Research On The Role And Mechanism Of Microglia On Brain Metastatic Colonization Of Non-small Cell Lung Cancer

Part Ⅰ The interactions between microglia and non-small cell lung cancer cells promote brain metastatic colonizationObjective: Brain metastasis is one of the leading causes of death in patients with non-small cell lung cancer.The mechanism of brain metastasis is unclear,and there is still a lack of effective treatment.In view of the increasingly significant role of tumor-associated macrophages in tumors,therapeutic approaches targeting macrophages have also attracted extensive attention.As native macrophages in the brain,the roles and molecular mechanism of microglia in brain metastasis from non-small cell lung cancer cells have not been revealed.This research aims to study the interactions between microglia and lung cancer cells in the brain metastatic microenvironment.Methods: Immunohistochemical staining was performed on the brain metastatic sections of non-small cell lung cancer from human and nude mice.Activated microglia cells were stained and analyzed with IBA1.In vitro,Transwell invasion and migration were used to select the lung cancer cell lines for further researches.Treated with conditioned medium of A549 cells,the expression of M1(i NOS and CD86)and M2(Arg1 and CD206)markers in microglia were detected by western blot,q RT-PCR and immunofluorescence assay.Then,we induced mesenchymal lung cancer cells with TGFβ1 and the supernatant of specific activated microglia was used to treat mesenchymal lung cancer cells.We analyzed the differences between epithelial(E-cadherin)and mesenchymal(Vimentin)molecules at the transcriptional as well as protein level.Results: In vivo study,we have observed the presence of large numbers of activated microglia in brain metastases from non-small cell lung cancer both from humans and nude mice.Further research on phenotypic molecular markers of macrophages indicated that microglia prominently existed with M2-phenotype in brain metastases.In vitro experiments,we have observed that A549 cells were with stronger invasion as well as migration ability,and were selected for further study.Treated with the supernatant of A549 cells,the microglia began to exist with M1-phenotype and the microglia were dominated by M2-phenotype after a certain period of time.The conditioned medium of M2-phenotype microglia can reverse the phenotype of mesenchymal A549 and H292 cells and induce mesenchymal-epithelial transformation.Conclusion: The interaction between microglial and tumor cells in brain metastases of nonsmall cell lung cancer is crucial for the brain metastatic colonization of tumor cells that have undergone epithelial-mesenchymal transformation in the primary lesion.Part Ⅱ Brain metastatic non-small cell lung cancer cells induced M2 microglia by activating the IL6/JAK2/STAT3 pathwayObjective: The interactions between tumor microenvironment and tumor cells are essential for the progression of metastasis,but the detailed mechanism has not been studied so far.Considering the promoting role of M2-phenotype tumor-associated tumor cells in tumors,we hope to investigate at the molecular level on how the microglial phenotype is affected by brain metastatic lung cancer cells.In this part,we plan to find interventions for controlling the phenotype of microglia shaped by brain metastatic lung cancer cells.Method: A549 cells were used as parents to construct cell line with highly propensity for brain metastases through in vivo circulation.Their epithelial / mesenchymal status as well as invasion and migration ability were analyzed by western blot and Transwell assay,respectively.Single-cell RNA sequencing was used to analyze the distribution of subsets of microglial cells and the related signaling pathways in brain metastases.The transcriptome microarray was used to analyze the differences of each gene in the high-propensity brain metastatic cells or in the co-culture system and find the key gene for the interaction.Then,the GO and KEGG pathways enriched analyses were used to find the signaling pathways involved in the formation of M2-phenotype microglia.After intervention with small molecule inhibitors,the activation status of signaling pathway were detected by western blot.Finally,dual luciferase reporter genes analysis was used to predict the regulation mechanism of M2-phenotype microglia at transcription level.Result: The epithelial molecular expression of brain metastatic A549-F3 cells was significantly decreased while their invasion and migration ability were enhanced.In addition,the rate of brain metastasis for A549-F3 cell in vivo was significantly increased.The results of single cell RNA sequencing showed that the microglia cells were in a state of dynamic balance and the microglia cells were mainly M2-phenotype in the brain metastases.Combined with transcriptome microarray analysis,the IL6,secreted by A549-F3 cells,was the key factor to induce the formation of M2-type microglia.Further analysis showed that IL6 secreted by A549-F3 cells increased downstream STAT3 phosphorylation by activating JAK2/STAT3 signaling in microglia.Then,p-STAT3 can directly regulate the expression of Arg1 at the transcriptional level and affect the phenotype of microglia.It can inhibit the formation of M2-phenotype microglia by intervening the pathway with inhibitors(tocilizumab and fedratinib).Conclusion: A549-F3 cells with high propensity of brain metastases may induce the formation of M2-phenotype microglia by secreting IL6 to activate the JAK2/STAT3 pathway.Part Ⅲ In vivo study of brain metastasis from non-small cell lung cancer by intervening the IL6/JAK2/STAT3 pathwayObjective: At present,the treatment effect of brain metastases is still unsatisfactory and there is a lack of effective prediction for brain metastases.In previous studies,we confirmed the important role of M2-phenotype microglia in brain metastasis of non-small cell lung cancer,and the activation of M2-phenotype microglia may be induced by the IL6/JAK2/STAT3 pathway.Therefore,it is urgent to carry out clinical translational research on molecular targets and find new therapeutic targets.Now,small molecule targeting drugs are promising therapeutic strategies for non-small cell lung cancer with brain metastases.Here,in vivo studies were conducted to explore whether molecular interventions targeting the pathway could achieve the effect of inhibiting the occurrence of brain metastasis.In addition,clinical samples were used to analyze the IL6 level in plasma from non-small cell lung cancer patients.Method: Knocking down the expression of IL6 in A549-F3 cells with lentivirus transfection or the application of inhibitors for IL6/JAK2/STAT3 pathway were used in vivo studies.The in vivo imaging technique was used to observe the incidence of brain metastasis from nude mice model constructed by left ventricular injection.Peripheral blood samples were collected from patients with non-small cell lung cancer and nude mice.The level of IL6 in plasma was analyzed by ELISA kit.The IL6 m RNA expression and survival data of non-small cell lung cancer patients from the TCGA databases were used to analyze the relationship between the expression of IL6 and the survival prognosis in non-small cell lung cancer patients.Result: Results of in vivo study suggested that either directly down-regulation of IL6 expression or the use of inhibitors(tocilizumab and fedratinib)for the IL6/JAK2/STAT3 pathway can reduce the occurrence and size of brain metastatic lesions(P < 0.05).In the nude mouse model,the level of IL6 in group with brain metastases was increased.In the NSCLC patients,the IL6 in plasma from group with brain metastases was 2.6 times higher than that with non-brain metastasis(P = 0.0295).The TCGA database analysis showed that the NSCLC patients with low expression of IL-6 had longer survival time(P = 0.0025).Conclusion: Intervening the IL6/JAK2/STAT3 pathway to influence the microglial phenotype can affect the occurrence of brain metastasis in lung cancer cells with high expression of IL6.The IL6 level in plasma from non-small cell lung cancer patients has a certain predictive effect on the occurrence of brain metastasis and the survival prognosis.