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Michael Ozawa, MD

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Specialties

Anatomic & Clinical Pathology

Work and Education

Professional Education

University of Texas Medical School at Houston Registrar, Houston, TX, 5/28/2012

Residency

Stanford University Pathology Residency, Stanford, CA, 6/30/2016

Fellowship

Stanford University Cytopathology Fellowship, Stanford, CA, 6/30/2018

Stanford University Surgical Pathology Fellowship, Stanford, CA, 6/30/2017

Board Certifications

Anatomic & Clinical Pathology, American Board of Pathology

Cytopathology, American Board of Pathology

All Publications

Impact of a deep learning assistant on the histopathologic classification of liver cancer. NPJ digital medicine Kiani, A. n., Uyumazturk, B. n., Rajpurkar, P. n., Wang, A. n., Gao, R. n., Jones, E. n., Yu, Y. n., Langlotz, C. P., Ball, R. L., Montine, T. J., Martin, B. A., Berry, G. J., Ozawa, M. G., Hazard, F. K., Brown, R. A., Chen, S. B., Wood, M. n., Allard, L. S., Ylagan, L. n., Ng, A. Y., Shen, J. n. 2020; 3 (1): 23

Abstract

Artificial intelligence (AI) algorithms continue to rival human performance on a variety of clinical tasks, while their actual impact on human diagnosticians, when incorporated into clinical workflows, remains relatively unexplored. In this study, we developed a deep learning-based assistant to help pathologists differentiate between two subtypes of primary liver cancer, hepatocellular carcinoma and cholangiocarcinoma, on hematoxylin and eosin-stained whole-slide images (WSI), and evaluated its effect on the diagnostic performance of 11 pathologists with varying levels of expertise. Our model achieved accuracies of 0.885 on a validation set of 26 WSI, and 0.842 on an independent test set of 80 WSI. Although use of the assistant did not change the mean accuracy of the 11 pathologists (p=0.184, OR=1.281), it significantly improved the accuracy (p=0.045, OR=1.499) of a subset of nine pathologists who fell within well-defined experience levels (GI subspecialists, non-GI subspecialists, and trainees). In the assisted state, model accuracy significantly impacted the diagnostic decisions of all 11 pathologists. As expected, when the model's prediction was correct, assistance significantly improved accuracy (p=0.000, OR=4.289), whereas when the model's prediction was incorrect, assistance significantly decreased accuracy (p=0.000, OR=0.253), with both effects holding across all pathologist experience levels and case difficulty levels. Our results highlight the challenges of translating AI models into the clinical setting, and emphasize the importance of taking into account potential unintended negative consequences of model assistance when designing and testing medical AI-assistance tools.

View details for DOI 10.1038/s41746-020-0232-8

View details for PubMedID 33594170

CD22-Directed CAR T-Cell Therapy Induces Complete Remissions in CD19-Directed CAR-Refractory Large B-Cell Lymphoma. Blood Baird, J. H., Frank, M. J., Craig, J. n., Patel, S. n., Spiegel, J. Y., Sahaf, B. n., Oak, J. S., Younes, S. n., Ozawa, M. n., Yang, E. n., Natkunam, Y. n., Tamaresis, J. S., Ehlinger, Z. n., Reynolds, W. D., Arai, S. n., Johnston, L. n., Lowsky, R. n., Meyer, E. n., Negrin, R. S., Rezvani, A. R., Shiraz, P. n., Sidana, S. n., Weng, W. K., Davis, K. L., Ramakrishna, S. n., Schultz, L. n., Mullins, C. D., Jacob, A. P., Kirsch, I. R., Feldman, S. A., Mackall, C. L., Miklos, D. B., Muffly, L. n. 2020

Abstract

The prognosis for patients with large B-cell lymphoma (LBCL) progressing after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first three consecutive patients with autologous CAR19-refractory LBCL treated with a single infusion of autologous 1106 CAR+ T-cells/kg targeting CD22 (CAR22) as part of a phase I dose escalation study. CAR22 therapy was relatively well tolerated, without any observed non-hematologic adverse events higher than grade 2. Following infusion, all three patients achieved complete remission, with all responses ongoing at the time of last follow up (mean 7.8 months, range 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range 85.4-350 cells/L), and persisted beyond three months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA (ctDNA) beyond six months post-infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients who have failed prior CAR T-cell therapies. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT04088890).

View details for DOI 10.1182/blood.2020009432

View details for PubMedID 33512414

Determining the Optimal Number of Core Needle Biopsy Passes for Molecular Diagnostics CARDIOVASCULAR AND INTERVENTIONAL RADIOLOGY Hoang, N. S., Ge, B. H., Pan, L. Y., Ozawa, M. G., Kong, C. S., Louie, J. D., Shah, R. P. 2018; 41 (3): 48995

Abstract

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

A study of the mutational landscape of pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma. Modern pathology Ozawa, M. G., Bhaduri, A., Chisholm, K. M., Baker, S. A., Ma, L., Zehnder, J. L., Luna-Fineman, S., Link, M. P., Merker, J. D., Arber, D. A., Ohgami, R. S. 2016; 29 (10): 1212-1220

