Advertisement

Metabolic Labeling of Cultured Mammalian Cells for Stable Isotope-Resolved Metabolomics: Practical Aspects of Tissue Culture and Sample Extraction

  • Daniel R. Crooks
  • Teresa W.-M. FanEmail author
  • W. Marston Linehan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1928)

Abstract

Stable isotope-resolved metabolomics (SIRM) methods are used increasingly by cancer researchers to probe metabolic pathways and identify vulnerabilities in cancer cells. Analytical and computational advances are being made constantly, but tissue culture and sample extraction procedures are often variable and not elaborated in the literature. This chapter discusses basic aspects of tissue culture practices as they relate to the use of stable isotope tracers and provides a detailed metabolic labeling and metabolite extraction procedure designed to maximize the amount of information that can be obtained from a single tracer experiment.

Key words

Tissue culture Stable isotopes Metabolomics Metabolite extraction Glutamine Glucose 

Notes

Acknowledgments

This work was supported by the Intramural Research Program of the National Cancer Institute, NIH, NIH 1U24DK097215-01A1, 1P01CA163223-01A1, and 3R01ES022191-04S1.

References

  1. 1.
    Lane AN, Higashi RM, Fan TW (2016) Preclinical models for interrogating drug action in human cancers using stable isotope resolved metabolomics (SIRM). Metabolomics 12(7).  https://doi.org/10.1007/s11306-016-1065-y
  2. 2.
    Buescher JM, Antoniewicz MR, Boros LG, Burgess SC, Brunengraber H, Clish CB, DeBerardinis RJ, Feron O, Frezza C, Ghesquiere B, Gottlieb E, Hiller K, Jones RG, Kamphorst JJ, Kibbey RG, Kimmelman AC, Locasale JW, Lunt SY, Maddocks OD, Malloy C, Metallo CM, Meuillet EJ, Munger J, Noh K, Rabinowitz JD, Ralser M, Sauer U, Stephanopoulos G, St-Pierre J, Tennant DA, Wittmann C, Vander Heiden MG, Vazquez A, Vousden K, Young JD, Zamboni N, Fendt SM (2015) A roadmap for interpreting (13)C metabolite labeling patterns from cells. Curr Opin Biotechnol 34:189–201.  https://doi.org/10.1016/j.copbio.2015.02.003CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Mackay GM, Zheng L, van den Broek NJ, Gottlieb E (2015) Analysis of cell metabolism using LC-MS and isotope tracers. Methods Enzymol 561:171–196.  https://doi.org/10.1016/bs.mie.2015.05.016CrossRefPubMedGoogle Scholar
  4. 4.
    Sellers K, Fox MP, Bousamra M 2nd, Slone SP, Higashi RM, Miller DM, Wang Y, Yan J, Yuneva MO, Deshpande R, Lane AN, Fan TW (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. J Clin Invest 125(2):687–698.  https://doi.org/10.1172/JCI72873CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hensley CT, Faubert B, Yuan Q, Lev-Cohain N, Jin E, Kim J, Jiang L, Ko B, Skelton R, Loudat L, Wodzak M, Klimko C, McMillan E, Butt Y, Ni M, Oliver D, Torrealba J, Malloy CR, Kernstine K, Lenkinski RE, DeBerardinis RJ (2016) Metabolic heterogeneity in human lung tumors. Cell 164(4):681–694.  https://doi.org/10.1016/j.cell.2015.12.034CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hui S, Ghergurovich JM, Morscher RJ, Jang C, Teng X, Lu W, Esparza LA, Reya T, Le Z, Yanxiang Guo J, White E, Rabinowitz JD (2017) Glucose feeds the TCA cycle via circulating lactate. Nature 551(7678):115–118.  https://doi.org/10.1038/nature24057CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sun RC, Fan TW, Deng P, Higashi RM, Lane AN, Le AT, Scott TL, Sun Q, Warmoes MO, Yang Y (2017) Noninvasive liquid diet delivery of stable isotopes into mouse models for deep metabolic network tracing. Nat Commun 8(1):1646.  https://doi.org/10.1038/s41467-017-01518-zCrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Faubert B, Li KY, Cai L, Hensley CT, Kim J, Zacharias LG, Yang C, Do QN, Doucette S, Burguete D, Li H, Huet G, Yuan Q, Wigal T, Butt Y, Ni M, Torrealba J, Oliver D, Lenkinski RE, Malloy CR, Wachsmann JW, Young JD, Kernstine K, DeBerardinis RJ (2017) Lactate metabolism in human lung tumors. Cell 171(2):358–371 e359.  