Enhancing Exosome-activated Dendritic Cells With PD-1 Antibody In Prostate Cancer Treatment

Prostate cancer(PCa)is one of the common malignant tumors in developed countries with high morbidity and mortality.Once prostate cancer has metastasized,the prognosis of patients will be adverse.Patients with metastasis usually receive significant therapeutic efficacy after undergoing androgen deprivation therapy(ADT),but eventually biochemical recurrence occurs and the disease progresses,progressing to castration-resistant prostate cancer(CRPC).Most prostate cancer-related deaths occur in metastatic castration-resistant prostate cancer.In many treatments for prostate cancer,immunotherapy has gradually become an established treatment,such as recombinant virus-based tumor vaccine PROSTVAC,tumor vaccine Sipuleucel-T and immune checkpoint inhibitors(PD-1,CTLA-4,etc.)It has been proven to be effective in prolonging overall survival(OS)in patients with prostate cancer.However,tumor vaccines that use fusion proteins based on tumor-associated antigens,can effectively improve overall patient survival,have limited effect on clinical progression.At the same time,a single immune checkpoint inhibitor has a limited efficacy in the treatment of prostate cancer.Therefore,it is important to find a novel tumor-specific antigen,and efficiently promote the uptake and presentation of dendritic cells to T cells,thereby producing an effective and specific anti-tumor immune response;on the other hand,prostate cancer tumor vaccine combined with immune checkpoint inhibitors,while recruiting tumor-specific cytotoxicity T cells,can increase the effector function of T cells,thus showing great potential in improving the therapeutic effect of prostate cancer.This study aimed to explore whether tumor cell derived-exosomes(TEX)can be used as a novel antigen to stimulate and activate dendritic cells(Dendritic cell pulsed with TEX,DC-TEX).Its effect on killing tumor cells in vitro and in vivo was observed.At the same time,PD-1/PD-L1 was a classical pathway of exhausting T cell function,so PD-1 antibody was evaluated whether it can improve the efficacy of DC-TEX,it will provide theoretical support for the combined application of two different immunotherapies.Objective: 1.To isolate the exosomes of tumor cells,and to investigate the antitumor immunity of DC-TEX in vitro and in vivo;2.Verify whether PD-1 antibody can enhance the effect of DC-TEX on prostate cancer in vitro and in vivo.Methods: 1.The exosomes derived by tumor cells were isolated by ultracentrifugation,and the exosomes were identified by Western blot,transmission electron microscopy and Nanoparticle Tracking Analysis(NTA).Fluorescence staining was conducted to observe whether it could be taken up by DC,and the activation of DC-TEX and the killing of tumor cells by T cells were determined by flow cytometry,ELISA and cytotoxicity assay;2.Western blot and flow cytometry was used to explore the expression of PD-L1 in tumor cells and the expression of PD-1 on the surface of Activated-T cells.Cytotoxicity assay and ELISA assay was used to explore whether PD-1 antibody can reverse the exhausting condition of activated CD8+PD-1+ T cells by DC-TEX;3.To establish an economical and rapid tumorforming wild-type C57/BL6 mouse prostate cancer tumor model by transplanting tumor tissues,and measure tumor volume?tumor growth rate and overall survival of mice to evaluate the therapeutic effects of DC-TEX treatment and PD-1 antibody combined with DC-TEX treatment,flow cytometry and immunohistochemistry was evaluated the DC-TEX recruitment and activation abilities of T cells,ELISA assay was conducted to analyze the function of activated-T cells.Results: 1.The TEX derived from PCa cells was isolated successfully,and its morphology,size and molecular markers was verified which are rich in various PCa immune related antigens;2.The TEX can promote the activation of DC 2.4 cells.Activated DC-TEX can promote the transformation of naive T cells into cytotoxic T cells;3.PD-1 antibody can reverse the exhausted state of DC-TEX-activated CD8+T cells due to expression of PD-1 receptor was demonstrated by flow cytometry?ELISA and cytotoxicity assay;4.Subcutaneous prostate tumor model of C57/BL6 mice was established successfully.We found that DC-TEX combined with PD-1 antibody could prolong the overall survival of the mice,and the tumor growth rate of the mice was significantly slowed down.The immune system was found to be activated by using flow cytometry?ELISA assay and IHC.Conclusion: 1)Exosomes-activated dendritic cells have stronger anti-tumor immunity compared with tumor lysates;2)PD-1 antibodies can enhance the effect of DC-TEX on killing tumors,PD-1 combined with DC-TEX can play a synergistic antitumor effect and has great potential for improving the clinical prognosis of patients with prostate cancer.