Abstract 1943: Exploring cancer stem cells heterogeneity via single cell multiplex gene expression analysis
Authors: Azizi Ebrahim , Fouladdel Shamileh , Deol Yadwinder S. , Bender Jonathan , McDermott Sean , Jiang Hui , Sehl Mary , Clouthier Shawn G. , Nagrath Sunitha , Wicha Max S. 
Published: May 2015

Abstract
A considerable body of evidence indicates that many cancers are hierarchically organized and driven by cancer stem cells (CSCs) that have the ability to self-renew and to generate heterogeneous populations of tumor bulk. Preclinical studies have also demonstrated that CSCs mediate tumor metastasis and resistance to chemotherapy and radiation therapy. As a result of substantial studies to identify CSCs markers and related pathways, agents developed to target CSCs are in early stage clinical trials. Given the clinical importance of CSCs, there is an urgency to develop the methodology to assay these cells. In this study, we explored CSCs heterogeneity at the single cell level using C1 and BioMark HD technologies. Cancer stem cells from MCF7 and SUM159 breast cancer cell lines were sorted by MoFlo XDP based CSCs marker including CD44, CD24 and ALDH1 immunophenotyping. Sorted cells were then separately loaded onto the C1 chip and studied by C1 instrument that isolates up to 96 single cells and performs lysis, RNA release, reverse transcription, and finally cDNA preamplification of up to 96 gene transcripts for each single cell. We selected the 96 genes based on important genetic pathways, EMT and CSCs markers, and also genes identified in the expression array data to be differentially expressed in CD44+/CD24- and or ALDH+ primary breast tumor samples. Following C1 preamplification, we analyzed the mRNA expression levels of 96 genes on each of the isolated single cells by BioMark HD instrument which generates nearly 10,000 qPCR data points in a single run using a 96x96 chip. Results showed that more than one population exist with significant differences in mRNA expression levels of certain genes such as CAPRIN2, TM4SF1, NECAB3, PPL, TSPAN6, STAP2, FAM46C, TCEA3, TPSB2 and FCN1, as well as IL6, GATA3, KI67, PCNA, EZH2, and MET in seemingly homogenous populations of ALDH+ and ALDH-(CD44+/CD24- in MCF7 cells) CSCs. In addition, a distinct pattern of gene expression for sorted luminal MCF7 in comparison to basal SUM159 cells was observed for epithelial versus mesenchymal markers such as CDH1, CDH2, KRT7, KRT19, EpCAM, and Vimentin genes. We have recently reported that breast cancer stem cells (BCSCs) exist in inter-convertible EMT/MET states characterized by distinct genes and biomarker expression. Using this methodology of single cell analysis, we found that these CSC populations still display significant heterogeneity in mRNA expression. This technique of single cell multiplex gene expression analysis, along with a microfluidic method for a label-free cell sorting is currently under optimization to be used for isolation and molecular characterization of circulating tumor cells (CTCs) from blood samples of breast cancer patients as a means of liquid biopsy. Altogether, results of this and similar studies on single CSCs have considerable clinical implications in cancer diagnosis and prognosis, as well as in the successful targeted therapy of cancer stem cells.
Citation Format: Ebrahim Azizi, Shamileh Fouladdel, Yadwinder S. Deol, Jonathan Bender, Sean McDermott, Hui Jiang, Mary Sehl, Shawn G. Clouthier, Sunitha Nagrath, Max S. Wicha. Exploring cancer stem cells heterogeneity via single cell multiplex gene expression analysis. [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 1943. doi:10.1158/1538-7445.AM2014-1943