Abstract Background Diffuse midline glioma (DMG) and glioblastoma (GBM) are aggressive brain tumors with limited treatment options. Macrophage phagocytosis is a complex, tightly regulated process governed by competing pro-phagocytic and anti-phagocytic signals. CD47-SIRPα signaling inhibits macrophage activity, while radiotherapy (RT) can enhance tumor immunogenicity. How RT and CD47 blockade together modulate macrophage “appetite” and activation states remains poorly understood, particularly in the context of glioma immune evasion and therapy resistance. Methods Human and mouse glioma cell lines were exposed to fractionated RT, anti-CD47 monoclonal antibody, or both. Flow cytometry and ELISA quantified the induction of immunogenic cell death (ICD) and expression of damage-associated molecular patterns (DAMPs). In vitro, phagocytosis assays were performed using peripheral blood mononuclear cell-derived and bone marrow-derived macrophages. Single-cell RNA sequencing (scRNA-seq) was used to analyze transcriptional changes in macrophage subsets that phagocytosed (“eaters”) or did not phagocytose (“non-eaters”) glioma cells. In vivo, efficacy of combination therapy was assessed using orthotopic xenograft and syngeneic mouse models of DMG and GBM. Results RT induced ICD in glioma cells, evidenced by dose-dependent increases in DAMPs such as phosphatidylserine, calreticulin, HSP70/90, and HMGB1. RT and anti-CD47 each promoted macrophage-mediated phagocytosis, with a synergistic effect observed when combined. scRNA-seq of phagocytic macrophages revealed transcriptionally distinct subpopulations associated with each treatment, characterized by enrichment in inflammatory, metabolic, and antigen presentation pathways. In vivo, combination therapy significantly reduced tumor burden, extended survival, and polarized tumor-associated macrophages toward a pro-inflammatory (M1-like) phenotype. Distinct macrophage markers (CLEC7A, CD44, CD63) validated scRNA-seq findings in vivo. Conclusions This study highlights that macrophage fate is intimately linked to the molecular properties of what they phagocytose. Phagocytosis is not a singular, uniform process but a dynamic and context-dependent event that drives macrophage specialization and plasticity. By demonstrating that RT and anti-CD47 therapy shape distinct macrophage phenotypes through their effects on tumor immunogenicity, this study provides a framework for understanding how to harness and reprogram macrophage activity for therapeutic benefit. These findings underscore the potential of targeting macrophage plasticity as a strategy to enhance antitumor immunity and improve outcomes in malignant gliomas and other diseases. Keywords: Combination therapy, Immunotherapy, Innate, Macrophage __________________________________________________________________ WHAT IS ALREADY KNOWN ON THIS TOPIC * Radiation therapy promotes immunogenic cell death in glioma cells, and its combination with anti-CD47 blockade elicits a more robust therapeutic response than either modality alone. WHAT THIS STUDY ADDS * This study establishes that macrophage phagocytosis is a context-specific process that actively reprograms macrophage fate rather than serving as a terminal immune function. By integrating flow cytometric isolation of phagocytic versus non-phagocytic macrophages with single-cell transcriptomics, it reveals that radiotherapy and CD47 blockade induce distinct, non-overlapping macrophage activation states. These phenotypes correlate with therapeutic efficacy in glioma models, highlighting phagocytosis as a tunable immunologic driver with implications for rational, macrophage-targeted therapies. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY * By uncovering distinct macrophage subsets shaped by phagocytic stimuli, this study lays the foundation for identifying specialized antigen-presenting myeloid cells that may drive adaptive immune responses in gliomas. It opens avenues for engineering or enriching macrophage populations with defined functional states to modulate the tumor microenvironment. The transcriptional plasticity observed suggests a path toward reprogramming innate immunity in situ. These findings may ultimately inform next-generation immunotherapies that exploit macrophage versatility to enhance antigen presentation, promote immune infiltration, and overcome resistance in cold tumors. Introduction Diffuse midline gliomas (DMGs) and glioblastoma (GBM) are two of the most aggressive brain tumors that affect children and adults. The outlook for patients with DMG is extremely poor, as over 90% succumb to the disease within 2 years following diagnosis, with the median survival period ranging from 9 to 12 months.[50]^1 The strategic location in the brainstem and the tumor’s tendency to spread into nearby healthy tissue greatly restrict the options for surgical intervention, positioning DMG as one of the most dire and complex cancers affecting children. Similarly, despite aggressive therapeutic interventions the prognosis of GBM remains poor, with a median survival ranging from 12 to 18 months. This grim outlook underscores the urgent need for the development of effective treatment strategies. Radiotherapy (RT) remains the cornerstone of treatment for both DMG and GBM, offering temporary control of tumor progression but ultimately failing to produce durable responses. RT primarily works by causing immediate damage to the DNA of cancer cells, leading to their death.[51]^2 Innovations such as intensity-modulated RT and image-guided RT have improved the precision of treatment, allowing for increased radiation doses to be delivered to tumors while minimizing exposure to adjacent healthy tissues.[52]^3 Consequently, RT has become safer and more effective for a broader range of patients. Additionally, the destruction of cancer cells by RT releases tumor antigens, potentially enhancing the activation of antigen-specific T cells and triggering an adaptive immune response to target residual cancer cells.[53]^4 This potential has sparked numerous clinical trials exploring the synergy between RT and immunotherapy.[54]^5 However, RT’s impact on the immune system is complex, as it can provoke both protective and harmful immune responses, influencing tumor growth in varying ways. Phagocytosis is a critical function of the immune system, involving immune cells that encapsulate and break down large external pathogens or dying cells to preserve the body’s balance.[55]^6 Macrophages play a pivotal role in this process by detecting signals on cells undergoing apoptosis or cancerous cells that indicate they should be engulfed and digested.[56]^7 These signals include “find-me” and “eat-me” cues that trigger the phagocytic process.[57]^8 Conversely, healthy cells and some cancer cells can emit “don’t-eat-me” signals that deter phagocytic activity.[58]^9 A key interaction in this mechanism is between CD47 on the cell surface and SIRPα on macrophages, leading to the activation of processes that facilitate phagocytosis, such as cytoskeletal changes and membrane movement.[59]^10 Beyond the CD47-SIRPα interaction,[60]^11 an increasing number of signals have been identified that inhibit phagocytosis, including interactions between CD24-Siglec 10,[61]^12 PD-L1-PD-1,[62]^13 MHC-1-LILRB1,[63]^14 and APMAP-GPR84.[64]^15 Given that tumors often contain high levels of macrophages, inhibiting these “don’t-eat-me” signals presents a promising approach for enhancing cancer immunotherapy. Several studies have demonstrated that combining RT with CD47 immune checkpoint blockade is an effective strategy to promote macrophage-mediated phagocytosis of tumor cells.[65]^16 17 However, despite the observed increase in phagocytic activity following combination therapy, a subset of tumor-associated macrophages fails to engage in phagocytosis. The mechanisms underlying this heterogeneity remain unclear, and to date, the transcriptional responses of macrophages following phagocytosis have not been systematically examined. In this study, we investigated the effects of RT and CD47 blockade in DMG and GBM. We found that RT induces immunogenic cell death in glioma cells and, when combined with CD47 blockade, significantly enhances in vitro phagocytosis. Single-cell RNA sequencing revealed distinct macrophage subpopulations that either facilitated or suppressed the phagocytosis of glioma cells treated with RT, anti-CD47, or both. Notably, this combination therapy also extended survival in multiple glioma mouse models by promoting diverse macrophage activation states and enhancing tumoricidal activity. Together, these findings highlight the dynamic heterogeneity of macrophages in response to therapy and support the potential of RT plus CD47 blockade as a promising strategy for improving the outcome for patients with malignant glioma. Results Radiotherapy induces immunogenic cell death in diffuse midline glioma and glioblastoma RT is known to cause immunogenic cell death (ICD) in different cancer types.