Experimental Study On Enhancing The Antihepatoma Immunity Mediated By Dendritic Cells And Their Related Exosomes

The treatment for hepatocellular carcinoma(HCC)has shown poor outcomes, therefore,researchers are striving for new approaches to overcome tumor relaps, prolonge survival and even eradicate malignant cells.Advancements in the field of immunology and cellular biology have demonstrated that biotherapy is a promising strategy for tumor treatment and is attracting intense attention in recent years. Dendritic cell(DC)vaccine,as a regimen of antitumor biotherapy,has demonstrated progresses in vitro and in vivo.However,outcomes of its clinical application are far beyond expectation.To improve the situation,scientists have tried multi-ways to enhance the DC-induced antitumor specific immunity,such as inducing DC from different sources and find optimal precursors for preparing DC vaccine,administering the combination of DC and herbs,cytokines or monoclonal antibody.Other consideration includes the subcellular vaccine,exosome,which may regulate or substitute DC function.Exosome,which carries the functional proteins of parent cells, is a kind of membrane microcapsule secreted by tumor cells,DCs or other cells.It is relatively stable under temperature variation and feasible to quality control.Treating melanoma and lung cancer with exosome have achieved progresses while exosome-based hepatoma therapy is still in preliminary stage.In this study, monocytes from peripheral blood of healthy donors,cord blood and lost blood of HCC patients undergoing hepatectomy were cultured to induce DCs in vitro. Exosomes from hepatoma cell line SMMC7721(Tumor-derived exosome,Texo)and sensitized DCs(DC-derived exosome,Dexo)were prepared,their morphology, phenotypic features and their roles in anti-hepatoma immunity were compared, respectively.Moreover,we preliminarilly investigated the role of AstrogalosideⅣ(ASI)in regulating DC's immune activities and the exosomes they secrete.ASI may be a new vaccinal adjuvant for clinical application.Objective:1.Attempt to obtain abundant and mature DCs from intraoperative lost blood of liver cancer and other different sources via simple and effective ways.2. To compare the morphology,phenotype of Texo and Dexo.And to study their antihepatoma effects as non-cellular vaccine.3.To investigate the role of ASI in up-regulating the activities of DCs and their exosomes in antihepatoma immunity.Methods:1.Isolated monocytes from peripheral blood of healthy donors,cord blood and the lost blood of hepatoma patients undergoing hepatectomy.The monocytes were induced with rhGM-CSF,rhIL-4,TNF-α,then loaded with SMMC7721 antigen.Cell morphology were observed under light microscope and electric microscope,cell phenotypes were identified by FACS.2.Extracted exosomes from supematant of SMMC7721(Texo)and DCs loaded with antigens of SMMC7721(Dexo)with multi-steps of centrifugation,ultrafilter and ultracentrifugation.Then observed exosomes under electric microscope,determined their carrying proteins with Western blot.Autologous naive T lymphocytes(NTL) was cocultured with Texo,Dexo,sensitized DCs and nonsensitized DCs,respectively. T cell proliferation and IL-2 release were measured.MTT assay was used to measure the cytotoxicities of effector cells against SMMC7721.3.ASI was supplemented during DC induction.The DC morphology,phenotype,IL-12 release and MHCⅡmolecule were detected.Then lymphocytes proliferation and their cytotoxicities on SMMC7721 induced by DC were analyzed.Results:1.DCs with typical morphology and phenotype can be obtained from the three different sources of precursors.The levels of cell surface markers on DCs from lost blood were lower than that on DCs from the other two sources,but obviously higher than their precursors.2.Obtained exosomes from mini-scale quantity of supernatants about 60 to 80 mL.Dexo and Texo posessed similar morphology.Dexo expressed CD40,CD80,CD81,CD54,MHCⅠand MHCⅡmolecule(HLA-DR).Texo lacks CD40,and its CD80,CD86 and MHCⅡ(HLA-DR) were lower than Dexo.In the presence of DC,their abilities to promote lymphocyte proliferation and initiate specific cytotoxicity on SMMC 7721 were superior to that of sensitized DCs group.Dexo alone could stimulate lymphocytes more effectively and induce potent specific cytotoxicities(%)than Texo(30.93±11.1 vs 10.24±7.37,P= 0.004),but minor than that of sensitized DCs(30.93±11.1%vs 43.76±8.93%,P= 0.050).3.No dramatic variations of DCs growth and morphology were observed under the addition of ASI.DCs under the ASI modulation showed upregulation of cell surface markers,particularly the expression of CD83,a hallmark of DC's maturity (73.5%vs 41.3%).Herb(ASI)-regulated group showed a higher level of IL-12 (632.65±14.26 vs 442.85±38.96,P=0.000)and the increased lymphocyte proliferation.ASI can promote the release of exosome from imature DCs,induce the higher cytotoxicity on SMMC7721 than DCs alone(59.98±5.23 vs 46.50±2.31,P= 0.002),but showed no significant effects on Dexo's.Conclusions:1.DCs with typical morphology and mature phenotype could be obtained in vitro from different sources and is suitable for further research.DCs induced from lost blood showed lower levels of mature markers and may be further raised by additional cytokines.2.Dexo,as a subcellular structure with tumor antigens, could promote lymphocyte proliferation,induced specific immunity which was significantly enhanced in the presence of DCs.The subcellular complex,as a tumor vaccine or natural antitumor adjuvant,may completely or partly replace the role of DCs.In the absence of DC,Texo alone could not promote the proliferation and failed to induce antitumor cytotoxicity,nevertheless,it still may be a potential vaccine in eliciting anti-hepatoma immunity in vivo.3.ASI could enhance the DC-induced CTL cytotoxicity through promoting DC maturation,increasing antigen-presenting and releasing more IL-12.It may be a potential adjuvant for the DC-mediated antitumor immunity.