The Mechanism Of Phagocytosis Checkpoint Gene PROCR Promoting Malignant Progression And Phagocytosis Escape In Glioma

Background: Glioma,as the most common primary malignant tumor in intracranial,is the main cause of poor prognosis due to its strong malignant proliferative ability and invasiveness,which is recognized as a difficult disease in the current field of tumor treatment,especially glioblastoma(GBM).Glioblastoma has a high recurrence rate and a short survival period,with the 5-year survival rate often less than 5%.At present,the clinical treatment of glioma is mainly through surgery and postoperative concurrent chemoradiotherapy,but the therapeutic effect is still unsatisfactory.In the past,the treatment of tumor often focused on the tumor cells themselves,but ignored the influence of tumor microenvironment(TME)on the occurrence and development of tumor.The introduction of immune microenvironment(TIME)makes people have a further understanding of the occurrence and development of tumor.There is interaction between immune cells and tumor cells,and immune cells participate in the occurrence and development of tumor cells.Meanwhile,tumor cells can act on immune cells by secreting signaling molecules.The end result is a microenvironment of local immunosuppression around the tumor.Therefore,immunosuppression is expected to be an important breakthrough in cancer treatment.At present,various immunotherapies for tumor have been gradually developed,among which cancer immunotherapy targeting adaptive immune checkpoint has significantly improved the prognosis of some patients with metastatic and refractory cancer types.However,compared with the therapeutic effect of glioma,the therapeutic effect of adaptive immune checkpoint is not good,which may be because glioblastoma is a cold tumor,T cells are few in the central nervous system,and glioblastoma infiltrates the innate immune response cells represented by macrophages.The research on innate immune checkpoint is relatively late.But there have also been significant advances in recent years.The present study found that innate immune checkpoints,which interfere with the detection and clearance of malignant cells through phagocytosis and suppress innate immune responses,also play a key role in tumormediated immune escape,Therefore,we consider the identification of signal targets and regulatory mechanisms related to innate immunity in glioblastoma,which has important theoretical significance and clinical practical value in understanding the immune escape of glioblastoma and developing related targeted therapy.Based on our research group’s long-term research on immunosuppression of glioblastoma,combined with a number of domestic and foreign glioma databases and our own glioma cell sequencing data,we established an individualized phagocytic checkpoint risk score.Later,we found that phagocytic checkpoint risk score was correlated with tumor pathological grade of glioblastoma patients.Survival prognosis,biological function,immune microenvironment and immune process are closely related.Later,we identified PROCR,the core gene of glioblastoma phagocytosis checkpoint.Based on the analysis results of multiple glioma data platforms and our own glioma database,we clarified the biological process of PROCR in glioma blasts through cell functional experiments,analyzed the specific mechanism of action and signal regulation involved,and explored the phagocytosis escape mechanism accordingly.It provides a new solution for the immunotherapy of glioblastoma.Purpose:1.To clarify the phagocytosis checkpoint gene set effect in glioblastoma.2.To explore the effect of phagocytic checkpoint key molecule PROCR on the function of glioblastoma and the related mechanism of promoting the malignant progression of glioma.3.To explore the regulation of PROCR related pathway on immune microenvironment of glioblastoma.Research methods;(1)Data bank selection and clinical sample information: This study involved sample information from the Chinese glioma Genome Atlas(CGGA)cohort,Cancer Genome Atlas(TCGA)cohort.Including expression profile data,patient clinical information data and corresponding molecular information data.Meanwhile,the collection of gene sets also draws on two authoritative literatures.All the clinical tissue samples used in this study were obtained from the First Affiliated Hospital of China Medical University,and the histological diagnosis of the included samples was confirmed by two neuropathologists according to the 2016 WHO classification Guidelines.The corresponding experiment has been reviewed and approved by the Ethics committee of the unit.