Microfluidic Platform for the Isolation of Cancer-Cell Subpopulations Based on Single-Cell Glycolysis

Abstract

High rates of glycolysis in tumors have been associated with cancer metastasis, tumor recurrence, and poor outcomes. In this light, single cells that exhibit high glycolysis are specific targets for therapy. However, the study of these cells requires efficient tools for their isolation. We use a droplet microfluidic technique developed in our lab, Sorting by Interfacial Tension (SIFT), to isolate cancer cell subpopulations based on glycolysis without the use of labels or active sorting components. By controlling the flow conditions on chip, the threshold of selection can be modified, enabling the isolation of cells with different levels of glycolysis. Hypoxia in tumors, that can be simulated with treatment with CoCl₂, leads to an increase in glycolysis, and more dangerous tumors. The device was used to enrich CoCl₂ treated MDA-MB 231 breast cancer cells from an untreated population. It is also used to sort K562 human chronic myelogenous leukemia cells that have either been treated or untreated with 2-deoxy-d-glucose (2DG), a pharmaceutical that targets cell metabolism. The technique provides a facile and robust way of separating cells based on elevated glycolytic activity; a biomarker associated with cancer cell malignancy.

Figure 1.

Selection of droplets of different pH using SIFT. Image shows the end of incubator and sorting region. K562 cells (a few circled in yellow) are labeled with Calcein AM for better visibility. Droplet pH is analyzed at the end of incubator before entering into the sorting region. Droplets containing a highly glycolytic cell (circled in green) ride the rail laterally up, leaving the rail at the top (selected). Droplets containing no cell or less active cells (circled in red) are at higher pH. They do not ride the rail or are only slightly deflected up the sorting rail (unselected).

Figure 2.

A) pH of droplets containing K562 cells plotted vs applied flow rate (μL/min) of Oil Entrainment Inlet. Square, red markers represent unselected; circular, green markers represent selected droplets. A small amount of noise (jitter) was added to the x-value to avoid overlap of data points. B) Estimated pH threshold as determined by logistic regression fits. 95% confidence interval is indicated in grey.

Figure 3.

SIFT device sorting of hypoxia treated and untreated MDA-MB 231 cells. Hypoxic cells are labelled with Calcein AM to enable differentiation of cell populations. Cell grown under hypoxic conditions (circled in orange) with low pH, pH 6.82, rides the rail laterally up (selected). Control cell (circled in white) grown under normoxic conditions with higher pH (pH 7.19) is only slightly deflected by the rail (unselected).

Figure 4.

pH of droplets containing either control (grey) or hypoxia treated (orange) MDA-MB 231 cells. Square markers annotate unselected, circular markers selected droplets. pH values were determined via a fluorescence intensity ratio. A) Droplets presented as control and hypoxia cell populations. Average pH values, indicated by a black line, are 7.14 ± 0.02 (N = 66) for control and 6.95 ± 0.02 (N = 54) for hypoxia cells. B) Droplets presented as unselected and selected populations. Blue line indicates the pH of empty droplets (pH 7.37 ± 0.02). The pH threshold of selection was determined by a logistic regression fit and was found to be 6.96 ± 0.02 (threshold ± 95% confidence interval) (fit shown in Figure S5a). The sorting of droplets leads to an enrichment of hypoxia treated cells from 45 % (N = 54) before sorting to 75 % (N = 29) of selected cells.

Figure 5.

pH of droplets containing either control (grey) or 2DG treated (orange) K562 cells. Square markers annotate unselected, circular markers selected droplets. A) Droplets presented as control and 2DG treated cell populations. Average pH values, indicated by a black line, are 6.97 ± 0.03 (N = 46) for control and 7.23 ± 0.02 (N = 41) for 2DG treated cells. B) Droplets presented as unselected and selected populations. Blue line indicates the pH of empty droplets (pH 7.41 ± 0.04). The selection threshold was determined to be 6.96 ± 0.06 (fit shown in Figure S5b). While 2DG treated cells make up 47 % of cells prior to sorting, selected cells contain exclusively control cells (N = 22) excluding all the 2DG cells.