Carmel Chan

All Publications

• Engineering of a novel subnanomolar affinity fibronectin III domain binder targeting human programmed death-ligand 1. Protein engineering, design & selection : PEDS Ramakrishnan, S., Natarajan, A., Chan, C. T., Panesar, P. S., Gambhir, S. S. 2019

  Abstract

  The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5mg/L and a protein mass of 12kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-gamma-induced states, with a binding affinity of 2.38±0.26nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders.

  View details for DOI 10.1093/protein/gzz030

  View details for PubMedID 31612217

• Microglia are effector cells of CD47-SIRPalpha antiphagocytic axis disruption against glioblastoma. Proceedings of the National Academy of Sciences of the United States of America Hutter, G., Theruvath, J., Graef, C. M., Zhang, M., Schoen, M. K., Manz, E. M., Bennett, M. L., Olson, A., Azad, T. D., Sinha, R., Chan, C., Assad Kahn, S., Gholamin, S., Wilson, C., Grant, G., He, J., Weissman, I. L., Mitra, S. S., Cheshier, S. H. 2019

  Abstract

  Glioblastoma multiforme (GBM) is a highly aggressive malignant brain tumor with fatal outcome. Tumor-associated macrophages and microglia (TAMs) have been found to be major tumor-promoting immune cells in the tumor microenvironment. Hence, modulation and reeducation of tumor-associated macrophages and microglia in GBM is considered a promising antitumor strategy. Resident microglia and invading macrophages have been shown to have distinct origin and function. Whereas yolk sac-derived microglia reside in the brain, blood-derived monocytes invade the central nervous system only under pathological conditions like tumor formation. We recently showed that disruption of the SIRPalpha-CD47 signaling axis is efficacious against various brain tumors including GBM primarily by inducing tumor phagocytosis. However, most effects are attributed to macrophages recruited from the periphery but the role of the brain resident microglia is unknown. Here, we sought to utilize a model to distinguish resident microglia and peripheral macrophages within the GBM-TAM pool, using orthotopically xenografted, immunodeficient, and syngeneic mouse models with genetically color-coded macrophages (Ccr2 RFP) and microglia (Cx3cr1 GFP). We show that even in the absence of phagocytizing macrophages (Ccr2 RFP/RFP), microglia are effector cells of tumor cell phagocytosis in response to anti-CD47 blockade. Additionally, macrophages and microglia show distinct morphological and transcriptional changes. Importantly, the transcriptional profile of microglia shows less of an inflammatory response which makes them a promising target for clinical applications.

  View details for PubMedID 30602457

• Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection. Nanotheranostics Li, K., Chan, C. T., Nejadnik, H., Lenkov, O. D., Wolterman, C., Paulmurugan, R., Yang, H., Gambhir, S. S., Daldrup-Link, H. E. 2018; 2 (4): 306?19

  Abstract

  Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo. Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence "light-up" feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies. Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively. Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence "light-up" signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.

  View details for DOI 10.7150/ntno.26389

  View details for PubMedID 29977742

  View details for PubMedCentralID PMC6030766

• Multimodal assessment of SERS nanoparticle biodistribution post ingestion reveals new potential for clinical translation of Raman imaging BIOMATERIALS Campbell, J. L., SoRelle, E. D., Ilovich, O., Liba, O., James, M. L., Qiu, Z., Perez, V., Chan, C. T., de la Zerda, A., Zavaleta, C. 2017; 135: 42-52

  Abstract

  Despite extensive research and development, new nano-based diagnostic contrast agents have faced major barriers in gaining regulatory approval due to their potential systemic toxicity and prolonged retention in vital organs. Here we use five independent biodistribution techniques to demonstrate that oral ingestion of one such agent, gold-silica Raman nanoparticles, results in complete clearance with no systemic toxicity in living mice. The oral delivery mimics topical administration to the oral cavity and gastrointestinal (GI) tract as an alternative to intravenous injection. Biodistribution and clearance profiles of orally (OR) vs. intravenously (IV) administered Raman nanoparticles were assayed over the course of 48 h. Mice given either an IV or oral dose of Raman nanoparticles radiolabeled with approximately 100 ?Ci (3.7MBq) of (64)Cu were imaged with dynamic microPET immediately post nanoparticle administration. Static microPET images were also acquired at 2 h, 5 h, 24 h and 48 h. Mice were sacrificed post imaging and various analyses were performed on the excised organs to determine nanoparticle localization. The results from microPET imaging, gamma counting, Raman imaging, ICP-MS, and hyperspectral imaging of tissue sections all correlated to reveal no evidence of systemic distribution of Raman nanoparticles after oral administration and complete clearance from the GI tract within 24 h. Paired with the unique signals and multiplexing potential of Raman nanoparticles, this approach holds great promise for realizing targeted imaging of tumors and dysplastic tissues within the oral cavity and GI-tract. Moreover, these results suggest a viable path for the first translation of high-sensitivity Raman contrast imaging into clinical practice.

