Abstract 1546: Differential gene expression analysis by NGS of primary tumor xenograft cells, circulating tumor cells, and metastasis cells purified from the same mouse hosts to identify drivers of cancer dissemination
Authors: Xu Tong , Morrison Gareth , Xu Yucheng , Triche Timothy , Buckley Jonathan , Goldkorn Amir 
Published: July 2016

Abstract
Introduction: Cancer dissemination involves ongoing dynamic transit of tumor cells between primary and metastatic sites via a hematogenous or “liquid phase”. To identify molecular mechanisms related to the liquid phase of cancer dissemination, we developed a tractable mouse xenograft model wherein GFP-labeled pure primary tumor (PT) cells, circulating tumor cells (CTCs) and lung metastasis (LM) cells are isolated and characterized from the same host.
Methods: GFP+ MDA-MB-231 human breast cancer cells were inoculated subcutaneously into NOD/SCID/IL2r-γnull (NSG) mice. When a 1cm diameter primary tumor xenograft formed, 500 μl blood were collected by intracardiac puncture, and CTCs were enriched by wbc/rbc depletion (StemCell Tech). PT and LM samples were fluorescence microdissected from the same mouse hosts and disaggregated into single cell suspensions. Using a micromanipulator pipette, matched PT, CTC, and LM samples (100 pure GFP+ cells each) were isolated, lysed for RNA extraction (Ambion), and analyzed using the Ion AmpliSeq Transcriptome Human Gene Expression workflow for library preparation and pooled sequencing (Ion Proton). Sequence data from biologic triplicates was aligned and curated using the Genetrix bioinformatics suite, and candidate genes were further analyzed using Ingenuity Pathway Analysis (IPA).
Results: The sequence panel provides gene-level expression information from over 20,000 genes covering &gt; 95% of the RefSeq gene database. Initially, we focused on genes that were up- or down- regulated in CTCs compared with both PT and LM (i.e. differentially expressed in the liquid phase: 402 genes, 184 up-regulated in CTCs, 218 down-regulated in CTCs, fold change range 1.2x-22x). After further filtering for false discovery, 4 up-regulated and 39 down-regulated genes were selected for IPA analysis, which revealed associations with cellular movement pathways (16 genes) and cancer pathways (24 genes). Of these, the top-ranked 12 differentially expressed genes in CTCs relative to PT and LM (9 down-regulated and 3 up-regulated) were selected for validation by qPCR and further functional characterization in the mouse xenograft model.
Conclusions: Here we leveraged new techniques for pure CTC capture, AmpliSeq transcriptome amplification and sequencing to identify and validate new candidate drivers of cancer dissemination. This model holds the potential to uncover key mechanisms engaged by cells that leave the primary and metastatic tumor niche and enter the cancer's liquid phase. Further validation and functional analysis of these candidates in vitro, in vivo and in patient samples may identify new therapeutic targets that specifically disrupt cancer dissemination.
Citation Format: Tong Xu, Gareth Morrison, Yucheng Xu, Timothy Triche, Jonathan Buckley, Amir Goldkorn. Differential gene expression analysis by NGS of primary tumor xenograft cells, circulating tumor cells, and metastasis cells purified from the same mouse hosts to identify drivers of cancer dissemination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1546.