[66]18,[67]20 However, it is unclear if RT can elicit ICD in malignant gliomas (H3K27M-DMG and GBM). To address this question, we exposed several patient-derived human DMG (BT-245, SU-DIPGXVII, SU-DIPGXXV) and murine GBM cell lines (SB28 and CT-2A) to increasing doses of RT for three consecutive days (0 Gy, 2 Gy×3, 4 Gy×3, 8 Gy×3, 16 Gy×3) and evaluated their response. We find the half-maximal inhibitory concentration (IC[50]) of human DMG lines to range from 10 to 12 Gy (BT-245, SU-DIPGXVII, SU-DIPGXXV), whereas the IC[50] for murine GBM cell lines were 9.3 Gy (CT-2A) and 14.2 Gy (SB28) ([68]online supplemental figure S1). The cells undergoing ICD are characterized by the increased expression of damage-associated molecular patterns (DAMPs).[69]^21 We, therefore, measured the surface expression of common DAMP molecules such as phosphatidylserine (PS), calreticulin (CRT), heat shock protein 70 (HSP70) and 90 (HSP90)[70]^22 at 24 hours after the last treatment with RT. In both the human DMG and mouse glioma cell lines, there is a dose-dependent increase in the expression of PS ([71]figure 1A and [72]online supplemental figure S2A), CRT ([73]figure 1B and [74]online supplemental figure S2B), HSP70 ([75]figure 1C and [76]online supplemental figure S2C), and HSP90 ([77]figure 1D and [78]online supplemental figure S2D) compared with non-irradiated (0 Gy) cells. We further measured the extracellular high mobility group protein 1 (HMGB1), a DAMP molecule released by dying cells[79]^23 in the supernatants of DMG and GBM cell lines that were collected at 48 hours after the last treatment with RT. In line with the findings for the surface expression of DAMPs, we observed a dose-dependent increase in the release of HMGB1 ([80]figure 1E and [81]online supplemental figure S2E) compared with non-irradiated cell lines. Collectively, these findings indicate that fractionated RT induces ICD in both DMG and GBM cell lines as demonstrated by a dose-dependent increase in the surface expression and extracellular release of DAMPs. Figure 1. Radiation therapy induces surface expression and release of damage-associated molecular patterns in human DMG/DIPG. Human patient-derived DMG/DIPG cell lines were exposed to increasing doses of RT for three consecutive days. (A–D) Representative overlay histograms and median fluorescence intensity (MFI) values displaying the expression levels of phosphatidylserine, calreticulin, heat shock protein (HSP70), and HSP 90 on the surface of BT245, SU-DIPGXVII and SU-DIPGXXV cells. Results are expressed as mean+SD (n=3 technical replicates). Unpaired Student’s t-test: p<0.05, **p<0.01. (E) 24 hours post the final RT treatment, HMGB1 released in the supernatants of BT245, SU-DIPGXVII and SU-DIPGXXV cells were quantified. Results are expressed as mean+SD (n=3 technical replicates); Unpaired Student’s t-test: p<0.05, **p<0.01. DMG, diffuse midline glioma; HMGB1, high mobility group protein 1; RT, radiotherapy. [82]Figure 1 [83]Open in a new tab A combination of radiotherapy and anti-CD47 therapy enhances the in vitro phagocytosis of malignant glioma cell lines DAMPs are known to activate the cells of the innate immune system and enhance adaptive immune responses either directly or indirectly.[84]^24 25 To examine if the increased expression of DAMPs on DMG and GBM cell lines can promote the clearance of DMG and GBM cell lines by macrophages, we performed an in vitro phagocytosis assay. Briefly, the DMG cell lines were exposed to fractionated RT as described before and co-cultured with human peripheral blood mononuclear cell (PBMC)-derived macrophages at a 2:1 ratio in the presence or absence of blocking human anti-CD47 monoclonal antibody (mAb). The phagocytosis of tumor cells by macrophages were determined by flow cytometry (gating strategy is shown in [85]online supplemental figure S3) as previously published.[86]^26 Anti-CD47 mAb treatment increased the phagocytosis of three different DMG cell lines, SU-DIPG17, SU-DIPG25, and BT-245 compared with IgG treatment ([87]figure 2A–F). However, RT (4 Gy×3) significantly increased the phagocytosis of SU-DIPG25, but not SU-DIPG17 and BT-245 compared with control ([88]figure 2A–F). Combining RT (4 Gy×3) and anti-CD47 treatment significantly enhanced the phagocytosis of DMG cells compared with individual treatments ([89]figure 2A–F). We further assessed the phagocytosis of murine glioblastoma cell lines that were either irradiated or non-irradiated and co-cultured with mouse bone marrow-derived macrophages in the presence or absence of anti-msCD47 mAb (clone MIAP301). Anti-CD47 treatment significantly increased the phagocytosis of SB28 cells, but not CT-2A cells compared with control ([90]figure 2G–J). RT did not increase the phagocytosis of either SB28 or CT-2A cells ([91]figure 2H–J). However, combining RT (3 Gy×3) and anti-CD47 mAb treatment significantly enhanced the phagocytosis of both CT-2A and SB28 compared with either control, anti-CD47 mAb or RT treatment alone ([92]figure 2G–J). These findings suggest that a combination of fractionated RT and anti-CD47 treatment significantly promotes the in vitro phagocytosis of human DMG/DIPG and murine glioblastoma cell lines. Notably, this combination enabled consistent phagocytosis even in cell lines that invariably escaped monotherapy. Figure 2. Combining radiation therapy with anti-CD47 mAb treatment enhances in vitro phagocytosis of human DIPG and mouse glioma cell lines. Human patient-derived DMG cell lines (BT-245, SU-DIPGXVII, and SU-DIPGXXV) were exposed to either 0 or 4 Gy×3 and incubated with human peripheral blood-derived macrophages in the presence of anti-CD47 mAb, HU5F9-G4. Flow cytometry (A, C, and E) as well as histogram (B, D, and F) plots show that combining fractionated irradiation and anti-CD47 antibody treatment increases the phagocytosis of DIPG/DMG cells by macrophages compared with individual treatments alone. (G–J) Mouse glioblastoma cell lines (SB28 and CT2A) were exposed to either 0 or 4 Gy×3 and incubated with mouse bone marrow-derived macrophages in the presence of anti-CD47 mAb, MIAP301. Data shown are consistent with two independent experiments (n=3) and are shown as mean+SD. Unpaired Student’s t-test. *p<0.05, **p<0.01 and ***p<0.0001. One-way analysis of variance, ****p<0.0001 (BT245), ***p<0.001 (DIPGXVII) and ****p<0.0001 (DIPGXXV). ***p<0.001 (CT2A) and **p<0.001 (SB28). DMG, diffuse midline glioma; mAb, monoclonal antibody. [93]Figure 2 [94]Open in a new tab Single-cell RNA-seq identifies distinct macrophage subsets induced by DMG cells following radiotherapy and anti-CD47 treatment To understand the response of macrophages to different phagocytic stimuli, we performed an unbiased and comprehensive overview of the macrophages that had phagocytosed (“eaters”) tumor cells versus those that had not (“non-eaters”) in response to either control, RT, anti-CD47, or combo treatments. To this end, we co-cultured PBMC-derived macrophages with either irradiated (4 Gy×3) or non-irradiated (0 Gy) DMG (BT-245) cells in the presence or absence of anti-CD47 mAb for 24 hours and physically segregated the “eaters” from the “non-eaters” using flow cytometry as shown in [95]figure 3A. Consistent with our earlier results, we observed more “eaters” and less “non-eaters” in RT plus anti-CD47 treatment compared with either control or monotherapy treatments ([96]online supplemental figure S4A–D). To assess the durability of the phagocytic phenotype, both “eaters” and “non-eaters” were subsequently rechallenged with fresh tumor cells. “Eaters” retained a robust phagocytic capacity even in the absence of additional anti-CD47 blockade, consistent with a stably reprogrammed effector phenotype. In contrast, “non-eaters” fail to engage in significant phagocytosis without anti-CD47 treatment but exhibit