Ovarian Cancer Derived Exosomes Facilitate Metastasis Of Ovarian Cancer Through Activating The Tumor Microenvironment

Objectives1.To explore the influences of ovarian cancer ascites derived exosomes(ADE)on the epithelial-mesenchymal transition(EMT)and metastasis of ovarian cancer.2.To explore the functions of ovarian cancer derived exosomes(OCDE)on the pre-metastatic niche formation in omentum and metastasis of ovarian cancer.3.To identify the key factors and mechanisms by which OCDE activate the tumor microenvironment and promote metastasis of ovarian cancer.Methods1.The exosomes derived from the culture media of ovarian cancer cells and ascites of ovarian cancer patients were isolated by ultracentrifugation;the size and shape of OCDE were observed under an electron microscope;the expressions of protein markers of exosomes were detected by western blot;the particle diameter of OCDE was measured by DLS and NTA;the expressions of surface protein markers of OCDE were detected by flow cytometry and whether OCDE can be taken in by the effector cells was evaluated by immunofluorescence assay.2.After the ovarian cancer cells were treated with ADE,the abilities of migration,invasion and proliferation were assessed using transwell assay,wound-healing assay,EdU assay and clone formation assay,and the expressions of E-cadherin,N-cadherin and Vimentin,which were well-known markers of EMT,were detected by western blot and immunofluorescence assay;3.Primary ovarian cancer cells were isolated from ovarian cancer tissues and treated with ADE;their capacities of migration,proliferation and EMT were measured;4.The ovarian orthotopic mouse models were established,and ADE were intraperitoneal injected.The growth and metastasis of tumors were monitored by bioluminescent imaging.The expressions of E-cadherin,N-cadherin and Vimentin in xenografts were detected by immunohistochemical staining.5.We collected the omentum tissues from ovarian cancer patients with or without omentum metastasis.The expressions of a-SMA in omentum tissues were assccessed by immunohistochemical staining,and the correlation between a-SMA and metastasis was analyzed;6.Primarily isolated and cultured cancer-associated fibroblast(CAFs)and adipose-derived mesenchymal stem cells(ADSCs)from omentum which had no metastasis were treated with OCDE in vitro,then the expression of a-SMA was examined by western blot and immunofluorescence assays;7.OCDE were intraperitoneal injected in nude mice which had no tumor burden,and the expressions of a-SMA in the omentum of mice were detected by immunohistochemical staining;8.OCDE were intraperitoneal injected in mice with ovarian orthotopic xenografts.The growth and metastasis of tumors were evaluated by bioluminescent imaging and the expressions of a-SMA in the omentum of mice were detected by immunohistochemical staining after the animals were killed.9.We selected the miRNAs which were high-expressed in high metastatic SKOV3-ipl and HEYA8 cells,and low-expressed in low metastatic A2780 cells.;10.The expression of seven miRNAs in ovarian cancer cells and exosomes,cancer cells,CAFs and ADSCs treated by OCDE was assessed by RT-PCR;11.The expression of miR-6780b-5p in ADE was measured by RT-PCR and the correlation between miR-6780b-5p and omentum metastasis was analyzed;12.The ovarian cancer cells were treated with ADE,containing different miR-6780b-5p levels.The expression of EMT markers were detected by western blot,RT-PCR,immunofluorescence assays.The expression of mi-R-6780b-5p in ovarian cancer cells was up-regulated or down-regulated by transfection of agomir or antagomir,and the expression of EMT markers were detected by western blot,RT-PCR and immunofluorescence assays.Results1.OCDE showed as a kind of circular double membrane vesicles under the electron microscope with a particle diameter of 50 to 140 nm;OCDE positively expressed CD63 and CD9;OCDE could be taken in by ovarian cancer cells,CAFs and ADSCs.2.ADE promoted the migration,invasion and proliferation of ovarian cancer cells in a dose-dependent manner.ADE increased the expressions of N-cadherin and Vimentin in ovarian cancer cells,while decreased the expression of E-cadherin.3.The migration and proliferation,as well as EMT,of primary ovarian cancer cells were promoted by ADE.4.In the ovarian orthotopic mouse models,intraperitoneal injection of ADE facilitated EMT of ovarian cancer cells and motivated the growth and metastasis of tumor.5.There were numerious ?-SMA positive cells in most omentum from ovarian cancer patients who had no metastasis.The expression of a-SMA was much higher in omentum with metastasis than that without metastasis.6.OCDE increased the expression of a-SMA in CAFs and ADSCs in vitro.7.OCDE made the omentum of nude mice which had no tumor burden express a-SMA.8.In the mice with ovarian orthotopic xenografts,the tumor growth and metastasis and the a-SMA in omentum were promoted by OCDE injection.9.There were seven miRNAs differently expressed in the exosomes of high metastatic SKOV3-ipl and HEYA8 cells and that of low metastatic A2780 cell.10.The expression of miR-6780b-5p was elevated in ovarian cancer,CAFs and ADSCs after treatment with OCDE,which was chosen to study in subsequent experiments.11.The expressions of miR-6780b-5p in ADE varied and correlated with metastasis of ovarian cancer.12.ADE with different miR-6780b-5p content promoted ovarian cancer cells to EMT in different degree.Up-regulation of miR-6780b-5 promoted EMT of cancer cells,while downregulation of miR-6780b-5p inhibited EMT of cancer cells.ConclusionsOCDE promote the pre-metastatic niche formation in omentum and metastasis of ovarian cancer by delivery of miR-6780b-5p.