Abstract 1847: Evaluating the metastatic potential and the molecular heterogeneity of patient-derived orthotopic xenograft models of triple-negative breast cancer
Authors: Ramani Vishnu C. , Gupta Rakhi , Quon Gerald , Triboulet Melanie , Renier Corinne , Greene Cassandra , Sanada Chad , Lu Tracy , Szpankowski Lukasz , Ramalingam Naveen , Salahudeen Ameen A. , O Sean de la , Rajapaksa Ranjani , Levy Shoshana , Leyrat Anne A. , West Jay A. , Sollier-Christen Elodie , Kuo Calvin J. , Sledge George W. , Jeffrey Stefanie S. 
Published: July 2017

Abstract
We report an in-depth characterization of patient-derived orthotopic xenograft (PDOX) models of triple-negative breast cancer (TNBC) regarding their molecular profile at the single cell level, tumor heterogeneity, 3D organoid generation, and ability to generate circulating tumor cells (CTCs). A panel of seven TNBC PDOX tumors were grown orthotopically in Nod scid gamma mice and used in this study. Blood obtained via cardiac puncture from tumor bearing animals was processed on the Vortex microfluidic platform, for label-free, size-based enrichment of circulating tumor cells (CTCs). Enriched cell populations were stained for human-specific cytokeratin (CK) and Vimentin (Vim), mouse-specific CD45, and DAPI; CTCs were identified as cells that were CD45 negative and positive for CK or Vim. Bulk tumor growing in the mammary fat pads was dissociated to single cells and characterized using Fluidigm’s® PolarisTM platform for single cell biological experimentation and cDNA generation within an integrated fluidic circuit (IFC). From the cell suspension, Polaris identified single cells that were then processed for mRNA-seq. The resulting cDNA libraries were then multiplexed using Nextera XT® (Illumina®) and sequenced on Illumina systems. Data generated from mRNA-seq was processed to correct for confounding factors such as cell size, cell cycle and read depth and then analyzed to screen for heterogeneity between different populations of cells. Tumors were analyzed by flow cytometry for both tumor and immune cells and additionally the single cell suspension was seeded into 3-D culture to generate organoids. Finally, organs from tumor bearing animals were analyzed for metastases. With the Vortex platform, we detected CTCs from a majority of our PDOX tumor-bearing mice. The total number of CTCs varied over a wide range between different PDOX tumors. There was a clear heterogeneity in CTCs in terms of CK and Vim expression. In CTCs from one of the PDOX tumors, we detected a small population of CTCs that were either CK+ or Vim+ but the major fraction that was double positive (Vim+ CK+). Probing the bulk tumor from different PDOX models revealed heterogeneity in the levels and number of cells positive for cell surface markers like EpCAM and a difference in the levels of infiltrating myeloid cells (CD11b+). mRNA-seq analyses of individual tumor cells from the bulk tumor belonging to different PDOX models will be described. Additionally, lung and brain metastases were identified. 3D organoid cultures from our PDOX models were successfully grown and their gene expression profiles will be analyzed. In summary, PDOX models of TNBC will help advance our understanding of the molecular basis of this deadly cancer.
Citation Format: Vishnu C. Ramani, Rakhi Gupta, Gerald Quon, Melanie Triboulet, Corinne Renier, Cassandra Greene, Chad Sanada, Tracy Lu, Lukasz Szpankowski, Naveen Ramalingam, Ameen A. Salahudeen, Sean de la O, Ranjani Rajapaksa, Shoshana Levy, Anne A. Leyrat, Jay A. West, Elodie Sollier-Christen, Calvin J. Kuo, George W. Sledge, Stefanie S. Jeffrey. Evaluating the metastatic potential and the molecular heterogeneity of patient-derived orthotopic xenograft models of triple-negative breast cancer [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 1847. doi:10.1158/1538-7445.AM2017-1847