In vivo Passage of Human Prostate Cancer Cells in Mice Results in Stable Gene Expression Changes Affecting Numerous Cancer-Associated Biological Processes Lavarnan Sivanathan, 1 Annabelle Chow, 1 Amy Wong, 1 Van C. Hoang, 1 and Urban Emmenegger 1,2 * 1 Biological Sciences Platform, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, University of Toronto,Toronto,Ontario,Canada 2 Division of Medical Oncology,Odette Cancer Centre, Sunnybrook Health Sciences Centre, University of Toronto,Toronto,Ontario,Canada BACKGROUND. While therapeutic resistance is difficult to model in vitro in its entirety, in vivo passage and re-derivation of treatment resistant prostate cancer cell variants is a strategy to study therapeutic resistance more comprehensively. However, the process of in vivo passage itself may result in gene expression changes that could confound the analysis of such resistant cell variants compared to their parental cell lines. METHODS. We compared the expression profiles of parental PC-3 human prostate cancer cells and PC-3 cells re-derived after in vivo passage in athymic nude mice. Whole tran- scriptome information was obtained using the SOLiD 4 system (Applied Biosystems). Differentially expressed genes were mapped to genes in the Database for Annotation, Visualization and Integrated Discovery for gene enrichment and functional annotation analysis. The expression of a panel of these genes was validated using quantitative RT-PCR. RESULTS. Altogether, 21,032 distinct transcripts were found in PC-3 and/or NS1.1. Of these, 906 were differentially regulated (2-fold) in NS1.1 versus PC-3. 337 transcripts were upregulated, and 569 were downregulated, including genes previously associated with various aspects of prostate carcinogenesis such as TLR4 and IGFBP5, respectively. Gene ontology analysis of the differentially expressed transcripts revealed enrichment for biological processes such as cell adhesion, migration, and angiogenesis. CONCLUSIONS. When using in vivo as opposed to in vitro derived prostate cancer cell variants for comparative genetic studies of complex traits such as therapeutic resistance, one may be better served to use similarly in vivo passaged control cell variants instead of parental cell lines. Prostate 74:537–546, 2014. # 2014 Wiley Periodicals, Inc. KEY WORDS: human prostate cancer xenografts; in vivo passage; whole transcriptome analysis; therapeutic resistance INTRODUCTION Angiogenesis is among the hallmarks of neoplastic growth [1]. While agents targeting the vascular endo- thelial growth factor (VEGF) pathway (i.e., bevacizu- mab and aflibercept) have failed to significantly improve the survival of patients with castration-resis- tant prostate cancer (CRPC) when combined with docetaxel chemotherapy [2,3], the antiangiogenic agents tasquinimod and cabozantinib are currently being tested as single agents in phase III CRPC trials [4]. Potent antiangiogenic effects can also be Grant sponsor: Prostate Cancer Canada Clinician-Scientist Award; Grant sponsor: University of Toronto. Disclosure Statement: The authors declare no conflict of interest. Lavarnan Sivanathan and Annabelle Chow contributed equally to this work.  Correspondence to: Urban Emmenegger, MD, Clinician Scientist, Division of Medical Oncology, Odette Cancer Centre and Biological Sciences Platform, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, University of Toronto, T2-054, 2075 Bayview Avenue, Toronto, ON, Canada M4N3M5. E-mail: urban.emmenegger@sunnybrook.ca Received 8 July 2013; Accepted 23 December 2013 DOI 10.1002/pros.22774 Published online 16 January 2014 in Wiley Online Library (wileyonlinelibrary.com). The Prostate 74:537^546 (2014) ß 2014 Wiley Periodicals, Inc.