Abstract 1197: Differential gene expression analysis by RNA-seq of primary tumors, circulating tumor cells, and metastases from a mouse xenograft model of cancer dissemination
Authors: Rochefort Holly M. , Xu Tong , Xu Yucheng , Li Meng , Chen Yibu , Hu Brian , Goldkorn Amir 
Published: May 2015

Abstract
Tumor dissemination and metastasis is the root cause of cancer mortality, and informative models are needed to identify key drivers of this process. We developed a tractable mouse xenograft model of cancer dissemination, wherein GFP-labeled pure primary tumor (PT) cells, circulating tumor cells (CTCs) and lung metastasis (LM) cells can be isolated from the same host and studied to map drivers of cancer progression.
GFP-labeled SCC351 human squamous cell carcinoma cells were inoculated subcutaneously into NOD/SCID/IL2r-γnull (NSG) mice. When subcutaneous xenografts approximately 1cm in diameter were palpable (4-8 weeks), 500-1000 µl of blood was collected by intracardiac puncture and Ficoll-centrifuged to remove red blood cells. At the same time, PT and LM were resected and digested into single cell suspensions. An XYZ micromanipulator was used to pick up individual GFP+ CTCs, PTs and LMs for RNA extraction, amplification with Ovation RNA-Seq System V2 (Nugen), and library generation for RNA-Seq on a HiSeq2000 (Illumina). RNA-Seq data was analyzed with Partek Flow and mapped to human genome (Ensembl GRCh37) using TopHat2. Gencode V17 annotation was used to quantify the aligned reads to genes using Partek E/M, and read counts per gene were analyzed for differential expression using DESeq2. Gene expression validation was accomplished by qPCR.
Comparing CTC gene expression to PT and to LM, a differential expression of at least 2x (with BH adjusted p-value &lt;0.05) was seen in 166 genes. Of these, 135 genes encoded known proteins and 31 corresponded to pseudogenes and lincRNAs. The 166 candidate genes were further ranked based on their degree of differential expression, read depth, and functional analysis (using Next Bio for annotated genes) suggesting involvement in pathways of cell survival, proliferation or tumor growth. Based on this analysis flow, 17 genes were chosen for validation by qPCR: 8 with an expression level that went up from PT to CTCs and then down in LM, 6 that went down from PT to CTCs and then up in LMs, and 3 that were lincRNAs. To date, differential expression has been validated for five of these genes by qPCR, and additional validation currently is ongoing.
Here we leveraged new techniques for rare CTC capture, whole transcriptome amplification and next generation sequencing to identify and validate new candidate drivers of dissemination and metastasis in a tractable xenograft mouse model of cancer progression. This model holds the potential to uncover novel functions of lincRNAs, in addition to new roles for known genes in the context of cancer dissemination. Functional analysis of these candidates in vitro and in vivo is planned, with the goal of identifying and characterizing new drivers of cancer progression for therapeutic targeting.
Citation Format: Holly M. Rochefort, Tong Xu, Yucheng Xu, Meng Li, Yibu Chen, Brian Hu, Amir Goldkorn. Differential gene expression analysis by RNA-seq of primary tumors, circulating tumor cells, and metastases from a mouse xenograft model of cancer dissemination. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2014-1197