A Potent Microglial Response to Blocking the CD47-Sirpα Anti-Phagocytic Axis Overcomes Deficient Macrophage Recruitment during Anti-CD47 Immunotherapy Against Glioblastoma

Aims: Modulation of tumor-associated macrophages and microglia (TAMs) in glioblastoma (GBM) poses a strategy to overcome the disease. But little is known about the composition of the TAM-pool and the role of GBM subtypes in shaping their immunologic environment. We sought to decipher the contribution of microglia and macrophages to the GBM-TAM-pool and their response to myeloid modulation such as CD47-Sirpa disruption with CD47 blocking antibodies.

Methods: We generated immunodeficient mice with color-coded macrophages and microglia termed Ccr2Rfp/+Cx3cr1Gfp+- NSG-mice as well as Ccr2Rfp/RfpCx3cr1Gfp+- NSG-mice devoid of macrophage influx into the brain. Mice were grafted with GBM cell lines expressing EBFP2-luciferase, and treated with humanized anti-CD47 antibodies. At endpoint, tumors FACS analyzed and both TAM subsets sorted for RNAseq analysis. Plasma cytokines were assessed. Mice were imaged using cranial window technology and 2-photon microscopy to monitor microglial and macrophage phagocytosis upon anti-CD47 treatment in real time.

Results: We identified a GBM subtype dependent composition of the TAM pool. While microglial TAMs were dominant in slow growing tumors, high passaged GBM cell lines of various subtypes induced a mixed pool still depending on GBM subtype. Anti-CD47 treatment let to macrophage and microglial phagocytosis and a microglial morphology change assessed by intracranial in vivo imaging and FACS. Furthermore, anti-CD47 treatment led to macrophage recruitment and vascular egress to the tumor site in a Ccr2 independent manner. Sirpa-CD47 blockade in context of Ccr2 deficiency was accompanied by a strong proinflammatory cytokine profile. Anti-CD47 treatment significantly improved survival of tumor burdened mice in presence or absence of peripheral macrophages. RNAseq analysis of sorted macrophages and microglia after anti-CD47 treatment revealed a transcriptional profile change toward a proinflammatory and M1-polarized signature in macrophages, whereas microglia displayed a loss of M2-related genes.

Conclusion: These results emphasize the importance of both resident microglia and invading macrophages in GBM biology. Microglia by themselves are amenable to anti-CD47 treatment and act as phagocytic effector cells that reduce GBM growth and improves survival. Moreover, CD47-Sirpa disruption caused an important phenotypic and functional status change of resident microglia, which will have implications for CNS immunotherapies in the future.