a delayed and measurable response when re-exposed to the antibody ([97]online supplemental figure S5). Figure 3. Single-cell RNA sequencing reveals the enrichment of distinct macrophage subsets following co-culture with diffuse midline glioma cells pretreated with either control, RT, anti-CD47 therapy, or combination of RT and anti-CD47 therapy. (A) Schematic diagram of the workflow used for in vitro phagocytosis and single-cell RNA sequencing. Briefly, BT245 cells were irradiated (4Gy×3) treated for three consecutive days and treated with either PBS or anti-CD47 mAb for 30 min at 37°C and co-cultured with PBMC-derived macrophages for 24 hours. BT245 cells not exposed to radiation and treated with PBS served as controls. Macrophages that either phagocytose (“eaters”) or do not phagocytose (“non-eaters”) tumor cells were sorted using flow cytometry and subjected to single-cell RNA-sequencing. (B) UMAP projection displaying 11 distinct cell clusters from the eater’s cohort. Each dotted line and arrow indicate the identity of that specific cell cluster. (C–D) UMAP plots and proportion of cells in each cluster from four treatment groups: control, 4 Gy×3, anti-CD47, or 4 Gy×3+anti-CD47. Dotted lines indicate the expansion/enrichment of distinct macrophage clusters in that treatment condition. Note the expansion/enrichment of two distinct cell clusters (1 and 3) in the combination treatment. (E) Heatmap of marker genes in each cell cluster. Representative genes with higher gene expression for each cluster are outlined on the left. Bubble plots demonstrating expression of marker genes associated with antigen presenting (F), inflammatory (G), M1-like (H), M2-like (I), proliferation (J), and tumor cell signature (K) by the various cell clusters. The dotted box indicates the cell clusters with higher average marker gene expression. The size of the bubble dot is proportional to the percentage of cells in a cluster expressing the marker gene and the color intensity is proportional to average scaled marker gene expression within a cluster. mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; RT, radiotherapy; UMAP, Uniform Manifold Approximation and Projection. [98]Figure 3 [99]Open in a new tab Flow-sorted “eaters” and “non-eaters” were subjected to single-cell RNA-sequencing (scRNA-seq). After quality control and filtering out low-quality cells from scRNA-seq datasets (see Materials and methods), we obtained 6,641 “eater” cells from the control, 2,790 cells from RT, 2,014 cells from anti-CD47, and 5,732 cells from RT plus anti-CD47 ([100]figure 3D). Dimension reduction and clustering of the cells based on their gene expression profiles identified 11 distinct cell clusters ([101]figure 3B), of which 8 were identified as macrophage clusters and 3 as contaminating tumor cell clusters. Intriguingly, our analysis revealed that certain clusters exhibited striking preferential enrichment depending on the treatment condition of DMG cells, highlighting previously unappreciated immune responses induced by combination therapy. Cluster 0 was enriched in the control, Cluster 1 was enriched in the anti-CD47 treatment, and Cluster 3 was enriched in the RT treatment. There was an enrichment of both Cluster 1 and Cluster 3 following RT plus anti-CD47 treatment ([102]figure 3C,D). Cluster 2 was characterized by the expression of the antigen-presenting genes (HLA-DRA, HLA-DQB1, and CD74) ([103]figure 3B, E and F, and [104]online supplemental table S3); Cluster 4 expressed inflammatory genes (CCL3, CCL3L1, CCL4, CCL4L2, and CSF1) ([105]figure 3B, E and G, and [106]online supplemental table S3); Cluster 5 expressed anti-inflammatory M2-like signature genes (CD163, MRC1, IL-10) ([107]figure 3B, E and I, and [108]online supplemental table S3). Interestingly, all the clusters expressed M1-like signature genes (CD86, TLR4, TLR2, IL1R1, NOS2) ([109]figure 3B, E and H). Clusters 6 and 7 had higher expression of proliferation-related genes (KI67, PCNA, and TOP2A) ([110]figure 3B, E and J, and [111]online supplemental table S3); Clusters 8, 9, and 10 expressed tumor-associated genes (PDGFRA and MYC) ([112]figure 3B, E and K, and [113]online supplemental table S3). Tumor cells were identified by the exogenous expression of green flourescent protein (GFP), blasticidin, and luciferase (Luc2) (transduced) ([114]online supplemental figure S6A,C and E). Lastly, Cluster 11 had upregulation of ribosomal genes (RPS and RPL genes) ([115]figure 3B and E, and [116]online supplemental table S3). We next performed quality control (QC) and filtering of the scRNA-seq data obtained from the “non-eaters” ([117]figure 3A). In total, there were 6,252 cells from the control, 4,537 cells from the anti-CD47 treatment, 3,541 cells from the RT, 2,060 cells from the RT plus anti-CD47 treatment ([118]online supplemental figure S7C). Dimension reduction and clustering of the cells based on their gene expression profiles identified 13 different clusters ([119]online supplemental figure S7A). Seven major macrophage clusters were identified and like the eaters, we saw a preferential enrichment of certain macrophage clusters depending on the treatment condition. For instance, Cluster 0 was enriched in the control, Cluster 1 was enriched in the RT treatment, and Cluster 2 was enriched in the control treatment, and Cluster 4 was enriched in the anti-CD47 treatment. Importantly, there was enrichment of both Cluster 11 and Cluster 4 following RT plus anti-CD47 treatment ([120]online supplemental figure S7B,C). Clusters 0 and 5 expressed inflammatory genes (CCL3, CCL3L1, CCL4, CCL4L2, and CSF1) ([121]online supplemental figure S7A,D and E, and [122]online supplemental table S4); Cluster 3 expressed signature genes associated with lipid metabolism (CEBPA, APOC1, CYP27A1, GM2A, PLIN2, and ASAH1) ([123]online supplemental figures S6A and S7D,E and [124]online supplemental table S4); Clusters 6, 9 and 10 expressed high levels of MKI67, PCNA, and TOP2A and were identified as proliferating cells ([125]online supplemental figure S7A, D and G, and [126]online supplemental table S4); Clusters 7, 10 and 13 expressed T-cell signature genes (CD247, CD3D, CD3E, and CD3G) ([127]online supplemental figure S7A,D and I and [128]online supplemental table S4); Cluster 8 had high expression of ribosomal biogenesis genes (FAU, RPS, and RPL proteins) ([129]online supplemental figure S7A,D and H, and [130]online supplemental table 4). Surprisingly, the cells in Clusters 11 and 12 expressed tumor-associated genes such as PDGFRA, MYC, GFP, Luc2, and blasticidin ([131]online supplemental figures S6B,D,F and S7A,D and J). This could be due to the carry-over of tumor cells from “eater” samples at the time of flow sorting or a subset of macrophages that had “eaten” tumor cells earlier is not double-positive for CD45 and Calcein-AM at the time of flow sorting ([132]online supplemental figure S4). Distinct macrophage subsets exhibit treatment-specific transcriptional and functional adaptations in response to DMG cell treatment-induced phagocytosis To identify the genes and pathways that are altered in “eaters”, specifically in control-enriched (Cluster 0), anti-CD47 enriched (Cluster 1) and RT-enriched (Cluster 3) macrophages, we first determined the marker genes that were differentially expressed and distinguished control-enriched from anti-CD47 enriched and RT-enriched using the “FindMarkers” function in Seurat.