Determining the Optimal Number of Core Needle Biopsy Passes for Molecular Diagnostics
CARDIOVASCULAR AND INTERVENTIONAL RADIOLOGY
2018; 41 (3): 489?95
A study of the mutational landscape of pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma.
2016; 29 (10): 1212-1220
The number of core biopsy passes required for adequate next-generation sequencing is impacted by needle cut, needle gauge, and the type of tissue involved. This study evaluates diagnostic adequacy of core needle lung biopsies based on number of passes and provides guidelines for other tissues based on simulated biopsies in ex vivo porcine organ tissues.The rate of diagnostic adequacy for pathology and molecular testing from lung biopsy procedures was measured for eight operators pre-implementation (September 2012-October 2013) and post-implementation (December 2013-April 2014) of a standard protocol using 20-gauge side-cut needles for ten core biopsy passes at a single academic hospital. Biopsy pass volume was then estimated in ex vivo porcine muscle, liver, and kidney using side-cut devices at 16, 18, and 20 gauge and end-cut devices at 16 and 18 gauge to estimate minimum number of passes required for adequate molecular testing.Molecular diagnostic adequacy increased from 69% (pre-implementation period) to 92% (post-implementation period) (p < 0.001) for lung biopsies. In porcine models, both 16-gauge end-cut and side-cut devices require one pass to reach the validated volume threshold to ensure 99% adequacy for molecular characterization, while 18- and 20-gauge devices require 2-5 passes depending on needle cut and tissue type.Use of 20-gauge side-cut core biopsy needles requires a significant number of passes to ensure diagnostic adequacy for molecular testing across all tissue types. To ensure diagnostic adequacy for molecular testing, 16- and 18-gauge needles require markedly fewer passes.
View details for PubMedID 29279975
Synchronous Hepatoblastoma, Neuroblastoma, and Cutaneous Capillary Hemangiomas: A Case Report
PEDIATRIC AND DEVELOPMENTAL PATHOLOGY
2016; 19 (1): 74-79
Pediatric-type follicular lymphoma and pediatric marginal zone lymphoma are two of the rarest B-cell lymphomas. These lymphomas occur predominantly in the pediatric population and show features distinct from their more common counterparts in adults: adult-type follicular lymphoma and adult-type nodal marginal zone lymphoma. Here we report a detailed whole-exome deep sequencing analysis of a cohort of pediatric-type follicular lymphomas and pediatric marginal zone lymphomas. This analysis revealed a recurrent somatic variant encoding p.Lys66Arg in the transcription factor interferon regulatory factor 8 (IRF8) in 3 of 6 cases (50%) of pediatric-type follicular lymphoma. This specific point mutation was not detected in pediatric marginal zone lymphoma or in adult-type follicular lymphoma. Additional somatic point mutations in pediatric-type follicular lymphoma were observed in genes involved in transcription, intracellular signaling, and cell proliferation. In pediatric marginal zone lymphoma, no recurrent mutation was identified; however, somatic point mutations were observed in genes involved in cellular adhesion, cytokine regulatory elements, and cellular proliferation. A somatic variant in AMOTL1, a recurrently mutated gene in splenic marginal zone lymphoma, was also identified in a case of pediatric marginal zone lymphoma. The overall non-synonymous mutational burden was low in both pediatric-type follicular lymphoma and pediatric marginal zone lymphoma (4.6 mutations per exome). Altogether, these findings support a distinctive genetic basis for pediatric-type follicular lymphoma and pediatric marginal zone lymphoma when compared with adult subtypes and to one another. Moreover, identification of a recurrent point mutation in IRF8 provides insight into a potential driver mutation in the pathogenesis of pediatric-type follicular lymphoma with implications for novel diagnostic or therapeutic strategies.Modern Pathology advance online publication, 24 June 2016; doi:10.1038/modpathol.2016.102.
View details for DOI 10.1038/modpathol.2016.102
View details for PubMedID 27338637
Dasatinib-related Follicular Hyperplasia: An Underrecognized Entity With Characteristic Morphology.
