently used molecular imaging technique in body imaging is currently fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET). FDG PET has become the method of choice for the staging and restaging of many of the most common cancers, including lymphoma, lung cancer, breast cancer, and colorectal cancer. FDG PET has also become extremely valuable in monitoring the response to therapeutic drugs in many cancers. New PET agents, such as fluorothymidine and acetate, have also shown promise in the evaluation of response to therapy and in the staging of prostate cancer. Magnetic resonance (MR) spectroscopy has shown promise in the evaluation of prostate cancer. Breast cancer evaluation benefits from advances in spectroscopic imaging and contrast-enhanced kinetic evaluation of vascular permeability, which is altered in neoplastic processes because of release of angiogenic factors. Superparamagnetic iron oxide (SPIO) particles represent the first of an expanding line of MR contrast agents that target specific cellular processes. SPIO particles have also been used in the evaluation of the cirrhotic liver and at MR lymphangiography.

View details for DOI 10.1148/radiol.2452061117

View details for Web of Science ID 000250343800007

View details for PubMedID 17940297
Multimodality imaging of T-cell hybridoma trafficking in collagen-induced arthritic mice: image-based estimation of the number of cells accumulating in mouse paws JOURNAL OF BIOMEDICAL OPTICS Yaghoubi, S. S., Creusot, R. J., Ray, P., Fathman, C. G., Gambhir, S. S. 2007; 12 (6)
Abstract

Appropriate targeting of therapeutic cells is essential in adoptive cellular gene therapy (ACGT). Imaging cell trafficking in animal models and patients will guide development of ACGT protocols. Collagen type II (C-II)-specific T cell hybridomas are transduced with a lentivirus carrying a triple fusion reporter gene (TFR) construct consisting of a fluorescent reporter gene (RG), a bioluminescent RG (hRluc), and a positron emission tomography (PET) RG. Collagen-induced arthritic (CIA) mice are scanned with a bioluminescence imaging camera before and after implantation of various known cell quantities in their paws. Linear regression analysis yields equations relating two parameters of image signal intensity in mice paws to the quantity of hRluc expressing cells in the paws. Afterward, trafficking of intravenously injected cells is studied by quantitative analysis of bioluminescence images. Comparison of the average cell numbers does not demonstrate consistently higher accumulation of T-cell hybridomas in the paws with higher inflammation scores, and injecting more cells does not cause increased accumulation. MicroPET images illustrate above background signal in the inflamed paws and chest areas of CIA mice. The procedures described in this study can be used to derive equations for cells expressing other bioluminescent RGs and in other animal models.

View details for DOI 10.1117/1.2821415

View details for Web of Science ID 000252851100046

View details for PubMedID 18163841
Design and evaluation of a variable aperture collimator for conformal radiotherapy of small animals using a microCT scanner MEDICAL PHYSICS Graves, E. E., Zhou, H., Chatterjee, R., Keall, P. J., Gambhir, S. S., Contag, C. H., Boyer, A. L. 2007; 34 (11): 4359-4367
Abstract

Treatment of small animals with radiation has in general been limited to planar fields shaped with lead blocks, complicating spatial localization of dose and treatment of deep-seated targets. In order to advance laboratory radiotherapy toward what is accomplished in the clinic, we have constructed a variable aperture collimator for use in shaping the beam of microCT scanner. This unit can image small animal subjects at high resolution, and is capable of delivering therapeutic doses in reasonable exposure times. The proposed collimator consists of two stages, each containing six trapezoidal brass blocks that move along a frame in a manner similar to a camera iris producing a hexagonal aperture of variable size. The two stages are offset by 30 degrees and adjusted for the divergence of the x-ray beam so as to produce a dodecagonal profile at isocenter. Slotted rotating driving plates are used to apply force to pins in the collimator blocks and effect collimator motion. This device has been investigated through both simulation and measurement. The collimator aperture size varied from 0 to 8.5 cm as the driving plate angle increased from 0 to 41 degrees. The torque required to adjust the collimator varied from 0.5 to 5 N x m, increasing with increasing driving plate angle. The transmission profiles produced by the scanner at isocenter exhibited a penumbra of approximately 10% of the collimator aperture width. Misalignment between the collimator assembly and the x-ray source could be identified on the transmission images and corrected by adjustment of the collimator location. This variable aperture collimator technology is therefore a feasible and flexible solution for adjustable shaping of radiation beams for use in small animal radiotherapy as well as other applications in which beam shaping is desired.

View details for DOI 10.1118/1.2789498

View details for Web of Science ID 000251145900029

View details for PubMedID 18072501
Dual-function probe for PET and near-infrared fluorescence imaging of tumor vasculature JOURNAL OF NUCLEAR MEDICINE Cai, W., Chen, K., Li, Z., Gambhir, S. S., Chen, X. 2007; 48 (11): 1862-1870
Abstract

To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function PET/near-infrared fluorescence (NIRF) probe can allow for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs.A QD with an amine-functionalized surface was modified with RGD peptides and 1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelators for integrin alpha(v)beta(3)-targeted PET/NIRF imaging. A cell-binding assay and fluorescence cell staining were performed with U87MG human glioblastoma cells (integrin alpha(v)beta(3)-positive). PET/NIRF imaging, tissue homogenate fluorescence measurement, and immunofluorescence staining were performed with U87MG tumor-bearing mice to quantify the probe uptake in the tumor and major organs.There are about 90 RGD peptides per QD particle, and DOTA-QD-RGD exhibited integrin alpha(v)beta(3)-specific binding in cell cultures. The U87MG tumor uptake of (64)Cu-labeled DOTA-QD was less than 1 percentage injected dose per gram (%ID/g), significantly lower than that of (64)Cu-labeled DOTA-QD-RGD (2.2 +/- 0.3 [mean +/- SD] and 4.0 +/- 1.0 %ID/g at 5 and 18 h after injection, respectively; n = 3). Taking into account all measurements, the liver-, spleen-, and kidney-to-muscle ratios for (64)Cu-labeled DOTA-QD-RGD were about 100:1, 40:1, and 1:1, respectively. On the basis of the PET results, the U87MG tumor-to-muscle ratios for DOTA-QD-RGD and DOTA-QD were about 4:1 and 1:1, respectively. Excellent linear correlation was obtained between the results measured by in vivo PET imaging and those measured by ex vivo NIRF imaging and tissue homogenate fluorescence (r(2) = 0.93). Histologic examination revealed that DOTA-QD-RGD targets primarily the tumor vasculature through an RGD-integrin alpha(v)beta(3) interaction, with little extravasation.We quantitatively evaluated the tumor-targeting efficacy of a dual-function QD-based probe with PET and NIRF imaging. This dual-function probe has significantly reduced potential toxicity and overcomes the tissue penetration limitation of optical imaging, allowing for quantitative targeted imaging in deep tissue.

View details for DOI 10.2967/jnumed.107.043216

View details for Web of Science ID 000252894900024

View details for PubMedID 17942800
Quantum dot imaging for embryonic stem cells BMC BIOTECHNOLOGY Lin, S., Xie, X., Patel, M. R., Yang, Y., Li, Z., Cao, F., Gheysens, O., Zhang, Y., Gambhir, S. S., Rao, J. H., Wu, J. C. 2007; 7
Abstract

Semiconductor quantum dots (QDs) hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES) cells labeled with Qtracker delivered quantum dots (QDs).Murine embryonic stem (ES) cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS). Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background)/exposure time in millisecond was 11 +/- 2 for cells labeled with QD 525, 12 +/- 9 for QD 565, 176 +/- 81 for QD 605, 176 +/- 136 for QD 655, 167 +/- 104 for QD 705, and 1,713 +/- 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested.In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy non-invasively in vivo.

View details for DOI 10.1186/1472-6750-7-67

View details for Web of Science ID 000252448600001

View details for PubMedID 17925032
Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Levi, J., De, A., Cheng, Z., Gambhir, S. S. 2007; 129 (39): 11900-?
View details for DOI 10.1021/ja073936h

View details for Web of Science ID 000249887700013

View details for PubMedID 17850082
Molecular imaging can accelerate anti-angiogenic drug development and testing NATURE CLINICAL PRACTICE ONCOLOGY Lagaru, A., Chen, X., Gambhir, S. S. 2007; 4 (10): 556-557
View details for DOI 10.1038/ncponc0929

View details for Web of Science ID 000249708800002

View details for PubMedID 17726490
Osseous and soft tissue sarcomas: When can F-18 FDG PET/CT evaluation provide useful information? Iagaru, A., Quon, A., Jacobs, C., Marina, N., McDougall, I., Gambhir, S. S. SPRINGER. 2007: S152-S152
View details for Web of Science ID 000253283900155
Differentiation, survival, and function of embryonic stem cell-derived endothelial cells for ischemic heart disease CIRCULATION Li, Z., Wu, J. C., Sheikh, A. Y., Kraft, D., Cao, F., Xie, X., Patel, M., Gambhir, S. S., Robbins, R. C., Cooke, J. P., Wu, J. C. 2007; 116 (11): I46-I54
Abstract

Embryonic stem (ES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of ES cell-derived endothelial cells (ESC-ECs), use molecular imaging techniques to examine their survival in vivo, and determine the therapeutic efficacy of ESC-ECs for restoration of cardiac function after ischemic injury.Murine ES cells were transfected with a construct composed of a vascular endothelial cadherin promoter driving enhanced green fluorescence protein (pVE-cadherin-eGFP). Differentiation of ES cells to ECs was detected by FACS analysis using Flk-1 (early EC marker at day 4) and VE-cadherin (late EC marker at day 8). After isolation, these ESC-ECs express endothelial cell markers similar to adult mouse lung endothelial cells, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). For in vivo imaging, ES cells were transduced with an ubiquitin promoter driving firefly luciferase and monomeric red fluorescence protein (pUb-Fluc-mRFP). A robust correlation exists between Fluc signals and cell numbers by ex vivo imaging analysis (R2=0.98) and by in vitro enzyme assay (R2=0.94). Afterward, 5x10(5) ESC-ECs or PBS (as control) was injected into the hearts of mice undergoing LAD ligation (n=15 per group). Bioluminescence imaging showed longitudinal survival of transplanted ESC-ECs for approximately 8 weeks. Echocardiogram demonstrated significant functional improvement in the ESC-EC group compared with control (P=0.04). Finally, postmortem analysis confirmed increased presence of small capillaries and venules in the infarcted zones by CD31 staining.This is the first study to track the fate and function of transplanted ESC-ECs in the heart. With further validation, these ESC-ECs could become a valuable source of cell therapy for induction of angiogenesis in the treatment of myocardial ischemia.

View details for DOI 10.1161/CIRCULATIONAHA.106.680561

View details for Web of Science ID 000249364500007

View details for PubMedID 17846325
2-Deoxy-2-[F-18]fluoro-D-glucose accumulation in ovarian carcinoma cell lines MOLECULAR IMAGING AND BIOLOGY Lutz, A. M., Ray, P., Willmann, J. K., Drescher, C., Gambhir, S. S. 2007; 9 (5): 260-266
Abstract

To evaluate 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) accumulation in human ovarian carcinoma cell lines compared with control tumor cell lines known to accumulate FDG.FDG accumulation assays were performed in 15 different ovarian carcinoma cell lines at 1, 2, and 3 hours after incubation with 1 microCi of FDG. Results were compared with FDG accumulation in six different control tumor cell lines. 2-deoxy-2-[F-18]fluoro-D-glucose accumulation was expressed as counts per minute (cpm) in cells and normalized to initial cpm in medium and total protein content of cell lysates.FDG accumulation in all 15 ovarian carcinoma cell lines was equal to or higher than 0.0005 +/- 8.6 10(-5) cpm in cells/cpm in medium/mug protein at all three different time points. In two ovarian carcinoma cell lines (ES-2, poorly differentiated clear cell carcinoma, and OVCAR-3, poorly differentiated papillary adenocarcinoma), FDG accumulation was not statistically, significantly different compared to the control cell line with the highest FDG accumulation (LS 174T human colorectal adenocarcinoma) at two or more time points (P > or = 0.07). In 2 of 15 (13%) ovarian carcinoma cell lines (OVCAR5 epithelial carcinoma and SKOV3 clear cell carcinoma), FDG accumulation was lower than that in the control cell line with the lowest FDG accumulation (HT-29 human colorectal adenocarcinoma) at one or more time points (P < 0.05).Most human ovarian carcinoma cell lines showed comparable FDG accumulations with control cell lines known to accumulate FDG. This study lays the foundations for further comparisons with other ovarian cancer cell lines and for other positron emission tomography tracers.

View details for DOI 10.1007/s11307-007-0105-4

View details for Web of Science ID 000248865200002

View details for PubMedID 17610017
Fusion of Gaussia luciferase to an engineered anti-carcinoembryonic antigen (CEA) antibody for in vivo optical imaging MOLECULAR IMAGING AND BIOLOGY Venisnik, K. M., Olafsen, T., Gambhir, S. S., Wu, A. M. 2007; 9 (5): 267-277
Abstract

The bioluminescent protein Gaussia luciferase (GLuc) was fused to an anti-carcinoembryonic antigen (CEA) antibody fragment, the diabody, for in vivo optical tumor imaging. A 15-amino acid N-terminal truncation (GLDelta15) resulted in a brighter protein. Fusions of the anti-CEA diabody to full-length GLuc and GLDelta15 retained high affinity for the antigen, emitted light, and exhibited excellent enzymatic stability. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-GLDelta15 to CEA-positive xenografts, with a tumor/background ratio of 3.8 +/- 0.4 at four hours after tail-vein injection, compared to antigen-negative tumors at 1.3 +/- 0.1 (p = 0.001). MicroPET imaging using (124)I-diabody-GLDelta15 demonstrated specific uptake in the CEA-positive tumor (2.6% ID [injected dose]/g) compared to the CEA-negative tumor (0.4% ID/g) at 21 hours. Although further optimization of this fusion protein may be needed to improve in vivo performance, the diabody-GLDelta15 is a promising optical imaging probe for tumor detection in vivo.

View details for DOI 10.1007/s11307-007-0101-8

View details for Web of Science ID 000248865200003

View details for PubMedID 17577599
Molecular imaging: Integration of molecular imaging into the musculoskeletal imaging practice RADIOLOGY Biswal, S., Resnick, D. L., Hoffman, J. M., Gambhir, S. S. 2007; 244 (3): 651-671
Abstract

Chronic musculoskeletal diseases such as arthritis, malignancy, and chronic injury and/or inflammation, all of which may produce chronic musculoskeletal pain, often pose challenges for current clinical imaging methods. The ability to distinguish an acute flare from chronic changes in rheumatoid arthritis, to survey early articular cartilage breakdown, to distinguish sarcomatous recurrence from posttherapeutic inflammation, and to directly identify generators of chronic pain are a few examples of current diagnostic limitations. There is hope that a growing field known as molecular imaging will provide solutions to these diagnostic puzzles. These techniques aim to depict, noninvasively, specific abnormal cellular, molecular, and physiologic events associated with these and other diseases. For example, the presence and mobilization of specific cell populations can be monitored with molecular imaging. Cellular metabolism, stress, and apoptosis can also be followed. Furthermore, disease-specific molecules can be targeted, and particular gene-related events can be assayed in living subjects. Relatively recent molecular and cellular imaging protocols confirm important advances in imaging technology, engineering, chemistry, molecular biology, and genetics that have coalesced into a multidisciplinary and multimodality effort. Molecular probes are currently being developed not only for radionuclide-based techniques but also for magnetic resonance (MR) imaging, MR spectroscopy, ultrasonography, and the emerging field of optical imaging. Furthermore, molecular imaging is facilitating the development of molecular therapies and gene therapy, because molecular imaging makes it possible to noninvasively track and monitor targeted molecular therapies. Implementation of molecular imaging procedures will be essential to a clinical imaging practice. With this in mind, the goal of the following discussion is to promote a better understanding of how such procedures may help address specific musculoskeletal issues, both now and in the years ahead.

View details for DOI 10.1148/radiol.2443060295

View details for Web of Science ID 000248993500006

View details for PubMedID 17709823
MicroPET-based biodistribution of quantum dots in living mice JOURNAL OF NUCLEAR MEDICINE Schipper, M. L., Cheng, Z., Lee, S., Bentolila, L. A., Iyer, G., Rao, J., Chen, X., Wu, A. M., Weiss, S., Gambhir, S. S. 2007; 48 (9): 1511-1518
Abstract

This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice.(64)Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis.Both methods show rapid uptake by the liver (27.4-38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0-12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5-6.9 %ID/g). No evidence of clearance from these organs was observed.Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.

View details for DOI 10.2967/jnumed.107.040071

View details for Web of Science ID 000252894700039

View details for PubMedID 17704240
Cardiovascular molecular imaging RADIOLOGY Wu, J. C., Bengel, F. M., Gambhir, S. S. 2007; 244 (2): 337-355
Abstract

The goal of this review is to highlight how molecular imaging will impact the management and improved understanding of the major cardiovascular diseases that have substantial clinical impact and research interest. These topics include atherosclerosis, myocardial ischemia, myocardial viability, heart failure, gene therapy, and stem cell transplantation. Traditional methods of evaluation for these diseases will be presented first, followed by methods that incorporate conventional and molecular imaging approaches.

View details for Web of Science ID 000248821400005

View details for PubMedID 17592037
Red-shifted Renilla reniformis luciferase variants for imaging in living subjects NATURE METHODS Loening, A. M., Wu, A. M., Gambhir, S. S. 2007; 4 (8): 641-643
Abstract

The use of R. reniformis luciferase (RLuc) as a reporter gene in small-animal imaging has been hampered by its 481 nm peaked emission spectrum, as blue wavelengths are strongly attenuated in biological tissues. To overcome this, we generated variants of RLuc with bathochromic (red) shifts of up to 66 nm (547 nm peak) that also had greater stability and higher light emission than native RLuc.

View details for DOI 10.1038/NMETH1070

View details for Web of Science ID 000248443900017

View details for PubMedID 17618292
An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects CANCER RESEARCH De, A., Loening, A. M., Gambhir, S. S. 2007; 67 (15): 7175-7183
Abstract

Bioluminescence resonance energy transfer (BRET) is currently used for monitoring various intracellular events, including protein-protein interactions, in normal and aberrant signal transduction pathways. However, the BRET vectors currently used lack adequate sensitivity for imaging events of interest from both single living cells and small living subjects. Taking advantage of the critical relationship of BRET efficiency and donor quantum efficiency, we report generation of a novel BRET vector by fusing a GFP(2) acceptor protein with a novel mutant Renilla luciferase donor selected for higher quantum yield. This new BRET vector shows an overall 5.5-fold improvement in the BRET ratio, thereby greatly enhancing the dynamic range of the BRET signal. This new BRET strategy provides a unique platform to assay protein functions from both single live cells and cells located deep within small living subjects. The imaging utility of the new BRET vector is shown by constructing a sensor using two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. This new BRET vector should facilitate high-throughput sensitive BRET assays, including studies in single live cells and small living subjects. Applications will include anticancer therapy screening in cell culture and in small living animals.

View details for DOI 10.1158/0008-5472.CAN-06-4623

View details for Web of Science ID 000248529300018

View details for PubMedID 17671185
Oxygen sensitivity of reporter genes: Implications for preclinical imaging of tumor hypoxia MOLECULAR IMAGING Cecic, I., Chan, D. A., Sutphin, P. D., Ray, P., Gambhir, S. S., Giaccia, A. J., Gravcs, E. E. 2007; 6 (4): 219-228
Abstract

Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ. Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing beta-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-galactoside (DDAOG). Generation of signal in in vivo tumor models expressing bioluminescent or beta-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas beta-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining beta-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.

View details for DOI 10.2310/7290.2007.00017

View details for Web of Science ID 000249349100001

View details for PubMedID 17711777
Molecular imaging: The vision and opportunity for radiology in the future RADIOLOGY Hoffman, J. M., Gambhir, S. S. 2007; 244 (1): 39-47
Abstract

Molecular imaging is being hailed as the next great advance for imaging. This introductory article in the molecular imaging series to be published over the next several months in Radiology sets the stage for the subsequent set of articles by providing relevant definitions and background information and traces the evolution of molecular imaging to its current state of research and clinical practice. It discusses in detail the evolution of molecular imaging and the role that the National Cancer Institute and the National Institutes of Health have had in the funding and development of many of the important molecular imaging research programs that are in existence today. The article also provides basic information about the complex biology of the cell and details of the pathogenesis of cancer and how molecular imaging will be critical for earlier detection and management of cancer in the future. The article lays the foundation for the subsequent articles in the series and describes how and why molecular imaging will be critical and integral for clinical care of patients in the future. The introductory article also discusses the relevance of molecular imaging to clinical radiology practice and why it is critical for the practicing radiologist to understand these evolving techniques, as they will be the future of imaging.

View details for DOI 10.1148/radiol.2441060773

View details for Web of Science ID 000247436500005

View details for PubMedID 17507723
Small-animal PET of melanocortin 1 receptor expression using a F-18-labeled alpha-melanocyte-stimulating hormone analog JOURNAL OF NUCLEAR MEDICINE Cheng, Z., Zhang, L., Graves, E., Xiong, Z., Dandekar, M., Chen, X., Gambhir, S. S. 2007; 48 (6): 987-994
Abstract

(18)F-Labeled small synthetic peptides have emerged as attractive probes for imaging various molecular targets with PET. The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor [MC1R]) is overexpressed in most murine and human melanomas. It is a promising molecular target for diagnosis and therapy of melanomas. However, (18)F compounds have not been successfully developed for imaging the MC1R.In this study, an alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH(2) (NAPamide), was radiolabeled with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB). The resulting radiopeptide was evaluated as a potential molecular probe for small-animal PET of melanoma and MC1R expression in melanoma xenografted mouse models.The binding affinity of (19)F-SFB-conjugated NAPamide, (19)F-FB-NAPamide, was determined to be 7.2 +/- 1.2 nM (mean +/- SD) using B16/F10 cells and (125)I-(Tyr(2))-[Nle(4),D-Phe(7)]-alpha-MSH [(125)I-(Tyr(2))-NDP] as a radioligand. The biodistribution of (18)F-FB-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16/F10 melanoma tumors with high expression of MC1Rs and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Biodistribution experiments showed that tumor uptake values (percentage injected dose per gram of tumor [%ID/g]) of (18)F-FB-NAPamide were 1.19 +/- 0.11 %ID/g and 0.46 +/- 0.11 %ID/g, in B16/F10 and A375M xenografted melanoma at 1 h after injection, respectively. Furthermore, the B16/F10 tumor uptake was significantly inhibited by coinjection with excess alpha-MSH peptide (P < 0.05), indicating that (18)F-FB-NAPamide specifically recognizes the MC1R in living mice. Small-animal PET of (18)F-FB-NAPamide in mice bearing B16/F10 and A375M tumors at 1 h after tail vein injection revealed good B16/F10 tumor-to-background contrast and low A375M tumor-to-background ratios.(18)F-FB-NAPamide is a promising molecular probe for alpha-MSH receptor-positive melanoma PET and warrants further study.

View details for DOI 10.2967/jnumed.107.039602

View details for Web of Science ID 000247054800024

View details for PubMedID 17504880
Evaluation of herpes simplex virus 1 thymidine kinase-mediated trapping of I-131 FIAU and prodrug activation of ganciclovir as a synergistic cancer radio/chemotherapy MOLECULAR IMAGING AND BIOLOGY Schipper, M. L., Goris, M. L., Gambhir, S. S. 2007; 9 (3): 110-116
Abstract

Evaluation of selective killing of Herpes Simplex Virus 1 thymidine kinase (HSV1-tk) expressing tumors by radiolabeled (131)I-fialuridine (FIAU), and of synergy between (131)I-FIAU and Ganciclovir (GCV).HSV1-tk-expressing cell lines and parental cell lines were exposed to (131)I-FIAU alone, GCV alone, or combinations. Activity and concentration were varied widely, concurrent and sequential administrations tested, and dose rate effects were studied.HSV1-tk-expressing cells accumulated up to 15.7-fold more (131)I-FIAU, were growth inhibited by 2 muCi/ml, or 5 muCi/ml (131)I-FIAU, and were inhibited by two log orders lower concentrations of GCV than parental cells. However, no synergy or additive effect was observed. Dose rate variations, or sequential treatment, did not alter outcome.Radioisotope therapy of HSV1-tk-expressing tumor cells with (131)I-FIAU is reported for the first time. Lack of synergy between (131)I-FIAU and GCV does not warrant further investigation of combination treatment with the two agents.

View details for DOI 10.1007/s11307-007-0078-3

View details for Web of Science ID 000246175500003

View details for PubMedID 17294333
Integrating noninvasive molecular imaging into molecular medicine: an evolving paradigm TRENDS IN MOLECULAR MEDICINE Massoud, T. F., Gambhir, S. S. 2007; 13 (5): 183-191
Abstract

Molecular imaging is a rapidly emerging field, providing noninvasive visual quantitative representations of fundamental biological processes in intact living subjects. Fundamental biomedical research stands to benefit considerably from advances in molecular imaging, with improved molecular target selection, probe development and imaging instrumentation. The noninvasiveness of molecular imaging technologies will also provide benefit through improved patient care. Molecular imaging endpoints can be quantified, and therefore are particularly useful for translational research. Integration of the two disciplines of molecular imaging and molecular medicine, combined with systems-biology approaches to understanding disease complexity, promises to provide predictive, preventative and personalized medicine that will transform healthcare.

View details for DOI 10.1016/j.molmed.2007.03.003

View details for Web of Science ID 000247166100002

View details for PubMedID 17403616
Fluorescent fructose derivatives for imaging breast cancer cells BIOCONJUGATE CHEMISTRY Levi, J., Cheng, Z., Gheysens, O., Patel, M., Chan, C. T., Wang, Y., Namavari, M., Gambhir, S. S. 2007; 18 (3): 628-634
Abstract

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.

View details for DOI 10.1021/bc060184s

View details for Web of Science ID 000246485500005

View details for PubMedID 17444608
Cu-64-Labeled alpha-melanocyte-stimulating hormone analog for MicroPET imaging of melanocortin 1 receptor expression BIOCONJUGATE CHEMISTRY Cheng, Z., Xiong, Z., Subbarayan, M., Chen, X., Gambhir, S. S. 2007; 18 (3): 765-772
Abstract

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess alpha-MSH peptide. MicroPET imaging of 64Cu-DOTA-NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that 64Cu-DOTA-NAPamide is a promising molecular probe for alpha-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice.

View details for DOI 10.1021/bc060306g

View details for Web of Science ID 000246485500021

View details for PubMedID 17348700
In vivo bioluminescence tumor imaging of RGD peptide-modified adenoviral vector encoding firefly luciferase reporter gene MOLECULAR IMAGING AND BIOLOGY Niu, G., Xiong, Z., Cheng, Z., Cai, W., Gambhir, S. S., Xing, L., Chen, X. 2007; 9 (3): 126-134
Abstract

The goal of this study is to demonstrate the feasibility of chemically modified human adenovirus (Ad) vectors for tumor retargeting.E1- and E3-deleted Ad vectors carrying firefly luciferase reporter gene under cytomegalovirus promoter (AdLuc) was surface-modified with cyclic arginine-glycine-aspartic acid (RGD) peptides through a bifunctional poly(ethyleneglycol) linker (RGD-PEG-AdLuc) for integrin alpha(v)beta(3) specific delivery. The Coxsackie and adenovirus viral receptor (CAR) and integrin alpha(v)beta(3) expression in various tumor cell lines was determined by reverse transcriptase PCR and fluorescence-activated cell sorting. Bioluminescence imaging was performed in vitro and in vivo to evaluate RGD-modified AdLuc infectivity.RGD-PEG-AdLuc abrogated the native CAR tropism and exhibited significantly enhanced transduction efficiency of integrin-positive tumors than AdLuc through intravenous administration.This approach provides a robust platform for site-specific gene delivery and noninvasive monitoring of the transgene delivery efficacy and homing.

View details for DOI 10.1007/s11307-007-0079-2

View details for Web of Science ID 000246175500005

View details for PubMedID 17297551
Detection of bone metastases: Assessment of integrated FDG PET/CT imaging RADIOLOGY Taira, A. V., Herfkens, R. J., Gambhir, S. S., Quon, A. 2007; 243 (1): 204-211
Abstract

To retrospectively evaluate the positive predictive value (PPV) of fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) in the identification of malignant bone lesions when the PET and CT findings are discordant and concordant.The study conformed to HIPAA standards, and the need for informed consent was waived by the institutional review board that approved the study. FDG PET/CT reports of 712 patients were reviewed to identify patients with malignant bone lesions. Fifty-nine patients (30 female and 29 male patients; age range, 10-82 years) with 113 lesions were analyzed. With use of confirmation from histopathologic examination or clinical follow-up, the PPVs of the integrated examination and of the stand-alone CT and PET components of the examination were calculated. The results were stratified according to cancer type, chemotherapy status, and number of bone lesions and were compared by using Fisher exact tests.Of 47 lesions with positive findings at both PET and CT, 46 were malignant and one was benign, for a PPV of 98%. Of 31 lesions with positive findings at PET and negative findings at CT, 19 were malignant and 12 were benign, for a PPV of 61%. Of 35 lesions with negative findings at PET and positive findings at CT, six were malignant and 29 were benign, for a PPV of 17%. Independently, the PPV of all lesions with positive findings at PET was significantly higher than that of all lesions with positive findings at CT. Chemotherapy status for lesions with positive findings at CT and the number of lesions per patient had a statistically significant effect on the PPV of examinations (P = .02 and P < .001, respectively).PET/CT has a very high PPV for bone metastases (98%) when the findings at PET and CT are concordant; however, in lesions with discordant PET and CT findings at the integrated examination, PPV is markedly diminished.

View details for DOI 10.1148/radiol.2431052104

View details for Web of Science ID 000245312500025

View details for PubMedID 17392254
Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects CANCER RESEARCH Ray, P., Tsien, R., Gambhir, S. S. 2007; 67 (7): 3085-3093
Abstract

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, d-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase (fl)] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes (jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 +/- 0.1 versus 1.9 +/- 0.1) x (10(6) p/s/cm(2)/sr)] but similar level of fluorine-18-labeled 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 +/- 0.15 versus 1.37 +/- 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttk-expressing tumors. Both tumors showed 4- to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques.

View details for DOI 10.1158/0008-5472.CAN-06-2402

View details for Web of Science ID 000245622900025

View details for PubMedID 17409415
Reproducibility of F-18-FDG microPET studies in mouse tumor xenografts JOURNAL OF NUCLEAR MEDICINE Dandekar, M., Tseng, J. R., Gambhir, S. S. 2007; 48 (4): 602-607
Abstract

(18)F-FDG has been used to image mouse xenograft models with small-animal PET for therapy response. However, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FDG small-animal PET studies.Mouse tumor xenografts were formed with B16F10 murine melanoma cells. A 7-min small-animal PET scan was performed 1 h after a 3.7- to 7.4-MBq (18)F-FDG injection via the tail vein. A second small-animal PET scan was performed 6 h later after reinjection of (18)F-FDG. Twenty-five sets of studies were performed. Mean injected dose per gram (%ID/g) values were calculated from tumor regions of interest. The coefficient of variation (COV) from studies performed on the same day was calculated to determine the reproducibility. Activity from the second scans performed after 6 h were adjusted by subtracting the estimated residual activity from the first (18)F-FDG injection. For 7 datasets, an additional scan immediately before the second injection was performed, and residual activity from this additional delayed scan was subtracted from the activity of the second injection. COVs of both subtraction methods were compared. Blood glucose values were measured at the time of injection and used to correct the %ID/g values.The COV for the mean %ID/g between (18)F-FDG small-animal PET scans performed on the same day 6 h apart was 15.4% +/- 12.6%. The delayed scan subtraction method did not produce any significant change in the COV. Blood glucose correction increased the COV. The injected dose, tumor size, and body weight did not appear to contribute to the variability of the scans.(18)F-FDG small-animal PET mouse xenograft studies were reproducible with moderately low variability. Therefore, serial small-animal PET studies may be performed with reasonable accuracy to measure tumor response to therapy.

View details for DOI 10.2967/jnumed.106.036608

View details for Web of Science ID 000245647000024

View details for PubMedID 17401098
Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions ANALYTICAL CHEMISTRY Paulmurugan, R., Gambhir, S. S. 2007; 79 (6): 2346-2353
Abstract

Split reporter-based bioluminescence imaging is a useful strategy for studying protein-protein as well as other intracellular interactions. We have used a combinatorial strategy to identify a novel split site for firefly luciferase with improved characteristics over previously published split sites. A combination of fragments with greater absolute signal with near-zero background signals was achieved by screening 115 different combinations. The identified fragments were further characterized by using five different interacting protein partners and an intramolecular folding strategy. Cell culture studies and imaging in living mice was performed to validate the new split sites. In addition, the signal generated by the newly identified combination of fragments (Nfluc 398/ Cfluc 394) was compared with different split luciferase fragments currently in use for studying protein-protein interactions and was shown to be markedly superior with a lower self-complementation signal and equal or higher postinteraction absolute signal. This study also identified many different combinations of nonoverlapping and overlapping firefly luciferase fragments that can be used for studying different cellular events such as subcellular localization of proteins, cell-cell fusion, and evaluating cell delivery vehicles, in addition to protein-protein interactions, both in cells and small living animals.

View details for DOI 10.1021/ac062053q

View details for Web of Science ID 000244867100020

View details for PubMedID 17295448
In vivo optical bioluminescence imaging of collagen-supported cardiac cell grafts JOURNAL OF HEART AND LUNG TRANSPLANTATION Kutschka, I., Chen, I. Y., Kofidis, T., von Degenfeld, G., Sheikh, A. Y., Hendry, S. L., Hoyt, G., Pearl, J., Blau, H. M., Gambhir, S. S., Robbins, R. C. 2007; 26 (3): 273-280
Abstract

Histology-based survival assessment of cell grafts does not allow for in vivo follow-up. In this study we introduce two new experimental models for longitudinal in vivo survival studies of cardiac cell grafts using optical bioluminescence imaging.H9c2 cardiomyoblasts expressing both firefly luciferase (fluc) and green fluorescent protein (GFP) reporter genes were implanted into Lewis rats. In Model 1, H9c2-fluc-IRES-GFP cells (0.5 x 10(6)) were implanted into a cryoinjured abdominal wall muscle. Cells were injected using either liquid collagen (Matrigel [MG]) or phosphate-buffered saline (PBS) suspension. Cell survival was evaluated in vivo using bioluminescence imaging on days 1, 5 and 10 post-operatively. In model 2, rats underwent ligation of the left anterior descending (LAD) artery. The donor hearts were harvested, and the infarcted region was restored ex situ using 1 x 10(6) H9c2-fluc-IRES-GFP cells seeded in collagen matrix (Gelfoam [GF]) or suspended in PBS (n = 8/group). Hearts were then transplanted into the abdomen of syngeneic recipients. Optical bioluminescence imaging was performed on Days 1, 5, 8 and 14 post-operatively. After 4 weeks, immunohistologic studies were performed.For model 1, at day 5, bioluminescence signals were markedly higher for the H9c2/MG group (449 +/- 129 photons/second x 10(3)) compared with the H9c2/PBS group (137 +/- 82 photons/second x 10(3)) (p < 0.05). For model 2, bioluminescence signals were significantly (p < 0.04) higher in the H9c2/GF group compared with plain cell injection on days 5 (534 +/- 115 vs 219 +/- 34) and 8 (274 +/- 34 vs 180 +/- 23). Data were in accordance with GFP immunohistology.Optical bioluminescence is a powerful method for assessment of cardiac cell graft survival in vivo. Collagen matrices support early survival of cardiomyoblasts after transplantation into injured musculature.

View details for Web of Science ID 000244979000010

View details for PubMedID 17346630
Standardized uptake value atlas: Characterization of physiological 2-deoxy-2-[F-18]fluoro-D-glucose uptake in normal tissues MOLECULAR IMAGING AND BIOLOGY Wang, Y., Chiu, E., Rosenberg, J., Gambhir, S. S. 2007; 9 (2): 83-90
Abstract

The purpose of this study was to map the distribution of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) uptake in organs of patients with no known abnormalities in those tissues.We measured maximum and mean standardized uptake values (SUV) from FDG-positron emission tomography (PET)/computed tomography (CT) obtained from 98 patients (48 males and 50 females).Significant uptake (mean SUVmean>2.5) was visualized in the cerebellum (8.0+/-2.2), soft palate (2.92+/-0.86), palatine tonsils (3.45+/-1.4), lingual tonsils (3.08+/-1.05), sublingual glands (3.3+/-1.5), and testes (2.57+/-0.56). Negative correlation for FDG uptake versus age was observed for the palatine tonsils, sublingual glands, and lungs (P<0.001).Better understanding of physiological uptake throughout the body is valuable for improved interpretive accuracy and should be useful for future semi-automated comparisons to a normal SUV database.

View details for DOI 10.1007/s11307-006-0075-y

View details for Web of Science ID 000244866700005

View details for PubMedID 17225983
Impact of integrated PET/CT on variability of target volume delineation in rectal cancer TECHNOLOGY IN CANCER RESEARCH & TREATMENT Patel, D. A., Chang, S. T., Goodman, K. A., Quon, A., Thorndyke, B., Gambhir, S. S., McMillan, A., Loo, B. W., Koong, A. C. 2007; 6 (1): 31-36
Abstract

Several studies have demonstrated substantial variability among individual radiation oncologists in defining target volumes using computed tomography (CT). The objective of this study was to determine the impact of combined positron emission tomography and computed tomography (PET/CT) on inter-observer variability of target volume delineation in rectal cancer. We also compared the relative concordance of two PET imaging tracers, 18F-fluorodeoxyglucose (FDG) and 18F-fluorodeoxythymidine (FLT), against conventional computed tomography (CT). Six consecutive patients with locally advanced rectal cancer were enrolled onto an institutional protocol involving preoperative chemoradiotherapy and correlative studies including FDG- and FLT-PET scans acquired in the treatment position. Using these image data sets, four radiation oncologists independently delineated primary and nodal gross tumor volumes (GTVp and GTVn) for a hypothetical boost treatment. Contours were first defined based on CT alone with observers blinded to the PET images, then based on combined PET/CT. An inter-observer similarity index (SI), ranging from a value of 0 for complete disagreement to 1 for complete agreement of contoured voxels, was calculated for each set of volumes. For primary gross tumor volume (GTVp), the difference in estimated SI between CT and FDG was modest (CT SI = 0.77 vs. FDG SI = 0.81), but statistically significant (p = 0.013). The SI difference between CT and FLT for GTVp was also slight (FLT SI = 0.80) and marginally non-significant (p < 0.082). For nodal gross tumor volume, (GTVn), SI was significantly lower for CT based volumes with an estimated SI of 0.22 compared to an estimated SI of 0.70 for FDG-PET/CT (p < 0.0001) and an estimated SI of 0.70 for FLT-PET/CT (p < 0.0001). Boost target volumes in rectal cancer based on combined PET/CT results in lower inter-observer variability compared with CT alone, particularly for nodal disease. The use of FDG and FLT did not appear to be different from this perspective.

View details for Web of Science ID 000244732600005

View details for PubMedID 17241098
Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-animal CT, and bioluminescence imaging JOURNAL OF NUCLEAR MEDICINE Deroose, C. M., De, A., Loening, A. M., Chow, P. L., Ray, P., Chatziioannou, A. F., Gambhir, S. S. 2007; 48 (2): 295-303
Abstract

Recent developments have established molecular imaging of mouse models with small-animal PET and bioluminescence imaging (BLI) as an important tool in cancer research. One of the disadvantages of these imaging modalities is the lack of anatomic information. We combined small-animal PET and BLI technology with small-animal CT to obtain fusion images with both molecular and anatomic information.We used small-animal PET/CT and BLI to detect xenografts of different cell lines and metastases of a melanoma cell line (A375M-3F) that had been transduced with a lentiviral vector containing a trimodality imaging reporter gene encoding a fusion protein with Renilla luciferase, monomeric red fluorescent protein, and a mutant herpes simplex virus type 1 thymidine kinase.Validation studies in mouse xenograft models showed a good coregistration of images from both PET and CT. Melanoma metastases were detected by 18F-FDG PET, 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) PET, CT, and BLI and confirmed by ex vivo assays of Renilla luciferase and mutant thymidine kinase expression. 18F-FHBG PET/CT allowed detection and localization of lesions that were not seen on CT because of poor contrast resolution and were not seen on 18F-FDG PET because of higher background uptake relative to 18F-FHBG.The combination of 18F-FHBG PET, small-animal CT, and BLI allows a sensitive and improved quantification of tumor burden in mice. This technique is potentially useful for the study of the biologic determinants of metastasis and for the evaluation of novel cancer treatments.

