| Hepatocellular carcinoma(HCC)is one of the common malignant tumors with strong invasiveness and high mortality.Because of the progress of tumors,the tumor microenvironment(TME)is changed,and the anti-tumor immune response is reduced,which makes the tumor cells more prone to immune escape.Therefore,the current clinical treatment measures can't achieve the expected therapeutic effect.With the progress of medical technology,immunotherapy has made excellent achievements in the field of tumor therapy.Immunotherapy aims to accurately select the target of tumor cells,apply antibodies corresponding to the target to tumor cells,improve TME,enhance the body's anti-tumor immune response,and kill tumor cells.The CpG oligodeoxynucleotides(CpG-ODN)has been used as an immunopotentiator in HCC treatment research,and it can be used as a therapeutic adjuvant to improve the therapeutic effect.However,studies have shown that the use of CpG-ODN alone is not effective in the treatment of HCC metastases.The 4-1BB molecule is a novel immunological checkpoint in tumor immunotherapy research,and its corresponding agonistic antibody has significant therapeutic effects on breast cancer,melanoma,lymphoma,and homologous metastases.In the early period,some scholars detected the high expression of 4-1BB molecule in tumor tissues and adjacent tissues of HCC patients,and the use of CpG-ODN could promote its expression.These research results provide a new idea for HCC immunotherapy.In this experiment,we first established mouse hepatocellular carcinoma multiple tumor-bearing models.We used a combination therapy regimen of CpG-ODN and anti-4-1BB agonistic antibodies for the treatment of mouse models.We monitored the growth of all tumor-bearing mice in the model and the prognosis of the mice and evaluated the effect of the anti-tumor immune response in mice by detecting changes in some indicators.Objective:To investigate the effect of topical application of CpG-ODN combined with anti-4-1BB agonistic antibodies on mouse HCC multiple tumor-bearing models,and the degree of improvement of anti-tumor immune response in mice.Methods:After the H22 cells were taken out from the liquid nitrogen,they were cultured by resuscitation,and adjusted to a cell suspension having a concentration of 1×107 cells/ml.0.2 ml of H22 cell suspension was injected subcutaneously into the extremities of 100 BALB/c male mice.After 7 days,40 mice with similar tumor volume were screened and randomly divided into model control group,CpG group,4-1BB group and CpG+4-1BB group,with 10 mice in each group.On the 7th,9th,and 11 th day after the inoculation of the tumor,the intratumoral injection was performed on the tumor-bearing site of the left lower limb of the mouse.The model control group was given 10?l of physiological saline.CpG-ODN was administered to each mouse in the CpG group at 60?g/10?l.The 4-1BB group received anti-4-1BB m Ab 60?g/10?l per mouse.CpG+4-1BB group received CpG-ODN 60?g/10?l and anti-4-1BB m Ab 60?g/10?l per mouse.Ten normal mice in the same batch were selected as the normal control group for comparison of partial detection indexes.The long diameter and the broad diameter of the tumors of the four groups of mice(except the normal control group)were measured before the administration on the 7th day after the tumor-injection,and the measurement was performed every 4 days from the 9th day to compare the changes in the tumor-bearing volume.The proportions of spleen CD8+T lymphocytes and CD4+CD25+Foxp3+ regulatory T cells(Tregs)in spleen lymphocytes of five groups of mice were detected by flow cytometry.The concentrations of interleukin(IL)-12 and interferon(IFN)-? in the serum of the five groups of mice were determined by ELISA.The weights of heart,lung,liver and kidney of mice in each group(except the model control group)were weighed and the organ coefficients were calculated.Cytotoxic T lymphocyte(CTL)killing activity of spleen of four groups of mice(except normal control group)was measured by CCK-8 method at 5:1,25:1 and 50:1,respectively.The survival status and survival time of the four groups of mice(except the normal control group)after administration were observed and recorded,and the difference in survival rate was compared.Statistical analysis was performed using statistical software SPSS19.0.P < 0.05 indicated that the difference was statistically significant.Results:1 Comparison results of tumor volume in four groups of mice1.1 Comparison of tumor-bearing volume in the treatment of left lower limbs of four groups of miceThe tumor-bearing volume of mice in the model control group increased progressively.The tumor-bearing volume of mice in the CpG group,the 4-1BB group,and the CpG+4-1BB group decreased compared with the model control group(P < 0.05).The tumor-bearing volume of mice in the 4-1BB group and the CpG+4-1BB group was reduced compared with the CpG group(P < 0.05).The tumor-bearing volume of the mice in the CpG+4-1BB group was smaller than that in the 4-1BB group(P < 0.05).It can be seen that the three treatment schemes have shown a good therapeutic effect on the tumor at the treatment site,and the combination of CpG-ODN and anti-4-1BB antibodies has a better therapeutic effect.1.2 Comparison results of distant untreated tumor-bearing volume in four groups of miceThe volume of distant tumor-bearing mice in the model control group and CpG group showed a progressive increase trend,and the difference was not statistically significant(P > 0.05),indicating that the local application of CpG-ODN alone didn't affect distant tumor-bearing tumors.However,among