Abstract

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

Synchronous Hepatoblastoma, Neuroblastoma, and Cutaneous Capillary Hemangiomas: A Case Report PEDIATRIC AND DEVELOPMENTAL PATHOLOGY Ozawa, M. G., Cooney, T., Rangaswami, A., Hazard, F. K. 2016; 19 (1): 74-79

Abstract

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

Synchronous Hepatoblastoma, Neuroblastoma, and Cutaneous Capillary Hemangiomas: A Case Report. Pediatric and developmental pathology Ozawa, M. G., Cooney, T., Rangaswami, A., Hazard, F. K. 2016; 19 (1): 74-79

Abstract

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 PubMedID 26368548

Dasatinib-related Follicular Hyperplasia: An Underrecognized Entity With Characteristic Morphology. American journal of surgical pathology Ozawa, M. G., Ewalt, M. D., Gratzinger, D. 2015; 39 (10): 1363-1369

Abstract

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

Correlation of percutaneously biopsied axillary lymph nodes marked with black tattoo ink prior to neoadjuvant chemotherapy with sentinel lymph nodes in breast cancer patients Choy, N., Lipson, J., Pal, S., Ikeda, D., Trinh, L., Allison, K., Ozawa, M., Wheeler, A., Wapnir, I. AMER ASSOC CANCER RESEARCH. 2015
The utility of IgM, CD21, HGAL and LMO2 in the diagnosis of pediatric follicular lymphoma HUMAN PATHOLOGY Karnik, T., Ozawa, M. G., Lefterova, M., Luna-Fineman, S., Alvarez, E., Link, M., Zehnder, J. L., Arber, D. A., Ohgami, R. S. 2015; 46 (4): 629-633

Abstract

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

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 Mintz, P. J., Rietz, A. C., Cardo-Vila, M., Ozawa, M. G., Dondossola, E., Do, K., Kim, J., Troncoso, P., Logothetis, C. J., Sidman, R. L., Pasqualini, R., Arap, W. 2015; 112 (8): 2515-2520

Abstract

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

Initial results with preoperative tattooing of biopsied axillary lymph nodes and correlation to sentinel lymph nodes in breast cancer patients. Annals of surgical oncology Choy, N., Lipson, J., Porter, C., Ozawa, M., Kieryn, A., Pal, S., Kao, J., Trinh, L., Wheeler, A., Ikeda, D., Jensen, K., Allison, K., Wapnir, I. 2015; 22 (2): 377-382

Abstract

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 nodeswas 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 0.1to0.5ml 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

Initial Results With Black Ink Tattooing of Biopsied Axillary Lymph Nodes Choy, N. S., Lipson, J., Kieryn, A., Porter, C., Ikeda, D., Pal, S., Trinh, L., Ozawa, M., Allison, K., Jensen, K., Wapnir, I. SPRINGER. 2014: 3536
Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells NATURE COMMUNICATIONS Rangel, R., Guzman-Rojas, L., Le Roux, L. G., Staquicini, F. I., Hosoya, H., Barbu, E. M., Ozawa, M. G., Nie, J., Dunner, K., Langley, R. R., Sage, E. H., Koivunen, E., Gelovani, J. G., Lobb, R. R., Sidman, R. L., Pasqualini, R., Arap, W. 2012; 3

Abstract

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

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 Staquicini, F. I., Ozawa, M. G., Moya, C. A., Driessen, W. H., Barbu, E. M., Nishimori, H., Soghomonyan, S., Flores, L. G., Liang, X., Paolillo, V., Alauddin, M. M., Basilion, J. P., Furnari, F. B., Bogler, O., Lang, F. F., Aldape, K. D., Fuller, G. N., Hook, M., Gelovani, J. G., Sidman, R. L., Cavenee, W. K., Pasqualini, R., Arap, W. 2011; 121 (1): 161-173

Abstract

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

Combinatorial targeting and nanotechnology applications BIOMEDICAL MICRODEVICES Souza, G. R., Staquicini, F. I., Christianson, D. R., Ozawa, M. G., Miller, J. H., Pasqualini, R., Arap, W. 2010; 12 (4): 597-606

Abstract

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

Three-dimensional tissue culture based on magnetic cell levitation NATURE NANOTECHNOLOGY Souza, G. R., Molina, J. R., Raphael, R. M., Ozawa, M. G., Stark, D. J., Levin, C. S., Bronk, L. F., Ananta, J. S., Mandelin, J., Georgescu, M., Bankson, J. A., Gelovani, J. G., Killian, T. C., Arap, W., Pasqualini, R. 2010; 5 (4): 291-296

Abstract

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

Cracking the code for compartment-specific dual functionality proteins in cancer The case for CRKL CELL CYCLE Ozawa, M. G., Cardo-Vila, M., Mintz, P. J., Arap, W., Pasqualini, R. 2010; 9 (1): 8-9

View details for Web of Science ID 000273236800004

View details for PubMedID 20009577

The Interleukin-11 Receptor a as a Candidate Ligand-Directed Target in Osteosarcoma: Consistent Data from Cell Lines, Orthotopic Models, and Human Tumor Samples CANCER RESEARCH Lewis, V. O., Ozawa, M. G., Deavers, M. T., Wang, G., Shintani, T., Arap, W., Pasqualini, R. 2009; 69 (5): 1995-1999