View details for Web of Science ID 000244115600037

View details for PubMedID 17268028
PET imaging of colorectal cancer in xenograft-bearing mice by use of an F-18-labeled T84.66 anti-carcinoembryonic antigen diabody JOURNAL OF NUCLEAR MEDICINE Cai, W., Olafsen, T., Zhang, X., Cao, Q., Gambhir, S. S., Williams, L. E., Wu, A. M., Chen, X. 2007; 48 (2): 304-310
Abstract

In this study, we investigated the 18F-labeled anti-carcinoembryonic antigen (CEA) T84.66 diabody, a genetically engineered noncovalent dimer of single-chain variable fragments, for small-animal PET imaging of CEA expression in xenograft-bearing mice.18F labeling of the anti-CEA T84.66 diabody (molecular mass, 55 kDa) was achieved with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB). The biodistribution of the 18F-fluorobenzyl-T84.66 diabody (18F-FB-T84.66 diabody) was evaluated in athymic nude mice bearing subcutaneous LS 174T human colon carcinoma and C6 rat glioma tumors. Serial small-animal PET imaging studies were performed to further evaluate in vivo targeting efficacy and pharmacokinetics.Radiolabeling required 35 +/- 5 (mean +/- SD) min starting from 18F-SFB, and the tracer 18F-FB-T84.66 diabody was synthesized with a specific activity of 1.83 +/- 1.71 TBq/mmol. The decay-corrected radiochemical yield was 1.40% +/- 0.16% (n = 4), and the radiochemical purity was greater than 98%. The radioimmunoreactivity was 57.1% +/- 2.0%. The 18F-FB-T84.66 diabody showed rapid and high tumor uptake and fast clearance from the circulation in the LS 174T xenograft model, as evidenced by both small-animal PET imaging and biodistribution studies. High-contrast small-animal PET images were obtained as early as 1 h after injection of the 18F-FB-T84.66 diabody, and only a background level of activity accumulation was found in CEA-negative C6 tumors. The tracer exhibited predominantly renal clearance, with some activity in the liver and spleen at early time points.The 18F-labeled diabody represents a new class of tumor-specific probes for PET that are based on targeting cell surface antigen expression. The 18F-FB-T84.66 diabody can be used for high-contrast small-animal PET imaging of CEA-positive tumor xenografts. It may be translated to the clinic for PET of CEA-positive malignancies.

View details for Web of Science ID 000244115600038

View details for PubMedID 17268029
Drug Delivery: Keeping Tabs on Nanocarriers. Nature Nanotechnology de la Zerda, A., Gambhir, SS 2007; 2: 745-746
Osseous and Soft Tissue Sarcomas: When Can F-18 FDG PET/CT Evaluation Provide Useful Information? Osseous and Soft Tissue Sarcomas: When Can F-18 FDG PET/CT Evaluation Provide Useful Information? Iagaru, A. 2007; 34 (2)
Development of a Bicistronic Vector for Multimodality Imaging of Estrogen Receptor Activity in a Breast Cancer Model; Preliminary Application. European Journal of Nuclear Medicine and Molecular Imaging Ottobrini L, Ciana P, Moresco R Lecchi M, Belloli S, Martelli C, Todde S, Fazio F, Gambhir SS, Maggi A, Lucignani G. 2007; 35(2): 365-378
Follicular Dendritic Sarcoma within a Focus of Castleman?s Disease. Serial FDG PET/CT in the follow up of Recurrence with Histopatholigic Confirmation. Revista Espanola Medicina Nuclear Journa Iagaru A, Mari C, Gambhir SS 2007; 26(1): 40-45
Endothelial progenitor cells from murine embryonic stem cells: isolation and transplantation for myocardial infarction Li, Z., Wu, J., Sheikh, A., Kraft, D., Cao, F., Xie, X., Patel, M., Gambhir, S., Robbins, R., Cooke, J., Wu, J. SAGE PUBLICATIONS LTD. 2007: 152-152
View details for Web of Science ID 000247470400044
Noninvasive monitoring of ligand-dependent VEGF receptor-2 dimerization with split firefly luciferase Lee, P., Chan, C., Hua, A., Paulmurugan, R., Chan, D., Gambhir, S., Le, Q., Giaccia, A. ELSEVIER SCIENCE INC. 2007: S96-S97
View details for Web of Science ID 000249950200173
Studying the biodistribution of positron emission tomography reporter probes in mice NATURE PROTOCOLS Yaghoubi, S. S., Berger, F., Gambhir, S. S. 2007; 2 (7): 1752-1755
Abstract

Positron emission tomography (PET) reporter probes (PRPs) are used to detect PET reporter gene (PRG) expression in living subjects. This article details protocols for analyzing the biodistribution of a PRP used to detect herpes simplex virus 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk PRG expression. However, the methods described are generalizable to other beta- or gamma/positron-emitting probes. Accumulation of PRPs in animal tissues can be determined by counting PRP activity of isolated tissues, whereas digital whole-body autoradiography (DWBA) provides high-resolution images of PRP biodistribution in 5- to 45-microm tissue slices of killed research animals at a single time point. Biodistribution assay results may be obtained in less than a week after beginning the assay, and DWBA image acquisitions can take up to 3 months depending on the probe's radioisotope.

View details for DOI 10.1038/nprot.2007.228

View details for Web of Science ID 000253139200020

View details for PubMedID 17641641
Reporter Gene Imaging of Protein-Protein Interactions in Living Subjects. Current Opinion in Biotechnology Massoud T, Paulmurugan R, De A, Ray P, Gambhir SS 2007; 18: 31-37
Noninvasive imaging of molecular events with bioluminescent reporter genes in living subjects. Methods in molecular biology (Clifton, N.J.) Ray, P., Gambhir, S. S. 2007; 411: 131-144
Abstract

Bioluminescence imaging has become a very popular tool for noninvasive monitoring of fundamental biological and molecular processes in small living subjects. Luciferases are light-emitting enzymes that can generate light (known as bioluminescence) after reacting with specific substrates. The emitted light is used as a detection system for luciferase activity, which acts as a "reporter" for the activity of any regulatory elements that control its expression. These enzymes are isolated from various organisms, conveniently modified for expression in mammalian cells, and are extensively used in molecular biology and cell culture experiments. Recent advances in optical technology have opened a new dimension for in vivo application of luciferase enzymes in biomedical research. The most commonly utilized luciferases for in vivo bioluminescence are isolated from two very different sources: firefly luciferase (or beetle luciferase) and renilla luciferase (isolated from sea pansy). Although both these luciferases can produce light following interaction with the substrates, structurally and biochemically they are very different. Here we describe the methods and applications of firefly and renilla luciferases in molecular imaging using small animals.

View details for DOI 10.1007/978-1-59745-549-7_10

View details for PubMedID 18287643
2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography/computed tomography in the management of melanoma MOLECULAR IMAGING AND BIOLOGY Iagaru, A., Quon, A., Johnson, D., Gambhir, S. S., McDougall, I. R. 2007; 9 (1): 50-57
Abstract

2-Deoxy-2-[F-18]fluoro-D-glucose (FDG)-positron emission tomography (PET)/computed tomography (CT) is widely available as a powerful imaging modality, combining the ability to detect active metabolic processes and their morphologic features in a single exam. The role of FDG-PET is proven in a variety of cancers, including melanoma, but the estimates of sensitivity and specificity are based in the majority of the published studies on dedicated PET, not PET/CT. Therefore, we were prompted to review our experience with FDG-PET/CT in the management of melanoma.This is a retrospective study on 106 patients with melanoma (20-87 years old; average: 56.8 +/- 15.9), who had whole-body FDG-PET/CT at our institution from January 2003 to June 2005. Thirty-eight patients (35.9%) were women and 68 patients (64.1%) were men. Reinterpretation of the imaging studies for accuracy and data analysis from medical records were performed.All patients had the study for disease restaging. The primary tumor depth (Breslow's thickness) at initial diagnosis was available for 76 patients (71.7%) and ranged from 0.4 to 25 mm (average: 3.56 mm). The anatomic level of invasion in the skin (Clark's level) was determined for 70 patients (66%): 3, level II; 13, level III; 43, level IV; 11, level V. The administered dose of (18)F FDG ranged from 9.8 to 21.6 mCi (average: 15.4 +/- 1.8 mCi). FDG-PET/CT had a sensitivity of 89.3% [95% confidence interval (CI): 78.5-95] and a specificity of 88% (95% CI: 76.2-94.4) for melanoma detection.This study confirms the good results of FDG-PET/CT for residual/recurrent melanoma detection, as well as for distant metastases localization. PET/CT should be an integral part in evaluation of patients with high-risk melanoma, prior to selection of the most appropriate therapy.

View details for DOI 10.1007/s11307-006-0065-0

View details for Web of Science ID 000243545600007

View details for PubMedID 17051322
F-18FDG PET/CT evaluation of osseous and soft tissue sarcomas CLINICAL NUCLEAR MEDICINE Iagaru, A., Quon, A., McDougall, T. R., Gambhir, S. S. 2006; 31 (12): 754-760
Abstract

Osseous and soft tissue sarcomas (OSTS) represent a histologic heterogeneous group of malignant tumors. Most of the current clinical data on the role of F-18 FDG PET in sarcomas come from patients studied with dedicated PET and less frequently with hardware fusion PET/CT. Therefore, we were prompted to review our experience with F-18 FDG PET/CT in OSTS.This is a retrospective study (January 2003-December 2005) of 44 patients with histologic diagnoses of OSTS who had F-18 FDG PET/CT at our institution. The group included 22 men and 22 women with an age range of 2 of 84 years (average, 37 +/- 20.2 years). The administered doses of F-18 FDG range 4.1 to 19.5 mCi (average, 14.3 +/- 3 mCi). Reinterpretation of the imaging studies for accuracy and data analysis from medical records was performed.The sensitivity and specificity of combined F-18 FDG PET/CT were 100% (95% confidence interval [CI] = 75.7-100) and 93.3% (95% CI = 78.7-98.1) for the primary OSTS, and 80% (95% CI = 58.4-91.9) and 86.4% (95% CI = 66.7-95.2) for metastases. When interpreted separately, CT outperformed PET for pulmonary metastases detection: CT was 76.5% sensitive and 88% specific, whereas PET was only 57.1% sensitive but 96.4% specific. For detection of other metastases, CT was 82.3% sensitive and 76% specific, with PET demonstrating 78.6% sensitivity and 92.8% specificity.Relatively similar results (except better specificity for PET and PET/CT) were noted when examining the rate of metastases detection, excluding pulmonary lesions. However, CT had a better detection rate for pulmonary metastases when compared with PET alone. A negative PET scan in the presence of suspicious CT findings in the chest cannot reliably exclude pulmonary metastases from OSTS.

View details for Web of Science ID 000242481400004

View details for PubMedID 17117068
Quantitative micro positron emission tomography (PET) imaging for the in vivo determination of pancreatic islet graft survival NATURE MEDICINE Kim, S., Doudet, D. J., Studenov, A. R., Nian, C., Ruth, T. J., Gambhir, S. S., McIntosh, C. H. 2006; 12 (12): 1423-1428
Abstract

Islet transplantation is an attractive approach for treating type-1 diabetes, but there is a massive loss of transplanted islets. It is currently only possible to estimate islet mass indirectly, through measurement of circulating C-peptide and insulin levels. This type of estimation, however, is not sufficiently sensitive or reproducible for follow-up of individuals who have undergone islet transplantation. Here we show that islet graft survival could be assessed for 1 month in diabetic NOD mice using 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG)-positron emission tomography (PET) technology, the PET signal reflecting insulin secretory capacity of transplanted islets. Expression of the gene encoding viral interleukin-10 (vIL-10), was measurable in real time with PET scanning. Additionally, we addressed the clinical potential of this approach by visualizing transplanted islets in the liver, the preferred clinical transplantation site. We conclude that quantitative in vivo PET imaging is a valid method for facilitating the development of protocols for prolonging islet survival, with the potential for tracking human transplants.

View details for DOI 10.1038/nm1458

View details for Web of Science ID 000242618200024

View details for PubMedID 17143277
Long-term monitoring of transplanted islets using positron emission tomography MOLECULAR THERAPY Lu, Y., Dang, H., Middleton, B., Campbell-Thompson, M., Atkinson, M. A., Gambhir, S. S., Tian, J., Kaufman, D. L. 2006; 14 (6): 851-856
Abstract

Islet transplantation can restore glucose homeostasis in those with type 1 diabetes; however, most recipients eventually lose graft function. A noninvasive method to monitor islets following transplantation would enable assessment of their survival and aid the development of therapeutics to prolong graft survival. Here, we show that recombinant lentivirus can be used to engineer human islets to express a positron emission tomography (PET) reporter gene. Following transplantation into mice, transduced islets could be imaged in vivo using microPET and a radiolabeled probe approved by the FDA for clinical use in humans. The magnitude of signal from engineered islets implanted into the axillary cavity reflected the implanted islet mass. Signals from implanted islets decreased by approximately one-half during the first few weeks following transplantation, which may reflect islet cell death shortly after transplantation. Thereafter, the magnitude of signals from the implanted islets remained fairly constant when the recipients were repetitively reimaged over 90 days. Histological analysis of the implants showed healthy islets with PET reporter-expressing cells distributed throughout the islet architecture. These studies suggest that PET imaging of lentivirus-transduced islets could provide a safe and feasible method for long-term monitoring of islet graft survival.

View details for DOI 10.1016/j.ymthe.2006.08.007

View details for Web of Science ID 000242723300011

View details for PubMedID 16982215
Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells PROTEOMICS Wu, J. C., Cao, F., Dutta, S., Xie, X., Kim, E., Chungfat, N., Gambhir, S., Mathewson, S., Connolly, A. J., Brown, M., Wang, E. W. 2006; 6 (23): 6234-6249
Abstract

Study of stem cells may reveal promising treatment for diseases. The fate and function of transplanted stem cells remain poorly defined. Recent studies demonstrate that reporter genes can monitor real-time survival of transplanted stem cells in living subjects. We examined the effects of a novel and versatile triple fusion (TF) reporter gene construction on embryonic stem (ES) cell function by proteomic analysis. Murine ES cells were stably transduced with a self-inactivating lentiviral vector containing fluorescence (firefly luciferase; Fluc), bioluminescence (monomeric red fluorescence protein; mRFP), and positron emission tomography (herpes simplex virus type 1 truncated thymidine kinase; tTK) reporter genes. Fluorescence-activated cell sorting (FACS) analysis isolated stably transduced populations. TF reporter gene effects on cellular function were evaluated by quantitative proteomic profiling of control ES cells versus ES cells stably expressing the TF construct (ES-TF). Overall, no significant changes in protein quantity were observed. TF reporter gene expression had no effect on ES cell viability, proliferation, and differentiation capability. Molecular imaging studies tracked ES-TF cell survival and proliferation in living animals. In summary, this is the first proteomic study, demonstrating the unique potential of reporter gene imaging for tracking ES cell transplantation non-invasively, repetitively, and quantitatively.

View details for DOI 10.1002/pmic.200600150

View details for Web of Science ID 000242879000011

View details for PubMedID 17080479
PET of vascular endothelial growth factor receptor expression JOURNAL OF NUCLEAR MEDICINE Cai, W., Chen, K., Mohamedali, K. A., Cao, Q., Gambhir, S. S., Rosenblum, M. G., Chen, X. 2006; 47 (12): 2048-2056
Abstract

For solid tumors and metastatic lesions, tumor vascularity is a critical factor in assessing response to therapy. Here we report the first example, to our knowledge, of (64)Cu-labeled vascular endothelial growth factor 121 (VEGF(121)) for PET of VEGF receptor (VEGFR) expression in vivo.VEGF(121) was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and then labeled with (64)Cu for small-animal PET of mice bearing different sized U87MG human glioblastoma xenografts. Blocking experiments and ex vivo histopathology were performed to confirm the in vivo results.There were 4.3 +/- 0.2 DOTA molecules per VEGF(121), and the VEGFR2 binding affinity of DOTA-VEGF(121) was comparable to VEGF(121). (64)Cu labeling of DOTA-VEGF(121) was achieved in 90 +/- 10 min and the radiolabeling yield was 87.4% +/- 3.2%. The specific activity of (64)Cu-DOTA-VEGF(121) was 3.2 +/- 0.1 GBq/mg with a radiochemical purity of >98%. Small-animal PET revealed rapid, specific, and prominent uptake of (64)Cu-DOTA-VEGF(121) in small U87MG tumors (high VEGFR2 expression) but significantly lower and sporadic uptake in large U87MG tumors (low VEGFR2 expression). No appreciable renal clearance of (64)Cu-DOTA-VEGF(121) was observed, although the kidney uptake was relatively high likely due to VEGFR1 expression. Blocking experiments, immunofluorescence staining, and western blot confirmed the VEGFR specificity of (64)Cu-DOTA-VEGF(121).Successful demonstration of the ability of (64)Cu-DOTA-VEGF(121) to visualize VEGFR expression in vivo may allow for clinical translation of this radiopharmaceutical for imaging tumor angiogenesis and guiding antiangiogenic treatment, especially patient selection and treatment monitoring of VEGFR-targeted cancer therapy.

View details for Web of Science ID 000242563900047

View details for PubMedID 17138749
Imaging mitogen-activated protein kinase function in xenograft models of prostate cancer CANCER RESEARCH Ilagan, R., Pottratz, J., Le, K., Zhang, L., Wong, S. G., Ayala, R., Iyer, M., Wu, L., Gambhir, S. S., Carey, M. 2006; 66 (22): 10778-10785
Abstract

Mitogen-activated protein kinases (MAPK) play important roles in malignancy. The ability to detect and quantitate MAPKs in live animal models of cancer will facilitate an understanding of disease progression. We have developed a gene expression-based imaging system that detects and quantifies MAPK activity in prostate cancer tumors implanted into severe combined immunodeficient mice. The imaging technology uses a modified version of two-step transcriptional amplification (TSTA). The tissue specificity of gene expression is imparted by an enhanced version of the prostate-specific antigen regulatory region that expresses GAL4-ELK1. GAL4-ELK1 confers MAPK specificity by activating a firefly luciferase (FLuc) reporter gene when the Ets-like transcription factor (ELK) 1 activation domain is phosphorylated by MAPK. FLuc activity in live animals was detected using the Xenogen In vivo Imaging System. We validated the TSTA-ELK1 system by analyzing its response to epidermal growth factor treatment in transfected tissue culture cells and in adenovirus (AdTSTA-ELK1)-injected prostate cancer xenograft tumors. We measured MAPK activity in two well-characterized xenograft models, CWR22 and LAPC9. Although no significant differences in MAPK levels were detected between androgen-dependent and androgen-independent xenografts, the CWR22 models display significantly higher levels of AdTSTA-ELK1 activity versus LAPC9. Western blots of tumor extracts showed that the elevated imaging signal in CWR22 xenografts correlated with elevated levels of phosphorylated extracellular signal-regulated kinase 1/2 but not p38 or c-Jun NH(2)-terminal kinase. We conclude that a gene expression-based optical imaging system can accurately detect and quantify MAPK activity in live animals.

View details for DOI 10.1158/0008-5472.CAN-05-3577

View details for Web of Science ID 000242264400019

View details for PubMedID 17108114
How molecular imaging is speeding up antiangiogenic drug development MOLECULAR CANCER THERAPEUTICS Cai, W., Rao, J., Gambhir, S. S., Chen, X. 2006; 5 (11): 2624-2633
Abstract

Drug development is a long process that generally spans about 10 to 15 years. The shift in recent drug discovery to novel agents against specific molecular targets highlights the need for more robust molecular imaging platforms. Using molecular probes, molecular imaging can aid in many steps of the drug development process, such as providing whole body readout in an intact system, decreasing the workload and speeding up drug development/validation, and facilitating individualized anticancer treatment monitoring and dose optimization. The main focus of this review is the recent advances in tumor angiogenesis imaging, and the targets include vascular endothelial growth factor and vascular endothelial growth factor receptor, integrin alpha(v)beta(3), matrix metalloproteinase, endoglin (CD105), and E-selectin. Through tumor angiogenesis imaging, it is expected that a robust platform for understanding the mechanisms of tumor angiogenesis and evaluating the efficacy of novel antiangiogenic therapies will be developed, which can help antiangiogenic drug development in both the preclinical stage and the clinical settings. Molecular imaging has enormous potential in improving the efficiency of the drug development process, including the specific area of antiangiogenic drugs.

View details for DOI 10.1158/1535-7163.MCT-06-0395

View details for Web of Science ID 000242138000004

View details for PubMedID 17121909
Noninvasive imaging of bone marrow stem cell homing, survival, and proliferation in. ischemic myocardium Lin, S., Sheikh, A. Y., Cao, F., Gambhir, S. S., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2006: 660-660
View details for Web of Science ID 000241792804305
An intramolecular folding sensor for imaging estrogen receptor-ligand interactions PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Paulmurugan, R., Gambhir, S. S. 2006; 103 (43): 15883-15888
Abstract

Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla/firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.

View details for DOI 10.1073/pnas.0607385103

View details for Web of Science ID 000241568500029

View details for PubMedID 17043219
Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumors PROTEIN ENGINEERING DESIGN & SELECTION Venisnik, K. M., Olafsen, T., Loening, A. M., Iyer, M., Gambhir, S. S., Wu, A. M. 2006; 19 (10): 453-460
Abstract

An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 +/- 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 +/- 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using (124)I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.

View details for DOI 10.1093/protein/gzl030

View details for Web of Science ID 000240544900003

View details for PubMedID 16882674
F-18FDG PET/CT imaging of extramammary Paget disease of the perianal region CLINICAL NUCLEAR MEDICINE Niederkohr, R. D., Gambhir, S. S. 2006; 31 (9): 561-563
View details for Web of Science ID 000240122400015

View details for PubMedID 16921286
Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output PROTEIN ENGINEERING DESIGN & SELECTION Loening, A. M., Fenn, T. D., Wu, A. M., Gambhir, S. S. 2006; 19 (9): 391-400
Abstract

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

View details for DOI 10.1093/protein/gzl023

View details for Web of Science ID 000240544600001

View details for PubMedID 16857694
F-18 fluorodeoxyglucose PET/CT as an imaging tool for staging and restaging cutaneous angiosarcoma of the scalp CLINICAL NUCLEAR MEDICINE Vasanawala, M. S., Wang, Y., Quon, A., Gambhir, S. S. 2006; 31 (9): 534-537
Abstract

Cutaneous angiosarcoma of the scalp is a rare highly aggressive malignant tumor that typically afflicts elderly patients and commonly presents with extensive local spread and distant metastasis. Distant metastases favor lung, liver, lymph nodes, and skin. Overall, the prognosis is poor. It differs from other soft tissue sarcomas in that the size of the lesion at presentation instead of tumor grade is the important prognostic factor. Optimal treatment is yet to be determined. Wide-margin complete excision with postoperative radiotherapy has been the most effective therapy. Chemotherapy and gene therapy have been used with some success. Local extent is critical in surgical planning, especially in the head and face, and is difficult to determine accurately with clinical examination and morphologic imaging tools. We report the case of a 70-year-old man diagnosed with multifocal angiosarcoma of the scalp. PET/CT imaging with F-18 2-fluoro-2-deoxyglucose (F-18 FDG) not only showed avid FDG uptake by an angiosarcoma (SUVmax = 10.7), but also simultaneously showed local extension of multifocal lesions with periosteal involvement and excluded metastatic abdominal nodal disease. PET/CT imaging after chemotherapy and before radiation therapy showed complete resolution of FDG uptake in the scalp and osseous lesions. Evaluation of more cases of this subset of soft tissue sarcoma with FDG PET/CT may suggest a possible role in not only staging angiosarcomas to determine the extent of local as well as distant disease, but also to potentially help determine response to therapy and early recognition of local or distant recurrence.

View details for Web of Science ID 000240122400005

View details for PubMedID 16921276
Noninvasive imaging of islet grafts using positron-emission tomography PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lu, Y., Dang, H., Middleton, B., Zhang, Z., Washburn, L., Stout, D. B., Campbell-Thompson, M., Atkinson, M. A., Phelps, M., Gambhir, S. S., Tian, J., Kaufman, D. L. 2006; 103 (30): 11294-11299
Abstract

Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. However, islet transplantation is not routinely successful because most islet recipients gradually lose graft function. Furthermore, serological markers of islet function are insensitive to islet loss until the latter stages of islet graft rejection. A noninvasive method of monitoring islet grafts would aid in the assessment of islet graft survival and the evaluation of interventions designed to prolong graft survival. Here, we show that recombinant adenovirus can engineer isolated islets to express a positron-emission tomography (PET) reporter gene and that these islets can be repeatedly imaged by using microPET after transplantation into mice. The magnitude of signal from engineered islets implanted into the axillary cavity was directly related to the implanted islet mass. PET signals attenuated over the following weeks because of the transient nature of adenovirus-mediated gene expression. Because the liver is the preferred site for islet implantation in humans, we also tested whether islets could be imaged after transfusion into the mouse liver. Control studies revealed that both intrahepatic islet transplantation and hyperglycemia altered the biodistribution kinetics of the PET probe systemically. Although transplanted islets were dispersed throughout the liver, clear signals from the liver region of mice receiving PET reporter-expressing islets were detectable for several weeks. Viral transduction, PET reporter expression, and repeated microPET imaging had no apparent deleterious effects on islet function after implantation. These studies lay a foundation for noninvasive quantitative assessments of islet graft survival using PET.

View details for DOI 10.1073/pnas.0603909103

View details for Web of Science ID 000239353900035

View details for PubMedID 16868090
Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts CIRCULATION Kutschka, I., Chen, I. Y., Kofidis, T., Arai, T., von Degenfeld, G., Sheikh, A. Y., Hendry, S. L., Pearl, J., Hoyt, G., Sista, R., Yang, P. C., Blau, H. M., Gambhir, S. S., Robbins, R. C. 2006; 114: I167-I173
Abstract

Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices.H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1 x 10(6) genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 microg/mL), or (5) GF/MG/FGF (10 microg/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function.Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.

View details for DOI 10.1161/CIRCULATIONAHA.105.001297

View details for Web of Science ID 000238688200028

View details for PubMedID 16820568
Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts CIRCULATION Kutschka, I., Kofidis, T., Chen, I. Y., von Degenfeld, G., Zwierzchoniewska, M., Hoyt, G., Arai, T., Lebl, D. R., Hendry, S. L., Sheikh, A. Y., Cooke, D. T., Connolly, A., Blau, H. M., Gambhir, S. S., Robbins, R. C. 2006; 114: I174-I180
Abstract

Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts.H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1x10(6) mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a "working heart" model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21+/-0.03; mH9c2: 0.21+/-0.04; control: 0.15+/-0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0+/-6.2; mH9c2: 48.7+/-6.1; control: 34.3+/-6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology.Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.

View details for DOI 10.1161/CIRCULATIONAHA.105.001370

View details for Web of Science ID 000238688200029

View details for PubMedID 16820569
Evaluation of firefly luciferase biolurninescence mediated photodynarnic toxicity in cancer cells MOLECULAR IMAGING AND BIOLOGY Schipper, M. L., Patel, M. R., Gambhir, S. S. 2006; 8 (4): 218-225
Abstract

This work investigated whether fLuc-catalyzed oxidation of D-luciferin generates sufficient light to induce photodynamic toxicity in cancer cells.Light emission was assessed via cooled CCD (charge-coupled device) camera. Parental and fLuc expressing cancer cells were exposed to subtoxic concentrations of photosensitizers (Rose Bengal or hypericin) and D-luciferin, sunlight, or lamplight. Toxicity was assessed by MTT assay.fLuc expressing cells emitted up to 500-fold higher levels of photons than parental cell lines. Although exposure to photosensitizer and sunlight reduced survival of various cell lines, survival of fLuc expressing cells incubated with photosensitizer and D-luciferin, or photosensitizer and lamplight, did not differ significantly from parental or untreated cells.Contesting recent reports, fLuc bioluminescence does not generate sufficient photons to induce Rose Bengal or hypericin photodynamic toxicity in a range of malignant and nonmalignant cell lines, and is not suitable as a generalizable approach to antineoplastic therapy.

View details for DOI 10.1007/s11307-006-0048-1

View details for Web of Science ID 000239124800004

View details for PubMedID 16791748
Merkel cell carcinoma: Is there a role for 2-deoxy-2-[F-18]fluoro-D-glucose-positron emission tomography/computed tomography? MOLECULAR IMAGING AND BIOLOGY Iagaru, A., Quon, A., McDougall, I. R., Gambhir, S. S. 2006; 8 (4): 212-217
Abstract

2-Deoxy-2-[F-18]fluoro-D-glucose (FDG)-positron emission tomography (PET)/computed tomography (CT) is becoming widely available as a powerful imaging modality, combining the ability to detect active metabolic processes and their morphologic features in a single study. The role of FDG-PET/CT is proven in lymphoma, melanoma, colorectal carcinoma, and other cancers. However, there are rare malignancies such as Merkel cell carcinoma that can potentially be evaluated with PET/CT. We were therefore prompted to review our experience with FDG-PET/CT in the management of patients with Merkel cell carcinoma.This is a retrospective case series of six patients with Merkel cell carcinoma, 58-81 years old (average 69 +/- 8.3), who had whole-body PET/CT at our institution from January 1st, 2003 to August 31st, 2005. Two patients were women and four were men. Reinterpretation of the imaging studies for accuracy and data analysis from medical records were performed.Twelve examinations were acquired for the six patients (one patient had six PET/CT, one patient had two PET/CT, and four patients had one PET/CT). The injected FDG doses ranged 381.1-669.7 MBq (average 573.5 +/- 70.3). Four patients had the PET/CT as part of initial staging, and two patients had the exam for restaging (after surgery and XRT). A total of six Merkel lesions (pancreas, adrenal, lip, submandibular lymph nodes, cervical lymph nodes, and parapharyngeal soft tissue) were identified in three patients and confirmed on histopathological examination. The FDG uptake in these areas was intense, with maximum standardized uptake value (SUVmax) values of 5-14 (average 10.4 +/- 3.8). In one patient, the PET/CT scan identified abnormal focal distal sigmoid uptake that was biopsied and diagnosed as adenocarcinoma. Two patients had negative scans and had no clinical evidence of disease on follow-up office visits (up to one year after PET/CT).This case series suggests that FDG-PET/CT may have a promising role in the management of patients with Merkel cell carcinoma.

View details for DOI 10.1007/s11307-006-0047-2

View details for Web of Science ID 000239124800003

View details for PubMedID 16724293
"Flying through" and "flying around" a PET/CT scan: Pilot study and development of 3D integrated F-18-FDG PET/CT for virtual bronchoscopy and colonoscopy JOURNAL OF NUCLEAR MEDICINE Quon, A., Napel, S., Beaulieu, C. F., Gambhir, S. S. 2006; 47 (7): 1081-1087
Abstract

The objective of this pilot project was to devise a new image acquisition and processing technique to produce PET/CT images rendered in 3-dimensional (3D) volume that can then be reviewed in several 3D formats such as virtual bronchoscopy and colonoscopy "fly-throughs" and external "fly-arounds."We tested the new imaging and processing protocol on 24 patients with various malignancies to determine whether it could dependably acquire and reformat standard tomographic 2-dimensional PET/CT images into 3D renderings.This new technique added helpful information to the diagnostic interpretation for 2 of the 24 patients. Further, in the 6 patients undergoing mediastinoscopy, bronchoscopy, or endoscopy, 3D imaging helped in preprocedural planning.In this initial study, we demonstrated both the feasibility of rendering PET/CT images into 3D volumes and the potential clinical utility of this technique for diagnostic lesion characterization and preprocedural planning.

View details for Web of Science ID 000238879300008

View details for PubMedID 16818940
Significance of one-bead-one-compound combinational chemistry NATURE CHEMICAL BIOLOGY Chen, X., Gambhir, S. S. 2006; 2 (7): 351-352
View details for Web of Science ID 000238376400004

View details for PubMedID 16783336
Near-infrared fluorescent deoxyglucose analogue for tumor optical imaging in cell culture and living mice BIOCONJUGATE CHEMISTRY Cheng, Z., Levi, J., Xiong, Z., Gheysens, O., Keren, S., Chen, X., Gambhir, S. S. 2006; 17 (3): 662-669
Abstract

2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging, and therapy monitoring of cancer and other diseases. Nonradioactive glucose analogues enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A nonradioactive fluorescent deoxyglucose analogue may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated D-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of the Cy5.5-D-glucosamine (Cy5.5-2DG) conjugate for NIR fluorescence imaging of tumors in a preclinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and D-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 and 4 degrees C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 degrees C incubation, while they exhibit marginal uptake at 4 degrees C. The tumor cell uptake of Cy5.5-2DG cannot be blocked by the 50 mM D-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization was clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min postinjection (pi), and the highest U87MG tumor/muscle ratios of 2.81 +/- 0.10 and 3.34 +/- 0.23 were achieved 24 h pi for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micropositron emission tomography imaging study shows that [18F]FDG preferentially localizes to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h postadministration of the probe. In conclusion, the NIR fluorescent glucose analogues, Cy5.5-2DG and Cy5.5-NHS, both demonstrate tumor-targeting abilities in cell culture and living mice. More studies are warranted to further explore their application for optical tumor imaging. To develop NIR glucose analogues with the ability to target GLUTs/hexokinase, it is highly important to select NIR dyes with a reasonable molecular size.

View details for DOI 10.1021/bc050345c

View details for Web of Science ID 000237576000013

View details for PubMedID 16704203
Noninvasive evaluation of immunosuppressive drug efficacy on acute donor cell survival MOLECULAR IMAGING AND BIOLOGY Gheysens, O., Lin, S., Cao, F., Wang, D., Chen, I. Y., Rodriguez-Porcel, M., Min, J. J., Gambhir, S. S., Wu, J. C. 2006; 8 (3): 163-170
Abstract

The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death. PROCEDURES AND RESULTS: Embryonic rat H9c2 myoblasts were stably transduced to express firefly luciferase reporter gene (H9c2-Fluc). H9c2-Fluc cells (3x10(6)) were injected into thigh muscles of Sprague-Dawley rats (N=30) treated with cyclosporine, dexamethasone, mycophenolate mofetil, tacrolimus, or saline from day -3 to day +14. Longitudinal optical bioluminescence imaging was performed over two weeks. Fluc activity was 40.0+/-12.1% (dexamethasone), 30.5+/-12.5% (tacrolimus), and 21.5+/-3.5% (mycophenolate) vs. 12.0+/-5.0% (control) and 8.3+/-5.0% (cyclosporine) at day 4 (P<0.05). However, by day 14, cell signals had decreased drastically to <10% for all groups despite drug therapy.This study demonstrates the ability of optical molecular imaging for tracking cell survival noninvasively and raises important questions with regard to the overall efficacy of immunosuppressives for prolonging transplanted cell survival.

View details for DOI 10.1007/s11307-006-0038-3

View details for Web of Science ID 000237754300003

View details for PubMedID 16555032
Preclinical safety evaluation of F-18-FHBG: A PET reporter probe for Imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk's expression JOURNAL OF NUCLEAR MEDICINE Yaghoubi, S. S., Couto, M. A., Chen, C. C., Polavaram, L., Cui, G. G., Sen, L. Y., Gambhir, S. S. 2006; 47 (4): 706-715
Abstract

9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) is a sensitive and specific PET reporter probe for imaging the PET reporter genes, herpes simplex 1 thymidine kinase (HSV1-tk) and its mutant HSV1-sr39tk. (18)F-FHBG has suitable pharmacokinetics and dosimetry for clinical applications and imaging of HSV1-TK has been demonstrated in the livers of hepatocellular cancer patients.Male and female Sprague-Dawley rats and New Zealand White rabbits were divided into equal groups receiving either 14 microg/kg cold FHBG or carrier solution, for a 14-d acute toxicity assessment. We monitored body weight, food and water consumption, body temperature, cardiovascular electrical and functional indices, respiratory performance and oxygen saturation, comprehensive blood chemistry, complete blood count (CBC), and urinalysis. We conducted daily cage-side examinations for the detection of any clinical abnormalities. Tissues of the animals that were euthanized and necropsied on day 14 were prepared for histopathologic examination.No significant differences in cardiovascular and respiratory parameters, food consumption, body weight, urine components, or clinical signs attributable to test article toxicity were observed between the treatment and control groups. Any differences noted in the blood chemistry and CBC parameters were deemed to be incidental findings unrelated to the administration of the FHBG.Acute toxicity evaluation of FHBG at 100 times the expected human dose does not indicate harm to organ function or tissues. The Food and Drug Administration has approved FHBG as an Investigational New Drug.

View details for Web of Science ID 000236593600034

View details for PubMedID 16595506
Monitoring the antitumor response of naive and memory CD8 T cells in RAG1(-/-) mice by positron-emission tomography JOURNAL OF IMMUNOLOGY Su, H., Chang, D. S., Gambhir, S. S., Braun, J. 2006; 176 (7): 4459-4467
Abstract

Therapeutic antitumor immunity depends on a highly migratory CTL population capable of activation and trafficking between lymphoid and tumor-bearing microanatomic sites. We recently adapted positron-emission tomography gene expression imaging for noninvasive, longitudinal localization and quantitation of antitumor T lymphocyte migration in vivo. In this study, we apply this system to enumerate the temporal accumulation of naive vs memory T cells. Naive or memory OT-1 CD8(+) T cells, retrovirally marked with the sr39TK gene, were adoptively transferred into RAG1(-/-) animals bearing EL-4 or EG.7 (an OVA-expressing subline), and repetitively imaged by microPET over several weeks. Memory cells demonstrated early accumulation and apparent proliferation, with large T cell numbers at the Ag-positive tumor as early as day 1 after T cell transfer. Naive T cells did not accumulate in the E.G7 tumor until day 8, and reached only 25% of the peak levels achieved by memory T cells. Both naive and memory cells eradicated the Ag-expressing tumor at a comparable density of intratumoral T cells (2-4 x 10(6)/g). However, due to the slower rate of T cell expansion and continued tumor growth, naive cells required approximately 10-fold higher Ag-specific precursor frequency to reach a tumoricidal cell density. As recently reported, memory but not naive T cells accumulated in local lymph nodes and lungs, where they persisted as a resident population after tumor eradication. Positron-emission tomography-based immunologic imaging is a noninvasive modality providing unique and meaningful information on the dynamics of the antitumor CTL response.

View details for Web of Science ID 000238769300073

View details for PubMedID 16547284
Bioluminescent-inescent monitoring of NIS-mediated I-131 ablative effects in MCF-7 Xenografts MOLECULAR IMAGING Ghosh, M., Gambhir, S. S., De, A., Nowels, K., Goris, M., Wapnir, I. 2006; 5 (2): 76-84
Abstract

Optical imaging has made it possible to monitor response to anticancer therapies in tumor xenografts. The concept of treating breast cancers with (131)I is predicated on the expression of the Na(+)/I- symporter (NIS) in many tumors and uptake of I- in some. The pattern of (131)I radioablative effects were investigated in an MCF-7 xenograft model dually transfected with firefly luciferase and NIS genes. On Day 16 after tumor cell implantation, 3 mCi of (131)I was injected. Bioluminescent imaging using d-luciferin and a cooled charge-coupled device camera was carried out on Days 1, 2, 3, 7, 10, 16, 22, 29, and 35. Tumor bioluminescence decreased in (131)I-treated tumors after Day 3 and reached a nadir on Day 22. Conversely, bioluminescence steadily increased in controls and was 3.85-fold higher than in treated tumors on Day 22. Bioluminescence in (131)I-treated tumors increased after Day 22, corresponding to tumor regrowth. By Day 35, treated tumors were smaller and accumulated 33% less (99m)TcO(4)(-) than untreated tumors. NIS immunoreactivity was present in <50% of (131)I-treated cells compared to 85-90% of controls. In summary, a pattern of tumor regression occurring over the first three weeks after (131)I administration was observed in NIS-expressing breast cancer xenografts.

