Synergistic Effect Of Radiofrequency Ablation And Immune Checkpoint Inhibitors On Advanced Kidney Cancer And Its Imaging Evaluation

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The Effect of Radiofrequency Ablation Combined with Immune Checkpoint Inhibitors on Renal Tumor MetastasisObjective Radiofrequency ablation can regulate the immune function of the body while reducing the tumor burden,so there is a potential synergy with the immunological checkpoint inhibitor.This experiment is to establish a model of subcutaneous renal tumor in bilateral flank of mice.Radiofrequency ablation is used to treat one side of the tumor and combined with PD-1 blockade.The effect of the combination of the two modalities on the other tumor is observed.At the same time,to explore the synergistic mechanism of radiofrequency ablation combined with PD-1 inhibitor in advanced renal cell carcinoma,and provide a theoretical basis for clinical application.Materials and Methods A BALB/c mouse bilateral flank abdomen subcutaneous renal tumor model was constructed,and the successfully modeled mice were randomly grouped according to a predetermined protocol.On the 14th day after inoculation of the left flank tumors of the mice,when the tumor volume reached about 500mm~3,the left tumors of the mice randomly assigned to the radiofrequency ablation group and the combined treatment group were subjected to radiofrequency ablation,and the combined treatment group was intraperitoneally injected with 200?g anti-PD-1 monoclonal antibody.The anti-PD-1 monoclonal antibody was injected every three days for three times.The size of the subcutaneous tumor on the right side was dynamically measured,and the diet,activity,and body weight of the mice after treatment was observed.Flow cytometry,enzyme-linked immunosorbent assay,enzyme-linked immunospot assay,Western blotting and other methods were used to detect the expression of cytokines and others in peripheral blood,spleen,tumor draining lymph nodes and tumor cells.The tumor challenge test was performed to observe the immunological memory effect of the combination therapy.Results Two weeks after radiofrequency ablation,the contralateral tumor tissues were excised and Western blot analysis was performed.The results showed that radiofrequency ablation could up-regulate the expression of PD-L1 in tumor cells(P<0.001),and the level was about1.5 times that of the control group.Compared with the control group,both sequential therapy and simultaneous treatment significantly inhibited the growth of distant tumors(P<0.001).The complete response rate of the sequential treatment group was 62.5%(5/8),while the complete response rate was 50.0%(4/8)in the simultaneous treatment group.Compared with the control group,the surgical resection group,and the IgG2b group,the PD-1 inhibitor alone inhibited tumor growth to a certain extent,but similar to radiofrequency ablation alone,the inhibition was not obvious,and the tumor growth continued to be persistent.When PD-1inhibitors were combined with radiofrequency ablation,the growth of contralateral tumors was significantly inhibited(P=0.0023).Radiofrequency ablation combined with PD-1inhibitors relied on CD8~+T cells to exert synergistic anti-tumor effects.Combination therapy can greatly increase the proportion of mature dendritic cells in tumor-draining lymph nodes.Radiofrequency ablation combined with PD-1 inhibitor significantly increased the proportion of Th1 cells(15.67%±0.81%,P<0.0001),about 1.5 times that of the control group,and decreased the proportion of Th2 cells(1.60%±0.05%,P=0.0001),the ratio of Th1/Th2 cells in the combination group was significantly higher than that in the control group(9.81±0.34,P<0.0001),which was about twice that of the control group.Radiofrequency ablation combined with PD-1 inhibitor can further increase the ratio of IFN-g~+CD8~+TIL and TNF-a~+CD8~+TIL in distant tumors based on single PD-1 monoclonal antibody therapy.The ratio of IFN-g~+CD8~+TIL was 3.83%(±0.12%),which was about 8 times that of the control group(P<0.0001),and the ratio of TNF-a~+CD8~+TIL was 1.60%(±0.06%),which was about4 times that of the control group(P<0.0001).Combination therapy can not only significantly increase the infiltration of lymphocytes in the tumor,but also reduce the proportion of tumor infiltrating regulatory T cells(CD4~+CD25~+Foxp3~+)(3.83%(±0.13%),P<0.0001),The ratio of CD4 effect T and