The Role And Mechnism Of Regulation Of Cisplatin On Macrophages In The Process Of Ovarian Cancer Metastasis

Ovarian cancer is one of the most lethal malignancy of the female reproductive tract,and seriously threatens women's health and life.Despite the high response rate after surgery and platinum-combination chemotherapy,treatment of ovarian cancer remains challenging due to drug resistance,tumor recurrence and metastasis.The latest research shows that in addition to the direct effect of chemotherapeutic drugs on tumor cells during chemotherapy,they can also regulate macrophages in the tumor microenvironment,thereby affecting the malignant biological activity of tumor cells and in turn affects the efficacy of chemotherapy drugs.The relevant mechanisms have not been fully elucidated.This project is divided into three parts:(1)Study the effect of cisplatin-treated macrophage on the metastatic capacity of ovarian cancer cells;(2)Explore the related mechanism of effect of cisplatin-treated macrophage on the metastatic capacity of ovarian cancer cells;(3)Investigate the role of CCL20-CCR6axis in ovarian cancer metastasis.Objective(1)Investigate the effect of cisplatin-treated macrophage on ovarian cancer cell metastasis ability;(2)Screen out candidate genes involved in cisplatin-treated macrophage to affect ovarian cancer metastasis and explore its related mechanisms;(3)Explore the effect of CCL20-CCR6 axis on ovarian cancer cell metastasis ability,and the clinical relationship between CCR6 expression and tumor metastasis and prognosis of ovarian cancer patients.Methods(1)THP1 and human peripheral blood mononuclear cells(PBMC)were differentiated into naive inactivated macrophage(M?)by treatment with PMA and M-CSF,respectively.The induction effect was detected by flow cytometry.CAM were generated by treating M?with LPS/IFN-?and verified by Real-time PCR.Transwell assay was used to detect the effect of conditioned medium(CM)of chemotherapy drugs-treated(cisplatin,carboplatin,cyclophosphamide and paclitaxel)macrophage on the invasion and migration of ovarian cancer cells(A2780 and SKOV3),and Western blot was adopted to detect the expression effect of cisplatin-treated M?(Cis-M?-CM)and CAM(Cis-CAM-CM)on epithelial-mesenchymal transition(EMT)markers in A2780 cells.The expression changes of polarization markers in CAM treated with cisplatin was detected by Real-time PCR,and the proliferation and apoptosis of ovarian cancer cells treated with CM of macrophage were detected respectively by CCK8 method and flow cytometry.After cultured in CM of macrophage,the sensitivity of ovarian cancer cells to cisplatin was detected by CCK8method.(2)The concentration of cytokines in CM of macrophage treated with or without cisplatin was measured by using luminex liquid suspension chip detection.Real-time PCR and ELISA were adopted to verify the expression of differential genes and candidate proteins were found out.CCK8 method and transwell assay were used to examine the proliferation and migration of ovarian cancer cells treated by human recombinant candidate proteins.Neutralizing antibody of candidate protein was added to CM of macrophage,and its effect on the invasion and migration of ovarian cancer cells was detected by transwell method.We further constructed CCR6 specific si RNA and tranfected it into A2780 and SKOV3 cells,real-time PCR and Western blot were used to verify the knockdown effect,and the proliferation,migration and EMT markers of CCR6-KD ovarian cancer cells were detected by CCK8 method,transwell assay and Western blot,respectively.