Microglial stimulation of glioblastoma invasion involves EGFR and CSF-1R signaling

Glioblastoma multiforme is a deadly cancer whose current treatment options are limited. The ability of glioblastoma tumor cells to infiltrate the surrounding brain parenchyma critically limits the effectiveness of current treatments. We investigated how microglia, the resident macrophages of the brain, stimulate glioblastoma cell invasion. We first examined the ability of normal microglia from C57Bl/6J mice to stimulate GL261 glioblastoma cell invasion in vitro. We found that microglia stimulate the invasion of GL261 glioblastoma cells by approximately 8 fold in an in vitro invasion assay. Pharmacological inhibition of EGFR strongly inhibited microglia-stimulated invasion. Furthermore, blockade of CSF-1R signaling using RNA interference or pharmacological inhibitors, completely inhibited microglial enhancement of glioblastoma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which were unaffected by EGF stimulation, EGFR inhibition or coculture with microglia. CSF-1 only stimulated microglia invasion, whereas EGF only stimulated glioblastoma cell migration demonstrating a synergistic interaction between these two cell types. Finally, using PLX3397 (a CSF-1R inhibitor which can cross the blood brain barrier) in live animals, we discovered that blockade of CSF-1R signaling in vivo reduced the number of tumorassociated microglia and glioblastoma invasion. These data indicate that glioblastoma and microglia interactions mediated by EGF and CSF-1 can enhance glioblastoma invasion, and demonstrate the possibility of inhibiting glioblastoma invasion by targeting glioblastoma-associated microglia via inhibition of the CSF-1R. 2 U N C O R R E C TE D P R O O F Molecular Medicine