CD8~+T cells to regulatory T cells was approximately 5 and 4 times higher than that of the control group,respectively(P<0.0001).Tumor challenge test results showed that radiofrequency ablation combined with PD-1 inhibitor can induce durable tumor antigen-specific CD8~+T cell immune response.Conclusions Radiofrequency ablation combined with PD-1 inhibitor can enhance the systemic anti-tumor immune response while destroying the primary tumor lesion,and then inhibit the growth of distant lesions.Radiofrequency ablation can increase the expression of PD-L1 on the surface of tumor cells.Radiofrequency ablation combined with immunological checkpoint inhibitors can promote the maturation of dendritic cells in tumor-draining lymph nodes,enhance the body's Th1 immune response,increase the number of tumor infiltrating lymph cells in distant tumors and up-regulate of the ratio of T cells to regulatory T cells,while inducing the body to produce a durable tumor-specific anti-tumor immune response.Part ? Radiofrequency Ablation Combined with Immune Checkpoint Inhibitors for Advanced Kidney Cancer and Inhibition of Tumor MetastasisObjective To observe the effect of radiofrequency ablation on renal tumors and combined with different mechanisms of immune checkpoint inhibitors on lung mimetic metastases in a mouse model of orthotopic renal tumor and lung metastasis.To explore different combination therapy strategies and find the most excellent joint program to improve the effect of combined treatment and reduce systemic side effects.Materials and Methods The BALB/c mouse orthotopic renal tumor and lung simulated metastasis model were constructed.The successfully modeled mice were randomly divided according to the predetermined protocol.The left kidney tumor of the mice was randomly distributed about 10 days after inoculation.Radiofrequency ablation was performed on left kidney tumors in the RFA group and the combination treatment group.According to the predetermined protocol,each mouse was intraperitoneally injected with 200 ?g of anti-PD-1 monoclonal antibody(or 200 ?g of anti-CTLA-4 m Ab,or a combination of both),and the halved group was injected with 100 ?g of the above monoclonal antibody,the injection was performed every three days for three times,and the change in body weight was measured dynamically and the survival state of the mice was observed.The lesions of the lungs from different treatment groups were evaluated by weighing and HE staining.Results The mouse model of orthotopic renal carcinoma and simulated lung metastasis were successfully constructed by injecting Renca cells into the subcapsular and tail vein.When radiofrequency ablation combined with two different mechanisms of immune checkpoint inhibitors,the survival time of mice can be further prolonged.The survival time of this group of mice is(40.63±4.34)days.Compared with radiofrequency ablation combined with single immunological checkpoint inhibitor group,the survival extension was about 10 days(P<0.0001).The effect of combined therapy on lung metastases was evaluated by weighing the lungs.The results showed that radiofrequency ablation combined with dual immune checkpoint inhibitors significantly inhibited the formation of lung tumor lesions in mice.Radiofrequency ablation combined with low-dose dual-immunization checkpoint inhibitors significantly prolonged survival.The mean survival time of this group of mice was(39.75±5.01)days,which was not significantly different from radiofrequency ablation combined with conventional dose dual immune checkpoint inhibitors(P=0.714).At the same time,radiofrequency ablation combined with low-dose dual-immune checkpoint inhibitors can inhibit the formation of lung metastases.The average lung weight of this group of mice is(0.27±0.05)grams.There was no significant difference compared with radiofrequency ablation combined with conventional dose dual immune checkpoint inhibitors(P=0.302).The use of lower dose dual immune checkpoint inhibitors can effectively inhibit tumorigenesis,prolong survival,and reduce the incidence of adverse events.Conclusions Radiofrequency ablation combined with dual immune checkpoint inhibitors can enhance the body's anti-tumor immune response,inhibit tumor metastasis and prolong survival time.Radiofrequency ablation and low-dose dual-immune checkpoint inhibitors still have a synergistic effect,which does not reduce the efficacy of combination