Abstract 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