Table 3_Integrated multi-omics reveals GABARAP-mediated mitophagy and pyruvate metabolism as key drivers of osteosarcoma progression.xlsx

BackgroundOsteosarcoma is a highly aggressive bone malignancy characterized by frequent metastasis and therapy resistance. Although mitophagy and pyruvate metabolism are increasingly recognized as critical metabolic regulators, their interaction in osteosarcoma remains poorly understood. The autophagy-related protein GABARAP, central to mitochondrial quality control, has not been systematically evaluated in osteosarcoma.
MethodsSingle-cell RNA sequencing (scRNA-seq) datasets (GSE162454, GSE237070) were analyzed to delineate cellular heterogeneity and malignant states, with prognostic clusters identified by Scissor and inferCNV. Tumor microenvironment (TME) composition and intercellular signaling were profiled using CellChat. Pathway enrichment and multi-omics integration across TARGET, GSE21257, and GSE32981 highlighted mitophagy-pyruvate coupling, which were further validated by spatial transcriptomics and in vitro functional assays.
ResultsWe mapped the osteosarcoma ecosystem and identified two malignant subpopulations, Ost_1 and Cho_2 (Mal_Ost/Cho), exhibiting high genomic instability, stemness, and poor prognosis. The osteosarcoma TME displayed profound immune remodeling, characterized by infiltration of T/NK cells alongside enrichment of immunosuppressive Tregs and M2-polarized macrophages. Enhanced MIF-mediated signaling between Mal_Ost/Cho and T/NK compartments suggested a key mechanism of immune evasion. Both malignant subtypes demonstrated coordinated activation of mitophagy and pyruvate metabolism, sustaining metabolic adaptation and tumor progression. Multi-omics integration pinpointed GABARAP as a central hub regulating this mitophagy-metabolism axis, spatially enriched within metabolic hotspots and immunosuppressive niches. Functionally, GABARAP depletion disrupted mitophagy flux, mitochondrial integrity, and energy production, thereby impairing osteosarcoma cell proliferation and migration.
ConclusionThese findings reveal that GABARAP links mitophagy-driven metabolic adaptation with immune evasion, representing a key regulator and potential therapeutic target in osteosarcoma.

背景 骨肉瘤是一种高度侵袭性的骨恶性肿瘤，以频繁转移与治疗耐药为典型特征。尽管线粒体自噬与丙酮酸代谢作为关键代谢调控通路日益受到学界重视，但二者在骨肉瘤中的相互作用仍未得到充分阐明。作为线粒体质量控制核心的自噬相关蛋白GABARAP（GABARAP），在骨肉瘤中尚未得到系统性评估。
方法 本研究通过分析单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据集GSE162454与GSE237070，以阐明细胞异质性与恶性表型，并借助Scissor与inferCNV算法鉴定预后亚型。利用CellChat工具对肿瘤微环境（tumor microenvironment, TME）的组成及细胞间信号通路进行刻画。通过整合TARGET、GSE21257、GSE32981队列的多组学数据开展通路富集分析，筛选出线粒体自噬-丙酮酸代谢耦合通路，并通过空间转录组学与体外功能实验对该通路进行验证。
结果 本研究绘制了骨肉瘤的肿瘤生态系统图谱，并鉴定出两种恶性亚群Ost_1与Cho_2（合称Mal_Ost/Cho），二者均表现出高基因组不稳定性、干细胞样特性与不良预后结局。骨肉瘤肿瘤微环境存在显著的免疫重塑，表现为T/NK细胞浸润，同时伴随免疫抑制性调节性T细胞（Tregs）与M2型极化巨噬细胞的富集。Mal_Ost/Cho亚群与T/NK细胞间存在增强的巨噬细胞迁移抑制因子（macrophage migration inhibitory factor, MIF）介导的信号通路，提示这是肿瘤免疫逃逸的关键机制。两种恶性亚群均呈现出线粒体自噬与丙酮酸代谢的协同激活，以维持代谢适应性与肿瘤进展。多组学整合分析确定GABARAP为调控该线粒体自噬-代谢轴的核心枢纽蛋白，其在代谢热点区域与免疫抑制微环境区域呈空间富集状态。功能实验显示，敲低GABARAP会破坏线粒体自噬流、线粒体完整性与能量产生过程，从而损害骨肉瘤细胞的增殖与迁移能力。
结论 本研究结果揭示GABARAP可将线粒体自噬介导的代谢适应性与免疫逃逸联系起来，是骨肉瘤的关键调控因子与潜在治疗靶点。