Effects Of Mesenchymal Stem Cells On Immunotherapy Sensitivity In Non-small Cell Lung Cancer And Underlying Mechanisms

Lung cancer is one of the most common malignancies and the leading cause of cancer-related deaths worldwide.According to pathological types,lung cancer can be divided into small cell lung cancer and non-small cell lung cancer(NSCLC),of which NSCLC is the most common pathological type,accounting for about 85%of the total number of lung cancer patients.Because lung cancer lacks specific clinical symptoms when it is in the early stage,most NSCLC patients are already diagnosed at the advanced stage.In recent years,immunotherapy represented by anti-PD-1/PD-L1 has developed rapidly,providing new treatments for patients with advanced NSCLC,especially patients without driver gene mutation,and significantly improves the prognosis.Although anti-PD-1/PD-L1 immunotherapy can significantly prolong the progressive free survival and overall survival of advanced NSCLC,and it has the benefits of long-term survival,its efficient is low.Therefore,clarifying the factors and participation mechanisms which affect the sensitivity of immunotherapy has important theoretical research significance and clinical value.Studies have shown that the various biological behaviors of tumors,including their sensitivity to anti PD-1/PD-L1 immunotherapy,are not only related to the characteristics of tumor cells themselves,but the tumor microenvironment.Mesenchymal stem cells(MSCs)are a type of multipotentially differentiated stem cells that almost exist in all tissues and participate in tissue damage and repair processes.Recent studies have shown that,MSCs could migrate into tumor tissues and become the important components of the tumor microenvironment which played a key role in the regulation of tumor cell apoptosis,epithelial-mesenchymal transition,stem cell proliferation,angiogenesis and local immune response.In recent years,numerous studies have been conducted on the interaction of MSCs and tumors.Studies have shown that MSCs could not only promote the growth of tumor cells,but suppress immune response and promote tumor metastasis by releasing the immunosuppressive factors such as IL-10,nitric oxide and kynurerine.MSCs also play an important role in anti-tumor therapy resistance such as chemotherapy,radiotherapy,and targeted therapy.However,little research has been done on the effects of MSCs in the tumor microenvironment on the sensitivity of immunotherapy and related mechanisms.In this study,we systematically studied the effects of MSCs on the sensitivity of anti PD-1 immunotherapy in NSCLC and its related mechanisms through in animal experiments,and further verified by clinical data analysis.Part ? Mechanisms of mesenchymal stem cells decreasing the sensitivity of anti PD-1 immunotherapy in non-small cell lung cancerObjective:To study the effect of MSCs on the sensitivity of immunotherapy of NSCLC through lung cancer tumor-bearing mouse model,and explore its possible participation mechanisms.Methods:1.MSCs and Lewis lung carcinoma(LLC)cells are routinely cultured.2.The tumor-bearing mouse model was established by subcutaneous injection,and meanwhile MSCs was also injected subcutaneously.The anti-PD-1 antibody was injected intraperitoneally.Tumor size was regularly measured,and the effect of anti-PD-1 antibody on tumor growth was recorded.3.The mice were killed as planned,and then tumor size and weight were measured.Tumor tissue and peripheral blood of the mice was taken for subsequent experiments.4.Flow cytometry was used to analyzed the immunosuppressive cells in peripheral blood.5.Flow cytometry were used to analyzed the immunosuppressive cells in tumor tissue.Results:1.Successfully cultivate MSCs,observe and record their shape using the confocal microscope.2.In the LLC tumor-bearing mouse models,anti-PD-1 antibody significantly suppressed tumor growth in vivo(anti-PD-1 group vs control group,P<0.05),and MSCs significantly reversed the tumor suppressive effect of anti-PD-1 antibody(MSCs+ anti-PD-1 group vs anti-PD-1 group,P<0.05).3.Flow cytometry showed that in peripheral blood,the proportion of PMN-MDSCs,M-MDSCs,TAMs in LLC+MSCs+anti-PD-1 Ab group was significantly increased compared to LLC+anti-PD-1 Ab group,P<0.05;the proportion of PMN-MDSCs in LLC+anti-PD-1 Ab group was significantly decreased compared to LLC control group,P<0.05.4.Flow cytometry showed that in tumor tissues,the proportion of PMN-MDSCs in LLC+MSCs+anti-PD-1 Ab group was significantly increased compared to LLC+anti-PD-1 Ab group,P<0.05.However M-MDSCs