View details for DOI 10.2310/7290.2006.00008

View details for Web of Science ID 000239285900004

View details for PubMedID 16954021
Using radiolabeled DNA as an imaging agent to recognize protein targets JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S. 2006; 47 (4): 557-558
View details for Web of Science ID 000236593600014

View details for PubMedID 16595486
Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects NANO LETTERS Cai, W. B., Shin, D. W., Chen, K., Gheysens, O., Cao, Q. Z., Wang, S. X., Gambhir, S. S., Chen, X. Y. 2006; 6 (4): 669-676
Abstract

We report the in vivo targeting and imaging of tumor vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled quantum dots (QDs). Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered QD705-RGD intravenously. The tumor fluorescence intensity reached maximum at 6 h postinjection with good contrast. The results reported here open up new perspectives for integrin-targeted near-infrared optical imaging and may aid in cancer detection and management including imaging-guided surgery.

View details for DOI 10.1021/nl052405t

View details for Web of Science ID 000236916200015

View details for PubMedID 16608262
Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation PHYSIOLOGICAL GENOMICS Wu, J. C., Spin, J. M., Cao, F., Lin, S. A., Xie, X. Y., Gheysens, O., Chen, I. Y., Sheikh, A. Y., Robbins, R. C., Tsalenko, A., Gambhir, S. S., Quertermous, T. 2006; 25 (1): 29-38
Abstract

Stem cell therapy offers exciting promise for treatment of ischemic heart disease. Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively. Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles. Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes. Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations. Microarray studies comparing gene expression in nontransduced control ES cells vs. stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability. Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes. Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability. Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging. Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells.

View details for DOI 10.1152/physiolgenomics.00254.2005

View details for Web of Science ID 000236722700004

View details for PubMedID 16390873
Self-illuminating quantum dot conjugates for in vivo imaging NATURE BIOTECHNOLOGY So, M. K., Xu, C. J., Loening, A. M., Gambhir, S. S., Rao, J. H. 2006; 24 (3): 339-343
Abstract

Fluorescent semiconductor quantum dots hold great potential for molecular imaging in vivo. However, the utility of existing quantum dots for in vivo imaging is limited because they require excitation from external illumination sources to fluoresce, which results in a strong autofluorescence background and a paucity of excitation light at nonsuperficial locations. Here we present quantum dot conjugates that luminesce by bioluminescence resonance energy transfer in the absence of external excitation. The conjugates are prepared by coupling carboxylate-presenting quantum dots to a mutant of the bioluminescent protein Renilla reniformis luciferase. We show that the conjugates emit long-wavelength (from red to near-infrared) bioluminescent light in cells and in animals, even in deep tissues, and are suitable for multiplexed in vivo imaging. Compared with existing quantum dots, self-illuminating quantum dot conjugates have greatly enhanced sensitivity in small animal imaging, with an in vivo signal-to-background ratio of > 10(3) for 5 pmol of conjugate.

View details for DOI 10.1038/nbt1188

View details for Web of Science ID 000235868600037

View details for PubMedID 16501578
In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery CIRCULATION Cao, F., Lin, S., Xie, X. Y., Ray, P., Patel, M., Zhang, X. Z., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X. Y., Weissman, I. L., Gambhir, S. S., Wu, J. C. 2006; 113 (7): 1005-1014
Abstract

Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities.Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1x10(7) of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7x10(7)+/-5.8x10(6) photons.s(-1).cm(-2) per steradian (sr) and 0.08+/-0.03% injected dose/g, respectively (P<0.05 versus control). Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (1 mL/kg).This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.

View details for DOI 10.1161/CIRCULATIONHA.105.588954

View details for Web of Science ID 000235403900015

View details for PubMedID 16476845
Visualization of telomerase reverse transcriptase (hTERT) promoter activity using a trimodality fusion reporter construct JOURNAL OF NUCLEAR MEDICINE Padmanabhan, P., Otero, J., Ray, P., Paulmurugan, R., Hoffman, A. R., Gambhir, S. S., Biswal, S., Ulaner, G. A. 2006; 47 (2): 270-277
Abstract

Our goal was to noninvasively measure chemotherapy-induced changes in the expression of critical tumor growth genes. To achieve this goal, we used radionuclide and optical methods to measure changes in human telomerase reverse transcriptase (hTERT) gene expression in tumor cells before and after 5-fluorouracil treatment.A fusion reporter construct, containing humanized Renilla luciferase (hrl, for bioluminescent imaging), monomeric red fluorescence protein 1 (mrfp1, for fluorescent imaging), and a truncated thymidine kinase (ttk, for imaging of radiolabeled acycloguanosines), was placed under the control of hTERT promoter fragments. These constructs were introduced into tumor cell lines with and without hTERT expression. Transfected cells were treated with 5-fluorouracil, a chemotherapeutic that decreases hTERT gene expression, and treatment-induced changes in hTERT promoter activity were imaged.When the fusion construct is introduced into cell lines that express hTERT, all 3 reporter systems are highly expressed and hTERT promoter activity can be visualized. Cell lines lacking hTERT transcription show no significant reporter expression. Decreases in hTERT gene expression caused by 5-fluorouracil treatment could be visualized in living 293T cells by both fluorescent microscopy and bioluminescent imaging.hTERT promoter activity can be monitored by 1 radionuclide and 2 optical reporter systems using a single reporter construct. This in vitro study provides evidence that our multimodality reporter construct can be used to study the expression of a critical tumor growth gene in living subjects.

View details for Web of Science ID 000235283500027

View details for PubMedID 16455633
Diagnosis of aseptic deep venous thrombosis of the upper extremity in a cancer patient using fluorine-18 fluorodeoxyglucose positron emission tomography/computerized tomography (FDG PET/CT) ANNALS OF NUCLEAR MEDICINE Do, B., Mari, C., Biswal, S., Kalinyak, J., Quon, A., Gambhir, S. S. 2006; 20 (2): 151-155
Abstract

We describe a patient with a history of recurrent squamous cell carcinoma of the tongue and abnormal FDG uptake in the left arm during a re-staging FDG PET/CT. After revision of the patient's clinical history, tests and physical exam, the abnormal FDG uptake was found to correspond to an extensive aseptic deep venous thrombosis of the upper extremity.

View details for Web of Science ID 000236242700010

View details for PubMedID 16615425
Noninvasive indirect imaging of vascular endothelial growth factor gene expression using bioluminescence imaging in living transgenic mice PHYSIOLOGICAL GENOMICS Wang, Y. L., Iyer, M., Annala, A., Wu, L., Carey, M., Gambhir, S. S. 2006; 24 (2): 173-180
Abstract

Vascular endothelial growth factor (VEGF) plays a critical role in the early activation of stromal tissues during wound healing and tumor growth. We report the use of a two-step transcriptional amplification (TSTA) approach to augment the transcriptional activity of the relatively weak VEGF promoter (pVEGF) using firefly luciferase (fl) reporter gene and bioluminescence imaging (BLI). In cell culture, we demonstrate that TSTA-based fl gene expression can be significantly enhanced over the direct one-step system. Using a transgenic mouse model (pVEGF-TSTA-fl), we demonstrate the induction of VEGF gene expression using a wound-healing model and a subcutaneous mammary tumor model. In skin-wounding experiments, pVEGF-induced fl expression in the wound lesion is detected on days 4 and 5 and peaks on days 15-22. Furthermore, the bioluminescence signal shows good correlation with the endogenous VEGF protein levels in the wound tissue (r2 = 0.70). In the mammary tumor model, fl expression is detected on day 3, peaks at day 17, and declines thereafter. These results support the use of noninvasive BLI for the longitudinal monitoring of VEGF induction during wound healing and tumor progression, and this mouse model should find use in various applications in which it is important to noninvasively study VEGF gene expression.

View details for DOI 10.1152/physiolgenomics.00308.2004

View details for Web of Science ID 000234590300011

View details for PubMedID 16410544
Quantitative PET imaging of tumor integrin alpha(v)beta(3) expression with F-18-FRGD2 JOURNAL OF NUCLEAR MEDICINE Zhang, X. Z., Xiong, Z. M., Wu, Y., Cai, W. B., Tseng, J. R., Gambhir, S. S., Chen, X. Y. 2006; 47 (1): 113-121
Abstract

The development of noninvasive methods to visualize and quantify integrin alpha(v)beta(3) expression in vivo appears to be crucial for the success of antiangiogenic therapy based on integrin antagonism. Precise documentation of integrin receptor levels will allow appropriate selection of patients who will most likely benefit from an antiintegrin treatment regimen. Imaging can also be used to provide an optimal dosage and time course for treatment based on receptor occupancy studies. In addition, imaging integrin expression will be important to evaluate antiintegrin treatment efficacy and to develop new therapeutic drugs with favorable tumor targeting and in vivo kinetics. We labeled the dimeric RGD peptide E[c(RGDyK)](2) with (18)F and evaluated its tumor-targeting efficacy and pharmacokinetics of (18)F-FB-E[c(RGDyK)](2) ((18)F-FRGD2).E[c(RGDyK)](2) was labeled with (18)F by conjugation coupling with N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) under a slightly basic condition. The in vivo metabolic stability of (18)F-FRGD2 was determined. The diagnostic value after injection of (18)F-FRGD2 was evaluated in various xenograft models by dynamic microPET followed by ex vivo quantification of tumor integrin level.Starting with (18)F(-) Kryptofix 2.2.2./K(2)CO(3) solution, the total reaction time for (18)F-FRGD2, including final high-performance liquid chromatography purification, is about 200 +/- 20 min. Typical decay-corrected radiochemical yield is 23% +/- 2% (n = 20). (18)F-FRGD2 is metabolically stable. The binding potential extrapolated from graphical analysis of PET data and Logan plot correlates well with the receptor density measured by sodium dodecyl sulfate polyacrylamide electrophoresis and autoradiography in various xenograft models. The tumor-to-background ratio at 1 h after injection of (18)F-FRGD2 also gives a good linear relationship with the tumor tissue integrin level.The dimeric RGD peptide tracer (18)F-FRGD2, with high integrin specificity and favorable excretion profile, may be translated into the clinic for imaging integrin alpha(v)beta(3) expression. The binding potential calculated from simplified tracer kinetic modeling such as the Logan plot appears to be an excellent indicator of tumor integrin density.

View details for Web of Science ID 000234679300022

View details for PubMedID 16391195
Creating self-illuminating quantum dot conjugates Creating self-illuminating quantum dot conjugates So, M., et al 2006; 3: 1160-1164
Split luciferase complementation assay for studying interaction of proteins x and y in living mice. CSH protocols Paulmurugan, R., Ray, P., De, A., Chan, C. T., Gambhir, S. S. 2006; 2006 (6)
Abstract

This protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), as described below, or with tumor models grown from tumor cells stably expressing the complete reporter system. For optical imaging, the number of cells implanted can be relatively low (~1-5 × 10(6)), and imaging can begin even before the tumors are palpable. When using a regulated reporter gene, it may be necessary to perform a dose-dependent pilot experiment with the inducer or repressor before performing the primary experiments. Animal models other than mice can be used, including rats and, in theory, transgenic animals in which the reporter constructs have been stably integrated into the genome. Animals larger than the rat would be difficult to image due to poor penetration of light. For these larger-animal models, the use of other imaging technologies such as microPET should be considered.

View details for DOI 10.1101/pdb.prot4595

View details for PubMedID 22485982
Bioluminescence imaging of systemic tumor targeting using a prostate-specific lentiviral vector HUMAN GENE THERAPY Iyer, M., Salazar, F. B., Wu, L., Carey, M., Gambhir, S. S. 2006; 17 (1): 125-132
Abstract

Developments in vector design using tissue-specific and tumor-specific promoters have led to significant improvements in tumor-targeting strategies. These developments combined with the ability to monitor gene expression by molecular imaging have facilitated the detection and prolonged monitoring of disease progression in small-animal models. Bioluminescence imaging offers a convenient and sensitive platform for monitoring gene expression patterns in preclinical models of gene therapy. Targeting a specific subset of cells/tissues via systemic delivery of vectors would be highly beneficial in gene therapy protocols. Using a two-step transcriptional amplification (TSTA)-based lentiviral vector (LV-TSTA), we demonstrate specific targeting of prostate tumors in vivo after systemic administration of lentivirus. Four days after intravenous administration of LV-TSTA into adult severe combined immunodeficient (SCID) mice (n=5) carrying subcutaneous prostate tumors, we found significant levels of transduction at the tumor site when compared with other organs (p<0.05). Gene expression was sustained in the tumor for up to 3 weeks (7.3x10(4)+/-2x10(4) photons/ sec/cm2/steradian (p/sec/cm2/sr) on day 4 and 7.0x10(4)+/-4x10(4) p/sec/cm2/sr on day 21). Low levels of transduction were also observed in the spleen and liver (5.0x10(2)+/-1.7x10(2) p/sec/cm2/sr). The results from this study support the use of TSTA-based lentiviral vectors for prostate tumor targeting after systemic delivery. Noninvasive imaging using such vectors should be useful for monitoring long-term gene expression in gene therapy applications.

View details for Web of Science ID 000234720400012

View details for PubMedID 16409131
Long-Term Monitoring of Transplanted Islets using Positron Emission Tomography. Molecular Therapy Lu Y, Dang H, Middleton B, Thompson M, Atkinson M, Gambhir SS, Tian J, Kaufman D. 2006; 14: 851-856
Molecular imaging of HIF-1 alpha and pVHL interaction in living subjects using firefly luciferase complementation - Improvement over the Renilla luciferase system Choi, C. Y., Paulmurugan, R., Chan, D. A., Sutphin, P. D., Le, Q., Koong, A., Gambhir, S. S., Giaccia, A. J. ELSEVIER SCIENCE INC. 2006: S48-S49
View details for Web of Science ID 000241221600083
HaloTag protein-mediated site-specific conjugation of bioluminescent proteins to quantum dots ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Zhang, Y., So, M., Loening, A. M., Yao, H., Gambhir, S. S., Rao, J. 2006; 45 (30): 4936-4940
View details for DOI 10.1002/anie.200601197

View details for Web of Science ID 000239543300010

View details for PubMedID 16807952
Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro NATURE PROTOCOLS Yaghoubi, S. S., Gambhir, S. S. 2006; 1 (4): 2137-2142
Abstract

The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.

View details for DOI 10.1038/nprot.2006.334

View details for Web of Science ID 000251155500058

View details for PubMedID 17487205
Bifunctional Antibody-Renilla Luciferase Fusion Protein for in Vivo Optical Detection of Tumors. Protein Engineering, Design & Selection Venisnik KM, Olafsen T, Loening AM, Iyer M, Gambhir SS, Wu AM 2006; 19: 453-460
Consensus Guided Mutagenesis of Renilla Luciferase Yields Enhanced Stability and Light Output. Protein Engineering, Design & Selection Loening AM, Fenn TD, Wu, AM, Gambhir SS 2006; 19: 391-400
Monitoring the Anti-tumor Response of Naive and Memory CD8 T Cells in RAG1-/-Mice by Positron-Emission Tomography. The Journal of Immunology Su H, Chang DS, Gambhir SS, Braun J 2006; 176(7): 4459-4467
PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [F-18] FHBG NATURE PROTOCOLS Yaghoubi, S. S., Gambhir, S. S. 2006; 1 (6): 3069-3075
Abstract

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.

View details for DOI 10.1038/nprot.2006.459

View details for Web of Science ID 000251155700069

View details for PubMedID 17406570
Imaging chemically modified adenovirus for targeting tumors expressing integrin alpha(v)beta(3) in living mice with mutant herpes simplex virus type 1 thymidine kinase PET reporter gene JOURNAL OF NUCLEAR MEDICINE Xiong, Z. M., Cheng, Z., Zhang, X. Z., Patel, M., Wu, J. C., Gambhir, S. S., Chen, X. Y. 2006; 47 (1): 130-139
Abstract

The aim of this study was to change adenovirus tropism by chemical modification of the fiber knobs with PEGylated RGD peptide for targeting integrin alpha(v)beta(3) that is uniquely or highly expressed in tumor cells and neovasculature of tumors of various origins.The first generation Ad (Ad) vector, which expresses the herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) gene under the control of cytomegalovirus (CMV) promoter was conjugated with poly(ethylene glycol) (PEG) or RGD-PEG. The transduction efficiency of Ads (Adtk, PEG-Adtk, and RGD-PEG-Adtk) into different types of cells (293T, MCF7, MDA-MB-435, and U87MG) was analyzed and quantified by thymidine kinase (TK) assay using 8-(3)H-penciclovir (8-(3)H-PCV) as substrate. The in vivo infectivity of the Ad vectors after intravenous administration into integrin alpha(v)beta(3)-positive U87MG and MDA-MB-435 tumor-bearing athymic nude mice was measured by both noninvasive microPET using 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) as a reporter probe and ex vivo TK assay of the tumor and tissue homogenates.PEGylation completely abrogated coxsackievirus and adenovirus receptor (CAR)-knob interaction and the infectivity of PEG-Adtk is significantly lower than that of unmodified Adtk in CAR-positive cells. RGD-PEG-modified virus (RGD-PEG-Adtk) had significantly higher infectivity than PEG-Adtk and the extent of increase is related to both CAR and integrin alpha(v)beta(3) expression levels. (18)F-FHBG had minimal nonspecific uptake in the liver and tumors that are void of sr39tk. Mice preinjected intravenously with unmodified Adtk resulted in high hepatic uptake and moderate tumor accumulation of the tracer. In contrast, RGD-PEG-Adtk administration resulted in significantly lower liver uptake without compromising the tumor accumulation of (18)F-FHBG. Expression of TK in the liver and tumor homogenates corroborated with the magnitude of (18)F-FHBG uptake quantified by noninvasive microPET. Analysis of liver and tumor tissue integrin level confirmed that RGD-integrin interaction is responsible for the enhanced tumor infectivity of RGD-PEG-Adtk.The results of this study suggest that RGD-PEG conjugation is an effective way to modify Ad vector tropism for improved systemic gene delivery. Noninvasive PET and (18)F-FHBG are able to monitor in vivo transfectivity of both Adtk and RGD-PEG-Adtk vectors in the liver and tumors after intravenous injection.

View details for Web of Science ID 000234679300024

View details for PubMedID 16391197
Image-guided cardiac cell delivery using high-resolution small-animal ultrasound MOLECULAR THERAPY Rodriguez-Porcel, M., Gheysens, O., Chen, I. Y., Wu, J. C., Gambhir, S. S. 2005; 12 (6): 1142-1147
Abstract

Open-chest cardiac injection is the preferred delivery method for cardiac gene and stem cell therapy in small animals, but it is invasive and the operator is unable to see the actual delivery site. High-resolution ultrasound has recently been developed for small-animal imaging. We tested the hypothesis that image-guided cardiac cell delivery using high-resolution ultrasound guidance is feasible and reproducible. Sprague-Dawley rats (n = 11) were imaged using high-resolution ultrasound, and stably transfected cardiomyoblasts (plasmid-CMV-firefly luciferase) were injected into the anterior cardiac wall under ultrasound guidance (parasternal long-axis view), using a 28-gauge needle. After injection, bioluminescence imaging was performed using a cooled charged-coupled camera. Injection was successful in all animals and was associated with no mortality. The signal detected was positively correlated with the amount of cells transplanted (R(2) = 0.94, P = 0.03) and highly correlated with ex vivo assays (R(2) = 0.82). In addition, the optical signal could be followed longitudinally using bioluminescence imaging. Ultrasound image-guided cardiac cell delivery is an effective, safe, and reproducible way to perform cell delivery to a specific myocardial region and can be combined with assessment of cardiac function. We are confident that the use of these technologies will play a significant role in the future of gene and cell therapy.

View details for DOI 10.1016/j.ymthe.2005.07.532

View details for Web of Science ID 000233864700017

View details for PubMedID 16111921
Clinical molecular imaging and therapy - moving ahead together EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Bengel, F. M., Gambhir, S. S. 2005; 32: S323-S323
View details for DOI 10.1007/s00259-005-1959-9

View details for Web of Science ID 000203011200001

View details for PubMedID 16258761
Applications of molecular imaging in cancer gene therapy CURRENT GENE THERAPY Iyer, M., Sato, M., Johnson, M., Gambhir, S. S., Wu, L. 2005; 5 (6): 607-618
Abstract

Gene-based therapy is a promising and flexible therapeutic approach to manage diverse types of cancer. The lack of convincing therapeutic success of current gene therapy protocols in part, can be attributed to the inability to monitor gene expression at the targeted site in the living subject. Linking molecular imaging to gene therapy will enable real-time assessment of the therapeutic process and the refinement of treatment protocols. This review will cover two common imaging modalities, positron emission tomography (PET) and bioluminescence imaging (BLI), used in pre-clinical and clinical gene therapy applications. Strategies to develop more specific and robust cancer gene therapy and imaging approaches will be discussed. Coupling PET to gene therapy of cancer has already been implemented in several clinical studies. This approach would help to improve the efficacy and safety of future gene therapy clinical trials.

View details for Web of Science ID 000233920100007

View details for PubMedID 16457650
Gene therapy imaging in patients for oncological applications EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Penuelas, I., Haberkorn, U., Yaghoubi, S., Gambhir, S. S. 2005; 32: S384-S403
Abstract

Thus far, traditional methods for evaluating gene transfer and expression have been shown to be of limited value in the clinical arena. Consequently there is a real need to develop new methods that could be repeatedly and safely performed in patients for such purposes. Molecular imaging techniques for gene expression monitoring have been developed and successfully used in animal models, but their sensitivity and reproducibility need to be tested and validated in human studies. In this review, we present the current status of gene therapy-based anticancer strategies and show how molecular imaging, and more specifically radionuclide-based approaches, can be used in gene therapy procedures for oncological applications in humans. The basis of gene expression imaging is described and specific uses of these non-invasive procedures for gene therapy monitoring illustrated. Molecular imaging of transgene expression in humans and evaluation of response to gene-based therapeutic procedures are considered. The advantages of molecular imaging for whole-body monitoring of transgene expression as a way to permit measurement of important parameters in both target and non-target organs are also analyzed. The relevance of this technology for evaluation of the necessary vector dose and how it can be used to improve vector design are also examined. Finally, the advantages of designing a gene therapy-based clinical trial with imaging fully integrated from the very beginning are discussed and future perspectives for the development of these applications outlined.

View details for DOI 10.1007/s00259-005-1928-3

View details for Web of Science ID 000203011200005

View details for PubMedID 16180032
Multimodality tumor imaging targeting integrin alphavbeta3. BioTechniques Cai, W., Sam Gambhir, S., Chen, X. 2005; 39 (6): S14-25
Abstract

The cell adhesion molecule integrin alphavbeta3 is an important player in the process of tumor angiogenesis and metastasis. Antibodies, peptides, peptidomimetics, and small molecule antagonists against integrin alphavbeta3 have been shown to induce endothelial apoptosis, to inhibit tumor angiogenesis, and to increase endothelial permeability. The ability to quantitatively image integrin alphavbeta3 expression in vivo in a noninvasive manner may shed new light into the mechanism of angiogenesis and antiangiogenic treatment efficacy based on integrin antagonism. Tumor integrin expression imaging will also aid in lesion detection, patient stratification, new anti-integrin drug development/validation, as well as treatment monitoring and optimization. This review summarizes the recent advances in multimodality imaging of tumor integrin alphavbeta3 expression using magnetic resonance imaging (MRI), ultrasound, near-infrared (NIR) fluorescence, single photon emission computed tomography (SPECT), and positron emission tomography (PET).

View details for DOI 10.2144/000112091

View details for PubMedID 20158499
Visualization of a primary anti-tumor immune response by positron emission tomography PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shu, C. Y., Guo, S. L., Kim, Y. J., Shelly, S. M., Nijagal, A., Ray, P., Gambhir, S. S., Radu, C. G., Witte, O. N. 2005; 102 (48): 17412-17417
Abstract

Current methodologies that monitor immune responses rely on invasive techniques that sample tissues at a given point in time. New technologies are needed to elucidate the temporal patterns of immune responses and the spatial distribution of immune cells on a whole-body scale. We describe a noninvasive, quantitative, and tomographic approach to visualize a primary anti-tumor immune response by using positron emission tomography (PET). Bone marrow chimeric mice were generated by engraftment of hematopoietic stem and progenitor cells transduced with a trifusion reporter gene encoding synthetic Renilla luciferase (hRluc), EGFP, and Herpes virus thymidine kinase (sr39TK). Mice were challenged with the Moloney murine sarcoma and leukemia virus complex (M-MSV/M-MuLV), and the induced immune response was monitored by using PET. Hematopoietic cells were visualized by using 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG), a radioactive substrate specific for the sr39TK PET reporter protein. Immune cell localization and expansion were seen at the tumor and draining lymph nodes (DLNs). 2-[(18)F]fluoro-2-deoxy-D-glucose ([(18)F]FDG), which is sequestered in metabolically active cells, was used to follow tumor growth and regression. Elevated glucose metabolism was also seen in activated lymphocytes in the DLNs by using the [(18)F]FDG probe. When M-MSV/M-MuLV-challenged mice were treated with the immunosuppressive drug dexamethasone, activation and expansion of immune cell populations in the DLNs could no longer be detected with PET imaging. The method we describe can be used to kinetically measure the induction and therapeutic modulations of cell-mediated immune responses.

View details for DOI 10.1073/pnas.0508698102

View details for Web of Science ID 000233762000028

View details for PubMedID 16293690
Reproducibility of 3 '-deoxy-3 '-F-18-fluorothymidine MicroPET studies in tumor xenografts in mice JOURNAL OF NUCLEAR MEDICINE Tseng, J. R., Dandekar, M., Subbarayan, M., Cheng, Z., Park, J. M., Louie, S., Gambhir, S. S. 2005; 46 (11): 1851-1857
Abstract

3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been used to image tumor proliferation in preclinical and clinical studies. Serial microPET studies may be useful for monitoring therapy response or for drug screening; however, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FLT microPET studies.C6 rat glioma xenografts were implanted into nude mice (n = 9) and grown to mean diameters of 5-17 mm for approximately 2 wk. A 10-min acquisition was performed on a microPET scanner approximately 1 h after (18)F-FLT (1.9-7.4 MBq [50-200 muCi]) was injected via the tail vein. A second microPET scan was performed approximately 6 h later on the same day after reinjection of (18)F-FLT to assess for reproducibility. Most of the mice were studied twice within the same week (for a total of 17 studies). Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility after subtraction of the estimated residual tumor activity from the first (18)F-FLT injection.The coefficient of variation (mean +/- SD) for %ID/g values between (18)F-FLT microPET scans performed 6 h apart on the same day was 14% +/- 10%. The difference in %ID/g values between scans was -0.06% +/- 1.3%. Serum thymidine levels were mildly correlated with %ID/g values (R(2) = 0.40). Tumor size, mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, controlling body temperature, the time of imaging after injection, and the ROI size.(18)F-FLT microPET mouse tumor xenograft studies are reproducible with moderately low variability. Serial studies may be performed to assess for significant changes in therapy response or for preclinical drug development.

View details for Web of Science ID 000233095800017

View details for PubMedID 16269599
Small animal imaging center design: The facility at the UCLA Crump Institute for Molecular Imaging MOLECULAR IMAGING AND BIOLOGY Stout, D. B., Chatziioannou, A. F., Lawson, T. P., Silverman, R. W., Gambhir, S. S., Phelps, M. E. 2005; 7 (6): 393-402
Abstract

The growing number of mouse and rat experiments, coupled with advances in small-animal imaging systems such as microPET, optical, microCAT, microMR, ultrasound and microSPECT, has necessitated a common technical center for imaging small animals.At the UCLA Crump Institute for Molecular Imaging, we have designed and built a facility to support the research interests of a wide range of investigators from multiple disciplines. Requirements to satisfy both research and regulatory oversight have been critically examined. Support is provided for investigator training, study scheduling, data acquisition, archiving, image display, and analysis.The center has been in operation for more than 18 months, supporting more than 13,000 individual imaging procedures.We have created a facility that maximizes our resource utilization while providing optimal investigator support, as well as the means to continually improve the quality and diversity of the science by integrating physical and biological sciences.

View details for DOI 10.1007/s11307-005-0015-2

View details for Web of Science ID 000234151200003

View details for PubMedID 16261425
Imaging androgen receptor function during flutamide treatment in the LAPC9 xenograft model MOLECULAR CANCER THERAPEUTICS Ilagan, R., Zhang, L. Q., Pottratz, J., Le, K., Salas, S., Iyer, M., Wu, L., Gambhir, S. S., Carey, M. 2005; 4 (11): 1662-1669
Abstract

The current understanding of the response of androgen receptor to pharmacologic inhibitors in prostate cancer is derived primarily from serum prostate-specific antigen (PSA) levels. In this study, we test whether a novel androgen receptor-specific molecular imaging system is able to detect the action of the antiandrogen flutamide on androgen receptor function in xenograft models of prostate cancer. Adenoviruses bearing an optical imaging cassette containing an androgen receptor-responsive two-step transcriptional amplification system were injected into androgen-dependent and hormone-refractory tumors of animals undergoing systemic time-controlled release of the antiandrogen flutamide. Imaging of tumors with a cooled charge-coupled device camera revealed that the response of AdTSTA to flutamide is more sensitive and robust than serum PSA measurements. Flutamide inhibits the androgen signaling pathway in androgen-dependent but not refractory tumors. Analysis of androgen receptor and RNA polymerase II binding to the endogenous PSA gene by chromatin immunoprecipitation revealed that flutamide treatment and androgen withdrawal have different molecular mechanisms. The application of imaging technology to study animal models of cancer provides mechanistic insight into antiandrogen targeting of androgen receptor during disease progression.

View details for DOI 10.1158/1535-7163.MCT-05-0197

View details for Web of Science ID 000233265900003

View details for PubMedID 16275987
Near-infrared fluorescent RGD peptides for optical imaging of integrin alpha(v)beta 3 expression in living mice BIOCONJUGATE CHEMISTRY Cheng, Z., Wu, Y., Xiong, Z. M., Gambhir, S. S., Chen, X. Y. 2005; 16 (6): 1433-1441
Abstract

Near-infrared fluorescence optical imaging is a powerful technique for studying diseases at the molecular level in preclinical models. We recently reported that monomeric RGD peptide c(RGDyK) conjugated to the NIR fluorescent dye specifically targets integrin receptor both in cell culture and in living subjects. In this report, Cy5.5-conjugated mono-, di-, and tetrameric RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model in order to investigate the effect of multimerization of RGD peptide on integrin avidity and tumor targeting efficacy. The binding affinities of Cy5.5-conjugated RGD monomer, dimer, and tetramer for alpha(v)beta(3) integrin expressed on U87MG cell surface were determined to be 42.9 +/- 1.2, 27.5 +/- 1.2, and 12.1 +/- 1.3 nmol/L, respectively. All three peptide-dye conjugates had integrin specific uptake both in vitro and in vivo. The subcutaneous U87MG tumor can be clearly visualized with each of these three fluorescent probes. Among them, tetramer displayed highest tumor uptake and tumor-to-normal tissue ratio from 0.5 to 4 h postinjection. Tumor-to-normal tissue ratio for Cy5.5-conjugated RGD monomer, dimer, and tetramer were found to be 3.18 +/- 0.16, 2.98 +/- 0.05, and 3.63 +/- 0.09, respectively, at 4 h postinjection. These results suggest that Cy5.5-conjugated monomeric, dimeric, and tetrameric RGD peptides are all suitable for integrin expression imaging. The multmerization of RGD peptide results in moderate improvement of imaging characteristics of the tetramer, compared to that of the monomer and dimeric counterparts.

View details for DOI 10.1021/bc0501698

View details for Web of Science ID 000233393800015

View details for PubMedID 16287239
Tracking embryonic stem cell transplant in the heart using a novel triple fusion reporter gene Cao, F., Lin, S., Krishnan, M., Drukker, M. E., Patel, M. R., Ray, P., Zhang, X. Z., Chen, X. Y., Gambhir, S. S., Weissman, I., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2005: U144-U145
View details for Web of Science ID 000232956400464
microPET imaging of glioma integrin alpha(V)beta(3) expression using Cu-64-labeled tetrameric RGD peptide JOURNAL OF NUCLEAR MEDICINE Wu, Y., Zhang, X. Z., Xiong, Z. M., Cheng, Z., Fisher, D. R., Liu, S., Gambhir, S. S., Chen, X. Y. 2005; 46 (10): 1707-1718
Abstract

Integrin alpha(v)beta(3) plays a critical role in tumor-induced angiogenesis and metastasis and has become a promising diagnostic indicator and therapeutic target for various solid tumors. Radiolabeled RGD peptides that are integrin specific can be used for noninvasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy.In this study we developed a tetrameric RGD peptide tracer (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) for PET imaging of integrin alpha(v)beta(3) expression in female athymic nude mice bearing the subcutaneous UG87MG glioma xenografts.The RGD tetramer showed significantly higher integrin binding affinity than the corresponding monomeric and dimeric RGD analogs, most likely due to a polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 +/- 0.01 %ID/g at 30 min and 0.21 +/- 0.01 %ID/g at 4 h after injection, respectively [%ID/g is percentage injected dose per gram]) and predominantly renal excretion. Tumor uptake was rapid and high, and the tumor washout was slow (9.93 +/- 1.05 %ID/g at 30 min after injection and 4.56 +/- 0.51 %ID/g at 24 h after injection). The metabolic stability of (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70%, 58%, 51%, and 26%, respectively. Noninvasive microPET studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution results. Integrin alpha(v)beta(3) specificity was demonstrated by successful blocking of tumor uptake of (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) in the presence of excess c(RGDyK) at 1 h after injection. The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.262 mGy/MBq), kidneys (0.0296 mGy/MBq), and liver (0.0242 mGy/MBq). The average effective dose resulting from a single (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) injection was estimated to be 0.0164 mSv/MBq.The high integrin and avidity and favorable biokinetics make (64)Cu-DOTA-E{E[c(RGDfK)](2)}(2) a promising agent for peptide receptor radionuclide imaging and therapy of integrin-positive tumors.

View details for Web of Science ID 000232452700033

View details for PubMedID 16204722
Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer FASEB JOURNAL De, A., Gambhir, S. S. 2005; 19 (12): 2017-?
View details for DOI 10.1096/fj.05-4628fje

View details for Web of Science ID 000232991100029
Micro-PET/CT monitoring of herpes thymidine kinase suicide gene therapy in a prostate cancer xenograft: the advantage of a cell-specific transcriptional targeting approach. Molecular imaging Johnson, M., Sato, M., Burton, J., Gambhir, S. S., Carey, M., Wu, L. 2005; 4 (4): 463-472
Abstract

Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols.

View details for PubMedID 16285908
Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging FASEB JOURNAL Krishnan, M., Park, J. M., Cao, F., Wang, D. X., Paulmurugan, R., Tseng, J. R., Gonzalgo, M. L., Gambhir, S. S., Wu, J. C. 2005; 19 (12): 106-?
View details for DOI 10.1096/fj.05-4551fje

View details for Web of Science ID 000232991100011
Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals CANCER RESEARCH Paulmurugan, R., Gambhir, S. S. 2005; 65 (16): 7413-7420
Abstract

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

View details for DOI 10.1158/0008-5472.CAN-05-0588

View details for Web of Science ID 000231188600049

View details for PubMedID 16103094
Optimizing radiolabeled engineered anti-p185(HER2) antibody fragments for in vivo imaging CANCER RESEARCH Olafsen, T., Kenanova, V. E., Sundaresan, G., Anderson, A. L., Crow, D., Yazaki, P. J., Li, L., Press, M. F., Williams, L. E., Wong, J. Y., Raubitschek, A. A., Shively, J. E., Wu, A. M. 2005; 65 (13): 5907-5916
Abstract

We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-C(H)3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 +/- 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 +/- 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 +/- 4.0% ID/g). A minibody derived from a second anti-p185(HER2) antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 +/- 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 +/- 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-C(H)2-C(H)3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 +/- 2.4% ID/g) and reduced kidney uptake (13.1 +/- 1.5% ID/g). Thus, by manipulating the size and format of anti-p185(HER2) antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.

View details for Web of Science ID 000230165000055

View details for PubMedID 15994969
Noninvasive imaging of ex vivo intracoronarily delivered nonviral therapeutic transgene expression in heart MOLECULAR THERAPY Sen, L., Gambhir, S. S., Furukawa, H., Stout, D. B., Lam, A. L., Laks, H., Cui, G. 2005; 12 (1): 49-57
Abstract

We developed a clinically applicable approach for noninvasive monitoring of reporter-therapeutic linked gene expression in the whole heart of large animals using PET imaging and further validated the efficacy and cardiac adverse effects of reporter-therapeutic linked gene transfer in a rabbit cervical heterotopic functional heart transplant model. Cationic liposome complexed with a vector containing a herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) as the reporter gene and a recombinant human immunosuppressive cytokine, interleukin-10 (hIL-10), as the therapeutic gene was ex vivo intracoronarily delivered into cardiac allografts before implantation. Long-term HSV1-sr39tk and hIL-10 transgene and protein overexpression associated with myocardial PET reporter probe 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) accumulation was observed in the allografts. The expression of the HSV1-sr39tk gene was significantly correlated with the hIL-10 gene expression and the total myocardial [18F]FHBG accumulation quantified as a percentage of intravenously injected [18F]FHBG dose. A homogeneous distribution of [18F]FHBG accumulation was seen in the whole heart similar to the distribution of [18F]fluorodeoxyglucose, a PET glucose metabolism probe. The immunosuppressive therapeutic efficacy remained the same in allografts treated with reporter-therapeutic linked gene and therapeutic gene only. No cardiac adverse effect was found. Our results demonstrate for the first time that PET reporter-therapeutic linked gene imaging is applicable for noninvasively monitoring ex vivo intracoronarily delivered therapeutic transgene expression in the whole heart.

View details for DOI 10.1016/j.ymthe.2005.03.004

View details for Web of Science ID 000230282200012

View details for PubMedID 15963920
Comparison of [C-14]FMAU, [H-3]FEAU, [C-14]FIAU, and [H-3]PCV for monitoring reporter gene expression of wild type,and mutant herpes simplex virus type 1 thymidine kinase in cell culture MOLECULAR IMAGING AND BIOLOGY Kang, K. W., Min, J. J., Chen, X. Y., Gambhir, S. S. 2005; 7 (4): 296-303
Abstract

To assess the optimal reporter probe/reporter gene combination for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression, we compared the cellular uptake of 1-(2'-fluoro-2'-deoxy-D-arabinofuranosyl)-5-methyluracil (FMAU), 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), 2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl-5-iodouracil (FIAU) and penciclovir (PCV) in both HSV1-tk and HSV1-sr39tk expressing cells.For stably transfected cell studies, C6 rat glioma cells, C6 HSV1-tk transfectant, C6 mutant HSV1-sr39tk transfectant, rat Morris hepatoma cells (MH3924A), and MH3924A HSV1-tk transfectant cells were used. For adenoviral infection studies, C6 rat glioma cells were exposed to serial titers of AdCMV-HSV1-tk, AdCMV-HSV1-sr39tk, or AdCMV-fluc for 24 hours. These cells were incubated with [(14)C]FMAU, [(3)H]FEAU, [(14)C]FIAU, and [(3)H]PCV, and cellular uptake of radioactivity was measured.[(3)H]FEAU exhibited the highest or second highest accumulation and the most selectivity regardless of the mode of gene transfer for both HSV1-tk and mutant HSV1-sr39tk reporter genes.This combination of high accumulation and high selectivity for both HSV1-tk and HSV1-sr39tk makes suitably radiolabeled FEAU a promising candidate as a radiotracer for imaging HSV1-tk/HSV1-sr39tk gene expression in living subjects.

