A Clinically Validated Multiplex Immunofluorescence Assay For The Quantitative Assessment Of Changes In Emt Phenotypes In Ffpe Tumor Tissues In Response To Cancer Therapeutics

Epithelial-mesenchymal transition (EMT) is a critical process implicated in metastasis. Despite extensive research, the clinical significance of EMT remains unclear, and a quantitative evaluation of this process in human tumors has yet to be demonstrated. We previously reported a validated EMT immunofluorescence assay (EMT-IFA), that utilizes β-catenin as a tumor segmentation marker to delineate tumor tissue from surrounding stroma in FFPE tumor biopsies. The assay accurately quantifies individual expression and co-localization of E-cadherin (E) and vimentin (V) in tumor cells (Navas et al. NCI-EORTC 2015). We used this assay to measure changes in both the total number and the corresponding EMT phenotypes of tumor cells in xenograft tissues following treatment with various anticancer agents currently in clinical trials at the NCI. Daily treatment for 14 days with the multikinase inhibitor pazopanib caused significant tumor regression and delayed regrowth in the epithelial (E+) gastric cancer xenograft model MKN45, and the tumor cells remaining after treatment were significantly shifted toward a mesenchymal (V+) phenotype. In contrast, pazopanib (QDx15) had less anti-tumor efficacy in the mesenchymal gastric cancer xenograft model SNU5. In another instance, daily treatment of the MDA-MB-468 breast cancer xenograft model with the BCR-Abl kinase inhibitor nilotinib for 19 days did not demonstrate any significant anti-tumor efficacy or change in the predominance of the E+ phenotype, whereas a cycle of treatment with the anti-tubulin agent paclitaxel (Q3Dx4) led to MDA-MB-468 tumor regression and delayed tumor regrowth after completion of the treatment cycle. Furthermore, the combination of nilotinib with paclitaxel not only significantly diminished the total MDA-MB-468 tumor cell number compared to single-agent arms, but also effectively transformed the EMT phenotype of the tumor, with only a subset of cancer cells surviving by the last day of treatment, which were mostly CD44+CD133+ mesenchymal cells and potential cancer stem cells. The changes in EMT phenotype brought on by effective drug treatments occurred via preferential killing of cells with E+ phenotype, suggesting that V+ and V+E+ tumor cells may be more resistant to therapy. The EMT-IFA provides a much-needed analysis tool suitable for clinical investigation of the proposed role of EMT in tumor progression, metastasis, and acquired drug resistance, and is able to detect changes in EMT signature which may serve as early indicators of treatment efficacy and tumor resistance. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Melinda G. Hollingshead, Suzanne Borgel, John P. Carter, Angelena Millione, Brad A. Gouker, Donna Butcher, Susan Holbeck, Apurva K. Srivastava, Robert J. Kinders, Donald P. Bottaro, Shivaani Kumar, Alice Chen, James H. Doroshow, Ralph E. Parchment. A clinically validated multiplex immunofluorescence assay for the quantitative assessment of changes in EMT phenotypes in FFPE tumor tissues in response to cancer therapeutics [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 845. doi:10.1158/1538-7445.AM2017-845