Abstract 4072: Molecular characterization of single tumor cells isolated from blood samples using immunomagnetic enrichment and dielectrophoretic cell sorting: A feasibility study
Authors: Peeters Dieter J. , Van den Eynden Gert G. , Van Laere Steven J. , Salgado Roberto , Huget Philippe , van Dam Peter A. , Peeters Marc , Pauwels Patrick , Vermeulen Peter B. , Dirix Luc Y. 
Published: August 2012

Abstract
Introduction Molecular characterization of CTC holds considerable promise for the identification and monitoring of therapeutic targets in cancer patients under systemic treatment. Molecular profiling of CTC is however frustrated by white blood cell (WBC) signatures overwhelming those emanating from the CTC minority. In this study, we investigated the feasibility to isolate and molecularly characterize single tumor cells (TC) and small pools of up to 10 pure TC from immunomagnetically enriched blood samples using a semi-automated platform for dielectrophoretic cell sorting (DEPArray, Silicon Biosystems, Bologna, IT). Methods MDA-MB-231 human breast cancer cells were spiked into healthy donor blood at a concentration of 1000 cells/7.5 ml blood. Clinical patient samples were obtained from patients with metastatic breast cancer. Blood samples were subjected to EpCAM based immunomagnetic enrichment using the CellSearch Profile Kit (CellSearch, Veridex, Raritan, NJ, USA). Enriched samples were immunofluorescently stained for EpCAM-PE, CD45-APC and Hoechst and subsequently further purified using a DEPArray cell sorter. Single TC, pools of 5 TC, pools of 10 TC and pools of 20 WBC were recovered into individual reaction tubes. Molecular analyses consisting of whole genome amplification followed by K-ras mutation analysis and RT-qPCR for an in-house selected panel of breast cancer related transcripts were performed. Results Genomic DNA (gDNA) was succesfully amplified and detected in 3/5 (60%) single MDA-MB-231 TC and 4/4 (100%) pools of 5-10 TC and 20 WBC. K-ras mutation analysis revealed the G13D mutation - heterozygously present in the MDA-MB-231 cell line - in all TC samples and in none of two WBC samples, indicating 100% purity of the sorted cell samples. In addition, gDNA was succesfully detected and amplified in 5/9 (55%) single TC and 5/6 (83%) pools of 5-10 TC and 20 WBC from a clinical patient sample. Transcriptional profiles of pools of 5-10 MDA-MB-231 TC samples, were consistently correlated with publicly available gene expression profiles of MDA-MB-231 cells (Spearman R2=0.36±0.03) and inversely correlated with gene expression profiles of MCF-7 cells (Spearman R2=−0.09±0.03), indicating correct classification according to molecular subtypes. In a clinical patient sample, transcriptional profiles of pools of CTC could be correctly distinguished from WBC. Discussion We show the feasibility of an integrated workflow for the molecular characterization of single TC and small pools of up to 10 pure TC isolated from immunomagnetically enriched blood samples using a semi-automated dielectrophoretic cell sorting technique. Further optimization is being undertaken to allow for single cell transcriptional profiling and results will be expanded in clinical patient samples in order to gain insight into in vivo CTC heterogeneity.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4072. doi:1538-7445.AM2012-4072