  View details for DOI 10.1016/j.biomaterials.2017.04.045

  View details for PubMedID 28486147

• A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant. Cancer research Ronald, J. A., Kim, B., Gowrishankar, G., Namavari, M., Alam, I. S., D'Souza, A., Nishikii, H., Chuang, H., Ilovich, O., Lin, C., Reeves, R., Shuhendler, A., Hoehne, A., Chan, C. T., Baker, J., Yaghoubi, S. S., VanBrocklin, H. F., Hawkins, R., Franc, B. L., Jivan, S., Slater, J. B., Verdin, E. F., Gao, K. T., Benjamin, J., Negrin, R., Gambhir, S. S. 2017; 77 (11): 2893-2902

  Abstract

  A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-?-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

  View details for DOI 10.1158/0008-5472.CAN-16-2953

  View details for PubMedID 28572504

• Detection of Stem Cell Transplant Rejection with Ferumoxytol MR Imaging: Correlation of MR Imaging Findings with Those at Intravital Microscopy. Radiology Daldrup-Link, H. E., Chan, C., Lenkov, O., Taghavigarmestani, S., Nazekati, T., Nejadnik, H., Chapelin, F., Khurana, A., Tong, X., Yang, F., Pisani, L., Longaker, M., Gambhir, S. S. 2017: 161139-?

  Abstract

  Purpose To determine whether endogenous labeling of macrophages with clinically applicable nanoparticles enables noninvasive detection of innate immune responses to stem cell transplants with magnetic resonance (MR) imaging. Materials and Methods Work with human stem cells was approved by the institutional review board and the stem cell research oversight committee, and animal experiments were approved by the administrative panel on laboratory animal care. Nine immunocompetent Sprague-Dawley rats received intravenous injection of ferumoxytol, and 18 Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice received rhodamine-conjugated ferumoxytol. Then, 48 hours later, immune-matched or mismatched stem cells were implanted into osteochondral defects of the knee joints of experimental rats and calvarial defects of Jax mice. All animals underwent serial MR imaging and intravital microscopy (IVM) up to 4 weeks after surgery. Macrophages of Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice express enhanced green fluorescent protein (GFP), which enables in vivo correlation of ferumoxytol enhancement at MR imaging with macrophage quantities at IVM. All quantitative data were compared between experimental groups by using a mixed linear model and t tests. Results Immune-mismatched stem cell implants demonstrated stronger ferumoxytol enhancement than did matched stem cell implants. At 4 weeks, T2 values of mismatched implants were significantly lower than those of matched implants in osteochondral defects of female rats (mean, 10.72 msec for human stem cells and 11.55 msec for male rat stem cells vs 15.45 msec for sex-matched rat stem cells; P = .02 and P = .04, respectively) and calvarial defects of recipient mice (mean, 21.7 msec vs 27.1 msec, respectively; P = .0444). This corresponded to increased recruitment of enhanced GFP- and rhodamine-ferumoxytol-positive macrophages into stem cell transplants, as visualized with IVM and histopathologic examination. Conclusion Endogenous labeling of macrophages with ferumoxytol enables noninvasive detection of innate immune responses to stem cell transplants with MR imaging. (©) RSNA, 2017 Online supplemental material is available for this article.

