We have previously reported the development of ELISA assays for intact MET, pY1235-MET, and pY1356-MET in tumor lysates (Srivastava et al, ASCO 2011). Here we report the development of a multiplex quantitative immunofluorescence assay (qIFA) for simultaneous detection of pY1235-MET and total MET in formalin-fixed paraffin-embedded (FFPE) tissues. We utilized a rabbit monoclonal antibody specific to pY1235-MET (with no detectable crossreactivity to pY1234-MET) to develop a highly specific and sensitive IFA assay with total MET (MET4 Mab; from G. Vande Woude, Van Andel Research Institute). Specificity of the IFA was demonstrated by HGF stimulation of A549 cells showing increased intensity of pY1235-MET which was completely inhibited by crizotinib but not by the non-MET specific multi-kinase inhibitor sorafenib, in vitro. The pY1235-MET epitope is stable in FFPE, and IFA detection was enhanced by EDTA (vs. Citrate) for antigen retrieval. Quantitative assessment of pY1235-MET was determined in xenograft models that demonstrate very high IFA expression of pY1235-MET and total MET via gene amplification (GTL-16 and SNU-5), lower expression in HGF-induced paracrine or autocrine cell lines (A549 and HT29), and no detectable staining for pY1235-MET in negative controls (SNU-1 and MDA-MB-231). We have demonstrated assay fitness for purpose using SNU-5 FFPE xenograft tumor samples from animals treated with increasing doses of crizotinib in vivo. Quantitative measurement by Definiens analysis of pY1235-MET and total MET in tissue regions of interest (ROI) showed 50% and 95% pY1235-MET inhibition with 12.5 mg/kg and 25 mg/kg crizotinib, respectively. There were no significant changes in total MET by IFA. A high correlation (R=0.899) was observed between % pY1235-MET/total MET ratio measured by IFA levels (expressed as the marker area/# nuclei for pY1235-MET and total MET per ROI) and % pY1235-MET/total MET ratio determined by ELISA levels (pM/ug protein). This quantitative MET IFA assay is currently being applied in conjunction with a previously developed EMT IFA assay (Navas et al, AACR 2013) for focal tissue analysis of pY1235-MET and total MET IFA expression as well as changes in epithelial to mesenchymal transition (EMT) in gastric tumor xenograft tissues treated with the VEGF inhibitor pazopanib, the MET inhibitor tivantinib (ARQ197), or the drug combination in vivo. These studies will determine whether pY1235-MET or total MET is induced by anti-VEGF inhibitors via increased EMT transition, and whether this effect can be reversed by combination with tivantinib. Funded by NCI Contract No. HHSN261200800001E Citation Format: Tony Navas, Scott L. Lawrence, Donna Butcher, Lindsay M. Dutko, Melinda G. Hollingshead, Robert J. Kinders, Ralph E. Parchment, Donald P. Bottaro, W. Marston Linehan, Joseph E. Tomaszewski, Apurva K. Srivastava, James H. Doroshow. Quantitative immunofluorescence assessment of MET and epithelial to mesenchymal transition (EMT) biomarker modulation by antiangiogenic inhibitors in xenograft tumor tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1049. doi:10.1158/1538-7445.AM2014-1049

We previously reported the generation of rabbit monoclonal antibodies to twelve EMT (epithelial-to-mesenchymal transition) transcription factors and cancer stem cell (CSC) markers for the development of pharmacodynamic assays to inform clinical trials of new anticancer therapies (Pfister et al., AACR 2013). Here we demonstrate the functional utility of some of these reagents in detecting HGF-induced changes in EMT and CSC biology in a xenograft tumor model. Initial antibody characterization was performed in vitro and a subset [including SNAIL, SLUG, SOX9, Goosecoid (GSC), NANOG and CD133] was selected for further testing of functional utility in FFPE tissues by quantitative multiplex IFA. The antibodies were applied to xenograft tissues derived from the non-small cell lung cancer tumor line, NCI-H596, implanted in hHGFscid/scid, hHGFki/scid or hHGFki/ki mice to examine HGF-induced changes in EMT factors, CSC markers, as well as pY1235-MET expression in vivo. H596 tumors grown in either hHGFki/scid or hHGFki/ki mice exhibited enhanced EMT