Study On Combined Therapy Based On Tumor Immunogenic Death Induced By Chemotherapy In Breast Cancer

Background:As the second leading cause of cancer death in women,breast cancer remains the most common cancer,while the primary causes of death are distant metastasis and recurrence.Up to now,chemotherapy and surgery are still the main treatment of breast cancer,yet the effectiveness of chemotherapy is hindered by multidrug resistance,recurrence,and metastasis.In recent years,immunotherapy has become a new strategy for a variety of tumor treatments.However,breast cancer itself is a cold tumor with low immunogenicity,single drug immunotherapy can’t obtain effective anti-tumor immune response.Tumor immunogenic cell death(ICD)can not only transform cancer into a "therapeutic vaccine" to make dying cancer cells immunogenic,but also trigger immune response by activating dendritic cells(DC)and then activating specific T cell response.Some traditional chemotherapeutic drugs can induce ICD.This chemical immunotherapy makes up for the shortcomings of traditional chemotherapy and immunotherapy.CD47 expression in breast cancer being so high enables tumor cells to escape the attack of innate immune system,which is closely related to the low immunogenicity of tumors.Inhibition of CD47 expression can enhance the anti-tumor immunity of breast cancer.Toll-like receptor(TLR)plays a connecting role in innate and adaptive immunity.TLR7/8 agonist resiquimod(R848)can regulate tumor immune microenvironment by polarizing M2 macrophages into M1 macrophages and activating DC,and further enhance the anti-tumor effect of immune system.In this study,we envisage that chemotherapy-induced ICD can be combined with other immunotherapy methods,such as inhibition of CD47 expression,combination of R848,to regulate the immune microenvironment,enhance tumor immunogenicity,and use the immune system to inhibit tumor growth,recurrence and metastasis.Nanotechnology can effectively deliver the best dose of ICD inducer to specific sites,load multiple drugs simultaneously,protect the payload from degradation and premature release,and realize combined immunotherapy.Objective:It is verified that paclitaxel(PTX)can induce breast tumor ICD.On this basis,a kind of nanoparticles which can efficiently deliver different drug components simultaneously is constructed.The purpose of this study is for the sake of exploring the scheme of chemotherapy-induced ICD combined with other immunotherapy to enhance tumor immunogenicity and inhibit the growth,metastasis and recurrence of breast tumor,so as to provide a research basis for solving the difficult problem of breast cancer treatment.Methods:Flow cytometry revealed CRT expression on the cell membrane after PTX treatment of 4T1 breast cancer cells,and ELISA confirmed HMGB1 secretion in the supernatant to ascertain if PTX can induce immunogenic death in breast cancer.Nanodrugs loaded with PTX,small interference RNA that suppresses CD47 expression(CD47si RNA,si CD47)and immunomodulator R848 were prepared by double emulsification method.The particle size and electric potential of NP-PTX /si CD47/R848 were characterized.Establishment of the mouse model of breast cancer(4T1)in situ,observe the effect of intravenous injection of NP-PTX/si CD47/R848 on the growth of 4T1 tumor in situ,and flow cytometry was used to detect the effect of drugs on tumor immune cells.Results:1.NP-PTX/si CD47/R848 nano-drug with tumor therapeutic potential were successfully prepared by double emulsification method,with particle size of 71.9±3.6 nm and surface potential of 40.4±0.8 m V.2.After 4T1 cells were treated with PTX,the calreticulin on the surface of cell membrane increased.3.The weight and volume of tumor in situ in the breast cancer model mice injected with nano-drugs through tail vein were significantly lower than those in PBS group.4.The ratio of CD4/CD8 in draining lymph nodes and the percentage of dendritic cells in peripheral blood increased significantly in breast cancer model mice injected with nano-drugs through tail vein.Conclusion:To sum up,the chemotherapeutic drug paclitaxel can induce breast cancer to produce ICD.The nano-drug which can deliver PTX,CD47 si RNA and R848 at the same time was prepared by double emulsification.NP-PTX/si CD47/R848 nano-drug can be enriched in the tumor site.The experiment of 4T1 cell tumor-bearing mice shows that the nano-drug can enhance tumor immunogenicity and inhibit breast tumor growth,which provides a new scheme for breast cancer treatment.