Study Of CD8~+T Cell Exhaustion Driven By Exosome Released By P53 R249S Mutant Hepatocellular Carcinoma Cells

Objective:Hepatocellular carcinoma(HCC)accounts for the majority of primary liver cancer and is currently the third leading cause of cancer-related deaths worldwide.HCC occurs after years of chronic liver disease,and mutations in the genome are the pathogenic and determining feature of cancer.Chronic liver disease,especially in the stage of cirrhosis,can lead to the progressive accumulation of mutations,thereby promoting the development of cancer.Therefore,understanding the causes of specific mutations and how they interact to lead to the occurrence and development of cancer is a core issue in understanding,preventing,and responding to liver cancer.The p53 protein encoded by the TP53 gene is a tumor suppressor transcription factor that can serve as a genetic guardian against the occurrence and development of various cancers.The TP53gene undergoes mutations in approximately half of human cancer cases.P53 mutations regulate many cellular processes,including proliferation,migration,invasion,survival,metabolism,and chemotherapy resistance,to promote tumor progression.It is worth noting that previous studies have shown that TP53 mutation is closely related to the immune microenvironment of HCC.CD8~+T cells are the main killer cells in the immune microenvironment.Under the influence of many factors,such as continuous stimulation of tumor antigen,suppression of immunosuppressive cells,and imbalance of physical and chemical status,CD8~+T will gradually degenerate into a functional disorder,called CD8~+T cell exhaustion,where the proliferation ability is weakened,the level of Effector cell factors is reduced,and the expression of inhibitory receptors is increased.The research on the promotion of malignant biological behavior of tumors by p53 mutations has mostly focused on the dependence of tumor cells on autonomous signals.However,more and more studies have emphasized the involvement of p53 mutations in regulating tumor cell secretomics and promoting cancer progression.Therefore,it is worth exploring whether p53 mutant liver cancer cells can affect CD8~+T cell exhaustion through exosomes and the specific mechanism.Therefore,this study first clarified whether p53 R249S mutant liver cancer cells promote CD8~+T cell exhaustion.Secondly,it explored whether exosomes are involved in the promotion of CD8~+T cell exhaustion by p53 R249S mutant liver cancer cells.Finally,it explored the specific mechanism by which exosomes released by p53 R249S mutant liver cancer cells promote CD8~+T cell exhaustion.Methods:1.Materials:Human liver cancer cell lines Hep3B and PLC/PRF/5,mouse liver cancer cell lines Hepa1-6,and C57BL/6 mice.2.Methods:(1)Cell culture:Culture human liver cancer cell lines Hep3B,PLC/PRF/5,and mouse liver cancer cell line Hepa1-6.(2)Virus transfection:p53 R249S mutation is transfected in Hep3B cell line,shp53 is transfected in PLC cell line,and p53 R249S mutation is transfected in Hepa1-6 cell line.(3)Western blot:Used to detect virus transfection efficiency,verify the purity of exosomes,and levels of exosome release related proteins(RAB35,RAB27A,RAB27B,RAB10,SNAP23,VAMP3)in liver cancer cells,as well as SHP2 levels in liver cancer cells,exosomes,and CD8~+T cells.(4)Cell co-culture experiment:Human liver cancer cells transfected with the virus were co-cultured with CD8~+T cells for subsequent flow cytometry analysis.(5)Subcutaneous tumor formation experiment in mice:Used to study the effects of p53R249S mutation on tumor weight,volume,and growth rate in mice.(6)Immunohistochemistry:Used to study the effect of p53 R249S mutation on the number of CD8~+T cells in mouse tumors.(7)Differential ultracentrifugation:Used to extract exosomes.(8)NTA:Used to detect the particle size distribution of exosomes.(9)Transmission electron microscopy:Used to observe the particle size and morphology of exosomes.(10)Exosomes-cells co-culture:Exosomes extracted from liver cancer cells are co-cultured with CD8~+T cells for subsequent flow cytometry analysis.(11)Subcutaneous tumor formation and tail vein injection of exosomes in mice:Used to study the effect of exosomes derived from p53 R249S mutant liver cancer cells on the volume of tumor weight in mice.(12)Flow cytometry:Used to detect the levels of Gzm B~+,Perforin~+,PD-1~+,and Tim-3~+in CD8~+T cells in tumor tissue and spleen,as well as in co-culture of cells and exosomes.(13)Immunofluorescence:Used to detect the uptake of exosomes by CD8~+T cells.(14)Real-time PCR:Used to detect the m RNA level of SHP2 in liver cancer cells.