[133]^27 28 We found control-enriched expressed higher levels of NUPR1, LYZ, and JAML and lower levels of IL1RN, SPP1, FABP4, TM4SF19, and RGCC ([134]figure 4A and [135]online supplemental table S5). anti-CD47 enriched when compared with control-enriched and RT-enriched expressed higher levels of BLVRB, CHIT1, and HAMP and lower levels of IL1RN, SPP1, and ALCAM ([136]figure 4C and [137]online supplemental table S6). RT-enriched compared with anti-CD47 enriched and control-enriched expressed higher levels of ILIRN, SPP1, FABP4, RGCC, and ALCAM and lower levels of CHIT1, ALOX5AP, GLUL, CD52, and PTGDS ([138]figure 4E and [139]online supplemental table S7). Strikingly, genes upregulated in RT-enriched are downregulated in anti-CD47 enriched ([140]figure 4A,E). Next, to determine the pathways that are altered in control-enriched compared with anti-CD47 enriched and RT-enriched, we performed gene set enrichment analysis using clusterProfiler (an R package designed to perform over-representation analysis), enrichGO, and DAVID bioinformatic analysis on the significant marker genes (p value adj <0.01 and average log[2] fold change >0.25). Biological process in gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway from DAVID identified lymphocyte proliferation, phagosome, endosome, and antigen processing and presentation via major histocompatibility complex (MHC) class II genes are upregulated in control-enriched macrophages ([141]figure 4B, [142]online supplemental figure S9A,B). Similar pathway analysis on differentially expressed significant marker genes in anti-CD47 enriched showed that ATP metabolic process, oxidative phosphorylation, electron transport chain, NADH dehydrogenase, mitochondrial ATP synthesis, reactive oxygen species, and antigen processing and presentation pathways via MHC class I are upregulated in anti-CD47 mAb treatment-enriched macrophages ([143]figure 4D, [144]online supplemental figure S10A,B). In contrast, response to interferon-gamma, ERK signaling, leukocyte cell–cell adhesion, focal adhesion and monocyte chemotaxis pathways are upregulated in radiation-enriched macrophages ([145]figure 4E and F, [146]online supplemental figure S11A,B). Figure 4. Characterization of macrophages that are enriched following phagocytosis of diffuse midline glioma cells pretreated with Control, anti-CD47 therapy or RT. (A) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with control treated BT245 cells (cell Cluster 0) compared with macrophages that were enriched after co-culture with either anti-CD47 (cell Cluster 1) or RT (cell Cluster 3) treated BT245 cells. (B) Gene Ontology enrichment analysis of biological process for significantly upregulated genes between control-enriched macrophages versus anti-CD47-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. (C) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with anti-CD47 treated BT245 cells (cell Cluster 1) compared with macrophages that were enriched after co-culture with either control (cell Cluster 0) or RT (cell Cluster 3) treated BT245 cells. (D) Gene Ontology enrichment analysis of biological processes for significantly upregulated genes between anti-CD47-enriched macrophages versus control-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. (E) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with RT treated BT245 cells (cell Cluster 3) compared with macrophages that were enriched after co-culture with either control (cell Cluster 0) or anti-CD47 (cell Cluster 1) treated BT245 cells. (F) Gene Ontology enrichment analysis of biological processes for significantly upregulated genes between control-enriched macrophages versus anti-CD47-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. BP, biological process; FC, fold change; NS, not significant; RT, radiotherapy. [147]Figure 4 [148]Open in a new tab To identify the genes and pathways altered in “non-eaters,” we performed the same approach as used for “eaters”. In comparison to RT-enriched (C1) and anti-CD47-enriched (C4) macrophages, control-enriched macrophages (C2) expressed higher levels of PTGDS, CHIT1, CLU, MDM2, and CYP1B1 and lower levels of IL1RN, TM4SF19, RGCC, FABP4 ([149]online supplemental figure S8A and [150]online supplemental table S8). RT-enriched compared with control-enriched and anti-CD47-enriched macrophages expresses higher levels of MARCO, NUPR1, C1QA, CD14, and MARCKS and lower levels of IL1RN, RGCC, TM4SF19, CRABP2, and SPP1 ([151]online supplemental figure S8C and [152]online supplemental table S9). anti-CD47-enriched compared with RT-enriched and control-enriched macrophages expressed higher levels of IL1RN, RGCC, TM4SF19, SPP1, CRABP2 and lower levels of CD14, MARCKS, CTSC, NUPR1, and HLA-DMB ([153]online supplemental figure S8E and [154]online supplemental table S10). The pathway analysis on significant (p value adj <0.01 and average log[2] FC>0.25) marker genes in control-enriched “non-eaters” revealed the upregulation of apoptotic signaling, p53 signaling, DNA damage checkpoint and oxidative phosphorylation ([155]online supplemental figure S8B and S12A,B). Upregulation of antigen processing and presentation, T-cell receptor and CD4 receptor binding genes were observed in radiation-enriched “non-eaters” ([156]online supplemental figure S8D and S13A,B). Lastly, upregulation of purine ribonucleotide metabolic process, ATPase and GTPase activity genes are upregulated in anti-CD47-enriched “non-eaters” ([157]online supplemental figure S8F and S14A,B). Collectively, our analysis revealed distinct transcriptional adaptations in macrophages depending on their exposure to glioma cells pretreated with either radiation or anti-CD47 therapy. Radiation-enriched macrophages exhibited upregulation of interferon-gamma response and ERK signaling, which may be attributed to radiation-induced DNA damage and ICD. ICD leads to the release of DAMPs, including cytosolic DNA and ATP, which activate the STING pathway, subsequently inducing type I interferons and pro-inflammatory cytokines. This response can further enhance ERK signaling, a key pathway involved in macrophage activation, cytokine production, and inflammatory responses. In contrast, macrophages exposed to anti-CD47-treated tumor cells demonstrated significant upregulation of oxidative phosphorylation, ATP synthesis, and NADH dehydrogenase activity, suggesting that active live-cell phagocytosis is a metabolically demanding process. Engulfment and degradation of tumor cells require high ATP turnover to fuel cytoskeletal rearrangements, phagosome acidification, and lysosomal processing, leading to increased mitochondrial respiration. Notably, in macrophages that failed to phagocytose tumor cells, we observed upregulation of p53 signaling and DNA damage checkpoint pathways, which may indicate a stress response associated with prolonged tumor cell interaction without successful engulfment. The inability to clear tumor cells might lead to persistent DNA damage accumulation or oxidative stress, triggering cell-cycle arrest and apoptotic priming via p53 activation. These findings suggest that successful phagocytosis reprograms macrophage metabolism toward an oxidative state, whereas ineffective phagocytosis engages stress and DNA repair mechanisms, potentially altering immune functionality in the tumor microenvironment. Combination of radiotherapy and anti-CD47 treatment reduced tumor growth and prolonged the survival of human and mouse DMG/DIPG The promising results from the in vitro phagocytosis assay and scRNA-seq prompted us to test the efficacy of combining RT plus anti-CD47 in vivo models of DMG/DIPG. To this end, we implanted BT245-Luc2 and SU-DIPGXVII-Luc2 in NOD-SCID IL2R gamma^null (NSG) mice. The schema for tumor establishment and RT and anti-CD47 treatments for the two respective models are outlined in [158]figure 5A and [159]online supplemental figure S16A. The above-mentioned lines were inoculated into the mouse pons and bioluminescence (BLI) imaging was used to monitor tumor establishment and tumor growth. Once tumors were established, mice were randomized into four groups (control, RT, anti-CD47, and RT plus anti-CD47) and subjected to indicated treatment regimens and doses as shown in [160]figure 5A and [161]online supplemental figure S16A. The combination treatment of RT followed by anti-CD47 treatment reduced tumor growth ([162]figure 5B, [163]online supplemental figure S15 and S16B,C). The mice receiving monotherapy (fractionated RT or anti-CD47 treatment) did not show any effect on tumor growth ([164]figure 5B and 1, [165]online supplemental figure S16B,C). The survival analysis suggested that the combination treatment significantly improved the survival compared with monotherapy, with the median survival increasing from 46 to 58 days in the BT245-bearing mice and from 170 to 210 days in the SU-DIPG17-bearing mice (p<0.005). Importantly, in the less aggressive, SU-DIPG17 model, we see a complete regression of the tumor in 3/7 mice (S16D). There was no increase in the median survival of the mice that received either RT or anti-CD47 in either of the models ([166]figure 5C and [167]online supplemental figure S16D). Lastly, we assessed the tumor-bearing brain tissue at endpoint for its macrophage surface marker profile. In BT245 tumor-bearing mice that received anti-CD47 were found to have an increased number of F4/80^+ macrophages when compared with all other treatments ([168]figure 5D). However, the mice that received the combination treatment showed an increased percentage of M1-like and decreased percentage of M2-like macrophages compared with all other treatments ([169]figure 5E and F). In the SU-DIPG17 model, the mice that received the combination treatment were found to have an increased number of F4/80^+ and M1-like and decreased percentage of M2-like macrophages compared with all other treatments ([170]online supplemental figure S16E,F and G). Figure 5. Combination of RT and anti-CD47 treatment reduces tumor burden and prolongs the survival of mice bearing BT245 xenografts compared with monotherapy. (A) Schematic diagram showing the experimental treatment plan followed. (B) Quantification of total IVIS flux values over time course. (C) Kaplan-Meier survival analysis of BT245 xenografts with indicated treatments, control, n=10; RT, n=9; anti-CD47, n=10; and RT+anti-CD47, n=10. The log-rank test was used to calculate statistical significance. *p<0.05, **p<0.01. (D–F) Bar graphs demonstrating the relative percentages of F4/80^+, CD80^+ (M1-like) and CD206^+ (M2-like) tumor-associated macrophages in control, RT, anti-CD47, or RT+anti-CD47 treated mice bearing BT245 xenografts. