Abstract Diffuse Midline Glioma (DMG) remains one of the most lethal pediatric brain tumors, characterized by near-universal therapeutic resistance. Tumor-associated macrophages and resident microglia comprise the dominant immune population in DMG, yet the functional consequences of their engagement with tumor cells following therapy remain poorly defined. Here, we investigate how distinct modes of tumor cell clearance shape macrophage activation states and immune function, with direct relevance to pediatric DMG. Using complementary in vivo glioma models, ex vivo phagocytosis assays, and single-cell transcriptomic profiling, we demonstrate that macrophage responses are not uniformly pro-inflammatory following tumor cell uptake. Instead, the qualitative nature of phagocytosis, particularly the cellular context in which tumor cells are engulfed, drives divergent and durable macrophage activation programs. Phagocytosis of therapy-damaged tumor cells, such as those induced by radiotherapy, preferentially activates innate immune signaling pathways including cGAS–STING and NF-κB, promoting antigen presentation, interferon signaling, and cross-priming potential. In contrast, engulfment of apoptotic or metabolically intact tumor cells induces a transcriptional program enriched for scavenger receptors, lipid metabolism, oxidative stress responses, and immune suppressive pathways characteristic of dysfunctional tumor-associated macrophages. Single-cell RNA sequencing reveals that these phagocytosis-dependent programs coexist spatially and temporally within the tumor microenvironment, suggesting that macrophage heterogeneity in DMG may be driven by differential access to distinct tumor cell substrates. Importantly, blockade of the CD47–SIRPα axis amplifies phagocytic flux but does not uniformly reprogram macrophages toward immunostimulatory states, highlighting the need to consider phagocytic context rather than magnitude alone. Collectively, these findings establish phagocytosis as a fate-determining event for macrophages in pediatric DMG and identify tumor cell clearance as a critical regulator of myeloid-driven immune outcomes. Notably, the act of phagocytosis itself appears to directly drive an immunosuppressive scavenger signature under specific tumor and therapy contexts, with important implications for the rational design of macrophage-targeted immunotherapies in DMG.
Mitra et al. (Tue,) studied this question.