Fibrosis Of Mesothelial Cell Induced Peritoneal Implantation Of Ovarian Cancer Cells

Objective:Ovarian cancer is the most lethal gynecological malignancy.About 70% ovarian cancer patients are diagnosed when tumors have widely spread within the peritoneal cavity,which ultimately leads to mortality.However,the mechanism underlying ovarian cancer spread has not been thoroughly explored.Therefore,to further study the occurrence and the development of peritoneal metastasis of ovarian cancer,clarify its molecular pathological mechanism,and find the key therapeutic targets are the fundamental ways to further improve the survival rate of ovarian cancer patients.The peritoneal metastasis of ovarian cancer is the concurrent results of ovarian cancer cells and peritoneal microenvironment.Most studies focused on ovarian cancer cells,while few studies have been conducted regarding the changes in peritoneal microenvironment.Mesothelial cells are an important part of peritoneal microenvironment.The current study investigated the morphological changes and mechanism underlying peritoneal mesothelial cells in the process of peritoneal metastasis of ovarian cancer.Our results may provide new molecular targets and a theoretical basis for the prevention and treatment of ovarian cancer peritoneal metastasis.Methods:Patients and specimens:This study was approved by the local organ Review Committee of China Medical University.Tissue specimens were collected from 60 patients who underwent surgery in Shengjing Hospital of China Medical University from 2016 to 2017.Our study selected 40 cases of peritoneal specimen from ovarian cancer patients with peritoneal metastasis and 20 cases of normal peritoneal specimen.The morphologic changes and the arrangement of peritoneal mesothelial cells were observed using light and electron microscope.The human mesothelial cell line HMRSV5 were cultured in serum-free medium,normal medium and supernatant of ovarian cancer cell line SKOV3,respectively.Then the expression of E-cadherin and α-SMA in mesothelial cells were detectd by Western-blot.ELISA was used to detect the expression levels of TGF-β1,IL-10,IL-8,TNF-α,VEGF,EGF and MMPs in supernetants of ovarian cancer cell lines SKOV3 and HO8910,to find the possible cytokines that causing fibrosis of mesothelial cells and get the optimal dose and action time.MTT cell proliferation test and transwell invasion test were applied to observe the ability of MFT promoting the proliferation and metastasis of ovarian cancer cells.The expression of some chemokines(CXCL12,CXCL16,CCL19 and CCL21)in the supernatant of MFT were detected by ELISA and the expression of some chemokine receptors(CXCR4,CXCR6 and CCR7)in ovarian cancer cells were detected by Western-blot.Results:1.Under light microscopy,we observed that the normal peritoneum surface was one single layer of mesothelial cells,which were cubical and tightly attached to each other,however,the mesothelial cells were spindle and absent with fibrosis-transformed changes in the pathological metastasis peritoneum.The normal and metastatic peritoneal mesothelial cells were also observed under a SEM.The mesothelial cells were arranged tightly on the normal peritoneum surface,and the mesothelial cells were undergoing MFT with mesothelial cells suffering from injury and fibrosis,cell becoming spindle,intercellular space enlarging and local bare area forming.Compared with the normal medium and serum-free medium cultured mesothelial cell line HMrSV5,the levels of E-cadherin protein were down regulated and the levels of α-SMA protein were upregulated in the HMrSV5 cells that were cultured with supernatants from ovarian cancer cell line SKOV3.2.ELISA results reveals that the levels of TGF-β1 and IL-10 concentration were relatively higher in SKOV3 cell lines and HO8910 cell lines.In vitro experiments,we verified that TGF-β1 induced MFT in a time and dose dependent manner.3.The ovarian cancer cells SKOV3 and HO8910 were cultured with the supernatant medium of MFT model(experimental group),normal mesothelial cell culture medium and normal serum-free medium(control groups)respectively for 48 hours.There was no difference in cell proliferation rate of SKOV3 cells between the two control groups.The survival rates