particularly in tumor microenvironments adjacent to mouse stroma containing the HGF knockin gene, compared to those in hHGFscid/scid mice. By quantitative immunofluorescence, H596 tumors showed increased Vimentin:E-cadherin ratio when grown in hHGFki/scid (P<0.0006) or hHGFki/ki (P<0.022) vs. HGFscid/scid mice. Moreover, significant increases in nuclear pY1235-MET, measured by%NAP (percentage nuclear area positive), were observed in H596 tumors in hHGFki/scid (P<0.0041) or hHGFki/ki (P<0.0058) vs. hHGFscid/scid. We detected varying levels of EMT/CSC marker expression, including CD44, CD133, ALDH, and GSC in the membrane or cytoplasm of noninvasive regions of H596 tumors in hHGFscid/scid mice. Sox9 was also co-expressed with GSC in some tumor cells but was predominantly in the nuclei. In tissues collected from hHGFki/scid or hHGFki/ki mice, Slug, Snail and Sox9 expression were increased in transitioning tissue regions, adjacent to HGF-containing stroma, coincident with diminished E-Cadherin expression and enhanced Vimentin expression. While Slug expression was predominantly cytoplasmic in invading tumor fronts, the expression was mutually exclusive with CD133 within non-invasive regions. Snail and Sox9 showed enhanced nuclear expression in tumor cells undergoing EMT. These markers are currently being investigated in additional tumor samples from human TNBC or CRC xenograft models treated with various drug combinations. To our knowledge, this is the first demonstration of EMT and changes in cancer stem cell biology in NSCLC induced by an HGF-knockin stromal microenvironment. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Thomas D. Pfister, Scott M. Lawrence, Apurva K. Srivastava, Robert J. Kinders, Suzanne Borgel, Sergio Alcoser, Melinda G. Hollingshead, Lindsay M. Dutko, Brad A. Gouker, Donna Butcher, Elinor Ng-Eaton, Naoko Takebe, Young H. Lee, Donald P. Bottaro, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow. Impact of HGF knockin microenvironment on epithelial-mesenchymal transition and cancer stem cells in a non-small cell lung cancer xenograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5082. doi:10.1158/1538-7445.AM2015-5082

Cancer Organoids are discrete multicellular structures that recapitulate tumor microanatomy (1). These reagents can be generated by extended culture of partially or fully dissociated tumor samples in three-dimensional matrices. By maintaining tumor and accessory cells in an appropriate context, they provide a biosimilar platform for studying disease pathogenesis and cellular pharmacology (2). Similarly, cancer organoid culture is useful for propagating slow growing tumors or those requiring heterotypic cell-cell interactions. Here, preliminary data will be presented regarding generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiative. This initiative aims to develop a national repository of patient-derived cancer models (PDMs) consisting of clinically annotated patient-derived xenografts (PDXs) and patient-derived tumor cell cultures (PDCs) prepared from primary and metastatic tumors (3). A standardized panel of different organoid media formulations was constructed to optimize culture conditions for disease subsets. Using this approach, organoids were generated for colon, prostate, pancreatic, breast, melanoma, NSCLC, and bladder tumors. Although some samples were refractory to organoid generation, in several instances, samples that failed to generate 2D cultures thrived as organoids. A further finding was that direct implantation of organoid cultures was an efficient means of generating xenografts. Indeed, work will be presented detailing the exact number of organoids required to establish xenograft tumors. Protocols were developed for routine culture, passaging and long-term storage in liquid nitrogen. Similarly, organoids were amenable to characterization by FACS analysis, ICC/IHC and qRT-PCR to evaluate metrics such as tumor type, histological similarity with patient tumor, cell viability, percentage stroma and whether mouse cells persist in PDX-derived organoids. In summary, growth, expansion, analysis and storage of tumor organoids is feasible for a wide range of tumor types. Importantly, for certain samples, generation of cancer organoids appears to be a useful intermediary step for subsequent PDX model and 2D culture generation. Funded by NCI Contract No. HHSN261200800001E. References: 1. Baker LA, Tiriac H, Clevers H, Tuveson DA. Modeling pancreatic cancer with organoids. Trends Cancer. 