Proteomic analysis of malignant ascites and its impact on ovarian cancer spheroids.

Abstract

Background: Most advanced ovarian cancer patients develop malignant ascites, which describes a buildup of fluid in the peritoneal cavity caused by increased vascular permeability and obstructed lymphatic drainage. Malignant ascites contains cancer cells, which can aggregate as spheroids, as well as stromal cells, cancer-associated fibroblasts, and blood cells that create a complex tumor microenvironment. This study explores the proteome of ascites and how this environment affects the viability, phenotypes, proteomes, and treatment responses of ovarian cancer cells.

Methods: Using label-free proteomics, we compared the proteomes of cell-free malignant ascites from ovarian cancer patients with those of serum. Additionally, we examined the ex vivo chemotherapy responses of cancer spheroids cultured in ascites. Through detailed proteomic analysis of cells grown as 2D or 3D in tissue culture medium or ascites, we identified biological pathways and specific proteins induced by ascites. Finally, we performed orthogonal validation of a candidate marker, TGM2, using immunofluorescent staining.

Results: Proteomics of cell-free ascites identified increased levels of extracellular, secreted, and membrane proteins when compared to serum. Ascites enhanced the baseline cell viability and spheroid formation of immortalized ovarian cancer cell lines compared to standard cell culture medium. However, chemotherapy-induced cell death of spheroids grown in standard cell culture medium remained proportional to changes observed in ascites-cultured spheroids. Ascites-driven phenotypic changes were not recapitulated by adding selected chemokines nor periostin to the cell culture medium, suggesting that additional factors are required. Notably, ascites induced similar ECM, secreted, and membrane proteins across 2D and 3D models, including TGM2, which was validated in spheroids through immunofluorescent staining.

Conclusions: This study contributes to our understanding of the role of ascites in the regulation of the proteome and viability of cancer cells. It provides evidence for the induction of TGM2 expression by ascites. Results from this pilot study warrant further study in a larger cohort.