Role And Mechanism Of Incomplete Microwave Ablation In The Treatment Of Advanced Oligometastatic Non-Small Cell Lung Cancer

About 80% of lung cancer patients were Non small cell lung cancer(NSCLC).Nearly half of NSCLC patients had metastasis at the first diagnosis,could not be treated with radical surgery and be cured.Systemic therapy was the main treatment for metastatic advanced NSCLC.Metastatic advanced NSCLC included a special population.Could the people with a few metastases(small number and small range of metastases)achieved better clinical benefits by adding local treatment on the basis of systemic antitumor treatment.In recent years,ablation technology had developed rapidly.Among them,microwave ablation(MWA)had the advantages of faster temperature rise,simpler operation and easier clinical promotion than other ablation methods,so it had more prospects in the clinical application of lung cancer.MWA could be divided into complete MWA to clinical necrosis and incomplete MWA to reduce the tumor load,alleviate the symptoms caused by tumor and improve the quality of patients life.At present,the research of the effect and mechanism of incomplete MWA in the treatment of advanced oligometastatic NSCLC is still in a relatively blank stage.Therefore,it is a great practical significance to clarify the effect of incomplete MWA in the treatment of advanced oligometastatic NSCLC and explore the relationship between the thermal effect of incomplete MWA and the immune function and prognosis of advanced oligometastatic NSCLC.The main content of this study is divided into the three parts:Part One Clinical efficacy and immune function of incomplete microwave ablation in the treatment of advanced oligometastatic non-small cell lung cancerObjective: By analyzing the local control rate,progression free survival(PFS)and overall survival(OS)of incomplete MWA in the treatment of advanced oligometastatic NSCLC,to explore the correlation between different immune cell peripheral blood levels and the clinical and prognosis of patients with advanced oligometastatic NSCLC.Methods: Patients with advanced oligometastatic NSCLC with negative driver gene were selected as the research object model.They were randomly divided into two groups.The experimental group was treated with primary incomplete MWA combined with radiotherapy and chemotherapy ±immunotherapy,and the control group was treated with radiotherapy and chemotherapy ± immunotherapy.The local control rate,progression free survival,complications and corresponding immune cell expression between the two groups were compared.Results: The local control rates of the experimental group and the control group were 94.44%(34/36)vs 66.67%(24/36),the difference of local control rate was significant(P<0.05).The PFS of the experimental group and the control group was 7.8vs6.2 months,the difference was statistically significant(P<0.05).There was no significant difference in OS between the experimental group and the control group(P>0.05).T lymphocytes from patients on the first day after incomplete MWA were detected and analyzed.CD3+T,CD4+T and CD8+T cells were divided into high and low expression group according to the median value.There was no significant difference in PFS between CD3+,CD4+T and CD8+T cell low and high expression group(all P>0.05).According to the subgroup analysis of treatment methods,the PFS of low and high expression group of CD3+T cells in the experimental group was 6.2vs9.3months,the difference was statistically significant(P<0.05).There was no significant difference in PFS between CD4+T cell low and high expression group(P>0.05).The PFS of low and high expression group of CD8+T cells in the experimental group was 5.9vs9.7 months,the difference was statistically significant(P<0.05).There was no significant difference in PFS between the low and high expression group of CD3+T,CD4+T and CD8+T cells in the control group(all P>0.05).In terms of OS,there was no significant difference in OS between low and high expression group of CD3+T,CD4+T and CD8+T cells(all P>0.05).According to the treatment method,the OS of low and high CD3+T cell expression groups in the experimental group was 11.5vs15.4months,the difference was statistically significant(P<0.05).There was no significant difference in OS between CD4+T cell low and high expression group(P>0.05).The OS of low and high expression group of CD8+T cells in the experimental group was 11.5vs15.3 months,the difference was statistically significant(P<0.05).There was no significant difference in OS between the low and high expression group of CD3+T,CD4+T and CD8+T cells in the control group(all P>0.05).The incidence of complications of lung injury in the experimental group was 27.78%,all of which were grade I-II pneumothorax,pulmonary hemorrhage and radiation pneumonia.The overall incidence of radiation-induced pneumonia was 16.67% in the control group.There was no significant difference in the degree of lung injury between the experimental group and the control group(P>0.05).Conclusion: For NSCLC with few metastase≤3 late oligometastases and negative driving genes,combined with incomplete MWA for primary lesions on the basis of radiotherapy and chemotherapy ± immunotherapy,it can significantly improve the local control rate of tumor and prolong PFS.The high expression of CD8+T in blood after incomplete MWA suggests a better prognosis for patients combined with radiotherapy and chemotherapy ±immunotherapy.At the same time,the incidence of complications did not increase significantly.Part Two Effect of incomplete microwave ablation on HSP70 and CD8+ T cells in lung cancer microenvironmentObjective: The clinical value of incomplete MWA was preliminarily discussed by comparing the short-term effect of complete and incomplete MWA,CD8+T cell infiltration and HSP70 expression in tumor tissue and serum at different time points.Methods: A mouse model of primary lung cancer was established to simulate the treatment state of complete and incomplete MWA.It was randomly divided into three groups.The complete MWA group was completely covered with MWA of tumor lesions,the incomplete MWA group was completely ablated with incomplete MWA of tumor lesions,and the control group was not treated.The tumor growth was observed and the changes of CD8+T cells in each group were detected according to the time relationship,Then according to the time