Study Of Antigen-modified Dendritic Cell-derived Exosomes In Hepatocellular Carcinoma Immunotherapy

Hepatocellular carcinoma（HCC） is the third most common maligancy with the incidence rate second to stomach cancer and esophagus cancer worldwide,and poses great risk on human health.However there is no effective treatment available in the clinic.Although dendritic cell（DC）-based immunotherapy has shown promise,the effect is very limited.Particularly,DCs as cell vaccines,have a short shelf-life with a high demand on the availability of patients’DCs.Recently,exosomes,microvesicles secreted by most mammalian cells,have emerged as a new form of cell-free vaccines.In particular,DC-derived exosomes（DEXs）bear large amounts of functional molecules from their parental cells,such as major histocompatibility complex I/II（MHC-I/MHC-II）and CD80,CD86 and intercellular adhesion molecule（ICAM）.Thus DEX can serve as surrogates for DCs to activate na?ve T cells and elicit cytotoxic T cells（CTLs）and suppress tumor growth.Importantly,DEX can be stored at-80°C for half year with a much longer shelf-life than DCs.It was demonstrated that antigenic peptide-pulsed DCs-derived exosomes can elicit strong anti-tumor effect in melanoma and other tumor models;however its potential in HCC remains to be investigated.Therefore,in our current study,we have investigated the feasibility and potency of HCC antigen（alpha-fetoprotein,AFP）-modified DEX(DEX_AFP)in eliciting antigen-specific immunity in different HCC models systemically.Furthermore,we have explored the possible mechanisms underpinning DEX_AFP-mediated anti-tumore immune response.DCs（DC2.4）were infected with AFP-expressing lentiviruses to obtain AFP-expressing cell lines(DC_AFP).Western blot and Flow cytometry results confirmed the expression of AFP in DCs and higher levels of MHC-I,MHC-II,CD80,CD86 and ICAM molecules in DC_AFP compared to DC.The antigen-specific anti-tumor effect of DC_AFP was demonstrated in subcutaneous HCC mice.Examination of AFP and levels of MHC-I,MHC-II,CD80,CD86 and ICAM molecules on DEX_AFP revealed that the expression of AFP and higher levels of co-stimulatory molecules on DEX_AFP compared to DEX.The antigen-specific effect of DEX_AFP was verified with mixed lymphocyte reaction（MLR）with T cells derived from DEX_AFP-treated mice followed by stimulation with DEX_AFP.To optimize the anti-tumor effect of DEX_AFP,we tested different delivery routes（intravenous vs subcutaneous injection）,dosing regimens and doses in subcutaneous HCC mice.The results showed that intravenous injection is slightly better than subcutaneous injection as demonstrated by suppressed tumor growth and prolonged survival rate.Also multiple repeated injections were better than one single injection with a dosing regimen of 40μg/mouse/week for 3 weeks showing the optimal effect.Although subcutaneous HCC models are useful models for optimization studies,they fail to reflect the immune-suppressive tumor microenvironment observed in HCC patients.Orthotopic HCC mice can closely mimick HCC patients;therefore we have investigated the immunogenicity of DEX_AFP in orthotopic transplantation HCC mice.Two different dosing regimens（150μg/mouse/week for 3 weeks vs 150μg/mouse every 5 days for 4 injections）were tested in day-7-established orthtopic HCC mice.The results showed that the former was slightly better than the latter,though no significant difference was observed.Therefore,we selected the dosing regimen of150μg/mouse/week for 3 weeks for our following studies.To explore the correlation between the anti-tumor effect of DEX_AFP and the improvement in immune microenvironment,we monitored the tumor growth and immune microenvironment in a real-time manner in DEX_AFP-treated orthotopic HCC mice.A dynamic cumulative anti-tumor effect and improved immune microenvironment was established,suggesting that DEX_AFP can elicit sustainable anti-tumor effect and result in improved immune microenvironment.To further investigate the potency of DEX_AFP in a more clinicallyrelevantsetting,we established a diethylnitrosamine（DENA）-induced autochthonous HCC mouse model,a model can closely manifest the heterogeneity and complexicity and immune-suppressive microenvironment observed in HCC patients.Visible nodules and immunosuppressive milieu were detected 7 month after one intraperitoneal injection of 50mg/kg DENA.An identical dosing regimen（150μg/mouse/week for 3weeks）was applied in 8-month old DENA-induced autochthonous HCC mice.Ten weeks after the last injection,body-wide tissues were harvested.Examination of tumor-bearing mice showed that significantly fewer tumor nodules,decreased liver weight and tumor volume were observed in DEX_AFP-treated groups compared to other treatment groups.Measurement of immune and tumor microenvironment of DEX_AFP-treated mice revealed that significantly increased number of effector CD8 T cells and eleveated levels of IFN-γand IL-2 were detected compared to other treatment groups.These data support the conclusion that DEX_AFP can elicit competent anti-tumor immune response in autochthonous HCC mice.To further ecluicate the mechanisms underpinning DEX_AFP’s functionality,we administered DEX_AFP into athymic immune-deficient nude mice and the results showed no difference between DEX_AFP-and DEX-treated nude mice,suggesting that T cells play an important role in the functionality of DEX_AFP.Furthermore,examination of natural killer cells（NKs）in DEX_AFP-treated autochthonous HCC mice indicated that NKs might also contribute to the anti-tumor effect of DEX_AFP.Conclusions:Our findings provide evidence that AFP-enriched DEXs can trigger potent antitumor immune responses and reshape the tumor microenvironment in orthotopic HCC mice and thus provide a cell-free vaccine option for HCC immunotherapy.