Faculty of Health, Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia.
Translational Research Institute, Brisbane, QLD, Australia.
Methods Mol Biol. 2021;2179:327-340. doi: 10.1007/978-1-0716-0779-4_25.
The critical role of metabolism in facilitating cancer cell growth and survival has been demonstrated by a combination of methods including, but not limited to, genomic sequencing, transcriptomic and proteomic analyses, measurements of radio-labelled substrate flux and the high throughput measurement of oxidative metabolism in unlabelled live cells using the Seahorse Extracellular Flux (XF) technology. These studies have revealed that tumour cells exhibit a dynamic metabolic plasticity, using numerous pathways including both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to support cell proliferation, energy production and the synthesis of biomass. These advanced technologies have also demonstrated metabolic differences between cancer cell types, between molecular subtypes within cancers and between cell states. This has been exemplified by examining the transitions of cancer cells between epithelial and mesenchymal phenotypes, referred to as epithelial-mesenchymal plasticity (EMP). A growing number of studies are demonstrating significant metabolic alterations associated with these transitions, such as increased use of glycolysis by triple negative breast cancers (TNBC) or glutamine addiction in lung cancer. Models of EMP, including invasive cell lines and xenografts, isolated circulating tumour cells and metastatic tissue have been used to examine EMP metabolism. Understanding the metabolism supporting molecular and cellular plasticity and increased metastatic capacity may reveal metabolic vulnerabilities that can be therapeutically exploited. This chapter describes protocols for using the Seahorse Extracellular Flux Analyzer (XFe96), which simultaneously performs real-time monitoring of oxidative phosphorylation and glycolysis in living cells. As an example, we compare the metabolic profiles generated from two breast cancer sublines that reflect epithelial and mesenchymal phenotypes, respectively. We use this example to show how the methodology described can generate bioenergetic results that in turn can be correlated to EMP phenotypes. Normalisation of bioenergetic studies should be considered with respect to cell number, and to potential differences in mitochondrial mass, itself being an important bioenergetics endpoint.
代谢在促进癌细胞生长和存活中的关键作用已经通过多种方法得到证实，包括但不限于基因组测序、转录组学和蛋白质组学分析、放射性标记底物通量的测量以及使用 Seahorse 细胞外通量 (XF) 技术对未标记活细胞中的氧化代谢进行高通量测量。这些研究表明，肿瘤细胞表现出动态代谢可塑性，利用多种途径，包括糖酵解和线粒体氧化磷酸化 (OXPHOS)，以支持细胞增殖、能量产生和生物量合成。这些先进技术还证明了癌细胞类型之间、癌症内部的分子亚型之间以及细胞状态之间的代谢差异。这通过检查癌细胞在上皮和间充质表型之间的转变来证明，这种转变被称为上皮-间充质可塑性 (EMP)。越来越多的研究表明，与这些转变相关的代谢发生了显著改变，例如三阴性乳腺癌 (TNBC) 中糖酵解的增加或肺癌中的谷氨酰胺成瘾。EMP 的模型，包括侵袭性细胞系和异种移植物、分离的循环肿瘤细胞和转移性组织，已被用于研究 EMP 代谢。了解支持分子和细胞可塑性以及增加转移能力的代谢可能会揭示可用于治疗的代谢弱点。本章描述了使用 Seahorse 细胞外通量分析仪 (XFe96) 的协议，该分析仪可实时监测活细胞中的氧化磷酸化和糖酵解。作为一个例子，我们比较了反映上皮和间充质表型的两种乳腺癌亚系产生的代谢谱。我们使用这个例子来说明所描述的方法学如何生成生物能量学结果，这些结果反过来又可以与 EMP 表型相关联。应该考虑到细胞数量以及线粒体质量的潜在差异来对生物能量学研究进行归一化，线粒体质量本身就是一个重要的生物能量学终点。
