Abstract 5139: Cell sorting strategies for the isolation of cancer cells and associated fibroblasts from tumor biopsies, surgical resections, and patient-derived xenografts
Authors: Stockwin Luke H. , Mullendore Michael , Bonomi Carrie , Dougherty Kelly , Doroshaw James H. , Hollingshead Melinda G. , Newton Dianne L. 
Published: August 2015

Abstract
The ability of early passage patient-derived tumor xenografts (PDXs) to recapitulate human tumor biology makes them invaluable tools for pre-clinical drug development (1, 2). Similarly, clinically-annotated human tumor cell lines of low passage have potential as scalable biosimilar reagents for in vitro study. Here, we describe an integrated platform for the purification, expansion, and quality control of tumor cells and cancer-associated fibroblasts (CAFs) from patient biopsies, surgical resections, and mouse PDX models. Following dissociation of solid tumors, cells are cultured on Matrigel® in fibroblast conditioned media containing Y-27632 (a Rho-associated kinase inhibitor). Cells are then analyzed by FACS using a panel of species- or cell type-specific antibodies. This panel (anti-mCD9, hCD9, hCD24, hCD90, MHC, HLA, and hEPCAM) permits quantitation of the percentage of mouse/human cells, and the percentages of tumor cells and CAFs in each patient specimen. From these data, a cell sorting strategy can be selected (e.g. mCD9 and hCD90 with hEpCAM or CD24) that will permit purification of both tumor cells and CAFs, along with removal of any mouse fibroblast contamination (for PDX tumors). Samples are sorted multiple times until target populations reach 99.99% purity (typically 2-3 rounds). Purified cells are then returned to culture, expanded over several weeks, and frozen at low passage. The purity and identity of these cultures is quality-assured using 1) mouse-specific QPCR, 2) a cDNA array designed to differentiate fibroblasts from tumor cells, and 3) STR genotyping. Tumor lines and CAFs are then scaled and re-qualified at passages 10 and 20, an essential step given the propensity of low-frequency mouse and human fibroblasts to ‘overtake’ impure cultures with time. Lastly, cancer lines and CAF cultures undergo genomic characterization and are evaluated for in vivo tumorigenicity (or the lack thereof for CAFs). To date, the platform has been used to generate a growing number of purified cancer and CAF pairs from patient materials. In conclusion, our goal is to provide useful reagents to the extramural community that will accompany current efforts within the Division of Cancer Treatment and Diagnosis (DCTD, NCI) aimed at generating a large and extensively characterized repository of PDX models. Funded by NCI Contract No. HHSN261200800001E.
References
1.Hidalgo M, Amant F, Biankin AV, Budinska E, Byrne AT, Caldas C, et al. Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov. 2014;4:998-1013.
2.Rosfjord E, Lucas J, Li G, Gerber HP. Advances in patient-derived tumor xenografts: from target identification to predicting clinical response rates in oncology. Biochem Pharmacol. 2014;91:135-43.
Citation Format: Luke H. Stockwin, Michael Mullendore, Carrie Bonomi, Kelly Dougherty, James H. Doroshaw, Melinda G. Hollingshead, Dianne L. Newton. Cell sorting strategies for the isolation of cancer cells and associated fibroblasts from tumor biopsies, surgical resections, and patient-derived xenografts. [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 5139. doi:10.1158/1538-7445.AM2015-5139