Immunocytotoxicity Of Inhibition Of Immune Checkpoint B7-H3 On Hepatocellular Carcinoma In Vitro And In Vivo

ObjectiveThe expression level of B7-H3 protein in HCC tissues was assessed via immunohistochemistry,and the clinical significances of the expression quantity in HCC was analyzed.Then,the influences on T cell immunoactivity and immunotherapeutic effect on HCC was investigated with in vitro and in vivo experiments following anti-B7-H3 antibody treatment.Finally,The impact of anti-B7-H3 antibody therapy on tumor microenvironment was preliminarily studied.Methods1.A total of 61 clinical HCC tissue samples were collected from patients who underwent surgical resection and were diagnosed by pathologists.Then,clinicopathological data of 61 patients with HCC were collected through follow-up survey.2.The expression of B7-H3 protein in 61 HCC tissues was examined by IHC,and they were divided into high expression group of B7-H3 and low expression group based on the intensity of staining and range of positive cells.IBM SPSS 25.0 software was used to analyze the correlation between B7-H3 expression level and clinicopathological characteristics,T cell infiltration,postoperative recurrence and prognosis of patients which was combined with clinicopathological data.3.In vitro experiment,we divided the experiment into experimental group(anti-B7-H3antibody:MIH35)and control group(PBS).T cells were activated by CD3 and CD28antibody,and a proper amount of IL-2 was added to the medium to maintain its proliferation state in vitro.Hepatoma cells in the experimental group were pre-incubated with anti-B7-H3 antibody at 37℃for 2 h.Apoptosis Detection Kit and Lactate Dehydrogenase Assay Kit were used to compare the killing efficiency of T cells against cancer cells between the two groups after activated T cells were co-cultured with hepatoma cells at different effect-target ratios(1:1,3:1,5:1)for 24 h.The secretion of pro-inflammatory cytokines in the co-culture system of the two groups was detected by ELISA Kit.4.A subcutaneous tumor-bearing model of mouse hepatocellular carcinoma was established.Mice are randomised through a computer-generated randomisation sequence.They were the treatment group(anti-B7-H3 antibody,clone:MJ18),the isotype control group(isotype immunoglobulin Ig G1)and the blank group(PBS).The dosage of antibody was normalized according to the body weight of the mouse(MJ18:5mg/kg,Ig G1:5 mg/kg),which was injected through the tail vein once every 3 days,four times altogether.The ability of MJ18 to inhibit HCC cells in vivo was evaluated by continuous monitoring of subcutaneous tumor volume and survival of mice.5.FCM and IF were used to analyze the infiltration of T cells in tumor tissues after antibody treatment.The secretion of pro-inflammatory cytokines in tumor tissue of each group were detected by ELISA.The vital organs of mice were stained with HE and observed to evaluate the biosafety of the antibodies used.Results:1.Pearson’s Chi-squared test showed that the expression of B7-H3 was significantly associated with recurrence(within 1 year,***P<0.001)、TNM stage(*P=0.028)and the amount of T lymphocyte infiltration(*P=0.03)in patients with HCC.2.Kaplan-Meier survival analysis showed that patients with high B7-H3 expression had remarkably shorter RFS than those with low B7-H3 expression(***P<0.001).Cox’s proportional hazard regression model analysis confirmed that high expression of B7-H3 was an independent risk factor for RFS(1 year,*P=0.021).However,there was no significant difference in Overall survival(OS)between the two groups,which may be caused by too short follow-up time.3.In vitro killing assay,the killing efficiency of T cells to tumor cells increased with higher effect-target ratio(E:T ratio).when the E:T ratio was 1:1,3:1 and 5:1,T cells in the experimental group have a high level of killing activity against tumor cells.The killing efficiency were 54.26%,63.57%and 73.23%,respectively,which were higher than that of the control group(25.95%,49.09%and 56.47%).The results suggested that in vitro inhibition of B7-H3 could enhanced the killing ability of T cells to tumor cells,which was further confirmed by subsequent LDH experiments.In addition,higher levels of secretion of pro-inflammatory cytokines IFN-γand TNF-αwere detected in the co-culture system of the experimental group.The differences were statistically significant(*P<0.05).4.The subcutaneous model of mouse hepatocellular carcinoma was successfully established.The results showed that the tumor volume increased in each group.However,the average tumor volume of the treatment group(MJ18)was significantly smaller than that of isotype control group(Ig G1、**P=0.0083),and the growth rate of the tumor was also significantly slower.Compared with the control group,the survival time of mice in the treatment group was significantly prolonged(*P=0.0117),and there were more CD8~+T lymphocytes in the tumor tissues of the experimental group(***P<0.001).CD8~+tumor-infiltrating lymphocytes in the treated group produced more IFN-γand TNF-αcompared to tumor-infiltrating lymphocytes from nontreated mice(**P=0.0085,***P<0.001).HE staining indicated that therapeutic dose of antibodies had no damage to the important organs of mice.Conclusions1.The expression of B7-H3 was significantly correlated with postoperative recurrence(within 1 year),TNM stage and T cell infiltration in HCC patients.Patients with high expression of B7-H3 have poor prognosis after surgery,which can be used as one of the potential markers of poor prognosis.2.The vitro killing experiments have shown that inhibition of B7-H3 molecule can enhance the killing ability of T cells,and promote the secretion of IFN-γand TNF-α.3.The results of animal experiments in vivo showed that the injection of MJ18 could induce the infiltration of CD8~+T cells and promote the secretion of IFN-γand TNF-αin tumor immune microenvironment,thus exerting anti-tumor effect.