XRT-02. SINGLE-CELL RNA SEQUENCING IDENTIFIES FUNCTIONAL MACROPHAGE SUBSETS THAT ARE ENRICHED IN RESPONSE TO DIFFERENTIAL PHAGOCYTOSIS INDUCTION AGAINST GLIOMA

Pediatric diffuse midline gliomas (DMGs), are fatal brain tumors of childhood arising in the ventral pons. Currently, radiation therapy (RT) is the mainstay treatment for DIPG. However, RT is not a curative treatment and provides only temporary relief for most patients. Recent advances in immunotherapy have yielded some fantastic opportunities to effectively treat patients with high-grade pediatric brain tumors. In this study, we demonstrate that fractionated RT (4Gy X 3) induces immunogenic cell death and activates multiple damage-associated molecular patterns (DAMPs) on DMG/DIPG cells. Furthermore, combining 4Gy X 3 with anti-CD47 therapy enhances the in vitro phagocytosis of DIPG/DMG cells by peripheral blood mononuclear cell-derived macrophages. Next, using single-cell RNA sequencing (scRNA-seq), we investigated the transcriptomic profile of macrophages that were co-cultured with irradiated or non-irradiated DMG cells in the presence of anti-CD47 monoclonal antibodies for 24 hours. Our findings identified eleven distinct macrophage clusters displaying different gene expression patterns in all the treatment conditions. However, PBS treatment led to a marked increase in macrophages expressing antigen-presentation genes (HLA-DR and HLA-DQB1). 4Gy X 3 treatment led to the enrichment of macrophages expressing genes related to the oxidative phosphorylation pathway, whereas anti-CD47 treatment led to the enrichment of macrophages expressing genes related to interferon-gamma response and ERK-signaling pathway. Lastly, mice intracranially transplanted with DMG/DIPG that received 4Gy X 3 and anti-CD47 therapy showed a significant decrease in tumor growth and an increase in survival rate than those receiving either monotherapy alone. We are currently performing scRNA-seq validation studies on tumors obtained from orthotopic and syngeneic models of DMG. In summary, our results highlight the functional heterogeneity of macrophages and suggest that combining fractionated RT with anti-CD47 therapy has distinct anti-tumor effects and can be used as a therapeutic approach for treating DIPG patients.