(3)The expression of CCR6 in human immortalized ovarian epithelial cells IOSE and ovarian cancer cell lines(SKOV3,A2780,HO 8910,OVCAR3,OVISE,ES2,HEY)was detected by real-time PCR and Western blot.CCK8 method and transwell assay was adopted to detect the effects of rh CCL20 on the proliferation and migration of ovarian cancer cell lines.We further constructed CCR6-KD A2780 and SKOV3cells by si RNA and CCR6-KO A2780 cells by Crispr-cas9 technology,and examined their migration and rh CCL20-mediated migration by transwell assay.The activation of MAPK and Akt signal pathway in ovarian cancer cells after rh CCL20 stimulation was detected by Western blot.An intra-abdominal xenograft tumor model was set up to assess the role of CCL20-CCR6 axis in ovarian cancer metastasis in vivo.The correlation between CCR6 expression and prognosis of ovarian cancer patients was assessed by KM plotter,and the correlation between CCR6 expression and metastasis-related gene set expression in ovarian cancer tissues was evaluated by gene set enrichment analysisResults(1)After induction of PMA and M-CSF and subsequent LPS/IFN-?,THP1 and PBMC-derived naive inactivated macrophage(M?)and classical activated macrophage(CAM)were successfully induced.Compared to untreated macrophage,the CM of cisplatin-treated CAM(but not M?)significantly promoted the migration ability of A2780 and SKOV3 cells and the expression of EMT-related proteins,but had no significant effect on the invasion ability of A2780 and SKOV3 cells.Similarly,the CM of CAM treated with carboplatin and cyclophosphamide also significantly promoted the migration ability of A2780 and SKOV3.Cisplatin had no significant effect on the expression of polarization markers in CAM,and there were no significant changes in the proliferation,apoptosis,and sensitivity to cisplatin in ovarian cancer cells cultured with CAM-CM and Cis-CAM-CM.(2)After Luminex liquid suspension chip screening and real-time PCR and ELISA verification,IL-1?and CCL20 were confirmed to be significantly up-regulated in CAM treated with cisplatin.Between the two cytokines,only CCL20 was proved to promote the migration of ovarian cancer cells,without affecting their proliferation.In addition,cisplatin promoted the expression of CCL20 in CAM in a time-and concentration-dependent manner,and compared to M?,A2780 and SKOV3 cells hardly expressed CCL20.Pharmacological blockage of CCL20 on cisplatin-stimulated CAM and si RNA-mediated inactivation of CCR6 on cancer cells effectively abrogated ovarian cancer cell migration induced by cisplatin-stimulated CAM,among which the latter also inhibited the expression of mesenchymal marker promoted by cisplatin-stimulated CAM.(3)The expression of CCR6 in ovarian cancer cell lines(SKOV3,A2780,HO8910,OVCAR3,OVISE,ES2,HEY)was significantly increased compared to normal ovarian epithelial cell IOSE.Rh CCL20 obviously promoted the migration of CCR6^highovarian cancer cells A2780 and SKOV3,but not CCR6^lowovarian cancer cells ES2 and HEY,without affecting their proliferation.Knockdown or knockout of CCR6 had no effect on the migration of A2780 and SKOV3 cells,but significantly inhibited rh CCL20-mediated cancer cell migration.In addition,rh CCL20significantly promoted phosphorylated Akt level in A2780 and SKOV3 cells,but had no significant effect on phosphorylated Erk1/2 level.In vivo nude animal model showed that rh CCL20 significantly promoted the number and weight of metastatic tumors in nude mice inoculated with WT-A2780 cells,but not in that inoculated with CCR6 KO-A2780 cells.The patients with higher CCR6 expression in ovarian cancer tissues had a shorter overall survival and their gene expression profiles were highly enriched in the metastasis-related gene sets"Positive regulation of cell adhesion"and"Positive regulation of epithelial cell migration".Conclusions(1)Activated macrophage CAM(but not inactivated macrophage M?)can significantly promote epithelial-mesenchymal transition and migration of ovarian cancer cells after cisplatin treatment.(2)Cisplatin does not affect CAM polarity,anti-cancer phenotype,and CAM-mediated resistance of ovarian cancer cells,but it can significantly promote the expression and secretion of CCL20 in CAM.Pharmacological blockage of CCL20 on cisplatin-stimulated CAM and si RNA-mediated inactivation of CCR6 on cancer cells effectively abrogated ovarian cancer cell migration induced by cisplatin-stimulated CAM.(3)CCR6 expression is significantly increased in epithelial ovarian cancer cells.CCL20-CCR6 axis directly mediates ovarian cancer cell migration and metastasis in vitro and in vivo.Increased CCR6 expression indicates an increased likelihood of ovarian cancer metastasis and a poor prognosis.