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. IP, intraperitoneal; IVIS, In Vivo Imaging System; RT, radiotherapy. [171]Figure 5 [172]Open in a new tab To determine the safety and efficacy of combining RT and anti-CD47 treatment in a syngeneic immune-competent mouse model, we stereotactically implanted a GEMM-derived syngeneic mouse pHGG cell line, PKC-HA, into the pons of 6–8 weeks old C57BL/6 mouse. The schema for tumor establishment and RT and anti-CD47 treatments for the two respective models are outlined in [173]online supplemental figure S17A. Once tumors were established, mice were randomized into four groups (control, RT, anti-CD47, and RT plus anti-CD47) and subjected to indicated treatment regimens and doses as shown in [174]online supplemental figure S17A. In line with the findings from patient-derived xenograft (PDX) models, combining RT and anti-CD47 treatment reduced tumor growth compared with monotherapy ([175]online supplemental figure S17B,C and S18). Additionally, the survival analysis suggested that the combination treatment significantly improved the median survival compared with monotherapy. Importantly, complete tumor regression was observed in 6/10 mice treated with the combination treatment. RT and anti-CD47 treatment also significantly increased the median survival compared with control ([176]online supplemental figure S17D). Taken together, these results strongly support that combining RT and anti-CD47 treatment is highly effective, well-tolerated and prolongs survival in both immunodeficient and immunocompetent DMG animal models. Combination of radiotherapy and anti-CD47 treatment reduced tumor growth and prolonged the survival in immunocompetent GBM mouse models Given that pediatric and adult high-grade gliomas (HGG) have historically exhibited differential responses to immunotherapies—likely due to variations in immune cell infiltration, mutational burden, and myeloid composition—we sought to investigate whether the promising effects of combining RT and anti-CD47 therapy observed in our pediatric models could be translated into adult HGG. Previous studies have highlighted the relative paucity of T-cell infiltration in pediatric HGG compared with their adult counterparts, which may influence the efficacy of myeloid-targeted therapies.[177]^29 We have recently shown that combining a single dose of RT with anti-CD47 treatment significantly increased the survival in PDX models of GBM.[178]^16 Nevertheless, the safety and efficacy of combining fractionated RT with anti-CD47 in GBM mouse models with intact immune system was not investigated previously. To this end, we stereotactically implanted two well-characterized murine glioblastoma cell lines CT-2A and SB28 into the frontal cortex of 6–8 weeks old C57BL/6 mice. After implantation with CT-2A and SB28 luciferase-expressing cells, the tumor engraftment was confirmed by BLI, and mice were randomized into four groups (control, RT, anti-CD47, and RT plus anti-CD47) and subjected to indicated treatment regimens and doses as shown in [179]figure 6A and [180]online supplemental figure S19A. Similar to the DMG models, the combination treatment of RT followed by anti-CD47 treatment reduced tumor growth compared with monotherapy ([181]figure 6B, [182]online supplemental figure S19C,D). Additionally, the survival analysis of CT-2A-bearing mice suggested that the combination treatment significantly improved the median survival compared with monotherapy, with the median survival increasing from 36 to 72 days. RT alone and anti-CD47 alone also significantly increased the median survival to 58 and 57 days from 36 days ([183]figure 6C). In the more aggressive, SB28-bearing mice, the combination treatment increased the median to 35 days from 30 days. In contrast, either RT alone or anti-CD47 alone did not significantly increase the median survival in SB28-bearing mice ([184]online supplemental figure S19D). Next, we assessed the tumor-bearing brain tissue at the endpoint for its macrophage surface marker profile. In the CT-2A-bearing mice, anti-CD47 treatment increased the infiltration of F480^+ and M1-like macrophages and decreased the infiltration of M2-like macrophages compared with other treatments ([185]figure 6D–F). In the SB28-bearing mice, both anti-CD47 alone and RT plus anti-CD47 treatment increased the increased the infiltration of F480^+ and M1-like macrophages and decreased the infiltration of M2-like macrophages compared with other treatments ([186]online supplemental figure S19E–G). Collectively, these results strongly support that combining RT and anti-CD47 mAb treatment is highly effective, well-tolerated, and prolongs survival in the fully immunocompetent GBM mouse models. Figure 6. Combination of RT and anti-CD47 treatment reduces tumor burden and prolongs the survival of mice-bearing CT-2A intracranial allografts compared with monotherapy. (A) Schematic diagram showing the experimental treatment plan followed. (B) Quantification of total IVIS flux values over time course. (C) Kaplan-Meier survival analysis of CT-2A allografts with indicated treatments, control, n=8; RT, n=8; anti-CD47, n=8; and RT+anti-CD47, n=8. The log-rank test was used to calculate statistical significance. *p<0.05, **p<0.01, ***p<0.001. ns, not significant. (D–F) Bar graphs demonstrating the relative percentages of F4/80^+, CD80^+ (M1-like) and CD206^+ (M2-like) tumor-associated macrophages in control, RT, anti-CD47, or RT+anti-CD47 treated mice bearing CT-2A intracranial allografts. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. IP, intraperitoneal; IVIS, In Vivo Imaging System; RT, radiotherapy. [187]Figure 6 [188]Open in a new tab Distinct macrophage subpopulations are shaped by radiation and anti-CD47 therapy in gliomas scRNA-seq revealed the enrichment of distinct macrophage subsets in response to co-culture with either control, irradiated, or anti-CD47 treated DMG cells. To validate these findings in vivo and at the protein level, we first determined the marker genes that are expressed highly on the cell surface of control-enriched (C0), RT-enriched (C3), and anti-CD47-enriched (C1) macrophages. Several genes were expressed at higher levels; however, in comparison to all the cell clusters, CLEC7A showed higher expression in control-enriched ([189]figure 7A), CD44 showed higher expression in RT-enriched ([190]figure 7D), CD63 showed higher expression in anti-CD47-enriched ([191]figure 7G). Next, to determine if these cell surface marker genes are enriched in vivo, we performed high-dimensional flow cytometry on single-suspension cells harvested at endpoint from the brains of C57BL/6 mice bearing either CT-2A or PKC-HA cells and treated with different conditions as shown in [192]figure 6A and [193]online supplemental figure S17A. In agreement with the scRNA-seq data, we found higher expression of CLEC7A on the surface of F4/80^+ macrophages infiltrating the brain tumors of control-treated mice compared with either RT or anti-CD47 or RT plus anti-CD47 treated mice ([194]figure 7B,C). Similarly, CD44 was expressed highly on the surface of F4/80+macrophages infiltrating the brain tumors of RT and RT