Metabolism, and especially glucose uptake, is a key quantitative cell trait that is closely linked to cancer initiation and progression. Therefore, developing high-throughput assays for measuring glucose uptake in cancer cells would be enviable for simultaneous comparisons of multiple cell lines and microenvironmental conditions. This study was designed with two specific aims in mind: the first was to develop and validate a high-throughput screening method for quantitative assessment of glucose uptake in “normal” and tumor cells using the fluorescent 2-deoxyglucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and the second was to develop an image-based, quantitative, single-cell assay for measuring glucose uptake using the same probe to dissect the full spectrum of metabolic variability within populations of tumor cells in vitro in higher resolution.
The kinetics of population-based glucose uptake was evaluated for MCF10A mammary epithelial and CA1d breast cancer cell lines, using 2-NBDG and a fluorometric microplate reader. Glucose uptake for the same cell lines was also examined at the single-cell level using high-content automated microscopy coupled with semi-automated cell-cytometric image analysis approaches. Statistical treatments were also implemented to analyze intra-population variability.
Our results demonstrate that the high-throughput fluorometric assay using 2-NBDG is a reliable method to assess population-level kinetics of glucose uptake in cell lines in vitro. Similarly, single-cell image-based assays and analyses of 2-NBDG fluorescence proved an effective and accurate means for assessing glucose uptake, which revealed that breast tumor cell lines display intra-population variability that is modulated by growth conditions.
These studies indicate that 2-NBDG can be used to aid in the high-throughput analysis of the influence of chemotherapeutics on glucose uptake in cancer cells.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
DMEM without serum and supplements
Arbitrary fluorescence units
Epidermal growth factor
Fetal bovine serum
High-content automated microscopy
Positron emission tomography
Standard error of the mean
DMEM with serum and supplements
Warburg O (1956) On the origin of cancer cells. Science 123(3191):309–314
Tennant DA et al (2009) Metabolic transformation in cancer. Carcinogenesis 30(8):1269–1280
Warburg O (1956) On respiratory impairment in cancer cells. Science 124(3215):269–270
Deberardinis RJ et al (2008) Brick by brick: metabolism and tumor cell growth. Curr Opin Genet Dev 18(1):54–61
Hsu PP, Sabatini DM (2008) Cancer cell metabolism: Warburg and beyond. Cell 134(5):703–707
Czernin J, Phelps ME (2002) Positron emission tomography scanning: current and future applications. Annu Rev Med 53:89–112
Jones RG, Thompson CB (2009) Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev 23(5):537–548
Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324(5930):1029–1033
Christofk HR et al (2008) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452(7184):230–233
Auerbach R, Auerbach W (1982) Regional differences in the growth of normal and neoplastic cells. Science 215(4529):127–134
Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70
Franzius C et al (2000) Evaluation of chemotherapy response in primary bone tumors with F-18 FDG positron emission tomography compared with histologically assessed tumor necrosis. Clin Nucl Med 25(11):874–881
Kenny L et al (2007) Imaging early changes in proliferation at 1 week post chemotherapy: a pilot study in breast cancer patients with 3′-deoxy-3′-[18F]fluorothymidine positron emission tomography. Eur J Nucl Med Mol Imaging 34(9):1339–1347
Mankoff DA et al (2007) Tumor-specific positron emission tomography imaging in patients: [18F] fluorodeoxyglucose and beyond. Clin Cancer Res 13(12):3460–3469
Eary JF et al (2008) Spatial heterogeneity in sarcoma 18F-FDG uptake as a predictor of patient outcome. J Nucl Med 49(12):1973–1979
Yoshioka K et al (1996) Evaluation of 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]-2-deoxy-D-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans. Appl Microbiol Biotechnol 46(4):400–404
Yoshioka K et al (1996) Intracellular fate of 2-NBDG, a fluorescent probe for glucose uptake activity, in Escherichia coli cells. Biosci Biotechnol Biochem 60(11):1899–1901
Yoshioka K et al (1996) A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta 1289(1):5–9
Yamada K et al (2007) A real-time method of imaging glucose uptake in single, living mammalian cells. Nat Protoc 2(3):753–762
Natarajan A, Srienc F (1999) Dynamics of glucose uptake by single Escherichia coli cells. Metab Eng 1(4):320–333
Lloyd PG, Hardin CD, Sturek M (1999) Examining glucose transport in single vascular smooth muscle cells with a fluorescent glucose analog. Physiol Res 48(6):401–410
Yamada K et al (2000) Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic beta-cells. J Biol Chem 275(29):22278–22283