therapy.And in theory,this combination strategy can reduce treatment-related adverse effects.Part ? Bioluminescence Imaging Monitoring of Radiofrequency Ablation Combined with Immune Checkpoint Inhibitors for the Treatment of Advanced Kidney CancerObjective To establish a mouse model of orthotopic renal tumor and lung metastasis using Renca-Luc2-GFP cells,and to monitor the therapeutic effect of radiofrequency ablation combined with immune checkpoint inhibitors by bioluminescence imaging,and to evaluate the feasibility of bioluminescence imaging for early,real-time,dynamic,and non-invasive evaluation of radiofrequency ablation combined with immune checkpoint inhibitors.Materials and Methods The mouse kidney cancer cells(Renca-Luc2-GFP)stably expressing green fluorescent protein and luciferase were amplified in vitro.The expression of green fluorescence in tumor cells was observed by inverted fluorescence microscope.The activity of luciferase was detected by bioluminescence imaging.The proliferation,migration and chemotaxis of tumor cells were evaluated by cell proliferation experiments,scratch experiments and chemotaxis experiments.In situ renal tumor models were constructed using Renca-Luc2-GFP cells and Renca cells,respectively,to compare the tumorigenicity of the two cells in mice.The orthotopic renal tumor model was constructed by subcapsular injection of Renca-Luc2-GFP cells.Radiofrequency ablation of renal tumors was performed 10 days after inoculation,and bioluminescence imaging and magnetic resonance imaging were used to evaluate the early treatment effects.The above-mentioned orthotopic renal tumor model was constructed.On the 10 th day after modeling,tumor cells were injected from the tail vein to construct a simulated lung metastasis tumor model.The next day,renal tumor radiofrequency ablation was performed,and intraperitoneal injection of PD-1 m Ab was performed at 1,4,and 7 days after radiofrequency ablation.Bioluminescence imaging was performed every 3 days for the purpose of dynamic monitoring of radiofrequency ablation combined with PD-1 m Ab treatment.Results Renca cells and Renca-Luc2-GFP cells in logarithmic growth phase were observed under an inverted microscope.There was no significant difference in morphology between them.No expression of green fluorescent protein was observed in control Renca cells,while strong green fluorescence was observed on Renca-Luc2-GFP cells.The bioluminescence intensity of Renca-Luc2-GFP cells was positively correlated with the number of cells(R2=0.9981).Double reporter gene transfection did not affect tumor cell proliferation,migration and chemotaxis.The bioluminescence imaging system can stably detect the stable expression of luciferase in mice.As the tumor growed,the area of luciferase expression increased,and the number of detected fluorescent photons increased.On the 7th day after inoculation,the orthotopic kidney tumor was imaged by 7.0T magnetic resonance.Renca-Luc2-GFP cells and Renca cells were able to grow in mouse kidney,and the tumor model was successfully constructed,and there was no significant differenc between the tumor sizes.After single radiofrequency ablation,the bioluminescence intensity of the original kidney tumor area was significantly weakened,but a strong bioluminescence signal could be detected after one week,and increased with time,and the lung bioluminescence signal intensity gradually increased.After radiofrequency ablation combined with PD-1 inhibitor treatment,the bioluminescence signal intensity of the kidney and lung increased slowly and was significantly weaker than the single radiofrequency ablation group.The results showed that bioluminescence imaging can be used to dynamically monitor the residual of primary lesions and the growth of distant metastatic lesions after radiofrequency ablation combined with immune checkpoint inhibitors.Conclusions Renca-Luc2-GFP cells can be used to construct mouse orthotopic renal carcinoma and simulated lung metastasis tumor models,which provides a new tool for the study of combined immunotherapy strategies.Bioluminescence imaging can non-invasively,real-time and dynamically assess the occurrence and development of primary lesions and distant metastatic lesions during tumor treatment.It can be used to dynamically monitor the effects of radiofrequency ablation combined with immune checkpoint inhibitors in the treatment of advanced tumors.