| Objective:Lung cancer is the main tumor type causing cancer-related death in human,also the primary source of brain metastasis particularly the non-small cell lung cancer(NSCLC)almost accounts for half cases in all brain metastases.Despite a great advance has been made on the treatment of lung cancer,the survival prognosis of patients still dismal,especially the recurrence and metastasis of lung cancer significantly impair the quality of life and survival of patients.Though the application of immune checkpoint inhibitors dramatically fostered the development and extension of tumor immunotherapy,the significant response of immune-related side-effect and applied limitation impeded to some extent its further development and application.Thus,it’s necessary to further explore the interaction and related mechanism between tumor microenvironment(TME)and tumor cells,mining the microenvironment remodeling associated factors.Particularly,the interaction and influence between tumor immune microenvironment(TIME)and tumor cells have being gained increasing attention,for example,studies found that tumor-infiltrating lymphocyte(TIL)was significantly associated with the 5-year survival rate of non-small cell lung cancer(NSCLC)patients and the low density of infiltrated lymphocytes was the marker of gloomy prognosis of NSCLC in early stage.It is the clinical correlation study promoted the development of immunotherapy,boosting the application of immune checkpoint inhibitors on the treatment of NSCLC.Ibrutinib is a small molecule inhibitor specific to BTK,receiving admirable effect in the treatment of myeloid hematopoietic malignancies.In recent years,the application of ibrutinib has extended to the treatment of some solid tumors such as pancreatic cancer and lung cancer,however received no promising effect.As the advancing of science and technology,notably,the development of sequencing technology promoted greatly the studies towards the inheritance,expression,and function of genes,which to some extent accelerated the advancing speed of biology and medicine towards the era of big data.Sequencing technology,RNA-Seq for example,efficiently produced large amounts of data in a short time,consequently significantly promoted the widely application of bioinformatics characterized by typical comprehensive features of multidiscipline to the research fields of biology and medicine.Especially in the field of oncology research,along with the establishment and improvement of The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)database,increasing research data of genes associated with diseases and clinical informatic data of patients were collected and sorted by corresponding databases,which provided sufficient and objective data support for the comprehensive and in-depth research for diseases.Therefore,the comprehensive analysis by feat of bioinformatics towards the large amounts of clinical sequencing data would be of great benefit for understanding the rules of occurrence and development of the disease in the view of much more comprehensive and deeper level,which accordingly offer much more precise strategy and therapeutic targets for the prevention and treatment of tumor.Methods:In this study through the bioinformatic analysis towards the transcriptomic RNA-Seq data of clinical lung adenocarcinoma(LUAD)samples,aiming to mining the TME associated factors with the prognostic roles.The ESTIMATE algorithm was used to calculate content of immune and stromal components in TME of clinical samples.Combining the clinical informatic data of patients,the Kaplan-Meier method was used for the survival analysis of clinical samples with different amount of immune and stromal components with aim of exploring the correlation between the amount of immune and stromal components with the survival of patients.The differentially expressed gene(DEG)analysis was performed to the samples with different proportion of immune and stromal components in order to explore the genes associated with the immune and stromal components in microenvironment.After acquired the DEG sets associated with the immune and stromal components,by feat of annotation data in Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases,the enrichment analysis was performed to the DEG sets.Constructed the Protein-Protein Interaction(PPI)with the data of protein interaction in STRING database,followed by the reconstruction of core network with Cytoscape,extracting the core network.Through the univariate COX regression analysis to the DEGs for determining the genes associated with the survival of patients,and by the interaction analysis with the core nodes in the PPI network further exploring the factors correlated with the remodeling of TME.The differentially expressed gene analysis was carried out between the normal and tumor tissues for determining the different expression of BTK.Survival analysis was performed to explore the correlation of BTK expression with the survival and pathological staging of patients.Gene Set Enrichment Analysis(GSEA)was performed towards the whole genes respective in samples with different BTK expression with the purpose of exploring the association of BTK with the overall metabolic status of LUAD TME.Taking advantage of the CIBERSORT algorithm to calculate the types and proportions of tumor-infiltrating immune cell(TIC),and differentiation and correlation analysis were performed to determine the association of BTK with TICs.The gene expression data of GSE110495 in GEO database was downloaded followed by the differentiated expression analysis of BTK for exploring the correlation of BTK with the process of brain metastasis of lung tumor.Real-time PCR and Western Blot were used to resolve the expression of BTK both in the level of mRNA and protein.Establishing the brain metastatic animal model of lung cancers via mouse ICA injection operation.Transfecting the brain metastatic lung tumor cells with the recombinant lentivirus followed by the reinjection into the brain of the mouse via ICA injection operation,and then observed the intracranial neoplasia with the two-photo laser confocal scanning microscope.Co-culture of the brain metastatic lung tumor cells and the macrophage with Transwell was performed to explore the influence of lung tumor cells on the expression of BTK in the macrophage.Results:In this study,bioinformatic analysis was performed towards the transcriptome RNA-Seq data of lung adenocarcinoma samples downloaded from TCGA database,which revealed that BTK was the potential prognostic factor for the survival and disease progression of LUAD patients,besides was the potential indicator for the remodeling of TME,and the downregulation of BTK indicated the transition of TME from predominant immune activities to the remodeling characterized by proliferating metabolism.The differentiation and correlation analysis of BTK with TIC showed that the expression of BTK was associated with the type and proportion of multiple TICs,for example,BTK positively correlated with the proportion of CD8~+T and negatively with that of M0 macrophage,which further suggested that BTK was correlated with the remodeling of TME.Meanwhile the analysis to the RNA-Seq data of gene expression in the study regarding brain metastasis of lung cancer revealed that as the advancing of phases in the process of lung tumor brain metastasis,the expression of BTK was dramatically downregulated.The animal model of lung cancer brain metastasis was established via ICA injection operation,followed by the tumor tissue extraction from the mouse bearing intracranial tumor and the subsequential culture of the primary tumor cells.Expression analysis of BTK in brain metastatic lung tumor cells showed that along with the increasing of capability of the proliferation and intracranial tumorigenesis,the expression of BTK in tumor cells and the co-cultured macrophage were remarkably downregulated.The results of experiments and analyses to some extent explained the reasons that why the BTK specific inhibitor ibrutinib received no significant effect in the treatment of lung cancer,providing novel direction and therapeutic targets for the clinical treatment of lung cancer,meanwhile offering new strategy and prospective for the further study and exploration of the interaction between tumor cells and TME.Conclusion:1.The expression of BTK was associated with the remodeling of tumor microenvironment,the microenvironment with higher BTK expression mainly engaged in immune activities,as contrast the lower BTK expression microenvironment was characterized by tumor related metabolism.2.The expression of BTK was associated with the type and proportion of the infiltrating immune cells in the tumor microenvironment,for example,it was negatively correlated with the amount proportion of M0 macrophage and positively related to CD8~+T cells.3.The expression level of BTK was positively correlated with the survival prognosis of lung adenocarcinoma,median survival time of patients with higher BTK expression was larger than that of patients with lower BTK expression,meanwhile as the increasing of clinicopathological staging,the expression of BTK was decreasing,presenting with significantly negative correlation with stage and T in statistics.4.As the advancing of brain metastasis of lung cancer,from the primary tumor to the brain metastatic tumor,the expression of BTK was decreasing.5.Injecting lung tumor cells into the brain of mouse via ICA injection operation,as the iterative influence of intracranial microenvironment,the proliferative capability of the brain metastatic lung tumor cells was significantly enhanced,and the expressions of BTK in tumor cells and the co-cultured macrophage conditioned by tumor cells were dramatically decreased. |
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