Genetic changes through spheroid formation in ascites - interaction with ovarian cancer cell line, OV90, and human primary mesothelial cells (HPMC)-

More than 70% of patients with epithelial ovarian cancer (EOC) are diagnosed with peritoneal metastasis and ascites, which is the accumulation of intraperitoneal fluid containing non-malignant cells. However, the interactions between EOC and non-malignant cells before the development of peritoneal metastasis remain unclear. This study investigated the mechanism by which EOC cells survive through interactions with the surrounding cells in ascites. Whole EOC spheroids were observed, and their invasion into collagen layers and mesothelial monolayers was examined. RNA sequencing revealed the molecular mechanisms associated with aggressiveness of EOC cells. In malignant ascites, almost 100% of EOC cells were spheroids, 60% of which contained mesothelial cells. EOC cells quickly generated aggregated spheroids with mesothelial cells, and these aggregated cancer-mesothelial spheroids (ACMS) rapidly invaded collagen or mesothelial layers. Mesothelial cells forming ACMS initiated the invasion, with EOC cells following the route created by the mesothelial cells. RNA sequence revealed dramatically RNA expression changes in mesothelial cells, whereas the changes in EOC cells were relatively small. TGF-β1-stimulated mesothelial cells showed increased invadopodia formation along with fascin-1 upregulation. These findings suggest that EOC cells alter gene expression in mesothelial cells through ACMS formation, which explains the rapid spread of EOC in the abdominal cavity. We used OV90-GFP cells and three different kinds of primary human mesothelial cell (HPMC). To investigate the genetic changes through spheroid formation, three types of cell components were incubated for 72 hours to form spheroids in ultra-low attachement 96-well round-bottome plates; (i) OV90-GFP only (2,000 cells/well), (ii) HPMC cells only (4,000 cells/well), (iii) a co-culture of OV90-GFP and HPMC cells (2,000 cells/well each). Before analyzing, these spheroids were disaggregated with trypsin and collected by FACS. Comparative gene expression profile analysis of RNA-seq data for (i) OV90-GFP cells with or without interaction with HPMC, and (ii) HPMC cells with or without interaction with OV90-GFP.