Cell type specific gene expression analysis of prostate needle biopsies resolves tumor tissue heterogeneity

// Malte Kronig 1 , Max Walter 1 , Vanessa Drendel 2 , Martin Werner 2 , Cordula A. Jilg 1 , Andreas S. Richter 3, 4 , Rolf Backofen 3 , David McGarry 1 , Marie Follo 5 , Wolfgang Schultze-Seemann 1 , Roland Schule 1 1 Medical Center, University of Freiburg, Department of Urology, 79106 Freiburg, Germany 2 Medical Center, University of Freiburg, Department of Pathology, 79106 Freiburg, Germany 3 University of Freiburg, Department of Computer Science, 79110 Freiburg, Germany 4 Max Planck Institute of Immunbiology and Epigenetics, 79108 Freiburg, Germany 5 Medical Center, University of Freiburg, Department of Medicine I, 79106 Freiburg, Germany Correspondence to: Roland Schule, e-mail: roland.schuele@uniklinik-freiburg.de Keywords: prostate cancer, RNA detection, living cells, needle biopsy, gene expression, tumor heterogeneity Received: August 06, 2014      Accepted: November 11, 2014      Published: December 01, 2014 ABSTRACT A lack of cell surface markers for the specific identification, isolation and subsequent analysis of living prostate tumor cells hampers progress in the field. Specific characterization of tumor cells and their microenvironment in a multi-parameter molecular assay could significantly improve prognostic accuracy for the heterogeneous prostate tumor tissue. Novel functionalized gold-nano particles allow fluorescence-based detection of absolute mRNA expression levels in living cells by fluorescent activated flow cytometry (FACS). We use of this technique to separate prostate tumor and benign cells in human prostate needle biopsies based on the expression levels of the tumor marker alpha-methylacyl-CoA racemase (AMACR). We combined RNA and protein detection of living cells by FACS to gate for epithelial cell adhesion molecule (EPCAM) positive tumor and benign cells, EPCAM/CD45 double negative mesenchymal cells and CD45 positive infiltrating lymphocytes. EPCAM positive epithelial cells were further sub-gated into AMACR high and low expressing cells. Two hundred cells from each population and several biopsies from the same patient were analyzed using a multiplexed gene expression profile to generate a cell type resolved profile of the specimen. This technique provides the basis for the clinical evaluation of cell type resolved gene expression profiles as pre-therapeutic prognostic markers for prostate cancer.