View details for DOI 10.1007/s11307-005-0010-7

View details for Web of Science ID 000233422000005

View details for PubMedID 16041591
Bioluminescent imaging of melanoma in live mice JOURNAL OF INVESTIGATIVE DERMATOLOGY Craft, N., Bruhn, K. W., Nguyen, B. D., PRINS, R., Liau, L. M., Collisson, E. A., De, A., Kolodney, M. S., Gambhir, S. S., Miller, J. F. 2005; 125 (1): 159-165
Abstract

Melanoma is highly resistant to conventional chemotherapeutic agents and novel therapeutic approaches are needed. Current animal models of melanoma in animals are sub-optimal. The most commonly used homograft model is the B16 mouse melanoma. Evaluation of potential melanoma therapies with this model is limited by the inaccuracy of caliper measurement of subcutaneous tumors, of counting lung nodules in metastasis models, and the indirect nature of "survival" curves when studying brain metastases. We have developed and characterized an accurate, sensitive, and reproducible bioluminescent B16 melanoma model that allows for serial, real-time analyses of tumor burden in live mice. We demonstrate that this model is applicable to subcutaneous tumors, lung metastases, and intracranial tumors and offers a solution to many of the limitations of previous models. As proof of principle, we use this model to show the efficacy of a live, Listeria monocytogenes vaccine expressing the melanoma antigen tyrosinase-related protein-2 to protect mice against intravenous B16 melanoma challenge. Additionally, we extend our approach to include the human A375 melanoma model and are able to show in vivo differences between sub-lines with varying metastatic potential. These models represent an accurate and reproducible means for in vivo melanoma monitoring in preclinical studies.

View details for DOI 10.1111/j.0022-202X.2005.23759.x

View details for Web of Science ID 000230342000026

View details for PubMedID 15982316
Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients GASTROENTEROLOGY Penuelas, I., Mazzolini, G., Boan, J. F., SANGRO, B., Marti-Climent, J. P., Ruiz, M., Ruiz, J., Satyamurthy, N., Qian, C., Barrio, J. R., Phelps, M. E., Richter, J. A., Gambhir, S. S., Prieto, J. 2005; 128 (7): 1787-1795
Abstract

In gene-therapy protocols, imaging of gene expression is needed to evaluate the transduction efficiency of the vector, its tissue distribution, and the duration of transgene expression and to assess the feasibility of repeated vector administration.We have used positron emission tomography with a fluorine-18-labeled penciclovir analogue to monitor thymidine kinase gene expression after intratumoral injection of a first-generation recombinant adenovirus in patients with hepatocellular carcinoma. Patients were enrolled in a pilot clinical trial and treated with escalating doses of the vector. Two days after adenovirus inoculation, transgene expression was evaluated during the first hours after administration of the radiotracer both on the treated lesion and on a whole-body basis.Transgene expression in the tumor was dependent on the injected dose of the adenovirus and was detectable in all patients who received > or = 10(12) viral particles. However, when the study was repeated 9 days after vector injection, no expression could be observed. It is interesting to note that no specific expression of the transgene could be detected in distant organs or in the surrounding cirrhotic tissue in any of the cases studied.Our findings show the real possibility of imaging transgene expression in humans by using viral vectors. We show that hepatocarcinoma is a permissive tumor for adenoviral infection and that the nontumoral cirrhotic liver is spared from transduction when the vector is administered by intratumoral injection. These results show that positron emission tomography imaging may help in the design of gene-therapy strategies and in the clinical assessment of new-generation vectors.

View details for DOI 10.1053/j.gastro.2005.03.024

View details for Web of Science ID 000229662900005

View details for PubMedID 15940613
Comparison between PET and bioluminescence imaging for quantitative assessment of tumor burden Maxim, P., Thorndyke, B., Boyer, A., Contag, C., Gambhir, S., Xing, L. AMER ASSOC PHYSICISTS MEDICINE AMER INST PHYSICS. 2005: 2134-2134
View details for Web of Science ID 000229908601456
Functionality of androgen receptor-based gene expression imaging in hormone refractory prostate cancer CLINICAL CANCER RESEARCH Sato, M., Johnson, M., Zhang, L. Q., Gambhir, S. S., Carey, M., Wu, L. 2005; 11 (10): 3743-3749
Abstract

A highly augmented, prostate-specific two-step transcriptional amplification (TSTA) method was developed with the ultimate goal of delivering an effective and safe gene-based treatment to prostate cancer patients. Because very limited treatment options are available for recurrent hormone refractory prostate cancer (HRPC), it is imperative to assess whether the prostate-specific antigen (PSA) promoter-based TSTA gene therapy will be functional in HRPC.We tested the TSTA-driven adenovirus vector on three androgen-dependent and six HRPC models. Real-time gene expression was monitored by both optical imaging and the combined modality of positron emission tomography (PET) and computed tomography.The TSTA-driven firefly luciferase expressing adenoviral vector was active in all androgen receptor (AR)-expressing HRPC models, but inactive in AR- and PSA-negative lines. Interestingly, the TSTA-mediated gene expression was induced by hydrocortisone in MDA PCa 2b, a cell line with mutated AR that possesses altered ligand specificity. In animal models, the TSTA-mediated optical signal was more robust in the HRPC than androgen-dependent tumors. In a parallel trend, a TSTA vector that expresses the herpes simplex virus thymidine kinase PET reporter gene also displayed more robust PET signal in the HRPC tumor.The activity of TSTA system is AR dependent and it recapitulates the functional status of endogenous AR. These data support the conclusion that AR function is activated in HRPC despite castrated levels of androgen. Together with the fact that majority of recurrent prostate cancers express AR and PSA, we foresee that the TSTA approach can be a promising gene therapy strategy for the advanced stages of prostate cancer.

View details for Web of Science ID 000229086600019

View details for PubMedID 15897571
A new strategy to screen molecular imaging probe uptake in cell culture without radiolabeling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry JOURNAL OF NUCLEAR MEDICINE Cheng, Z., Winant, R. C., Gambhir, S. S. 2005; 46 (5): 878-886
Abstract

Numerous new molecular targets for diseases are rapidly being identified and validated in the postgenomic era, urging scientists to explore novel techniques for accelerating molecular probe development. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated as a potential tool for high-throughput screening and characterization of molecular imaging probes. Specifically, MALDI-TOF-MS was used to screen a small library of phosphonium cations for their ability to accumulate in cells.C6 cells incubated with phosphonium cations at room temperature were collected and lysed for experiments. Calibration curves for the internal standard, methyltriphenyl phosphonium, and for tetraphenylphosphonium bromide (TPP) and other phosphonium cations were first established. The time course of TPP uptake by C6 cells was then quantified using both MALDI-TOF-MS and liquid scintillation counting with (3)H-TPP. In addition, MALDI-TOF-MS was used to screen a library of 8 phosphonium cations and subsequently rank their ability to penetrate membranes and accumulate in cells. Finally, the accumulation of 4-fluorophenyltriphenyl phosphonium (FTPP) in the membrane potential-modulated cells was also measured by MALDI-TOF-MS.MALDI-TOF-MS spectra clearly revealed that TPP was easily identified from cell lysates even as early as 10 min after incubation and that levels as low as 0.11 fmol of TPP per cell could be detected, suggesting the high sensitivity of this technique. The time course of TPP influx determined by both MALDI-TOF-MS and radioactivity counting showed no statistically significant difference (P > 0.05 for all time points). These data validated MALDI-TOF-MS as an alternative approach for accurately measuring uptake of phosphonium cations by cells. TPP and FTPP demonstrated greater accumulation in cells than did the other cations evaluated in this study. Furthermore, uptake profiles suggested that FTPP preserves the membrane potential-dependent uptake property of TPP in cell cultures. Taken together, these data justify further synthesis and evaluation of (18)F-FTPP as a molecular probe for imaging mitochondrial dysfunction.These results demonstrate that MALDI-TOF-MS is a powerful analytic tool for rapid screening and characterization of phosphonium cations as molecular probes. This technique can potentially be applied to the evaluation of other imaging probes or drugs and thus may facilitate their rational design and development.

View details for Web of Science ID 000228952400030

View details for PubMedID 15872363
Imaging protein-protein interactions in living subjects TRAC-TRENDS IN ANALYTICAL CHEMISTRY Paulmurugan, R., Ray, P., De, A., CHAN, C. T., Gambhir, S. S. 2005; 24 (5): 446-458
View details for Web of Science ID 000234203000028
Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors JOURNAL OF NUCLEAR MEDICINE Wang, Y. L., Iyer, M., Annala, A. J., Chappell, S., Mauro, V., Gambhir, S. S. 2005; 46 (4): 667-674
Abstract

Indirect, noninvasive imaging of therapeutic gene expression based on levels of reporter gene expression is a powerful tool to devise improved therapeutic strategies in cancer gene therapy. The use of bicistronic vectors carrying internal ribosome entry sites (IRESs) allows the coexpression of multiple gene products from the same promoter but leads to considerable attenuation of the downstream gene. In this study, we describe the use of 10 linked copies of the Gtx (homeodomain protein) IRES (abbreviated as SIRES) in place of the encephalomyocarditis (EMCV) IRES in mediating downstream reporter gene expression in cell culture and in vivo.We constructed several plasmid vectors carrying different upstream and downstream reporter genes (herpes simplex virus type I thymidine kinase [tk], firefly luciferase [fl], and Renilla luciferase [rl]) placed between EMCV IRES and SIRES segments. RL, FL, and TK enzyme activities in N2a, C6, and 293 cells transiently transfected with these vectors were found to be significantly higher for the SIRES vectors than for the EMCV IRES vectors. For in vivo experiments, 4 stably transfected N2a cell lines were implanted in nude mice. The mice were imaged for rl and fl gene expression using a charged-coupled device (CCD) camera. For bioluminescence and microPET imaging of downstream gene expression of fl and tk genes, respectively, mice carrying 4 stably transfected xenografts were imaged using the CCD camera and microPET.In cell culture, using rl as the upstream gene, we demonstrate that the expression of the downstream tk gene is 12-fold greater using SIRES when compared with EMCV IRES. Furthermore, the expression of the 2 genes was highly correlated in N2a cells. In vivo bioluminescence imaging using 4 stably transfected N2a cell lines revealed increasing levels of rl and fl gene expression. Bioluminescence and microPET, respectively, of fl and tk reporter gene expression in nude mice bearing N2a tumor xenografts showed the gene expression mediated by SIRES to be 4- and 8-fold higher, respectively, than EMCV IRES.These findings support the use of SIRES bicistronic vectors for a better assessment of therapeutic gene expression based on reporter gene expression in living subjects.

View details for Web of Science ID 000228200000022

View details for PubMedID 15809490
FDG-PET and beyond: Molecular breast cancer imaging JOURNAL OF CLINICAL ONCOLOGY Quon, A., Gambhir, S. S. 2005; 23 (8): 1664-1673
View details for DOI 10.1200/JCO.2005.11.024

View details for Web of Science ID 000227587200017

View details for PubMedID 15755974
Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living subjects with positron emission tomography CANCER GENE THERAPY Yaghoubi, S. S., Barrio, J. R., Namavari, M., Satyamurthy, N., Phelps, M. E., Herschman, H. R., Gambhir, S. S. 2005; 12 (3): 329-339
Abstract

Molecular imaging of a suicide transgene's expression will aid the development of efficient and precise targeting strategies, and imaging for cancer cell viability may assess therapeutic efficacy. We used the PET reporter probe, 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)guanine ([18F]FHBG) to monitor the expression of a mutant Herpes Simplex Virus 1 thymidine kinase (HSV1-sr39tk) in C6 glioma tumors implanted subcutaneously in nude mice that were repetitively being treated with the pro-drug Ganciclovir (GCV). [18F]-Fluorodeoxyglucose ([18F]FDG), a metabolic tracer, was used to assess tumor cell viability and therapeutic efficacy. C6 glioma tumors stably expressing the HSV1-sr39tk gene (C6sr39) accumulated [18F]FHBG prior to GCV treatment. Significant declines in C6sr39 tumor volumes and [18F]FHBG and [18F]FDG accumulation were observed following 2 weeks of GCV treatment. However, 3 weeks after halting GCV treatment, the tumors re-grew and [18F]FDG accumulation increased significantly; in contrast, tumor [18F]FHBG concentrations remained at background levels. Therefore, [18F]FHBG can be used to detect tumors expressing HSV1-sr39tk, susceptible to regression in response to GCV exposure, and the effectiveness of GCV therapy in eradicating HSV1-sr39tk-expressing cells can be monitored by [18F]FHBG scanning. [18F]FHBG and [18F]FDG imaging data indicate that exposure of C6sr39 tumors to GCV causes the elimination of [18F]FHBG-accumulating C6sr39 cells and selects for re-growth of tumors unable to accumulate [18F]FHBG.

View details for DOI 10.1038/sj.cgt.7700795

View details for Web of Science ID 000227026700013

View details for PubMedID 15592447
Firefly luciferase enzyme fragment complementation for imaging in cells and living animals ANALYTICAL CHEMISTRY Paulmurugan, R., Gambhir, S. S. 2005; 77 (5): 1295-1302
Abstract

We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein-protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting d-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1-475)/Cfluc (245-550), Nfluc (1-475)/Cfluc (265-550), and Nfluc (1-475)/Cfluc (300-550). The Cfluc (265-550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1-475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering TAT-Cfluc (265-550) to cells stably expressing Nfluc (1-475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell-cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation.

View details for DOI 10.1021/ac0484777

View details for Web of Science ID 000227409800021

View details for PubMedID 15732910
Non-invasive imaging of a transgenic mouse model using a prostate-specific two-step transcriptional amplification strategy TRANSGENIC RESEARCH Iyer, M., Salazar, F. B., Lewis, X., Zhang, L., Wu, L., Carey, M., Gambhir, S. S. 2005; 14 (1): 47-55
Abstract

Non-invasive assessment of transgenic animals using bioluminescence imaging offers a rapid means of evaluating disease progression in animal models of disease. One of the challenges in the field is to develop models with robust expression to image repetitively live intact animals through solid tissues. The prostate-specific antigen (PSA) promoter is an attractive model for studying gene regulation due to its hormonal response and tissue-specificity permitting us to measure signaling events that occur within the native tissues. The use of the GAL4-VP16 activator offers a powerful means to augment gene expression levels driven by a weak promoter. We have used a two-step transcriptional amplification (TSTA) system to develop a transgenic mouse model to investigate the tissue-specificity and developmental regulation of firefly luciferase (fl) gene expression in living mice using bioluminescence imaging. We employed an enhanced prostate-specific promoter to drive the yeast transcriptional activator, GAL4-VP16 (effector). The reporter construct carries five Gal4 binding sites upstream of the fl gene. We generated a transgenic mouse model using a single vector carrying the effector and reporter constructs. The transgenic mice show prostate-specific expression as early as three weeks of age. The bioluminescence signal in the prostate is significantly higher than in other organs. We also demonstrate that blocking androgen availability can downregulate the fl expression in the prostate. The transgenic mice display normal physical characteristics and developmental behavior, indicating that the high level of GAL4 driven expression is well tolerated. These findings suggest that the GAL4-VP16 transactivator can be used to amplify reporter gene expression from a relatively weak promoter in a transgenic mouse model. The transgenic TSTA model in conjunction with other transgenic cancer models should also help to detect and track malignancies. The strategies developed will be useful for transgenic research in general by allowing for amplified tissue specific gene expression.

View details for DOI 10.1007/s11248-004-2836-1

View details for Web of Science ID 000227424200005

View details for PubMedID 15865048
Quantum dots for live cells, in vivo imaging, and diagnostics SCIENCE Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., Weiss, S. 2005; 307 (5709): 538-544
Abstract

Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

View details for DOI 10.1126/science.1104274

View details for Web of Science ID 000226694000036

View details for PubMedID 15681376
Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments CANCER RESEARCH Kenanova, V., Olafsen, T., Crow, D. M., Sundaresan, G., Subbarayan, M., Carter, N. H., Ikle, D. N., Yazaki, P. J., Chatziioannou, A. F., Gambhir, S. S., Williams, L. E., Shively, J. E., Colcher, D., Raubitschek, A. A., Wu, A. M. 2005; 65 (2): 622-631
Abstract

Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti-carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using (125)I- and (131)I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was approximately 12 days. Additionally, (124)I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn interaction provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting.

View details for Web of Science ID 000226334500033

View details for PubMedID 15695407
Studying molecular and cellular processes in the intact organism. Progress in drug research. Fortschritte der Arzneimittelforschung. Progrès des recherches pharmaceutiques Gheysens, O., Gambhir, S. S. 2005; 62: 117-150
View details for PubMedID 16329256
A New Strategy to Screen Molecular Imaging Probe Uptake in Cell Culture without Radiolabeling using Matrix Assisted Laser Desorption/Ionization time of Flight Mass Spectrometry A New Strategy to Screen Molecular Imaging Probe Uptake in Cell Culture without Radiolabeling using Matrix Assisted Laser Desorption/Ionization time of Flight Mass Spectrometry Cheng, Z. 2005; 46: 878-886
Imaging studies for evaluating gene therapy in translational research. Drug discovery today. Technologies Peñuelas, I., Gambhir, S. S. 2005; 2 (4): 335-343
Abstract

Molecular imaging of gene expression in living subjects is leading to significant advances in the field of gene therapy. Three different technologies have been utilized for monitoring gene expression: optical imaging, radionuclide imaging and magnetic resonance imaging. In the present review, we focus on the latest developments including multimodality approaches and recent imaging of human gene therapy to noninvasively determine the location(s), magnitude and time-variation of gene therapy. Continued development should lead to greater use of these technologies in gene therapy and also help to shed light towards optimizing gene therapy in preclinical models and in the clinical setting.:

View details for DOI 10.1016/j.ddtec.2005.11.006

View details for PubMedID 24982010
Comparison between PET and bioluminescence imaging for quantitative assessment of tumor burden Maxim, P. G., Thorndyke, B. R., Dandekar, M., Mayer, D., Contag, C. H., Gambhir, S. S., Xing, L. ELSEVIER SCIENCE INC. 2005: S490-S490
View details for Web of Science ID 000232083301348
Multimodality Radionuclide, Fluorescence, and Bioluminescence Small-Animal Imaging. IEEE Park J, Gambhir SS 2005; 93: 771-783
Quantum Dots for Molecular Imaging and Cancer Medicine. Discovery Medicine Bentolila LA, Michalet X, Pinaud Ff, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S 2005; 5(26): 213-218
Synthesis of (4-[18F]Fluorophenyl) triphenylphosphonium as a Potential Imaging Agent for Mitochondrial Dysfunction. Journal of Labeled Compound and Radiopharmaceuticals Cheng Z, Subbarayan M, Chen X, Gambhir SS 2005; 48: 131-137
Multimodality Tumor Imaging Targeting Integrin αvβ3 BioTechniques Cai W, Gambhir SS, Chen X 2005; 39: S1-S25
Clinical Molecular Imaging and Therapy-Moving Ahead Together. European Journal of Nuclear Medicine and Molecular Imaging Bengel FM, Gambhir SS. 2005; 32 Suppl 2: S323
Gene Therapy Imaging in Patients for Oncological Applications. European Journal of Nuclear Medicine and Molecular Imaging Panuelas I, Haberkorn U, Yaghoubi S, Gambhir SS. 2005; 32:Suppl 2: S384-403
A New Strategy to Screen Molecular Imaging Probe Uptake in Cell Culture without Radiolabeling using Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. Journal of Nuclear Medicine Cheng Z, Winant RC, Gambhir SS. 2005; 46: 878-886
PET of cardiac transgene expression: Comparison of 2 approaches based on herpesviral thymidine kinase reporter gene JOURNAL OF NUCLEAR MEDICINE Miyagawa, M., Anton, M., Haubner, R., Simoes, M. V., Stadele, C., Erhardt, W., Reder, S., Lehner, T., Wagner, B., Noll, S., Noll, B., Grote, M., Gambhir, S. S., Gansbacher, B., Schwaiger, M., Bengel, F. M. 2004; 45 (11): 1917-1923
Abstract

PET of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Previously, 2 approaches based on the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) have been successfully applied to the heart. Wild-type HSV1-tk was imaged with (124)I-labeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU), and a mutant HSV1-tk (HSV1-sr39tk) was imaged with (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG). The aim of this study was to compare these 2 combinations with regard to specificity, imaging contrast, and reporter probe kinetics using dynamic PET in small and large animals.Similar titers of adenovirus-expressing wild-type HSV1-tk (Ad(tk)), mutant HSV1-sr39tk (Ad(sr39tk)), or control genes were directly injected into the myocardium of 24 rats and 8 pigs. Two days later, dynamic PET was performed with a clinical scanner during the 120 min after injection of (124)I-FIAU (Ad(tk) animals and controls) or (18)F-FHBG (Ad(sr39tk) animals and controls). Imaging with (13)N-ammonia was performed to identify cardiac regions of interest.In rats, significant cardiac (124)I-FIAU accumulation occurred in images obtained early (10-30 min) after Ad(tk) injection. Because of tracer washout, however, no difference between Ad(tk)-injected animals and controls was seen in the images obtained later. For (18)F-FHBG, specific myocardial accumulation greater than background levels was detected in Ad(sr39tk)-injected animals at early imaging and, in contrast to (124)I-FIAU accumulation, increased over time until the latest imaging (105-120 min). At maximum, cardiac (18)F-FHBG concentration showed a 4.15 +/- 1.65-fold increase compared with controls (105-120 min), and cardiac (124)I-FIAU concentration reached a maximal increase of 1.34 +/- 0.38-fold compared with controls (10-30 min, P = 0.0014). Global cardiac reporter probe kinetics in rats were confirmed by regional myocardial analysis in pig hearts. Transgene expression was specifically visualized by both approaches. The highest target-to-background ratio of (124)I-FIAU in Ad(tk)-infected pig myocardium was 1.50 +/- 0.20, versus 2.64 +/- 0.49 for (18)F-FHBG in Ad(sr39tk)-infected areas (P = 0.01). In vivo results were confirmed by ex vivo counting and autoradiography.Both reporter gene/probe combinations were feasible for noninvasive imaging of cardiac transgene expression in different species. Specific probe kinetics suggest different myocardial handling of pyrimidine (FIAU) and acycloguanosine (FHBG) derivatives. The results favor (18)F-FHBG with mutant HSV1-sr39tk because of continuous accumulation over time and higher imaging contrast.

View details for Web of Science ID 000225086000026

View details for PubMedID 15534063
A tracer kinetic model for F-18-FHBG for quantitating herpes simplex virus type 1 thymidine kinase reporter gene expression in living animals using PET JOURNAL OF NUCLEAR MEDICINE Green, L. A., Nguyen, K., Berenji, B., Iyer, M., Bauer, E., Barrio, J. R., Namavari, M., Satyamurthy, N., Gambhir, S. S. 2004; 45 (9): 1560-1570
Abstract

Reporter probe 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG) and reporter gene mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) have been used for imaging reporter gene expression with PET. Current methods for quantitating the images using the percentage injected dose per gram of tissue do not distinguish between the effects of probe transport and subsequent phosphorylation. We therefore investigated tracer kinetic models for 18F-FHBG dynamic microPET data and noninvasive methods for determining blood time-activity curves in an adenoviral gene delivery model in mice.18F-FHBG (approximately 7.4 MBq [approximately 200 microCi]) was injected into 4 mice; 18F-FHBG concentrations in plasma and whole blood were measured from mouse heart left ventricle (LV) direct sampling. Replication-incompetent adenovirus (0-2 x 10(9) plaque-forming units) with the E1 region deleted (n = 8) or replaced by HSV1-sr39tk (n = 18) was tail-vein injected into mice. Mice were dynamically scanned using microPET (approximately 7.4 MBq [approximately 200 microCi] 18F-FHBG) over 1 h; regions of interest were drawn on images of the heart and liver. Serial whole blood 18F-FHBG concentrations were measured in 6 of the mice by LV sampling, and 1 least-squares ratio of the heart image to the LV time-activity curve was calculated for all 6 mice. For 2 control mice and 9 mice expressing HSV1-sr39tk, heart image (input function) and liver image time-activity curves (tissue curves) were fit to 2- and 3-compartment models using Levenberg-Marquardt nonlinear regression. The models were compared using an F statistic. HSV1-sr39TK enzyme activity was determined from liver samples and compared with model parameter estimates. For another 3 control mice and 6 HSV1-sr39TK-positive mice, the model-predicted relative percentage of metabolites was compared with high-performance liquid chromatography analysis.The ratio of 18F-FHBG in plasma to whole blood was 0.84 +/- 0.05 (mean +/- SE) by 30 s after injection. The least-squares ratio of the heart image time-activity curve to the LV time-activity curve was 0.83 +/- 0.02, consistent with the recovery coefficient for the partial-volume effect (0.81) based on independent measures of heart geometry. A 3-compartment model best described 18F-FHBG kinetics in mice expressing HSV1-sr39tk in the liver; a 2-compartment model best described the kinetics in control mice. The 3-compartment model parameter, k3, correlated well with the HSV1-sr39TK enzyme activity (r2 = 0.88).18F-FHBG equilibrates rapidly between plasma and whole blood in mice. Heart image time-activity curves corrected for partial-volume effects well approximate LV time-activity curves and can be used as input functions for 2- and 3-compartment models. The model parameter k3 from the 3-compartment model can be used as a noninvasive estimate for HSV1-sr39TK reporter protein activity and can predict the relative percentage of metabolites.

View details for Web of Science ID 000223809500025

View details for PubMedID 15347725
Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene MOLECULAR IMAGING AND BIOLOGY Kim, Y. J., Dubey, P., Ray, P., Gambhir, S. S., Witte, O. N. 2004; 6 (5): 331-340
Abstract

Previous work showed quantitative imaging of T-cell migration into a tumor site by positron emission tomography (PET), using retroviral transduction of mutated thymidine kinase (sr39TK) reporter genes into immunized T-lymphocytes. PROCEDURES AND RESULTS: In order to improve the sensitivity and flexibility of the imaging analysis, lentivirus, that expressed sr39TK, was used to transduce the lymphocytes that migrated to an immunogenic sarcoma site. In comparison to retrovirally transduced lymphocytes, the lentivirally transduced lymphocytes showed enhanced PET signal when equal numbers of transduced lymphocytes were transferred. Furthermore, in order to utilize multimodality in vivo imaging capability, a tri-fusion reporter gene containing sr39TK, synthetic Renilla luciferase (hRluc), and enhanced green fluorescent protein (eGFP) was inserted into a lentiviral transfer vector. Using the adoptive transfer model, tumor-specific lymphocytic migration was detected by both microPET scan and bioluminescence imaging.The multimodal imaging strategy coupled with lentiviral reporter construct delivery demonstrated here can facilitate future molecular imaging studies.

View details for DOI 10.1016/j.mibio.2004.06.009

View details for Web of Science ID 000224370600007

View details for PubMedID 15380743
Noninvasive Imaging of enhanced prostate-specific gene expression using a two-step transcriptional amplification-based lentivirus vector MOLECULAR THERAPY Iyer, M., Salazar, F. B., Lewis, X., Zhang, L. Q., Carey, M., Wu, L., Gambhir, S. S. 2004; 10 (3): 545-552
Abstract

Noninvasive evaluation of gene transfer to specific cells or tissues will allow for long-term, repetitive monitoring of transgene expression. Tissue-specific promoters that restrict the expression of a transgene to tumor cells play a vital role in cancer gene therapy imaging. In this study, we have developed a third-generation HIV-1-based lentivirus vector carrying a prostate-specific promoter to monitor the long-term, sustained expression of the firefly luciferase (fl) reporter gene in living mice. The fl gene in the transcriptionally targeted vector is driven by an enhanced prostate-specific antigen promoter in a two-step transcriptional amplification (TSTA) system. The efficiency of the lentivirus (LV-TSTA)-mediated gene delivery, cell-type specificity, and persistence of gene expression were evaluated in cell culture and in living mice carrying prostate tumor xenografts. In vivo bioluminescence imaging with a cooled charge-coupled device camera revealed significantly high levels of fl expression in prostate tumors. Injection of LV-TSTA directly into the prostate of male nude mice revealed efficient and long-term fl gene expression in the prostate tissue for up to 3 months. These studies demonstrate the significant potential of TSTA-based lentivirus vectors to confer high levels of tissue-specific gene expression from a weak promoter, while preserving cell-type specificity and the ability to image noninvasively the sustained, long-term expression of reporter genes in living animals.

View details for DOI 10.1016/j.ymthe.2004.06.118

View details for Web of Science ID 000224480600019

View details for PubMedID 15336654
Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium CIRCULATION Wu, J. C., Chen, I. Y., Wang, Y. L., Tseng, J. R., Chhabra, A., Salek, M., Min, J. J., Fishbein, M. C., Crystal, R., Gambhir, S. S. 2004; 110 (6): 685-691
Abstract

Angiogenic gene therapy is a promising treatment paradigm for patients with ischemic heart disease. In this study, we used micro-positron emission tomography (microPET) to monitor the transgene expression, function, and effects in a whole-body system.Adenovirus with cytomegalovirus promoter driving an angiogenic gene (vascular endothelial growth factor [VEGF]) linked to a PET reporter gene (herpes simplex virus type 1 mutant thymidine kinase; Ad-CMV-VEGF121-CMV-HSV1-sr39tk) was used to transfect rat embryonic cardiomyoblasts in vitro. Expression of both genes correlated strongly (r=0.98; P<0.001). Afterward, rats underwent ligation of the left anterior descending artery followed by injection of 1x10(10) pfu of Ad-CMV-VEGF121-CMV-HSV1-sr39tk (study; n=35) or Ad-null (control; n=15) at the peri-infarct region. Noninvasive microPET imaging was used to assess the uptake of 9-(4-[18F]-fluoro-hydroxymethylbutyl)guanine ([18F]-FHBG) PET reporter probe by cells expressing the HSV1-sr39tk PET reporter gene. Cardiac transgene expression peaked at day 1 and declined over the next 2 weeks. Repeat adenoviral injections at day 60 yielded no detectable signal. The in vivo reporter gene expression (% injected dose/g of [18F]-FHBG) correlated well with ex vivo gamma counting (r=0.92), myocardial tissue HSV1-sr39TK enzyme activity (r=0.95), and myocardial tissue VEGF level (r=0.94; P<0.001 for all). The VEGF121 isoform induced significant increases in capillaries and small blood vessels. However, the level of neovasculature did not translate into significant improvements in functional parameters such as myocardial contractility by echocardiography, perfusion by nitrogen-13 ammonia imaging, and metabolism by [18F]-fluorodeoxyglucose imaging.Taken together, these findings establish the feasibility of molecular imaging for monitoring angiogenic gene expression with a PET reporter gene and probe noninvasively, quantitatively, and repetitively. The principles demonstrated here can be used to evaluate other therapeutic genes of interest in animal models before future clinical trials are initiated.

View details for DOI 10.1161/01.CIR.0000138153.02213.22

View details for Web of Science ID 000223194700008

View details for PubMedID 15302807
Molecular imaging of cardiovascular gene products JOURNAL OF NUCLEAR CARDIOLOGY Wu, J. C., Tseng, J. R., Gambhir, S. S. 2004; 11 (4): 491-505
View details for DOI 10.1016/j.nuclcard.2004.04.004

View details for Web of Science ID 000223210200015

View details for PubMedID 15295418
Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression HUMAN GENE THERAPY Ray, S., Paulmurugan, R., Hildebrandt, I., Iyer, M., Wu, L., Carey, M., Gambhir, S. S. 2004; 15 (7): 681-690
Abstract

Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification (TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression.

View details for Web of Science ID 000222786900006

View details for PubMedID 15242528
Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bhaumik, S., Walls, Z., Puttaraju, M., Mitchell, L. G., Gambhir, S. S. 2004; 101 (23): 8693-8698
Abstract

Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.

View details for DOI 10.1073/pnas.0402772101

View details for Web of Science ID 000222037000037

View details for PubMedID 15161977
Spontaneous and controllable activation of suicide gene expression driven by the stress-inducible Grp78 promoter resulting in eradication of sizable human tumors HUMAN GENE THERAPY DONG, D. Z., Dubeau, L., Bading, J., Nguyen, K., Luna, M., Yu, H., Gazit-Bornstein, G., Gordon, E. M., Gomer, C., HALL, F. L., Gambhir, S. S., Lee, A. S. 2004; 15 (6): 553-561
Abstract

GRP78 is a stress-inducible chaperone protein with antiapoptotic properties that is overexpressed in transformed cells and cells under glucose starvation, acidosis, and hypoxic conditions that persist in poorly vascularized tumors. Previously we demonstrated that the Grp78 promoter is able to eradicate tumors using murine cells in immunocompetent models by driving expression of the HSV-tk suicide gene. Here, through the use of positron emission tomography (PET) imaging, we provide direct evidence of spontaneous in vivo activation of the HSV-tk suicide gene driven by the Grp78 promoter in growing tumors and its activation by photodynamic therapy (PDT) in a controlled manner. In this report, we evaluated whether this promoter can be applied to human cancer therapy. We observed that the Grp78 promoter, in the context of a retroviral vector, was highly activated by stress and PDT in three different types of human breast carcinomas independent of estrogen receptor and p53. Complete regression of sizable human tumors was observed after prodrug ganciclovir treatment of the xenografts in immunodeficient mice. In addition, the Grp78 promoter-driven suicide gene is strongly expressed in a variety of human tumors, including human osteosarcoma. In contrast, the activity of the murine leukemia virus (MuLV) long-terminal repeat (LTR) promoter varied greatly in different human breast carcinoma cell lines, and in some cases, stress resulted in partial suppression of the LTR promoter activity. In transgenic mouse models, the Grp78 promoter-driven transgene is largely quiescent in major adult organs but highly active in cancer cells and cancer-associated macrophages, which can diffuse to tumor necrotic sites devoid of vascular supply and facilitate cell-based therapy. Thus, transcriptional control through the use of the Grp78 promoter offers multiple novel approaches for human cancer gene therapy.

View details for Web of Science ID 000222258500003

View details for PubMedID 15212714
Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice JOURNAL OF BIOMEDICAL OPTICS Bhaumik, S., Lewis, X. Z., Gambhir, S. S. 2004; 9 (3): 578-586
Abstract

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents.

View details for Web of Science ID 000221304400019

View details for PubMedID 15189096
Molecular imaging applications for immunology CLINICAL IMMUNOLOGY Hildebrandt, I. J., Gambhir, S. S. 2004; 111 (2): 210-224
Abstract

The use of multimodality molecular imaging has recently facilitated the study of molecular and cellular events in living subjects in a noninvasive and repetitive manner to improve the diagnostic capability of traditional assays. The noninvasive imaging modalities utilized for both small animal and human imaging include positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), ultrasound, and computed tomography (CT). Techniques specific to small-animal imaging include bioluminescent imaging (BIm) and fluorescent imaging (FIm). Molecular imaging permits the study of events within cells, the examination of cell trafficking patterns that relate to inflammatory diseases and metastases, and the ability to rapidly screen new drug treatments for distribution and effectiveness. In this paper, we will review the current field of molecular imaging assays (especially those utilizing PET and BIm modalities) and examine how they might impact animal models and human disease in the field of clinical immunology.

View details for Web of Science ID 000221681800008

View details for PubMedID 15137954
Quantitation of cell number by a positron emission tomography reporter gene strategy MOLECULAR IMAGING AND BIOLOGY Su, H., Forbes, A., Gambhir, S. S., Braun, J. 2004; 6 (3): 139-148
Abstract

An important potential of positron emission tomography (PET) is the capacity for quantitation of cell signals in an anatomic regions of interest. However, little is known about the constraints and parameters for using PET signal detection to establish cell numbers in regions of interest. In this study, we determined the correlation of PET signal to cell number, and characterized the cellular limit of detection for PET imaging.Cells expressing the herpes simplex virus type I thymidine kinase PET reporter gene (HSV1-sr39TK) were detected following accumulation of [(18)F]FHBG (9-[4-[(18)F]-fluoro-3-(hydroxymethyl) butyl]guanine) by microPET scanning and quantitation.When cells were cultured with [(18)F]FHBG in vitro, and then transferred to a model vascularized site, 73% retention was observed one hour post-transfer. Using this information, and the measured attenuation of PET signal in whole mouse scans, we assessed the per-cell uptake of [(18)F]FHBG in the vascularized site following standard parenteral administration of the substrate. We observed a cell number-dependent signal, with a limit of detection calculated as 10(6) cells in a region of interest of 0.1 mL volume. Quantitatively similar parameters were observed with stably tranfected N2a glioma cells and retrovirally transduced primary T lymphocytes.These methods and findings provide a strategy for quantitation of cellularity using PET imaging that has implications for both experimental models and clinical diagnosis.

View details for DOI 10.1016/j.mibio.2004.02.001

View details for Web of Science ID 000223467100005

View details for PubMedID 15193248
Molecular imaging of homodimeric protein-protein interactions in living subjects FASEB JOURNAL Massoud, T. F., Paulmurugan, R., Gambhir, S. S. 2004; 18 (7): 1105-?
View details for DOI 10.1096/fj.03-1128fje

View details for Web of Science ID 000221883200025
MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene GENE THERAPY Sundaresan, G., Paulmurugan, R., Berger, F., Stiles, B., Nagayama, Y., Wu, H., Gambhir, S. S. 2004; 11 (7): 609-618
Abstract

Site-specific recombination tools such as the Cre-loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1 x 10(9) PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre-). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]-FHBG). The [(18)F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72+/-1.13 for the Cre+ mice and 0.10+/-0.02 for the Cre- mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre-loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre-loxP-mediated gene activation by imaging in a microPET scanner.

View details for DOI 10.1038/sj.gt.3302194

View details for Web of Science ID 000220281300006

View details for PubMedID 14724687
Tetraphenylphosphonium as a novel molecular probe for Imaging tumors JOURNAL OF NUCLEAR MEDICINE Min, J. J., Biswal, S., Deroose, C., Gambhir, S. S. 2004; 45 (4): 636-643
Abstract

Mitochondrial membrane potential (DeltaPsim)-dependent enhanced uptake of phosphonium salts, including (3)H-tetraphenylphosphonium ((3)H-TPP), in tumor cells, suggests the potential use of phosphonium salts as tracers for tumor imaging. In this study, we characterize the tumor accumulation of (3)H-TPP and compare it with (18)F-FDG in cell culture and in xenograft, metastatic, and inflammation models in living animals.(3)H-TPP and (3)H-FDG accumulation was compared in cell culture with a variety of cell lines in different glucose concentrations. Normal biodistribution and tumor uptake were assessed using nude mice with or without subcutaneous xenograft tumors (C6). To compare the accumulation of (3)H-TPP and (18)F-FDG in a metastatic tumor, severe combined immunodeficiency mice were tail-vein injected with human melanoma cell lines (A375-FL). To characterize the accumulation of (3)H-TPP and (18)F-FDG in inflammation, an inflammatory reaction was induced by subcutaneous injection of Complete Freund's Adjuvant in the left hind paw of Sprague-Dawley rat.The DeltaPsim data from a separate study and the current (3)H-TPP uptake data showed good correlation (r(2) = 0.82, P < 0.05). (3)H-TPP accumulation was significantly greater than that of (3)H-FDG for glucose >/=100 mg/dL. The biodistribution study of (3)H-TPP showed low uptake in most tissues but high accumulation in the heart and kidneys. (3)H-TPP accumulation in xenograft or metastatic tumors was comparable with that of (18)F-FDG, whereas (3)H-TPP accumulation in inflammatory tissues was markedly lower than that of (18)F-FDG.The sensitive tumor accumulation of (3)H-TPP with less propensity for inflammatory regions warrants further investigation of radiolabeled phosphonium analogs for tumor imaging in living subjects.

View details for Web of Science ID 000220729700023

View details for PubMedID 15073261
Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery CIRCULATION Chen, I. Y., Wu, J. C., Min, J. J., Sundaresan, G., Lewis, X., Liang, Q. W., Herschman, H. R., Gambhir, S. S. 2004; 109 (11): 1415-1420
Abstract

We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES).Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5x10(8) pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r2=0.96, P<0.001 for D2R80A; r2=0.98, P<0.001 for HSV1-sr39TK) and each other (r2=0.97; P<0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2x10(9) pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[18F]fluoroethyl)spiperone ([18F]-FESP) and HSV1-sr39tk-dependent sequestration of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG) using microPET. Longitudinal [18F]-FESP and [18F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r2=0.73; P<0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart.The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.

