RNA-seq for MMT-induced mesothelial cells

Malignant ascites accompanied with peritoneal dissemination contains various factors and cell populations as well as cancer cells, yet how tumor microenvironment is shaped in ascites remains unclear. A single-cell proteomic profiling and a comprehensive proteomic analysis were conducted to uncover the full picture of malignant ascites. Here, we found the defect of immune effectors along with immunosuppressive cell accumulation in ascites of gastric cancer (GC) patients and identified five distinct subpopulations in CD45(-)/EpCAM(-) cells. Mesothelial cells with mesothelial-mesenchymal transition (MMT) phenotype in CD45(-)/EpCAM(-) cells are predominant source of chemokines involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Moreover, MMT-induced mesothelial cells are highly expressed the extracellular matrix related genes, including tenascin-C (TNC) enhancing the metastatic colonization. These findings highlight the definite roles of mesenchymal cell population in the development of pro-tumorigenic microenvironment to promote peritoneal dissemination. Mesothelial cells from ascites of patients were stimulated with vehicle control, TGF-B1 and IL-1B. After 48-hour culture, total RNA of cells from each group was extracted and sequencing was performed by Nextseq500 (Illumina) or MinION (Oxford Nanopore Technologies).