Abstract 2000: Targeted characterization of tumor heterogeneity through RNA-Seq analysis of phenotypically defined subpopulations
Authors: Porter Warren , Snowden Eileen , Hahn Friedrich , Ferguson Mitchell , Tong Frances , Dillmore Shannon , Blaesius Rainer 
Published: July 2017

Abstract
Cancer progression and metastases have been linked to the complex heterogeneity found within many solid tumors. In order to understand the complexities within solid tumors there is a need for in-depth characterization of the tumor and the surrounding microenvironment, including the identification and measurement of the subpopulations found within the diseased tissue.
Here we describe a method to interrogate the heterogeneity within patient derived xenograft (PDX) derived solid tumors through tissue dissociation, single cell flow sorting, and expression analysis of the resulting subpopulations. Cell sorting enables the identification and capture of phenotypically defined subpopulations within the tumor microenvironment for additional downstream analysis. Whole transcriptome RNA-Seq was performed on samples collected throughout the workflow to characterize the expression changes in cells during the different steps of the process. Expression differences were measured before and after tissue dissociation, before and after incubation, and before and after cell sorting. Expression differences were also measured between phenotypically defined subpopulations sorted by the CD133 and CD49f surface markers.
Several significant differentially expressed gene combinations between samples were observed. These differences involved unique pathway characteristics between the different subpopulations sorted based on expression of CD133 and CD49f markers. In contrast, changes induced by the workflow process alone were restricted to a small and well-defined gene subset of immediate early genes (IEGs). Within the workflow, these changes were restricted to the tumor dissociation step and no changes were detected after incubating and/or cell sorting the dissociated cells.
Our results indicate that solid tumor dissociation followed by flow cytometric analysis and sorting enables the interrogation of phenotypically defined subpopulations found within solid tumors. Since workflow induced changes are minimal and defined, these methods reveal biological differences between subpopulations within and between cancers. Differential expression of numerous surface marker genes were identified by our method allowing discovery of other candidate surface markers that may extend the subpopulation structure. Once verified these additional surface markers could serve to elucidate the dynamic changes cancer tissue undergoes during progression as well as in response to selective pressures such as drug therapy.
Citation Format: Warren Porter, Eileen Snowden, Friedrich Hahn, Mitchell Ferguson, Frances Tong, Shannon Dillmore, Rainer Blaesius. Targeted characterization of tumor heterogeneity through RNA-Seq analysis of phenotypically defined subpopulations [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 2000. doi:10.1158/1538-7445.AM2017-2000