The Mechanism Of Soluble LRIG3 Regulating The M1/M2-like Phenotypes Of Tumor-associated Macrophage And The Tumor Immune Microenvironment In Glioma

Objective:Glioma is a primary malignant tumor of central nervous system with poor prognosis.The main reasons for poor prognosis of glioma patients are tumor invasion,recurrence and resistance to conventional therapy.These characteristics are closely related to the heterogeneity and dynamic plasticity of cell components in glioma tumor microenvironment.Our research group previously found soluble LRIG3(s LRIG3)in tumor cyst fluid of patients with cystic glioma,and its expression level was negatively correlated with the grade of glioma in patients with cystic glioma.However,the specific role and mechanism of s LRIG3 in the glioma microenvironment remain unclear and need to be further explored.Methods:Firstly,based on the data from the public glioma database,bioinformatics techniques were used to analyze the correlation between LRIG3 expression and immune cell components and signaling pathways in the tumor microenvironment.Subsequently,single-cell sequencing technology and protein western blotting were used to analyze the expression of LRIG3 in each cell component in glioma microenvironmen,and enzyme linked immunosorbent assay(ELASA)was further used.By knocking down special genes or applying specific protein agonists or inhibitors,the relevant regulatory mechanisms of s LRIG3 secretion were analyzed.The effect of s LRIG3 on tumor-associated macrophages(TAMs)was verified by quantitative real-time polymerase chain reaction,western blotting,ELISA and flow cytometry at the cellular level.Intracranial tumor formation model of mouse glioma was constructed by C57 mice and murine-derived glioma cell lines,and single cell suspension flow cytometry,tissue immunohistochemistry,and in vivo imaging of small animals were used to verify the M1/M2-like phenotype transformation and secreted cytokine changes of s LRIG3 on TAM in mouse tumor formation model.The effect and mechanism of CD8+T cell composition changes in tumor microenvironment.CRISPR and lentivirus transfection techniques were used to knocking out endogenous NETO2 of tumor-associated macrophage cell lines,and exogenous full-length and truncated NETO2 plasmids were used to detect the effects of specific domains on TAM phenotype and the changes of CD8+T cell components in tumor microenvironment and the corresponding mechanisms.Results:Herein,we reported that s LRIG3 derived from glioma tumor cells can block the M2 polarization of TAMs via interacting with NETO2 and reshape the anti-tumor microenvironment,thus suppressing glioma malignant progression.The expression or activity of ADAM17 in glioma cells was positively correlated with the expression of s LRIG3 in cell supernatant.Soluble LRIG3 can suppress the M2-like polarity transformation of TAMs,inhibit the secretion of tumor promoting cytokine IL-10 and promote the secretion of inflammatory cytokines i NOS of TAMs,and increase the number of CD8~+T cells in tumor microenvironment,thus inhibit the growth of tumor.Mass spectrometry and Co-immunoprecipitation showed that s LRIG3 interacts with the CUB1domain of NETO2 in TAMs.Silencing or knockout of NETO2 could block the effect of s LRIG3,which inhibited the M2-like polarity transformation of TAMs and promoted glioma tumor growth.However,overexpressing His-target NETO2 with CUB1 deletion mutation does not fully recover the suppressive effects of s LRIG3 on the NETO2-Knockout TAMs and the tumor microenvironment.Conclusions:Our study revealed vital molecular crosstalk between glioma tumor cells and TAMs.Glioma cells mediated the M2 polarization of TAMs through the s LRIG3-NETO2pathway and inhibited the progression of glioma,suggesting that s LRIG3-NETO2 may be a potential target for glioma treatment.