DMG-60. Deciphering the role of microglia in diffuse midline glioma tumor initiation and progression

Abstract Diffuse midline glioma (DMG) is among the most aggressive types of central nervous system tumors in children. Based on recent studies showing that diverse myeloid populations heavily infiltrate H3-mutant gliomas and that combining CSF1R inhibitor (CSF1Ri) with anti-PD1 therapy extended the overall survival (OS) in a syngeneic pre-clinical model, we hypothesized microglia aid in DMG tumor formation and progression. Using PLX3397 (CSF1Ri) to deplete microglia, we evaluated immune cell changes with an optimized spectral flow cytometry panel on Cytek Aurora and observed the effect of microglia depletion on DMG tumor formation. C57BL/6 mice were treated with PLX3397 i) 7 days before, ii) concurrent with, or iii) 7 days after injection of syngeneic K27M-4-hit DMG tumor cells and compared to tumor formation in control animals without PLX3397 treatment. Animals in which PLX3397 induced microglial depletion before tumor initiation showed the fastest tumor onset and significantly shortened median OS (28 days vs. 40 days for control mice; p 0.01). Mice treated with PLX3397 during or after tumor initiation showed no significant change in median OS (31 days and 41 days respectively, p=ns). All PLX3397 treatment groups had reduced microglia populations by flow cytometry and immunofluorescence. Similar to non-tumor bearing mice, no significant change in CD3+ cells was observed, although the proportion of regulatory T cells (Tregs, CD3+FoxP3+) decreased in PLX3397 treated mice (1.12 - 2.17% in PLX3397 treated groups versus 9.84% in tumor controls, p 0.01). These findings challenge our hypothesis that microglia promote tumor initiation and suggest microglia may serve an anti-tumor role during tumor formation, albeit not durably. Microglia may also support Treg infiltration, indicating their complex role in the tumor microenvironment. Ongoing studies are investigating tumor-microglia interactions to understand how signals change over time in order to capitalize on the potential therapeutic effect of these immune cells.