Study On The Effect And Mechanism Of Tumor-Associated Macrophages Induce Resistance To Chemotherapy In Triple Negative Breast Cancer Cells

Objective:To construct a model of Tumor associated macrophages(TAM)induced in vitro,and identify the cytophenotypic characteristics.Methods:In the first step,THP-1 cells were stimulated with Phorbol-12-myristate-13-acetate(PMA)for 48 h to induce the differentiation of THP-1 cells into M0 macrophages.IL-4)and IL-13 stimulated macrophages for 48 h to induce differentiation into TAM(M2 type).The expression levels of macrophage surface molecules CD14 and CD206 were analyzed by flow cytometry.The m RNA expression of cytokine IL-10 and chemokine CCL2 in macrophages of each group was analyzed by Real-time PCR.Results:Compared with M0 macrophages induced by PMA alone,the expressions of CD14 and CD206 on the surface of M2-type macrophages induced by IL-4 and IL-13 were significantly increased((P< 0.001)).Real-time PCR showed that the expression levels of IL-10 m RNA and CCL2 m RNA in M2-type macrophages were significantly increased(P<0.001).Conclusion:We successfully induced normal macrophages into TAM which was M2-type macrophages characterized by higher expression of CD14,CD206,IL-10 and CCL2.Objective:To investigate the role of tumor-associated macrophages(TAM)in resistance to albumin-binding paclitaxel(Nab-PTX)in triple negative breast cancer cells,and to analyze its potential molecular mechanism.Methods:First,we measured effective killing concentration of Nab-PTX on triple negative breast cancer cell line MDA-MB-231 by cell counting kit-8(CCK-8)method.The co-culture mode of TAM with breast cancer cells in vitro was established by Transwell cell culture method,The sensitivity of MDA-MB-231 cells to albumin-binding paclitaxel under different culture conditions was analyzed and compared by CCK-8 method.The apoptosis of MDA-MB-231 cells was detected by flow cytometry under different culture conditions.The expression of MDR1 and Caspase-3 gene in MDA-MB-231 cells was detected under different culture conditions was detected by RT-PCR.The activation of IGF-1R,p-IGF-1R,AKT,p-AKT,ERK and p-ERK proteins in MDA-MB-231 cells were detected under different culture conditions by western blot.IGF-1R pathway was then inhibited by Linsitinib,To detect the effect of inhibiting IGF-1R signaling pathway on the expression of IGF-1R,p-IGF-1R,AKT,p AKT,ERK,and p ERK proteins in tumor cells.cck8 was used to detect changes in tumor cell viability induced by albumin-paclitaxel,The apoptosis of tumor cells was analyzed by flow cytometry,The changes of MDR1 and Caspase3 gene expression in tumor cells were detected by Real-time PCR.Results:Compared with MDA-MB-231 cells cultured alone,MDA-MB-231 cells in TAM co-culture group showed decreased sensitivity to albumin paclitaxel,which indicated that TAM increased the survival rate of MDA-MB-231 cells under the condition of albumin-binding paclitaxel(p < 0.001).Compared with MDA-MB-231 cells cultured alone,the apoptosis of MDA-MB-231 cells treated with albumin paclitaxel in TAM coculture group was significantly reduced(p < 0.01),These results indicated that TAMs could increase the anti-apoptotic ability of MDA-MB-231 cells.Real-time PCR showed that MDR1 m RNA expression was significantly increased(p < 0.001)and caspase-3 m RNA expression was significantly decreased(p < 0.05)in MDA-MB-231 cells after TAM co-culture.Western blot results showed that P-IGF-1R,P-Akt and P-Er K were significantly activated(p < 0.001).Further study found that the addition of IGF-1R inhibitors decreased the expression of IGF-1R and its downstream signaling pathway key proteins in TAM activated MDA-MB-231cells(p < 0.001).The survival rate and the expression of MDR1 in TAM co-cultured tumor cells were decreased(p < 0.001),the apoptosis rate and the expression of caspase 3 were increased(p < 0.01),the sensitivity of cells to albuminpaclitaxel was improved,and the TAM-mediated drug resistance was partially reversed.Conclusion:TAM can induce albumin paclitaxel resistance in triple negative breast cancer cells,and the mechanism is related to the activation of IGF-1R signaling pathway in MDA-MB-231 cells by TAM.IGF-1R plays a role in promoting tumor resistance by regulating the downstream PI3K/Akt and MAPK/ERK pathways.TAM and IGF-1R may be potential new therapeutic targets for patients with white-violet resistant triple negative breast cancer.