Fibroblasts-derived COL1A1 Facilitates The Metastasis Of Ovarian Cancer By Activating The Tumor Microenvironment

Ovarian cancer occupies the leading cause for deaths related to gynecologic cancers among women.According to data released by the American Cancer Society in 2020,the overall 5-year survival rate of ovarian cancer patients is 48%,but only 29% in those with advanced stage,in the United States.Because the ovaries locate deep in the pelvic cavity,there are no typical clinical symptoms of the disease and specific tumor markers in the early stage,more than 75% of ovarian cancer patients present with abdominal dissemination and/or distant metastasis(stage III-IV)when they are primarily diagnosed.Therefore,it is urgent to understand the biological process of ovarian cancer metastasis,so as to find effective monitoring indicator and intervention strategy.In recent years,the role of tumor microenvironment in the development of tumors has been paid more and more attention.Ascites is a specific tumor microenvironment for advanced intraperitoneal cancers,including ovarian cancer,and closely related to disease prognosis.Ascites contains a large number of tumor cells,stromal cells,various active factors and proteins.The later is responsible for communication among cells.Fibroblasts,as the most prominent stromal cells in the microenvironment,secrete a variety of tumor-promoting factors that directly transmit signals to adjacent tumor cells to promote cellular proliferation,invasion,and metastasis,but the involved mechanism needs to be further explored.Preliminarily,ovarian epithelial cancer ascites and normal peritoneal fluids were collected and screened for differentially expressed proteins,using liquid chromatography(LC)-mass spectrometry(MS)/MS label-free quantitative proteomics and bioinformatics analysis.As a result,49 secreted proteins with significantly differential expression(fold change ?2 or ? 0.5)were found between the two groups.Of those,COL1A1 protein(Collagen type I alpha 1)showed 4.8-fold higher in cancer ascites than normal peritoneal fluids.On this basis,this study used a variety of molecular biological techniques to identify the main source of COL1A1,observe the effects of COL1A1 protein on the biological functions such as proliferation,migration and invasion of ovarian cancer cells and its related mechanisms,and explore the effects of ovarian cancer cells on the secretion of COL1A1 by fibroblasts.The aim of this study was to investigate the mechanism of fibroblasts-derived COL1A1 facilitating the metastasis of ovarian cancer by activating the tumor microenvironment,providing an experimental evidence for the development of new targeted drugs and new molecular markers.Part I The effect of COL1A1 on proliferation,migration and invasion in ovarian cancer cellsObjective: To confirm the effect of COL1A1 on the proliferation,migration and invasion in ovarian cancer cells and the effect on intraperitoneal metastasis of ovarian orthotopic xenograft tumor in immunodeficiency mice.Methods: Firstly,the protein levels of COL1A1 in 10 normal peritoneal fluids and 27 ovarian cancer ascites were detected by ELISA.Then,we detected the expression of COL1A1 m RNA and protein in cytoplasm by RT-PCR and Western blot,and the level of COL1A1 protein in culture supernatant from fibroblasts(IMR-90)and ovarian cancer cells(Caov3 and SKOV3)by ELISA,respectively.Next,CCK-8 assay was used to detect the effect of exogenous COL1A1 on the proliferation of ovarian cancer cells.The effects of COL1A1 or conditioned medium(CM)of IMR-90 on migration and invasion of Caov3 and SKOV3 cells were detected in the presence or absence of anti-COL1A1 neutralizing antibody by Transwell assays.Finally,the orthotopic xenograft model of ovarian cancer was constructed to observe the effect of intraperitoneal injection of COL1A1 protein on the growth and metastasis of xenograft.Results: 1.ELISA results confirmed that COL1A1 protein expression in ascites of epithelial ovarian cancer patients was significantly higher than that in normal peritoneal fluid(p<0.0001).2.IMR-90 cells more highly expressed COL1A1 m RNA and protein in the cytoplasm, and highly secreted COL1A1 protein in the supernatant,compared with Caov3 and SKOV3 cells.3.Exogenous COL1A1 protein had no effect on the proliferation of ovarian cancer cells.4.COL1A1 protein or IMR-90 conditioned medium promoted migration and invasion of ovarian cancer cells,such effects were partially eliminated by COL1A1 antibodies.5.Intraperitoneally injected COL1A1 accelerated intraperitoneal metastasis of ovarian cancer xenograft in immunodeficient mice.Conclusions: 1.COL1A1 is elevated in ascites from epithelial ovarian cancer patients,and mainly secreted by