Study Of Alarmin Modified Exosomes On The Immunotherapy Of Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma?HCC?is a highly malignant tumor with high mortality rate and poor prognosis.In recent years,tumor derived exosomes have ben extensively used as tumor antigen carriers for immunotherapy and shows great promise.In our previous study,weproved that hepatocellular carcionoma cell derived exosomes?TEXs?can be used as effective tumor antigen carriers to promote activation and matureation of dendritic cells?DCs?and thus result in strong anti-tumor effects in HCC tumor-bearing mice.However,to overcome the complicated immune and tumor microenvironment in HCC patients,novel strategies to strengthen the immunogenicity of DCs are urgently needed.It was shown that alarmin,especially high mobility group nucleosome-binding protein 1?HMGN1?,can recruit and activate DCs and thus has been used as novel immune adjuvants.However,its potential in HCC remains to be determined.Therefore,in the present study,we plan to use genetic approaches and our recently established exosome functionalization platform to investigate the feasibility and potential of HMGN1 in enhancing DCs'immunogeneicity in vitro and in vivo.Methods:Lentivirus transfection was used to establish HMGN1-expressing stable Hepa1-6 cells(Hepa1-6_HMGN1).The proliferation and growth rates of Hepa1-6_HMGN1 and Hepa1-6cells in vitro or in vivo were compared via cell counting and the measurement of tumor sizes.Transmission eletron microscopy,nanosight,western blotting and flow cytometry were utilized to characterize the morphology,size and surface biomarkers.Exosmal anchor peptide CP05 was used to link the functional domain of HMGN1?N1ND?on the surface of TEXs?N1ND-TEX?.N1ND-TEXs pulsed DCs(DC_N1ND-TEX)were systematically evaluated in vitro and in vivo including subcutaneous,orthtopic HCC mouse models.Flow cytometry,ELISA and immunostaining were used to monitor the immune microenvironmental changes dynamically.To elucidate the mechanism underping the functionality of N1ND-TEXs,we measured the number of memory T cells in DC_N1ND-TEX treated HCC mice and also induced the production of memory T cells prior to the tumor challenge with flow cytometry.Meanwhile,to investigate key pathways involved in the regulation of N1ND,we knock-down the related genes and measure the gene and protein expression with western blotting.Results:The proliferation rate of Hepa1-6_HMGN1was unaltered in vitro,whereas the tumor growth significantly decreased when Hepa1-6_HMGN1 was inoculated in C57BL6 mice subcutaneously,compared to Hepa1-6 cells under identical conditions,suggesting that HMGN1 can inhibit the tumor growth in vivo.Transmission eletron microscopic results showed that TEXs present as sauce-cup shape of nano-scaled vesicles with a size range of 30-100nm in diameter.DC_N1ND-TEX showed substantially up-regulated expression of major histocompatibility protein I and II?MHC I and II?and other co-stimulatory molecules and increased capacities to activate na?ve T cells compared to TEX-pulsed DCs(DC_TEX)in vitro.DC_N1ND-TEX demonstrated stronger immunogeneicity as revealed by significantly reduced tumor growth,extended survival rates and improved immune microenvironments in subcutaneous and orthotopic tumor-bearing mice,compared to DC_TEXEX under identical conditions.Importantly,DC_N1ND-TEX1ND-TEX indicated long-lasting anti-tumor immunity and reshaped the tumor microenvironments compared to DC_TEX in orthotopic HCC mice bearing large tumors.Examination on the change of memory T cells revealed that significantly increased numbers of meomory T cells in DC_N1ND-TEX-treated group compared to DC_TEX.Down-regulation of toll-like receptor 4?TLR4?in DC_N1ND-TEX abolished the enhancement of N1ND on activation and maturation of DCs.Also significantly increased numbers of DC_N1ND-TEX were observed in lymphoid tissues and spleens compared to DC_TEX,suggesting that N1ND functions through TLR4 to activate DCs and activated DCs show stronger homing capabilities to lymphoid tissues where they trigger activation of na?ve T cells and results more memory T cells,which are responsible for the long-lasting anti-tumor immunity detected in DC_N1ND-TEX.Likewise,N1ND loaded on different tumor cell-derived exosomes could enhance activation of DCs than corresponding parental exosomes.Examination of HMGN1expression in different HCC patient samples demonstrated that significantly reduced levels of HMGN1 expression in HCC tumor biospies than corresponding peri-tumoral tissues.Conclusions:1.HMGN1 can inhibit tumor growth in HCC tumor-bearing mice.2.TEXs function as effective biological nano-carriers for N1ND.3.N1ND-TEX effectively promotes DC activation,maturation and triggers T cell activation.4.DC_N1ND-TEX shows long-lasting anti-tumor immunity in different HCC mouse models.5.N1ND-TEX activates DCs via TLR4 and confers DCs stronger homing abilities,which result in the generation of memory T cells and the long-lasting anti-tumor effect.