Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by ¹³C-metabolic flux analysis (¹³C-MFA). Principal component analysis of ¹³C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. ¹³C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism.

癌细胞使其细胞内能量代谢适应缺氧的肿瘤微环境 (TME) 以确保肿瘤进展。这种适应性机制将注意力集中在缺氧 TME 下肿瘤细胞的代谢表型，以开发新的癌症疗法。虽然广泛使用的单层 (2D) 培养不能完全反映体内缺氧的 TME，但球体 (3D) 培养可以产生类似于体内TME 的环境。然而，与缺氧下的 2D 培养物相比，在模拟肿瘤缺氧的 3D 培养物中如何表达不同的代谢表型仍不清楚。^{为了解决这个问题，我们通过13} C-代谢通量分析研究了 2D 和 3D 培养的癌细胞的代谢表型( ¹³C-MFA)。¹³ C 质量同位素分布的主成分分析清楚地证明了 3D 培养细胞的不同代谢表型。¹³ C-MFA 阐明 3D 培养显着上调丙酮酸羧化酶通量与丙酮酸羧化酶蛋白表达水平一致。另一方面，3D 培养下调谷氨酰胺水解通量。与我们的研究结果一致，3D 培养的细胞比 2D 培养的细胞对谷氨酰胺酶抑制剂的抵抗力更强。这项研究表明了考虑用于癌症代谢研究的特定体外模型的代谢特征的重要性。