TRAIL-dependent apoptosis of human peritoneal mesothelial cells by activated NK cells promotes ovarian cancer cell invasion in vitro
Transcoelomic dissemination of tumor cells via the malignant peritoneal fluid (ascites) occurs at an early stage of high-grade serous ovarian carcinoma, enabling extensive tumor spreading within the peritoneum. A high quantity of malignant ascites arises during ovarian cancer progression, which is a...
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|Transcoelomic dissemination of tumor cells via the malignant peritoneal fluid (ascites) occurs at an early stage of high-grade serous ovarian carcinoma, enabling extensive tumor spreading within the peritoneum. A high quantity of malignant ascites arises during ovarian cancer progression, which is an important characteristic of the unique tumor microenvironment. The ascites is rich in soluble factors and different cell types, including immune cells, promoting the metastatic spread, in particular to the omentum as a first site of tumor implantation. To date, the exact mechanism of peritoneal invasion is still largely unknown. The omentum and further peritoneal organs are covered by a mesothelial layer, physiologically functioning as a protective shield, which is believed to prevent tumor invasion. Different mechanisms leading to a disruption of the protective mesothelium, favoring metastatic outspread, have been discussed. Interestingly, increased mesothelial cell apoptosis introduced during pathological processes, including inflammation and malignancy, have been reported. Strikingly, we are the first to pinpoint the induction of mesothelial cell apoptosis to tumor-associated lymphocytes present within the ovarian cancer tumor microenvironment. Thus, we determined NK cells as the main effectors promoting mesothelial clearance in vitro. Soluble factors secreted by activated T cells stimulate the NK cells’ cytotoxic potential. Here we discovered TRAIL-dependent killing of omentum-derived mesothelial cells as central mode of NK cell cytotoxicity, as demonstrated by applying a neutralizing anti-TRAIL antibody. Further evidence evolved from enhanced TRAIL expression on NK cells upon stimulation with T cell-secreted mediators, while the corresponding activating death cell receptors, DR4 and DR5, are expressed on mesothelial cells. By contrast, a significant contribution of Fas/FasL signaling or cytotoxic cytokines, including TNFα and IFNɣ, could be ruled out. Even though mesothelial apoptosis could generally be induced by granzyme B/perforin, immunosuppressive ascites completely blocked this pathway, which is therefore not important in our system. Affinity proteomic-based analysis (Olink) of T cell-soluble factors comparing the secretomes of untreated and activated T cells revealed mediators inducing NK cell activation and TRAIL up-regulation. Intriguingly, we detected a previously unknown secretome-based crosstalk between ascites-derived T and NK cells mediating TRAIL upregulation on NK cells mainly by T cell-secreted TNFα, demonstrated by gain and loss of function experiments. A more subordinate role was assigned to further T cell-secreted cytokines linked to NK cell activation and TRAIL signaling, including IL-2, IL-21 and IFNɣ, possibly cooperating with TNFα to activate the NK cell cytotoxic potential. In the following, we addressed whether TRAIL-dependent killing mediated by NK cells in the tumor milieu was selectively directed against mesothelial cells. By analyzing the potential of NK cells in killing high-grade serous ovarian carcinoma cells, we found that the primary tumor cells were resistant towards TRAIL-mediated killing, which was associated with decreased expression levels of the death receptors. To further investigate why especially mesothelial cells derived from the malignant tumor microenvironment were prone to TRAIL-dependent killing, mesothelial cells obtained from a benign background were compared and found to be less sensitive to apoptosis induction by NK cells, which was further TRAIL-independent. This could be reasoned by the lower expression of DR4 and indicates that the mesothelial cells might be educated by ascites-derived soluble factors to TRAIL sensitivity, thereby supporting tumor implantation.
A causal link between TRAIL-mediated disruption of the mesothelial barrier by NK cells and enhanced ovarian cancer cell invasion could be proven in vitro by a trans-mesothelial invasion assay. Finally, these findings could be clinically accredited by immunohistological analysis of patient samples showing a loss of the mesothelium in the close proximity of early metastatic lesions, while free-floating apoptotic mesothelial cells were present in the ascites.
In conclusion, these novel findings indicate that NK cells, activated primarily by T cell-derived TNFα, play an essential role in promoting transcoelomic dissemination by selectively destroying the protective mesothelial barrier in a TRAIL-dependent manner.