fibroblasts.2.COL1A1 promotes the migration and invasion of ovarian cancer cells and accelerated intraperitoneal metastasis of ovarian cancer xenograft in immunodeficient mice,suggesting that COL1A1 plays a cancer promotion role in ovarian cancer.Part II ITGB1/AKT signaling pathway is involved in the process of COL1A1 regulating migration and invasion in ovarian cancer cellsObjective: To identify intracellular signaling pathways participated in COL1A1 regulation of ovarian cancer migration and invasion.Methods: The Uniprot,String database analysis and literature review were used to screen the receptors that might transmit extracellular signals of COL1A1 to cancer cells,and RT-PCR was used to detect the m RNA relative expression in Caov3 and SKOV3 cells.Then,si RNA was used to regulate the receptors ITGB1 and CD44 expression in ovarian cancer cells,following treatment with COL1A1,the effect of COL1A1 protein on the migration and invasion of cancer cells after silencing the receptors was assessed by Transwell assay.Similarly,ovarian cancer cells were pretreated with ITGB1 blocking antibody(BV7),followed by treatment with COL1A1,the effect of COL1A1 protein on the migration and invasion of cancer cells after blocking the binding site of ITGB1 was assessed by Transwell assay.Moreover,phosphorylation of AKT,one of the intracellular integrin-related signaling pathway,was examined in ovarian cancer cells after treatment of COL1A1 by Western blot and immunofluorescence.Ovarian cancer cells were transfected with ITGB1-specific si RNA or pretreated with ITGB1 blocking antibody,followed by treatment with COL1A1,the phosphorylation levels of AKT were examined by Western blot.Finally,the phosphorylation of AKT was inhibited by the small-molecule inhibitor MK-2206,followed by treatment with COL1A1,the phosphorylation levels of AKT were examined by Western blot,the migration and invasion of ovarian cancer cells were assessed by Transwell assay.Results: 1.Silencing ITGB1 partially inhibited COL1A1 enhanced migration and invasion in ovarian cancer cells.2.Blocking ITGB1 partially inhibited COL1A1 enhanced migration and invasion in ovarian cancer cells.3.Western blot and cellular immunofluorescence assay showed that COL1A1 activated the phosphorylation of AKT.4.Silencing or blocking ITGB1 partially suppressed the increased phosphorylation levels of AKT induced by COL1A1.5.MK-2206,an inhibitor of AKT,suppressed the increased phosphorylation levels of AKT,and attenuated the enhanced migration and invasion of ovarian cancer cells mediated by COL1A1.Conclusions: COL1A1 promotes migration and invasion of ovarian cancer cells by activating the ITGB1/AKT signaling pathway.Part III Ovarian cancer ascites and fibrinogen regulate the secretion of COL1A1 in fibroblastsObjective: To investigate which factors influence COL1A1 over-secretion of fibroblasts in ovarian cancer ascites.Methods: Firstly,the ascites of three ovarian cancer patients were diluted to different percentages(0%,25%,50%,75%)by MEM medium to stimulate fibroblasts,and the levels of COL1A1 protein in culture supernatant of fibroblasts were detected by ELISA.Next,we re-checked the results of ascites proteomics,and found increased fibrinogen protein in ovarian cancer ascites.Further,we verified the level of fibrinogen protein in 10 normal peritoneal fluids and 27 ovarian cancer ascites by ELISA,and analyzed the correlation with COL1A1 expression.We then detected COL1A1 level in culture supernatant of fibroblasts,stimulated by different concentrations of fibrinogen.Further,to explore the cell source of fibrinogen,we determined the fibrinogen level in culture supernatant of ovarian cancer cells by ELISA,and it was undetectable.We next speculated that it originated from the extravasation of plasma fibrinogen,and in vitro endothelial permeability assay was performed to determine the effects of conditioned medium from ovarian cancer cells on vascular endothelial permeability.Results: 1.Ovarian cancer ascites promoted fibroblasts to secret COL1A1,and fibrinogen protein expression in ovarian cancer ascites was significantly higher than that in normal peritoneal fluid.2.There was a linear positive correlation between COL1A1 and fibrinogen expression in normal peritoneal fluid and ovarian cancer ascites,and fibrinogen promoted fibroblasts to secret COL1A1.3.Fibrinogen was undetectable in the supernatant of ovarian cancer cells,but the supernatant enhanced the permeability of vascular endothelial cells.Conclusions: 1.Ovarian cancer ascites and fibrinogen promote fibroblasts to secrete COL1A1 2.Elevated fibrinogen in ascites might be associated with extravasation of plasma fibrinogen,due to increased vascular permeability caused by ovarian cancer.