https://doi.org/10.1016/j.cell.2017.09.019CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Davidson SM, Papagiannakopoulos T, Olenchock BA, Heyman JE, Keibler MA, Luengo A, Bauer MR, Jha AK, O'Brien JP, Pierce KA, Gui DY, Sullivan LB, Wasylenko TM, Subbaraj L, Chin CR, Stephanopolous G, Mott BT, Jacks T, Clish CB, Vander Heiden MG (2016) Environment impacts the metabolic dependencies of Ras-driven non-small cell lung Cancer. Cell Metab 23(3):517–528.  https://doi.org/10.1016/j.cmet.2016.01.007CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Keibler MA, Wasylenko TM, Kelleher JK, Iliopoulos O, Vander Heiden MG, Stephanopoulos G (2016) Metabolic requirements for cancer cell proliferation. Cancer Metab 4:16.  https://doi.org/10.1186/s40170-016-0156-6CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kim J, Hu Z, Cai L, Li K, Choi E, Faubert B, Bezwada D, Rodriguez-Canales J, Villalobos P, Lin YF, Ni M, Huffman KE, Girard L, Byers LA, Unsal-Kacmaz K, Pena CG, Heymach JV, Wauters E, Vansteenkiste J, Castrillon DH, Chen BPC, Wistuba I, Lambrechts D, Xu J, Minna JD, DeBerardinis RJ (2017) CPS1 maintains pyrimidine pools and DNA synthesis in KRAS/LKB1-mutant lung cancer cells. Nature 546(7656):168–172.  https://doi.org/10.1038/nature22359CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lane AN, Tan J, Wang Y, Yan J, Higashi RM, Fan TW (2017) Probing the metabolic phenotype of breast cancer cells by multiple tracer stable isotope resolved metabolomics. Metab Eng 43(Pt B):125–136.  https://doi.org/10.1016/j.ymben.2017.01.010CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Liu L, Shah S, Fan J, Park JO, Wellen KE, Rabinowitz JD (2016) Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage. Nat Chem Biol 12(5):345–352.  https://doi.org/10.1038/nchembio.2047CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Crooks DR, Maio N, Lane AN, Jarnik M, Higashi RM, Haller RG, Yang Y, Fan TWM, Linehan M, Rouault TA (2018) Acute loss of iron-sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells. J Biol Chem 293:8297.  https://doi.org/10.1074/jbc.RA118.001885CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Fan TW, Lane AN (2011) NMR-based stable isotope resolved metabolomics in systems biochemistry. J Biomol NMR 49(3–4):267–280.  https://doi.org/10.1007/s10858-011-9484-6CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Higashi RM, Fan TW, Lorkiewicz PK, Moseley HN, Lane AN (2014) Stable isotope-labeled tracers for metabolic pathway elucidation by GC-MS and FT-MS. Methods Mol Biol 1198:147–167.  https://doi.org/10.1007/978-1-4939-1258-2_11CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Jang C, Chen L, Rabinowitz JD (2018) Metabolomics and isotope tracing. Cell 173(4):822–837.  https://doi.org/10.1016/j.cell.2018.03.055CrossRefPubMedGoogle Scholar
  18. 18.
    Bruntz RC, Lane AN, Higashi RM, Fan TW (2017) Exploring cancer metabolism using stable isotope-resolved metabolomics (SIRM). J Biol Chem 292(28):11601–11609.  https://doi.org/10.1074/jbc.R117.776054CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Fan TW-M (2012) The handbook of metabolomics. Springer, New YorkCrossRefGoogle Scholar
  20. 20.
    Lane AN, Fan TW, Xie Z, Moseley HN, Higashi RM (2009) Isotopomer analysis of lipid biosynthesis by high resolution mass spectrometry and NMR. Anal Chim Acta 651(2):201–208.  https://doi.org/10.1016/j.aca.2009.08.032CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lane AN, Arumugam S, Lorkiewicz PK, Higashi RM, Laulhe S, Nantz MH, Moseley HN, Fan TW (2015) Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by 1H-detected 15N nuclear magnetic resonance. Magn Reson Chem 53(5):337–343.  https://doi.org/10.1002/mrc.4199CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yang Y, Fan TW, Lane AN, Higashi RM (2017) Chloroformate derivatization for tracing the fate of amino acids in cells and tissues by multiple stable isotope resolved metabolomics (mSIRM). Anal Chim Acta 976:63–73.  https://doi.org/10.1016/j.aca.2017.04.014CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lee WN, Boros LG, Puigjaner J, Bassilian S, Lim S, Cascante M (1998) Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose. Am J Phys 274(5 Pt 1):E843–E851Google Scholar