American journal of surgical pathology
2015; 39 (10): 1363-1369
Multiple synchronous tumors presenting in infancy raise concern for inherited or sporadic cancer predisposition syndromes, which include Beckwith-Wiedemann syndrome, familial adenomatous polyposis syndrome, and Li-Fraumeni syndrome. We report a case of a 7-month-old previously healthy male born following an in vitro fertilization-assisted twin pregnancy who presented with new-onset refractory shock, severe acidosis, and rapid decline over several hours. An autopsy revealed a ruptured liver involved by hepatoblastoma, an adrenal gland involved by neuroblastoma, and multiple cutaneous capillary hemangiomas. Standard genetic testing demonstrated that both twins were Gaucher disease (GD) carriers without evidence of other known cancer predisposition syndromes. This report describes a unique association of multiple synchronous tumors, which underscores the utility and importance of the pediatric autopsy. Moreover, given that the reported child was a GD carrier, the possibility the tumors were the result of a GD-mediated cancer-associated phenotype or an unrecognized sporadic clinical syndrome remains an unanswered, but intriguing, question worthy of further investigation.
View details for DOI 10.2350/14-11-1573-CR.1
View details for Web of Science ID 000373286000012
The utility of IgM, CD21, HGAL and LMO2 in the diagnosis of pediatric follicular lymphoma
2015; 46 (4): 629-633
Dasatinib, a second-generation tyrosine kinase inhibitor with activity against BCR-ABL1 and other Src family tyrosine kinases, is approved as a first-line treatment option for Philadelphia chromosome-positive chronic myelogenous leukemia (CML) in the chronic phase. Recently, lymphadenopathy with morphologic features of reactive follicular hyperplasia was described in a cohort of patients with CML on long-term dasatinib therapy. However, the complete morphologic and immunophenotypic features of this previously underappreciated adverse effect have not been fully described. Herein, we report 3 cases of unexplained lymphadenopathy resulting in multiple diagnostic procedures in patients with CML and a history of long-term dasatinib therapy. Morphologic examination demonstrated preserved nodal architecture showing hybrid features of progressive transformation of germinal centers and Castleman-type changes in a background of florid follicular hyperplasia. Large germinal centers were disrupted by complex infolding of IgD mantle zones arranged as cuffs surrounding perforating capillaries. Other abnormalities variably present included decreased CD20 expression among polytypic B cells and increased Epstein-Barr virus reactivity in scattered paracortical cells and/or individual germinal centers. B-cell clonality studies showed no predominant clonal rearrangements. Consideration of dasatinib-related lymphadenopathy may pre-empt unnecessary repeat diagnostic procedures in patients with CML or other dasatinib-susceptible malignancies and persistent lymphadenopathy.
View details for DOI 10.1097/PAS.0000000000000488
View details for PubMedID 26360368
Discovery and horizontal follow-up of an autoantibody signature in human prostate cancer
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2015; 112 (8): 2515-2520
Pediatric follicular lymphoma (pFL) is a rare neoplasm with features differing from follicular lymphoma arising in adults. Here, we describe a rare case of pFL that showed morphologic features partially overlapping with progressive transformation of germinal centers and reactive follicular hyperplasia. As typical of pFL, neoplastic B cells within follicles did not express B-cell leukemia/lymphoma 2 (BCL2). However, this case showed additional distinctive abnormal findings, which contributed to the diagnosis: (1) diffuse and uniform staining of immunoglobulin M (IgM) on cells within and outside of follicles, (2) abnormally dim expression of CD21 on follicular dendritic cells, and (3) expression of human germinal center-associated lymphoma (HGAL) and LIM domain only 2 (LMO2) on B cells in interfollicular and follicular areas. This case demonstrates the utility of these abnormal features, which can be seen in adult- or usual-type follicular lymphoma, in the diagnosis of pFL. Further studies are necessary to evaluate the significance of these findings in other cases of pFL.
View details for DOI 10.1016/j.humpath.2014.12.016
View details for Web of Science ID 000352117000020
View details for PubMedID 25701230
Initial results with preoperative tattooing of biopsied axillary lymph nodes and correlation to sentinel lymph nodes in breast cancer patients.
