Calcipotriol-loaded Microparticles Derived From AFP-overexpressed Dendritic Cells For Liver Cancer Therapy

Hepatic carcinoma is one of the malignant tumors with the highest morbidity and mortality.Effective treatment of hepatic carcinoma is still a worldwide problem.Recently,immunotherapy has been widely used in the treatment of malignant tumors and achieved obvious therapeutic effects.Among them,dendritic cell（DC）-based immunotherapy has been widely used in tumor treatment.DC-based tumor vaccines inhibit tumor growth by activating specific anti-tumor immune responses.However,high cost of cultivation and storage of DCs in vitro limit further applications.Dendritic cell-derived microparticles（DC-MPs）are vesicles secreted by DCs with sizes of 100-1000 nm.DC-MPs inherit most membrane proteins,nucleic acids,lipids of DC.Therefore,DC-MPs are potential to present antigens and activate T lymphocytes.Meanwhile,the stability,simplicity of preparation and preservation of DC-MPs contribute to the applications in tumor therapy as vaccines.Infiltration of T cells and tumor immusuppressive microenvironment in tumor tissues are the critical factors determining the therapeutic effects of tumor vaccines.However,dense matrix microenvironment severely restricts tumor infiltration of T lymphocytes.As one of the dominant tumor stromal cells,cancer associated fibroblasts（CAFs）play a critical role in tumor development,metastasis and recurrence.Recently,many researches indicated that conversion of CAFs from an activation state to a quiescent state can significantly inhibit the function of CAFs,improve tumor matrix microenvironment,and finally inhibit tumor recurrence and metastasis.In this study,we constructed hepatic carcinoma associated antigenα-fetoprotein（AFP）-overexpressed DC2.4 cell-derived microparticles(DC^AFP+MPs)to load calcipotriol（Cal）,an agent for deactivating CAF(Cal@DC^AFP+MPs).Compared with DC-MPs,the expression of CD80,CD86 and MHC II in DC^AFP+MPs were significantly improved.Furthermore,T cells were effectively activated by DC^AFP+MPs and killed tumor cells.Cal@DC^AFP+MPs significantly deactivated CAFs,resulting in the reduced secretion of matrix proteins such as fibronectin.Meanwhile,improved infiltration of T cells into tumor tissues was observed and obvious tumor inhibition effects were also indicated.The main contents and results were as follows:（1）Preparation and characterization of Cal@DC^AFP+MPsDC2.4 cell line overexpressing AFP(DC2.4^AFP+)was constructed by lentivirus vector.Flow cytometry and western blot data showed that the expression of CD80,CD86,MHC II,and AFP protein in DC2.4^AFP+was significantly increased.After ultraviolet irradiation,DC2.4^AFP+-derived MPs(DC^AFP+MPs)were collected by ultracentrifugation and then Cal was loaded into DC^AFP+MPs(Cal@DC^AFP+MPs).Dynamic light scattering（DLS）and transmission electron microscopy（TEM）analysis indicated that Cal@DC^AFP+MPs were circular structure with uniform size of 350 nm and zeta potential of-11 m V.Compared to DC-MPs,the expression of costimulatory factors CD86,CD86,MHC II and tumor-associated antigen AFP in DC^AFP+MPs were significantly improved.These data indicate that DC^AFP+MPs have the potential of presenting antigens and stimulating T cells.（2）Immune activation effects of Cal@DC^AFP+MPs in vitroPrimary T cells were isolated from mice and treated with different MPs in vitro.After stimulation with DC^AFP+MPs and Cal@DC^AFP+MPs,the expression of interleukin 2（IL-2）,interferonγ（IFN-γ）,and granzyme B（Gr-B）in CD8⁺T cells was significantly upregulated.Meanwhle,the cytotoxicity of activated T cells by DC^AFP+MPs and cal@DC^AFP+MPs against mouse hepatocellular carcinoma Hepa1-6 cells was significantly enhanced by LDH assay.These data indicate that DC^AFP+MPs can activate T cells and enhance the cytotoxicity of activated T cells against tumor cells.（3）Deactivating effects of Cal@DC^AFP+MPs on CAFsPrimary fibroblasts were isolated from the skin of neonatal mice and then activated with TGF-βinto CAFs.Immunofluorescence analysis showed that after treatment with Cal@DC^AFP+MPs,the expression of fibroblast activation protein（FAP）,α-smooth muscle actin（α-SMA）and fibronectin,three markers of CAFs,were significantly reduced.In addition,the recruitment of CD8⁺T cells by CAFs treated with Cal@DC^AFP+MPs was significantly upregulated by transwell assay.These data indicate that Cal@DC^AFP+MPs deactivate CAFs and increase the recruitment of CD8⁺T cells by CAFs.（4）Tumor inhibition ability of Cal@DC^AFP+MPs in vivoHepa1-6 cells and fibroblasts were subcutaneously co-injected into C57/BL6 mice to construct hepatic carcinoma models.Cal@DC^AFP+MPs induced effective tumor inhibition after intravenous injection.Flow cytometric analysis revealed that Cal@DC^AFP+MPs could promote the maturation of DCs,and induced the activation of CD8⁺T cells in both tumors and lymph nodes.Meanwhile,the upregulated proportion of immune memory T cells and downregulated proportion of immune suppressive cells such as myeloid-derived suppressor cells（MDSCs）and regulatory T cells（Tregs）were also observed after Cal@DC^AFP+MPs treatment.Furthermore,Cal@DC^AFP+MPs significantly decreased the numbers of the activated CAFs after Cal@DC^AFP+MPs treatment.In summary,Cal@DC^AFP+MPs could significanlty improve the tumor immune microenvironment by inducing CD8⁺T cells activation and decreasing immune suppressive cells,exhibiting remarkable tumor inhibition ability.