  24. 24.
    Saxena N, Maio N, Crooks DR, Ricketts CJ, Yang Y, Wei MH, Fan TW, Lane AN, Sourbier C, Singh A, Killian JK, Meltzer PS, Vocke CD, Rouault TA, Linehan WM (2016) SDHB-deficient cancers: the role of mutations that impair Iron sulfur cluster delivery. J Natl Cancer Inst 108(1):djv287.  https://doi.org/10.1093/jnci/djv287CrossRefGoogle Scholar
  25. 25.
    Lewis CA, Parker SJ, Fiske BP, McCloskey D, Gui DY, Green CR, Vokes NI, Feist AM, Vander Heiden MG, Metallo CM (2014) Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol Cell 55(2):253–263.  https://doi.org/10.1016/j.molcel.2014.05.008CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Moseley HN, Lane AN, Belshoff AC, Higashi RM, Fan TW (2011) A novel deconvolution method for modeling UDP-N-acetyl-d-glucosamine biosynthetic pathways based on (13)C mass isotopologue profiles under non-steady-state conditions. BMC Biol 9:37.  https://doi.org/10.1186/1741-7007-9-37CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Moseley HN (2010) Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry. BMC Bioinformatics 11:139.  https://doi.org/10.1186/1471-2105-11-139CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Freshney RI (2005) Culture of animal cells: a manual of basic techniques, 5th edn. John Wiley & Sons, Inc., Hoboken, NJCrossRefGoogle Scholar
  29. 29.
    Lane AN, Fan TW, Bousamra M 2nd, Higashi RM, Yan J, Miller DM (2011) Stable isotope-resolved metabolomics (SIRM) in cancer research with clinical application to nonsmall cell lung cancer. OMICS 15(3):173–182.  https://doi.org/10.1089/omi.2010.0088CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Theobald U, Mailinger W, Baltes M, Rizzi M, Reuss M (1997) In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae: I. experimental observations. Biotechnol Bioeng 55(2):305–316.  https://doi.org/10.1002/(SICI)1097-0290(19970720)55:2<305::AID-BIT8>3.0.CO;2-MCrossRefPubMedGoogle Scholar
  31. 31.
    Zhang GF, Sadhukhan S, Tochtrop GP, Brunengraber H (2011) Metabolomics, pathway regulation, and pathway discovery. J Biol Chem 286(27):23631–23635.  https://doi.org/10.1074/jbc.R110.171405CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Vasilakou E, Machado D, Theorell A, Rocha I, Noh K, Oldiges M, Wahl SA (2016) Current state and challenges for dynamic metabolic modeling. Curr Opin Microbiol 33:97–104.  https://doi.org/10.1016/j.mib.2016.07.008CrossRefPubMedGoogle Scholar
  33. 33.
    Cantor JR, Abu-Remaileh M, Kanarek N, Freinkman E, Gao X, Louissaint A Jr, Lewis CA, Sabatini DM (2017) Physiologic medium rewires cellular metabolism and reveals uric acid as an endogenous inhibitor of UMP synthase. Cell 169(2):258–272 e217.  https://doi.org/10.1016/j.cell.2017.03.023CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Psychogios N, Hau DD, Peng J, Guo AC, Mandal R, Bouatra S, Sinelnikov I, Krishnamurthy R, Eisner R, Gautam B, Young N, Xia J, Knox C, Dong E, Huang P, Hollander Z, Pedersen TL, Smith SR, Bamforth F, Greiner R, McManus B, Newman JW, Goodfriend T, Wishart DS (2011) The human serum metabolome. PLoS One 6(2):e16957.  https://doi.org/10.1371/journal.pone.0016957CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Heeneman S, Deutz NE, Buurman WA (1993) The concentrations of glutamine and ammonia in commercially available cell culture media. J Immunol Methods 166(1):85–91CrossRefGoogle Scholar
  36. 36.
    Nikfarjam L, Farzaneh P (2012) Prevention and detection of mycoplasma contamination in cell culture. Cell J 13(4):203–212PubMedGoogle Scholar
  37. 37.
    Sanchez JF, Crooks DR, Lee CT, Schoen CJ, Amable R, Zeng X, Florival-Victor T, Morales N, Truckenmiller ME, Smith DR, Freed WJ (2006) GABAergic lineage differentiation of AF5 neural progenitor cells in vitro. Cell Tissue Res 324(1):1–8CrossRefGoogle Scholar