(2)Bioinformatics analysis: Single sample gene set enrichment analysis(ss GSEA)was used to calculate the activity scores of patients in different pathways.Gene ontology(GO),gene set enrichment analysis(GSEA)and pathway enrichment analysis(KEGG)were used for functional analysis.Estimate analysis,x Cell analysis and Cibersort were used to evaluate the immune risk score and the degree of immune cell infiltration.TIP analysis was used to analyze the correlation between phagocytic checkpoint score and different immunotherapy processes.Univariate and multivariate COX regression analysis was used to analyze the survival of the included genes.(3)Cell line origin: human macrophage cell line THP-1 was purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.GSC21,GSC63,GSC1 and GSC24 neurospheres were derived from patient-derived glioblastoma primary cell lines constructed by our laboratory team.Murine glioma cell line GL261 was obtained from the United States model culture collection(ATCC).(4)Molecular biology experiments: q PCR,Western Blot,immunohistochemistry and other experiments were used to detect molecular expression levels;The coimmunoprecipitation technique was used to search for potential ligand proteins that were bound to the target proteins.The self-renewal ability and proliferation ability of glioma cells were measured by limited dilution test and neurosphere size test.q PCR was used to detect the change of macrophage phenotype,and the co-culture chamber was used to explore the interaction between glioma cells and macrophages.The changes of macrophage migration ability were detected by Transwell assay.The mouse subcutaneous intracranial tumor assay was used to verify the phenotype and mechanism in vivo.(5)Statistical analysis: Kaplan-Meier survival curve was drawn and Log-rank test was performed to compare the survival differences among different patient groups;COX regression analysis was used to identify the independent prognostic factors.Graph Pad Prism 8,R 4.0.2 and SPSS were used for statistical analysis.A p value less than 0.05 was defined as a statistically significant difference.The result;Part Ⅰ: Establishment of phagocytosis checkpoint gene set in glioblastoma and description of its clinical value.Firstly,we combined relevant information from the public glioma gene Map database and our own glioma database with relevant authoritative literature to form phagocytosis checkpoint gene set.Subsequently,ss GSEA score was analyzed in CGGA and TCGA databases to form PCS risk score(PCP risk score).Using the data information from CGGA and TCGA databases,the corresponding relationship between phagocytic checkpoint risk score and clinical characteristics of each molecular subtype,grade and prognosis was demonstrated.Subsequently,the correlation between the immune risk score and tumor purity and the degree of immune cell infiltration in GBM and PCS score was calculated in the GBGA and CGGA databases of GBM samples.Finally,we found that the risk score was positively correlated with the pathological grade of glioma patients,and the survival time of patients with high score was significantly shortened,which could significantly improve the prognosis of patients with glioblastoma.Functional Enrichment and pathway Analysis based on GO(gene ontology),KEGG(Kyoto Encyclopedia of Genes and Genomes),Gene Set Enrichment Analysis(GSEA),genomes analysis Phagocytic checkpoints in glioblastoma are closely related to the NF-κB and IL-6 signaling pathways.We found that phagocytosis checkpoints in glioblastoma and IDH1 wild-type patients had a significantly higher risk score than IDH1 mutant glioblastoma patients.It was also found that PCS score was positively correlated with immune risk score and negatively correlated with tumor purity.It was found that macrophages had the highest correlation with phagocytic checkpoint risk score,showing a significant positive correlation.The second part;The PROC/PROCR/NF-κB signal promotes malignant progression of glioblastoma in glioblastoma.In the first step,we established the PCS score,and then conducted correlation analysis on the sequencing data of CGGA and our own glioma cells.The 7 phagocytosis checkpoint genes that play a regulatory function in glioblastoma.After biogenic analysis,It was found that PROCR was the most representative in the expression difference between low and low gliomas and its correlation with macrophages.Western Blot confirmed that the expression of PROCR increased with the increase of glioma grade in clinical tissue samples.At the same time,the correlation between PROCR and malignant degree of glioblastoma was clarified,which provided a