Cancer-associated Fibroblasts Induce Growth And Radioresistance Of Breast Cancer Cells Through Paracrine IL-6

IntroductionWith the deepening of the research on the tumor microenvironment,the concept that killing tumor cells alone is not the optimal solution for tumor treatment has received more and more attention.The relevant therapeutic targets of the tumor microenvironment are important to enhance the effect of tumor therapy.Tumorassociated fibroblasts are the most abundant stromal cells in breast cancer,which can regulate various malignant biological behaviors of tumor cells and participate in chemotherapy resistance,tumor progression and recurrence.However,few studies have explored the role of tumor-associated fibroblasts in the radiosensitivity of breast cancer cells.We speculate that tumor-associated fibroblasts play an important role in breast cancer radioresistance.Primary fibroblasts were isolated from fresh tumor tissues of breast cancer patients who underwent mastectomy to study the effect and molecular mechanisms of tumor-associated fibroblasts on breast cancer cell proliferation and radioresistance.ObjectiveThis study aimed to investigate the role and mechanism of cancer-associated fibroblasts in the induction of breast cancer cell proliferation and radioresistance.MethodsWe isolated primary fibroblasts from fresh tumor tissues of breast cancer patients undergoing mastectomy,and detected molecular markers of cancer-associated fibroblasts by Western blot and immunofluorescence to determine the phenotype of the isolated cells.The effects of cancer-associated fibroblasts on the proliferation ability and radioresistance of breast cancer cells were studied through clonogenic experiments and tumor xenograft mouse models in vitro and in vivo.Then,the cytokine antibody chip analysis was performed to screen the cytokines secreted by breast cancer cancer-associated fibroblasts in the regulation of the radioresistance of breast cancer cells,and the chip results were verified by immunohistochemistry of breast cancer specimens.The effects of IL-6 on the proliferation ability and radiosensitivity of breast cancer cells were detected by clonogenic assay.Rescued experiments were performed with IL-6 neutralizing antibodies or STAT inhibitors.Western blot was used to detect the phosphorylation level of STAT3 in each group,and clone formation experiments were performed to verify the roles of IL-6 in cancerassociated fibroblasts promoting breast cancer cell proliferation and radioresistance.Cancer-associated fibroblasts and human breast cancer cell line MD-MB-231 were mixed and inoculated into nude mice subcutaneously to establish a mouse xenograft model.The administration group was then given tocilizumab by intraperitoneal injection.Finally,we detected IL-6 and STAT3 phosphorylation levels in tumor specimens from 103 breast cancer patients who underwent postoperative radiotherapy,and performed statistical analysis through follow-up data.ResultWe successfully isolated primary fibroblasts from fresh tumor tissue from breast cancer patients undergoing mastectomy.The results showed that the conditioned medium of primary tumor-associated fibroblasts can promote breast cancer cell proliferation and radioresistance in vitro and in vivo.Microarray and immunohistochemical results showed that cancer-associated fibroblasts highly secreted IL-6.The results of rescued experiments showed that primary cancerassociated fibroblasts promoted breast cancer cell proliferation and radioresistance through IL-6.Compared with the control group,tocilizumab combined with radiation therapy significantly inhibited tumor growth in mice.The upregulation of IL-6 and STAT3 phosphorylation levels were negatively correlated with disease-free survival in breast cancer patients.ConclusionBreast cancer tumor-associated fibroblasts activate the STAT3 signaling pathway in tumor cells by producing IL-6,thereby inducing breast cancer cell proliferation and resistance to radiotherapy.The IL-6/STAT3 signaling pathway is an important therapeutic target for breast cancer treatment.