Application Of Exosomes-loaded Dendritic Cells Combined With PD-1 Antibody In The Treatment Of Mouse Hepatocellular Carcinoma

At present,the hepatocellular carcinoma has high malignancy grade and poor prognosis and effect of chemotherapy and radiotherapy on disease control.Therefore,there is a lack of effective treatment for advanced hepatocellular carcinoma.Sorafenib can only extend the survival time by about three months.In recent years,immunotherapy has played a role in the treatment of hepatocellular carcinoma,but it still needs to further improve its efficacy.In immunotherapy,the tumor antigens play a pivotal role in the treatment oriented by dendritic cells(DCs).It has been confirmed in a variety of solid tumors that tumor cell lysate can be absorbed by DCs and mature them,making these cells possess the T cells with tumor-activated specificity.DCs loaded with tumor cell lysates does not significantly benefit the hepatocellular carcinoma.In recent studies,however,as the source of tumor cells,Exosomes has been considered to be a highly immunogenic tumor antigen,which could be absorbed by DCs and mature them,and could be able to activate T cells with tumor-specificity to kill the tumor cells.Under the sustained stimulation of tumor antigens,the activated CD8+T cells would express an inhibitory receptor(programmed death-1,PD-1)on their surface,which could disable CD8+T cells’ ability to secrete and proliferate cytokines through combination with ligands;as a result,the activated CD8+ T cells would lose their effect on killing tumor cells.Since PD-1 is induced in response to the stimulation with antigen,its inductive production requires a certain amount of time.Although Exosomes,the source of tumor cells,has a better curative effect than tumor cell lysates in the treatment of primary tumors as the tumor antigens,the combined application and the optimal combination times among PD-1 and DCs loaded with Exosomes have not been reported yet in researches about the hepatocellular carcinoma.This study has further confirmed the anti-tumor effect of DCs loaded with Exosomes among the hepatocellular carcinoma.In the mean time,after the administration of DC-TEX,the CD8+T cells infiltrated in tumor tissues gradually expressed PD-1,and the administration of PD-1 antibody at different times could produce different anti-tumor effects.The combination among DC-TEX and PD-1 antibody was used to observe the therapeutic effect of sorafenib on hepatocellular carcinoma and can increase the therapeutic effect of sorafenib.In addition,Exosomes,the origin of cancerous ascites of hepatocellular carcinoma,could also be absorbed by DCs as tumor antigens and further produce anti-tumor effects.Finally,we compared Exosomes levels in the serum of patients suffered from advanced hepatocellular carcinoma and non-suffered patients,and found that the level of Exosomes in patients with advanced hepatocellular carcinoma was significantly higher.Objectives:1.Further confirm the anti-tumor effect of DC-TEX in hepatocellular carcinoma,and to observe whether Exosomes can induce PD-1’s expression as a tumor antigen.2.Determine whether DC-TEX combined with PD-1 antibody can produce the synergistic anti-tumor effect among the hepatocellular carcinoma,and their optimal time and anti-tumor mechanism.3.Determine whether DC-TEX combined with PD-1 antibody can increase the therapeutic effect of sorafenib on hepatocellular carcinoma.4.Determine whether the Exosomes,the origin of cancerous ascites of hepatocellular carcinoma,can be used as tumor antigens and its anti-tumor efficacy and mechanism.5.Determine whether Exosomes levels in patients with advanced hepatocellular carcinoma can be used as the tumor markers of liver cancer’s specificity.Methods:Firstly,build a subcutaneous tumor model with Hepal-6 cells.When the major diameter of the subcutaneous tumor grows to 1 cm,take the subcutaneous tumor under aseptic conditions,cut it into 1mm3 pieces,and pass through the tumor mass with a puncture needle to the left hepatic lobe of the mouse,and then observe the growth of orthotopic tumors with small animal MRI.An in situ model should be injected intraperitoneally with diethylnitrosamine(DENA)into 15 days old C57 mice.Take the H22 cell lines to build an ascites tumor model.Analyze the changes of CD8+T cells in tumor tissues with flow cytometry and observe the expression of PDL-1 in tumor tissues with immunohistochemistry.Detect changes of cytokines in the peripheral blood with ELISA.Co-culture DCs with Exosomes for 48h to render DCs ingest Exosomes and be mature.DC-TEX is injected through the tail vein and PD-1 antibody is injected intraperitoneally.Vivo blocking antibodies of CD4 and CD8 take the abdominal injection,and are used to study the anti-tumor mechanism of DC-TEX.The Exosomes shoule be extracted by kit from the serum of patients with advanced liver cancer,and its specific prote:in expression is to be detected by Western blot.Results:1.DC-TEX could produce a certain anti-tumor effect in hepatocellular carcinoma,but at the same time,the expression of PD-1 on the surface of CD8+ T cells also increased;after 72h of DC-TEX administration,the number of CD8+PD-1+ T cells reached the highest level.2.The administration of PD-1 antibody at different time points after DC-TEX administration produced different anti-tumor effects.The antitumor effect of PD-1 antibody was best at 72 h after the application of DC-TEX.In vivo blocking of CD4 and CD8 antibodies and the tumor-bearing model of nude mice,it was found that the anti-tumor effects of DC-TEX and PD-1 antibodies mainly depended on CD8+ T cells.3.DC-TEX combined with PD-1 antibody could reverse sorafenib’s resistance and increase the therapeutic effect of sorafenib in hepatocellular carcinoma.4.The Exosomes derived from the ascites,could also act as tumor antigens,which could be absorbed by DCs and mature them,and produce the certain anti-tumor effects.5.The level of Exosomes in serum of patients with advanced hepatocellular carcinoma was significantly higher than that of healthy people,and some specific surface proteins were expressed.Conclusions:As the origin of Hepal-6 cells,Exosomes can be absorbed by DCs as tumor antigens and thus induces anti-tumor immune responses.However,in the presence of tumor antigens,the PD-1 expression of CD8+T cells further inhibits the anti-tumor effects of CD8+T cells.The combination of DC-TEX and PD-1 antibodies can produce different anti-tumor effects,and can increase the therapeutic effect of sorafenib in hepatocellular carcinoma.In addition,Exosomes levels in serum of patients with advanced hepatocellular carcinoma go up.As the origin of cancerous ascites in hepatocellular carcinoma,Exosomes can also be treated as tumor-associated antigens.