Lakshmanachetty Senthilnath, Riemondy Kent, Sanford Bridget, Donson Andrew, Chen Vincent, Balakrishnan Ilango, Prince Eric W, Hankinson Todd, Dahl Nathan, Vibhakar Rajeev, Foreman Nicholas, Venkatraman Sujatha, Mitra Siddhartha S
Department of Pediatrics, Center for Childhood Cancer and Blood Disorders, Division of Heme/Onc and Bone Marrow Transplant, Children's Hospital Colorado, University of Colorado School of Medicine, Aurora, Colorado, USA.
Morgan Adams Foundation for Pediatric Brain Tumor Research Program, Children's Hospital Colorado, Aurora, Colorado, USA.
J Immunother Cancer. 2025 Sep 5;13(9):e012211. doi: 10.1136/jitc-2025-012211.
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.
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.
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.
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.
弥漫性中线胶质瘤（DMG）和胶质母细胞瘤（GBM）是侵袭性脑肿瘤，治疗选择有限。巨噬细胞吞噬作用是一个复杂的、受到严格调控的过程，由相互竞争的促吞噬和抗吞噬信号所支配。CD47-SIRPα信号传导抑制巨噬细胞活性，而放射治疗（RT）可增强肿瘤免疫原性。RT与CD47阻断联合作用如何调节巨噬细胞的“吞噬能力”和激活状态仍知之甚少，尤其是在胶质瘤免疫逃逸和治疗耐药的背景下。
将人源和小鼠源胶质瘤细胞系暴露于分次RT、抗CD47单克隆抗体或两者联合处理。流式细胞术和酶联免疫吸附测定法（ELISA）定量免疫原性细胞死亡（ICD）的诱导情况以及损伤相关分子模式（DAMPs）的表达。在体外，使用外周血单核细胞来源和骨髓来源的巨噬细胞进行吞噬试验。单细胞RNA测序（scRNA-seq）用于分析吞噬（“吞噬者”）或未吞噬（“非吞噬者”）胶质瘤细胞的巨噬细胞亚群的转录变化。在体内，使用DMG和GBM的原位异种移植和同基因小鼠模型评估联合治疗的疗效。
RT诱导胶质瘤细胞发生ICD，表现为磷脂酰丝氨酸、钙网蛋白、热休克蛋白70/90和高迁移率族蛋白B1等DAMPs呈剂量依赖性增加。RT和抗CD47各自均促进巨噬细胞介导的吞噬作用，联合使用时观察到协同效应。对吞噬性巨噬细胞进行scRNA-seq分析发现，与每种治疗相关的转录上不同的亚群，其特征是在炎症、代谢和抗原呈递途径中富集。在体内，联合治疗显著降低肿瘤负荷，延长生存期，并使肿瘤相关巨噬细胞向促炎（M1样）表型极化。不同的巨噬细胞标志物（CLEC7A、CD44、CD63）在体内验证了scRNA-seq的结果。
本研究强调巨噬细胞的命运与它们所吞噬物质的分子特性密切相关。吞噬作用不是一个单一、统一的过程，而是一个动态的、依赖于环境的事件，它驱动巨噬细胞的特化和可塑性。通过证明RT和抗CD47治疗通过对肿瘤免疫原性的影响塑造不同的巨噬细胞表型，本研究提供了一个框架，用于理解如何利用和重新编程巨噬细胞活性以获得治疗益处。这些发现强调了将巨噬细胞可塑性作为一种策略来增强抗肿瘤免疫力并改善恶性胶质瘤和其他疾病治疗结果的潜力。