Annals of surgical oncology
2015; 22 (2): 377-382
In response to an urgent need for improved diagnostic and predictive serum biomarkers for management of metastatic prostate cancer, we used phage display fingerprinting to analyze sequentially acquired serum samples from a patient with advancing prostate cancer. We identified a peptide ligand, CTFAGSSC, demonstrating an increased recovery frequency over time. Serum antibody reactivity to this peptide epitope increased in the index patient, in parallel with development of deteriorating symptoms. The antigen mimicking the peptide epitope was identified as alpha-2-Heremans-Schmid glycoprotein, also known as fetuin-A. Metastatic prostate cancer cell lines and bone metastasis samples displayed robust fetuin-A expression, and we demonstrated serum immune reactivity to fetuin-A with concomitant development of metastatic castrate-resistant disease in a large cohort of prostate cancer patients. Whereas fetuin-A is an established tumor antigen in several types of cancer, including breast cancer, glioblastoma, and pancreas cancer, this report is to our knowledge the first study implicating fetuin-A in prostate cancer and indicating that autoantibodies specific for fetuin-A show utility as a prognostic indicator for prostate cancer patients prone to progress to metastatic disease.
View details for DOI 10.1073/pnas.1500097112
View details for Web of Science ID 000349911700059
View details for PubMedID 25675522
View details for PubMedCentralID PMC4345585
Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells
Pretreatment evaluation of axillary lymph nodes (ALNs) and marking of biopsied nodes in patients with newly diagnosed breast cancer is becoming routine practice. We sought to test tattooing of biopsied ALNs with a sterile black carbon suspension (Spot?). The intraoperative success of identifying tattooed ALNs and their concordance to sentinel nodes was determined.Women with suspicious ALNs and newly diagnosed breast cancer underwent palpation and/or ultrasound-guided fine needle aspiration or core needle biopsy, followed by injection of 0.1 to 0.5 ml of Spot? ink into the cortex of ALNs and adjacent soft tissue. Group I underwent surgery first, and group II underwent neoadjuvant therapy followed by surgery. Identification of black pigment and concordance between sentinel and tattooed nodes was evaluated.Twenty-eight patients were tattooed, 16 in group I and 12 in group II. Seventeen cases had evidence of atypia or metastases, 8 (50 %) in group I and 9 (75 %) in group II. Average number of days from tattooing to surgery was 22.9 (group I) and 130 (group II). Black tattoo ink was visualized intraoperatively in all cases, except one case with microscopic black pigment only. Fourteen group I and 10 group II patients had black pigment on histological examination of ALNs. Sentinel nodes corresponded to tattooed nodes in all except one group I patient with a tattooed non-sentinel node.Tattooed nodes are visible intraoperatively, even months later. This approach obviates the need for additional localization procedures during axillary staging.
View details for DOI 10.1245/s10434-014-4034-6
View details for PubMedID 25164040
Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma
JOURNAL OF CLINICAL INVESTIGATION
2011; 121 (1): 161-173
Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.
View details for DOI 10.1038/ncomms1773
View details for Web of Science ID 000303455200025
View details for PubMedID 22510693
View details for PubMedCentralID PMC3337985
Combinatorial targeting and nanotechnology applications
2010; 12 (4): 597-606
The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.
View details for DOI 10.1172/JCI44798
View details for Web of Science ID 000285892300022
View details for PubMedID 21183793
View details for PubMedCentralID PMC3007161
Three-dimensional tissue culture based on magnetic cell levitation
2010; 5 (4): 291-296
The development of improved methods for targeted cell detection is of general interest in many fields of research and drug development. There are a number of well-established techniques for the study and detection of biomarkers expressed in living cells and tissues. Many of them rely on multi-step procedures that might not meet ideal assay requirements for speed, cost, sensitivity, and specificity. Here we report and further validate an approach that enables spontaneous molecular assembly to generate biologically active networks of bacteriophage (phage) assembled with gold (Au) nanoparticles (termed Au-phage nanoshuttles). Here, the nanoshuttles preserve the cell binding and internalization attributes mediated by a displayed peptide targeted to a cell surface receptor. The organization of such targeted assemblies can be further manipulated to be used as a multimodal detection assembly, and they can be characterized as fractal nanostructures by angle-dependent light scattering fractal dimension analysis. Targeted Au-phage nanoshuttles offer multiple functionalities for nanotechnology-based sensing and reporting, including enhanced fluorescence and improved contrast for darkfield microscopy.
