Development of high-throughput quantitative assays for glucose uptake in cancer cell lines

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.

Fig. 1

2-NBDG uptake correlates with cell number. Population-level 2-NBDG uptake was quantified using a fluorometric plate reader. MCF10A or CA1d cells were seeded at a range of densities (0–30,000/well) and 2-NBDG (100 µM) was added and allowed to incubate for 10 min prior to stopping reactions. All raw values were normalized to background levels obtained when seeding no cells, and are presented as the mean±SEM of all experiments performed (N=5). Fluorescence levels indicate a linear response of 2-NBDG uptake for both cell lines in a density-dependent manner. CA1d consistently exhibited a higher level of 2-NBDG uptake, although not significantly different than MCF10A cells.

Fig. 2

2-NBDG uptake is dose-dependent. Population-level glucose uptake was quantified in response to increasing doses of 2-NBDG (0–300 µM) using a fluorometric plate reader. All raw values were normalized to background levels obtained for “blank” wells that were treated the same as samples, and are presented as the mean±SEM of all experiments performed. a Both normal MCF10A cells and CA1d cancer cells absorbed 2-NBDG in a dose-dependent manner; not surprisingly, uptake levels were fairly low at low concentrations of the probe, while higher levels of uptake were observed at high concentrations. In summary, CA1d displayed consistently consumed more 2-NBDG than MCF10A (N=5). b Similarly, MCF7 human breast cancer cells and HepG2 human liver carcinoma cell lines exhibited a steady increase in fluorescence signal in a dose-dependent manner (N=3).

Fig. 3

d-Glucose and Phloretin inhibit 2-NBDG uptake. a To determine if 2-NBDG uptake is specific to GLUTs for MCF10A and CA1d cells, population-level 2-NBDG uptake was assessed in the presence of increasing doses of d-glucose (0–10 mM). All raw values were normalized to background levels obtained for “blank” wells that were treated the same as samples, and the data are presented as the mean±SEM of all experiments performed (N=3). Both cell lines exhibited a steady decrease in 2-NBDG uptake in response to increasing d-glucose concentration. *P<0.05, significant differences. b Specificity to transportation via GLUTs was also validated by pharmacological inhibition of 2-NBDG uptake by Phloretin (0–1,000 µM; N=3). Similarly, both cell lines exhibited a steady decrease in 2-NBDG uptake in response to increasing Phloretin concentrations. *P<0.05, significant differences.

Fig. 4

Receptor tyrosine kinase small molecule inhibitors reduce 2-NBDG uptake. a Treatment of MCF10A and CA1d with a Lapatinib or b Erlotinib for 24 h resulted in reduction of 2-NBDG uptake in both cell lines in a dose-dependent manner (N=3). CA1d showed more pronounced decrease at concentrations of ≥10 nM compared to MCF10A. *P<0.05, significant differences.

Fig. 5

Single-cell 2-NBDG uptake is variable within cell line populations. a Representative microscopic images of MCF10A and CA1d are shown. Images include from left to right: unlabeled cells (fluorescent channel) and 2-NBDG-labeled cells (both brightfield and fluorescent channels). Both cell lines displayed intra-population variability of 2-NBDG uptake, both at cellular and sub cellular levels. From inspection of images, three patterns of labeling can be seen: 1) cells with no label (fluorescence), 2, diffusively stained cells which were the most predominate pattern in MCF10A and third, locally punctuate pattern that was abundantly detected in CA1d to less extinct in MCF10A (see bottom arrows in CA1d after adding 2-NBDG). b Schematic of image processing workflow for single-cell analysis using CellProfiler. Briefly original fluorescence images were exported as a pair of Hoechst stained nuclei (blue) and 2-NBDG stained cells (green). Hoechst staining was used to identify primary objects (nuclei). Once the primary objects were identified, the OTSU adaptive propagation software was used to identify 2-NBDG labeled cell bodies (secondary objects). Images were used to extract cell measurements, including: nuclei count, nuclei intensity, 2-NBDG labeled cell count, 2-NBDG intensity. 2-NBDG was used as a quantifier of glucose uptake per cell (in a.f.u.), and other measurements were used to calculate the percentage of cells that were 2-NBDG labeled (based on 2-NBDG count) out of total cells (based on nuclei count). All extracted data generated were exported to Excel spreadsheets for analysis.

Fig. 6

Single-cell-based HCAM experiments reveal variability in 2-NBDG uptake. Average population-level data were obtained by taking the average image intensity (a.f.u.) of 2-NBDG (green channel) with increasing doses of 2-NBDG, data for glucose competition and dose–response (a, d, respectively) the data are presented as the mean±SEM of all experiments performed (N=3). The percentage of 2-NBDG-labeled cells for d-glucose competitive inhibition assay (b) and dose–response studies (e) were analyzed using HCAM single-cell assay. c The distribution of single-cell uptake measurements expressed as mean intensity/cell in MCF10A and CA1d cell populations for glucose competition assay are shown, the data are representative of two independent trials. Auto fluorescence background was excluded from the raw data by applying threshold value (gray dotted line) that correspond to non-labeled cell controls as illustrated by sub-window (c) for MCF10 A and CA1d cell populations. f The single-cell distribution of 2-NBDG uptake in dose–response assay after background correction.

Fig. 7

Inter-population cell variability is modulated by growth conditions. MCF10A and CA1d cells were cultured for 24 h in optimal (S/S), suboptimal (EGF) or deprived media (0/0), and were subsequently double-labeled with Hoescht and 2-NBDG for measurements of total cell count and glucose uptake, respectively. a The average glucose uptake was determined as fluorescence intensity (a.f.u.), which showed that under suboptimal and deprived growth conditions, MCF10A consumed less 2-NBDG than CA1d under the same conditions. *P<0.05, significant differences. b The percentage of single cells that absorbed 2-NBDG was also calculated. CA1d consistently exhibited a higher percentage of cells that consumed 2-NBDG than MCF10A; however, no significant changes were obtained. c Statistical analysis of single-cell measurements of 2-NBDG uptake revealed the presence of two subpopulations, of low and high glucose uptake, in both cell lines in full culture conditions (S/S). Interestingly, the presence of these two subpopulations persisted in CA1d regardless of growth conditions; however, they disappeared in MCF10A when grown under suboptimal (EGF) or deprived conditions (0/0).