and TAMs in LLC+MSCs+anti-PD-1 Ab group was not in significant difference compared to LLC+anti-PD-1 Ab group,P>0.05;the proportion of PMN-MDSCs,M-MDSCs and TAMs in LLC control group was not in significant difference compared to LLC+anti-PD-1 Ab group,P>0.05.Conclusion:The tumor-bearing mouse model showed that MSCs,an important component of the tumor microenvironment,significantly reduced the antitumor effect of anti-PD-1 antibodies in vivo,and further testing found that the proportion of these immunosuppressive cells such as PMN-MDSCs,M-MDSCs,TAMs in tumor tissues and peripheral blood had significantly increased,which suggested that MSCs could reduce the sensitivity of immunotherapy by promoting the recruitment of these immunosuppressive cells.Part ? The significance and mechanisms of angiotensin ?receptor gene expression in the prognosis of non-small cell lung cancerObjective:The formation of inhibitory tumor immune microenvironment is the main reason for the failure of immunotherapy.In the first part of the study,we found that the proportion of MDSCs in tumor microenvironment had increased,but the mechanism was still unclear.Studies had shown that angiotensin ? was the main cause of infiltration of MDSCs,TAMs and Tregs,and it played a key role in tumor microenvironment through its receptor AT1R.At present,the relationship between angiotensin ? or its receptors and the infiltration of MDSCs,TAMs and Tregs in tumor microenvironment of NSCLC is unclear.In this part of the study,The Cancer Genome Atlas(TCGA)database was used to explore the correlation between the expression of angiotensin ? receptor(AGTR)gene and the prognosis of NSCLC patients,and to study the factors affecting the prognosis from the perspective of tumor infiltrating lymphocytes.Methods:1.We obtained the data of angiotensin ? receptor,type 1 gene(AGTR1),angiotensin ? receptor,type 2 gene(AGTR2)expression level,prognosis,and tumor infiltrating lymphocytes(TILs)of NSCLC patients from the TCGA database.2.Using the online analysis website GEPIA to analyze the relationship between the expression level of AGTR1,AGTR2 and the overall survival of NSCLC.3.Using the online analysis website TISIDB to analyze the correlation between AGTR1,AGTR2 expression level and the TILs in lung squamous cell carcinoma(LUSC)and lung adenocarcinoma(LUAD).TILs include MDSCs,TAMs,and Tregs.4.Statistical analysis,Spearman correlation test was used to analyze the correlation between AGTR1,AGTR2 expression and TILs.OS analyses was performed using the Kaplan-Meier method,and the differences between groups were analyzed by using the log-rank test.P-values<0.05 was considered to have a significant statistical difference.Results:1.Cut-off value and survival analysis of AGTR1 expression level in NSCLC patientsThe cut-off value of AGTR1 expression level was calculated by OS.By using the cut-off value,the AGTR1 expression level was divided into AGTR1 low expression group(low AGTR1)(?55%)and AGTR1 high expression group(high AGTR1)(>55%).For lung squamous cell carcinoma,the prognosis of patients with low AGTR1 was better than that with high AGTR1(P=0.034).But for lung adenocarcinoma the level of AGTR1 expression had no significance to the prognosis(P=0.11).2.The correlation between AGTR1 expression level and TILs in NSCLCThe expression level of AGTR1 was positively correlated with the infiltration of MDSCs(P<0.001 for LUSC,P=0.0387 for LUAD),TAMs(P<0.001 for LUSC,P<0.001 for LUAD)and Tregs(P<0.001 for LUSC,P<0.001 for LUAD)in NSCLC tumor tissue.3.Cut-off value and survival analysis of AGTR2 expression level in NSCLC patientsThe cut-off value of AGTR2 expression level was calculated by OS.By using the cut-off value,the AGTR2 expression level was divided into AGTR2 low expression group(AGTR2 low)(?52%)and AGTR2 high expression group(AGTR2 high)(>52%).The level of AGTR2 expression had no significance to the prognosis in NSCLC(P=0.063 for LUSC,P=0.11 for LUAD.4.The correlation between AGTR2 expression level and TILs in NSCLCThe correlation between the expression level of AGTR2 gene and the infiltration of MDSCs,TAMs,Tregs in lung cancer tissues was weak.Conclusion:AGTR1 as the coding gene of AT1R was significantly associated with poor prognosis of lung squamous cell carcinoma,and it could significantly increase the infiltration of immunosuppressive cells such as MDSCs,TAMs and Tregs in the tumor microenvironment.But for lung adenocarcinoma,there is no significant correlation with the prognosis.AGTR2 as the coding gene of AT2R had no significant correlation with the prognosis of NSCLC.