View details for DOI 10.1007/s10544-009-9340-6
View details for Web of Science ID 000279500200004
View details for PubMedID 19669890
Cracking the code for compartment-specific dual functionality proteins in cancer The case for CRKL
2010; 9 (1): 8-9
The Interleukin-11 Receptor a as a Candidate Ligand-Directed Target in Osteosarcoma: Consistent Data from Cell Lines, Orthotopic Models, and Human Tumor Samples
2009; 69 (5): 1995-1999
Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.
View details for DOI 10.1038/nnano.2010.23
View details for Web of Science ID 000276460600017
View details for PubMedID 20228788
View details for PubMedCentralID PMC4487889
An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (7): 2182-2187
The interleukin-11 receptor alpha (IL-11Ralpha) is a functional target in bone metastasis. However, its role in primary bone tumors has not been established. As such, here, we evaluated IL-11Ralpha as a candidate target in primary and metastatic human osteosarcoma. First, in an orthotopic mouse model, we showed that IL-11Ralpha protein is markedly expressed in primary osseus and pulmonary metastatic osteosarcoma but absent from control normal tibia and lung. Moreover, systemic administration of an IL-11Ralpha-targeting phage displaying the cyclic nonapeptide CGRRAGGSC resulted in strong and selective accumulation of IL-11Ralpha-homing phage particles in the osteosarcoma but not in several control organs. Finally, IL-11Ralpha expression in a large panel of human primary and metastatic osteosarcoma samples was remarkably consistent with the observations in the orthotopic mouse model. These data establish IL-11Ralpha as a candidate target in human osteosarcoma and provide leads for the development of novel imaging and therapeutic agents for the management of this malignant tumor.
View details for DOI 10.1158/0008-5472.CAN-08-4845
View details for Web of Science ID 000263937800040
View details for PubMedID 19244100
Ligand-directed Cancer Gene Therapy to Angiogenic Vasculature
TISSUE-SPECIFIC VASCULAR ENDOTHELIAL SIGNALS AND VECTOR TARGETING, PART A
2009; 67: 103-121
Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.
View details for DOI 10.1073/pnas.0807543105
View details for Web of Science ID 000263516100021
View details for PubMedID 19168626
View details for PubMedCentralID PMC2630201
Beyond receptor expression levels: The relevance of target accessibility in ligand-directed pharmacodelivery systems
TRENDS IN CARDIOVASCULAR MEDICINE
2008; 18 (4): 126-133
Gene therapy strategies in cancer have remained an active area of preclinical and clinical research. One of the current limitations to successful trials is the relative transduction efficiency to produce a therapeutic effect. While intratumoral injections are the mainstay of many treatment regimens to date, this approach is hindered by hydrostatic pressures within the tumor and is not always applicable to all tumor subtypes. Vascular-targeting strategies introduce an alternative method to deliver vectors with higher local concentrations and minimization of systemic toxicity. Moreover, therapeutic targeting of angiogenic vasculature often leads to enhanced bystander effects, improving efficacy. While identification of functional and systemically accessible molecular targets is challenging, approaches, such as in vivo phage display and phage-based viral delivery vectors, provide a platform upon which vascular targeting of vectors may become a viable and translational approach.
