Study On The Killing Effect Of CIK Combined With OK432 On Ovarian Cancer Cells In Vitro

Background:The mortality rate of ovarian cancer ranks the first among gynecological malignant tumors.In addition to the traditional treatment method of advanced ovarian cancer combined with tumor cell reduction combined with platinum-based chemotherapy,immunotherapy to improve the anti-tumor activity of ovarian cancer patients is an important research direction of current tumor treatment.A large number of studies have reported that cytokine-induced killer(CIK)treatment in non-specific immunotherapy has been widely used in hematological tumors and solid tumors such as lung cancer,gastric cancer,and ovarian cancer,and its efficacy is remarkable.A large number of clinical studies on CIK in the treatment of ovarian cancer have shown that as maintenance therapy after first-line treatment of patients with advanced ovarian cancer can significantly prolong the survival period and improve the quality of life.However,CIK still faces many problems in the treatment of ovarian cancer.Patients with advanced ovarian cancer have poor immune function status,cannot effectively activate the anti-tumor immune response.The tumor tissue has an immunosuppressive micro-environment,and CIK is difficult to infiltrate the tumor tissue to function locally.OK432(picibanil)is a group A hemolytic streptococcus preparation,which can promote the maturation of dendritic cells(DC),enhance antigen presentation,activate anti-tumor immune response;change the tumor micro-environment,induce T lymphocytes infiltrate the tumor tissue locally.Several clinical studies have shown that OK432 can effectively control ovarian cancer ascites and reduce the recurrence rate of ovarian cancer.So,can OK432 improve the therapeutic effect of CIK on ovarian cancer?CIK combined with OK432 therapy has not been reported in ovarian cancer and other tumors.Objective:Using in vitro experiments to investigate whether OK432 can enhance the killing effect of CIK on ovarian cancer cells,to preliminary explore its mechanism of action,to provide a safe and effective immunotherapy strategy for patients with ovarian cancer,thereby prolonging the patient’s survival time and improving the patient’s quality of life.Methods:1.The Peripheral blood mononuclear cell(PBMC)was collected from healthy people,and the CIK primary cells were prepared in vitro under the influence of interferon-y.The CIK cells initiated amplification under the influence of CD3 monoclonal antibody,interleukine-1α and interleukine-2.After bacteria detection,endotoxin detection,cell counting and cell state observation,the immunophenotype of CIK cells was determined by Flow Cytometry,and CIK cells were used as effector cells for the killing experiment.2.Ovarian cancer cells SKOV3,ES2 and OVCAR3 were resuscitated,cultured and passaged.Lentiviral vectors were used to construct ovarian cancer cells SKOV3-LUC,OVCAR3-LUC and ES2-LUC that were stably expressed luciferase,using log-grown ovarian cancer cells as target cells for the killing experiment.3.The experiment is divided into five groups,including CIK group(single application of CIK),OK432 group(single application of OK432),OK432 pretreatment group(OK432 overnight treatment of tumor cells,discard the supernatant and apply CIK),combined group(CIK combined with OK432)and control group.After pretreatment of target cells,co-cultivation with effector cells for 4h as the effect-target ratios were 5:1,10:1 and 20:1 and then adding D-luciferin(substrate),the photon number generated by substrate oxidation and luminescence is positively related to the concentration of luciferase.The killing effects of each group on the ovarian cancer cells were detected by the enzyme-labeled instrument,and the killing rate was calculated.4.The expression levels of IFN-γ,IL-1β,IL-2,IL-6,IL-10 and IL-12 of ovarian cancer cells of CIK group,OK432 pretreatment group and combined group were detected by real-time quantitative PCR.Results:1.PBMCs were isolated and purified from healthy human peripheral blood.Under the influence of cytokines,primary CIK cells were prepared and CIK cells expansion were initiated.After the detection of bacteria and endotoxin were negative,CD4-CD8+69.6%,CD3+CD56+18%and CD3+CCR7+32.5%of CIK cells immunophenotype were detected by flow cytometry.With the extension of the culture time,the percentage of CD4-CD8+and CD3+CD56+T lymphocytes gradually increased,and the percentage of CD3+CCR7+T lymphocytes increased rapidly,gradually decreasing after the sixth day,indicating that the cells continued to differentiate into CIK