(15)Statistical analysis:Statistical analysis was completed using the software SPSS 22.0.All data were presented in the form of mean±standard deviation,and all experiments were completed with 3 independent repeated experiments.Histogram quantization and line chart are completed by Prism 9(Graph Pad Software).Statistical significance was determined through t-test and one-way ANOVA,and when P<0.05,the data was considered to have statistical differences.3.Protocol:3.1 Clarify that p53 R249S mutant liver cancer cells promote CD8~+T cell exhaustion(1)Evaluation of CD8~+T cell exhaustion:Flow cytometry was used to detect Gzm B~+,Perforin~+,PD-1~+and Tim-3~+in CD8~+T cells.The percentage of Gzm B~+CD8~+T cells and Perforin~+CD8~+T cells decreased,while the percentage of PD-1~+CD8~+T cells and Tim-3~+CD8~+T cells increased,indicating CD8~+T cell exhaustion.(2)Construction of p53 R249S mutant liver cancer cell line:Lentivirus transfection technology was used to transfect PLVX in Hep3B human hepatoma cell line as the control group,and overexpression of p53 R249S was used as the experimental group;In PLC human liver cancer cell lines,PLKO was transfected as the control group,and shp53,also known as p53 knockout,was transfected as the experimental group.PLVX was transfected into Hepa1-6 mouse liver cancer cell lines as the control group,and overexpression of p53 R249S was transfected into the experimental group.(3)Cell co-culture system construction:The above constructed human liver cancer cell line(p53 R249S mutant liver cancer cells and p53 null liver cancer cells were compared with each other)was co-cultured with CD8~+T cells.(4)Animal experiments:Hepa1-6 mouse liver cancer cells were constructed and injected subcutaneously into C57BL/6 mice for tumor formation.The tumor volume and weight were recorded,and the infiltration level of CD8~+T cells in liver cancer tissue and the exhaustion of CD8~+T cells in mouse tumor tissue and spleen were evaluated to determine.3.2 Explore the exosomes released by p53 R249S mutant liver cancer cells promoting CD8~+T cell exhaustion(1)Extraction and validation of exosomes:Differential ultracentrifugation method was used to extract exosomes from cell supernatant,and the purity of exosomes was identified by transmission electron microscopy,NTA,and western blot.(2)Construction of exosomes and cell co-culture system:Collect exosomes from different states of p53 liver cancer cells and co-culture with CD8~+T cells.Flow cytometry is used to detect CD8~+T cell exhaustion.(3)Animal experiments:A mouse subcutaneous tumor model was established by injecting exosomes of p53 liver cancer cells in different states into the tail vein of the mouse.The tumor volume,size,and weight were recorded,and the exhaustion of CD8~+T cells in the tumor and spleen was detected by flow cytometry.3.3 Explore the specific mechanism of exosomes released by p53 R249S mutant liver cancer cells promoting CD8~+T cell exhaustion(1)Exosome Release Experiment:Compare p53 R249S mutant liver cancer cells with p53 null liver cancer cells,and detect exosome release related proteins using western blot.Collect exosomes released by liver cancer cells,and detect changes in the size and release amount of exosomes using NTA.To comprehensively determine whether the p53R249S mutation can affect the release of exosomes in liver cancer cells.(2)CD8~+T cell uptake of exosomes experiment:Di D dye-labeled exosomes were co-cultured with CD8~+T cells,and immunofluorescence was used to verify the uptake of exosomes by CD8~+T cells.(3)Exploring the key components that cause CD8~+T cell exhaustion in exosomes:Proteomic analysis screening differential proteins.Western blot and PCR validation of SHP2 levels in liver cancer cells and their secreted exosomes.Co-culture exosomes with CD8~+T cells,and verify the SHP2 level in CD8~+T cells with western blot.Results:1.In vitro and in vivo experiments have demonstrated that p53 R249S mutant liver cancer cells promote CD8~+T cell exhaustion.2.In vitro and in vivo experiments have demonstrated that the exosomes released by p53R249S mutant liver cancer cells promote CD8~+T cell exhaustion3.The p53 R249S mutation does not affect the release of exosomes and related proteins in liver cancer cells,and CD8~+T cells can uptake exosomes from different sources.The p53 R249S mutation in liver cancer cells can release exosomes carrying SHP2 and deliver them to CD8~+T cells,promoting their exhaustion.Conclusion:1.p53 R249S mutant liver cancer cells can promote CD8~+T cell exhaustion.2.The exosomes derived from p53 R249S mutant liver cancer cells promote the exhaustion of CD8~+T cells.3.p53 R249S mutant liver cancer cells promote CD8~+T cell exhaustion by releasing exosomes carrying SHP2 and delivering them to CD8~+T cells.