Tumor Associated Microglioma/Macrophages Regulate The Heterogeneity Of Glioma Stem Cell Via TGF? Induced

Objective: To investigate the possible mechanism of tumor-associated microglia/macrophages regulating glioma stem cell heterogeneity through TGFBI and promoting malignant progression of glioblastoma.Methods: This project is based on NCH-421 K human glioma stem cell line and glioblastoma specimens,combined with open source tumor databases such as TCGA,Rembrandt,Oncomine,and RNA-seq results of NCH-421 K cells before and after rhTGFBI stimulation.Our project is aimed to investigate TAM/Ms regulation on the heterogeneity of glioma stem cell via TGFBI:1.Immunohistochemical staining of the TAM/Ms marker Iba-1 in human GBM specimens;2.Immunofluorescence staining of glioma stem cell marker Nestin in human GBM specimens;3.We retrieve the expression of TGFBI,ITGAM and the patient survival in different grades of glioma from open source tumor databases such as TCGA,Rembrandt,and Oncomine;4.RNA-seq was used to detect the expression of markers of M1 and M2 TAM/Ms in different parts of GBM specimens,qPCR was used to detect the TGFBI expression of TAM/Ms in different parts of the tumor specimens;5.Co-localization and Immunohistochemical was used to detect the relationship between TAM/Ms marker CD163,stem cell marker SOX2 and TGFBI in GBM specimens;6.When NCH-421 K were stimulated by rhTGFBI protein,we detected the changes of stem cell characteristics and mRNA expression of NCH-421 K by colony formation assay,invasion assay and RNA-seq.Results: 1.Immunohistochemistry and immunofluorescence staining of tumor-associated macrophage marker Iba-1 and stem cell marker Nestin in GBM specimens showed that TAM/Ms and GSCs aggregated in tumor angiogenesis,necrosis and invasive brain regions.The distribution of the two cells is consistent,suggesting that TGFBI is involved in the regulation of glioma malignant progression;2.By searching the TCGA,Rembrandt,and Oncomine databases,we found that TGFBI expression is associated with prognosis,higher in GBM than in normal brain tissue and lower grade glioma,and higher in mesenchymal GBM,indicating that TAM/Ms may promote the malignant progression of GBM through TGFBI;3.Through RNA-seq,we found that cells in the central of tumor expressed M2 phenotype molecular markers more than the peripheral;TAM/Ms in the tumor center expressed TGFBI more than the periphery,and we also found the co-localization between TGFBI and CD163 and SOX2 by immunofluorescence.These suggest that TGFBI is a key molecule regulating the heterogeneity of GSCs.4.When the NCH-421 K was stimulated by rhTGFBI protein,we found that the invasive ability(P<0.01)and self-renewal ability(P<0.01)of the cell were enhanced,and through sequencing,we also found that 875 genes were significantly up-regulated mainly in intercellular adhesion,cell and extracellular matrix adhesion,exogenous apoptosis signaling pathway,TGF? receptor binding,and monocarboxylic acid transmembrane transporter and other related biological processes.Conclusion: 1.In GBM,TAM/Ms in different infiltration regulate the heterogeneity of GSCs.2.TGFBI is secreted by TAM/Ms and enhances stem cell characteristics such as invasion and self-renewal of GSCs,these up-regulated genes are concentrated on: intercellular adhesion,cell-to-extracellular matrix adhesion,exogenous apoptotic signaling pathways,TGF? receptor binding,and monocarboxylic acid transmembrane transporters.