basis for further research.Firstly,we established stable cell lines with PROCR lentivirus knockdown and overexpression respectively.The results of functional phenotype experiments confirmed that PROCR knockdown can significantly inhibit the malignant proliferation and selfrenewal ability of glioma cells as well as intracranial tumor formation ability.On the contrary,PROCR overexpression can promote the malignant proliferation and self-renewal ability of glioma cells.These results suggest that PROCR plays a role in regulating the malignant progression of glioma cells.In order to clarify the molecular mechanism of PROCR’s malignant progression in glioblastoma,GO and KEGG functional enrichment and pathway analysis were used to detect enrichment of NF-κB pathway and IL6 pathway in glioblastoma with high expression of PROCR related genes,which was consistent with the results of phagocytic checkpoint related gene set analysis.The GSEA results show similar results.Subsequently,PROCR was knocked down and PROCR was overexpressed in the previously identified cell lines,and it was found that the total p65 protein in the NF-κB pathway was not significantly changed,and phosphorylated p65 was significantly changed with the change of Procr expression,and IL6 was also significantly changed.Furthermore,IL6 cytokine was added into the cells with PROCR knockdown,and it was found that the inhibition of self-renewal and proliferation of tumor cells caused by PROCR knockdown was restored,indicating that PROCR’s effect on the self-renewal and proliferation of tumor cells was caused by the activation of NF-κB pathway through the production of IL6.Then we analyzed PROCR related ligand,and the data analysis in the CGGA RNA-seq dataset showed that PROCR expression was closely related to PROCR expression.In order to further confirm this result,we confirmed the direct combination of Proc R and Procr by immunoprecipitation in glioma cells.We then added the PROC recombinant protein into glioma cell lines and found that PROC could regulate the activation of NFKB/IL6,the downstream pathway of PROCR.Therefore,PROCR activated the downstream pathway by binding to PROC.The third part;The PROC/PROCR pathway in glioma cells regulates glioma cell and TAMs interactions.Western blotting was used to verify that PROCR activated the NF κpathway and increased IL-6 secretion at the same time.Then,after tumor cells knocked down and overexpressed PROCR,In co-culture with Th P1-induced macrophages,the phenotypic transformation of macrophages was found,indicating that tumor cell PROCR can promote the phenotypic transformation of macrophages.Transwell experiment showed that the PROCR expression of glioma cells had a regulatory effect on the chemotaxis of macrophages in the co-culture of glioma cells and macrophages.We also found that the proliferation of glioma cells co-cultured with M2-type macrophages was active.Considering the interaction between macrophages and glioma cells,we hypothesized that macrophages may have secreted proteins that promote the regulation of malignant progression of tumor cells.PROC was a ligand of PROCR,and according to the published single-cell database analysis,PROC was mainly expressed in macrophages.Western Blot analysis showed that PROC was significantly highly expressed in M2 macrophages,indicating that macrophages co-cultured with tumor cells could secrete PROC and promote the malignant phenotype of tumor cellsConclusion:Part Ⅰ: The phagocytic checkpoint risk score(PCS)established by us can be used as an important marker to judge the survival status and adverse molecular events of GBM patients.PCS was positively correlated with immune risk score and negatively correlated with tumor purity,and macrophages were significantly positively correlated with the score.At the same time,the score was closely related to immune progression.Part Ⅱ: PROCR can serve as a core representative molecule for phagocytic checkpoint and a malignant marker molecule for glioblastoma.PROCR has a significant regulatory effect on the proliferation and self-renewal ability of glioma cells.This regulatory effect is mainly achieved through PROC/PROCR binding and activation of classical NF-κ The B pathway plays a role in increasing the expression and secretion of IL-6 by glioma cellsPart Ⅲ: Our experiments demonstrated that PROCR can induce tumor-associated macrophages to differentiate into M2 phenotypes and promote Th P1-derived macrophages to migrate to tumors through PROCR /proc/NF-κB /IL-6 signaling.At the same time,M2-type macrophages express and secrete more PROC to promote further malignant progression of glioma cells.