View details for DOI 10.1161/01.CIR.0000121727.59564.5B

View details for Web of Science ID 000220364700015

View details for PubMedID 15007006
Molecular imaging of drug-modulated protein-protein interactions in living subjects CANCER RESEARCH Paulmurugan, R., Massoud, T. F., Huang, J., Gambhir, S. S. 2004; 64 (6): 2113-2119
Abstract

Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor kappaB promoter/enhancer elements using tumor necrosis factor alpha, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.

View details for Web of Science ID 000220249100033

View details for PubMedID 15026351
Bioluminescent monitoring of islet graft survival after transplantation MOLECULAR THERAPY Lu, Y. X., Dang, H., Middleton, B., Zhang, Z., Washburn, L., Campbell-Thompson, M., Atkinson, M. A., Gambhir, S. S., Tian, J., Kaufman, D. L. 2004; 9 (3): 428-435
Abstract

Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. A method to image transplanted islets noninvasively and repeatedly would greatly assist studies of islet transplantation. Using recombinant adenovirus, we show that isolated rodent and human islets can be genetically engineered to express luciferase and then imaged after implantation into NOD-scid mice using a cooled charge-coupled device. The magnitude of the signal was dependent on the islet dose. Adenovirus-directed luciferase expression, however, rapidly attenuated. We next tested lentivirus vectors that should direct the long-term expression of reporter genes in transduced islets. Transplanted lentivirus-transduced islets restored euglycemia long term in streptozotocin-treated NOD-scid mice. The signal from implanted lentivirus-transduced islets was related directly to the implanted islet mass, and the signal did not attenuate over the observation period. Viral transduction, luciferase expression, and repeated imaging had no apparent long-term deleterious effects on islet function after implantation. These data demonstrate that the introduction of reporter genes into an isolated tissue allows the long-term monitoring of its survival following implantation. Such imaging technologies may allow earlier detection of graft rejection and the adjustment of therapies to prolong graft survival posttransplantation.

View details for DOI 10.1016/j.mythe.2004.01.008

View details for Web of Science ID 000220170100019

View details for PubMedID 15006610
Imaging tri-fusion multimodality reporter gene expression in living subjects CANCER RESEARCH Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. 2004; 64 (4): 1323-1330
Abstract

Imaging reporter gene expression in living subjects with various imaging modalities is a rapidly accelerating area of research. Applications of these technologies to cancer research, gene therapy, and transgenic models are rapidly expanding. We report construction and testing of several triple fusion reporter genes compatible with bioluminescence, fluorescence and positron emission tomography (PET) imaging. A triple fusion reporter vector harboring a bioluminescence synthetic Renilla luciferase (hrl) reporter gene, a reporter gene encoding the monomeric red fluorescence protein (mrfp1), and a mutant herpes simplex virus type 1 sr39 thymidine kinase [HSV1-truncated sr39tk (ttk); a PET reporter gene] was found to preserve the most activity for each protein component and was therefore investigated in detail. After validating the activities of all three proteins encoded by the fusion gene in cell culture, we imaged living mice bearing 293T cells transiently expressing the hrl-mrfp-ttk vector by microPET and using a highly sensitive cooled charge-coupled device camera compatible with both bioluminescence and fluorescence imaging. A lentiviral vector carrying the triple fusion reporter gene was constructed and used to isolate stable expressers by fluorescence-activated cell sorting. These stable 293T cells were further used to show good correlation (R(2) approximately 0.74-0.85) of signal from each component by imaging tumor xenografts in living mice with all three modalities. Furthermore, metastases of a human melanoma cell line (A375M) stably expressing the triple fusion were imaged by microPET and optical technologies over a 40-50-day time period in living mice. Imaging of reporter gene expression from single cells to living animals with the help of a single tri-fusion reporter gene will have the potential to accelerate translational cancer research.

View details for Web of Science ID 000189245200019

View details for PubMedID 14973078
Postnatal hypoxic-ischemic brain injury alters mechanisms mediating neuronal glucose transport AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY Zovein, A., Flowers-Ziegler, J., Thamotharan, S., Shin, D., Sankar, R., Nguyen, K., Gambhir, S., Devaskar, S. U. 2004; 286 (2): R273-R282
Abstract

We examined the effect of hypoxic ischemia and hypoxia vs. normoxia on postnatal murine brain substrate transporter concentrations and function. We detected a transient increase in the neuronal brain glucose transporter isoform (GLUT-3) in response to hypoxic ischemia after 4 h of reoxygenation. This increase was associated with no change in GLUT-1 (blood-brain barrier/glial isoform), monocarboxylate transporter isoforms 1 and 2, synapsin I (neuronal marker), or Bax (proapoptotic protein) but with a modest increase in Bcl-2 (antiapoptotic mitochondrial protein) protein concentrations. At 24 h of reoxygenation, the increase in GLUT-3 disappeared but was associated with a decline in Bcl-2 protein concentrations and the Bcl2:Bax ratio, an increase in caspase-3 enzyme activity (apoptotic effector enzyme), and extensive DNA fragmentation, which persisted later in time (48 h) only in the hippocampus. Hypoxia alone in the absence of ischemia was associated with a transient but modest increase in GLUT-3 and synapsin I protein concentrations, which did not cause significant apoptosis and/or necrosis. Assessment of glucose transporter function by 2-deoxyglucose (2-DG) uptake using two distinct techniques, namely positron emission tomography (PET) and the modified Sokoloff method, revealed a discrepancy due to glucose uptake by extracranial Harderian glands that masked the accurate detection of intracranial brain glucose uptake by PET scanning. The modified Sokoloff method assessing 2-DG uptake revealed that the transient increase in GLUT-3 was critical in protecting against a decline in brain glucose uptake. We conclude that hypoxic-ischemic brain injury is associated with transient compensatory changes targeted at protecting glucose delivery to fuel cellular energy metabolism, which then may delay the processes of apoptosis and cell necrosis.

View details for DOI 10.1152/ajpregu.00160.2003

View details for Web of Science ID 000187791500007

View details for PubMedID 14525722
Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications PROTEIN ENGINEERING DESIGN & SELECTION Olafsen, T., Cheung, C. W., Yazaki, P. J., Li, L., Sundaresan, G., Gambhir, S. S., Sherman, M. A., Williams, L. E., Shively, J. E., Raubitschek, A. A., Wul, A. M. 2004; 17 (1): 21-27
Abstract

An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti-CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti-CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modification of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site-specific conjugation approaches can direct modifications to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti-CEA diabody at three different locations, to provide specific thiol groups for chemical modification. One version (with a C-terminal Gly-Gly-Cys) existed exclusively as a disulfide-bonded dimer. This cysteine-modified diabody (Cys-diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non-covalent diabody. Furthermore, following reduction of the disulfide bond, the Cys-diabody could be chemically modified using a thiol-specific bifunctional chelating agent, for radiometal labeling. Thus, the Cys-diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modified site-specifically. This format will provide a versatile platform for targeting a variety of agents to CEA-positive tumors.

View details for DOI 10.1093/protein/gzh009

View details for Web of Science ID 000220905100003

View details for PubMedID 14985534
PET of Cardiac Transgene Expression: Comparison of 2 Approaches Based on Herpesviral Thymidine Kinase Reporter Gene PET of Cardiac Transgene Expression: Comparison of 2 Approaches Based on Herpesviral Thymidine Kinase Reporter Gene Miyagawa, M. 2004; 45 (11): 1917-1923
Gene therapy progress and prospects: Noninvasive imaging of gene therapy in living subjects GENE THERAPY Min, J. J., Gambhir, S. S. 2004; 11 (2): 115-125
Abstract

Recent progress in the development of noninvasive imaging technologies should allow molecular imaging to play a major role in the field of gene therapy. These tools have recently been validated in gene therapy models for continuous quantitative monitoring of the location(s), magnitude, and time variation of gene delivery and/or expression. This article reviews the use of radionuclide, magnetic resonance, and optical imaging technologies, as they have been used in imaging gene delivery and gene expression for gene therapy applications. The studies published to date lend support that noninvasive imaging tools will help to accelerate preclinical model validation, as well as allow for clinical monitoring of human gene therapy.

View details for DOI 10.1038/sj.gt.3302191

View details for Web of Science ID 000187909100001

View details for PubMedID 14712295
I-124-labeled engineered Anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice JOURNAL OF NUCLEAR MEDICINE Sundaresan, G., Yazaki, P. J., Shively, J. E., Finn, R. D., Larson, S. M., Raubitschek, A. A., Williams, L. E., Chatziioannou, A. F., Gambhir, S. S., Wu, A. M. 2003; 44 (12): 1962-1969
Abstract

Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C(H)3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with (124)I, for imaging tumor-bearing mice using a high-resolution small-animal PET system.Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of (124)I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or >90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9-3.1 MBq (53-85 microCi) of (124)I-minibody or with 3.1 MBq (85 microCi) of (124)I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using (18)F-FDG 2 d before imaging with (124)I-minibody.PET images using (124)I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using (124)I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the (124)I-diabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the (124)I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (<3 mm in diameter) could be imaged, and (124)I-anti-CEA minibodies, compared with (18)F-FDG, demonstrated highly specific localization.(124)I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.

View details for Web of Science ID 000187219400024

View details for PubMedID 14660722
Synthesis of a new heterobifunctional linker, N-[4-(Aminooxy)butyl]maleimide, for facile access to a thiol-reactive F-18-Labeling agent BIOCONJUGATE CHEMISTRY Toyokuni, T., Walsh, J. C., Dominguez, A., Phelps, M. E., Barrio, J. R., Gambhir, S. S., Satyamurthy, N. 2003; 14 (6): 1253-1259
Abstract

A new heterobifunctional linker containing an aldehyde-reactive aminooxy group and a thiol-reactive maleimide group, namely N-[4-(aminooxy)butyl]maleimide, was synthesized as a stable HCl salt by O-alkylation of either N-hydroxyphthalimide or N-(4-monomethoxytrityl)hydroxylamine, followed by N-alkylation of maleimide, in an overall yield of 18% (seven steps) or 29% (five steps), respectively. This heterobifunctional linker allowed a simple and efficient synthesis of a maleimide-containing thiol-reactive (18)F-labeling agent. Thus, N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide (specific activity: approximately 3000 Ci/mmol at end of synthesis) was synthesized in two steps involving the preparation of 4-[(18)F]fluorobenzaldehyde, followed by its aminooxy-aldehyde coupling reaction to the heterobifunctional linker, with an overall radiochemical yield of approximately 35% (decay corrected) within approximately 60 min from end of bombardment. Initial (18)F-labeling experiments were carried out using a thiol-containing tripeptide glutathione (GSH) and a 5'-thiol-functionalized oligodeoxynucleotide (5'-S-ODN) in phosphate-buffered saline (PBS, pH 7.5). After standing at room temperature for 10 min, the (18)F-labeled GSH and 5'-S-ODN were obtained in (18)F-labeling yields of approximately 70% and approximately 5% (decay-corrected), respectively. The heterobifunctional linker is easy to synthesize and provides a facile access to the maleimide-containing thiol-reactive (18)F-labeling agent, which could be advantageously employed in the development of (18)F-labeled biomomolecules for use with positron emission tomography.

View details for DOI 10.1021/bc034107y

View details for Web of Science ID 000186725700025

View details for PubMedID 14624642
Optimization of adenoviral vectors to direct highly amplified prostate-specific expression for imaging and gene therapy MOLECULAR THERAPY Sato, M., Johnson, M., Zhang, L. Q., Zhang, B. H., Le, K., Gambhir, S. S., Carey, M., Wu, L. 2003; 8 (5): 726-737
Abstract

Gene expression-based imaging coupled to gene therapy will permit the prediction of therapeutic outcome. A significant challenge for successful gene therapy is to achieve a high-level of specific gene expression; however, tissue-specific promoters are weak. We postulate that if the weak activity of tissue-specific promoters can be amplified to the levels of strong viral promoters, which have been successful in preclinical scenarios, while retaining specificity, the therapeutic index of gene therapy can be greatly augmented. With this in mind, we developed a two-step transcriptional activation (TSTA) system. In this two-tiered system, a modified prostate-specific antigen promoter was employed to drive a potent synthetic transcriptional activator, GAL4-VP2. This, in turn, activated the expression of a GAL4-dependent reporter or therapeutic gene. Here we demonstrate that recombinant adenoviral vectors (Ads) in which we have incorporated prostate-targeted TSTA expression cassettes retain cell specificity and androgen responsiveness in cell culture and in animal models, as measured by noninvasive optical bioluminescence imaging. We investigated the mechanism of TSTA in different adenoviral configurations. In one configuration, both the activator and the reporter components are inserted into a single Ad (AdTSTA-FL). The activity of AdTSTA-FL exceeds that of a cytomegalovirus promoter-driven vector (AdCMV-FL), while maintaining tissue specificity. When the activator and reporter components are placed in two separate Ads, androgen induction is more robust than for the single AdTSTA-FL. Based on these findings, we hope to refine the TSTA Ads further to improve the efficacy and safety of prostate cancer gene therapy.

View details for DOI 10.1016/j.ymthe.2003.08.016

View details for Web of Science ID 000186597500010

View details for PubMedID 14599805
Comparison of [F-18]FHBG and [C-14]FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection vs stable transfection EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Min, J. J., Iyer, M., Gambhir, S. S. 2003; 30 (11): 1547-1560
Abstract

Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1-beta- d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells-stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [(18)F]FHBG, [(3)H]PCV, and [(14)C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [(14)C]FIAU accumulation was greater than that of [(3)H]PCV and [(18)F]FHBG in control cells and in HSV1-tk stably transfected cells ( P<0.001). After infection of C6 cells with AdCMV- HSV1-tk (1.5x10(8) pfu), [(18)F]FHBG and [(3)H]PCV accumulation was significantly greater than that of [(14)C]FIAU ( P<0.01). [(18)F]FHBG and [(3)H]PCV accumulated to a significantly greater extent than [(14)C]FIAU in C6-stb-sr39tk+ and AdCMV- HSV1-sr39tk infected C6 cells ( P<0.001). Results from the nude mice supported the results in cell culture. [(14)C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [(18)F]FHBG ( P<0.05); however, compared with [(14)C]FIAU, [(18)F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [(18)F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A-stb-tk+). [(14)C]FIAU accumulated in HSV1-tk stably expressing cells to a greater extent than either [(3)H]PCV or [(18)F]FHBG. However, the accumulation of [(3)H]PCV and [(18)F]FHBG in adenoviral infected C6 cells or hepatocytes was equivalent to or greater than that of [(14)C]FIAU. These results may be due to intracellular biochemical changes (e.g., thymidine) when cells are infected with adenovirus. For adenoviral studies, the [(18)F]FHBG/ HSV1-sr39tk combination was shown to be more sensitive than the [(14)C]FIAU/ HSV1-tk combination HSV1-tk.

View details for DOI 10.1007/s00259-003-1238-6

View details for Web of Science ID 000186139700017

View details for PubMedID 14579096
Atorvastatin prevents RhoC isoprenylation, invasion, and metastasis in human melanoma cells MOLECULAR CANCER THERAPEUTICS Collisson, E. A., Kleer, C., Wu, M., De, A., Gambhir, S. S., Merajver, S. D., Kolodney, M. S. 2003; 2 (10): 941-948
Abstract

Melanoma is a deadly cancer due to its propensity to metastasize. Pharmacological inhibition of cell motility may benefit patients with cutaneous melanoma by preventing metastasis to internal organs. The Rho GTPases are signaling molecules that drive metastasis by controlling cell motility. We found RhoC to be expressed in clinical melanoma specimens and hypothesized that inhibiting its activation might prevent metastasis. Some Rho proteins, such as RhoC, depend on posttranslational geranylgeranylation for biological activity. We investigated the effect that Atorvastatin, a 3-hydroxy 3-methylglutaryl CoA (HMG-CoA) reductase inhibitor that prevents Rho geranylgeranylation, had on subcellular localization and activity of Rho proteins as well as the metastatic ability of melanoma cells. Atorvastatin inhibited Rho activation and reverted the metastatic phenotype of human melanoma cells in vitro. Moreover, Atorvastatin, at plasma levels comparable to those used to treat of hypercholesterolemia, inhibited in vivo metastasis of melanoma cells overexpressing RhoC. These results support further examination of statins for primary prophylaxis of melanoma metastasis.

View details for Web of Science ID 000186054800002

View details for PubMedID 14578459
Molecular imaging of cardiac cell transplantation in living animals using optical bioluminescence and positron emission tomography CIRCULATION Wu, J. C., Chen, I. Y., Sundaresan, G., Min, J. J., De, A., Qiao, J. H., Fishbein, M. C., Gambhir, S. S. 2003; 108 (11): 1302-1305
Abstract

The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques.For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3x10(6) to 5x10(5)) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3x10(6) cells: day 1 (627 000+/-15%), day 2 (346 100+/-21%), day 4 (112 800+/-20%), day 8 (78 860+/-24%), day 12 (67 780+/-12%), and day 16 (62 200+/-5% p x s(-1) x cm(2-1) x sr(-1)). For micro-positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3x10(6)) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48+/-0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01+/-0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays.The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.

View details for DOI 10.1161/01.CIR.0000091252.20010.6E

View details for Web of Science ID 000185328800006

View details for PubMedID 12963637
Treatment of metastatic melanoma with an orally available inhibitor of the Ras-Raf-MAPK cascade CANCER RESEARCH Collisson, E. A., De, A., Suzuki, H., Gambhir, S. S., Kolodney, M. S. 2003; 63 (18): 5669-5673
Abstract

The Ras-Raf-MAPK pathway is constitutively activated in the majority of melanomas because of a mutation in the BRAF gene. It has been hypothesized that activation of this pathway is crucial for the genesis and maintenance of melanoma and therefore represents an attractive clinical target for metastatic disease. We synthesized a previously characterized MAP kinase kinase inhibitor to test the effect that blocking the Ras-Raf-MAPK pathway would have on the establishment and maintenance of melanoma metastases. Oral administration of CI 1040 inhibited formation of pulmonary metastases and caused rapid regression of established pulmonary metastases in the mouse. Our findings indicate that Ras-Raf-MAPK activation provides crucial signals for the survival of melanoma cells at ectopic sites and that the pharmacological inhibition of this pathway is a promising target for melanoma therapy.

View details for Web of Science ID 000185672600003

View details for PubMedID 14522881
LABAZ1: A metastatic tumor model for renal cell carcinoma expressing the carbonic anhydrase type 9 tumor antigen CANCER RESEARCH Zisman, A., Pantuck, A. J., Bui, M. H., Said, J. W., Caliliw, R. R., Rao, N., Shintaku, P., Berger, F., Gambhir, S. S., Belldegrun, A. S. 2003; 63 (16): 4952-4959
Abstract

A metastatic renal cell carcinoma (RCC) tumor model xenograft that expresses the targetable, membrane-bound tumor-associated antigen carbonic anhydrase type 9 (CA IX) is described. The xenograft, established from a high-grade type-2 chromophil RCC (cRCC), has been serially transplanted in immune compromised mice, in which it grows orthotopically under the renal capsule, doubling its size every 9 weeks and sending metastases to the lung and liver at approximately 20 weeks. Tumors were capable of being imaged using a micro-PET (micro-positron emission tomograph) with an 18-fluorodeoxyglucose (18-FDG) tracer. Subsequent xenograft generations have conserved immunohistochemical and ultrastructural properties typical for malignant renal epithelium-derived neoplasia (vimentin+, CK-19+, CA IX+ with hypoxia-inducible factor (HIF)-1 alpha constitutive expression) and have demonstrated extensive proliferation, lack of apoptosis, severe genetic alterations, and molecular expression alterations; transforming growth factor beta 1 (TGF-beta 1), hepatocyte growth factor (HGF), proto-oncogene (c-met), matrix metalloproteinase (MMP)-1, and vascular endothelial growth factor (VEGF) C and D were overexpressed, whereas human epidermal growth factor receptor (HER)-2, MMP-2 and MMP-9, VEGF-R3, p53, and p27 were severely down-regulated, suggesting a proangiogenic environment, local invasiveness, and facilitated lymphatic metastasis. Altogether, LABAZ1 provides a relevant and flexible model to study the biology of cRCC, the role of CA IX in RCC tumorigenesis, progression, and metastasis, and a platform for testing new targeted therapeutic strategies.

View details for Web of Science ID 000184948400032

View details for PubMedID 12941820
Interrogating androgen receptor function in recurrent prostate cancer CANCER RESEARCH Zhang, L. Q., Johnson, M., Le, K. H., Sato, M., Ilgan, R., Iyer, M., Gambhir, S. S., Wu, L., Carey, M. 2003; 63 (15): 4552-4560
Abstract

The early androgen-dependent (AD) phase of prostate cancer is dependent on the androgen receptor (AR). However, it is unclear whether AR is fully functional in recurrent prostate cancer after androgen withdrawal. To address this issue we interrogated AR signaling in AD and recurrent prostate cancer xenografts using molecular imaging, chromatin immunoprecipitation, and immunohistochemistry. In the imaging experiments, an adenovirus bearing a two-step transcriptional activation cassette, which amplifies AR-dependent firefly luciferase reporter gene activity, was injected into tumors implanted into severe combined immunodeficiency mice. A charge-coupled device optical imaging system detected the initial loss and then resumption of AR transcriptional activity in D-luciferin-injected mice as tumors transitioned from AD to recurrent growth. The results of chromatin immunoprecipitation and immunohistochemical localization experiments correlated with the Ad two-step transcriptional activation imaging signal. AR localized to the nucleus and bound to the endogenous prostate-specific antigen enhancer in AD tumors but exited the nucleus and dissociated from the enhancer upon castration. However, AR reentered the nucleus and rebound the prostate-specific antigen enhancer as the cancer transitioned into the recurrent phase. Surprisingly, RNA polymerase II and the general factor TFIIB remained bound to the gene throughout the transition. Our data support the concept that AR is fully functional in recurrent cancer and suggest a model by which a poised but largely inactive transcription complex facilitates reactivation by AR at castrate levels of ligand.

View details for Web of Science ID 000184562500039

View details for PubMedID 12907631
AMIDE: a free software tool for multimodality medical image analysis. Molecular imaging Loening, A. M., Gambhir, S. S. 2003; 2 (3): 131-137
Abstract

Amide's a Medical Image Data Examiner (AMIDE) has been developed as a user-friendly, open-source software tool for displaying and analyzing multimodality volumetric medical images. Central to the package's abilities to simultaneously display multiple data sets (e.g., PET, CT, MRI) and regions of interest is the on-demand data reslicing implemented within the program. Data sets can be freely shifted, rotated, viewed, and analyzed with the program automatically handling interpolation as needed from the original data. Validation has been performed by comparing the output of AMIDE with that of several existing software packages. AMIDE runs on UNIX, Macintosh OS X, and Microsoft Windows platforms, and it is freely available with source code under the terms of the GNU General Public License.

View details for PubMedID 14649056
Optical imaging of transferrin targeted PEI/DNA complexes in living subjects GENE THERAPY Hildebrandt, I. J., Iyer, M., Wagner, E., Gambhir, S. S. 2003; 10 (9): 758-764
Abstract

Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf-PEI-PEG, Tf-PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (P<0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R(2)=0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf-PEI and Tf-PEI-PEG polyplex targeting of N2A tumors in A/J mice.

View details for DOI 10.1038/sj.gt.3301939

View details for Web of Science ID 000182482200005

View details for PubMedID 12704414
Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice MOLECULAR THERAPY De, A., Lewis, X. Z., Gambhir, S. S. 2003; 7 (5): 681-691
Abstract

Lentiviral-mediated gene delivery holds significant promise for sustained gene expression within living systems. Vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 1-based lentiviral vectors can be used to introduce transgenes in a broad spectrum of dividing as well as nondividing cells. In the current study, we construct a lentiviral vector carrying two reporter genes separated by an internal ribosomal entry site and utilize that virus in delivering both genes into neuroblastoma cells in cell culture and into cells implanted in living mice. We utilize two reporter genes, a mutant herpes simplex virus type 1 (HSV1) sr39tk as a reporter gene compatible with positron emission tomography (PET) and a bioluminescent optical reporter gene, firefly luciferase (Fluc), to image expression in living mice by an optical charge-coupled device (CCD) camera. By using this lentivirus, neuroblastoma (N2a) cells are stably transfected and a high correlation (R(2) = 0.91) between expressions of the two reporter genes in cell culture is established. Imaging of both reporter genes using microPET and optical CCD camera in living mice is feasible, with the optical approach being more sensitive, and a high correlation (R(2) = 0.86) between gene expressions is again observed in lentiviral-infected N2a tumor xenografts. Indirect imaging of HSV1-sr39tk suicide gene therapy utilizing Fluc is also feasible and can be detected with increased sensitivity by using the optical CCD. These preliminary results validate the use of lentiviral vectors carrying reporter genes for multimodality imaging of gene expression and should have many applications, including imaging of xenografts, metastasis, and cell trafficking as well as noninvasive monitoring of lentiviral-mediated gene delivery and expression.

View details for DOI 10.1016/S1525-0016(03)00070-4

View details for Web of Science ID 000182645800016

View details for PubMedID 12718911
Positron emission tomography in diagnosis and management of invasive breast cancer: current status and future perspectives. Clinical breast cancer Wu, D., Gambhir, S. S. 2003; 4: S55-63
Abstract

[18F]fluorodeoxyglucose positron emission tomography (FDG-PET) is a metabolic imaging modality that has increasing applications in oncology, neurology, and cardiology. Among the oncology applications, breast cancer is one of the most extensively studied diseases. FDG-PET has been performed for diagnosis, staging, and restaging of invasive breast cancer and for monitoring responsiveness to therapies. At the present time, the results of FDG-PET in detection of primary breast cancer and axillary staging are mixed and inconclusive. However, results demonstrating the superiority of FDG-PET over anatomic imaging modalities in detection of distant metastasis, recurrence, and monitoring therapies are relatively well documented. These applications have been accepted by medical professionals and the public, as evidenced by a recent decision by the Centers for Medicare and Medicaid Services (formerly Health Care Financing Agency) to provide coverage for the procedure. Future trends in this exciting area include development of novel breast cancer-specific PET radiopharmaceuticals and use of dedicated breast PET technologies for scans of breast/axillary lesions. PET/computed tomography technology, which combines anatomic and molecular/biochemical information, is also rapidly proliferating and should help to further improve the management of patients with breast cancer. The role of FDG-PET in breast cancer is increasing and evolving, and this metabolic imaging modality, in conjunction with newer tracers and other anatomic imaging methods, should improve diagnosis and management of patients with breast cancer

View details for PubMedID 12756080
Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation ANALYTICAL CHEMISTRY Paulmurugan, R., Gambhir, S. S. 2003; 75 (7): 1584-1589
Abstract

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

View details for DOI 10.1021/ac020731c

View details for Web of Science ID 000181993600013

View details for PubMedID 12705589
MicroPET imaging of prostate cancer in LNCAP-SR39TK-GFP mouse xenografts PROSTATE Yang, H. H., Berger, F., Tran, C., Gambhir, S. S., Sawyers, C. L. 2003; 55 (1): 39-47
Abstract

The aim of this study was to develop models that allow serial, noninvasive imaging of human prostate cancer cells in immunodeficient mice using a dedicated small animal positron emission tomography scanner (microPET).LNCaP tumor cells were stably transduced ex-vivo with the mutant herpes simplex virus type 1 thymidine kinase (HSV-sr39tk) PET reporter gene and green fluorescent protein (GFP). The stably transduced LNCaP cells were then enriched via fluorescent cell sorting and implanted into SCID mice. Beginning 2 weeks after tumor cell inoculation, mice were repeatedly scanned by microPET performed 1 hr after tail-vein injection of approximately 200 muCi Fluorine-18 labeled penciclovir ((18)F-FHBG). PET-images were correlated to tumor size, % injected dose (ID)/g tumor tissue, PSA levels, autoradiography, and histology.Monitoring LNCaP xenografts using microPET and our reporter gene approaches is feasible. MicroPET was capable of detecting subcutaneous tumors as small as 3 mm in diameter (approximately 0.2% ID/g). The magnitude of (18)F-FHBG-uptake in PET-images correlated with the tumor volumes and the serum PSA levels. Other non-HSV1-TK-specific tracers were also studied. While (18)F-flurodeoxyglucose ((18)F-FDG) gave poor imaging results in LNCaP cells, (11)C-acetate gave satisfactory images.We demonstrated the feasibility of monitoring prostate cancer xenografts in a mouse model using microPET and the HSV1-sr39tk PET reporter gene/(18)F-FHBG reporter probe system. Extension of this approach may allow repetitive imaging of tumor metastases.

View details for DOI 10.1002/pros.10208

View details for Web of Science ID 000181973300005

View details for PubMedID 12640659
Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice CANCER RESEARCH Ray, P., Wu, A. M., Gambhir, S. S. 2003; 63 (6): 1160-1165
Abstract

Noninvasive imaging of reporter gene expression using various imaging modalitiesis playing an increasingly important role in defining molecular events in the field of cancer biology, cell biology, and gene therapy. In this study, a novel reporter vector was constructed encoding a fusion protein comprised of a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) (tk), a positron emission tomography (PET) reporter gene, and renilla luciferase (rl), a bioluminescence optical reporter gene joined by a 20 amino acid long spacer sequence. We validated the activity of the two enzymes encoded by the fusion protein (tk(20)rl) in cell culture. Then, tumors stably expressing the tk(20)rl fusion gene were imaged both by microPET and optically using a cooled charge coupled device camera in xenograft-bearing living mice. Using a single fusion reporter (PET/optical) gene should accelerate the validation of reporter gene approaches developed in cell culture for translation into preclinical and clinical models.

View details for Web of Science ID 000181702300003

View details for PubMedID 12649169
Molecular imaging in living subjects: seeing fundamental biological processes in a new light GENES & DEVELOPMENT Massoud, T. F., Gambhir, S. S. 2003; 17 (5): 545-580
View details for DOI 10.1101/gad.1047403

View details for Web of Science ID 000181502300001

View details for PubMedID 12629038
Quantitative imaging of the T cell antitumor response by positron-emission tomography PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dubey, P., Su, H., Adonai, N., Du, S. Y., Rosato, A., Braun, J., Gambhir, S. S., Witte, O. N. 2003; 100 (3): 1232-1237
Abstract

We describe a noninvasive, quantitative, and tomographic method to visualize lymphocytes within the whole animal. We used positron-emission tomography (PET) to follow the localization of adoptively transferred immune T lymphocytes. Splenic T cells from animals that had rejected a Moloney murine sarcoma virus/Moloney murine leukemia virus (M-MSV/M-MuLV)-induced tumor were marked with a PET reporter gene, injected into tumor-bearing mice, and imaged in a microPET by using a substrate specific for the reporter. Specific localization of immune T cells to the antigen-positive tumor was detected over time, by sequential imaging of the same animals. Naive T cells did not localize to the tumor site, indicating that preimmunization was required. Autoradiography and immunohistochemistry analysis corroborated the microPET data. The method we have developed can be used to assess the effects of immunomodulatory agents intended to potentiate the immune response to cancer, and can also be useful for the study of other cell-mediated immune responses, including autoimmunity.

View details for DOI 10.1073/pnas.0337418100

View details for Web of Science ID 000180838100084

View details for PubMedID 12547911
Positron-emission tomography reporter gene expression imaging in rat myocardium CIRCULATION Inubushi, M., Wu, J. C., Gambhir, S. S., Sundaresan, G., Satyamurthy, N., Namavari, M., Yee, S., Barrio, J. R., Stout, D., Chatziioannou, A. F., Wu, L. L., Schelbert, H. R. 2003; 107 (2): 326-332
Abstract

This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[18F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([18F]-FHBG) and a small-animal PET (microPET).In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1x10(6) to 1x10(9) pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [13N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [18F]-FHBG. The total myocardial [18F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [18F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (r2=0.981, P<0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (r2=0.790, P<0.0001). Myocardial [18F]-FHBG accumulation was identified at viral titers down to 1x10(7) pfu. In 6 rats serially imaged up to day 17, myocardial [18F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17.HSV1-sr39tk reporter gene expression can be monitored with [18F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.

View details for DOI 10.1161/01.CIR.0000044385.60972.AE

View details for Web of Science ID 000180785700022

View details for PubMedID 12538436
Molecular Imaging with Positron Emission Tomography. Discussions in PET Imaging. Diagnostic Imaging Gambhir SS 2003: 1-4
Quantitation of the regional blood flow in the interventricular septum using positron emission tomography and nitrogen-13 ammonia EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Hove, J. D., Gambhir, S. S., Kofoed, K. F., Freiberg, J., Kelbaek, H. 2003; 30 (1): 109-116
Abstract

The purpose of the present study was to evaluate the effect of spillover of activity from the right ventricle (RV) on quantitation of the regional myocardial blood flow in the septum. Thirty-one healthy volunteers, 31 patients with ischemic heart disease, 7 patients with severe congestive heart failure, and 6 heart transplant patients underwent positron emission tomography (PET) with nitrogen-13 ammonia. Quantitation of the regional myocardial blood flow in the septum was performed using both a conventional two-compartment model and a previously validated two-compartment model taking RV spillover into account. Unaccounted RV spillover resulted in significant underestimation of the regional myocardial blood flow in the septum. The amount of underestimation was primarily dependent on the magnitude of spillover and the dispersion between the right and the left ventricular input functions. In healthy volunteers, the flow error was small but significant: on average 6% (range 5%-29%, P<0.00001), compared with 27% (range 0%-88%, P<0.002) in the group of patients with severe congestive heart failure, who had the most considerable amount of RV spillover. In the group of patients with ischemic heart disease and the group of heart transplant patients the flow errors were 10% (range 0%-55%, P<0.00001) and 6% (range 1%-19%, P<0.01), respectively. It is concluded that flow quantitation in the septum is significantly affected by RV spillover, resulting in a considerable underestimation of the septal blood flow unless correction is performed.

View details for DOI 10.1007/s00259-002-1014-z

View details for Web of Science ID 000180691800016

View details for PubMedID 12483417
6th ASNC Invitational Conference on Nuclear Cardiology ? Panel on Gene Product Imaging. Journal of Nuclear Cardiology Bengel FM, Dewanjee M, Dichek D, Gambhir SS, Gelovani J, Zaret B. 2003; 10: 223-257
Monitoring adenoviral DNA delivery, using a mutant herpes simplex virus type 1 thymidine kinase gene as a PET reporter gene GENE THERAPY Liang, Q., Nguyen, K., Satyamurthy, N., Barrio, J. R., Phelps, M. E., Gambhir, S. S., Herschman, H. R. 2002; 9 (24): 1659-1666
Abstract

Current gene therapy protocols often suffer from an inability to monitor the site, level and persistence of gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a reporter gene for in vivo imaging of reporter gene expression. The presence of the HSV1-tk reporter gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET reporter gene system, both alternative substrates and mutations in the HSV1-tk gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET reporter gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.

View details for Web of Science ID 000179898900003

View details for PubMedID 12457279
Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Paulmurugan, R., Umezawa, Y., Gambhir, S. S. 2002; 99 (24): 15608-15613
Abstract

In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.

View details for DOI 10.1073/pnas.242594299

View details for Web of Science ID 000179530000065

View details for PubMedID 12438689
Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice MOLECULAR THERAPY Iyer, M., Berenji, M., Templeton, N. S., Gambhir, S. S. 2002; 6 (4): 555-562
Abstract

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

View details for DOI 10.1006/mthe.2002.0700

View details for Web of Science ID 000178472200018

View details for PubMedID 12385291
Whole-body skeletal imaging in mice utilizing microPET: optimization of reproducibility and applications in animal models of bone disease EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Berger, F., Lee, Y. P., Loening, A. M., Chatziioannou, A., Freedland, S. J., Leahy, R., Lieberman, J. R., Belldegrun, A. S., Sawyers, C. L., Gambhir, S. S. 2002; 29 (9): 1225-1236
Abstract

The aims were to optimize reproducibility and establish [(18)F]fluoride ion bone scanning in mice, using a dedicated small animal positron emission tomography (PET) scanner (microPET) and to correlate functional findings with anatomical imaging using computed tomography (microCAT). Optimal tracer uptake time for [(18)F]fluoride ion was determined by performing dynamic microPET scans. Quantitative reproducibility was measured using region of interest (ROI)-based counts normalized to (a) the injected dose, (b) integral of the heart time-activity curve, or (c) ROI over the whole skeleton. Bone lesions were repetitively imaged. Functional images were correlated with X-ray and microCAT. The plateau of [(18)F]fluoride uptake occurs 60 min after injection. The highest reproducibility was achieved by normalizing to an ROI over the whole skeleton, with a mean percent coefficient of variation [(SD/mean) x 100] of <15%-20%. Benign and malignant bone lesions were successfully repetitively imaged. Preliminary correlation of microPET with microCAT demonstrated the high sensitivity of microPET and the ability of microCAT to detect small osteolytic lesions. Whole-body [(18)F]fluoride ion bone imaging using microPET is reproducible and can be used to serially monitor normal and pathological changes to the mouse skeleton. Morphological imaging with microCAT is useful to display correlative changes in anatomy. Detailed in vivo studies of the murine skeleton in various small animal models of bone diseases should now be possible.

View details for DOI 10.1007/s00259-002-0850-1

View details for Web of Science ID 000178150400020

View details for PubMedID 12418463
Molecular imaging of cancer with positron emission tomography NATURE REVIEWS CANCER Gambhir, S. S. 2002; 2 (9): 683-693
Abstract

The imaging of specific molecular targets that are associated with cancer should allow earlier diagnosis and better management of oncology patients. Positron emission tomography (PET) is a highly sensitive non-invasive technology that is ideally suited for pre-clinical and clinical imaging of cancer biology, in contrast to anatomical approaches. By using radiolabelled tracers, which are injected in non-pharmacological doses, three-dimensional images can be reconstructed by a computer to show the concentration and location(s) of the tracer of interest. PET should become increasingly important in cancer imaging in the next decade.

View details for DOI 10.1038/nrc882

View details for Web of Science ID 000180447400014

View details for PubMedID 12209157
CL1-SR39: A noninvasive molecular imaging model of prostate cancer suicide gene therapy using positron emission tomography JOURNAL OF UROLOGY Pantuck, A. J., Berger, F., Zisman, A., Nguyen, D., Tso, C. L., Matherly, J., Gambhir, S. S., Belldegrun, A. S. 2002; 168 (3): 1193-1198
Abstract

We developed a prostate cancer tumor model capable of being noninvasively imaged using positron emission tomography (PET) based on expression of the herpes simplex virus thymidine kinase (HSV1-tk) reporter gene.The androgen independent, metastatic prostate cancer cell lines CL1 and CL1-GFP were stably transfected with the mutant HSV1-tk gene pcDNA3.1/pCMV-sr39tk, which has increased ability to phosphorylate penciclovir. The presence of the sr39tk gene product was analyzed by Western blot analysis and relative thymidine kinase enzyme activity was assessed by a functional thymidine kinase enzyme activity assay. Subcutaneous and orthotopic CL1 and CL1-SR39 tumor xenografts were established in SCID mice. The ability to image CL1-SR39 was assessed using fluorodeoxyglucose and F-penciclovir ( F-FHBG) micro-PET (a rodent PET scanner). To investigate the systemic distribution of intratumoral sr39tk injections established CL1 tumors were transiently injected with first generation adenoviral vectors carrying the sr39tk gene under control of the strong cytomegalovirus promoter Ad-CMV-HSV1-sr39tk and imaged using micro-PET.Transfection of sr39tk into CL1 cells was successful. CL1-SR39 thymidine kinase enzyme activity was greater than twice the activity of the glioma cell line C6-SR39 control and above the threshold necessary for micro-PET detection. Fluorodeoxyglucose micro-PET in SCID mice was positive for CL1 and CL1-SR39 tumors. Selective micro-PET of subcutaneous CL1-SR39 tumors was done using F-FHBG. Micro-PET imaging after systemic and intratumoral injection of Ad-CMV-HSV1-sr39tk revealed significant systemic transgene leakage with significant hepatic expression of sr39TK protein.Molecular based imaging of sr39tk transfected prostate cancer tumors and adenoviral delivered HSV1-tk suicide gene therapy based on the selective conversion and intracellular trapping of F-FHBG by sr39tk is feasible. Potential applications include noninvasive monitoring of the location, duration and intensity of gene constructs, which may contribute to the safety of clinical gene therapy protocols, and noninvasive imaging of the prostate cancer xenograft response to experimental therapy.

