Inhibit Mouse Liver Cancer By Thermal Ablation Combined With Peritumoral Injected CpG ODN

Objective: To investigate the therapeutic effect of thermal ablation combined with peritumoral injected CpG ODN on mouse liver cancer, and to observe the antitumor immune response against mouse liver cancer.Methods Subcutaneous liver cancer model was established in mice.Mice were randomly divided into 4 groups when the tumor long diameter reached 6-10mm.①Combination therapy group: subcutaneous tumor were given ablation with 0.1ml 100℃saline solution, then 0.1ml 1mg/ml CpG ODN was injected multiple peritumoral at 10mins and 7 days after thermal ablation;②Thermal ablation therapy group: Subcutaneous tumor were given ablation with 0.1ml 100℃saline solution, then 0.1ml saline solution was injected multiple peritumoral at 10mins and 7 days after thermal ablation;③CpG ODN therapy group: 0.1ml 1mg/ml CpG ODN was multiple peritumoral injected into the subcutaneous tumor, when the tumor long diameter reached 6-10mm, then the same treatment was given at 7days after 1st treatment;④Control group: No treatment was given. The parameters were detected as follow(1) Eight mice were selected randomly in each group;①tumor volume were measured in 15 days. and tumor inhibition rate were calculated.②morality rate were observed in 60 days after the mice received the first treatment.⑵Eight mice was selected randomly in each group, were killed at 24 hours after 1st treatment, HSP70 expression in the tumor tissue were were detected by immunohistochemistry,and label of HSP70 was caculated in each group.⑶Eight mice were selected randomly in each group, were killed at 15 days after 1st treatment,①tumor residual rate was calculated by HE stain of tomor tissue.②the number of CD4+T and CD8+T were measured by immunohischemistry,and the ratio of CD4+T/CD8+T was calculated (4)Eight mice in each group that survival at 30 days after 1st treatment were choosed, they were rechallenged with the same tumor cells subcutaneously at the contralateral, the tumor emerging rate were observed in each group.Results:⑴The tumor volume and tumor inhibition rate: compared with tumor volume before treatment, tumor volume in combination therapy group has reduced 140.94mm3 in 15 days after treatment , which has reduced 44.42mm3 in thermal ablation therapy group and 87.68mm3 in CpG ODN therapy group, but it has increased 1263.94mm3 in control group. Every experimental group has reduced significant than control group(P<0.05), and combination therapy group has reduced more significant. But it has no significant difference between each experimental group. The tumor inhibition rate was 98.43% in combination therapy group, 91.05% in thermal ablation therapy group and 96.64% in CpG ODN therapy group. Tumor inhibition rate in each experimental group are higher than control group, tumor inhibition rate in combination therapy group was higher than thermal ablation therapy group and CpG ODN therapy group.⑵Mortality rate: the mortality rate in combination therapy group was 12.5% (1/8),which is 37.5%(3/8)in thermal ablation therapy group and CpG ODN therapy group, 87.5%(7/8)in control group. the mortality rate in each experimental group was significantly lower than control group (P<0.05) . The mortality rate in combination therapy group was significantly lower than thermal ablation therapy group and CpG ODN therapy group(P<0.05).⑶Labeling index of HSP70 (LI HSP70): LI HSP70 is(22.07±2.64)% in combination therapy group, (16.37±2.03)% in thermal ablation therapy group, (10.74±3.60)% in CpG ODN therapy group and (4.25±1.84)% in control group. Every experimental group has more HSP70 than control group, and combination therapy group has more HSP70 than other groups. Compared with thermal ablation therapy group and CpG ODN therapy group,LI HSP70 in combination therapy group has significant difference(P<0.05), combination therapy group has more HSP70 expression.⑷Tumor residue rate: Tumor residue rate in combination therapy group is 12.5%(1/8), which is 25%(2/8)in thermal ablation therapy group , 87.5%(7/8) in CpG OND therapy group and 100%(8/8)in control group. Tumor residue rate in combination therapy group has no significant difference compared with thermal ablation therapy group. Tumor residue rate in combination therapy group was significant lower than CpG ODN therapy group, and the significant difference also existed between therapy ablation therapy group and CpG ODN therapy group(P<0.05).⑸The number of CD4+ T cells and CD8+ T cells in tumor tissue:①CD4+ T cells number is 52.75±4.15 in combination therapy group, 26.00±1.79 in thermal ablation therapy group ,34.13±1.67 in CpG ODN therapy group and 12.25±1.03 in control group. Compared with thermal ablation therapy group and CpG ODN therapy group , CD4+ cell number in combination therapy increased significantly(P<0.05).②CD8+ T cells number was 10.50±1.17 in combination therapy group, 9.50±1.87 in thermal ablation therapy group,13.63±1.12 in CpG ODN therapy group and 10.63±1.3 in control group.There are no significant differenc between each experimental group and control group③The ratio of CD4+T/CD8+T was 5.02 in combination therapy group, 2.74 in thermal ablation therapy group,2.51 in CpG ODN therapy group and 1.15 in control group. The ratio of CD4+T/CD8+T in each experimental group were higher than control group, The ratio of CD4+T/CD8+T in combination therapy group has significant difference compared with thermal ablation therapy group and CpG ODN therapy group(P<0.05).⑹Tumor formation rate after tumor cells were rechallenged at opposite side of the mouse body: Tumor formation rate in combination therapy group is 12.5%(1/8), which is 25%(2/8)in thermal ablation therapy group , 37.5%(3/8)in CpG ODN therapy group and 100%(8/8)in control group. Compared with control group, Tumor formation rate in each experimental group are lower significantly(P<0.05). Tumor formation rate in combination therapy group decreased significantly than thermal ablation therapy group and CpG ODN therapy group (P<0.05), but there is no significant difference between thermal ablation therapy group and CpG ODN therapy group.Conclusion: 1. Peritumoral injection of CpG ODN in mouse subcutaneous liver cancer can enhance the local antitumor immunity response at some extent. 2. The expression of HSP 70 in subcutaneous liver cancer cells can be improved by thermal ablation. 3. Thermal ablation combined with peritumoral injection of CpG ODN can strengthen the host antitumor immunity effect, which can inhibit the growth of rechallenged tumor cells. These results indicate that thermal ablation combined with peritumoral injection of CpG ODN maybe a promising method to prevent the recurrence after tumor ablation.