| Introduction Glioma is the most common primary central nervous system tumors,more than 50 percent of central nervous system tumors.They often accompany with high recurrence rates and high mortality rates,especially for glioblastoma(GBM),which has the highest malignancy.The current standardized treatment of glioma uses surgery combined with chemotherapy and chemotherapy,but the treatment effect is not satisfactory.Traditional preclinical studies of gliomas often focus only on the tumor cells themselves,but ignore the mutual regulation between the tumor microenvironment and glioma cells,in fact,tumor cells and interstitial cells or immune cells can affect each other and transform each other.Previous research by our team found that glioma tumor purity and immune response intensity It can be used as an important indicator for predicting the survival prognosis of glioma patients,and also indicates the great potential of glioma therapy by modifying the glioma microenvironment.This study focus on the two main parts of non-tumor components,the stromal components and immune components,further explored the important clinical value and the regulation on microenvironment of the core stromal components(brain vascular endothelial cells)and interferon signaling in gliomas,and looked forward to screening potential targets.Which aimed at inhibiting the malignance of gliomas by transforming the microenvironment.Methods(1)Database information and tissue specimens: The data used for relevant bioinformatics analysis was obtained from CGGA,TCGA,and GEO platforms.We selected the CGGA(Chinese Glioma Genome Atlas)data platform as the main analysis database,then selected TCGA(The Cancer Genome Atlas)and GEO(Gene Expression Omnibus)data platform as external validation databases.The tissue specimens which used for molecular biology experimental verification were obtained by the surgery of the department with the informed consent of patients and their families.The relevant experimental plan was reviewed and approved by the unit's ethics committee.(2)Bioinformatic analysis: Principal components analysis(PCA)was conducted to describe expression patterns of grouped patients.The differential expressed genes were screened by limma R package.Analysis of biological processes and enriched pathways of differential genes through the DAVID website.Gene Set Enrichment Analysis(GSEA)is used to further verify the differential functions and pathways enriched.Apply Estimate R package to calculate the purity and immune score as well as stromal score of each tumor sample.The relative content of each non-tumor cell component in the sample was calculated by MCP Counter and Cibersort R package.(3)Cell lines model: Human brain normal astrocytes NHA,human glioma cell lines LN229,U87,U251 and brain microvascular endothelial cells(HBMEC)were purchased from Shanghai Cell Bank,Chinese Academy of Sciences.(4)Molecular and biology experiments: q PCR,WB,immunohistochemistry,and immunofluorescence are used to verify molecular expression;IP and liquid chromatography mass spectrometry are used to judge potential receptor proteins that have a binding relationship with the target protein;Transwell assay is used to detect the invasion or migration ability of glioma cells / brain microvascular endothelial cells;MTS is used to determine cell proliferation Viability;3D Matrigel tube formation test to detect the ability of vascular endothelial cells to form new blood vessels;intracranial orthotopic tumor formation in nude mice were used for in vivo verification of phenotype or mechanism.(5)Statistical analysis: The ANOVA test?Student's t test and chi-square test were performed to test the differences between variables.We used Kaplan-Meier curves to depict the survival conditions of patients,then Log-rank test was performed to evaluate the survival difference between groups.Cox regression analysis was conducted to identify survival related factors or targets.And the correlation between variables was calculated by pearson test.The relevant statistical analysis were conducted by SPSS 20.0,Graph Pad Prism 7,and R 3.6.1.A two-tailed p value of less than 0.05 was defined as a criterion for statistical significance.Results: Part 1: CTSD / HIF1 A signal axis mediates interactions between glioma cells and brain microvascular endothelial cellsBased on public TCGA and CGGA glioma and patient databases,we performed some bioinformatics analysis of transcription levels.We found that in all GBM patients,the effect of microvascular endothelial cell content on the prognosis of patients is not very obvious;then we divided the patients into two subgroups of low tumor purity and high tumor purity,and then survived according to the level of endothelial cells.The analysis found that the endothelial cell content was significantly correlated with survival in the high tumor purity group,that is,the higher the blood vessel density in high purity patients,the worse the survival prognosis of the patients.This phenomenon well illustrates that when glioma cells and vascular endothelial cells are present in high abundance at the same time,the malignant progress of gliomas will be more rapid.Therefore,we propose "the fate of glioma cells-vascular endothelial cells" Community ” concept.This also provides us with new insights: why the long-term effect of traditional anti-vascular drugs targeting VEGFA,bevacizumab,is not ideal.On the one hand,it may be related