View details for DOI 10.1079/01.ju.0000026576.46595.61

View details for Web of Science ID 000177539600086

View details for PubMedID 12187266
Visualization of advanced human prostate cancer lesions in living mice by a targeted gene transfer vector and optical imaging NATURE MEDICINE Adams, J. Y., Johnson, M., Sato, M., Berger, F., Gambhir, S. S., Carey, M., Iruela-Arispe, M. L., Wu, L. 2002; 8 (8): 891-896
Abstract

Non-invasive imaging and transcriptional targeting can improve the safety of therapeutic approaches in cancer. Here we demonstrate the ability to identify metastases in a human-prostate cancer model, employing a prostate-specific adenovirus vector (AdPSE-BC-luc) and a charge-coupled device-imaging system. AdPSE-BC-luc, which expresses firefly luciferase from an enhanced prostate-specific antigen promoter, restricted expression in the liver but produced robust signals in prostate tumors. In fact, expression was higher in advanced, androgen-independent tumors than in androgen-dependent lesions. Repetitive imaging over a three-week period after AdPSE-BC-luc injection into tumor-bearing mice revealed that the virus could locate and illuminate metastases in the lung and spine. Systemic injection of low doses of AdPSE-BC-luc illuminated lung metastasis. These results demonstrate the potential use of a non-invasive imaging modality in therapeutic and diagnostic strategies to manage prostate cancer.

View details for DOI 10.1038/nm743

View details for Web of Science ID 000177200900037

View details for PubMedID 12134144
Positron emission tomography imaging of cardiac reporter gene expression in living rats CIRCULATION Wu, J. C., Inubushi, M., Sundaresan, G., Schelbert, H. R., Gambhir, S. S. 2002; 106 (2): 180-183
Abstract

Imaging reporter gene expression is useful for noninvasive monitoring of gene therapy. In this study, we imaged cardiac reporter gene expression in living rats using micro positron emission tomography (microPET).Rats (n=10) underwent intramyocardial injection with 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 mutant thymidine kinase (Ad-CMV-HSV1-sr39tk) as PET reporter gene. Control rats (n=4) received 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (Ad-CMV-Fluc). On days 2 to 4, microPET images were obtained after a tail vein injection of nitrogen-13 ammonia ([13N]-NH3) as myocardial perfusion tracer, followed by 9-(4-[18F]-fluoro-3 hydroxymethylbutyl) guanine ([18F]-FHBG) to assess HSV1-sr39tk expression. After imaging, hearts were removed for ex vivo [18F] gamma counting and thymidine kinase enzyme assay. Results show homogenous myocardial distribution of [13N]-NH3 on all microPET images. Rats injected with Ad-CMV-HSV1-sr39tk have significant [18F]-FHBG uptake in the anterolateral wall compared with background signal in controls. Gamma counting shows 20.0+/-4.4-fold increase of radioactivity, whereas enzyme assay shows 22.1+/-6.1-fold increase of thymidine kinase activity in Ad-CMV-HSV1-sr39tk injected rats (P<0.05).Successful imaging of cardiac HSV1-sr39tk expression was performed in living rats with microPET. The presence of [18F]-FHBG uptake is confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay. Cardiac reporter gene imaging by PET may eventually be applied toward human gene therapy studies.

View details for DOI 10.1161/01.CIR.0000023620.59633.53

View details for Web of Science ID 000176820500016

View details for PubMedID 12105155
Noninvasive, repetitive, quantitative measurement of gene expression from a bicistronic message by positron emission tomography, following gene transfer with adenovirus MOLECULAR THERAPY Liang, Q. W., Gotts, J., Satyamurthy, N., Barrio, J., Phelps, M. E., Gambhir, S. S., Herschman, H. R. 2002; 6 (1): 73-82
Abstract

Gene therapy protocols are hampered by the inability to monitor the location, magnitude, and duration of ectopic gene expression following DNA delivery. Consequently, it is difficult to establish quantitative correlations and/or causal relationships between therapeutic gene expression and phenotypic responses in treated individuals. One approach to monitor "therapeutic gene" expression indirectly is to incorporate reporter genes that can be imaged in vivo into bicistronic transcription units, along with the therapeutic genes. Expression of the dopamine D2 receptor (D2R) and herpes simplex virus thymidine kinase (HSV1-TK) can both be monitored, in vivo, by positron-emission tomography (PET). We created ad.DTm, an adenovirus containing a cytomegalovirus (CMV) early promoter-driven transcription unit, in which the D2R gene is placed proximal to an encephalomyocarditis virus internal ribosomal entry site (IRES) and a modified HSV1-tk gene is placed distal to the IRES. Following intravenous ad.DTm injection into mice, correlated hepatic D2R and HSV1-sr39tk PET reporter gene expression was demonstrated. Repeated microPET scanning quantitated both D2R-dependent sequestration of a positron-emitting ligand and HSV1-TK-dependent sequestration of a positron-emitting product. It is possible, in living mice, to investigate noninvasively and to measure quantitatively and repeatedly correlated expression of two coding regions from a bicistronic transcription unit over a 3-month period following adenovirus delivery.

View details for DOI 10.1006/mthe.2002.0626

View details for Web of Science ID 000176578500012

View details for PubMedID 12095306
Towards in vivo nuclear microscopy: iodine-125 imaging in mice using micro-pinholes EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Beekman, F. J., McElroy, D. P., Berger, F., Gambhir, S. S., Hoffman, E. J., Cherry, S. R. 2002; 29 (7): 933-938
Abstract

Position-sensitive gamma-radiation detectors equipped with collimators have been used for in vivo imaging of the distribution of radiolabelled molecules in laboratory animals and humans for several decades. To date, the best image resolution achieved in a rodent is on the order of 1 mm. Here we demonstrate how a basic and compact gamma camera can be constructed for in vivo radionuclide imaging in small animals, at much higher spatial resolution. Resolution improvements were obtained by combining dense, shaped, micro-pinhole apertures with iodine-125, an isotope with low energy emissions, ease of incorporation into a wide range of molecules, and straightforward translation into the clinic via other isotopes of iodine that are suitable for nuclear medicine imaging. (125)I images of test distributions and a mouse thyroid have been obtained at a resolution of as high as 200 microm using this simple bench-top camera. Possible future applications and extension to ultra-high-resolution emission tomography are discussed.

View details for DOI 10.1007/s00259-002-0805-6

View details for Web of Science ID 000177001600014

View details for PubMedID 12111135
Added clinical benefit of incorporating 2-deoxy-2-[18F]fluoro-D-glucose with positron emission tomography into the clinical evaluation of patients with cognitive impairment. Molecular imaging and biology Silverman, D. H., Cummings, J. L., Small, G. W., Gambhir, S. S., Chen, W., Czernin, J., Phelps, M. E. 2002; 4 (4): 283-293
Abstract

Growing evidence indicates that appropriate incorporation of positron emission tomography (PET) into the evaluation of patients with early symptoms of cognitive decline can improve diagnostic and prognostic accuracy. In the present work, an explicitly defined role for PET and its associated impact on expected clinical outcomes were systematically examined.We compared the relative value of two strategies for assessing whether Alzheimer's disease (AD) was responsible for cognitive decline in geriatric patients, and in subsequently managing those patients according to the recommended standards of the American Academy of Neurology (AAN). The first strategy was based on an approach already endorsed by the AAN, following evidence-based reviews carried out by its quality standards subcommittee. The second approach was based on many of the same AAN recommendations-with respect to initial general medical and neurologic examination, structural imaging and laboratory tests, as well as ultimate management-but additionally incorporated PET in appropriate cases, to determine the presence or absence of a pattern of regional cerebral metabolism characteristic of AD. Clinical outcomes accruing to each strategy were calculated using formalized tools of decision analysis.The strategy making use of PET increased diagnostic accuracy, yielding decreased rates of both false negative (from 8.3 to 3.1%) and false positive (from 23.0 to 11.9%) diagnoses for AD, compared with the conventional strategy. When coupled with AAN treatment recommendations for patients having (or not having) non-severe AD, these differences in diagnostic accuracy corresponded to approximately a 62% decrease in avoidable months of nursing home care, and a 48% decrease in months of unnecessary drug therapy resulting from inaccurate diagnoses. The benefit in clinical outcome of the proposed strategy was maintained over a wide range of values for sensitivity, specificity, and projected impact on need for nursing home care.Use of PET for evaluating early cognitive decline in geriatric patients can add valuable information to the clinical assessment, resulting in a greater number of patients being accurately diagnosed and properly treated. PET can be used to diminish disease-related and treatment-related morbidity of dementia, through earlier institution of appropriate management.

View details for PubMedID 14537119
The impact of PET on the management of lung cancer: The referring physician's perspective JOURNAL OF NUCLEAR MEDICINE Seltzer, M. A., Yap, C. S., Silverman, D. H., Meta, J., Schiepers, C., Phelps, M. E., Gambhir, S. S., Rao, J., Valk, P. E., Czernin, J. 2002; 43 (6): 752-756
Abstract

(18)F-FDG PET is a molecular whole-body imaging modality that is increasingly being used for diagnosing, staging, and restaging cancer. The objective of this study was to determine referring physicians' perspectives on the impact of (18)F-FDG PET on staging and management of lung cancer.A questionnaire was sent to the 292 referring physicians of 744 consecutive patients with known or suspected lung cancer who were evaluated with PET. Questionnaires on 274 patients were returned (response rate, 37%). Management changes were categorized as intermodality (e.g., surgery to medical, surgery to radiation, and medical to no treatment) or intramodality (e.g., altered medical, surgical, or radiotherapy approach).The primary reasons for PET referral were staging of lung cancer in 61% of patients, diagnosis in 20%, and monitoring of therapy or the course of disease in 6%. Physicians reported that PET caused them to change their decision on clinical stage in 44% of all patients: The disease was upstaged in 29% and downstaged in 15%. PET resulted in intermodality management changes in 39% of patients, whereas 15% had an intramodality change.This survey-based study of referring physicians suggests that PET has a major impact on staging and management of lung cancer.

View details for Web of Science ID 000176001700011

View details for PubMedID 12050318
Positron emission tomography imaging analysis of G2A as a negative modifier of lymphoid leukemogenesis initiated by the BCR-ABL oncogene CANCER CELL Le, L. Q., Kabarowski, J. H., Wong, S., Nguyen, K., Gambhir, S. S., Witte, O. N. 2002; 1 (4): 381-391
Abstract

G2A is a lymphocyte-expressed G protein-coupled receptor whose genetic ablation results in the development of autoimmunity. Using HSV-TK reporter gene directed positron emission tomography (PET), we demonstrate that prior to any indication of the onset of illness, mice transplanted with BCR-ABL transduced G2A-deficient bone marrow harbor expanded populations of leukemic cells compared to recipients of wild-type bone marrow. The target cell type and anatomical locations of leukemia development are indistinguishable in animals transplanted with G2A+/+ or G2A-/- cells. Shorter disease latency in the G2A-deficient background is associated with an increased rate of cellular expansion. PET can be successfully applied to the temporal and spatial analysis of Bcr-Abl driven leukemic progression and should have utility for the study of other leukemias and lymphomas.

View details for Web of Science ID 000178352100012

View details for PubMedID 12086852
Optimizing prostate cancer suicide gene therapy using herpes simplex virus thymidine kinase active site variants HUMAN GENE THERAPY Pantuck, A. J., Matherly, J., Zisman, A., Nguyen, D., Berger, F., Gambhir, S. S., Black, M. E., Belldegrun, A., Wu, L. L. 2002; 13 (7): 777-789
Abstract

The herpes simplex virus (HSV) thymidine kinase gene (tk) forms the basis of a widely used strategy for suicide gene therapy. A library of HSV thymidine kinase enzyme (TK) active site mutants having different affinities for guanosine analog prodrugs was developed. We sought to determine the optimal combination of tk variant and prodrug specifically for prostate cancer gene therapy, using in vitro and in vivo studies of adenovirally infected CL1, DU-145, and LNCaP tumor lines carrying wild-type tk, tk30, tk75, and sr39tk mutants expressed by a strong, constitutive cytomegalovirus promoter and treated with ganciclovir and acyclovir. In vitro experiments involving prostate cancer (CaP) cell line infection were carried out with a broad range of prodrug concentrations, and cell killing was determined by limiting dilution (colony-forming), MTT, and propidium iodide assays. In vivo studies based on CL1-GFP xenograft experiments were carried out to examine the ability of each TK variant to prevent tumor formation and to inhibit tumor growth and development of metastases in established orthotopic and subcutaneous tumors in SCID mice. Both in vitro and in vivo studies suggest improved killing with the sr39tk variant. Thus, the results suggest that the use of sr39tk in future trials of prostate cancer tk suicide gene therapy may be beneficial.

View details for Web of Science ID 000175226600001

View details for PubMedID 11975845
Optical imaging of cardiac reporter gene expression in living rats CIRCULATION Wu, J. C., Inubushi, M., Sundaresan, G., Schelbert, H. R., Gambhir, S. S. 2002; 105 (14): 1631-1634
Abstract

Studies of cardiac gene transfer rely on postmortem analysis using histologic staining or enzyme assays. Noninvasive imaging of the temporal and spatial characteristics of cardiac gene expression in the same subject offers significant advantages.Rats underwent direct myocardial injection via left thoracotomy with adenovirus-expressing firefly luciferase (Ad-CMV-Fluc; n=30). The reporter substrate D-luciferin was injected intraperitoneally. Serial images were acquired by use of a cooled charged couple detector (CCD) camera. Results are expressed as relative light unit per minute (RLU/min). Rats transduced with 1x10(9) plaque-forming units show decremental cardiac luciferase activity over time: 152 070+/-21 170 (day 2), 195 806+/-62 630 (day 5), 7250+/-2941 (day 8), and 2040+/-971 RLU/min (day 14). To assess the detection sensitivity, serially diluted titers of Ad-CMV-Fluc were injected: 1x10(9) (195 393+/-14 896), 1x10(8) (33 777+/-18 179), 1x10(7) (417+/-91), 1x10(6) (185+/-64), 1x10(5) (53+/-1), and control (54+/-1) (P<0.05 for 1x10(9), 1x10(8), and 1x10(7) plaque-forming units versus control adenovirus-expressing mutant thymidine kinase [Ad-CMV-HSV1-sr39tk]; n=3). Finally, rats were euthanized, and in vitro luciferase activity correlated with in vivo CCD signals (r2=0.92).This study demonstrates for the first time the feasibility of imaging the location, magnitude, and time course of cardiac reporter gene expression in living rats. Cardiac gene therapy studies could be aided with wider application of this approach.

View details for DOI 10.1161/01.CIR.0000014984.95520.AD

View details for Web of Science ID 000174987300185

View details for PubMedID 11940538
Ex vivo cell labeling with Cu-64-pyruvaldehyde-bis(N-4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Adonai, N., Nguyen, K. N., Walsh, J., Iyer, M., Toyokuni, T., Phelps, M. E., McCarthy, T., MCCARTHY, D. W., Gambhir, S. S. 2002; 99 (5): 3030-3035
Abstract

We have used copper-64-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[18F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. 64Cu-PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64Cu-PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64Cu-PTSM than with FDG. However, both 64Cu-PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64Cu-PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64Cu-PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64Cu-PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64Cu-PTSM-labeled cells. Given the longer t(1/2) of 64Cu (12.7 h) relative to 18F (110 min), longer cell-tracking periods (up to 24-36 h) should be possible now with PET.

View details for DOI 10.1073/pnas.052709599

View details for Web of Science ID 000174284600079

View details for PubMedID 11867752
Noninvasive quantitative imaging of protein-protein interactions in living subjects PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ray, P., Pimenta, H., Paulmurugan, R., Berger, F., Phelps, M. E., Iyer, M., Gambhir, S. S. 2002; 99 (5): 3105-3110
Abstract

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-kappaB promoter through tumor necrosis factor alpha. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.

View details for DOI 10.1073/pnas.052710999

View details for Web of Science ID 000174284600092

View details for PubMedID 11854471
The impact of 2-deoxy-2[18F] fluoro-D-glucose whole body positron emission tomography for managing patients with melanoma: the referring physician's perspective. Molecular imaging and biology Wong, C., Silverman, D. H., Seltzer, M., Schiepers, C., Ariannejad, M., Gambhir, S. S., Phelps, M. E., Rao, J., Valk, P., Czernin, J. 2002; 4 (2): 185-190
Abstract

Whole body positron emission tomography (PET) imaging with 2-deoxy-2[18F]fluoro-D-glucose (FDG) has been used successfully to diagnose and stage melanoma. The impact of FDG-PET, however, on patient stage and management from the referring physicians' perspective is unknown.A questionnaire was sent to referring physicians to investigate whether and how PET altered clinical decision in treatment of melanoma patients. Surveys were sent to referring physicians of every melanoma patient who had a PET scan performed at UCLA or the Northern California PET Imaging Center (NCPIC). Data were used to evaluate the impact of FDG-PET on clinical management of melanoma patients based on pre-PET and post-PET staging. Management changes were classified as inter-modality if therapy changed from one modality to another or intra-modality if changes were made within a treatment modality.Fifty-one questionnaires (response rate of 35%) have been received to date. Referring physicians indicated that whole body FDG-PET changed the clinical stage in 15 out of 51 (29%) patients: 10 (20%) were up-staged and five (10%) were down-staged. The PET findings resulted in inter-modality management changes in 15 out of 51 patients (29%). Intra-modality management change occurred in nine patients (18%).From the referring physicians' perspective, FDG-PET has a major impact and results in management changes in 53% of patients with melanoma.

View details for PubMedID 14537142
Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer MOLECULAR THERAPY Zhang, L. Q., Adams, J. Y., Billick, E., Ilagan, R., Iyer, M., Le, K., Smallwood, A., Gambhir, S. S., Carey, M., Wu, L. L. 2002; 5 (3): 223-232
Abstract

Gene therapy is founded on the concept that tissue-specific promoters can express heterologous genes for molecular imaging or therapeutic applications. The engineering of cell-specific enhancers to improve potency and the development of two-step transcriptional activation (TSTA) approaches have significantly improved the efficacy of transgene expression. Here we combine these technologies to create a robust, titratable, androgen-responsive system targeted to prostate cancer cells. Our "chimeric" TSTA system uses a duplicated variant of the prostate-specific antigen (PSA) gene enhancer to express GAL4 derivatives fused to one, two, or four VP16 activation domains. We targeted the resulting activators to cells with reporter templates bearing one, two, or five GAL4 binding sites upstream of firefly luciferase. We monitored activity via firefly luciferase assays in transfected cell extracts and in live nude mice using a cooled charge-coupled device (CCD) imaging system. In this system, we found that firefly luciferase expression in prostate cancer cells can be varied over an 800-fold range. We also found that a single plasmid bearing the optimized enhancer, GAL4-VP16 derivative, and reporter expressed firefly luciferase at 20-fold higher levels than the cytomegalovirus enhancer. We discuss the implications of this strategy and its application to molecular imaging and therapy.

View details for DOI 10.1006/mthe.2002.0551

View details for Web of Science ID 000174192700005

View details for PubMedID 11863411
Gene expression tomography PHYSIOLOGICAL GENOMICS Brown, V. M., Ossadtchi, A., Khan, A. H., Gambhir, S. S., Cherry, S. R., Leahy, R. M., Smith, D. J. 2002; 8 (2): 159-167
Abstract

Gene expression tomography, or GET, is a new method to increase the speed of three-dimensional (3-D) gene expression analysis in the brain. The name is evocative of the method's dual foundations in high-throughput gene expression analysis and computerized tomographic image reconstruction, familiar from techniques such as positron emission tomography (PET) and X-ray computerized tomography (CT). In GET, brain slices are taken using a cryostat in conjunction with axial rotation about independent axes to create a series of "views" of the brain. Gene expression information obtained from the axially rotated views can then be used to recreate 3-D gene expression patterns. GET was used to successfully reconstruct images of tyrosine hydroxylase gene expression in the mouse brain, using both RNase protection and real-time quantitative reverse transcription PCR (QRT-PCR). A Monte-Carlo analysis confirmed the good quality of the GET image reconstruction. By speeding acquisition of gene expression patterns, GET may help improve our understanding of the genomics of the brain in both health and disease.

View details for DOI 10.1152/physiolgenomics.00090.2001

View details for Web of Science ID 000174252200011

View details for PubMedID 11875194
Decision analysis for the cost-effective management of recurrent colorectal cancer. Annals of surgery Park, K. C., Schwimmer, J., Gambhir, S. S. 2002; 235 (2): 309-310
View details for PubMedID 11807375
Evaluating early dementia with and without assessment of regional cerebral metabolism by PET: A comparison of predicted costs and benefits JOURNAL OF NUCLEAR MEDICINE Silverman, D. H., Gambhir, S. S., Huang, H. W., Schwimmer, J., Kim, S., Small, G. W., Chodosh, J., Czernin, J., Phelps, M. E. 2002; 43 (2): 253-266
Abstract

Evaluating dementia in patients with early symptoms of cognitive decline is clinically challenging. Growing evidence indicates that appropriate incorporation of PET into the clinical work-up can improve diagnostic and prognostic accuracy with respect to Alzheimer's disease (AD), the most common cause of dementia in the geriatric population. The precise diagnostic role of PET and its economic impact in this context, however, have not been systematically examined previously.We compared the relative value of 2 strategies for assessing whether early AD is responsible for cognitive symptoms in geriatric patients: (a) a conventional approach, based largely on establishing clinical criteria for the presence of dementia and excluding non-AD etiologies that could contribute to the patient's symptoms, and (b) a proposed approach using PET to examine regional cerebral metabolism and look for characteristic patterns of abnormal metabolism. The total costs (measured in dollars) and benefits (measured in number of accurate diagnoses) of diagnostic testing and clinical outcomes accruing to each strategy were calculated using formalized tools of decision analysis. The primary outcome measure by which the strategies were compared was the ratio of costs to benefits obtained following each approach.Following the proposed approach led to improved accuracy in identifying early AD, without adding to the overall costs of diagnosis and treatment ($3,433 vs. $3,564 per patient approached by the proposed or conventional algorithm, respectively). The strategy making use of PET was associated with a reduced rate of false-negative and false-positive findings compared with the conventional approach (3.1% vs. 8.2% and 12.0% vs. 23.0%, respectively, at a prevalence of 51.6% in the studied symptomatic population) and a cost savings of $1,138 per correct diagnosis rendered ($4,047 vs. $5,185). The lower cost per unit benefit for the proposed strategy was maintained over a wide range of tested values for variables of sensitivity, specificity, costs of PET and long-term care, and varying approaches to the use of structural neuroimaging.Appropriate use of PET for evaluating early dementia in geriatric patients can add valuable information to the clinical work-up, without adding to the overall costs of evaluation and management, resulting in a greater number of patients being accurately diagnosed for the same level of financial expenditure. Thus, the opportunity exists for diminishing the morbidity of dementia economically, with earlier institution of more appropriate management in evaluated patients.

View details for Web of Science ID 000173891900032

View details for PubMedID 11850493
Optical imaging of Renilla luciferase reporter gene expression in living mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bhaumik, S., Gambhir, S. S. 2002; 99 (1): 377-382
Abstract

Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme/protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme/protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, d-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes.

View details for Web of Science ID 000173233300068

View details for PubMedID 11752410
Welcome to the European Journal of Nuclear Medicine and Molecular Imaging. European journal of nuclear medicine and molecular imaging Gambhir, S. S., Ell, P. J. 2002; 29 (1): 1-2
View details for PubMedID 11807600
Molecular imaging of biological processes from micropet in mice to pet in patients 2002 IEEE INTERNATIONAL SYMPOSIUM ON BIOMEDICAL IMAGING, PROCEEDINGS Phelps, M. E., Chatziioannou, A., Cherry, S., Gambhir, S. 2002: 1-9
View details for Web of Science ID 000178000400001
Indirect monitoring of endogenous gene expression by positron emission tomography (PET) imaging of reporter gene expression in transgenic mice. Molecular imaging and biology Green, L. A., Yap, C. S., Nguyen, K., Barrio, J. R., Namavari, M., Satyamurthy, N., Phelps, M. E., Sandgren, E. P., Herschman, H. R., Gambhir, S. S. 2002; 4 (1): 71-81
Abstract

Repetitive imaging with microPET of endogenous albumin gene expression by using transgenic mice in which the Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene is driven by the albumin promoter (AL-HSV1-tk).Transgenic mice were imaged repeatedly on a microPET scanner with approximately 200 microCi of 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) (a substrate for HSV1-TK enzyme). Four transgenic mice were monitored for body weight, serum albumin, and imaged at the end of each of three dietary phases (17%, 0%, and 25% protein diet). Each phase last 14-21 days. The 0% protein diet has been reported previously to reduce albumin gene expression in rats. Twenty non-transgenic mice of the same strain followed a similar feeding schedule and were monitored for serum albumin, body weight, and sacrificed at various time points for determination of their GAPDH normalized albumin mRNA levels.Transgenic mice showed a relatively high FHBG signal from the liver region as expected. Variation of the mean FHBG signal in two mice with a fixed 17% protein diet over a four-month period was <19% s.d. The mean +/- s.e. FHBG liver standardized uptake value (SUV) in four transgenics went from 4.49 +/- 0.32 to 2.17 +/- 0.52 to 6.21 +/- 0.72 as the mice went through the three diets of 17%, 0%, and 25% sequentially. Non-transgenic mice showed GAPDH normalized albumin mRNA that went from 37.68 +/- 6.04 to 26.41 +/- 4.29 to 52.42 +/- 4.09. The FHBG SUV from transgenics was well correlated with GAPDH normalized albumin mRNA from non-transgenics (r(2) = 0.97) supporting that endogenous gene expression of albumin can be indirectly imaged with FHBG.Measuring correlated changes in albumin expression in wild type mice and HSV1-TK expression by microPET in transgenic mice in which the reporter gene is driven by the albumin promoter demonstrates that the HSV1-tk gene can be used to monitor, in living animals, modulated expression of transgenes.

View details for PubMedID 14538050
Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Iyer, M., Wu, L., Carey, M., Wang, Y., Smallwood, A., Gambhir, S. S. 2001; 98 (25): 14595-14600
Abstract

We are developing assays to image tissue-specific reporter gene expression in living mice by using optical methods and positron emission tomography. Approaches for imaging reporter gene expression depend on robust levels of mRNA and reporter protein. Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak relative to stronger but constitutively expressing viral promoters. In this study, we have validated methods to enhance the transcriptional activity of the prostate-specific antigen promoter for imaging by using a two-step transcriptional amplification (TSTA) system. We used the TSTA system to amplify expression of firefly luciferase (fl) and mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) in a prostate cancer cell line (LNCaP). We demonstrate approximately 50-fold (fl) and approximately 12-fold (HSV1-sr39tk) enhancement by using the two-step approach. The TSTA system is observed to retain tissue selectivity. A cooled charge-coupled device optical imaging system was used to visualize the amplified fl expression in living mice implanted with LNCaP cells transfected ex vivo. These imaging experiments reveal a approximately 5-fold gain in imaging signal by using the TSTA system over the one-step system. The TSTA approach will be a valuable and generalizable tool to amplify and noninvasively image reporter gene expression in living animals by using tissue-specific promoters. The approaches validated should have important implications for study of gene therapy vectors, cell trafficking, transgenic models, as well as studying development of eukaryotic organisms.

View details for Web of Science ID 000172576900074

View details for PubMedID 11734653
Positron emission tomography in evaluation of dementia - Regional brain metabolism and long-term outcome JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Silverman, D. H., Small, G. W., Chang, C. Y., Lu, C. S., de Aburto, M. A., Chen, W., Czernin, J., Rapoport, S. I., Pietrini, P., Alexander, G. E., Schapiro, M. B., Jagust, W. J., Hoffman, J. M., Welsh-Bohmer, K. A., Alavi, A., Clark, C. M., Salmon, E., de Leon, M. J., Mielke, R., Cummings, J. L., Kowell, A. P., Gambhir, S. S., Hoh, C. K., Phelps, M. E. 2001; 286 (17): 2120-2127
Abstract

Deficits in cerebral glucose utilization have been identified in patients with cognitive dysfunction attributed to various disease processes, but their prognostic and diagnostic value remains to be defined.To assess the sensitivity and specificity with which cerebral metabolic patterns at a single point in time forecast subsequent documentation of progressive dementia.Positron emission tomography (PET) studies of [(18)F]fluorodeoxyglucose in 146 patients undergoing evaluation for dementia with at least 2 years' follow-up for disease progression at the University of California, Los Angeles, from 1991 to 2000, and PET studies in 138 patients undergoing evaluation for dementia at an international consortium of facilities, with histopathological diagnoses an average of 2.9 years later, conducted from 1984 to 2000.Regional distribution of [(18)F]fluorodeoxyglucose in each patient, classified by criteria established a priori as positive or negative for presence of a progressive neurodegenerative disease in general and of Alzheimer disease (AD) specifically, compared with results of longitudinal or neuropathologic analyses.Progressive dementia was detected by PET with a sensitivity of 93% (191/206) and a specificity of 76% (59/78). Among patients with neuropathologically based diagnoses, PET identified patients with AD and patients with any neurodegenerative disease with a sensitivity of 94% and specificities of 73% and 78%, respectively. The negative likelihood ratio of experiencing a progressive vs nonprogressive course over the several years following a single negative brain PET scan was 0.10 (95% confidence interval, 0.06-0.16), and the initial pattern of cerebral metabolism was significantly associated with the subsequent course of progression overall (P<.001).In patients presenting with cognitive symptoms of dementia, regional brain metabolism was a sensitive indicator of AD and of neurodegenerative disease in general. A negative PET scan indicated that pathologic progression of cognitive impairment during the mean 3-year follow-up was unlikely to occur.

View details for Web of Science ID 000172032100031

View details for PubMedID 11694153
Quantitative imaging of gene induction in living animals GENE THERAPY Sun, X., Annala, A. J., Yaghoubi, S. S., Barrio, J. R., Nguyen, K. N., Toyokuni, T., Satyamurthy, N., Namavari, M., Phelps, M. E., Herschman, H. R., Gambhir, S. S. 2001; 8 (20): 1572-1579
Abstract

Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D(2)R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r(2) = 0.98) between 'target' and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.

View details for Web of Science ID 000172014800008

View details for PubMedID 11704818
Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice MOLECULAR THERAPY Wu, J. C., Sundaresan, G., Iyer, M., Gambhir, S. S. 2001; 4 (4): 297-306
Abstract

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

View details for Web of Science ID 000171403200006

View details for PubMedID 11592831
Monitoring gene therapy with reporter gene imaging SEMINARS IN NUCLEAR MEDICINE Ray, P., Bauer, E., Iyer, M., Barrio, J. R., Satyamurthy, N., Phelps, M. E., Herschman, H. R., Gambhir, S. S. 2001; 31 (4): 312-320
Abstract

Rapid advances in imaging technologies and gene transfer strategies offer a great opportunity to optimize clinical trials of human gene therapy. Reporter genes are emerging as very powerful tools to monitor the delivery, magnitude, and time variation of therapeutic gene transfer in vivo. Several reporter genes, such as the herpes simplex virus type 1 thymidine kinase, the dopamine type 2 receptor, and the somatostatin receptor type 2, are currently being successfully used with gamma camera, single photon emission computed tomography, and positron emission tomography imaging. These reporter genes can be coupled with a therapeutic gene of interest to indirectly monitor the expression of the therapeutic gene. Finally, applications of the reporter gene technology to other areas, such as cell trafficking studies and transgenic animal models, are now possible.

View details for DOI 10.1053/snuc.2001.26209

View details for Web of Science ID 000172025700010

View details for PubMedID 11710773
Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction GENE THERAPY Liang, Q., Satyamurthy, N., Barrio, J. R., Toyokuni, T., Phelps, M. P., Gambhir, S. S., Herschman, H. R. 2001; 8 (19): 1490-1498
Abstract

The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2'-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind [3H]spiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent [3H]spiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene.

View details for Web of Science ID 000171217000007

View details for PubMedID 11593362
Impact of whole-body F-18-FDG PET on staging and managing patients with breast cancer: The referring physician's perspective JOURNAL OF NUCLEAR MEDICINE Yap, C. S., Seltzer, M. A., Schiepers, C., Gambhir, S. S., Rao, J., Phelps, M. E., Valk, P. E., Czernin, J. 2001; 42 (9): 1334-1337
Abstract

FDG PET has emerged as an important clinical imaging modality for diagnosing and staging cancer. However, the impact of FDG PET on staging and managing patients with breast cancer from the referring physician's point of view is unknown.The referring physicians of 160 breast cancer patients received standardized questionnaires inquiring if and how PET findings altered their patient's stage and their clinical management decisions. Management changes were classified as intermodality if the change was from one modality to another (e.g., medical to surgical, surgical to radiation, medical to no treatment, and vice versa) or as intramodality if the change was within the same modality (e.g., altered medical or radiotherapy approach).Fifty of the 160 surveys were completed (31% response rate). PET changed the clinical stage in 36% of patients (28% upstaged, 8% downstaged) and resulted in intermodality changes in 28% of patients and intramodality changes in 30% of patients.The results of this prospective survey show that FDG PET has a major impact on the management of breast cancer patients, influencing both clinical stage and management in more than 30% of patients.

View details for Web of Science ID 000170841000018

View details for PubMedID 11535721
Human pharmacokinetic and dosimetry studies of [F-18]FHBG: A reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression JOURNAL OF NUCLEAR MEDICINE Yaghoubi, S., Barrio, J. R., Dahlbom, M., Iyer, M., Namavari, M., Goldman, R., Herschman, H. R., Phelps, M. E., Gambhir, S. S. 2001; 42 (8): 1225-1234
Abstract

9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) has been used as a reporter probe to image expression of herpes simplex virus type-1 thymidine kinase (HSV1-tk) reporter gene in living animals. Our aim was to study the kinetics, biodistribution, stability, dosimetry, and safety of [(18)F]FHBG in healthy human volunteers, preparatory to imaging patients undergoing HSV1-tk gene therapy.[(18)F]FHBG was synthesized with a specific activity of 37,000--444,000 GBq/mmol and a radiochemical purity > 99%. Ten healthy volunteers consented to participate in the study. A transmission scan was obtained before bolus injection of 70.3--229.4 MBq [(18)F]FHBG into a hand vein, followed by dynamic PET imaging with 4 consecutive emission scans. Warmed hand-vein blood was withdrawn at various times after injection for blood time--activity measurements. Electrocardiography, blood pressure, and blood and urine pharmacologic parameters were measured before and after injection of the [(18)F]FHBG tracer (n = 5). The stability of [(18)F]FHBG in the urine was analyzed. Attenuation-corrected images were reconstructed using the ordered-subsets expectation maximization algorithm. Image region-of-interest time-activity data were used with the MIRD program to estimate absorbed radiation dosages.[(18)F]FHBG had rapid blood clearance; only 8.42% +/- 4.76% (mean +/- SD) of the peak blood activity remained at approximately 30 min. The average ratio of plasma activity to whole-blood activity during the study was 0.91 +/- 0.04. Penetration of [(18)F]FHBG across the blood-brain barrier was not observed. The primary routes of clearance were renal and hepatobiliary. High activities were observed in the bladder, gut, liver, and kidneys, but <0.0002% of the injected dose per gram was observed in other tissues. In the urine, 83% of activity 180 min after injection was stable [(18)F]FHBG. Blood and urine pharmacologic parameters did not change significantly after injection of the [(18)F]FHBG tracer. The bladder absorbed the highest radiation dose.[(18)F]FHBG has the desirable in vivo characteristics of stability, rapid blood clearance, low background signal, biosafety, and acceptable radiation dosimetry in humans. This study forms the foundation for using [(18)F]FHBG in applications to monitor HSV1-tk reporter gene expression.

View details for Web of Science ID 000170311300017

View details for PubMedID 11483684
Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors GENE THERAPY Yaghoubi, S. S., Wu, L., Liang, Q., Toyokuni, T., Barrio, J. R., Namavari, M., Satyamurthy, N., Phelps, M. E., Herschman, H. R., Gambhir, S. S. 2001; 8 (14): 1072-1080
Abstract

Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.

View details for Web of Science ID 000170288800004

View details for PubMedID 11526454
A fast nonlinear method for parametric imaging of myocardial perfusion by dynamic N-13-ammonia PET JOURNAL OF NUCLEAR MEDICINE Golish, S. R., Hove, J. D., Schelbert, H. R., Gambhir, S. S. 2001; 42 (6): 924-931
Abstract

A parametric image of myocardial perfusion (mL/min/g) is a quantitative image generated by fitting a tracer kinetic model to dynamic (13)N-ammonia PET data on a pixel-by-pixel basis. There are several methods for such parameter estimation problems, including weighted nonlinear regression (WNLR) and a fast linearizing method known as Patlak analysis. Previous work showed that sigmoidal networks can be used for parameter estimation of mono- and biexponential models. The method used in this study is a hybrid of WNLR and sigmoidal networks called nonlinear regression estimation (NRE). The purpose of the study is to compare NRE with WNLR and Patlak analysis for parametric imaging of perfusion in the canine heart by (13)N-ammonia PET.A simulation study measured the statistical performance of NRE, WNLR, and Patlak analysis for a probabilistic model of time-activity curves. Four canine subjects were injected with 740 MBq (13)N-ammonia and scanned dynamically. Images were reconstructed with filtered backprojection and resliced into short-axis cuts. Parametric images of a single midventricular plane per subject were generated by NRE, WNLR, and Patlak analysis. Small regions of interest (ROIs) were drawn on each parametric image (8 ROIs per subject for a total of 32).For the simulation study, the median absolute value of the relative error for a perfusion value of 1.0 mL/min/g was 16.6% for NRE, 17.9% for WNLR, 19.5% for Patlak analysis, and 14.5% for an optimal WNLR method (computable by simulation only). All methods are unbiased conditioned on a wide range of perfusion values. For the canine studies, the least squares line fits comparing NRE (y) and Patlak analysis (z) with WNLR (x) for all 32 ROIs were y = 1.02x - 0.028 and z = 0.90x + 0.019, respectively. Both NRE and Patlak analysis generate 128 x 128 parametric images in seconds.The statistical performance of NRE is competitive with WNLR and superior to Patlak analysis for parametric imaging of myocardial perfusion. NRE is a fast nonlinear alternative to Patlak analysis and other fast linearizing methods for parametric imaging. NRE should be applicable to many other tracers and tracer kinetic models.

View details for Web of Science ID 000169143000021

View details for PubMedID 11390558
A tabulated summary of the FDG PET literature JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S., Czernin, J., Schwimmer, J., Silverman, D. H., Coleman, R. E., Phelps, M. E. 2001; 42 (5): 1S-93S
View details for Web of Science ID 000168669400001

View details for PubMedID 11483694
Impact of F-18-FDG PET on managing patients with colorectal cancer: The referring physician's perspective JOURNAL OF NUCLEAR MEDICINE Meta, J., Seltzer, M., Schiepers, C., Silverman, D. H., Ariannejad, M., Gambhir, S. S., Phelps, M. E., Valk, P., Czernin, J. 2001; 42 (4): 586-590
Abstract

Whole-body PET imaging with 18F-FDG has been used successfully to stage colorectal cancer. However, the impact of FDG PET on patient management from the referring physician's point of view has not been determined.A questionnaire was sent to referring physicians to determine whether and how PET altered the management of colorectal cancer patients. Management changes, when present, were classified as intermodality (e.g., medical to surgical, surgical to radiation, medical to no treatment) or intramodality (e.g., altered medical, surgical, or radiotherapy approach).Of 60 responses from referring physicians, changes in clinical stage were reported for 25 patients (42%). Among these, the disease was upstaged in 20 patients (80%) and downstaged in 5 patients (20%). The PET findings contributed to intermodality management changes in 22 of the 60 patients (37%), intramodality changes in 11 patients (18%), a combination of management changes in 4 patients (7%), and no change in 19 patients (32%). Two of the 60 patients (3%) had other changes, and no response to this question was received for the remaining 2 patients (3%). As a result of PET findings, physicians avoided major surgery in 41% of patients for whom surgery was the intended treatment.This survey-based study of referring physicians shows that FDG PET had a major impact on the management of colorectal cancer patients and contributed to changes in clinical stage and major management decisions in >40% of patients.