  View details for DOI 10.1148/radiol.2017161139

  View details for PubMedID 28128708

• Longitudinal Monitoring of Antibody Responses against Tumor Cells Using Magneto-nanosensors with a Nanoliter of Blood. Nano letters Lee, J. R., Chan, C. T., Ruderman, D., Chuang, H. Y., Gaster, R. S., Atallah, M., Mallick, P., Lowe, S. W., Gambhir, S. S., Wang, S. X. 2017; 17 (11): 6644?52

  Abstract

  Each immunoglobulin isotype has unique immune effector functions. The contribution of these functions in the elimination of pathogens and tumors can be determined by monitoring quantitative temporal changes in isotype levels. Here, we developed a novel technique using magneto-nanosensors based on the effect of giant magnetoresistance (GMR) for longitudinal monitoring of total and antigen-specific isotype levels with high precision, using as little as 1 nL of serum. Combining in vitro serologic measurements with in vivo imaging techniques, we investigated the role of the antibody response in the regression of firefly luciferase (FL)-labeled lymphoma cells in spleen, kidney, and lymph nodes in a syngeneic Burkitt's lymphoma mouse model. Regression status was determined by whole body bioluminescent imaging (BLI). The magneto-nanosensors revealed that anti-FL IgG2a and total IgG2a were elevated and sustained in regression mice compared to non-regression mice (p < 0.05). This platform shows promise for monitoring immunotherapy, vaccination, and autoimmunity.

  View details for PubMedID 28990786

• 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(?/?)/p23 interactions by BLI. In this manner, we identified compound 1A12 as a dose-dependent inhibitor of Hsp90(?/?)/p23 interactions, UKE-1 myeloid cell proliferation, p21(waf1) upregulation, and acetylated histone H3 levels. 1A12 was efficacious in tumor xenografts expressing Hsp90(?)/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

• 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

  View details for PubMedCentralID PMC3602312

• 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

• 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 PubMedID 22895790

• Overcoming Trastuzumab resistance with the novel pan-erbb inhibitor AZD8931 Balachandran, B., Chabot, C., Hamel, S., Chan, C. T., Kane, S., Buchanan, M., Aguilar-Mahecha, A., Basik, M. AMER ASSOC CANCER RESEARCH. 2012

  View details for DOI 10.1158/1538-7445.AM2012-770

  View details for Web of Science ID 000209701602457

• 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

  View details for PubMedCentralID PMC3288571

• 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

  View details for PubMedCentralID PMC4657136

• 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 PubMedID 21730157

• 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 PubMedID 20713710

• 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

  View details for PubMedCentralID PMC4154800

• 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

  View details for PubMedCentralID PMC4146344

• 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

  View details for PubMedCentralID PMC4145876

• 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

• Differential sensitivities of trastuzumab (Herceptin (R))-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors BREAST CANCER RESEARCH AND TREATMENT Chan, C. T., Metz, M. Z., Kane, S. E. 2005; 91 (2): 187?201

  Abstract

  Her2 (erbB2/neu) is overexpressed in 25-30% of human breast cancers. Herceptin is a recombinant humanized Her2 antibody used to treat breast cancer patients with Her2 overexpression. Over a 5-month selection process, we isolated clones of BT474 (BT) human breast carcinoma cells (BT/Her(R)) that were resistant to Herceptin in vitro. In BT/Her(R) subclones, cell-surface, phosphorylated and total cellular Her2 protein remained high in the continuous presence of Herceptin. Likewise, the levels of cell-surface, phosphorylated, and total cellular Her3 and EGFR were either unchanged or only slightly elevated in BT/Her(R) subclones relative to BT cells. One BT/Her(R) subclone had substantially upregulated cell-surface EGFR, but this did not correlate with a higher relative resistance to Herceptin. In looking at the downstream PI-3K/Akt signaling pathway, phosphorylated and total Akt levels and Akt kinase activities were all sustained in BT/Her(R) subclones in the presence of Herceptin, but significantly downregulated in BT cells exposed to Herceptin. Whereas BT cells lost sensitivity to the PI-3K inhibitor LY294002 in the presence of Herceptin, BT/Her(R) subclones were equally sensitive to this agent in the presence and absence of Herceptin. This suggests that BT/Her(R) subclones acquired a Herceptin-resistant mechanism of PI-3K signaling. BT/Her(R) subclones were also sensitive to the EGFR kinase inhibitor AG1478 in the presence of Herceptin, to the same extent as BT cells. The BT/Her(R) subclones provide new insights into mechanisms of Herceptin resistance and suggest new treatment strategies in combination with other inhibitors targeted to signal transduction pathways.

  View details for DOI 10.1007/s10549-004-7715-1

  View details for Web of Science ID 000228867600011

  View details for PubMedID 15868447

• 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

• Characterization of Three-Dimensional Tissue Cultures Using Electrical Impedance Spectroscopy Biophysical Journa Kyle, A. H., Chan, C. T., Minchinton, A. I. 1999; 76 (5): 2640-2648