Transcriptional dissection by single-cell RNASeq analysis of the metastasis settings from a highly metastatic Patient-Derived Xenograft model of pancreatic ductal adenocarcinoma

Single cells were isolated from the main scenarios during tumor dissemination of a highly metastatic Patient-Derived Xenograft model (Panc-265) in order to discover potential therapeutic targets against the metastatic settings of PDAC. Primary tumor (n=37), liver metastasis (n=23) cells and circulating tumor cells (CTCs, n=10, in bloodstream) were subjected of single-cell RNAseq. Single-cell RNA sequencing analysis revealed that CTCs clustered separately from their matched primary and metastatic tumors and were characterized by low expression of ECM associated genes, high expression of cell cycle genes, a metabolic switch to oxidative phosphorylation and high expression of genes involved in ribosome biogenesis. Overall design: Cells enriched from primary tumor, mouse blood and liver of PDX metastatic model (Panc-265) were individually separated on the C1™ Single-Cell Auto Prep System (Fluidigm, South San Francisco, CA,USA) and cDNA amplification was generated using the SMARTer®Ultra™ Low RNA kit for the Fluidigm C1™ System (Clontech, Mountain View, CA, USA). In total, 137 cells were captured on three C1 array chips for mRNA sequencing (17–25 µm Fluidigm). Cells that were negative for the human membrane marker and positive for a viability marker (DAPI) were excluded from further analysis. In total 70 samples passed the criteria. cDNA quantity and quality was measured with a 2100 Bioanalyzer (Agilent). Libraries were generated from 300 pg of amplified cDNA using the Nextera XT DNA Sample Prep Kit (Illumina) and sequenced on the HiSeq 2500 sequencer (Illumina) to generate 50 bases single reads at an average depth of 13.5 million reads per sample.