View details for Web of Science ID 000168000500013

View details for PubMedID 11337546
Decision Analysis for the Cost-Effective Management of Recurrent Colorectal Cancer. Annals of Surgery Park KC, Schwimmer J, Shepherd JE, Phelps ME, Czernin JR, Schiepers C, Gambhir SS 2001; 233(3): 310-319
Comparison of Helical Computerized Tomography, Positron Emission Tomography and Monoclonal Antibody Scans for Evaluation of Lymph Node Metastases in Patients with Prostate Specific Antigen Relapse after Treatment for Localized Prostate Cancer. Journal of Urology Seltzer M, Barbaric Z, Belldegrun A, Naitoh J, Dorey F, Phelps ME, Gambhir SS, Hoh C. 2001; 162(4): 1322-1328
Bar Harbor Panel on Molecular Imaging. Journal of Nuclear Cardiology Zaret B, Leppo J, Bender J, Carrio I, Echelman W, Gambhir SS, Gree A, Narula J. 2001; 8(2): 256-266
Use of positron emission tomography in animal research. ILAR journal Cherry, S. R., Gambhir, S. S. 2001; 42 (3): 219-232
Abstract

Among the several imaging technologies applied to in vivo studies of research animals, positron emission tomography (PET) is a nuclear imaging technique that permits the spatial and temporal distribution of compounds labeled with a positron-emitting radionuclide to be determined noninvasively. It can be viewed as an in vivo analog of classic autoradiographic methods. Many different positron-labeled compounds have been synthesized as tracers that target a range of specific markers or pathways. These tracers permit the measurement of quantities of biological interest ranging from glucose metabolism to gene expression. PET has been extensively used in imaging studies of larger research animals such as dogs and nonhuman primates. Now, using newly developed high-resolution dedicated animal PET scanners, these types of studies can be performed in small laboratory animals such as mice and rats. The entire whole-body biodistribution kinetics can be determined in a single imaging study in a single animal. This technique should enable statistically significant biodistribution data to be obtained from a handful of animals, compared with the tens or hundreds of animals that might be required for a similar study by autoradiography. PET also enables repeat studies in a single subject, facilitating longitudinal study designs and permitting each animal to serve as its own control in experiments designed to evaluate the effects of a particular interventional strategy. This paper provides a basic overview of the methodology of PET imaging, a discussion of the advantages and drawbacks of PET as a tool in animal research, a description of the latest generation of dedicated animal PET scanners, and a review of a few of the many applications of PET in animal research to date.

View details for PubMedID 11406721
Recent advances in imaging endogenous or transferred gene expression utilizing radionuclide technologies in living subjects: applications to breast cancer BREAST CANCER RESEARCH Berger, F., Gambhir, S. S. 2001; 3 (1): 28-U1
Abstract

A variety of imaging technologies is being investigated as tools for studying gene expression in living subjects. Two technologies that use radiolabeled isotopes are single photon emission computed tomography (SPECT) and positron emission tomography (PET). A relatively high sensitivity, a full quantitative tomographic capability, and the ability to extend small animal imaging assays directly into human applications characterize radionuclide approaches. Various radiolabeled probes (tracers) can be synthesized to target specific molecules present in breast cancer cells. These include antibodies or ligands to target cell surface receptors, substrates for intracellular enzymes, antisense oligodeoxynucleotide probes for targeting mRNA, probes for targeting intracellular receptors, and probes for genes transferred into the cell. We briefly discuss each of these imaging approaches and focus in detail on imaging reporter genes. In a PET reporter gene system for in vivo reporter gene imaging, the protein products of the reporter genes sequester positron emitting reporter probes. PET subsequently measures the PET reporter gene dependent sequestration of the PET reporter probe in living animals. We describe and review reporter gene approaches using the herpes simplex type 1 virus thymidine kinase and the dopamine type 2 receptor genes. Application of the reporter gene approach to animal models for breast cancer is discussed. Prospects for future applications of the transgene imaging technology in human gene therapy are also discussed. Both SPECT and PET provide unique opportunities to study animal models of breast cancer with direct application to human imaging. Continued development of new technology, probes and assays should help in the better understanding of basic breast cancer biology and in the improved management of breast cancer patients.

View details for Web of Science ID 000167344300008

View details for PubMedID 11250742
8-[F-18]fluoropenciclovir: An improved reporter probe for imaging HSV1-tk reporter gene expression in vivo using PET JOURNAL OF NUCLEAR MEDICINE Iyer, M., Barrio, J. R., Namavari, M., Bauer, E., Satyamurthy, N., Nguyen, K., Toyokuni, T., Phelps, M. E., Herschman, H. R., Gambhir, S. S. 2001; 42 (1): 96-105
Abstract

We have synthesized and evaluated 8-[18F]fluoropenciclovir (FPCV) and compared it with 8-[18F]fluoroganciclovir (FGCV) for monitoring the expression of herpes simplex virus type 1 thymidine kinase (HSV1 -tk) reporter gene in cell culture and in vivo.C6 rat glioma cells stably transfected with HSV1-tk (C6-stb-tk+) and control C6 cells were evaluated for their ability to accumulate FGCV versus FPCV. For in vivo studies, 15 mice were injected by tail vein with increasing levels of an adenoviral vector carrying HSV1-tk. Forty-eight hours later the mice were injected with FPCV and killed 3 h later. The percentage injected dose per gram (%ID/g) liver was then determined. Two additional mice were studied by microPET and autoradiography using FPCV to image adenoviral-mediated hepatic HSV1-tk reporter gene expression. A tumor-bearing mouse (C6 control and C6-stb-tk+) was imaged with FDG, FGCV, and FPCV. Two mice carrying tumors expressing two different reporter genes, HSV1-tk and dopamine type 2 receptor (D2R), were also imaged by microPET using FPCV (day 1) and 3-(2'-[18F]fluoroethyl)spiperone (FESP) (day 2).FPCV shows a significantly greater accumulation in C6-stb-tk+ cells than does FGCV (P < 0.05). Over identical ranges of adenoviral administration, mouse liver shows a higher %ID/g liver for FPCV (0%-9%) compared with our previously reported results with FGCV (0%-3%). In C6 control and C6-stb-tk+ tumor-bearing mice, FPCV has a greater accumulation than does FGCV for equal levels of HSV1-tk gene expression. In mice carrying tumors expressing either HSV1-tk or D2R reporter genes, there is a corresponding retention of FPCV and FESP, respectively.These results indicate that FPCV is a better reporter probe than is FGCV for imaging lower levels of HSV1 -tk gene expression in vivo. The results also reveal the ability to monitor the expression of two distinct reporter genes in the same animal using reporter probes specific for each gene.

View details for Web of Science ID 000166429400038

View details for PubMedID 11197989
PET imaging of transgene expression BIOLOGICAL PSYCHIATRY MacLaren, D. C., Toyokuni, T., Cherry, S. R., Barrio, J. R., Phelps, M. E., Herschman, H. R., Gambhir, S. S. 2000; 48 (5): 337-348
Abstract

A vital step in transgenic animal study and gene therapy is the ability to assay the extent of transgene expression. Unfortunately, classic methods of assaying transgene expression require biopsies or death of the subject. We are developing techniques to noninvasively and repetitively determine the location, duration, and magnitude of transgene expression in living animals. This will allow investigators and clinicians to assay the effectiveness of their particular experimental and therapeutic paradigms. Of radionuclide (single photon emission computed tomography, positron emission tomography [PET]), optical (green fluorescent protein, luciferase), and magnetic (magnetic resonance imaging) approaches, only the radionuclide approach has sufficient sensitivity and quantitation to measure the expression of genes in vivo. We describe the instrumentation involved in high resolution PET scanning. We also describe the principles of PET reporter gene/reporter probe in vivo imaging, the development of two in vivo reporter gene imaging systems, and the validation of our ability to noninvasively, quantitatively, and repetitively image gene expression in murine viral gene transfer and transgenic models. We compare the two reporter gene systems and discuss their utility for the study of transgenic animals and gene therapies. Finally, we mention alternative approaches to image gene expression by using radiolabeled antibody fragments to image specific proteins and radiolabeled oligonucleotides to image RNA messages directly.

View details for Web of Science ID 000089232700001

View details for PubMedID 10978717
Quantification of target gene expression by imaging reporter gene expression in living animals NATURE MEDICINE Yu, Y. J., Annala, A. J., Barrio, J. R., Toyokuni, T., Satyamurthy, N., Namavari, M., Cherry, S. R., Phelps, M. E., Herschman, H. R., Gambhir, S. S. 2000; 6 (8): 933-937
View details for Web of Science ID 000165473800040

View details for PubMedID 10932234
Prevalence of myocardial viability as detected by positron emission tomography in patients with ischemic cardiomyopathy - Response CIRCULATION Czernin, J., Auerbach, M. A., Schoder, H., Hoh, C., Gambhir, S. S., Yaghoubi, S., Sayre, J. W., Silverman, D., Phelps, M. E., Schelbert, H. R. 2000; 102 (4): E31-E31
View details for Web of Science ID 000088374400006
High-resolution microPET imaging of carcino-embryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wu, A. M., Yazaki, P. J., Tsai, S. W., Nguyen, K., Anderson, A. L., MCCARTHY, D. W., Welch, M. J., Shively, J. E., Williams, L. E., Raubitschek, A. A., Wong, J. Y., Toyokuni, T., Phelps, M. E., Gambhir, S. S. 2000; 97 (15): 8495-8500
Abstract

Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-C(H)3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7, 10-tetraazacyclododecane-N,N',N", N"'-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t(1/2) = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with (64)Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram +/- 3.79) compared with control tumor (6.0% injected dose per gram +/- 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3-4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.

View details for Web of Science ID 000088273900057

View details for PubMedID 10880576
A meta-analysis of the literature for whole-body FDG PET detection of recurrent colorectal cancer JOURNAL OF NUCLEAR MEDICINE Huebner, R. H., Park, K. C., Shepherd, J. E., Schwimmer, J., Czernin, J., Phelps, M. E., Gambhir, S. S. 2000; 41 (7): 1177-1189
Abstract

A meta-analysis of the literature for the use of FDG PET in the detection of recurrent colorectal cancer (CRC) was conducted to evaluate the quality of the reported studies. Overall values for the sensitivity and specificity of whole-body FDG PET and an overall FDG PET-directed percentage change in management were also determined through this analysis.Guidelines to evaluate the articles were formulated on the basis of the U.S. medical payer source criteria for assessing studies that report information on usage of new medical technology. A metaanalysis was conducted using methodology described in the peer-reviewed literature.On the basis of the guidelines established for our review, the availability of necessary information for assessing the reliability of the FDG PET data for diagnosing recurrent CRC was less than ideal. Through a meta-analysis of 11 articles, we determined, within a 95% confidence level, an overall sensitivity of 97% (95% confidence level, 95%-99%) and an overall specificity of 76% (95% confidence level, 64%-88%) for FDG PET detecting recurrent CRC throughout the whole body. Furthermore, through pooling of the change-in-management data, an overall FDG PET-directed change in management was calculated to be 29% (95% confidence level, 25%-34%).Our review suggests that improvements can be made to more effectively report the results of these FDG PET studies. The overall values determined through the meta-analysis indicate the potential benefits of using FDG PET as a diagnostic or management tool. Furthermore, these values should prove to be useful to assess the cost-effectiveness of using FDG PET in the management of patients with recurrent CRC.

View details for Web of Science ID 000089888700021

View details for PubMedID 10914907
Decision analysis for the cost effectiveness of Sestamibi Scintimammography in minimizing unnecessary biopsies QUARTERLY JOURNAL OF NUCLEAR MEDICINE Allen, M. W., Hendi, P., Schwimmer, J., Bassett, L., Gambhir, S. S. 2000; 44 (2): 168-185
Abstract

The purpose of this study was to assess if breast cancer screening using sestamibi scintimammography (SSMM) in conjunction with mammography (MM) is cost effective in avoiding biopsies in healthy patients.Quantitative decision tree sensitivity analysis was used to compare the conventional MM alone strategy (strategy A) with two decision strategies for screening with SSMM; SSMM after an indeterminate mammogram (strategy B) or SSMM after both a positive and an indeterminate mammogram (strategy C). Cost effectiveness was measured by calculating the expected cost per patient and the average life expectancy per patient for baseline values as well as over a range of values for all of the variables of each strategy.Based on Medicare reimbursement values, strategies B and C showed a cost savings of $9 and $20 per patient respectively as compared to strategy A. This translates into respective savings of $189 and $420 million per year assuming 21 million females undergo screening each year. Strategies B and C did however have a loss of mean life expectancy of 0.000178 and 0.000222 years respectively as compared to strategy A due to interval progression of breast cancer in a small number of women. Strategies B and C significantly lowered the number of biopsies performed on healthy patients in the screening population by 750,063 and 1,557,915 biopsies respectively as compared to strategy A.These results quantitatively verify the potential utility of using SSMM in avoiding unnecessary biopsies.

View details for Web of Science ID 000088264000007

View details for PubMedID 10967626
InternetQuestion and Answer (iQ&A): A Web-based survey technology IEEE TRANSACTIONS ON INFORMATION TECHNOLOGY IN BIOMEDICINE Dennis, R. A., Gambhir, S. S. 2000; 4 (2): 116-125
Abstract

This paper presents InternetQuestion and Answer, a Web-based survey development and implementation technology, which has been designed for constructing on-line surveys for educational, medical, or administrative purposes. The system, called iQ&A, is a three-tiered database-backed Web system that has been developed to support a wide range of applications. Surveys are considered as general data collection instruments and include a wide field of application. iQ&A facilitates rapid survey construction and administration which is ideally suited for biomedical research as well as other research and educational activities. Full report management capabilities provide the survey publisher on-line access to current information on survey responses. Current implementations of this technology in the areas of biomedical applications of clinical trials, longitudinal research, and other research-related systems are presented. Further refinement of the current system should lead to a powerful general survey technology for broad-based applications.

View details for Web of Science ID 000087552400005

View details for PubMedID 10866410
A review of the literature for whole-body FDG PET in the management of patients with melanoma QUARTERLY JOURNAL OF NUCLEAR MEDICINE Schwimmer, J., Essner, R., Patel, A., Jahan, S. A., Shepherd, J. E., Park, K., Phelps, M. E., Czernin, J., Gambhir, S. S. 2000; 44 (2): 153-167
Abstract

A review and meta-analysis of the literature on the use of 2-[18F]fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) in the detection of recurrent melanoma was conducted. The goals were to evaluate the quality of data reporting and to determine the overall values for the sensitivity and specificity of whole body FDG PET and management changes.Guidelines to evaluate reporting within articles were formulated based on the United States medical payer source criteria for assessing studies reporting information on the utilization of new medical technology. A meta-analysis was conducted using methodology described in the peer reviewed literature.Our MEDLINE PLUS search resulted in a universe of 89 total articles. Within these 89, 19 were categorized in our targeted content area of which 13 were selected for analysis in our targeted subset, with the remaining 70 covering 24 different related content areas. Five of 13 (38%) articles in the target subset reported data which was adequate for incorporation into modeling objectives based on PET sensitivity and specificity values, with 1 of 13 (8%) in the same target subset reporting data adequate for modeling based on change-in-management data. Through a meta-analysis of the 13 target articles we determined, within a 95% confidence level, an overall sensitivity of 92% (95% confidence level 88.41%-95.82%) and an overall specificity of 90% (95% confidence level 83.26%-96.05%) as calculated by number of lesions, for FDG PET detecting recurrent melanoma throughout the whole body. Furthermore, limited data available for change-in-management suggests an overall FDG PET directed change-in-management value of 22%.Our review suggests that improvements can be made to more effectively report the results of these FDG PET studies. The overall values determined through the meta-analysis indicate the potential benefits of using FDG PET as a diagnostic/management tool. Furthermore, these values should prove useful to assessing the cost effectiveness of utilizing FDG PET in the management of recurrent melanoma.

View details for Web of Science ID 000088264000006

View details for PubMedID 10967625
Economic evaluation studies in nuclear medicine: a methodological review of the literature QUARTERLY JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S., Schwimmer, J. 2000; 44 (2): 121-137
Abstract

The growing need for evaluation of the utility of new nuclear medicine technologies has spawned a few economic studies ranging from preliminary indications of cost savings to complete decision analysis models incorporating costs and quality of life. The objective of the current study was to evaluate the methodological quality of economic analyses of nuclear medicine procedures which targeted cost-effectiveness or cost-utility issues published in the medical literature during the years 1985-1999.A computerized literature search was used to identify original investigations from the medical literature which included an economic analysis of a nuclear medicine procedure. Each economic analysis article was evaluated by two independent reviewers for adherence to ten accepted methodological criteria.Of the 29 articles meeting the search criteria, only six (21%) conformed to all ten methodological criteria.Published economic analyses of nuclear medicine procedures usually do not meet accepted methodological standards and could be significantly improved to achieve overall better quality relative to similar analyses in the literature from other medical fields. Continued improvement in the number and quality of economic studies is critically needed for the future competitiveness of nuclear medicine studies.

View details for Web of Science ID 000088264000004

View details for PubMedID 10967623
Economics of nuclear medicine - Introduction QUARTERLY JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S. 2000; 44 (2): 103-104
View details for Web of Science ID 000088264000001

View details for PubMedID 10967620
Seeing is believing: Non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography JOURNAL OF NEUROSCIENCE RESEARCH Herschman, H. R., MacLaren, D. C., Iyer, M., Namavari, M., Bobinski, K., Green, L. A., Wu, L., Berk, A. J., Toyokuni, T., Barrio, J. R., Cherry, S. R., Phelps, M. E., Sandgren, E. P., Gambhir, S. S. 2000; 59 (6): 699-705
Abstract

The ability to monitor reporter gene expression in living animals and in patients will permit longitudinal examinations both of somatically transferred DNA in experimental animals and patients and of transgenic constructs expressed in experimental animals. If investigators can non-invasively monitor the organ and tissue specificity, the magnitude and the duration of gene expression from somatically transferred DNA and from transgenes, conceptually new experimental paradigms will be possible. If clinicians can non-invasively monitor the location, extent and duration of somatically transferred genes, they will be better able to determine the correlations between expression of therapeutic genes and clinical outcomes. We have developed two reporter gene systems for in vivo reporter gene imaging in which the protein products of the reporter genes sequester positron-emitting reporter probes. The "PET reporter gene" dependent sequestration of the "PET reporter probes" is subsequently measured in living animals by Positron Emission Tomography (PET). We describe here the principles of PET reporter gene/PET reporter probe in vivo imaging, the development of two imaging systems, and the validation of their ability to non-invasively, quantitatively and repetitively image reporter gene expression in murine viral gene transfer and transgenic models.

View details for Web of Science ID 000085698200001

View details for PubMedID 10700006
A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gambhir, S. S., Bauer, E., Black, M. E., Liang, Q. W., Kokoris, M. S., Barrio, J. R., Iyer, M., Namavari, M., Phelps, M. E., Herschman, H. R. 2000; 97 (6): 2785-2790
Abstract

We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8-(3)H]penciclovir ([8-(3)H]PCV), and 8-[(18)F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of approximately 2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET reporter gene and FPCV as a PET reporter probe results in significantly enhanced sensitivity for imaging reporter gene expression in vivo.

View details for Web of Science ID 000085941400069

View details for PubMedID 10716999
Synthesis of 8-[F-18]fluoroguanine derivatives: In vivo probes for imaging gene expression with positron emission tomography NUCLEAR MEDICINE AND BIOLOGY Namavari, M., Barrio, J. R., Toyokuni, T., Gambhir, S. S., Cherry, S. R., Herschman, H. R., Phelps, M. E., Satyamurthy, N. 2000; 27 (2): 157-162
Abstract

A new method for the preparation of 8-[(18)F]fluoroguanine derivatives based on a direct radiofluorination reaction has been developed. The radiofluorination of ganciclovir (1a) with [(18)F]F(2) was carried out in absolute ethanol in the presence of tetraethylammonium hydroxide at room temperature to give 8-[(18)F]fluoroganciclovir (3a) in an approximately 1% radiochemical yield. Similarly, 8-[(18)F]fluoropenciclovir (3b), 8-[(18)F]fluoroacyclovir (3c), and 8-[(18)F]fluoroguanosine (3d) were synthesized from penciclovir (1b), acyclovir (1c), and guanosine (1d), respectively, using [(18)F]F(2). The structural analyses of the final products (3a, 3b, 3c, and 3d) were carried out after (18)F decay by (1)H, (13)C, and (19)F nuclear magnetic resonance and high resolution mass spectroscopy.

View details for Web of Science ID 000086680000007

View details for PubMedID 10773544
Imaging transgene expression with radionuclide imaging technologies NEOPLASIA Gambhir, S. S., Herschman, H. R., Cherry, S. R., Barrio, J. R., Satyamurthy, N., Toyokuni, T., Phelps, M. E., Larson, S. M., Balatoni, J., Finn, R., Sadelain, M., Tjuvajev, J., Blasberg, R. 2000; 2 (1-2): 118-138
Abstract

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.

View details for Web of Science ID 000086519700009

View details for PubMedID 10933072
Progress Toward in Vivo Imaging of Reporter Gene Expression, Using Positron Emission Tomography in Cancer Gene Therapy. ASCO Spring Educational Book (M. Perry, Ed) American Society of Clinical Oncology Herschman HR, Barrio JR, Satyamurthy S, Toyokuni T, Cherry SR, Phelps ME, Gambhir SS. 2000: 169-177
ROC and localization ROC analyses of lesion detection in whole-body FDG PET: Effects of acquisition mode, attenuation correction and reconstruction algorithm JOURNAL OF NUCLEAR MEDICINE Farquhar, T. H., Llacer, J., Hoh, C. K., Czernin, J., Gambhir, S. S., Seltzer, M. A., Silverman, D. H., Qi, J. Y., Hsu, C. H., Hoffman, E. J. 1999; 40 (12): 2043-2052
Abstract

Receiver operating characteristic (ROC) and localization ROC (LROC) studies were performed to compare lesion detection at the borderline of detectability on images reconstructed with two-dimensional filtered backprojection (FBP) without attenuation correction (a common clinical protocol), three-dimensional FBP without attenuation correction, two-dimensional FBP with segmented attenuation correction and a two-dimensional iterative maximum a posteriori (MAP) algorithm using attenuation correction. Lung cancer was the model for the study because of the prominent role of 18F-fluorodeoxyglucose PET in the staging of lung cancer and the importance of lesion detection for staging.Simulated lung cancer lesions were added to two-dimensional and three-dimensional PET data from healthy volunteers. Data were reconstructed using the four methods. Four nuclear medicine physicians evaluated the images. Detection performance with each method was compared using ROC and LROC analysis. Jackknife analysis provided estimates of statistical significance for differences across all readers for the ROC results.ROC and LROC results indicated statistically significant degradation in detection performance with three-dimensional acquisition (average area under ROC curves [Az] 0.51; average area under LROC curves [A(z,LROC)] 0.13) and segmented attenuation correction (average Az 0.59; average Az,LROC 0.29) compared with two-dimensional FBP without attenuation correction (average Az 0.79; average A(z,LROC) 0.54). ROC and LROC results indicated an improvement in detection performance with iterative MAP reconstruction (average Az 0.83; average A(z,LROC) 0.64) compared with two-dimensional FBP reconstruction; this improvement was not statistically significant.Use of segmented attenuation correction or three-dimensional acquisition with FBP reconstruction is not expected to improve detection of lung lesions on whole-body PET images compared with images with two-dimensional FBP without attenuation correction. The potential improvement in detection obtained with an iterative MAP reconstruction method is small compared with that obtained with two-dimensional FBP without attenuation correction.

View details for Web of Science ID 000084468600018

View details for PubMedID 10616885
Comparison of helical computerized tomography, positron emission tomography and monoclonal antibody scans for evaluation of lymph node metastases in patients with prostate specific antigen relapse after treatment for localized prostate cancer JOURNAL OF UROLOGY Seltzer, M. A., Barbaric, Z., Belldegrun, A., Naitoh, J., Dorey, F., Phelps, M. E., Gambhir, S. S., Hoh, C. K. 1999; 162 (4): 1322-1328
Abstract

We compare the detection of metastatic disease by helical computerized tomography (CT), positron emission tomography (PET) with F-18 fluorodeoxyglucose and monoclonal antibody scan with 111indium capromab pendetide in patients with an elevated prostate specific antigen (PSA) after treatment for localized prostate cancer.A total of 45 patients with an elevated PSA (median 3.8 ng./ml.) were studied following definitive local therapy with radical prostatectomy in 33, radiation therapy in 9 and cryosurgery in 3. CT of the abdomen and pelvis, and whole body PET were performed in all patients, of whom 21 also underwent monoclonal antibody scan. Lymph nodes 1 cm. in diameter or greater on CT were considered abnormal and were sampled by fine needle aspiration in 12 patients.PET and CT were positive for distant disease in 50% of 22 patients with PSA greater than 4, and in 4 and 17%, respectively, of 23 with PSA less than 4 ng./ml. The detection rate for metastatic disease was similar for CT and PET, and higher overall than that for monoclonal antibody scan. Monoclonal antibody scan was true positive in only 1 of 6 patients, while PET was true positive in 6 of 9 with CT guided fine needle aspiration proved metastases.CT and PET each detected evidence of metastatic disease in 50% of all patients with a high PSA or PSA velocity (greater than 4 ng./ml. or greater than 0.2 ng./ml. per month, respectively). Both techniques are limited for detecting metastatic disease in patients with a low PSA or PSA velocity. Our data suggest that monoclonal antibody scan has a lower detection rate than CT or PET.

View details for Web of Science ID 000082510700020

View details for PubMedID 10492189
Decision analysis in nuclear medicine JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S. 1999; 40 (9): 1570-1581
Abstract

This review focuses primarily on the methodology involved in properly reviewing the literature for performing a meta-analysis and on methods for performing a formal decision analysis using decision trees. Issues related to performing a detailed metaanalysis with consideration of particular issues, including publication bias, verification bias and patient spectrum, are addressed. The importance of collecting conventional measures of test performance (e.g., sensitivity and specificity) and of changes in patient management to model the cost-effectiveness of a management algorithm is detailed. With greater utilization of the techniques discussed in this review, nuclear medicine researchers should be well prepared to compete for the limited resources available in the current health care environment. Furthermore, nuclear medicine physicians will be better prepared to best serve their patients by using only those studies with a proven role in improving patient management.

View details for Web of Science ID 000082514900028

View details for PubMedID 10492381
Assays for noninvasive imaging of reporter gene expression NUCLEAR MEDICINE AND BIOLOGY Gambhir, S. S., Barrio, J. R., Herschman, H. R., Phelps, M. E. 1999; 26 (5): 481-490
Abstract

Repeated, noninvasive imaging of reporter gene expression is emerging as a valuable tool for monitoring the expression of genes in animals and humans. Monitoring of organ/cell transplantation in living animals and humans, and the assessment of environmental, behavioral, and pharmacologic modulation of gene expression in transgenic animals should soon be possible. The earliest clinical application is likely to be monitoring human gene therapy in tumors transduced with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) suicide gene. Several candidate assays for imaging reporter gene expression have been studied, utilizing cytosine deaminase (CD), HSV1-tk, and dopamine 2 receptor (D2R) as reporter genes. For the HSV1-tk reporter gene, both uracil nucleoside derivatives (e.g., 5-iodo-2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil [FIAU] labeled with 124I, 131I) and acycloguanosine derivatives [e.g., 8-[18F]fluoro-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (8-[18F]-fluoroganciclovir) ([18F]FGCV), 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG)] have been investigated as reporter probes. For the D2R reporter gene, a derivative of spiperone [3-(2'-[18F]-Fluoroethyl)spiperone ([18F]FESP)] has been used with positron emission tomography (PET) imaging. In this review, the principles and specific assays for imaging reporter gene expression are presented and discussed. Specific examples utilizing adenoviral-mediated delivery of a reporter gene as well as tumors expressing reporter genes are discussed.

View details for Web of Science ID 000081586800001

View details for PubMedID 10473186
Prevalence of myocardial viability as detected by positron emission tomography in patients with ischemic cardiomyopathy CIRCULATION Auerbach, M. A., Schoder, H., Hoh, C., Gambhir, S. S., Yaghoubi, S., Sayre, J. W., Silverman, D., Phelps, M. E., Schelbert, H. R., Czernin, J. 1999; 99 (22): 2921-2926
Abstract

Detection of myocardial viability is important in patients with ischemic cardiomyopathy. Restoration of blood flow to viable myocardium is associated with improved left ventricular function and improved patient prognosis. However, the prevalence of viable myocardium in patients with ischemic cardiomyopathy is unknown.To determine the prevalence of myocardial viability, clinical [13N]ammonia/18F-deoxyglucose PET studies performed in 283 patients (age, 63+/-10 years) with ischemic heart disease (mean ejection fraction, 26+/-8%) were visually analyzed for the presence and extent of viable and nonviable myocardium. The myocardium was divided into 19 segments. The extent of viable myocardium was considered "functionally" significant if >/=5 segments ( approximately 25% of the left ventricular myocardium) exhibited a blood flow/metabolism mismatch and "prognostically" significant if 1 to 4 left ventricular segments did so. Of all patients, 41% had no evidence of viable myocardium, 55% had viable myocardium, and 4% had normal blood flow and metabolism within an enlarged left ventricle. Functionally significant viability was found in 27% and prognostically significant viability in 28% of the patients. Multivariate analysis revealed the presence of angina to be the only clinical parameter associated with the presence of functionally significant viability.Revascularization might improve patient prognosis in 55% and result in improved left ventricular function in 27% of all patients with ischemic cardiomyopathy.

View details for Web of Science ID 000080662900014

View details for PubMedID 10359737
A study on the cost effectiveness of sestamibi scintimammography for screening women with dense breasts for breast cancer BREAST CANCER RESEARCH AND TREATMENT Allen, M. W., Hendi, P., Bassett, L., Phelps, M. E., Gambhir, S. S. 1999; 55 (3): 243-258
Abstract

The potential impact of Sestamibi scintimammography (SSMM) on the cost effective management of women with dense breasts is not known. This study addresses this issue quantitatively by examining the impact of SSMM based screening strategies on the approximately 3,000,000 women over 40 with very dense breasts (DY patterns) without palpable masses and who have had one or more prior mammograms, who undergo routine screening each year. Quantitative decision tree sensitivity analysis was used to compare the conventional mammography (MM) strategy (strategy A), which does not subject patients with negative mammograms to any further examination until their next screening, with two decision strategies for screening with SSMM; SSMM after a negative mammogram (strategy B) or SSMM as the only screening test for women already identified as having dense breasts by a previous mammogram (strategy C). Cost effectiveness was measured by calculating the incremental cost effectiveness ratio (ICER) of strategies B and C, which is the cost of achieving an additional year of life in the screening population by choosing a SSMM based decision strategy rather than the conventional strategy. Strategies B and C reduced the number of false negative diagnoses by 62% and 8%, respectively. The ICER was $632,000 and $3.18M per life year for strategy B and C, respectively. To be cost effective, the pre-test probability of cancer in the study population must be greater than 3% for strategy B or the cost of SSMM must be less than $50 for strategy C. These results show the ICER of an SSMM based breast cancer screening strategy in the management of patients with dense breasts is not currently within the range (approximately $50,000 per year life saved) of other commonly performed medical interventions that are considered cost effective.

View details for Web of Science ID 000082271900005

View details for PubMedID 10517169
Repetitive, non-invasive imaging of the dopamine D-2 receptor as a reporter gene in living animals GENE THERAPY MacLaren, D. C., Gambhir, S. S., Satyamurthy, N., Barrio, J. R., Sharfstein, S., Toyokuni, T., Wu, L., Berk, A. J., Cherry, S. R., Phelps, M. E., Herschman, H. R. 1999; 6 (5): 785-791
Abstract

Reporter genes (e.g. beta-galactosidase, chloramphenicol-acetyltransferase, green fluorescent protein, luciferase) play critical roles in investigating mechanisms of gene expression in transgenic animals and in developing gene delivery systems for gene therapy. However, measuring expression of these reporter genes requires biopsy or death. We now report a procedure to image reporter gene expression repetitively and non-invasively in living animals with positron emission tomography (PET), using the dopamine type 2 receptor (D2R) as a reporter gene and 3-(2'-[18F]fluoroethyl)spiperone (FESP) as a reporter probe. We use a viral delivery system to demonstrate the ability of this PET reporter gene/PET reporter probe system to image reporter gene expression following somatic gene transfer. In mice injected intravenously with replication-deficient adenovirus carrying a D2R reporter gene, PET in vivo measures of hepatic [18F] retention are proportional to in vitro measures of hepatic FESP retention, D2R ligand binding and D2R mRNA. We use tumor-forming cells carrying a stably transfected D2R gene to demonstrate imaging of this PET reporter gene/PET reporter probe system in 'tissues'. Tumors expressing the transfected D2R reporter gene retain substantially more FESP than control tumors. The D2R/FESP reporter gene/reporter probe system should be a valuable technique to monitor, in vivo, expression from both gene therapy vectors and transgenes.

View details for Web of Science ID 000080186700011

View details for PubMedID 10505102
Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gambhir, S. S., Barrio, J. R., Phelps, M. E., Iyer, M., Namavari, M., Satyamurthy, N., Wu, L., Green, L. A., Bauer, E., MacLaren, D. C., Nguyen, K., Berk, A. J., Cherry, S. R., Herschman, H. R. 1999; 96 (5): 2333-2338
Abstract

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the HSV1-tk reporter gene in living mice. There is a significant positive correlation between the percent injected dose of FGCV retained per gram of liver and the levels of hepatic HSV1-tk reporter gene expression (r2 > 0.80). Over a similar range of HSV1-tk expression in vivo, the percent injected dose retained per gram of liver was 0-23% for ganciclovir and 0-3% for FGCV. Repeated, noninvasive, and quantitative imaging of PET reporter gene expression should be a valuable tool for studies of human gene therapy, of organ/cell transplantation, and of both environmental and behavioral modulation of gene expression in transgenic mice.

View details for Web of Science ID 000078956600095

View details for PubMedID 10051642
Imaging gene expression: Principles and assays JOURNAL OF NUCLEAR CARDIOLOGY Gambhir, S. S., Barrio, J. R., Herschman, H. R., Phelps, M. E. 1999; 6 (2): 219-233
View details for Web of Science ID 000079762000009

View details for PubMedID 10327107
Nonlinear Function Estimation and Other Numerical Methods for Imaging Myocardial Perfusion by N-13 Ammonia PET. Computers in Cardiology. Golish SR, Hove J, Schelbert HR, Gambhir SS 1999; 26: 651-654
The lag of cerebral hemodynamics with rapidly alternating periodic stimulation: Modeling for functional MRI MAGNETIC RESONANCE IMAGING Hathout, G. M., Gopi, R. K., Bandettini, P., Gambhir, S. S. 1999; 17 (1): 9-20
Abstract

A mathematical model that characterizes the response of venous oxygenation to changes in cerebral blood flow (rCBF) and oxygen consumption has been previously presented. We use this model to examine the dampening phenomenon in functional MRI (fMRI) signals with rapidly alternating periodic stimulation bursts. Using a mass balance approach, the equations for an input-output model are derived and solved using Matlab (the Math Works Inc.). Changes in venous oxygenation are related to the results of fMRI experiments using progressively shorter periods of stimulation. An impulse-response function for the model is derived in an attempt to explore the source of the lag in cerebral hemodynamics. Increasing the frequency of stimulation bursts eventually produces a dampening in the fMRI signal. The dampening phenomenon in fMRI signals occurs with stimulation of high frequency on-off alternation. The dynamics of signal dampening, as well as the impulse-response function of a blood oxygen level-dependent model, lend strong indirect support to the hypothesis that blood oxygen level-dependent contrast at the level of the venous blood pool, rather than R1 inflow effects or changes in oxygenation at the level of the capillary bed, underlies the observed signal changes in fMRI.

View details for Web of Science ID 000077953600002

View details for PubMedID 9888394
Prevalence of Myocardial Viability as Detected by Positron Emission Tomography in Patients with Ischemic Cardiomyopathy Circulation Auerbech MA, Schoder H, Hoh C, Gambhir SS, Yaghoubi S, Sayre JW, Silverman D, Phelps ME, Schelbert HR, Czernin J. 1999; 99: 2921-2926
Cost-effectiveness of FDG-PET for staging non-small cell lung cancer: A decision analysis ANNALS OF THORACIC SURGERY Scott, W. J., Shepherd, J., Gambhir, S. S. 1998; 66 (6): 1876-1883
Abstract

Preliminary studies have shown that thoracic positron emission tomography (PET) is more accurate than thoracic computed tomography (CT) for the staging of non-small cell lung carcinoma. In the present study the cost-effectiveness, as measured by national Medicare reimbursed costs, and patient life expectancy are used to compare several thoracic PET-based strategies with a conventional thoracic CT-based strategy for preoperative staging.Five decision strategies for selection of potential surgical candidates were compared; thoracic CT alone or four different strategies that use thoracic CT plus thoracic PET. The various paths of each strategy are dependent on numerous variables that were determined from a review of the medical literature. Life expectancy was calculated using the declining exponential approximation of life expectancy and reduced on the basis of procedural morbidity and mortality. Costs were based on national Medicare reimbursed costs. For all possible outcomes of each strategy, the expected cost and projected life expectancy were determined. The effects of changing one or more variables on the expected cost and life expectancy were studied using sensitivity analysis.A strategy that uses PET only after a negative CT study is shown to be a cost-effective alternative to the CT-alone strategy ($25,286 per life-year saved).These results show through rigorous decision tree analysis the potential cost-effectiveness of using thoracic PET in the management of non-small cell lung carcinoma. Greater use of thoracic PET for nonsmall cell lung carcinoma staging is warranted, and further clinical trials should help to validate the analytic results predicted from this study.

View details for Web of Science ID 000078294900002

View details for PubMedID 9930463
Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S., Barrio, J. R., Wu, L., Iyer, M., Namavari, M., Satyamurthy, N., Bauer, E., Parrish, C., MacLaren, D. C., Borghei, A. R., Green, L. A., Sharfstein, S., Berk, A. J., Cherry, S. R., Phelps, M. E., Herschman, H. R. 1998; 39 (11): 2003-2011
Abstract

We are developing procedures to repeatedly and noninvasively image the expression of transplanted reporter genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase gene (HSV1-tk) as a reporter gene and [8-14C]-ganciclovir as a reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8-14C]-ganciclovir. As a result, specific accumulation of phosphorylated [8-14C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk gene.An adenoviral vector was constructed carrying the HSV1-tk gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8-3H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8-14C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies.Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a reporter gene and [8-14C]-ganciclovir as an imaging reporter probe. A good correlation (r2 = 0.86) between the [8-14C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8-18F]-fluoroganciclovir and microPET imaging supports further investigation of [8-18F]-fluoroganciclovir as a PET reporter probe for imaging HSV1-tk gene expression.These results demonstrate the feasibility of using [8-14C]-ganciclovir as a reporter probe for the HSV1-tk reporter gene, using an in vivo adenoviral mediated gene delivery system in a murine model. The results form the foundation for further investigation of [8-18F]-fluoroganciclovir for noninvasive and repeated imaging of gene expression with PET.

View details for Web of Science ID 000076784400039

View details for PubMedID 9829598
Evaluating tumor biology and oncological disease with positron-emission tomography SEMINARS IN RADIATION ONCOLOGY Silverman, D. H., Hoh, C. K., Seltzer, M. A., Schiepers, C., Cuan, G. S., Gambhir, S. S., Zheng, L., Czernin, J., Phelps, M. E. 1998; 8 (3): 183-196
Abstract

The usefulness of positron-emission tomography (PET) for noninvasive assessment of several biological parameters of neoplastic tissue has been reviewed. Numerous radiotracers have been developed, whose particular distribution in the presence of cancer in vivo serves to distinguish medically relevant properties of the tumor cells with which they associate. That distribution is most accurately determined through use of a PET scanner, to localize and quantify the tracer molecules, in which have been incorporated positron-emitting isotopes. These tracers include hypoxia markers, receptor ligands, substrates for enzymatic modification by the products of expression of specific genes, and precursors of protein anabolism and carbohydrate catabolism. In addition, application of PET to evaluation of patients with some particular cancers has been examined, while placing special emphasis on the level of scientific rigor of the evidence underlying conclusions about appropriate use of PET in oncology.

