Metabolic pathway for the universal fluorescent recognition of tumor cells

Abstract

Quantification of circulating tumor cells (CTCs) in blood samples from cancer patients is a non-invasive approach to monitoring the status of the disease. Most of the methods proposed in the recent years are phenomenological and rely on the use of antibodies labelled with fluorophores, magnetic particles, or immobilized on surfaces to capture the CTCs. Herein, we designed and optimized a method that employs a glucose analogue labelled with a fluorophore which takes advantage of the different metabolic pathways of cancer cells to discern them from normal ones. Notably, we demonstrate that fluorescence signal in tumor cells can be greatly maximized by applying hyperoxia conditions without damaging the cells. These results are demonstrated by means of confocal fluorescence and flow-cytometry measurements in peripheral blood mononuclear cells (PBMC) extracted after Ficoll of human blood samples and spiked with a known concentration of MCF-7 tumor cells.

Keywords: breast cancer; circulating tumor cells; glucose uptake; hyperoxia; optical sensing.

Figure 1

(A) Absorption and emission profiles of 2-NBDG and APC. Dotted arrows indicate the excitation lines. Molecular structure of 2-NBDG. (B) Confocal fluorescence microscopy images of 2-NBDG uptake for PBMC and MCF-7 incubated with 300 μM 2-NBDG for 30 minutes in samples containing cell ratios of 1:10 MCF-7:PBMC. The bars indicate 40 μm.

Figure 2

(A) Optimization of 2-NBDG incubation time under different microenvironments: hypoxia, normoxia, and hyperoxia. 3D Walls graph with the oxygen condition for each cell line in z-axis, incubation time (min) in x-axis, and intensity of 2-NBDG/a.u. in y-axis. The results are presented as mean ± standard deviation of three independent experiments (n = 3) for samples containing cell ratios of 1:10 MCF-7:PBMC. The median values of 10,000 cytometry events were recorded for each sample. The Kruskal-Wallis test revealed a significant difference between cancer (MCF-7) and normal (PBMC) cells in optimized conditions, p < 0.01. (B) Ratiometric difference in 2-NBDG fluorescence between PBMC and MCF-7 obtained by dividing the signal intensity of the tumor cell by that of the normal cells as a function of the oxygen conditions.

Figure 3. Confocal fluorescence microscopy images of 2-NBDG uptake for PBMC and MCF-7 under optimized conditions for samples containing cell ratios of 1:10 MCF-7:PBMC. Scale bars = 40 μm

Figure 4

(A–E) Flow cytometry plots of MCF-7 and PBMCs samples with cell ratios (A) 1:10, (B) 1:100, (C) 1:1000, (D) 1:10000 and (E) only PBMCs; upon incubation with 2-NBDG and CD45-APC for 30 minutes under hyperoxia conditions. (F–I) Distributions of fluorescence intensities for (F) 2-NBDG and (G) CD45-APC, in a sample with a 1:1000 MCF-7:PBMC ratio; and (H) 2-NBDG and (I) CD45-APC, in a sample of PBMC. Over 10⁶ single-cell events were collected for each experiment.

Figure 5

(A) Comparison between the expected and measured MCF-7:PBMC ratios for the samples incubated with 2-NBDG and CD-45 for 30 min under hyperoxia. (B) Normalized fluorescence intensities per cell for PBMC and MCF-7 treated with 2-NBDG under optimized oxygen content and incubation time conditions. Samples with cell ratios of 1:10, 1:100, 1:1000, and 1:10000.