Experimental Study On Treatment Of Colorectal Cancer With Immunocytes Induced By ?-gal Engineered Tumor Derived Exosomes

Background:Exosomes are microvesicles with a lipid bilayer membrane structure and secreted by most living cells,diameters of exosomes are between 30-150 nm.Exosomes carry a variety of proteins,mRNA and other molecules,and play an important role in cell-to-cell communication,affect cell function and are related to the occurrence and development of various diseases.Tumor-associated antigens are present in exosomes secreted by tumor cells,which can be taken up and presented by antigen presenting cells to induce tumor-specific T cells.The exosomes can also be engineered to further enhance their immune function.The a-gal epitopes are widely present in cells of non-primate mammals,and there are no a-gal epitopes in the human body but anti-gal antibodies are present in the serum.After the antibody specifically binds to the a-gal epitopes,an immune response can be induced.The strong immunogenicity of the a-gal epitopes provides new insights into the immunotherapy of colorectal cancer.The immune cell function in colorectal cancer patients is inhibited and does not produce sufficient anti-tumor immune response.This is also the reason why results of many immunotherapy clinical trials are not satisfactory.Adoptive cellular therapy may solve this problem.Objective:To express a-gal epitopes in parental tumor cells by lentiviral vector,tumor cell derived exosomes with a-gal epitopes were isolated,combining with DC-CTL cell culture techniques,to explore the feasibility for treatment of colorectal cancer with stronger CTLs induced by a-gal engineered tumor derived exosomes.Methods:Lentiviral vector that regulates the expression of a1,3GT gene was conducted and colorectal cancer cell SW480 was transfected to establish a cell line stably expressing a-gal epitopes.2)The exosomes of SW480 and SW480-a-gal cell lines were separated by ultracentrifugation,and the extracted exosomes were identified by electron microscopy,NTA and Western blot and a-gal epitopes were identified by ELISA.3)Peripheral blood of healthy volunteers was taken to separate PBMC,DC and CTL were cultured in vitro.SW480 tumor cell lysate(Lys),SW480 tumor cell derived exosomes(TEX),and SW480-a-gal tumor cell derived exosomes(TEX-gal)were used as antigens to load DCs and induce different CTLs respectively.The phenotypes of DC and CTL cells were detected by flow cytometry.The Thl response related cytokines IL-12p70,IFN-y,Th2 response related cytokines IL-13,IL-10 and pro-inflammatory response related cytokines IL-6,TNF-a secreted by DC cells,IFN-y,IL-6,IL-4 and IL-13 secreted by CTLs were detected by AimPlex technology.4)The in vitro killing ability of different CTL cells was detected by LDH release method.A subcutaneous tumor-bearing model of nude mice was established,and CTL cells induced by different antigens were intratumoral injected.The tumor inhibition rate of each group was compared.HE and immunohistochemical staining of tumor tissues was performed to observe the infiltration of lymphocytes inside the tumor.Results:Lentiviral vector was successfully constructed and confirmed by identification of enzyme digestion and squencing.SW480-a-gal cell line stably expressing a-gal epitope was successfully established by infecting SW480 cells with lentiviral vector at optimal MOI 150.2)The SW480 and SW480-a-gal tumor cell derived exosomes were successfully extracted by ultracentrifugation.A typical double-layer membrane structure was observed under transmission electron microscopy.The NTA results showed that the isolated exosomes had a relatively concentrated diameter distribution.The results of Western blot demonstrated the expression of exosomes specific markers CD9,CD63,and EGFR,ELISA results demonstrated a-gal epitopes existed in SW480-a-gal tumor cell derived exosomes 3)The DC cells loaded with TEX-gal had better maturity,the levels of secreted Thl response related cytokines IL-12p70 and IFN-y were increased,the level of secreted Th2 response related cytokines IL-13 and IL-10 was decreased,and the level of secreted proinflammatory response related cytokines IL-6 was decreased,but the level of TNF-alpha was increased.The proportions of CTL and NKT cell subsets in CTL-TEX-gal were increased,the proportion of NK cell subset was decreased,and the level of IFN-y secreted by CTL-TEX-gal was increased.4)The cytotoxicity of CTL-TEX-gal was significantly stronger in vitro,and the growth of subcutaneous tumor in nude mice was inhibited significantly.HE and immunohistochemical staining results showed that the internal structure of tumors injected with CTL-TEX-gal was massively damaged,there were large amount of infiltrated CD8+CTL and residual tumor tissue were less.Conclusions:1)The a-gal epitopes were successfully synthesized on the surface of colorectal cancer SW480 cells using the lentiviral vector,and the a-gal epitope was present in the exosomes secreted by the transfected SW480-a-gal cells.2)TEX-gal-loaded DCs demonstrated better maturity,secretion of Thl response related cytokines increased,proportion of CTL subset in CTL-TEX-gal and IFN-y levels secreted by CTL-TEX-gal increased.3)CTL-TEX-gal induced by DCs loaded with TEX-gal demonstrated stronger cytotoxicity in vitro and in vivo.