Lymphocyte Specific Protein 1 Expression Is Involved In The Regulation Of Leukocyte Migration And Immunosuppressive Microenvironment In Glioblastoma

Objective: glioblastoma(GBM)is the most common primary malignant tumor in adult central nervous system,which is accompanied with a high recurrence rate and mortality rate.In recent years,researchers have made significant progress in the field of cancer immunotherapy,providing new options for cancer treatment,but not all tumors are sensitive to current immunotherapy.Therefore,it is necessary to identify the mechanisms of tumor’s response and resistance to immunotherapy,and design a rational combination strategy.Leukocyte-specific protein 1(LSP1)is an F-actin binding protein that can regulate the phagocytosis of macrophages and the migration of immune cells.Many studies have focused on LSP1 regulating neutrophils and T cell migration to affect neutrophil actin dysfunction,rheumatoid arthritis and other diseases.In melanoma,LSP1 in T cells can regulate tumor growth by affecting the migration and infiltration of T cells into tumors However,there is currently no report on LSP1 in the field of glioma,and the expression of LSP1 and its role in glioma and its regulation of tumor microenvironment(TME)still need to be discussed further.Materials and Methods: Firstly,using CGGA,TCGA RNAseq database to analyze the genes that regulate leukocyte migration in glioma.From January 2011 to May 2019,140 clinical tumor specimens of patients with glioma diagnosed as pathologically diagnosed by surgical resection were collected at the First Affiliated Hospital of China Medical University,and normal brain tissues of 8 patients with acute brain injury were collected during the same period.Western blot and immunohistochemical were used to verify the expression level of LSP1 in different grades of glioma.By immunohistochemistry and immunofluorescence,the relationship between the expression level of LSP1 and tumor infiltrating immune cell types was verified.On this basis,using the Chinese Glioma Genome Atlas(CGGA)and Cancer Genome Atlas(TCGA)RNAseq database,we analyzed the expression level of LSP1 in different grades and subtypes of glioma,and its impact on survival times and tumor immune microenvironment.Then by using small-interference RNA and overexpression plasmid,knock down or overexpression of LSP1 in macrophages,and using transwell experiment to detect whether LSP1 regulates the migration ability of macrophages.By western blotting,the correlation between LSP1 and immunosuppressive molecules,including LAIR1,OSMR,PD1,LILRB3,was verified in tumor samples.Results: 1.Through CGGA and TCGA RNAseq database analysis,it is found that only LSP1 is negatively correlated with tumor purity,and its expression is significantly different in low-grade and high-grade gliomas,and it has a negative impact on the prognosis of glioma patients.Immunohistochemical on clinical samples of GBM verified that patients with high LSP1 expression had a poor prognosis.2.By CGGA,TCGA RNAseq database,western blot and immunohistochemistry,We found that compared with normal brain tissue and other grades of glioma,LSP1 is high expression in GBM.We further analyzed GBM samples in the CGGA and TCGA RNAseq databases to find that LSP1 is high expression in the mesenchymal subtype GBM and IDH1 wild-type GBM.3.By analyzing GBM samples in the CGGA and TCGA RNAseq databases,the low LSP1 group had a survival advantage compared to high group in GBM patients receiving radiotherapy,but not those without radiotherapy,and the low LSP1 group had a survival advantage compared to high group with methylated MGMT promoter.4.Using CGGA,TCGA RNAseq database,GO analysis,GSEA and GSVA showed that LSP1 is related to immune response,inflammation,leukocyte migration and other immune pathways.Further analysis showed that with the increase of LSP1 expression level,the infiltration of monocyte-derived macrophages,neutrophils and Tregs increased,and toxic lymphoid T cells decreased in the tumor microenvironment.It was verified by immunohistochemistry that the number of macrophages and neutrophils in the tumor microenvironment was positively correlated with the expression of LSP1.At the same time,we proved that in GBM samples,LSP1 was expressed in macrophages and neutrophils by immunofluorescence.Furthermore,through transwell migration experiments,we found that low LSP1 expression in macrophages inhibited their migration ability.5.We proved that LSP1 is related to the immunosuppressive factors LAIR1,OSMR,PD1,and LILRB3 by Western blotting.Conclusion: The high expression of LSP1 is significantly related to the poor prognosis of patients with glioblastoma,and its expression level shows a significant increasing in the malignant progression of glioma.The high expression of LSP1 is not only significantly related to the enrichment of inhibitory immune-related phenotypes,but also to the expression of immunosuppressive factors,involving LAIR1,OSMR,PD1,and LILRB3,as well as the high infiltration of macrophages,neutrophils,and Treg cells in the tumor microenvironment.LSP1 provides a new therapeutic target for immunotherapy.