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Development of High-Throughput Quantitative Assays for Glucose Uptake in Cancer Cell Lines

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Abstract

Purpose

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.

Procedure

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.

Results

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.

Conclusions

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.

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Abbreviations

0/0:

DMEM without serum and supplements

2-DG:

2-Deoxglucose

2-NBDG:

2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose

a.f.u.:

Arbitrary fluorescence units

CI:

Confidence interval

EGF:

Epidermal growth factor

FDG:

2-Deoxy2-[F-18] fluoro-d-glucose

FBS:

Fetal bovine serum

GLUTs:

Glucose transporters

HCAM:

High-content automated microscopy

PBS:

Phosphate-buffered saline

PET:

Positron emission tomography

SEM:

Standard error of the mean

S/S:

DMEM with serum and supplements

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Acknowledgments

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.

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Authors and Affiliations

  1. Vanderbilt Integrative Cancer Biology Center, Vanderbilt University Medical Center, 2220 Pierce Avenue, 771 Preston Research Building, Nashville, TN, 37232, USA

    Mohamed Hassanein, Brandy Weidow, Shawn Garbett & Vito Quaranta

  2. Department of Biostatistics, Vanderbilt University Medical Center, Nashville, TN, 37232, USA

    Elizabeth Koehler & Yu Shyr

  3. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA

    Naimish Bakane

Authors
  1. Mohamed Hassanein
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  2. Brandy Weidow
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  3. Elizabeth Koehler
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  4. Naimish Bakane
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  5. Shawn Garbett
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  6. Yu Shyr
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  7. Vito Quaranta
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Corresponding author

Correspondence to Vito Quaranta.

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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

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