Abstract

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

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 Mintz, P. J., Cardo-Vila, M., Ozawa, M. G., Hajitou, A., Rangel, R., Guzman-Rojas, L., Christianson, D. R., Arap, M. A., Giordano, R. J., Souza, G. R., Easley, J., Salameh, A., Oliviero, S., Brentani, R. R., Koivunen, E., Arap, W., Pasqualini, R. 2009; 106 (7): 2182-2187

Abstract

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

Ligand-directed Cancer Gene Therapy to Angiogenic Vasculature TISSUE-SPECIFIC VASCULAR ENDOTHELIAL SIGNALS AND VECTOR TARGETING, PART A Driessen, W. H., Ozawa, M. G., Arap, W., Pasqualini, R. 2009; 67: 103-121

Abstract

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

Beyond receptor expression levels: The relevance of target accessibility in ligand-directed pharmacodelivery systems TRENDS IN CARDIOVASCULAR MEDICINE Ozawa, M. G., Zurita, A. J., Dias-Neto, E., Nunes, D. N., Sidman, R. L., Gelovani, J. G., Arap, W., Pasqualini, R. 2008; 18 (4): 126-133

Abstract

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

The original PathologischeAnatomie Leiden-Endothelium monoclonal antibody recognizes a vascular endothelial growth factor-binding site within neuropilin-1 CANCER RESEARCH Jaalouk, D. E., Ozawa, N. G., Sun, J., Lahdenranta, J., Schlingemann, R. O., Pasqualini, R., Arap, W. 2007; 67 (20): 9623-9629

Abstract

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

Impaired angiogenesis in aminopeptidase N-null mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rangel, R., Sun, Y., Guzman-Rojas, L., Ozawa, M. G., Sun, J., Giordano, R. J., Van Pelt, C. S., Tinkey, P. T., Behringer, R. R., Sidman, R. L., Arap, W., Pasqualini, R. 2007; 104 (11): 4588-4593

Abstract

Aminopeptidase N (APN, CD13; EC 3.4.11.2) 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

Techniques to decipher molecular diversity by phage display. Methods in molecular biology (Clifton, N.J.) Christianson, D. R., Ozawa, M. G., Pasqualini, R., Arap, W. 2007; 357: 385-406

Abstract

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

A hybrid vector for ligand-directed tumor targeting and molecular imaging CELL Hajitou, A., Trepel, M., Lilley, C. E., Soghomonyan, S., Alauddin, M. M., Marini, F. C., Restel, B. H., Ozawa, M. G., Moya, C. A., Rangel, R., Sun, Y., Zaoui, K., Schmidt, M., von Kalle, C., Weitzman, M. D., Gelovani, J. G., Pasqualini, R., Arap, W. 2006; 125 (2): 385-398

Abstract

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

Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels CANCER RESEARCH Yao, V. J., Ozawa, M. G., Varner, A. S., Kasman, I. M., Chanthery, Y. H., Pasqualini, R., Arap, W., McDonald, D. M. 2006; 66 (5): 2639-2649

Abstract

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

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 Souza, G. R., Christianson, D. R., Staquicini, F. I., Ozawa, M. G., Snyder, E. Y., Sidman, R. L., Miller, J. H., Arap, W., Pasqualini, R. 2006; 103 (5): 1215-1220

Abstract

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

Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries CANCER RESEARCH Kolonin, M. G., Bover, L., Sun, J., Zurita, A. J., Do, K. A., Lahdenranta, J., Cardo-Vila, M., Giordano, R. J., Jaalouk, D. E., Ozawa, M. G., Moya, C. A., Souza, G. R., Staquicini, F. I., Kunyiasu, A., Scudiero, D. A., Holbeck, S. L., SAUSVILLE, E. A., Arap, W., Pasqualini, R. 2006; 66 (1): 34-40

Abstract

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

Angiogenesis with pericyte abnormalities in a transgenic model of prostate carcinoma CANCER Ozawa, M. G., Yao, V. J., Chanthery, Y. H., Troncoso, P., Uemura, A., Varner, A. S., Kasman, I. M., Pasqualini, R., Arap, W., McDonald, D. M. 2005; 104 (10): 2104-2115

Abstract

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

Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection AMERICAN JOURNAL OF PATHOLOGY Yao, V. J., Ozawa, M. G., Trepel, M., Arap, W., McDonald, D. M., Pasqualini, R. 2005; 166 (2): 625-636

Abstract

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

Aminopeptidase A is a functional target in angiogenic blood vessels CANCER CELL Marchio, S., Lahdenranta, J., Schlingemann, R. O., Valdembri, D., Wesseling, P., Arap, M. A., Hajitou, A., Ozawa, M. G., Trepel, M., Giordano, R. J., Nanus, D. M., Dijkman, H. B., Oosterwijk, E., Sidman, R. L., COOPER, M. D., Bussolino, F., Pasqualini, R., Arap, W. 2004; 5 (2): 151-162

Abstract

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