relationship,the changes of HSP70 mRNA in CD8+T cells were detected by q PCR,and the changes of HSP70 in tumor tissues were detected by Western blot.The expression levels of CD8+T cells in blood after complete and incomplete MWA were compared by flow cytometry.The expression levels of HSP70 in blood after complete and incomplete MWA were compared by ELISA.QPCR was used to detect the relative expression of perforin and granzyme B mRNA in CD8+T cells in blood,and the changes of mRNA in different time groups were compared.Results: The tumor volume of the three groups had no significant difference before treatment(P>0.05).After treatment,the tumor volume of the complete MWA group did not grow,and the tumor volume and tumor growth of the incomplete MWA group were significantly lower than those of the control group.In the complete MWA group,there were no positive CD8+T cells and HSP70 protein on day 1 or day 14.On the first day,CD8+T cells in the control group were significantly higher than those in the incomplete MWA group(P<0.05).On the 14 th day,there were a large number of CD8+T cells in the control group,but it was still less than that in the incomplete MWA group(P<0.05).On the first day,the expression of HSP70 in CD8+T cells in tumor tissue in incomplete MWA group was significantly higher than that in control group(P<0.05);On the 14 th day,the expression of HSP70 in CD8+T cells in tumor tissue in incomplete MWA group was significantly lower than that in control group(P<0.05);On the first day,the expression of HSP70 in tumor tissue in incomplete MWA group was significantly higher than that in control group(P<0.05);On the 14 th day,the expression of HSP70 in tumor tissue in incomplete MWA group was significantly lower than that in control group(P<0.05);No matter on day 1 or day 14,the expression of CD8+T cells in serum of incomplete MWA group was the highest,that of complete MWA group was the second,and that of control group was the lowest.On the first day,the expression of HSP70 in incomplete MWA group was the highest,followed by complete MWA group and the lowest in control group;On the14 th day,the expression of HSP70 in the control group was the highest,that in the complete MWA group was the second,and that in the incomplete MWA group was the lowest.The relative expressions of perforin and granzyme B mRNA in CD8+T cells in incomplete MWA group on day 1 and day 14 were significantly higher than those in control group(all P<0.05).Conclusion: Incomplete MWA can also inhibit tumor proliferation.Incomplete MWA has an immediate killing effect on CD8+T cells in tumor tissue,while CD8+T cells in serum continue to increase.Incomplete MWA stimulated the transient increase of HSP70 in tumor tissue,serum and CD8+T cells.The chemotaxis of CD8+T cells in serum can further kill tumor cells.Part Three Effects of changes of HSP70 and CD8+T induced by incomplete microwave ablation on tumor and icroenvironmentObjective: By studying the effects of HSP70 on the proliferation and migration of LLC cells in vitro cell culture system,the direct contact and indirect contact between CD8+T and LLC cells(target cells)after incomplete MWA,and the effects of cell killing(target cell death)and non cell killing(cytokine secretion)of CD8+T cells after incomplete MWA in co culture system,the immune activation value and preliminary mechanism of incomplete MWA were discussed.Methods: The effects of HSP70 on the proliferation and migration of LLC tumor cells were studied by transfection of HSP70 with CCK8 to detect cell proliferation,clone,apoptosis,cycle,invasion and scratch.The direct and indirect contact co culture system of CD8+T cells in serum and LLC tumor cells was established to observe the changes of CD8+T cells from tumor tissue in the incomplete MWA.The direct cell killing function of CD8+T cells was evaluated by CCK8 cell proliferation test,and the TNF secreted by CD8+T cells was detected by ELISA-α、 IL-1β And IL-6 expression to evaluate the non cytotoxic function of CD8+T cells stimulated by incomplete MWA.Results: The interfering RNA of HSP70 was synthesized and transfected into cells to simulate the overexpression and deletion of HSP70.The cell proliferation experiment,plate cloning experiment,flow apoptosis experiment,flow cycle experiment,cell invasion experiment and cell scratch experiment showed that the overexpression of HSP70 could inhibit the proliferation,invasion and migration of tumor cells and promote the dormancy and apoptosis of tumor cells(all P<0.05).The direct and indirect contact culture systems of CD8+T cells from tumor tissue in the incomplete MWA group and LLC cells were successfully established.In the direct contact culture system,incomplete MWA can enhance the cell killing function of CD8+T cells in the tumor tissue,mainly manifested in the decrease of cell activity and the increase of death rate of LLC cells and inflammatory factor of TNF-α 、IL-1βand IL-6 decreased significantly.In the indirect contact culture system,although The levels of inflammatory factors of TNF-α 、 IL-1βand IL-6increased,but the death rate of LLC cells did not change significantly.Conclusion: The increase of HSP70 and the chemotaxis of CD8+T cells caused by incomplete MWA can further kill tumor cells.After incomplete MWA,the killing function of CD8+T cells is mainly cell killing.Conclusions:1.For advanced NSCLC with few metastases≤3 and negative driver gene,combined with incomplete MWA for primary lesions on the basis of radiotherapy,chemotherapy ± immunotherapy,it can significantly improve the local control rate of tumor and prolong PFS.The high expression of CD8+T in blood after incomplete MWA suggests that patients combined with radiotherapy,chemotherapy ± immunotherapy have a better prognosis.The incidence of complications did not increase significantly.2.Incomplete MWA can also inhibit tumor proliferation.Incomplete MWA has immediate killing effect on CD8+T cells in tumor tissue.The increase of HSP70 and chemotaxis of CD8+T cells caused by incomplete MWA can further kill tumor cells.The immune effect of HSP70 and CD8+T cells produced by incomplete MWA is better than that of complete MWA.3.The immune effect of incomplete MWA was mainly inhibited by HSP70 stimulated by thermal injury in the early stage and chemotactic CD8+T cells in the later stage.After incomplete MWA,the killing function of CD8+T cells is mainly cell killing.