Knockdown of milk-fat globule EGF factor-8 suppresses glioma progression in GL261 glioma cells by repressing microglial M2 polarization

Tumor-associated microglial cells promote glioma growth, invasion, and chemoresistance by releasing inflammatory factors. Milk fat globule EGF factor 8 protein (MFG-E8), a secreted glycoprotein, is closely related to tissue homeostasis and anti-inflammation. In the present study, we investigated the role of MFG-E8 in microglial polarization and glioma progression in vitro and in vivo. We found that glioma cells secrete comparable amounts of MFG-E8 in culture media to astrocytes. Recombinant MFG-E8 triggered microglia to express the M2 polarization markers, such as arginase-1 (ARG-1), macrophage galactose-type C-type lectin-2 (MGL-2), and macrophage mannose receptor (CD206). Forced expression of MFG-E8 in BV-2 microglia cells not only promoted IL-4-induced M2 polarization but also inhibited lipopolysaccharide (LPS)-induced M1 microglial polarization. Mechanistic studies demonstrated that recombinant MFG-E8 markedly induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the STAT3 inhibitor stattic significantly blocked MFG-E8-induced ARG-1 expression. Administration of antibody against MFG-E8 and knockdown of its receptor, integrin beta 3, significantly attenuated MFG-E8-induced ARG-1 expression. Similarly, knockdown of MFG-E8 also markedly reduced IL-4-induced M2 marker expression and increased LPS-induced M1 marker expression in microglia cells. Moreover, the knockdown of MFG-E8 in GL261 glioma cells inhibited cell proliferation and enhanced chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), which was likely associated with the downregulation of FAK/AKT activation and STAT3/cyclin D1 signaling. The murine GL261 glioma experimental model demonstrated that knockdown of MFG-E8 significantly reduced tumor size and extended survival times. Additionally, attenuated CD11b(+) cell infiltration and reduced CD206(+) expression in CD11b(+) cells were also observed in an MFG-E8 knockdown GL261 murine glioma model. These results suggested that inhibition of MFG-E8 might hamper the immunosuppressive microenvironment in gliomas and therefore ameliorate tumor progression.