Abstract
A large amount of energy used for nutrient processing and cellular functions is essential for tumorigenesis. Total intracellular adenosine triphosphate (ATP) is mainly generated by glycolysis and mitochondrial oxidative phosphorylation. Here, we provide a protocol for measurements of energy metabolism in cancer cells by using Seahorse XF24 Extracellular Flux analyzer. Specifically, this machine measures glycolysis by analyzing the extracellular acidification rate (ECAR) and measures mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR), through real-time and live cell analysis. This protocol is provided for researchers who are unfamiliar with the method and to aid them in carrying out the technique successfully.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Reference
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. https://doi.org/10.1016/j.cell.2011.02.013
Ward PS, Thompson CB (2012) Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer Cell 21(3):297–308. https://doi.org/10.1016/j.ccr.2012.02.014
Pavlova NN, Thompson CB (2016) The emerging hallmarks of cancer metabolism. Cell Metab 23(1):27–47. https://doi.org/10.1016/j.cmet.2015.12.006
Herst PM, Tan AS, Scarlett DJ, Berridge MV (2004) Cell surface oxygen consumption by mitochondrial gene knockout cells. Biochim Biophys Acta 1656(2-3):79–87. https://doi.org/10.1016/j.bbabio.2004.01.008
Warburg O, Posener K, Negelein E (1924) Über den Stoffwechsel der Carcinomzelle. Biochem Z 152:309–344
Wallace DC (2012) Mitochondria and cancer. Nat Rev Cancer 12(10):685–698. https://doi.org/10.1038/nrc3365
Guppy M, Leedman P, Zu X, Russell V (2002) Contribution by different fuels and metabolic pathways to the total ATP turnover of proliferating MCF-7 breast cancer cells. Biochem J 364(Pt 1):309–315
Nakashima RA, Paggi MG, Pedersen PL (1984) Contributions of glycolysis and oxidative phosphorylation to adenosine 5′-triphosphate production in AS-30D hepatoma cells. Cancer Res 44(12 Pt 1):5702–5706
Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4(11):891–899. https://doi.org/10.1038/nrc1478
Moreno-Sanchez R, Rodriguez-Enriquez S, Marin-Hernandez A, Saavedra E (2007) Energy metabolism in tumor cells. FEBS J 274(6):1393–1418. https://doi.org/10.1111/j.1742-4658.2007.05686.x
Zu XL, Guppy M (2004) Cancer metabolism: facts, fantasy, and fiction. Biochem Biophys Res Commun 313(3):459–465
Strohecker AM, Guo JY, Karsli-Uzunbas G, Price SM, Chen GJ, Mathew R, McMahon M, White E (2013) Autophagy sustains mitochondrial glutamine metabolism and growth of BrafV600E-driven lung tumors. Cancer Discov 3(11):1272–1285. https://doi.org/10.1158/2159-8290.CD-13-0397
Guo JY, Karsli-Uzunbas G, Mathew R, Aisner SC, Kamphorst JJ, Strohecker AM, Chen G, Price S, Lu W, Teng X, Snyder E, Santanam U, Dipaola RS, Jacks T, Rabinowitz JD, White E (2013) Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. Genes Dev 27(13):1447–1461. https://doi.org/10.1101/gad.219642.113
Weinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez M, Kalyanaraman B, Mutlu GM, Budinger GR, Chandel NS (2010) Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci U S A 107(19):8788–8793. https://doi.org/10.1073/pnas.1003428107
Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, Kamphorst JJ, Chen G, Lemons JM, Karantza V, Coller HA, Dipaola RS, Gelinas C, Rabinowitz JD, White E (2011) Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 25(5):460–470. https://doi.org/10.1101/gad.2016311
Qian W, Van Houten B (2010) Alterations in bioenergetics due to changes in mitochondrial DNA copy number. Methods 51(4):452–457. https://doi.org/10.1016/j.ymeth.2010.03.006
van der Windt GJ, Chang CH, Pearce EL (2016) Measuring bioenergetics in T cells using a seahorse extracellular flux analyzer. Curr Protoc Immunol 113:3 16B 11–13 16B 14. https://doi.org/10.1002/0471142735.im0316bs113
Zhang J, Nuebel E, Wisidagama DR, Setoguchi K, Hong JS, Van Horn CM, Imam SS, Vergnes L, Malone CS, Koehler CM, Teitell MA (2012) Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells. Nat Protoc 7(6):1068–1085. https://doi.org/10.1038/nprot.2012.048
Acknowledgment
We thank Yong Liu and Agilent agents for helpful discussions and suggestions. Jing Zhang is supported by a DoD BCRP Breakthrough Fellowship Award (W81XWH-17-1-0016). Qing Zhang is supported by grants from the National Cancer Institute (R01CA211732, R21CA223675) and American Cancer Society (RSG-18-059-01-TBE). Qing Zhang also received a career development award from the DoD (W81XWH-15-1-0599). Qing Zhang is a V Scholar, Kimmel Scholar, Komen Career Catalyst Awardee, and Mary Kay Foundation Awardee.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Zhang, J., Zhang, Q. (2019). Using Seahorse Machine to Measure OCR and ECAR in Cancer Cells. In: Haznadar, M. (eds) Cancer Metabolism. Methods in Molecular Biology, vol 1928. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9027-6_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9027-6_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9026-9
Online ISBN: 978-1-4939-9027-6
eBook Packages: Springer Protocols