to the compensatory increase of other angiogenic factors;on the other hand,it may be due to the limitation of targeting endothelial cells alone,which may cause the glioma cells themselves to compensate and open up a new path to reconstruct the vascular network.Including the new vascular formation model "vascular mimicry" and so on.Screening common functional molecules that can regulate glioma cells and brain microvascular endothelial cells in the microenvironment will provide new ideas for targeted antivascular therapy of gliomas.Previous basic experiments found that after the administration of HBMEC with different doses of bevacizumab,among the many classic pro-angiogenic factors,the up-regulation of CTSD was most pronounced,which is one of the members of the Cathepsin family.The changes of angiogenic factors are not obvious when treated glioma cells with bevacizumab.Through the analysis of database data and PCR results,CTSD is also found to be one of the few target molecules that are highly expressed in glioma cells and vascular endothelial cells.Experiments have shown that CTSD can affect gliomas Cell proliferation,invasion,migration as well as other phenotypes;CTSD can affect the proliferation and migration of HBMEC and angiogenesis ability;bioinformatics analysis suggests that CTSD is closely related to activation of pro-vascular related pathways.IP-mass spectrometry has identified HIF1 A as a CTSD binding protein.And experimentally confirmed that CTSD can bind to HIF1 A and maintain its stability and up-regulate its expression level to activate the HIF1A-VEGFA signal axis;we also conducted related experiments with CTSD-specific inhibitor Pepstatin-A in vivo and vitro,which have achieved some results.Part 2: Abnormally activated interferon signal and inducible IFI30 are important markers for judging GBM prognosis and unfavorable microenvironment componentsThe previous results of the research team found that excessive immune response and disordered immune microenvironment are important factors leading to GBM adverse outcomes and treatment resistance.Interferon,as one of the most important families for regulating the immune response,is involved in activating antigen-presenting cells and enhancing the effect of effector T cells on killing tumor cells.However,there is evidence that abnormal activation of interferon signals in gliomas can promote immune escape and lead to GBM immune regulatory point therapy resistance.Therefore,it is urgent to explore the clinical value of interferon in gliomas and establish a reliable typing method.We constructed an interferon risk score based on five prognostic interferon genes,which can reflect different interferon states.Survival analysis found that the survival time of patients with high interferon risk was significantly shortened.Multivariate COX analysis suggested that our interferon risk score could be used as an independent prognostic indicator.Functional enrichment analysis indicates that a subgroup of high-risk patients is associated with abnormally activated immune responses and chemotaxis of immune cells.CIBERSORT microenvironment analysis and a large number of clinical samples' immunohistochemical analysis found that the microenvironment of high-risk GBM patients tends to be immunosuppressive,which is characterized by significantly enriched M0 macrophages and neutrophils,but less infiltration of activated natural killer(NK)cells and M1-type macrophages(pro-inflammatory).The difference in microenvironment components may be one of the most important reasons for the significant difference in survival conditions between patients with high and low interferon risk GBM patients.In addition,we identified IFI30 as an inherent ?-IFN-induced gene,exerts an important role in the malignant progression of glioma,chemotherapy resistance,and immune microenvironment remodeling.Conclusions: Part I: In gliomas,CTSD can promote the malignant proliferation and invasion of glioma cells by binding HIF1 A to activate the HIF1A-VEGFA signal axis;CTSD could promote angiogenesis by a direct manner or mediate the paracrine effects of glioma cells.Bevacizumab-targeted VEGFA therapy may up-regulate CTSD by activating lysosomal autophagy in brain microvascular endothelial cells.To investigate the common regulatory mechanism of CTSD in glioma cells and brain microvascular endothelial cells,elucidate the mechanism of CTSD feedback increase after bevacizumab treatment,and develop a CTSD-specific inhibitor Pepstatin-A in combination with bevacizumab.Which will provide new ideas and methods for the vascular targeted treatment of gliomas.Part II: The interferon risk score we established can be used as an independent prognostic factor to determine the survival status of GBM patients.We found that a subpopulation of GBM patients with high interferon risk is accompanied by an enhanced immune response.Analysis of microenvironment components found that the GBM immune microenvironment in the high-risk group became more complex,characterized by significant enrichment of M0 macrophages and neutrophils.However,activated NK cells and M1-type macrophages have less infiltration.Moreover,we found that exogenous ?-IFN stimulation may reshape the microenvironment of glioma by up-regulating microenvironment-related targets such as IFI30,and then promote the malignant progress of GBM and treatment resistance. |
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