View details for DOI 10.1016/S0065-2660(09)67004-8
View details for Web of Science ID 000280563800004
View details for PubMedID 19914451
The original PathologischeAnatomie Leiden-Endothelium monoclonal antibody recognizes a vascular endothelial growth factor-binding site within neuropilin-1
2007; 67 (20): 9623-9629
For development of a new ligand-directed pharmacology, it is critical to measure delivery of targeted drug ligands via molecular imaging or diagnostic readouts (termed theranostics). Combinatorial peptide libraries serve as unbiased functional screens that can identify specific peptides targeting cell-surface receptors accessible to the circulation. As candidate drug leads, such peptides provide motifs likely to modify ligand-receptor interactions and downstream signal transduction pathways. This strategy is synergistic with genomic and proteomic approaches and has yielded insights into the specialized nature of the target tissue microenvironment. However, for this vision to be realized, one must look, as recent literature suggests, beyond receptor levels and critically analyze ligand accessibility as a key determinant in pharmacodelivery systems.
View details for Web of Science ID 000257280600003
View details for PubMedID 18555185
Impaired angiogenesis in aminopeptidase N-null mice
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (11): 4588-4593
For two decades, the antigen recognized by the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody, a standard vascular endothelial cell marker, has remained elusive. Here, we used a combinatorial phage display-based approach ("epitope mapping") to select peptides binding to the original PAL-E antibody. We found that a subset of the selected panel of peptides had motifs with strong homology to an exposed site within the b1 domain of human neuropilin-1 (NRP-1). We confirmed peptide binding by ELISA and by surface plasmon resonance. We also showed that the PAL-E antigen colocalizes with NRP-1 staining in endothelial cells. Crystal structure of the b1 domain in NRP-1 suggests that the PAL-E binding site overlaps with a vascular endothelial growth factor (VEGF) binding site. Taken together, these results indicate that NRP-1 is an endothelial cell antigen recognized by the true PAL-E antibody. The consistent biochemical, morphologic, and functional features between the PAL-E antigen and NRP-1 support our interpretation. Given that NRP-1 is a VEGF receptor, these results explain the attributes of the PAL-E antibody as a marker of vascular permeability and angiogenesis.
View details for DOI 10.1158/0008-5472.CAN-07-2737
View details for Web of Science ID 000250286300004
View details for PubMedID 17942890
Techniques to decipher molecular diversity by phage display.
Methods in molecular biology (Clifton, N.J.)
2007; 357: 385-406
Aminopeptidase N (APN, CD13; EC 184.108.40.206) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports of in vivo inactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases.
View details for DOI 10.1073/pnas.0611653104
View details for Web of Science ID 000244972700062
View details for PubMedID 17360568
View details for PubMedCentralID PMC1815469
A hybrid vector for ligand-directed tumor targeting and molecular imaging
2006; 125 (2): 385-398
Combinatorial phage display technology may be applied to decipher the molecular diversity of peptide binding specificity to isolated proteins, purified antibodies, cell surfaces, intracellular/cyto-domains, and blood vessels in vivo. The application of such a strategy ranges from identifying receptor-ligand pairs and antigen binding sites to understanding the progression of diseases by their differential expression patterns and developing therapeutic targeting strategies. Different strategies can be used to isolate peptides from diverse libraries displayed on the surface of bacteriophage by exposing the library to a target molecule or organ, washing away nonbinding phage, eluting and amplifying the bound phage for multiple round use, and then analyzing the peptide sequences of the enriched phage. The following methods first outline the construction of a phage library and then delineate various in vitro and in vivo biopanning applications to probe isolated integrins, purified antibodies, cell surface molecules, and vascular endothelial cells.
View details for PubMedID 17172704
Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels
2006; 66 (5): 2639-2649
Merging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.
