Background Osteosarcoma (OS) is a malignant bone tumor that often develops during the period of rapid growth associated with adolescence. Despite successful primary tumor control accompanied by adjuvant chemotherapy, death from pulmonary metastases occurs in approximately 30% of patients within 5 years. As overall survival in patients remains unchanged over the last 30 years, urgent needs for novel therapeutic strategies exist. Cancer metastasis is characterized by complex molecular events which result from alterations in gene and protein expression/function. Recent studies suggest that metabolic adaptations, or “metabolic reprogramming,” may similarly contribute to cancer metastasis. The goal of this study was to specifically interrogate the metabolic vulnerabilities of highly metastatic OS cell lines in a series of in vitro and in vivo experiments, in order to identify a tractable metabolically targeted therapeutic strategy for patients. Methods Nutrient deprivation and drug treatment experiments were performed in MG63.3, 143B, and K7M2 OS cell lines to identify the impact of glutaminase-1 (GLS1) inhibition and metformin treatment on cell proliferation. We functionally validated the impact of drug treatment with extracellular flux analysis, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry. 13 C-glucose and 13 C-glutamine tracing was employed to identify specific contributions of these nutrients to the global metabolic profiles generated with GLS1 inhibition and metformin treatment in vivo. Results Highly metastatic OS cell lines require glutamine for proliferation, and exposure to CB-839, in combination with metformin, induces both primary tumor growth inhibition and a distinct reduction in metastatic outgrowth in vivo. Further, combination-treated OS cells showed a reduction in cellular mitochondrial respiration, while NMR confirmed the pharmacodynamic effects of glutaminase inhibition in tumor tissues. We observed global decreases in glycolysis and tricarboxylic acid (TCA) cycle functionality, alongside an increase in fatty acid oxidation and pyrimidine catabolism. Conclusions This data suggests combination-treated cells cannot compensate for metformin-induced electron transport chain inhibition by upregulating glutaminolysis to generate TCA cycle intermediates required for cell proliferation, translating into significant reductions in tumor growth and metastatic progression. This therapeutic approach could be considered for future clinical development for OS patients presenting with or at high risk of developing metastasis.

中文翻译：

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谷氨酰胺酶-1 (GLS1) 抑制可限制骨肉瘤的转移进展


背景 骨肉瘤（OS）是一种恶性骨肿瘤，通常在青春期快速生长期间发生。尽管通过辅助化疗成功控制了原发肿瘤，但约 30% 的患者在 5 年内死于肺转移。由于患者的总生存率在过去 30 年中保持不变，因此迫切需要新的治疗策略。癌症转移的特点是复杂的分子事件，这些事件是由基因和蛋白质表达/功能的改变引起的。最近的研究表明，代谢适应或“代谢重编程”可能同样会导致癌症转移。本研究的目的是在一系列体外和体内实验中专门询问高度转移性 OS 细胞系的代谢脆弱性，以便为患者确定一种易于处理的代谢靶向治疗策略。方法在 MG63.3、143B 和 K7M2 OS 细胞系中进行营养剥夺和药物处理实验，以确定谷氨酰胺酶 1 (GLS1) 抑制和二甲双胍治疗对细胞增殖的影响。我们通过细胞外通量分析、核磁共振 (NMR) 光谱和质谱法对药物治疗的影响进行了功能验证。采用 13 C-葡萄糖和 13 C-谷氨酰胺示踪来确定这些营养素对体内 GLS1 抑制和二甲双胍治疗产生的总体代谢谱的具体贡献。结果高度转移的 OS 细胞系需要谷氨酰胺才能增殖，接触 CB-839 与二甲双胍联合可诱导原发性肿瘤生长抑制和体内转移性生长明显减少。 此外，联合处理的 OS 细胞显示细胞线粒体呼吸减少，而 NMR 证实了肿瘤组织中谷氨酰胺酶抑制的药效作用。我们观察到糖酵解和三羧酸（TCA）循环功能的整体下降，同时脂肪酸氧化和嘧啶分解代谢的增加。结论 该数据表明，联合治疗的细胞无法通过上调谷氨酰胺分解产生细胞增殖所需的 TCA 循环中间体来补偿二甲双胍诱导的电子传递链抑制，从而显着减少肿瘤生长和转移进展。这种治疗方法可以考虑用于患有转移或具有发生转移高风险的 OS 患者的未来临床开发。