cells.2.Observation of luciferase-expressing ovarian cancer cells constructed by lentiviral vectors under a fluorescent microscope showed extensive luminescence,indicating successful labeling.3.The results of in vitro killing experiments showed that the killing rate of CIK group,OK432 pretreatment group and combined group on ovarian cancer cells gradually increased with the increase of the effect-target ratio.Under the same effect-target ratio,the killing rates of the OK432 pretreatment group and the combined group were greater than that of the CIK group,and the differences were statistically significant(P<0.05).When the effect-target ratio of OVCAR3-LUC was 10:1 and 20:1,the killing rate of the combined group was greater than that of the OK432 pretreatment group,the difference was statistically significant(P<0.05).It shows that the OK432 pretreatment group and the combined group have stronger anti-tumor effects of lymphocytes than the CIK group,and the combined group has a stronger antitumor cell effect of lymphocytes than the OK432 pretreatment group.The killing rates of OK432 group on SKOV3-LUC,ES2-LUC,OVCAR3-LUC were 4.33%,5.40%,5.45%,respectively,indicating that OK432 has a direct killing effect on ovarian cancer cells.4.The results of real-time fluorescence quantitative PCR detection of cytokines showed that for SKOV3-LUC cells,regarding the expression of cytokines IFN-y,IL-12,IL-1β,compared with CIK group,OK432 pretreatment group and combined group showed high expression,the differences were statistically significant(P<0.05).Regarding the expression of cytokines IL-2 and IL-6,compared with CIK group,OK432 pretreatment group showed high expression,the difference was statistically significant(P<0.05).Regarding the expression of cytokine IL-6,compared with CIK group,the combined group showed low expression,the difference was statistically significant(P<0.05).Regarding the expression of cytokine IL-12,the combined group was higher than the OK432 pretreatment group,the difference was statistically significant(P<0.05).Regarding the expression of cytokine IL-10,compared with CIK group,the combined group showed high expression,the difference was statistically significant(P<0.05).For ES2-LUC cells,regarding the expression of cytokines IFN-y,IL-12,IL-2,compared with CIK group,OK432 pretreatment group and combined group showed high expression,the differences were statistically significant(P<0.05).Regarding the expression of cytokine IL-1β,compared with CIK group,the combined group showed high expression,the difference was statistically significant(P<0.05).Regarding the expression of cytokine IL-6,compared with CIK group,OK432 pretreatment group showed high expression,the difference was statistically significant(P<0.05).Regarding the expression of cytokines IFN-γ,IL-12,IL-1β,IL-2,the combined group was higher than OK432 pretreatment group,the difference was statistically significant(P<0.05).Regarding the expression of cytokine IL-10,compared with CIK group,the combined group showed high expression,the difference was statistically significant(P<0.05).For OVCAR3-LUC cells,regarding the expression of cytokines IL-12,IL-1β,IL-2,IL-6,compared with CIK group,OK432 pretreatment group and combined group showed high expression,the differences were statistically significant(P<0.05).Regarding the expression of cytokine IFN-γ,compared with CIK group,the combined group showed high expression,the difference was statistically significant(P<0.05).Regarding the expression of cytokines IL-12,IL-1β and IL-2,the combined group was higher than that of OK432 pretreatment group,the difference was statistically significant(P<0.05).Regarding the expression of cytokine IL-6,the OK432 pretreatment group was higher than the combined group,and the difference was statistically significant(P<0.05).Regarding the expression of cytokine IL-10,compared with CIK group,OK432 pretreatment group showed low expression,the combined group showed high expression,the differences were statistically significant(P<0.05).It showed that the OK432 pretreatment group had a stronger immune activation effect than the CIK group,and the combined group had a stronger immune activation effect than the OK432 pretreatment group.OK432 promotes the expression of immune activating factors.Conclusion:1.OK432 can enhance the killing effect of CIK on ovarian cancer cells,and with the increase of the effective target ratio,OK432 enhances the anti-tumor effect of CIK more obviously.2.OK432 enhances the killing effect of CIK on ovarian cancer cells,which may be related to OK432 promoting the secretion of immune active factors.