View details for DOI 10.1016/j.cell.2006.02.042
View details for Web of Science ID 000237241500026
View details for PubMedID 16630824
Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (5): 1215-1220
Tumor blood vessels normalized by antiangiogenic therapy may provide improved delivery of chemotherapeutic agents during a window of time but it is unknown how protein expression in tumor vascular endothelial cells changes. We evaluated the distribution of RGD-4C phage, which binds alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins on tumor blood vessels before and after antiangiogenic therapy. Unlike the control phage, fd-tet, RGD-4C phage homed to vascular endothelial cells in spontaneous tumors in RIP-Tag2 transgenic mice in a dose-dependent fashion. The distribution of phage was similar to alpha(v)beta(3) and alpha(5)beta(1) integrin expression. Blood vessels that survived treatment with AG-013736, a small molecule inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptors, had only 4% as much binding of RGD-4C phage compared with vessels in untreated tumors. Cellular distribution of RGD-4C phage in surviving tumor vessels matched the alpha(5)beta(1) integrin expression. The reduction in integrin expression on tumor vessels after antiangiogenic therapy raises the possibility that integrin-targeted delivery of diagnostics or therapeutics may be compromised. Efficacious delivery of drugs may benefit from identification by in vivo phage display of targeting peptides that bind to tumor blood vessels normalized by antiangiogenic agents.
View details for DOI 10.1158/0008-5472.CAN-05-1824
View details for Web of Science ID 000235826400022
View details for PubMedID 16510583
Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries
2006; 66 (1): 34-40
Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au-phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au-phage-imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.
View details for DOI 10.1073/pnas.0509739103
View details for Web of Science ID 000235094300013
View details for PubMedID 16434473
View details for PubMedCentralID PMC1346765
Angiogenesis with pericyte abnormalities in a transgenic model of prostate carcinoma
2005; 104 (10): 2104-2115
A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.
View details for DOI 10.1158/0008-5471CAN-05-2748
View details for Web of Science ID 000234529500006
View details for PubMedID 16397212
Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection
AMERICAN JOURNAL OF PATHOLOGY
2005; 166 (2): 625-636
Previous studies of the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model vasculature suggest that, as tumors develop, vessels invade the glandular epithelium. However, changes in the vasculature are difficult to study in conventional thin tissue sections. The authors used a new approach to characterize morphologic and architectural changes of blood vessels and pericytes during tumor development in TRAMP mice.Eighty-micron cryostat sections of normal prostate and three histopathologic stages of TRAMP tumor sections, classified by epithelial cell E-cadherin immunoreactivity, were immunostained with vascular endothelial cell and pericyte receptor antibodies and evaluated by confocal microscopy.In the normal mouse prostate, capillaries were most abundant in the fibromuscular tunica between the epithelium and smooth muscle of the ductules. In the prostatic intraepithelial neoplasia (PIN) stage, vessels accompanied epithelial cell protrusions into the ductule lumen but remained in the connective tissue at the basal side of the epithelium. Well differentiated tissues had extensive angiogenesis with five times the normal mean vascularity outside ductules. Vessels were of variable diameter, were associated with an increased number of pericytes, and some had endothelial sprouts. Angiogenic blood vessels from poorly differentiated adenocarcinomas were tortuous, variable in caliber, and lacked the normal hierarchy. Pericytes on these vessels had an abnormal phenotype manifested by alpha-smooth muscle actin expression and loose association with endothelial cells. Angiogenesis and loss of vascular hierarchy were also found in human prostate carcinoma.Vascular abnormalities, which begin at the PIN stage and intensify in well differentiated and poorly differentiated tumors, may be useful readouts for early detection and treatment assessment in prostate carcinoma.
View details for DOI 10.1002/cncr.21436
View details for Web of Science ID 000233103000009
View details for PubMedID 16208706
Aminopeptidase A is a functional target in angiogenic blood vessels
2004; 5 (2): 151-162
Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.
View details for Web of Science ID 000226743000027
View details for PubMedID 15681844
View details for PubMedCentralID PMC1602338
We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected angiogenic response to hypoxia or growth factors. We then isolated peptide inhibitors of APA from a peptide library and show that they specifically bind to and inhibit APA, suppress migration and proliferation of endothelial cells, inhibit angiogenesis, and home to tumor blood vessels. Finally, we successfully treated tumor-bearing mice with APA binding peptides or anti-APA blocking monoclonal antibodies. These data show that APA is a regulator of blood vessel formation, and can serve as a functional vascular target.
View details for Web of Science ID 000189286500010
View details for PubMedID 14998491