plus anti-CD47 treated mice compared with either control or anti-CD47 treated mice ([195]figure 7E,F). Finally, CD63 was expressed highly on the surface of F4/80^+ macrophages infiltrating the brain tumors of anti-CD47 and RT plus anti-CD47 treated mice compared with either control or RT treated mice ([196]figure 7H,I). In conclusion, these results validate the scRNA-seq data and confirm the existence as well as the enrichment of distinct macrophage subpopulations following treatment of tumor-bearing glioma mice with either control, RT, or anti-CD47 therapy. Strikingly, the combination of RT and anti-CD47 treatment results in the enrichment of macrophage subpopulations that were enriched in either RT alone or anti-CD47 alone treated mice. Figure 7. Validation of marker genes identified from single-cell RNA-sequencing using tumor-associated macrophages obtained from murine diffuse midline gliomas and glioblastoma intracranial allografts treated with either control, RT, anti-CD47, or RT+anti-CD47. (A) Dot plot indicates the average expression of CLEC7A, CD44 (D), and (G) for each cell cluster identified from single-cell RNA-sequencing. (B–C) Representative overlay histograms and median fluorescence intensity (MFI) values of CLEC7A, CD44 (E–F), and CD63 (H–I), expression in gliomas isolated from mice treated with either phosphate-buffered saline (control), RT, anti-CD47 therapy, or RT with anti-CD47 combination therapy. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. RT, radiotherapy. [197]Figure 7 [198]Open in a new tab Discussion This study for the first time reveals that macrophage fate is intricately linked to what they phagocytose, challenging the traditional notion of phagocytosis as a uniform process. Instead, phagocytosis emerges as a dynamic and multifaceted event that shapes macrophage transcriptional, metabolic, and functional states in response to the specific stimuli encountered in the tumor microenvironment. RT and anti-CD47 mAb treatment significantly alter the immunogenic landscape of tumor glioma cells and consequently, the identity and function of macrophages that interact with these cells. These findings highlight the importance of considering phagocytosis not only as an endpoint of immune activation but also as a driver of macrophage specialization and plasticity. The induction of ICD by RT, characterized by the dose-dependent upregulation of DAMPs such as PS, CRT, HSP70 and HSP90, and the extracellular release of HMGB1, profoundly influences macrophage function. By exposing or releasing these immunogenic signals, RT primes tumor cells for recognition and uptake by macrophages. However, the macrophage response to phagocytosis of these irradiated cells is not homogeneous. Instead, RT-induced tumor cell death shapes a distinct macrophage phenotype enriched in inflammatory and interferon-gamma-responsive pathways. These macrophages exhibit transcriptional upregulation of ERK signaling and antigen presentation pathways, suggesting that the nature of what macrophages “eat” directly programs their downstream functions and immune-stimulatory capacity. Similarly, anti-CD47 mAb treatment, which blocks the “don’t eat me” signal, enhances macrophage-mediated phagocytosis but elicits a different transcriptional and metabolic response. Macrophages that engulf tumor cells treated with anti-CD47 mAb upregulate metabolic pathways, including oxidative phosphorylation and ATP synthesis, and exhibit enhanced antigen presentation through MHC class I molecules. This metabolic reprogramming underscores that phagocytosis is not merely an endpoint event but a process that initiates distinct macrophage functional programs, driven by the immunologic and molecular properties of the ingested material. The combination of RT and anti-CD47 therapy revealed a remarkable synergy in macrophage-mediated tumor clearance, with macrophages adopting a hybrid phenotype that incorporates features of both RT-enriched and anti-CD47-enriched subsets. scRNA-seq revealed that macrophages in the combination-treated groups exhibited transcriptional signatures of metabolic activation, inflammatory signaling, and enhanced antigen presentation. These hybrid macrophages likely represent an optimal immune phenotype for antitumor activity, integrating the immune priming effects of RT with the enhanced phagocytic capacity enabled by anti-CD47 treatment. This finding reinforces the idea that macrophage fate is not only shaped by the process of phagocytosis but is also highly context-dependent, influenced by the molecular properties of the material being internalized and the broader microenvironmental signals. The in vivo data further supports the idea that macrophage fate is determined by the immunogenicity of the material they phagocytose. Tumor-associated macrophages from mice treated with RT and anti-CD47 therapy showed a marked shift in polarization toward an M1-like, pro-inflammatory phenotype, with reduced representation of M2-like, protumorigenic macrophages. Importantly, the enhanced antitumor macrophage activity was associated with a significant reduction in tumor growth and, in some models, complete tumor regression. These novel findings highlight the plasticity of macrophages and their ability to adopt distinct functional states based on the nature of the tumor cells they ingest. The study’s findings challenge the conventional view of phagocytosis as a singular process and instead present it as a critical determinant of macrophage identity and function. Macrophages are not passive scavengers but active participants in the immune response, with their fate and activity directly influenced by the molecular composition of what they phagocytose. For instance, macrophages that engulf irradiated tumor cells rich in DAMPs adopt an inflammatory, immune-activating phenotype, while those that internalize CD47-inhibited cells reprogram metabolically to optimize antigen processing and presentation. These context-dependent responses highlight how phagocytic inputs act as cues that influence downstream macrophage behavior and their capacity to engage in antitumor immunity. Importantly, our rechallenge experiments further support this concept of functional imprinting. Macrophages previously identified as “eaters” retained their phagocytic capacity on exposure to fresh tumor cells, even in the absence of additional anti-CD47 therapy suggesting stable reprogramming toward an effector state. Conversely, “non-eaters” were initially unresponsive in the absence of anti-CD47 but demonstrate a delayed phagocytic response on re-exposure to the antibody, indicating that they are not irreversibly inert but rather exist in a poised, signal-responsive state capable of activation on appropriate stimulation. This observed functional plasticity has important implications for immunotherapy. Therapeutic strategies that promote productive phagocytic engagement, such as the combination of radiation and CD47 blockade, may not only enhance immediate tumor clearance but also stably reprogram macrophages toward sustained antitumor activity. Understanding the molecular determinants of this reprogramming will be key to leveraging macrophage plasticity for durable therapeutic benefit. More broadly, these results underscore the importance of viewing phagocytosis as a context-dependent, instructive process that shapes the trajectory of innate and adaptive immune responses. The implications of these findings extend beyond gliomas, as the principles of macrophage plasticity and the dynamic nature of phagocytosis are likely relevant to other cancers and diseases involving macrophage activity. For example, macrophage phenotypes in infectious diseases, autoimmune conditions, and tissue repair may also be determined by the specific material they phagocytose. Understanding these context-dependent interactions could inform strategies to reprogram macrophages in various disease settings by altering the nature of the material they encounter. We are currently designing follow-up experiments involving depletion and gene perturbation strategies to elucidate the functional roles of distinct macrophage subsets identified in this study. Future research should aim to dissect the molecular mechanisms underlying the macrophage specialization in response to different treatments. For example, how do specific DAMPs or CD47 blockade signals program distinct transcriptional and metabolic pathways in macrophages? Additionally, the interplay between phagocytosis and other macrophage functions, such as cytokine production, antigen presentation, and tissue remodeling, warrants further investigation. Identifying the factors that influence macrophage fate could lead to novel therapeutic strategies to modulate macrophage activity in cancer and other diseases. In conclusion, this study highlights that macrophage fate is intimately linked to the molecular properties of what they phagocytose. Phagocytosis is not a singular, uniform process but a dynamic and context-dependent event that drives macrophage specialization and plasticity. By demonstrating that RT and anti-CD47 therapy shape distinct macrophage phenotypes through their effects on tumor immunogenicity, this study provides a framework for understanding how to harness and reprogram macrophages activity for therapeutic benefit. These findings underscore the potential of targeting macrophage plasticity as a strategy to enhance antitumor immunity and improve outcomes in malignant gliomas and other diseases. Materials and methods Cell lines and cell cultures DIPG/DMG cells were maintained as previously described.