PO.CL08.01 · 临床研究
M1与M2巨噬细胞的铁死亡脆弱性与食管腺癌适应性放射抵抗相关
Ferroptotic vulnerability in M1 and M2 macrophages is associated with adaptive radiation resistance in esophageal adenocarcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：放射抵抗仍是提高食管腺癌（EAC）放化疗（CRT）后治愈率的关键障碍。铁死亡是一种脂质过氧化驱动的细胞死亡途径，日益被认为是肿瘤-免疫相互作用的调节因子。我们使用单细胞测序及空间蛋白质组学分析，研究了EAC患者中放射应答者（GR）与无应答者（NR）巨噬细胞铁死亡易感性的差异。
方法：在CRT前、中、后对患者活检进行scRNA-seq及使用Lunaphore COMET的序贯多重免疫荧光（multiplex-IF），以在单细胞分辨率表征细胞亚群及铁死亡标志物。在THP-1及人PBMC来源的M1/M2巨噬细胞中，于0-12 Gy放射±RSL3或ferrostatin后进行体外铁死亡实验。通过CellTiter-Glo、BODIPY-C11氧化及GPX4染色（共聚焦、流式细胞术）评估铁死亡活性。
结果：scRNA-seq证实GR与NR中均有强劲的髓系细胞扩增，并揭示促铁死亡基因程序（BH4生物合成、铁利用及糖酵解途径）在NR中于基线及CRT期间均上调，尤其在CRT期间大量扩增的髓系群体中。相反，抗铁死亡途径（GPX4）在GR中富集而在NR中受抑制。COMET图谱分析显示，无应答者（NR）表现出更高的基线M2巨噬细胞密度及在M1与M2亚群中均显著升高的铁死亡标志物4-HNE。CRT期间，NR表现出M2巨噬细胞浸润的进一步增加，强化了免疫抑制性TME，而良好应答者（GR）维持更高的M1占比。体外，放射在人源及鼠源巨噬细胞样细胞中均显著增强RSL3诱导的铁死亡；ferrostatin挽救了活力，证实为铁死亡性细胞死亡。人巨噬细胞表现出极化依赖性易感性：M2巨噬细胞对放射诱导的铁死亡敏感，而M1巨噬细胞则具抵抗性。
结论：CRT应答似乎与巨噬细胞极化及铁死亡易感性相关。无应答者的特征为：(i) M2主导的巨噬细胞格局，(ii) 升高的脂质过氧化，(iii) 铁死亡预激的髓系亚群扩增，以及(iv) 促铁死亡途径的持续激活。总体而言，这些数据将巨噬细胞铁死亡确定为EAC放射增敏的潜在可靶向轴。
查看英文原文 English abstract
Background: Radiation resistance remains a critical barrier to enhancing cures after chemoradiation therapy (CRT) in esophageal adenocarcinoma (EAC). Ferroptosis, a lipid peroxidation-driven cell death pathway, is increasingly recognized as a regulator of tumor-immune interactions. We investigated how ferroptotic susceptibility of macrophages might differ in radiation responders (GR) and non-responders (NR) in EAC patients using both single cell sequencing and spatial proteomic analysis.
Methods: scRNA-seq and sequential multiplex-IF using Lunaphore COMET were performed on patient biopsies before, during and after CRT to characterize cellular subsets and ferroptosis markers at single-cell resolution. In vitro ferroptosis assays were performed in THP-1, and human PBMC-derived M1/M2 macrophages following 0-12 Gy radiation ± RSL3 or ferrostatin. Ferroptotic activity was assessed by CellTiter-Glo, BODIPY-C11 oxidation, and GPX4 staining (Confocal, flowcytometry).
Results: scRNA-seq confirmed robust myeloid cell expansion in both GR and NR and revealed that pro-ferroptosis gene programs (BH4 biosynthesis, iron utilization and glycolysis pathway) were upregulated in NR both at baseline and during CRT, particularly in the myeloid population, which greatly expands during CRT. Conversely, anti-ferroptosis pathways (GPX4) were enriched in GR and suppressed in NR. COMET profiling revealed that non-responders (NR) displayed higher baseline M2 macrophage density and significantly elevated ferroptosis marker 4-HNE in both M1 and M2 subsets. During CRT, NRs exhibited a further increase in M2 macrophage infiltration, reinforcing an immunosuppressive TME, whereas good responders (GR) maintained higher M1 representation. In vitro, radiation significantly potentiated RSL3-induced ferroptosis in both human and murine macrophage-like cells; ferrostatin rescued viability, confirming ferroptotic cell death. Human macrophages displayed polarization-dependent susceptibility: M2 macrophages were sensitive to radiation-induced ferroptosis, while M1 macrophages were resistant
Conclusion: CRT response appears to relate to macrophage polarization and ferroptosis susceptibility. Non-responders are characterized by (i) M2-dominant macrophage landscapes, (ii) elevated lipid peroxidation, (iii) expansion of ferroptosis-primed myeloid subsets, and (iv) persistent activation of pro-ferroptosis pathways. Collectively this data ascribes macrophage ferroptosis as a potential targetable axis for radio-sensitization in EAC.
利益披露 Disclosure
S. Aggarwal, None..
R. Ye, None..
J. K. Burks, None..
S. H. Lin, None.