Parental (F0) compared to MSC educated (F2) prostate cancer cell lines

Our studies demonstrated that bone marrow derived mesenchymal stromal cells (MSCs) drive the emergence of an apoptosis resistant population of prostate cancer cells that are also cross resistant to chemotherapy such as docetaxel. The goals of this RNAQuant study was to determine differential gene expression between the naïve and MSC educated prostate cancer cells in order to better understand the mechanisms underlying apoptosis resistance. Overall design: In triplicate prostate cacner cells PAIII and DU145 were exposed to conditioned media from mesenchymal stem cells (MSCs), primary murine MSCs for PAIII and human MSCs purchased from Lonza for DU145. After 72 hours of culture in the MSC conditioned media the cells were allowed to recover in complete media for 48 hours before a second round of 72 hours of culture in MSC conditioned media and the process repeated for a third time. At this point the cell lines were recovered and called F2 PAIII or F2 DU145. Total RNA was extracted using biological replicates collected in triplicate from each, F0 and F2 PaIII and F0 and F2 DU145 cell lines using the RNeasy kit (Qiagen Cat# 74104) followed by DNase treatment using the Max Kit (Qiagen Cat # 15200). RNA concentrations were measured using the Qubit RNA BR assay and RNA integrity was assessed using the Agilent 4200 Tapestation. 500ng of RNA per sample was processed by the Moffitt Molecular Genomics Core to generate libraries for gene expression analysis using the Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen Cat #015) following the manufacturer's protocol. At least 20 million 101-base single-end sequencing reads per sample were generated on the Illumina NextSeq 500 sequencer. The Bluebee Genomics Platform provided with the QuantSeq kit from Lexogen was used for read for alignment, counting, and differential expression analysis.