[199]^11 Briefly, SU-DIPXVII and SU-DIPGXXV cells were provided by Dr Michelle Monje (Stanford University, California, USA) and cultured in tumor stem media consisting of Neurobasal (-A), human basic fibroblast growth factor (FGFb; 20 ng/mL; Shenandoah Biotech), human epidermal growth factor (EGF; 20 ng/mL; Shenandoah Biotech), human PDGF-AA (20 ng/mL; Shenandoah Biotech), PDGF-BB (20 ng/mL; Shenandoah Biotech), and heparin sulfate (10 ng/mL) (Sigma). BT245 cells were provided by Dr Keith Ligon (Dana-Farber Cancer Institute, Boston) and maintained in human NeuroCult NS-A media (STEMCELL Technologies) supplemented with penicillin/streptomycin (100×), heparin (2 mg/mL), human EGF (20 ng/mL), and human FGFb (10 ng/mL). Mouse pediatric high-grade glioma cell lines, PKC-HA and PHC-HA were provided by Dr Oren Becher and grown in mouse NeuroCult NS-A media (STEMCELL Technologies) supplemented with penicillin/streptomycin, heparin (2 mg/mL, STEMCELL Technologies), human EGF (10 ng/mL), and human FGFb (20 ng/mL). Mouse glioma cell line, SB28 cells were provided by Dr Hideho Okada (University of California San Francisco) and grown in RPMI media 1640 (Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin (100×), GlutaMAX (100×), minimum essential media (MEM) non-essential amino acids (10 mM), HEPES buffer solution (1 M), Sodium Pyruvate (100 mM), and 2-mercaptoethanol (0.05 mM). The CT-2A cell line was purchased from Millipore Sigma and maintained in RPMI media 1640 as described above. Cells were grown in either tumor neurospheres (ultra-low attachment plates or flasks, Corning) or adherent monolayer conditions (CytoOne) as indicated. All patient-derived cell lines were authenticated using DNA fingerprinting (STR analysis) at the Molecular Biology service center (UCD-AMC). The cell lines were routinely tested for Mycoplasma contamination using Venor GeM Mycoplasma Detection Kit (Sigma-Aldrich, St. Louis, Missouri, USA). The list of cell lines used in this study is shown in [200]online supplemental table S1. Irradiation In vitro and IC[50] determination Cell lines used for in vitro assays were cultured on Geltrex-coated 96-well plate or T-25 flasks for 24 hours. Cells were subjected to irradiation (0 Gy×3, 2 Gy×3, or 4 Gy×3) for three consecutive days using ^137Cs irradiator. All irradiations were performed using a perpendicular 662 KeV γ-photon beam with dose coverage to uniformly irradiate all samples at a dose rate of 1.09 Gy/min and a source-to-surface distance of 30 cm. To determine the IC[50,] the cells were irradiated as described above and on day 5 after post-irradiation, cell viability was measured as the intracellular ATP content using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), following the manufacturer’s instructions. IC[50] was calculated using GraphPad Prism software. In vivo NOD-scid IL2rgnull (NSG) mice implanted with BT-245 and SU-DIPGXVII cells received fractionated doses of 1 Gy per day for three consecutive days over 2 weeks (a total of 6 Gy). C57BL/6 mice implanted with PKC-HA, SB28, and CT-2A received 3.3 Gy per day for three consecutive days (total 10 Gy). Under isoflurane anesthesia, each mouse was positioned in the prone orientation and aligned to the isocenter in two orthogonal planes by fluoroscopy. Each side of the mouse brain received half of the dose, which was delivered in opposing, lateral beams. Dosimetric calculation was done using a Monte Carlo simulation in SmART-ATP (SmART Scientific Solutions B.V.) for the midbrain+pons+frontal lobe receiving the prescribed dose. Treatment was administered using a 225 kV photon beam with 0.3 mm Cu filtration through a circular 10 mm diameter collimator. Flow cytometry Irradiated human DIPG/DMG and mouse glioma cell lines were dissociated by treatment with TrypLE and rinsed with cell staining buffer (BioLegend). Single-cell suspensions of tumor cells were incubated with Truestain FC blocking solution (BioLegend) for 10 min at 4°C. Next, the Zombie Fixable Aqua Dye (BioLegend) diluted (1:1,000) in phosphate-buffered saline (PBS) was added and samples were incubated for 20 min 4°C. Lastly, a cocktail of fluorophore-conjugated antibodies resuspended in 100 µL cell staining buffer (BioLegend) was added directly to each tube, and samples were incubated at 4°C for 30 min in the dark. All the antibodies used in this study are shown in [201]online supplemental table S2. Flow cytometry was performed using CytoFLEX LX (Beckman Coulter). FlowJo V.10.8 was used for data analysis. ELISA assay BT-245, SU-DIPGXVII, SU-DIPGXXV, CT-2A and SB28 were exposed to increasing doses for RT ([202]figure 1E and [203]online supplemental figure S1E) for three consecutive days. 48 hours post the final RT treatment, HMGB1 concentration in the supernatants of irradiated human DMG/DIPG and mouse glioma cell lines were quantified by using human and mouse HMGB1 ELISA kit (Novus Biologicals) according to the manufacturer’s instructions. In vitro phagocytosis In vitro phagocytosis assay with human and mouse macrophages were performed as described previously.[204]^11 16 30 To obtain human monocytes, leukopak from healthy individuals were obtained from Children’s Hospital of Colorado Blood Donor Center. The blood was diluted 1:1 with PBS (Gibco) and PBMCs separated on a Ficoll density gradient (GE HealthCare). CD14+monocytes were positively selected by using MojoSort Human CD14 Selection Kit (BioLegend), then plated at 1×10^6/mL in 150×25 mm (Corning) non-treated sterile tissue culture plates in RPMI with 10% FBS, 1×penicillin/streptomycin, 200 mM glutamine, and 25 mM HEPES (all from Corning). To generate monocyte-derived macrophages, monocytes were treated with human recombinant Macrophage Colony-Stimulating Factor (M-CSF; 50 ng/mL) (PeproTech) and human recombinant Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF; 10 ng/mL) (PeproTech). After 48 hours, non-adherent cells were removed and the cells were washed with either 1×PBS or 1×Hanks' Balanced Salt Solution (HBSS) two times, and above-mentioned media with growth factors were added, and the plates were returned into the tissue culture incubator for an additional 5 days. Mouse macrophages are obtained from 7 to 10 weeks old C57BL/6 mouse bone marrow. The mice were killed in a CO[2] chamber and femur and tibiae were isolated. The bones were kept in ice-cold PBS and sterilized in 70% ethanol. By flushing them with mouse macrophage medium (RPMI with 10% FBS, 1×penicillin/streptomycin, 200 mM glutamine, and 25 mM HEPES), bone marrow cells were collected and plated at 1×10^6/mL in 150×25 mm non-treated tissue culture plates in mouse macrophage medium containing mouse recombinant M-CSF (50 ng/mL) (PeproTech) and mouse recombinant GM-CSF (10 ng/mL) (PeproTech). The media was changed after 48 hours and both human and mouse macrophages were harvested using TrypLE (Gibco) for experiments on days 8–9. Unless mentioned otherwise, at the end of differentiation protocol, dissociated macrophages and Mock (0 Gy) or irradiated human DIPG/DMG, and mouse glioma cell lines stained with Carboxyfluorescein succinimidyl ester (CFSE) (BioLegend) were co-cultured in macrophage medium as described above with or without prior addition of 50 µg/mL anti-CD47 monoclonal antibody (H5F9-G4) at 37°C for 2 hours in ultra-low attachment round bottom plate (Corning) 96-well plates. The tumor cell to macrophage ratio was 2:1. Cells were analyzed with CytoFLEX LX (Beckman Coulter) using a high throughput autosampler. Gates were placed according to unstained and fluorescence-minus-one controls. 