restoring mitochondrial quantity and quality to reverse warburg effect and drive neuroblastoma differentiation

Reduced mitochondrial quality and quantity in tumors is associated with dedifferentiation and increased malignancy. However, it remains unclear how to restore mitochondrial quantity and quality in tumors, and whether mitochondrial restoration can drive tumor differentiation. Our study shows that restoring mitochondrial function using retinoic acid (RA) to boost mitochondrial biogenesis and a mitochondrial uncoupler to enhance respiration synergistically drives neuroblastoma differentiation and inhibits proliferation. U-13C-glucose/glutamine isotope tracing revealed a metabolic shift from the pentose phosphate pathway to oxidative phosphorylation, accelerating the TCA cycle and switching substrate preference from glutamine to glucose. These effects were reversed by ETC inhibitors or in ?0 cells lacking mtDNA, emphasizing the necessity of mitochondrial function for differentiation. Dietary RA and uncoupler treatment promoted tumor differentiation in an orthotopic neuroblastoma xenograft model, evidenced by neuropil production and Schwann cell recruitment. Single-cell RNA sequencing analysis of the orthotopic xenografts revealed that this strategy effectively eliminated the stem cell population, promoted differentiation, and increased mitochondrial gene signatures along the differentiation trajectory, which could potentially significantly improve patient outcomes. Collectively, our findings establish a mitochondria-centric therapeutic strategy for inducing tumor differentiation, suggesting that maintaining/driving differentiation in tumor requires not only ATP production but also continuous ATP consumption and sustained ETC activity. Overall design: Tumors with volumes ranging from 500 to 800 mm³ were collected from each group (CTRL, RA, NEN, and RA+NEN diet) within approximately 2 weeks of treatment. Two tumors per group were harvested for single cell RNA seq.

肿瘤中线粒体质量与数量的降低，与肿瘤去分化及恶性程度升高密切相关。然而，目前尚不清楚如何恢复肿瘤内线粒体的质量与数量，也尚不明确线粒体功能恢复是否能够诱导肿瘤分化。本研究表明，使用视黄酸（retinoic acid, RA）促进线粒体生物发生，联合线粒体解偶联剂增强呼吸作用，可协同诱导神经母细胞瘤分化并抑制其增殖。利用U-13C-葡萄糖/谷氨酰胺同位素示踪技术发现，该处理使肿瘤细胞的代谢途径从磷酸戊糖途径转向氧化磷酸化，加速了三羧酸（TCA）循环，并将底物偏好从谷氨酰胺切换为葡萄糖。电子传递链（electron transport chain, ETC）抑制剂或缺失线粒体DNA（mitochondrial DNA, mtDNA）的ρ⁰细胞可逆转上述效应，这凸显了线粒体功能在肿瘤分化中的必要性。在原位神经母细胞瘤异种移植模型中，饮食补充RA联合解偶联剂处理可促进肿瘤分化，这一现象可通过神经纤维网生成与施万细胞招募得到证实。对原位异种移植瘤的单细胞RNA测序分析显示，该干预策略可有效清除肿瘤干细胞群体，促进细胞分化，并沿分化轨迹上调线粒体基因特征表达，这有望显著改善患者预后。

综上，本研究结果确立了一种以线粒体为核心的诱导肿瘤分化治疗策略，提示维持或驱动肿瘤分化不仅需要ATP生成，还需要持续的ATP消耗与稳定的电子传递链活性。

总体实验设计：在治疗约2周内，从各组（对照组CTRL、RA组、NEN组及RA+NEN饮食组）中收集体积为500~800 mm³的肿瘤组织；每组选取2枚肿瘤组织用于单细胞RNA测序。