Fusion Of Dendritic Cells And Cancer-associated Fibroblasts For Activiation Of Anti-tumor Cytotoxic T Lymphocytes

Background:Surgery,radiotherapy,chemotherapy and immunotherapy are the four major approaches to treat malignant tumor.Among them,immunotherapy is becoming a new anti-tumor method by breaking the tumor immune tolerance and escape.Dendritic cells(DCs)-based tumor immunotherapy is one of the research focuses in recent years.Cancer associated fibroblasts(CAFs)is the central component in the tumor microenvironment,promoting the tumor genesis,development and transfer.Immunotherapy targeting CAFs has good application prospect.In this research,we fused DC with CAF basing on high efficiency of dendritic cells/tumor cell hybridization technology.The fused DC/CAF cells can not only express all CAF's antigen,but also retain the characteristics of dendritic cells,which can specifically present the complete antigen of CAF to T cells,thereby enhancing specific T cell-mediated immune response.The antitumor effect and its possible mechanism will be further verified by experiments in vitro and in vivo.In this study,a new fusion cell vaccine will be established,which provides a new strategy for cancer immunotherapy.Objective:In this study,we will explore a novel DC/CAF fusion cell vaccine based on DC/tumor fusion cells to amplify a large number of T lymphocytes that are specific to known and unknown antigens in the tumor microenvironment.This new vaccine can specifically target the tumor microenvironment in order to inhibit the growth of tumor cells,and explore an effective new strategy for tumor immunotherapy.Methods:We isolate and identify CAFs from H22 hepatocellular carcinoma and normal tissue fibroblasts(NFs)from mice tail,furtherly explore the differences of biological characters.Using polyethylene glycol 2000(PEG2000)to induced the fusion of hybrid cells vaccine(DC/(CAF).The fusion cells are observed with fluorescence microscope,and the expression of cell surface molecules are detected by flow cytometry;We use T cell proliferation and cytokine detection to analyze the function of T cells and the secretion of IFN-? and IFN-? after the stimulation of DC/CAF fusion cells.In vitro,fusion cells(DC/CAF)induced specific T lyphmcytes to kill CAFs.H22 hepatocellular mouse model is established to verify the anti-tumor effect of DC/CAF fusion cell induced T cells in vivo,and observe the tumor growth and survival time of mice.Results:1.Highly pure CAFs and NFs were successfully isolated and purified from mouse H22 hepatoma subcutaneously transplanted tumor tissue and mice tail tissue.Morphologically,CAF is spindle shaped or polygonal.CAFs highly expressed fibroblast activation protein(FAP)and ?-smooth muscle protein(?-SMA)with WB,flow cytometry and immunofluorescence methods.2.DC/CAF fusion cells were successfully synthesized under the induction of PEG 2000.With fluorescence confocal microscopy,the PKH-26-stained DCs showed red colour,the CFSE-stained CAF cells were green,and the DAPI-stained nucleus present blue.The fused cells appeared yellow.3.The fused cells can express higher CD80,CD86,and MHC-II than immature DC cells by flow cytometry,indicating that it has strong antigen presentation and immune response function;4.It showed that compared with DC,CAF,and DC mixed CAF groups,the DC/CAF fusion cell group secreted higher concentrations of cytokines(TNF-?,IL-1?,IL-6 and IL-12)by ELISA(P<0.0001),which was close to the positive control group of lipopolysaccharide(LPS).5.The proliferation index(PI)was obtained by flow cytometry.The PI of the DC/CAF fusion cell group reached 4.54,and the cell differentiation was concentrated in the fifth generation.The PI of the DC mixed CAF group was 3.77,and the cell differentiation was mostly concentrated in the third generation.However,in the other control groups,the PI were only about 1.5,and the cell differentiation was concentrated on the 1st and 2nd generation.The DC/CAF fusion cell group had stronger ability to stimulate T cell proliferation than the other groups(P<0.0001).6.Compared with T cell,DC + T cell,CAF + T cell and DC mixed CAF + T cell group,DC/CAF Fusion + T cell group secreted more cytokines IFN-? and IFN-?(P<0.0001).7.According to different target ratios(T:CAF=5:1,10:1,20:1),the killing rate of DC/CAF Fusion+T cell group were higer than the other groups in each target ratio.Compared with other groups(T cell,DC+T cell,CAF+T cell,DC mixed CAF+T cell),the killing rate of DC/CAF Fusion+T cell was significantly improved when the target ratio was 20:1.The killing efficiency of target cells reached to 60%;8.It showed that compared with the other groups(PBS,T cell,DC+T cell,CAF+T cell,DC mixed CAF+T cell),the tumor volume of mice in the DC/CAF Fusion+T cell group was smaller than the other groups.The difference was statistically significant(P<0.0001).PBS-treated groups were used as negative controls.However,weighed tumors after being sacrificed and measured tumor size were also smaller than other control groups;Compared with other groups,the survival time of DC/CAF Fusion+T cell group was longer(P<0.05).9.Compared with other groups(PBS,T cell,DC+T cell,CAF+T cell,and DC mixed CAF+T cell),the expression of Ki67 in tumor cells of tumor-bearing mice was significantly decreased after treated with DC/CAF fusion cell induced T cell.In DC/CAF Fusion+T cell group(P<0.001).Conclusion:In this study,we isolated and identified tumor-associated fibroblasts.Their ability to expressing FAP and ?-SMA proteins was verified.The DC/CAF fusion cell vaccine was successfully prepared under the induction of PEG,which confirmed that the fusion vaccine has the ability to express CD80,CD86 and MHC-II molecules of mature DC cells.The fusion cells can increase the secretion of TNF-?,IL-1?,IL-6,and IL-12 p.The DC/CAF fusion significantly stimulate the proliferation of T cells,enhancing the ability of T cells to secrete cytokines IFN-? and IFN-?.CTLs induced by DC/CAF fusion vaccine can specifically kill CAF cells in vitro.Tumor-bearing mice was treated by CTLs induced by DC/CAF fusion cells that can effectively kill CAFs,furtherly inhibited tumor cell proliferation and promoting tumor cell apoptosis.So that the growth of mice tumor was inhibited,and the survival time was prolonged.This study provides a new strategy for tumor treatment from the perspective of CAFs in the tumor microenvironment.