FTY720 Targets Glioma-associated Macrophages And Microglia To Exert Anti-glioma Effects

Glioblastoma multiforme(GBM)is a highly lethal tumor which is rapidly processing and infiltrative and takes up to 55.4% of the primary malignant brain tumors.The current therapy is mass surgical resection followed by radiotherapy or chemotherapy and only makes a poor prognosis.The 2-year survival rate is less than 3%-5% and the median survival is merely 14 months.Additional therapeutic strategies are needed to further improve prognosis and increasing evidences suggest that the glioma microenvironment may constitute extrinsic resistance mechanisms.The tumor microenvironment thus could be a target for anti-cancer therapy.The complex glioma microenvironment is composed with many types of cells,such as microglia,astrocytes,endothelial cells and so on.As the most predominant immune cells,microglial cells account for about 30-50% of the infiltrative cells in the glioma microenvironment.Glioma associated microglia and macrophages(GAMs)are recruited to the glioma environment and can release various growth factors and cytokines in response to those factors produced by cancer cells,facilitating tumor proliferation,migration and invasion.Several factors secreted by glioma cells,such as C-C motif chemokine ligand 2(CCL2),C-X-C motif chemokine 12(CXCL12),Colony stimulating factor 1(CSF-1),Macrophage migration inhibitory factor(MIF)etc.,acts on C-C chemokine receptor type 2(CCR2),C-X-C chemokine receptor type 4(CXCR4),Colony stimulating factor 1 receptor(CSF-1R),Cluster of Differentiation 74(CD74)on GAMs to recruit GAMs and push GAMs become immunosuppressive.In a word,blocking the chemoattractant receptors or ligands stands a chance for glioma strategy.Reportedly,the existence of an immune-suppressive glioma microenvironment pushes microglia to an immune-paralyzing and pro-tumorigenic phenotype.Therefore,shifting GAMs to anti-glioma phenotypes is considered to be a new strategy.FTY720 is an immunomodulator approved for oral treatment of multiple sclerosis(MS)by Food and Drug Administration(FDA).It is reported that FTY720 show anti-cancer effects for glioblastoma,lung cancer,melanoma and other cancers.but the exact mechanism of FTY720 on glioma remains unclear.In this study,we investigated the impacts and mechanism of FTY720 on the glioma microenvironment in vivo(glioma xenograft model)and in vitro(co-culture of glioma cells and microglial cells).We hope this can make sense for targets on the glioma microenvironment and provide academic basis for FTY720 in clinical use.AIM:To investigate and illustrate the modulations and mechanisms of FTY720 on the glioma microenvironment,providing academic basis for FTY720 on anti-glioma therapy.METHODS:1.Glioma xenograft models were established on 6-week-old male Wistar rats.After intraperitoneally injected with FTY720(2 mg/kg)for 14 days,Magnetic Resonance Imaging(MRI)and Hematoxylin and Eosin staining(H&E staining)were used to evaluate the therapeutic effects of FTY720 on glioma-bearing rats.2.Immunofluorescence(IF)assay was used to detect the amount and distribution of microglia and macrophages in the brain sections and the phenotypes of microglia in the brain sections and coculture system.3.Western Blot(WB)was used to analyze the expression of CCR2,CD74 and CXCR4 related to recruitment in the coculture system of C6 glioma cells and primary microglia.The effects of FTY720 on downstream Mitogen-activated Protein Kinase(MAPK)pathways of CXCR4 were measured by Western Blotting assay.4.Enzyme Linked Immunosorbent assay(ELISA)was used to detect the concentration of CXCL12 in the cell culture supernatants.The effects of FTY720 on TNF-α,IL-6 and IFN-γ secreted by microglial cells were analyzed by ELISA assay5.Wound healing assay and Matrigel Invasion assay were used to verify the effects of FTY720 in the coculture system between C6 glioma cells and microglia.RESULTS:1.FTY720 exerts anti-glioma effects in C6 Glioma Xenograft ModelMRI and H&E staining showed that FTY720 reduced the tumor volume of glioma-bearing rats and decreased the size of necrotic area.2.FTY720 blocks the chemoattraction of GAMs via internalizing CXCR4FTY720 reversed recruitment of glioma associated microglia,which were analyzed by immunofluorescence assay.Microglial recruitment related receptors,such as CCR2,CD74 and CXCR4,were analyzed western blots assay,and FTY720 mainly decreased the expression of CXCR4 and ELISA assay showed FTY720 had no effect for CXCL12 secreted by glioma cells.Moreover,FTY720 increased the internalization of CXCR4 to block the chemoattraction,exerting anti-glioma effects.3.FTY720 inhibits the activation of CXCR4-triggered intracellular signaling pathwayWestern blotting was used to detect the CXCR4-dependent intracellular MAPK signaling pathway,indicating FTY720 suppressed phosphorylation of MAPK pathways.The concentrations of TNF-α,IL-6 and IFN-γ were detected in the cell culture supernatants by ELISA assay,showing that FTY720 inhibited microglia to secrete IL-6(decreased 66%,compared to coculture group).4.FTY720 suppresses the migration and invasion of C6 cellsWound healing assay and Matrigel invasion assay showed that microglia could facilitate glioma migration and invasion in coculture system,which were reversed by FTY720.5.FTY720 polarizes GAMs from ‘M2’ to ‘M1’ phenotypeIF assay showed that FTY720 could decrease the amount of CD206 and increase iNOS-positive cells both in vivo and in vitro,polarizing microglia from ‘M2’to ‘M1’phenotype.CONCUSIONS:FTY720 reverses the recruitment of microglia,regulating the glioma microenvironment and suppresses glioma via internalizing CXCR4 and inhibiting activation of MAPK pathways,which decreasing the microglial secretion of IL-6.INNOVATIONS OF THE PRESENT STUDY:1.FTY720 targets on the glioma microenvironment to suppress glioma,providing evidence for further therapy targets on reprogramming the glioma microenvironment.2.FTY720 interacts the cross-talk between glioma cells and microglia via CXCR4-MAPK-IL-6 axis,exerting anti-glioma effects.