the three untreated tumors in the 4-1BB group,only a few tumors showed a slower growth rate,but there was no significant difference compared with the model control group and the CpG group(P > 0.05).From the overall results,there was no obvious therapeutic effect.The tumor-bearing volume of mice in the CpG+4-1BB group was reduced compared with the model control group,the CpG group,and the 4-1BB group(P < 0.05).It can be seen that the combined application of CpG-ODN and anti-4-1BB antibodies has a more obvious therapeutic effect on distant tumors.2 Comparison of the proportion of spleen CD4+CD25+Foxp3+Tregs in spleen lymphocytes of five groups of miceCompared with the normal control group,the proportion of CD4+CD25+Foxp3+Tregs in the spleen of the model control group was increased(P < 0.05).Tumor production changes the body's TME,and the activation of Tregs also increases,which promotes tumor progression.Compared with the model control group,the proportion of CD4+CD25+Foxp3+Tregs in the spleen of the CpG group,4-1BB group and CpG+4-1BB group decreased(P < 0.05).Compared with the CpG group,the proportion of CD4+CD25+Foxp3+Tregs in the spleen of the 4-1BB group and the CpG+4-1BB group decreased(P < 0.05).Compared with the 4-1BBgroup,the proportion of CD4+CD25+Foxp3+Tregs in the spleen of the CpG+4-1BB group decreased(P < 0.05).The combination of the two can enhance the anti-tumor immune response of mice and decrease the value of Tregs.3 Comparison of the proportion of spleen CD8+T lymphocytes in spleen lymphocytes in five groups of miceCompared with the normal control group,the proportion of spleen CD8+T cells in the model control group decreased(P < 0.05).Tumor progression changes TME,reducing the proportion of CD8+T cells.Compared with the model control group,the proportion of spleen CD8+T cells in the CpG group,4-1BB group and CpG+4-1BB group increased(P < 0.05).Compared with the CpG group,the proportion of spleen CD8+T cells in the 4-1BB group and the CpG+4-1BB group increased(P < 0.05).Compared with the 4-1BB group,the proportion of spleen CD8+T cells in the CpG+4-1BB group was increased(P < 0.05).Due to the combined application of the two,the anti-tumor immune response of the mouse body is strengthened,and the value of CD8+T cells is also enhanced.4 Comparison of serum IL-12 and IFN-? concentrations in five groups of miceCompared with the normal control group,the concentrations of IL-12 and IFN-? in the serum of the model control group were decreased(P < 0.05).It can be seen that tumor progression reduced the level of cytokines.Compared with the model control group,the serum concentrations of IL-12 and IFN-? in the CpG group,4-1BB group,and CpG+4-1BB group were all increased(P < 0.05);compared with the CpG group,the 4-1BB group and Serum IL-12 and IFN-? concentrations were increased in the CpG+4-1BB group(P < 0.05);compared with the 4-1BB group,the serum concentrations of IL-12 and IFN-? were increased in the CpG+4-1BB group(P < 0.05).It can be seen that the combined treatment regimen significantly increased the secretion of two cytokines and enhanced the anti-tumor immune effect in mice.5 Comparison results of organ coefficients of heart,lung,liver and kidney in four groups of miceThere were no significant differences in the organ coefficients between the CpG group,4-1BB group and CpG+4-1BB group compared with the normal control group.There was no significant difference between the three groups(P > 0.05).It can be seen that the intervention drugs used in the experiment did not cause immune-related damage to mice.6 Comparison results of CTL killing activity of spleen cells in four groups of miceCompared with before treatment,CTL killing activity was significantly increased in the CpG group,4-1BB group and CpG+4-1BB group(P < 0.05).The CTL killing activity of the 4-1BB group and CpG+4-1BB group was higher than that of CpG group,and CpG+4-1BB group was higher than the 4-1BB group(P < 0.05).The results of CTL killing experiments in vitro again demonstrated that the combination of the two drugs improved the anti-tumor immune effect of the body.There was no significant difference in CTL killing activity between the model control group and before treatment(P > 0.05).7 Comparison results of survival of four groups of miceThe mice in the CpG+4-1BB group had fewer deaths.The survival time of the 4-1BB group and the CpG+4-1BB group was higher than that of the model control group and the CpG group(P < 0.05),and the CpG+4-1BB group was longer than that of the 4-1BB group(P < 0.05).There was no significant difference between the model control group and the CpG group(P > 0.05),but the average survival time of the CpG group was slightly higher than that of the model control group.The survival time of mice was improved after the combination of the two drugs,which effectively improved the prognosis.Conclusions:1 When CpG-ODN or agonistic anti-4-1BB monoclonal antibody were administered alone to treat the mouse hepatocellular carcinoma models,the tumor growth at the treatment site was slow,but there was no significant therapeutic effect on the distant untreated tumor bearing,and the prognosis of the mice was not significantly improved.2 Combining CpG-ODN with anti-4-1BB monoclonal antibody in the treatment of mouse hepatocellular carcinoma model improves the anti-tumor immune response effect of mice,reduces the tumor-bearing capacity at the treatment site,and slows down the growth rate of untreated tumor-bearing at distant sites,thus prolonging the survival time of mice. |
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