The results from this study provide new insight into the mechanisms of glioblastoma invasion. Coculturing of glioblastoma cells with proportions of microglia similar to what has been observed in human patients results in a dramatic increase in glioblastoma invasion. Microglia-stimulated glioblastoma invasion was strongly inhibited by blockade of either EGFR or CSF-1R, implicating these receptors in mediating communication between these two cell types during invasion. We propose that the constitutive production of CSF-1 by glioblastoma cells can attract and stimulate microglia from the surrounding parenchyma. As microglia approach the glioblastoma cells in response to the CSF-1, microglia in turn stimulate glioblastoma cell invasion via, at least in part, EGFR activation.
In some breast cancer models, EGFR and CSF-1R stimulate production of the other receptor’s ligands (7
). In our studies, we find no evidence of stimulated secretion of the ligands by coculture or by stimulation with EGF or CSF-1. We conclude that production of CSF-1 by glio blastoma is necessary for the generation of interactions between microglia and glioblastoma cells that stimulate glioblastoma cell invasion. We observe that EGFR is necessary for microglia stimulation of glioblastoma invasion and that microglia express EGF. The most straightforward interpretation for these data is that microglia activate EGFR on glioblastoma. The EGF precursor is a membrane-spanning protein of a 150– 180 kDa that is expressed on the plasma membrane in an orientation that allows cleavage and release of EGF (20
). Because the precursor itself has EGF activity (21
), the surface expression of EGF raises the possibility that microglia can activate EGFR signaling on glioblastoma cells in a juxtacrine fashion. Alternatively, it is possible that proteases required for EGF processing are present on glioblastoma cells and EGFR activation is triggered by EGF release only when glioblastoma and microglia cells are in close proximity. Any EGF released in this fashion might be rapidly taken up by EGFR on the glioblastoma surface and endocytosed. This result perhaps explains our inability to detect EGF in the supernatant of cocultures using ELISA (data not shown). Alternatively, other ligands for EGFR, such as heparin-binding (HB)-EGF or TGF-α, might be expressed by microglia and required for the stimulation of glioblastoma invasion through activation of EGFR. The expression of these additional EGFR ligands by microglia is currently being investigated. Finally, it is possible that microglia induce glioblastoma cells to activate EGFR by an autocrine mechanism. For example, proteases expressed on the surface of microglia may process EGFR ligand precursors on glioblastoma cells once the two cell types are in proximity.
Several studies have observed an effect on GL261 tumor growth in vivo
when macrophages/microglia were ablated using the CD11b-TK
). Although treatment with PLX3397 substantially reduces the number of microglia/macrophages associated with the tumor to a level that is similar to that of gancyclovir-treated tumors in CD11-TK
mice (), we did not observe any obvious difference in tumor size between control and PLX3397-treated animals. Thus, it is possible the microglia/macrophages that remain in the CD11-TK
mice are also affected by the gancyclovir treatment and are unable to promote tumor growth. We also note that our investigation examines the effect of blocking CSF-1R signaling, which represents a more subtle treatment than eliminating these cells. Indeed, it is possible that blockade of CSF-1R signaling, while interfering with the ability of microglia to promote cell invasion into normal brain, has little or no effect on their support of tumor proliferation. Further studies will need to be carried out to elucidate the mechanisms of microglia-dependent invasion versus growth.