2016;2:176-90. 2. Cantrell MA, Kuo CJ. Organoid modeling for cancer precision medicine. Genome Med. 2015;7. 3. Doroshow J, Hollingshead M, Evrard Y, Williams M, Datta V, Das B, et al. NCI patient derived models repository. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA). Mol Cancer Ther. 2015;14(12 Suppl 2). Citation Format: Luke H. Stockwin, Jenna Moyer, Anna Wade, Carrie Bonomi, Kelly Dougherty, John Carter, Jesse Stottlemeyer, Kaitlyn Arthur, Vivekananda Datta, Lindsay Dutko, Michael Mullendore, James H. Doroshow, Melinda G. Hollingshead, Dianne L. Newton. Establishing a platform for the generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiave [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4827. doi:10.1158/1538-7445.AM2017-4827

Background: Patient-derived tumor xenografts (PDX) are powerful tools to study cancer biology, cancer genomics and developmental therapeutics. A common problem in the development of PDX models is proliferation of atypical lymphocytes at the implant site, which often overtake or limit the growth of the original tumor. This atypical proliferation has been described as Xenograft-Associated B cell Lymphoproliferative Disease (XABLD) in our PDX models. In this study, we characterized XABLD cases by morphology, immunophenotyping and genomic profiling. We hypothesize that XABLD tumors are morphologically and phenotypically similar to EBV-driven lymphoma of the elderly and may function as a surrogate model for that lymphoma. Materials and Methods: Models were generated from patient tissue collected under NCI Tissue Procurement Protocol (clincialtrials.gov: NCT00900198) and CIRB Tissue Procurement Protocol 9846 for development of models for NCI's Patient-Derived Models Repository (https://pdmr.cancer.gov). Specimens were implanted subcutaneously in NOD/SCID/IL2Rg null (NSG) mice and animal health was monitored throughout the study. Tumors in mice with suspected XABLD were harvested and reviewed by histology and immunohistochemical analysis for CD45, B and T cell markers and EBV status. All samples in this study were classified by the Lymph2Cx NanoString cell of origin assay and transcriptome profiling. Results: XABLD-associated mice had rapidly growing CD45-positive tumors at the implantation site. Histopathological features were consistent with EBV-driven diffuse large B-cell lymphoma (DLBCL) primarily of polymorphous subtype. All XABLD specimens were diffusely positive for CD20 and EBNA, and most cases contained tumor infiltrating CD8-positive T-cells. Out of 42 cases, 36 were PD-L1-positive and 26 were PD-1-positive by IHC. 39 cases exhibited an activated B cell (ABC) phenotype, which is predominant in EBV-positive DLBCL. Conclusion: XABLD development has been seen across multiple patient histologies from both solid tumor and circulating tumor cells tissues of origin. The clinical presentation, morphology and molecular characteristics of XABLD cases were similar to EBV-driven DLBCL. As DLBCL is an aggressive disease with limited treatment options, our early-passage XABLD models may be useful in the preclinical evaluation of new therapies for EBV-positive DLBCL. Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Citation Format: Tomas Vilimas, Gloryvee Rivera, Brandie Fullmer, Wiem Lassoued, Lindsay Dutko, William Walsh, Amanda Peach, Corinne Camalier, Li Chen, Rajesh Patidar, Suzanne Borgel, John Carter, Howard Stotler, Raymond Divelbiss, Jesse Stottlemyer, Margaret Defreytas, Michelle M. Gottholm-Ahalt, Michelle A. Crespo-Eugeni, Sean McDermott, Yvonne A. Evrard, Melinda G. Hollingshead, Biswajit Das, Chris Karlovich, Vivekananda Datta, James H. Doroshow, P. Mickey Williams. Xenograft-associated B cell lymphoproliferative disease as a surrogate model to study Epstein-Barr virus (EBV) driven lymphoma of the elderly [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1038.