4′,6-diamidino-2-phenylindole DAPI, Zombie Aqua and NIR fixable viability kit (BioLegend) were used to exclude dead cells. The flow cytometry gating strategy for determining the percentage of tumor cell phagocytosis by macrophages is shown in [205]online supplemental figure S3. Phagocytosis assays for each tumor type were performed in triplicates and repeated at least two times. RNA extraction from sorted cells, complementary DNA, and library preparation for single-cell RNA sequencing A live single-cell suspension of “eating” (phagocytic) and “non-eating” (non-phagocytic) macrophages as shown in [206]figure 3A were flow sorted, and single-cell library preparation and RNA-sequencing was performed at the University of Colorado Anschutz medical campus genomics and microarray core. Single-cell RNA sequencing data analysis, clustering, and visualization Raw sequencing reads were processed using Cell Ranger single-cell software suite (V.6.1.1) with default parameters. Reads were aligned to the human reference genome (GRCh38 V.3.0.0) from the 10x Genomics website. The resulting count table was filtered for features expressed by at least five cells and cells with at least 500 detected features. Cells with a small number of gene counts (<500) or high mitochondrial counts (mt-genes >0.2) were filtered out. After removing unwanted cells, gene expression measurements for each cell were normalized using the “LogNormalize” function in Seurat (V.4.0.4).[207]^31 Differentially expressed genes were identified using the “FindAllMarkers” function in Seurat (V.4.0.4).[208]^31 Genes with fold change more than 1.5 and adjusted p value<0.05 were considered differentially expressed genes. Cell clustering was performed using the “RunUMAP’’ and “FindClusters” function in Seurat with setting parameters dims=1:10 and resolution=0.6 and visualized using Uniform Manifold Approximation and Projection. Marker genes were used to assign identity to cell types. Pathway enrichment analysis To identify differentially expressed molecular pathways in enriched macrophage subpopulation, DAVID bioinformatics resources (V.6.8)[209]^32 was performed on differentially expressed significant marker genes. Gene sets from Gene Ontology biological process[210]^33 and KEGG[211]^34 35 were used. Cell cycle scoring Cell cycle phases of each cell were determined using the cell cycle scoring function in Seurat (V.4.0.4).[212]^31 The cell clusters that had high expression for KI67, PCNA, and TOP2A were considered proliferative. Intracranial xenograft and allograft models for diffuse midline glioma and glioblastoma In vivo mouse xenograft studies, including implantation, animal care, radiation, treatments, and euthanasia were performed under and approved by the University of Colorado Institutional Animal Care and Use Committee (IACUC) protocol #00777. Early passage BT-245 and SU-DIPG XVII cells that were transduced with Luc2-GFP are dissociated into single cells and orthotopically implanted in the pons of 8–10 weeks old female NSG mice. Briefly, mice were anesthetized and immobilized in a stereotactic frame. A midline incision was made on the skin to expose the scalp, and a microdrill was used to perform craniotomy 0.8 mm lateral to midline, 0.5 mm posterior to lambda, and 5.00 mm ventral to the surface of the skull. Next, a suspension of~1×10^5 cells in 2 mL serum-free media were stereotactically injected at the rate of 500 nL/min with a Hamilton syringe (ga26s/51 mm/pst2). For the syngeneic model of DMG/DIPG, a suspension of~5×10^5 PKC-HA cells were stereotactically injected into the pons of 6–10 weeks old female C57BL/6 mice. For the syngeneic model of GBM, ~SB28 (5×10^4) and GL261 (2.5×10^4) cells were stereotactically injected at the rate of 500 nL/min into the brain at a site of 2 mm lateral to bregma and 3 mm ventral to the surface of the skull. After leaving the needle in place for 1 min, it was retracted at 3 mm/min and the burr hole was closed with sterile bone wax (Ethicon), the skin was sutured and placed in a cage atop a surgical heat pad until ambulatory. Tumor formation was monitored by BLI using IVIS Xenogen 2500 imaging machine. After confirmation of intracranial tumor establishment with a total flux from BLI corresponding to~10^5 to 10^6 photons/second/cm^2, animals were assigned into different treatment groups using [213]www.randomizer.org. Following randomization, mice were injected with luciferin intraperitoneally and imaged for tumor progression once weekly until the end of the study. Body weight was measured once a week and mice were monitored daily and those reaching the endpoint were euthanized according to IACUC protocols by CO[2] asphyxiation, when they show signs of either neurologic deficit, failure to ambulate, body score less than 2, or weight loss greater than 15%. Antibody treatment Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use. All the NSG mice implanted with human glioma cells started receiving intraperitoneal (IP) injections of 16 mg/kg of anti-human CD47 mAb on day 18 until the end of the study as depicted in [214]figure 5A and [215]online supplemental figure S15A. All the C57BL/6 mice with human glioma cells started receiving IP injections of 32 mg/kg of anti-mouse CD47 ab (clone MIAP410) on day 18 until the end of the study as depicted in [216]figure 6A, [217]online supplemental figure S16A and S18A. Tumor tissue dissociation After resection of malignant gliomas from mice at endpoint, tumor samples were placed on ice and cut into small pieces and single-cell suspensions were made as described previously.[218]^11 Briefly, tumors were enzymatically dissociated by collagenase IV (1 mg/mL) (Worthington) in dissociation solution containing HBSS with calcium/magnesium (Gibco), non-essential amino acids (Gibco), sodium pyruvate (Gibco), sodium bicarbonate (Gibco), 25 mM HEPES (Gibco), 1X GlutaMAX-1 (Gibco), antibiotic-antimycotic (Gibco), DNase (Worthington) at 37°C. The suspension was washed two times with HBSS and filtered using 70 mM and 40 mM filters, respectively. The cells were further treated with ACK/RBC lysis buffer (Gibco), washed once with HBSS, resuspended in Bambanker (Fisher Scientific), and stored in liquid nitrogen until use. For multicolor flow cytometry, cells were thawed from liquid nitrogen, blocked, and stained as described under the flow cytometry section. Statistical analysis All statistical analysis was performed using GraphPad Prism V.9.2.0. Results were expressed as mean+SD. One-way analysis of variance followed by Bonferroni’s post hoc comparison tests were used for three or four group comparisons and two-tailed unpaired Student’s t-tests were used for comparing two conditions. Animal survival curves were analyzed using the Kaplan-Meier method and Grehan-Breslow-Wilcoxon tests, with groups compared by respective median survival or number of days taken to reach 50% morbidity. Differences were considered significant at *p<0.05. Supplementary material online supplemental file 1 [219]jitc-13-9-s001.jpg^ (998.8KB, jpg) DOI: 10.1136/jitc-2025-012211 online supplemental figure 1 [220]jitc-13-9-s002.docx^ (11.2MB, docx) DOI: 10.1136/jitc-2025-012211 online supplemental table 1 [221]jitc-13-9-s003.xlsx^ (9.6KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 2 [222]jitc-13-9-s004.xlsx^ (9.4KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 3 [223]jitc-13-9-s005.xlsx^ (555.6KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 4 [224]jitc-13-9-s006.xlsx^ (454.3KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 5 [225]jitc-13-9-s007.xlsx^ (33.3KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 6 [226]jitc-13-9-s008.xlsx^ (26.5KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 7 [227]jitc-13-9-s009.xlsx^ (56.1KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 8 [228]jitc-13-9-s010.xlsx^ (35.8KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 9 [229]jitc-13-9-s011.xlsx^ (44.2KB, xlsx) DOI: 10.1136/jitc-2025-012211 online supplemental table 10 [230]jitc-13-9-s012.xlsx^ (111.8KB, xlsx) DOI: 10.1136/jitc-2025-012211 Acknowledgements