were 81.3±3.6% and 78.6±2.3% respectively,p=1.398.However,SKOV3 cultured in supernatant from fibrotic HMrSV5 proliferated faster,and the 48-hour survival rate reached 99.6±4.2%.Compared with the former two,the survival rate of SKOV3 cultured in supernatant from fibrotic HMrSV5 was higher(p < 0.05).The same phenomenon was observed in another ovarian cancer cell line.The proliferation rate of ovarian cancer cell line HO8910 cultured in normal mesothelial medium and serum-free medium for 48 hours was not different.The survival rates were 75.3±3.1% and 70.6±1.3% respectively,p=1.472.The proliferation rate of HO8910 cultured in supernatant from fibrotic HMrSV5 was faster,and the 48-hour survival rate was 97.3±3.9%.Compared with the former two,the survival rate of HO8910 cultured in supernatant fromfibrotic HMrSV5 was higher(P<0.05).4.Transwell invasion experiment: HMrsV5 cell suspension in serum-free medium were used as control group,HMrsV5 cell suspension in SKOV3 medium and HMrsV5 cell suspension in serum-free medium +100 ng/ml of TGF-β1 were as experimental groups,After 16 h of incubation for invasion,the wells were harvested and the number of infiltrating SKOV3 cancer cells was counted.The number of invasive cancer cells was significantly increased in experimental groups compared with control group(P<0.05).5.ELISA was used to detect the expression of ovarian cancer related chemokines(CXCL12,CXCL16,CCL19 and CCL21)in the supernatants from fibrotic HMrSV5 cell(experimental group)and fromHMrSV5 cell that cultured in serum-free medium(control group).The results showed that there were higher expressions of CXCL12 and CXCL16 in the supernatant of the experimental group compared with the control group(P<0.05).The expressions of CCL19 and CCL21 in the supernatant of the experimental group were also higher than those in the control group,but there was no significant difference(P>0.05).Meanwhile,Western-blot was used to detect the expression of chemokine receptors(CXCR4,CXCR6 and CCR7)in the free serum cultured,normal cultured SKOV3 cells(control groups)and SKOV3 co-cultured with fibrotic HMrSV5 supernatants(experimental group).The results showed that the expression of CXCR4 and CXCR6 protein in the experimental group was significantly higher than that in the control groups(P<0.05),but there was no difference in the expression of CCR7(P>0.05).Conclusion:1.Morphological observation by light and electron microscopy confirmed that mesothelial cells of metastatic peritoneal tissues exhibited fibrotic change,which was an important change in peritoneal microenvironment.The fibrotic change of mesothelial cells was also validated from protein level.These results suggested that fibrosis of mesothelial cells may play an important role during the peritoneal metastasis of ovarian cancer cells.2.By detecting various cytokines in the supernatant from ovarian cancer cell lines,we found that there was high expression of TGF-β1,which suggested that there exist the factors promoting fibrosis of mesothelial cells in the microenvironment of ovarian cancer cells.3.It was confirmed that TGF-β1 could promote fibrosis of mesothelial cells by morphological observation of HMRSV5 and protein level detection.4.The results of cell proliferation and invasion experiments showed that fibrosis of mesothelial cells promoted the proliferation and invasion of ovarian cancer cells,which suggested that fibrotic transformation of mesothelial cells in peritoneal microenvironment plays an important role in the metastasis of ovarian cancer cells.5.The results of cytological experiments showed that fibrosis of mesothelial cells could promote the expression of chemokines CXCL12 and CXCL16 in microenvironment,which suggested that chemotaxis may be an important mechanism in peritoneal metastasis of ovarian cancer cells.6.Cytological experiments showed that fibrosis of mesothelial cells promoted the expression of chemokine receptors CXCR4 and CXCR6 in ovarian cancer cells,which suggested that CXCL12-CXCR4 and CXCL16-CXCR6 signaling pathways may be potential mechanisms of MFT promoting proliferation and metastasis of ovarian cancer cells,and they may become important therapeutic targets for inhibiting peritoneal metastasis of ovarian cancer in the future.