  38. 38.
    Liu X, Ser Z, Locasale JW (2014) Development and quantitative evaluation of a high-resolution metabolomics technology. Anal Chem 86(4):2175–2184.  https://doi.org/10.1021/ac403845uCrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Sheikh KD, Khanna S, Byers SW, Fornace A Jr, Cheema AK (2011) Small molecule metabolite extraction strategy for improving LC/MS detection of cancer cell metabolome. J Biomol Tech 22(1):1–4PubMedPubMedCentralGoogle Scholar
  40. 40.
    Wu JT, Wu LH, Knight JA (1986) Stability of NADPH: effect of various factors on the kinetics of degradation. Clin Chem 32(2):314–319PubMedGoogle Scholar
  41. 41.
    Nagana Gowda GA, Gowda YN, Raftery D (2015) Massive glutamine cyclization to pyroglutamic acid in human serum discovered using NMR spectroscopy. Anal Chem 87(7):3800–3805.  https://doi.org/10.1021/ac504435bCrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Purwaha P, Silva LP, Hawke DH, Weinstein JN, Lorenzi PL (2014) An artifact in LC-MS/MS measurement of glutamine and glutamic acid: in-source cyclization to pyroglutamic acid. Anal Chem 86(12):5633–5637.  https://doi.org/10.1021/ac501451vCrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Deng P, Higashi RM, Lane AN, Bruntz RC, Sun RC, Ramakrishnam Raju MV, Nantz MH, Qi Z, Fan TW (2017) Quantitative profiling of carbonyl metabolites directly in crude biological extracts using chemoselective tagging and nanoESI-FTMS. Analyst 143(1):311–322.  https://doi.org/10.1039/c7an01256jCrossRefPubMedGoogle Scholar
  44. 44.
    Kimball E, Rabinowitz JD (2006) Identifying decomposition products in extracts of cellular metabolites. Anal Biochem 358(2):273–280.  https://doi.org/10.1016/j.ab.2006.07.038CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Rabinowitz JD, Kimball E (2007) Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal Chem 79(16):6167–6173.  https://doi.org/10.1021/ac070470cCrossRefPubMedGoogle Scholar
  46. 46.
    Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 5(8):593–599.  https://doi.org/10.1038/nchembio.186CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Fan TW, Warmoes MO, Sun Q, Song H, Turchan-Cholewo J, Martin JT, Mahan A, Higashi RM, Lane AN (2016) Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator beta-glucan in a two-case ex vivo non-small-cell lung cancer study. Cold Spring Harb Mol Case Stud 2(4):a000893.  https://doi.org/10.1101/mcs.a000893CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Lorkiewicz P, Higashi RM, Lane AN, Fan TW (2012) High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS. Metabolomics 8(5):930–939.  https://doi.org/10.1007/s11306-011-0388-yCrossRefPubMedGoogle Scholar
  49. 49.
    Welshons WV, Wolf MF, Murphy CS, Jordan VC (1988) Estrogenic activity of phenol red. Mol Cell Endocrinol 57(3):169–178CrossRefGoogle Scholar
  50. 50.
    Bindal RD, Carlson KE, Katzenellenbogen BS, Katzenellenbogen JA (1988) Lipophilic impurities, not phenolsulfonphthalein, account for the estrogenic activity in commercial preparations of phenol red. J Steroid Biochem 31(3):287–293CrossRefGoogle Scholar
  51. 51.
    Villas-Bôas SG (2007) Metabolome analysis: an introduction. Wiley-Interscience series in mass spectrometry. Wiley-Interscience, Hoboken, NJCrossRefGoogle Scholar
  52. 52.
    Kelly AE, Ou HD, Withers R, Dotsch V (2002) Low-conductivity buffers for high-sensitivity NMR measurements. J Am Chem Soc 124(40):12013–12019CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Daniel R. Crooks
    • 1
  • Teresa W.-M. Fan
    • 2
    Email author
  • W. Marston Linehan
    • 1
  1. 1.Urologic Oncology BranchNational Cancer Institute, National Institutes of HealthBethesdaUSA
  2. 2.Department of Toxicology and Cancer BiologyCenter for Environmental and Systems Biochemistry, Markey Cancer Center, and University of KentuckyLexingtonUSA

Personalised recommendations