View details for Web of Science ID 000074767300006

View details for PubMedID 9634495
Rapid synthesis of a 5 '-fluorinated oligodeoxy-nucleotide: A model antisense probe for use in imaging with positron emission tomography (PET) BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Pan, D. F., Gambhir, S. S., Toyokuni, T., Iyer, M. R., Acharya, N., Phelps, M. E., Barrio, J. R. 1998; 8 (11): 1317-1320
Abstract

5'-Deoxy-5'-fluoro-O4-methylthymidine was synthesized by the reaction of the corresponding 5'-O-tosylate with KF in the presence of Kryptofix [222] and coupled to a 5'-phosphoramidite-activated CPG-bound oligodeoxynucleotide. The sequence of reactions and purifications were accomplished within 4 h, a necessary condition of the development of radiofluorinated antisense oligodeoxynucleotide probe for use with PET.

View details for Web of Science ID 000074157600010

View details for PubMedID 9871758
A new method to estimate parameters of linear compartmental models using artificial neural networks PHYSICS IN MEDICINE AND BIOLOGY Gambhir, S. S., Keppenne, C. L., Banerjee, P. K., Phelps, M. E. 1998; 43 (6): 1659-1678
Abstract

At present, the preferred tool for parameter estimation in compartmental analysis is an iterative procedure; weighted nonlinear regression. For a large number of applications, observed data can be fitted to sums of exponentials whose parameters are directly related to the rate constants/coefficients of the compartmental models. Since weighted nonlinear regression often has to be repeated for many different data sets, the process of fitting data from compartmental systems can be very time consuming. Furthermore the minimization routine often converges to a local (as opposed to global) minimum. In this paper, we examine the possibility of using artificial neural networks instead of weighted nonlinear regression in order to estimate model parameters. We train simple feed-forward neural networks to produce as outputs the parameter values of a given model when kinetic data are fed to the networks' input layer. The artificial neural networks produce unbiased estimates and are orders of magnitude faster than regression algorithms. At noise levels typical of many real applications, the neural networks are found to produce lower variance estimates than weighted nonlinear regression in the estimation of parameters from mono- and biexponential models. These results are primarily due to the inability of weighted nonlinear regression to converge. These results establish that artificial neural networks are powerful tools for estimating parameters for simple compartmental models.

View details for Web of Science ID 000074257600021

View details for PubMedID 9651032
Analytical decision model for the cost-effective management of solitary pulmonary nodules JOURNAL OF CLINICAL ONCOLOGY Gambhir, S. S., Shepherd, J. E., Shah, B. D., Hart, E., Hoh, C. K., Valk, P. E., Emi, T., Phelps, M. E. 1998; 16 (6): 2113-2125
Abstract

Multiple strategies are currently being used to manage patients who present with indeterminate solitary pulmonary nodules (SPN). We have used decision-analysis models to assess the cost-effectiveness of various strategies for the diagnosis and management of SPN. Four decision strategies were compared: a wait and watch strategy, a surgery strategy, a computed tomography (CT)-based strategy, and a CT-plus-positron emission tomography (PET) strategy. An incremental cost-effectiveness ratio (ICER) was used to compare all strategies to the wait and watch strategy.A CT-plus-PET strategy was the most cost-effective over a large pretest likelihood (probability of having a malignant nodule), with a range of 0.12 to 0.69. Furthermore, within this likelihood range, the potential cost savings of using the CT-plus-PET strategy over the CT strategy ranged from $91 to $2,200 per patient. This translates to a yearly national savings of approximately $62.7 million.Decision-analysis modeling indicates the potential cost-effectiveness of [18F]2-fluoro-2-deoxy-D-glucose (FDG)-PET in the management of SPN. Furthermore, the decision trees developed can be used to model various features of the management of SPN, including modeling the cost-effectiveness of other newly emerging technologies.

View details for Web of Science ID 000073919200016

View details for PubMedID 9626211
Noninvasive methods for quantitating blood time-activity curves from mouse PET images obtained with fluorine-18-fluorodeoxyglucose JOURNAL OF NUCLEAR MEDICINE Green, L. A., Gambhir, S. S., Srinivasan, A., Banerjee, P. K., Hoh, C. K., Cherry, S. R., Sharfstein, S., Barrio, J. R., Herschman, H. R., Phelps, M. E. 1998; 39 (4): 729-734
Abstract

The mouse model is currently being explored for various applications with PET imaging. Low resolution of current animal scanners relative to mouse size leads to difficulty in quantitating data from mouse PET images. We have, therefore, investigated methods for determining blood time-activity curves (TACs) from mouse PET studies done with fluorine-18-fluorodeoxyglucose (FDG).Eight mice were fasted, the tail vein was injected with 150-300 microCi of FDG and dynamic images were acquired with a CTI/Siemens (Knoxville, TN) animal tomograph for 64.5 min. Concurrently, 11-14 left ventricle (LV) blood samples were drawn directly from the LV chamber. Organ TACs were obtained by drawing circular regions of interest (ROIs) of various sizes on images of the heart, liver and brain. For each mouse, the FDG model parameter K = (K1 x k3)/(k2 + k3) was estimated by a Patlak algorithm with various estimates of the blood TAC and, as a reference tissue TAC, the brain TAC.Most partial-volume-corrected heart ROI TACs overestimated the LV samples. Blood TACs from heart images produced statistically different estimates of K than did the LV samples. The liver image-derived blood TACs yielded estimates of K that were comparable to those yielded by the LV samples. Estimates of K determined with two directly sampled LV points in conjunction with the liver image-derived TAC were not statistically different from the estimates obtained with the LV samples. The size and location of ROIs on images of the liver minimally affected the TACs.We have shown that it is experimentally possible to obtain a blood TAC from mouse studies by repeatedly sampling from the LV. We have also shown that images of the liver can be used to reliably estimate the blood TAC. Future FDG PET studies with the mouse model will benefit from this demonstrated ability to noninvasively quantitate blood TACs directly from FDG PET images.

View details for Web of Science ID 000072956600042

View details for PubMedID 9544690
Dual spillover problem in the myocardial septum with nitrogen-13-ammonia flow quantitation JOURNAL OF NUCLEAR MEDICINE Hove, J. D., Gambhir, S. S., Kofoed, K. F., Kelbaek, H., Schelbert, H. R., Phelps, M. E. 1998; 39 (4): 591-598
Abstract

Conventional cardiac PET modeling techniques for [13N]ammonia flow determination do not fully account for the effects of spillover of activity from the right ventricle (RV) onto the activity in the myocardial septum. The purpose of this study was to investigate and to quantitatively account and correct for this effect.Simulations were performed to determine the error introduced by conventional quantitation using septal time-activity curves, which only account for left ventricle (LV) spillover. Furthermore, we explored two separate methods to account for the dual spillover problem: direct estimation of the RV and LV spillover fractions incorporated into the [13N]ammonia model by using the LV and RV input functions in the fit and estimation of the relative dispersion and time shift between the LV and RV input functions by fitting using only the LV input function. The simulated curves were fitted using a two-compartment [13N]ammonia model. Flow estimates from the conventional model and the models including either of the two correction procedures were compared with canine microsphere data.The influence of RV spillover on flow estimation in the septum is determined by several parameters (e.g., dispersion between the RV and LV input function). Depending on the value of these parameters, the septal flow may be underestimated by 0%-30%. The applied methods for correction of the dual spillover problem were comparable and allow for more accurate quantitation in the septum. The canine microsphere data revealed that flow underestimation in the septum is small but significant.Dual spillover in the myocardial septum can introduce significant errors in the estimation of flow by the conventional [13N]ammonia model fitting method, which does not properly account for the RV spillover. Adjusting for the RV spillover in one of the two proposed methods allows for more accurate quantitation of myocardial septal flow with [13N]ammonia PET data.

View details for Web of Science ID 000072956600013

View details for PubMedID 9544662
Clinical Positron Imaging and the Transition Towards an On-Line Electronic Journal. Clinical Positron Imaging. Gambhir SS 1998; 1(1): 71-79
Positron Emission Tomography in Gynecologic Malignancies. Journal of Gynecologic Techniques Hoh CK, Seltzer MA, Yong TA, Czernin J, Silverman DHS, Gambhir SS, Phelps ME, Karlan BY. 1998; 4(1): 19-24
MD@: A physician-friendly decision analysis tool M D COMPUTING Gambhir, S. S., Gupta, P., Shepherd, J. E., Allen, M. A., Hoh, C., Maddahi, J., Phelps, M. E. 1998; 15 (1): 40-48
Abstract

The cost-effective use of medical resources is increasingly important in justifying strategies for medical diagnosis and management. Although some software is available to help with decision analysis, it can be difficult to use these tools for medical applications. We have developed a prototype package for modeling various medical decision strategies, which can be used with a Macintosh or Windows-based personal computer. The system is graphically based, intuitive, and user-friendly. The user constructs decision trees for comparing alternative strategies for diagnosis and management. Selecting blocks from a library, the user sets mean values for variables such as prevalence, sensitivity, specificity, cost, morbidity, and mortality. The system then generates the probabilities of various pathways, using Bayesian analysis, without requiring the user to enter equations. It displays the best strategy in terms of a particular criterion and, when appropriate, performs sensitivity analysis.

View details for Web of Science ID 000071517500005

View details for PubMedID 9458662
Cost-effectiveness analysis in nuclear medicine JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S. 1998; 39 (1): 17N-18N
View details for Web of Science ID 000071472400003

View details for PubMedID 9443723
Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters JOURNAL OF CLINICAL INVESTIGATION Agus, D. B., Gambhir, S. S., Pardridge, W. M., Spielholz, C., Baselga, J., Vera, J. C., Golde, D. W. 1997; 100 (11): 2842-2848
Abstract

Vitamin C concentrations in the brain exceed those in blood by 10-fold. In both tissues, the vitamin is present primarily in the reduced form, ascorbic acid. We identified the chemical form of vitamin C that readily crosses the blood-brain barrier, and the mechanism of this process. Ascorbic acid was not able to cross the blood-brain barrier in our studies. In contrast, the oxidized form of vitamin C, dehydroascorbic acid (oxidized ascorbic acid), readily entered the brain and was retained in the brain tissue in the form of ascorbic acid. Transport of dehydroascorbic acid into the brain was inhibited by d-glucose, but not by l-glucose. The facilitative glucose transporter, GLUT1, is expressed on endothelial cells at the blood-brain barrier, and is responsible for glucose entry into the brain. This study provides evidence showing that GLUT1 also transports dehydroascorbic acid into the brain. The findings define the transport of dehydroascorbic acid by GLUT1 as a mechanism by which the brain acquires vitamin C, and point to the oxidation of ascorbic acid as a potentially important regulatory step in accumulation of the vitamin by the brain. These results have implications for increasing antioxidant potential in the central nervous system.

View details for Web of Science ID 000071007300026

View details for PubMedID 9389750
Effect of intraluminal thrombus on abdominal aortic aneurysm wall stress JOURNAL OF VASCULAR SURGERY Mower, W. R., Quinones, W. J., Gambhir, S. S. 1997; 26 (4): 602-608
Abstract

Abdominal aortic aneurysms (AAAs) rupture when the wall stress exceeds the strength of the vascular tissue. Intraluminal thrombus may absorb tension and reduce AAA wall stress. This study was performed to test the hypothesis that intraluminal thrombus can significantly reduce AAA wall stress.AAA wall stresses were determined by axisymmetric finite element analysis. Model AAAs had external diameters ranging from 2.0 to 4.0 cm. Model parameters included: AAA length, 6 cm; wall thickness, 1.5 mm; Poisson's ratio, 0.49; Young's modulus, 1.0 MPa; and luminal pressure, 1.6 x 10(5) dyne/cm2. Stresses were calculated for each model without thrombus, and then were recalculated with thrombus filling 10% of the AAA cavity. Calculations were repeated as thrombus size was increased in 10% increments and as thrombus elastic modulus increased from 0.01 MPa to 1.0 MPa. Maximum wall stresses were compared between models that had intraluminal thrombus and the unmodified models. Stress reduction greater than 25% was considered significant.The maximum stress reduction of 51% occurred when thrombus with elastic modulus of 1.0 MPa filled the entire AAA cavity. Stresses were reduced by only 25% as modulus decreased to 0.2 MPa. Similarly, decreasing thrombus size by 70% resulted in stress reduction of only 28%. Large AAAs experienced greater stress reduction than small AAAs (48% vs 11%).Intraluminal thrombus can significantly reduce AAA wall stress.

View details for Web of Science ID A1997YE45200008

View details for PubMedID 9357460
PET in oncology: Will it replace the other modalities? SEMINARS IN NUCLEAR MEDICINE Hoh, C. K., Schiepers, C., Seltzer, M. A., Gambhir, S. S., Silverman, D. H., Czernin, J., Maddahi, J., Phelps, M. E. 1997; 27 (2): 94-106
Abstract

Medical imaging technology is rapidly expanding and the role of each modality is being redefined constantly. PET has been around since the early sixties and gained clinical acceptance in oncology only after an extreme number of scientific publications. Although PET has the unique ability to image biochemical processes in vivo, this ability is not fully used as a clinical imaging tool. In this overview, the role of PET in relation to other tumor imaging modalities will be discussed and the reported results in the literature will be reviewed. In predicting the future of PET, technical improvements of other imaging modalities need to be dealt with. The fundamental physical principles for image formation with computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), photon-emission tomography (PET), and single photon emission CT (SPECT) will not change. The potential variety of radiopharmaceuticals which may be developed is unlimited, however, and this provides nuclear imaging techniques with a significant advantage and adaptive features for future biologic imaging. The current applications of PET in oncology have been in characterizing tumor lesions, differentiating recurrent disease from treatment effects, staging tumors, evaluating the extent of disease, and monitoring therapy. The future developments in medicine may use the unique capabilities of PET not only in diagnostic imaging but also in molecular medicine and genetics. The articles discussed in this review were selected from a literature search covering the last 3 years, and in which comparisons of PET with conventional imaging were addressed specifically. PET studies with the glucose analogue fluorine-18-labeled deoxyglucose (FDG) have shown the ability of detecting tumor foci in a variety of histological neoplasms such as thyroid cancer, breast cancer, lymphoma, lung cancer, head and neck carcinoma, colorectal cancer, ovarian carcinoma, and musculoskeletal tumors. Also, the contribution of the whole body PET (WBPET) imaging technique in diagnosis will be discussed. In the current health care environment, a successful imaging technology must not only change medical management but also demonstrate that those changes improve patient outcome.

View details for Web of Science ID A1997WX00700003

View details for PubMedID 9144854
Cost-effective selection of patients for coronary angiography Maddahi, J., Gambhir, S. S. MOSBY-YEAR BOOK INC. 1997: S141-S151
Abstract

In patients suspected of having coronary artery disease (CAD), noninvasive testing has been playing an increasing role in selecting patients who would require coronary angiography for either the "definitive" diagnosis of CAD or as a prelude to planning myocardial revascularization. A mathematic model is presented that defines cost-effective utility of nuclear cardiology testing for diagnosis of CAD and selection of appropriate candidates for coronary angiography, according to quantitative methods of decision analysis. Clinical utility or effectiveness was defined in terms of percent correct diagnosis of CAD. Cost was defined as dollars of medical expenditure. Six competing strategies were compared in subsets of patients with different pretest likelihoods of CAD, based on age, sex, and symptoms. Nuclear cardiology testing was the most cost-effective initial modality of choice in patients with an intermediate pretest likelihood of CAD. In patients with a low pretest likelihood of CAD, nuclear cardiology testing was cost-effective in the subgroup of patients who had abnormal exercise treadmill electrocardiograms. In patients with a high pretest likelihood of CAD, direct referral to coronary angiography was the most cost-effective strategy for diagnosis of CAD. Coronary angiography, however, is performed most often as a prelude to myocardial revascularization. Because these invasive procedures are indicated only in patients who are at high risk with medical therapy, nuclear cardiology procedures, by virtue of incremental prognostic information, identify appropriate candidates for more invasive procedures, aimed at improving survival. Strategies for cost-effective prognostication of CAD depend on not only the patient's pretest likelihood of CAD but also the status of the rest electrocardiogram. In patients with a normal rest electrocardiogram, a low pretest likelihood of CAD indicates a low risk for cardiac events with medical therapy. Therefore coronary angiography is not indicated in these patients. Patients with an intermediate likelihood of CAD should first undergo exercise electrocardiographic testing; a negative response would indicate a low risk for cardiac events and a positive response would indicate the need for nuclear cardiology testing for further cost-effective risk stratification. In patients with a high pretest likelihood of CAD, the combined exercise electrocardiographic and nuclear cardiac testing is the most cost-effective strategy; a negative or a positive nuclear test result would imply low or high risk, respectively. The latter patients would then be candidates for coronary angiography. In all patients with an abnormal rest electrocardiogram, the most cost-effective strategy is uniform referral to nuclear cardiac testing (which is performed in conjunction with exercise electrocardiography), regardless of the pretest likelihood of CAD; a negative or a positive nuclear test result would indicate low or high risk for coronary events, respectively. The latter group would be proper candidates for referral to coronary angiography.

View details for Web of Science ID A1997WW68400002

View details for PubMedID 9115076
Cost-Effective Selection of Patients for Coronary Angiography Cost-Effective Selection of Patients for Coronary Angiography Maddahi, J. 1997; 4 (N2 PT2 SUPPS)
Decision tree sensitivity analysis for cost-effectiveness of FDG-PET in the staging and management of non-small-cell lung carcinoma JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S., Hoh, C. K., Phelps, M. E., Madar, I., Maddahi, J. 1996; 37 (9): 1428-1436
Abstract

Preliminary studies have shown that PET is more accurate than CT for the staging of non-small-cell lung carcinoma (NSCLC). However, the potential effect of PET on the management of these patients and its cost-effectiveness has not been rigorously studied. Thus, we have used decision tree sensitivity analysis to assess the cost-effectiveness of a PET based strategy for staging of NSCLC.Two decision strategies for selection of potential surgical candidates were compared; thoracic CT alone or thoracic CT and thoracic PET. The first decision tree was conservatively constructed by requiring mediastinoscopy (biopsy) to confirm imaging results so that no patient with surgically curable disease would miss the opportunity for surgery in either strategy. A second less conservative tree in which only nonconcordant results are biopsied was also tested. The various paths of each strategy are dependent on numerous parameters which were determined from a review of the medical literature. Life expectancy was calculated using the declining exponential approximation of life expectancy and reduced based on procedural mortality. Costs were based on mean costs at our institution. For all possible outcomes of each strategy, the expected cost and projected life expectancy were determined. The effect of changing one or more parameters on the expected cost and life expectancy were studied using a sensitivity analysis.The CT + PET strategy in the conservative decision tree showed a saving of $1154 per patient without a loss of life expectancy (increase of 2.96 days) as compared to the alternate strategy of CT alone. Both these effects were the result of improved staging of lung carcinoma prior to the decision for surgery. The CT + PET strategy in the less conservative decision tree showed a savings of $2267 per patient but misses 1.7% of potentially operable patients.These results show through rigorous decision tree analysis, the potential cost-effectiveness of using FDG PET in the management of NSCLC. These results form a basis for detailed study of the results obtained from multicenter trials on the accuracy of PET in NSCLC management. Furthermore, the techniques utilized for decision tree analysis have broad range of applicability to the entire field of nuclear medicine.

View details for Web of Science ID A1996VF79700010

View details for PubMedID 8790186
Symbolic Interactive Modeling Package and Learning Environment (SIMPLE), a New Compartmental Modeling Tool. Proceedings of the Society for Computer Simulation, Gambhir, S., Mahoney DK, Turner MS, Wong ATC, Rosenqvist G, Huang SC, Phelps ME. 1996: 173-180
A QUANTITATIVE PHYSIOLOGICAL MODEL OF BLOOD OXYGENATION FOR FUNCTIONAL MAGNETIC-RESONANCE-IMAGING INVESTIGATIVE RADIOLOGY Hathout, G. M., Gambhir, S. S., Gopi, R. K., KIRLEW, K. A., Choi, Y., So, G., Gozal, D., Harper, R., Lufkin, R. B., Hawkins, R. 1995; 30 (11): 669-682
Abstract

Variations in venous deoxyhemoglobin levels in response to neuronal activation represent a complex interplay between focal changes in cerebral blood flow (CBF), cerebral blood volume (CBV), and regional metabolism. The authors present a mathematic model that characterizes the response of venous oxygenation to changes in these variables.Using a mass balance approach, the equations for a simple input-output model are derived and solved using Matlab. Changes in blood oxygenation are related to available results from functional magnetic resonance imaging experiments.Increases in CBF produce declines in oxygen extraction fraction and venous deoxyhemoglobin according to Fick's law, and are quantitatively in agreement with available magnetic resonance and positron-emission tomography data. A flow-volume envelope defines the changes in CBF relative to CBV.It is possible to obtain a quantitative understanding of changes in blood oxygenation and to relate these changes to the observed dynamics of magnetic resonance signal change in the setting of functional stimulation.

View details for Web of Science ID A1995TE46900007

View details for PubMedID 8557508
PARAMETER-ESTIMATION OF CARDIAC GEOMETRY BY ECG-GATED PET IMAGING - VALIDATION USING MAGNETIC-RESONANCE-IMAGING AND ECHOCARDIOGRAPHY JOURNAL OF NUCLEAR MEDICINE Porenta, G., Kuhle, W., Sinha, S., Krivokapich, J., Czernin, J., Gambhir, S. S., Phelps, M. E., Schelbert, H. R. 1995; 36 (6): 1123-1129
Abstract

The purpose of this study was to apply and validate a previously developed model-based image analysis technique which derives estimates of regional myocardial wall thickness and the left ventricular radius directly from gated cardiac PET images.In 11 normal volunteers, gated myocardial 18F-deoxyglucose (FDG) images with 16 equal gates spanning the entire cardiac cycle were acquired for 20 min. To improve count statistics and thus image quality, 3 and 5 of 16 gates were summed to obtain systolic and diastolic images. Based on a five-parameter model, radial profiles from systolic and diastolic PET images were fit by nonlinear regression for myocardial wall thickness, left ventricular radius and tracer activities in the blood pool, the myocardial tissue and the extracardiac background. Echocardiography and gated magnetic resonance imaging (MRI) were performed in 11 and 7 volunteers, respectively.We observed a significant (p < 0.001) correlation between measurements obtained by gated PET imaging and the correlative imaging modalities for myocardial wall thickness and left ventricular radius. While good agreement was observed between measurements of average radial shortening, estimates of average wall thickening differed significantly.This model-based analysis offers accurate estimates of regional recovery coefficients directly from gated cardiac PET images and may also prove useful for the assessment of myocardial contractile function. These recovery coefficients are essential for the correction of partial volume effects when quantitative PET studies are performed.

View details for Web of Science ID A1995RB81600046

View details for PubMedID 7769438
REGIONAL MYOCARDIAL BLOOD-FLOW AND GLUCOSE-UTILIZATION IN SYMPTOMATIC PATIENTS WITH HYPERTROPHIC CARDIOMYOPATHY CIRCULATION Nienaber, C. A., Gambhir, S. S., Mody, F. V., Ratib, O., Huang, S. C., Phelps, M. E., Schelbert, H. R. 1993; 87 (5): 1580-1590
Abstract

Previous studies suggested the presence of myocardial ischemia in symptomatic patients with hypertrophic cardiomyopathy. Positron emission tomography, a technique that can identify metabolic consequences of ischemia in coronary artery disease, permits the noninvasive measurements of regional myocardial blood flow and glucose metabolism. This new quantitative imaging approach should therefore be suitable for detecting a possible enhancement of glucose utilization in myocardium of patients with hypertrophic cardiomyopathy and thus may help to elucidate the pathomechanism of ischemia in this disease.In 13 symptomatic patients with hypertrophic cardiomyopathy, myocardial blood flow and glucose utilization were measured with intravenous N-13-ammonia and F-18 deoxyglucose at rest and, in four patients, again during supine bicycle exercise. At rest, blood flow was significantly lower in hypertrophied than in normal myocardium (0.78 +/- 0.19 versus 0.99 +/- 0.13 mL.min-1.g-1, p < 0.025), whereas rates of glucose utilization were similar (0.88 +/- 0.31 versus 0.87 +/- 0.35 mumol.min-1.g-1). With exercise, blood flow and glucose utilization failed to increase in hypertrophic and normal segments but became more heterogeneously distributed throughout the left ventricular myocardium. Blood flow-metabolism mismatches indicative of myocardial ischemia were noted in three patients at rest and in three of the four patients during exercise and were due to reduced flow in the presence of maintained glucose uptake. The discordance between flow and glucose metabolism in hypertrophied myocardium was significantly more prominent in younger than in older patients.Normal or even elevated rates of glucose utilization and the presence of diminished blood flow in hypertrophied relative to normal myocardium suggest the presence of myocardial ischemia in symptomatic hypertrophic cardiomyopathy. The age dependence of blood flow metabolism disparity suggests differences in the underlying pathophysiology or severity of disease.

View details for Web of Science ID A1993LB08400019

View details for PubMedID 8491014
QUANTIFICATION OF REGIONAL MYOCARDIAL BLOOD-FLOW USING N-13 AMMONIA AND REORIENTED DYNAMIC POSITRON EMISSION TOMOGRAPHIC IMAGING CIRCULATION Kuhle, W. G., Porenta, G., Huang, S. C., Buxton, D., Gambhir, S. S., Hansen, H., Phelps, M. E., Schelbert, H. R. 1992; 86 (3): 1004-1017
Abstract

Regional myocardial blood flow has been quantified using transaxial positron emission tomographic (PET) imaging and tracer kinetic modeling. However, the use of transaxial images limits the accuracy of regional partial volume corrections and the localization of the quantified regional flow values. The purpose of the present study was to overcome both problems by calculating regional flows from reoriented short-axis PET images.Twelve experiments were performed in four dogs. 13N-ammonia was injected intravenously while microspheres were administered into the left atrium during baseline, hyperemic, and low-flow conditions. Serial transaxial frames were acquired with a 15-plane PET scanner and reoriented into short-axis frames. The arterial input function and eight regional myocardial tissue activity curves were derived from the reoriented frames. The arterial input functions were corrected for ammonia metabolites, and the myocardial tissue curves were corrected for spillover of activity, partial volume effects, and heterogeneities in the image's spatial resolution introduced during reorientation. Corrections for regional partial volume were based on estimates of the regional myocardial activity thickness derived from reoriented diastolic images of the heart. The myocardial 13N-ammonia kinetics were described with a two-pool compartmental model. Values of regional myocardial blood flow by PET correlated linearly with those by microspheres (slope, 0.94; y intercept, 0.06 ml/min/g; r = 0.93) over a wide range of flows.Regional myocardial blood flow can be measured accurately and noninvasively from serially acquired and reoriented short-axis 13N-ammonia images, thus overcoming limitations inherent to the use of transaxially acquired images and permitting a more complete evaluation of regional blood flows throughout the left ventricular myocardium.

View details for Web of Science ID A1992JM27000034

View details for PubMedID 1516170
USE OF THE ABDOMINAL-AORTA FOR ARTERIAL INPUT FUNCTION DETERMINATION IN HEPATIC AND RENAL PET STUDIES JOURNAL OF NUCLEAR MEDICINE Germano, G., Chen, B. C., Huang, S. C., Gambhir, S. S., Hoffman, E. J., Phelps, M. E. 1992; 33 (4): 613-620
Abstract

A method using the activity in the abdominal aorta of human and animal subjects to noninvasively estimate blood-pool input function in dynamic, abdominal PET scans is proposed and validated in this paper. Partial volume effects due to the aorta's dimensions are corrected by a semi-automated algorithm based on the transaxial resolution in the reconstructed images. The technique was validated by comparing PET measurements of abdominal aortic activity to well counter measurements of arterial blood samples (eight canine renal studies) and to PET measurements of left ventricular cavity activity (eight human hepatic studies). In renal studies, correlation analysis of the areas subtended by the two input functions yielded an essentially unitary slope (1.03 +/- 0.09), with high correlation (R2 greater than 0.95, p less than 0.001). In hepatic studies, similar values (0.99 +/- 0.03 and R2 greater than 0.85, p less than 0.001) were found. Correlation of the blood flow estimates based on the two input functions and a two-compartment model produced slopes of 1.07 +/- 0.16 and 1.03 +/- 0.07, and correlations of (R2 greater than 0.98, p less than 0.001) and (R2 greater than 0.97, p less than 0.001) for the renal and hepatic studies, respectively. We conclude that noninvasive, accurate measurements of the arterial input function by dynamic PET imaging are possible and represent a clinically viable alternative to arterial blood sampling.

View details for Web of Science ID A1992HM08200032

View details for PubMedID 1552350
METABOLIC AND FUNCTIONAL RECOVERY OF ISCHEMIC HUMAN MYOCARDIUM AFTER CORONARY ANGIOPLASTY JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Nienaber, C. A., Brunken, R. C., Sherman, C. T., Yeatman, L. A., Gambhir, S. S., Krivokapich, J., Demer, L. L., Ratib, O., Child, J. S., Phelps, M. E., Schelbert, H. R. 1991; 18 (4): 966-978
Abstract

Although revascularization of hypoperfused but metabolically active human myocardium improves segmental function, the temporal relations among restoration of blood flow, normalization of tissue metabolism and recovery of segmental function have not been determined. To examine the effects of coronary angioplasty on 13 asynergic vascular territories in 12 patients, positron emission tomography and two-dimensional echocardiography were performed before and within 72 h of revascularization. Ten patients underwent late echocardiography (67 +/- 19 days) and eight underwent a late positron emission tomographic study (68 +/- 19 days). The extent and severity of abnormalities of wall motion, perfusion and glucose metabolism were expressed as wall motion scores, perfusion defect scores and perfusion-metabolism mismatch scores. Angioplasty significantly increased mean stenosis cross-sectional area (from 0.95 +/- 0.9 to 2.7 +/- 1.4 mm2) and mean cross-sectional luminal diameter (from 0.9 +/- 0.6 to 1.9 +/- 0.5 mm) (both p less than 0.001). Perfusion defect scores in dependent vascular territories improved early after angioplasty (from 116 +/- 166 to 31 +/- 51, p less than 0.002) with no further improvement on the late follow-up study. The mean perfusion-metabolism mismatch score decreased from 159 +/- 175 to 65 +/- 117 early after angioplasty (p less than 0.01) and to 26 +/- 29 at late follow-up (p less than 0.001 vs. before angioplasty; p = NS vs. early after angioplasty). However, absolute rates of glucose utilization remained elevated early after revascularization, normalizing only at late follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)

View details for Web of Science ID A1991GH89700012

View details for PubMedID 1894871
PARAMETRIC IMAGES OF MYOCARDIAL METABOLIC-RATE OF GLUCOSE GENERATED FROM DYNAMIC CARDIAC PET AND 2-[F-18]FLUORO-2-DEOXY-D-GLUCOSE STUDIES JOURNAL OF NUCLEAR MEDICINE Choi, Y., HAWKINS, R. A., Huang, S. C., Gambhir, S. S., Brunken, R. C., Phelps, M. E., Schelbert, H. R. 1991; 32 (4): 733-738
Abstract

We describe a method for generating parametric images of the myocardial metabolic rate of glucose (MMRGlc) with positron emission tomography (PET). The method employs serially acquired images of 2-[18F]fluoro-2-deoxy-D-glucose (FDG) uptake and a Patlak graphical analysis of the image data. The arterial input function is derived from images of the left ventricular blood pool calibrated with 18F-plasma measurements. The approach is computationally fast enough to be used in a clinical environment. The MMRGlc parametric images improve myocardial contrast relative to non-parametric images, especially in studies with poor myocardial uptake of FDG. In addition, MMRGlc parametric images consolidate the large amount of data in a dynamic PET study into a clinically usable image set.

View details for Web of Science ID A1991FG79700031

View details for PubMedID 2013815
A QUANTITATIVE INDEX OF REGIONAL BLOOD-FLOW IN CANINE MYOCARDIUM DERIVED NONINVASIVELY WITH N-13 AMMONIA AND DYNAMIC POSITRON EMISSION TOMOGRAPHY JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY Nienaber, C. A., Ratib, O., Gambhir, S. S., Krivokapich, J., Huang, S. C., Phelps, M. E., Schelbert, H. R. 1991; 17 (1): 260-269
Abstract

To derive a quantitative index of regional myocardial blood flow, the arterial input function of the flow tracer N-13 ammonia and the regional myocardial N-13 activity concentrations were noninvasively determined in 29 experiments in eight dogs. N-13 ammonia was administered intravenously and cross-sectional images were acquired dynamically using an ECAT III positron emission tomograph with an effective in-plane resolution of 13.46 mm full-width half-maximum. Time-activity curves were derived from the serial images by assigning regions of interest to the left ventricular myocardium and left ventricular blood pool. Tracer net extractions were estimated from the myocardial time-activity concentrations at various times after tracer injection and the integral of the arterial input function. Myocardial blood flow was altered by intravenous dipyridamole, morphine, propranolol and partial or complete occlusion of the left anterior descending coronary artery, and ranged from 9 to 860 ml/min per 100 g. Estimates of tracer net extractions were most accurate when determined from the myocardial N-13 activity concentrations at 60 s divided by the integral of the arterial input function to that time. These estimates correlated with regional myocardial blood flows determined independently by the microsphere technique by y = x (1 - 0.64(e-114/x); SEE = 22.9; r = 0.94). First pass extraction fractions of N-13 ammonia determined noninvasively with this approach declined with higher flows in a nonlinear fashion and were similar to those determined invasively by direct intracoronary N-13 ammonia injections. The findings indicate that an accurate index of regional myocardial blood flow can be obtained noninvasively by high temporal sampling of arterial and myocardial tracer activity concentrations with positron emission tomography. They also provide a basis for the in vivo application of tracer kinetic principles to derive quantitatively and noninvasively regional rates of functional processes in human myocardium.

View details for Web of Science ID A1991EU23100040

View details for PubMedID 1987234
TRACER KINETIC MODELING APPROACHES FOR THE QUANTIFICATION OF HEPATIC-FUNCTION WITH TECHNETIUM-99M DISIDA AND SCINTIGRAPHY JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S., HAWKINS, R. A., Huang, S. C., Hall, T. R., Busuttil, R. W., Phelps, M. E. 1989; 30 (9): 1507-1518
Abstract

Serial scintigraphic images following injection of [99mTc]iminodiacetic acid compounds such as [99mTc]diisopropyl-iminodiacetic acid (DISIDA) provide qualitative information about liver function. We have investigated approaches for quantitatively describing liver function in terms of the kinetics of DISIDA extraction and excretion by the liver. Several compartmental model configurations were evaluated. A three-compartment model (blood, hepatic parenchyma, intrahepatic bile) was found to fit the data best and was used in conjunction with dynamic image data to obtain estimates of rate constants for liver extraction and excretion of DISIDA, and mean residence time (MRT) of DISIDA in the liver. A noncompartmental approach based on a parametric deconvolution technique was also used to estimate the noncompartmental mean residence time (MRTnc). To assess limitations of the noncompartmental approach, computer simulations were performed using the three-compartment model to generate time-activity curves followed by analysis of these curves by the noncompartmental method. The effect of plasma total bilirubin level on DISIDA uptake and MRT was also investigated. These techniques are readily adaptable to standard nuclear medicine computing facilities, and could be used in the clinical setting to numerically describe serial DISIDA studies (especially in liver transplant patients) efficiently and noninvasively.

View details for Web of Science ID A1989AP85000012

View details for PubMedID 2769404
SIMPLE NONINVASIVE QUANTIFICATION METHOD FOR MEASURING MYOCARDIAL GLUCOSE-UTILIZATION IN HUMANS EMPLOYING POSITRON EMISSION TOMOGRAPHY AND F-18 DEOXYGLUCOSE JOURNAL OF NUCLEAR MEDICINE Gambhir, S. S., Schwaiger, M., Huang, S. C., Krivokapich, J., Schelbert, H. R., Nienaber, C. A., Phelps, M. E. 1989; 30 (3): 359-366
Abstract

To estimate regional myocardial glucose utilization (rMGU) with positron emission tomography (PET) and 2-[18F]fluoro-2-deoxy-D-glucose (FDG) in humans, we studied a method which simplifies the experimental procedure and is computationally efficient. This imaging approach uses a blood time-activity curve derived from a region of interest (ROI) drawn over dynamic PET images of the left ventricle (LV), and a Patlak graphic analysis. The spillover of radioactivity from the cardiac chambers to the myocardium is automatically removed by this analysis. Estimates of rMGU were obtained from FDG PET cardiac studies of six normal human subjects. Results from this study indicate that the FDG time-activity curve obtained from the LV ROI matched well with the arterial plasma curve. The rMGU obtained by Patlak graphic analysis was in good agreement with direct curve fitting results (r = 0.90). The average standard error of the estimate of the Patlak rMGU was low (3%). These results demonstrate the practical usefulness of a simplified method for the estimation of rMGU in humans by PET. This approach is noninvasive, computationally fast, and highly suited for developing parametric images of myocardial glucose utilization rate.

View details for Web of Science ID A1989T600900015

View details for PubMedID 2786939
RADIONUCLIDE EVALUATION OF LIVER-TRANSPLANTS SEMINARS IN NUCLEAR MEDICINE HAWKINS, R. A., Hall, T., Gambhir, S. S., Busuttil, R. W., Huang, S. C., Glickman, S., Marciano, D., Brown, R. K., Phelps, M. E. 1988; 18 (3): 199-212
Abstract

Orthotopic liver transplantation is now an established technique for treating patients with various forms of end stage liver disease. The number of centers performing the procedure is increasing and, as the number of transplant recipients in the population increases, many institutions performing nuclear medicine studies will be confronted with requests to evaluate these patients. While a variety of radionuclides are proving useful in this evaluation, the 99mTc iminodiacetic acid (IDA) compounds, particularly 99mTc diisopropyl IDA (DISIDA), will probably account for the majority of radionuclide evaluations of these patients because they are well suited to monitor both structural and functional changes of the graft. The primary application of radionuclide studies is focused in the postoperative period, when problems with the vascular and biliary anastomoses, rejection, infections, and bile leaks all produce alterations in radionuclide hepatobiliary studies. Abnormalities such as rejection and infection produce primarily functional, rather than structural changes and are not easily differentiated based upon the kinetics of 99mTc-DISIDA extraction and excretion by the liver, serial imaging and correlation with clinical data is necessary in such situations. Quantitative analyses of kinetic 99mTc IDA (DISIDA) studies and quantitative approaches with other compounds such as 99mTc galactosyl-neoglycoalbumin (NGA) may permit better assessments of relatively subtle changes in liver function in the posttransplant period.

View details for Web of Science ID A1988P737200003

View details for PubMedID 3051393
A STUDY OF THE SINGLE COMPARTMENT TRACER KINETIC-MODEL FOR THE MEASUREMENT OF LOCAL CEREBRAL BLOOD-FLOW USING O-15-WATER AND POSITRON EMISSION TOMOGRAPHY JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Gambhir, S. S., Huang, S. C., HAWKINS, R. A., Phelps, M. E. 1987; 7 (1): 13-20
Abstract

The effects of varying the data collection time on the calculation of cerebral blood flow and distribution volume via the integrated projection technique were studied in four human subjects. The significance of these results in terms of the limitations of the single compartment model for 15O-water was explored using computer simulations. The simulations helped to account for causes for the variations seen in blood flow and distribution volume as a function of the data collection time. Two different compartmental models were explored for better quantitation of blood flow and distribution volume.

View details for Web of Science ID A1987G010000003

View details for PubMedID 3492506

Virginia and D.K. Ludwig Professor for Clinical Investigation in Cancer Research and Professor, by courtesy, of Bioengineering and of Materials Science and Engineering

Radiology - Nuclear Medicine

Bio

Clinical Focus

Academic Appointments

Administrative Appointments

Honors & Awards

Professional Education

Contact

Links

Research & Scholarship

Current Research and Scholarly Interests

Clinical Trials

Teaching

2015-16 Courses

Stanford Advisees

Graduate and Fellowship Programs

Publications

All Publications

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract

Abstract