Studies Of Tumor-derived Exosome In Hepatocellular Carcinoma Immunotherapy

Hepatocellular carcinoma(HCC)is a highly malignant and rapidly progressing disease with an extremely poor prognosis.There are about 600,000 new cases per year worldwide,which poses a major challenge for clinical treatment.In recent years,immunotherapy has drawn more and more’attention,in which dendritical cell(DC)mediated immunotherapy has made a great progress in treatment of some tumors such as melanoma,non-small cell lung cancer,urologic neoplasms,glioma and so on.However,for HCC,the progress is not smooth because of the unique immunobiology features of the liver.Immunotherapy,based on HCC cell lysates as antigens,dendritic cells as vaccines,has achieved a favourable effect on ectopic HCC mouse model.However,a further phase II clinical trial showed limited antitumor response rates in HCC patients.Recently,studies on tumor derived exosome(TEX)showed that TEX displays a series of tumor associated antigens and can be used as a natural source of antigens for tumor immunotherapy.And,further studies showed,compared with cell lysates,TEX can elicit much more robust immune response and get better tumor suppressor effect in renal cancer and myeloid leukemia models.But there is a lack of study on HCC.In this study,exosome derived from HCC cell line were used as antigens to activate DCs.The change of tumor volume was observed,the system immune response and the tumor immune microenvironment were detected in mouse ectopic and orthotopic HCC models to estimate the antitumor effect of TEX.In addition,the cytotoxicity to human HCC cells was further studied in vitro.By comparing with HCC cell lysates,we want to provide more choices of antigens for HCC immunotherapy and achieve better tumor suppressor effect.Objective: 1.To evaluate several different approaches commonly used to establish HCC mice to obtain uniform HCC mouse model with high take rates.And to explore the characteristic of immune microenvironment of HCC mice.2.To explore the specific lysis to HCC cells of TEX as antigen in vitro.3.To explore the anti-tumor effect and the change of immune microenvironment in vivo.4.To study the mechanism of TEX in vivo and test its immune function in human settings.Methods: 1.HCC mice were established with four different approaches including intravenous,intrasplenic,intrahepatic inoculation of tumor cells and intrahepatic tissue implantation.Four parameters—the latency period,take rates,pathological features and metastatic rates—were evaluated side-by-side.Meanwhile,immune cells and immune cytokines of mouse peripheral blood were detected with ELISA and flow cytometry(FCS).Immune cells in tumor microenvironment were stained with IHC.2.The morphology,diameter,purity and mark proteins of exosome extracted from cell supernatant were tested with scanning electron microscope,nanosight and western blot(Wb).Exosome uptake by DCs was observed by fluorescence staining and the maturation of DCs by FCS.The activation of T cells was evaluated by carboxyfluorescein diacetate succinimidyl ester(CFSE)staining and cytokines analysis.LDH assay was used to measure the specific cytotoxicity lysis againsit HCC cells of activated T cells.3.Tumor volume was calculated by measuring long and short diameter of the ectopic and orthotopic tumors,and life span of HCC mice was recorded.Serum and immune cells were isolated from peripheral blood and tumor.The activation and suppressive immune cytokines were detected with ELISA and the CD4+T and CD8+T cells were analyzed by FCS.Exosome derived from human HCC cells was also extracted and human DCs and lymph cells were isolated from peripheral blood to estimate its ability to human HCC cells.4.DC was tracked by fluorescence staining and the role of T lymph cells was studied by immunotherapy to immunodeficient mice.Results: Uniform,focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular,dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells,which suggest that intrahepatic tissue implantation was suitable for our study.Notably,T cell infiltration was evident in tumors,which was like the situation in HCC patients.2.Exosome secreted by HCC cell line displayed a series of tumor associated antigen like AFP,GPC3 and antigenic chaperone heat shock protein 70.And,AFP was enriched on exosome.TEX was taken in and induced DC maturation as antigens,which notably enhanced the ability of DCs to activate T cell.The effector T cells showed strong specific lysis to HCC.3.Strong tumor suppression,prolonged survival rate and improved immune microenvironment were observed in TEX as antigens in vivo.And,the immune response in vivo was T cell dependent.Meanwhile,TEX showed superiority to cell lysates in eliciting antitumor immunity.4.The cytoxicity to human HCC cells was confirmed in vitro.Conclusion: 1.Orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically,suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.2.TEX was an efficient antigen carrier of HCC,which can elicit robust antitumor immune response in vitro and in vivo.It may be a potential and important source of antigen for HCC immunotherapy.