The Role Of CD30L In The Pathogenesis Of Murine Glioma Microenvironment

Objective: The brain tumor microenvironment is a critical regulator of cancer progression in primary and metastatic brain malignancies.Histopathological and flow cytometry analyses of gliomas revealed tumor niche,composed of astrocytes,endothelial cells,numerous immune cells,and so on.Infiltrating immune cells consist of tumor-associated macrophages(TAMs),microglia,myeloid-derived suppressor cells(MDSCs),CD8+ T cells and so on.These immune cells play an important role in tumor progression and metastasis.CD30L(CD30 ligand)is belonging to the tumor necrosis factor superfamily(TNFSF).CD30 L is mainly expressed on the active CD4+ and CD8+ T cells,macrophages and dendritic cells.CD30 L can promote the growth of Hodgkin's lymphoma.Meanwhile,CD30 L has been reported to act as an immune regulator mainly in several autoimmune diseases.However,little is known about the regulation of CD30 L in the glioma microenvironment.Therefore,we used the GL261-injected glioma model in WT and CD30 LKO mice to test the role of CD30 L in the regulation of the formation of glioma microenvironment and the role of CD30 L in the development of glioma.Methods: C57BL/6(wiild-type,WT)and CD30 L knock-out(CD30LKO)mice with the same genetic background were used to establish the model of glioma by GL261-injected.1.The survival duration of the WT,WT with m CD30-Ig injection and CD30 LKO mice was monitored after GL261 cell line injection.2.The expression of CD30 L protein in the brain was detected by western blot in normal tissue,para-carcinoma tissue and tumor tissue at day 21 post injection;the expression of CD30 L protein in the GL261 cell line by immunofluorescence;the expression of CD30 L protein in the glioma-infiltrating TAMs by flow cytometry.3.The tumor size of GL261-bearing WT,WT with m CD30-Ig injection and CD30 LKO mice was calculated by HE staining at day 21 post injection.4.The glioma-infiltrating immune cells were isolated from WT and CD30 LKO mice.Compare the absolute numbers,cell subsets and cell functional proteins of TAMs,MDSCs and microglia between WT and CD30 LKO mice by flow cytometry at day 21 post injection.5.The glioma-infiltrating immune cells were isolated from WT and CD30 LKO mice.Compare cell phenotypes and functions of CD8+ T cells between WT and CD30 LKO mice by flow cytometry at day 21 post injection.Results:1.In the glioma model,comparing with WT,the deficiency of CD30 L or blocking CD30 L can decrease the survival rate.2.Comparing with WT,the expression of CD30 L was higher in para-carcinoma tissue and tumor tissue of GL261-bearing WT mice at day 21 post injection;GL261 cell line and glioma-infiltrating TAMs can express CD30 L.3.Comparing with WT,the deficiency of CD30 L or blocking CD30 L can increase the tumor volume at day 21 post injection.4.Comparing with WT,the CD30 L deficiency can increase the cell number of microglia,TAMs and CD45+ CD11b-cells;the CD30 L deficiency can increase cell frequencies and numbers of different TAM and MDSC subsets;CD30L deficiency can alter the phenotype of TAMs and microglia,which can promote the growth of glioma at day 21 post injection.5.Comparing with WT,CD30 L deficiency may lead to the increase of PD-1+ CD8+ T cells,decrease the proliferation ability of CD8+ T cells and decrease the secretion of IFN-gandTNF-ain CD8+ T cells at day 21 post injection.Conclusion: CD30 L signaling plays an important protective role in the formation of glioma by regulating the glioma-infiltrating immune cells.