Antineoplastic Effect Of Rapamycin Or Metformin Combined With PD-1 Monoclonal Antibody In Mice With Triple Negative Breast Cancer

BackgroundsBreast cancer is the most common malignant tumor in women and it is the main cause of cancer death in women which seriously threatens women's life and health.Triple negative breast cancer(TNBC)is the most malignant type of breast cancer.Due to the lack of effective chemotherapy and targeted drugs,the treatment is relatively simple,and finding new effective treatments is crucial for patients with TNBC.Recently,immunological checkpoint block therapy for PD-L1/PD-1(programmed death-ligand 1/programmed death-1)has achieved great success,and PD-1 or PD-L1 blocking drugs have been approved by the FDA for the treatment of patients with partial malignancies.Current studies on PD-L1 or PD-1 monoclonal antibody therapy in breast cancer patients suggest TNBC patients may benefit from PD-1/PD-L1 blocking therapy.However,due to the individual differences between patients and different mechanisms of tumor immune escape,PD1/PD-L1 blocking therapy is less effective.Thus increasing the efficiency of cancer patients with PD-1/PD-L1 blocking therapy has attracted more and more attention.Recent studies have indicated that part of targeted therapy can not only kill cancer cells directly,but also sensitize patients to immunotherapy.Therefore,the combination of targeted therapy and immunotherapy can achieve the combined effect of the two therapies,and play a more synergistic role.PI3K/AKT/mTOR pathway can regulate the expression of PD-L1 on the surface of tumors.Studies have shown that rapamycin,an inhibitor of mTOR,and metformin targeting AMPK/mTOR can play an anti-cancer role by regulating the immune system.So,we believe that the combination therapy of PD-1 antibody and Rapamycin or metformin is worth exploring.Our study will validate the therapeutic effect in TNBC and explore the synergism mechanism,so as to provide a new way for the treatment of triple-negative breast cancer.Objectives1.To evaluate the effects of mTOR inhibitors rapamycin and metformin on the expression of PD-L1 in breast cancer cells2.To establish a mouse model of TNBC and evaluate the effect of combination of PD-1 antibody and rapamycin or metformin in the treatment of triple negative breast cancer,and further explore its mechanism.Methods1.The expression of PD-L1 in TNBC and non-TNBC tissues was compared by analyzing TCGA database data.The expression of PD-L1 in TNBC and non-TNBC cell lines was compared by analyzing CCLE database data.2.RT-qPCR and Western Blot(WB)were used to detect the expression of PD-L1 in breast cancer cell lines;WB was used to detect the activation of mTOR pathway in breast cancer cell lines;MDA-MB-231 and 4T1 were screened for further experiments;and flow cytometry was used to detect the expression of PD-L1 on cell membrane surface.3.MDA-MB-231 and 4T1 cell lines were treated with rapamycin for different time.The changes of pS6,S6 and PD-L1 protein were detected by WB,and the changes of pS6,S6,pAMPK,AMPK and PD-L1 protein were detected by metformin for 24 hours.4.To construct a mouse model of breast cancer transplantation,which was divided into control group,rapamycin group and metformin group.After three weeks of treatment,the effects of rapamycin or metformin on the proliferation and metastasis of breast cancer in mice were counted,and the anti-tumor effects of rapamycin or metformin were evaluated.5.The transplanted tumor model of mice was established and divided into control group,rapamycin group,PD-1 group and rapamycin combined with PD-1 group.The effects of single and combined drugs on the growth of tumor were evaluated for three weeks.6.Morphological changes of tumor cells were evaluated by HE staining.The expressions of PD-L1,pS6,active casepase-3 and CD3 in mouse tumors were detected by immunofluorescence and immunohistochemistry.7.The transplanted tumor model of mice was established and divided into control group,metformin group,PD-1 group and metformin combined with PD-1 group.The effects of single and combined drugs on tumor growth were evaluated.8.Immunofluorescence and immunohistochemistry were used to detect the expression of PD-L1,pS6,active casepase-3 and CD3 in mouse tumors.Results1.The level of PD-L1 mRNA in TNBC tissue was significantly higher than that in non-TNBC tissue,and the difference was statistically significant.Although there was no statistical significance,the overall expression of PD-L1 in TNBC cell lines was higher than that in non-TNBC cells.2.In vivo and in vitro experiments showed that rapamycin and metformin could reduce the expression of PD-L1 both at the cellular level and in mice,which was related to the inhibition of mTOR pathway.3.In vivo experiments in mice showed that rapamycin and metformin could reduce the weight of tumors and lung metastasis,and the difference was statistically significant.4.In vivo experiments in mice showed that PD-1 monoclonal antibody,rapamycin and rapamycin combined with PD-1 monoclonal antibody could significantly inhibit the growth of tumors and reduce the weight of tumors,and the combined use of PD-1 monoclonal antibody,metformin and metformin combined with PD-1 monoclonal antibody could significantly inhibit the growth of tumors and reduce the weight of tumors.The anti-tumor effect was more significant,the difference was statistically significant;although the tumor weight of the combined drug group was lower than that of the PD-1 monoclonal antibody group,the difference was not statistically significant.5.HE staining of rapamycin combined with PD-1 showed that the tumor necrosis in rapamycin group was large,while the tumor necrosis in PD-1 group was slight,but the density of tumor cells was decreased,and fibrous tissue proliferation appeared.The combined drug group had both of the two manifestations and the necrosis area was larger.Immunofluorescence and immunohistochemical results showed that the combination of drugs increased the expression of CD3 and active casepase-3,indicating that the combination of drugs increased the expression of tumor infiltrating lymphocyte and cell apoptosis.6.The expression of CD3 and active casepase-3 increased in metformin group combined with PD-1,suggesting that the combination of metformin and PD-1 increased the expression of infiltrating lymphocyte and apoptosis.Conclusions1.Compared with using drug alone,rapamycin combined with PD-1 monoclonal antibody significantly inhibited the growth of TNBC mice by reducing the expression level of PD-L1,increasing the number of infiltrating lymphocytes and promoting apoptosis.2.Combination of metformin and PD-1 monoclonal antibody significantly enhanced the inhibitory effect of metformin on TNBC mice by decreasing the expression of PD-L1 and increasing the infiltrating lymphocyte.3.Rapamycin reduces the expression of PD-L1 by inhibiting the activity of mTOR.Metformin decreased the expression of PD-L1,which was consistent with rapamycin,suggesting that it might decrease the expression of PD-L1 by inhibiting mTOR through regulating AMPK/mTOR pathway.