Chapter 25
Studying the Metabolism of Epithelial-Mesenchymal
Plasticity Using the Seahorse XFe96 Extracellular Flux
Analyzer
Sugandha Bhatia, Erik W. Thompson, and Jennifer H. Gunter
Abstract
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.
Key words Epithelial-to-mesenchymal plasticity, Glycolysis, Oxidative phosphorylation, Respiration,
Metabolism, Metabolic phenotype, Mitochondrial CMxRos, Mitochondrial RedFM, Cellular bioenergetics, Extracellular acidification, Oxygen consumption, Seahorse Extracellular Flux Analyzer
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_25,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Erik W. Thompson and Jennifer H. Gunter shares senior authorship.
327
Studying the Metabolism of Epithelial-Mesenchymal
Plasticity Using the Seahorse XFe96 Extracellular Flux
Analyzer
Sugandha Bhatia, Erik W. Thompson, and Jennifer H. Gunter
Abstract
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.
Key words Epithelial-to-mesenchymal plasticity, Glycolysis, Oxidative phosphorylation, Respiration,
Metabolism, Metabolic phenotype, Mitochondrial CMxRos, Mitochondrial RedFM, Cellular bioenergetics, Extracellular acidification, Oxygen consumption, Seahorse Extracellular Flux Analyzer
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_25,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Erik W. Thompson and Jennifer H. Gunter shares senior authorship.
327