Preparation Of Ll/2 Lung Cancer Vaccine With Semi-bacterial Bacillus Pseudomallei Vector And Its Anti-tumor Effect

Objective:Lung cancer is the leading cause of malignant tumor death in the world.The immune system can recognize tumor cells,and tumor vaccine can enhance the body’s anti-tumor immunity,especially by activating anti-tumor immune-specific CD8~+T lymphocytes to eliminate tumor cells.Although great progress has been made in the research and development of tumor vaccine in the past decade,the effect of clinical practice is still not ideal,so it is necessary to find a better preparation method.Bacteria are born with the pathogen model molecule(PAMP),while immune cells have the pattern recognition receptor(PRR)of PAMP,so the immune system can produce an effective immune response to bacteria.In this study,using this characteristic of bacteria,Burkholderia pseudomallei cell wall was porous,and some of the contents were overflowed into a kind of drug carrier with gap hollow(called semi-bacteria).The lysate of mouse Lewis lung cancer cells(LL/2)was used as antigen to explore the feasibility of this"semi-bacteria"as vaccine carrier in mouse tumor model,and to find a new method to prepare tumor vaccine.Methods:In this study,Burkholderia pseudomallei cell wall was porous by means of radiation detoxification,proper heating and enzymatic hydrolysis,and a carrier(called"semi-bacterial",SB)which could permeate the cell lysate was prepared to determine its characteristics,activity and characterization.Then the lung cancer cell LL/2 tumor lysate(LTL)and vaccine adjuvant CpG,were observed by confocal microscope,which proved that the LL/2 tumor lysate and CpG could be effectively loaded into semi-bacteria,indicating that the LL/2 tumor lysate semi-bacterial vaccine(named SB-LC)was successfully prepared;The ability of SB-LC to mature and activate dendritic cells was evaluated in vitro.Dendritic cells were cultured with(DC).Flow cytometry was used to determine the expression of(DC)maturation-related(CD40,CD80 and CD86)and the secretion of cytokines(INF-γ,TNF-α).Whether it can effectively stimulate the maturation of dendritic cells.The LL/2 tumor model was constructed by homologous C57BL/6 mice,and tumor cells(1×10~6)were injected into the ribs of the right side as the prevention group and treatment group model.The growth of LL/2 tumor in mice was observed every 3 days to observe whether SB-LC vaccine could inhibit tumor growth.Spleen effector cells were prepared for cytotoxic T lymphoid reaction(CTL)to prove the specific anti-tumor immune effect,and the percentage of CD8~+cells expressing INF-γwas measured by flow cytometry.Similarly,in order to verify the effect of humoral immunity,Western Blot was used to detect serum-related antibodies,and enzyme-linked immunosorbent assay(ELISA)was used to determine the contents of cytokines in the supernatant of dendritic cells culture and serum specific antibodies.The content of specific antibody lymphocytes secreted by spleen was determined by enzyme-linked immunosorbent assay(ELISPOT).In addition,tumor infiltrating lymphocytes in mouse tumors were sorted,the proportion of CD8~+T cells expressing INF-γin different groups was analyzed by flow cytometry,and the number of(Tregs)expressing CD25~+and FOXP3~+double positive regulatory T cells was determined by CD4~+gating.To investigate whether LL/2 cell lysate semi-bacterial vaccine(SB-LC)can change the microenvironment of tumor immunosuppression.Results:In this study,we successfully prepared tumor cell lysate semi-bacterial vaccine(SB-LC).In vitro experiments showed that it could induce the maturation and activation of DC cells,induce the expression of maturation markers CD40,CD80 and CD86,and release cytokines.The mouse model of prevention group and treatment group proved that it could effectively inhibit tumor growth,and the effect of CTL reaction in SB-LC group was the most significant.Specific IgG antibody could be detected in the serum of tumor-bearing mice after treatment.The tumor infiltrating lymphocytes of tumor-bearing mice were isolated,and the(Treg)of regulatory T cells treated with SB-LC vaccine decreased and reversed the immune microenvironment of immunosuppressive tumor.Conclusions:LL/2 cell lysate hemibacterial vaccine(SB-LC)can effectively induce dendritic cell maturation in vitro.It can effectively inhibit tumor growth and kill tumor cells in preventive and therapeutic experimental groups.