Zou C, Wang Y, Shen Z (2005) 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys Methods 64(3):207–215
Barros LF et al (2009) Preferential transport and metabolism of glucose in Bergmann glia over Purkinje cells: a multiphoton study of cerebellar slices. Glia 57(9):962–970
Loaiza A, Porras OH, Barros LF (2003) Glutamate triggers rapid glucose transport stimulation in astrocytes as evidenced by real-time confocal microscopy. J Neurosci 23(19):7337–7342
Porras OH, Loaiza A, Barros LF (2004) Glutamate mediates acute glucose transport inhibition in hippocampal neurons. J Neurosci 24(43):9669–9673
Demartino NG, McGuire JM (1988) The multicatalytic proteinase: a high-Mr endopeptidase. Biochem J Lett 255:750–751
Gaudreault N et al (2008) Subcellular characterization of glucose uptake in coronary endothelial cells. Microvasc Res 75(1):73–82
Cheng Z et al (2006) Near-infrared fluorescent deoxyglucose analogue for tumor optical imaging in cell culture and living mice. Bioconjug Chem 17(3):662–669
Roman Y et al (2001) Confocal microscopy study of the different patterns of 2-NBDG uptake in rabbit enterocytes in the apical and basal zone. Pflugers Arch 443(2):234–239
Dimitriadis G et al (2005) Evaluation of glucose transport and its regulation by insulin in human monocytes using flow cytometry. Cytom A 64(1):27–33
Sheth RA, Josephson L, Mahmood U (2009) Evaluation and clinically relevant applications of a fluorescent imaging analog to fluorodeoxyglucose positron emission tomography. J Biomed Opt 14(6):064014
Jones TR et al (2008) Cell Profiler Analyst: data exploration and analysis software for complex image-based screens. BMC Bioinform 9:482
Pauley RJ et al (1993) The MCF10 family of spontaneously immortalized human breast epithelial cell lines: models of neoplastic progression. Eur J Cancer Prev 2(Suppl 3):67–76
Yang NS, Soule HD, McGrath CM (1977) Expression of murine mammary tumor virus-related antigens in human breast carcinoma (MCF-7) cells. J Natl Cancer Inst 59(5):1357–1367
Bouma ME et al (1989) Further cellular investigation of the human hepatoblastoma-derived cell line HepG2: morphology and immunocytochemical studies of hepatic-secreted proteins. In Vitro Cell Dev Biol 25(3 Pt 1):267–275
Vokes MS, Carpenter AE (2008) Using CellProfiler for automatic identification and measurement of biological objects in images. Curr Protoc Mol Biol (Chapter 14):Unit 14.17
O’Neil RG, Wu L, Mullani N (2005) Uptake of a fluorescent deoxyglucose analog (2-NBDG) in tumor cells. Mol Imaging Biol 7(6):388–392
Salter DW, Custead-Jones S, Cook JS (1978) Quercetin inhibits hexose transport in a human diploid fibroblast. J Membr Biol 40(1):67–76
Kobori M et al (1997) Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport. Cancer Lett 119(2):207–212
Kim MS et al (2009) Phloretin induces apoptosis in H-Ras MCF10A human breast tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase signaling. Ann NY Acad Sci 1171:479–483
Barros LF et al (2009) Kinetic validation of 6-NBDG as a probe for the glucose transporter GLUT1 in astrocytes. J Neurochem 109(Suppl 1):94–100
Cameron DA, Stein S (2008) Drug Insight: intracellular inhibitors of HER2–clinical development of lapatinib in breast cancer. Nat Clin Pract Oncol 5(9):512–520
Louzao MC et al (2008) “Fluorescent glycogen” formation with sensibility for in vivo and in vitro detection. Glycoconj J 25(6):503–510
Zhang Z et al (2004) Metabolic imaging of tumors using intrinsic and extrinsic fluorescent markers. Biosens Bioelectron 20(3):643–650
Nitin N et al (2009) Molecular imaging of glucose uptake in oral neoplasia following topical application of fluorescently labeled deoxy-glucose. Int J Cancer 124(11):2634–2642
Richardson AD et al (2008) Central carbon metabolism in the progression of mammary carcinoma. Breast Cancer Res Treat 110(2):297–307
Franzius C et al (2000) FDG-PET for detection of osseous metastases from malignant primary bone tumours: comparison with bone scintigraphy. Eur J Nucl Med 27(9):1305–1311
Sonveaux P et al (2008) Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J Clin Invest 118(12):3930–3942
Quaranta V et al (2009) Trait variability of cancer cells quantified by high-content automated microscopy of single cells. Methods Enzymol 467:23–57
We would like to acknowledge the following funding source for support of this work: NCI U54-CA113007 awarded to VQ. We would also like to thank Dr. David Degraff for his help with the revising the scientific content of this manuscript and for his insightful comments and critiques.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
(PDF 315 kb)
About this article
Cite this article
Hassanein, M., Weidow, B., Koehler, E. et al. Development of High-Throughput Quantitative Assays for Glucose Uptake in Cancer Cell Lines. Mol Imaging Biol 13, 840–852 (2011). https://doi.org/10.1007/s11307-010-0399-5