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

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.

Multiplexed Single-Cell Measurements of FDG Uptake and Lactate Release Using Droplet Microfluidics.

INTRODUCTION: Glucose utilization and lactate release are 2 important indicators of cancer metabolism. Most tumors consume glucose and release lactate at a higher rate than normal tissues due to enhanced aerobic glycolysis. However, these 2 indicators of metabolism have not previously been studied on a single-cell level, in the same cell. OBJECTIVE: To develop and characterize a novel droplet microfluidic device for multiplexed measurements of glucose uptake (via its analog 18F-fluorodeoxyglucose) and lactate release, in single live cells encapsulated in an array of water-in-oil droplets. RESULTS: Surprisingly, 18F-fluorodeoxyglucose uptake and lactate release were only marginally correlated at the single-cell level, even when assayed in a standard cell line (MDA-MB-231). While 18F-fluorodeoxyglucose-avid cells released substantial amounts of lactate, the reverse was not true, and many cells released high amounts of lactate without taking up 18F-fluorodeoxyglucose. DISCUSSION: These results confirm that cancer cells rely on multiple metabolic pathways in addition to aerobic glycolysis and that the use of these pathways is highly heterogeneous, even under controlled culture conditions. Clinically, the large cell-to-cell variability suggests that positron emission tomography measurements of 18F-fluorodeoxyglucose uptake represent metabolic flux only in an aggregate sense, not for individual cancer cells within the tumor.

Sorting by interfacial tension (SIFT): label-free selection of live cells based on single-cell metabolism.

Selection of live cells from a population is critical in many biological studies and biotechnologies. We present here a novel droplet microfluidic approach that allows for label-free and passive selection of live cells using the glycolytic activity of individual cells. It was observed that with the use of a specific surfactant utilized to stabilize droplet formation, the interfacial tension of droplets was very sensitive to pH. After incubation, cellular lactate release results in droplets containing a live cell to attain a lower pH than other droplets. This enables the sorting of droplets containing live cells when confined droplets flow over a microfabricated trench oriented diagonally with respect to the direction of flow. The technique is demonstrated with human U87 glioblastoma cells for the selection of only droplets containing a live cell while excluding either empty droplets or droplets containing a dead cell. This label-free sorting method, dubbed sorting by interfacial tension (SIFT) presents a new strategy to sort diverse cell types based on metabolic activity.

Toward a Droplet-Based Single-Cell Radiometric Assay.

Radiotracers are widely used to track molecular processes, both in vitro and in vivo, with high sensitivity and specificity. However, most radionuclide detection methods have spatial resolution inadequate for single-cell analysis. A few existing methods can extract single-cell information from radioactive decays, but the stochastic nature of the process precludes high-throughput measurement (and sorting) of single cells. In this work, we introduce a new concept for translating radioactive decays occurring stochastically within radiolabeled single-cells into an integrated, long-lasting fluorescence signal. Single cells are encapsulated in radiofluorogenic droplets containing molecular probes sensitive to byproducts of ionizing radiation (primarily reactive oxygen species, or ROS). Different probes were examined in bulk solutions, and dihydrorhodamine 123 (DHRh 123) was selected as the lead candidate due to its sensitivity and reproducibility. Fluorescence intensity of DHRh 123 in bulk increased at a rate of 54% per Gy of X-ray radiation and 15% per MBq/ml of 2-deoxy-2-[18F]-fluoro-d-glucose ([18F]FDG). Fluorescence imaging of microfluidic droplets showed the same linear response, but droplets were less sensitive overall than the bulk ROS sensor (detection limit of 3 Gy per droplet). Finally, droplets encapsulating radiolabeled cancer cells allowed, for the first time, the detection of [18F]FDG radiotracer uptake in single cells through fluorescence activation. With further improvements, we expect this technology to enable quantitative measurement and selective sorting of single cells based on the uptake of radiolabeled small molecules.

Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release.

Cancer cells release high levels of lactate that has been correlated to increased metastasis and tumor recurrence. Single-cell measurements of lactate release can identify malignant cells and help decipher metabolic cancer pathways. We present here a novel droplet microfluidic method that allows the fast and quantitative determination of lactate release in many single cells. Using passive forces, droplets encapsulated cells are positioned in an array. The single-cell lactate release rate is determined from the increase in droplet fluorescence as the lactate is enzymatically converted to a fluorescent product. The method is used to measure the cell-to-cell variance of lactate release in K562 leukemia and U87 glioblastoma cancer cell lines and under the chemical inhibition of lactate efflux. The technique can be used in the study of cancer biology, but more broadly in cell biology, to capture the full range of stochastic variations in glycolysis activity in heterogeneous cell populations in a repeatable and high-throughput manner.

Single-Cell Analysis of [18F]Fluorodeoxyglucose Uptake by Droplet Radiofluidics.

Radiolabels can be used to detect small biomolecules with high sensitivity and specificity without interfering with the biochemical activity of the labeled molecule. For instance, the radiolabeled glucose analogue, [18F]fluorodeoxyglucose (FDG), is routinely used in positron emission tomography (PET) scans for cancer diagnosis, staging, and monitoring. However, despite their widespread usage, conventional radionuclide techniques are unable to measure the variability and modulation of FDG uptake in single cells. We present here a novel microfluidic technique, dubbed droplet radiofluidics, that can measure radiotracer uptake for single cells encapsulated into an array of microdroplets. The advantages of this approach are multiple. First, droplets can be quickly and easily positioned in a predetermined pattern for optimal imaging throughput. Second, droplet encapsulation reduces cell efflux as a confounding factor, because any effluxed radionuclide is trapped in the droplet. Last, multiplexed measurements can be performed using fluorescent labels. In this new approach, intracellular radiotracers are imaged on a conventional fluorescence microscope by capturing individual flashes of visible light that are produced as individual positrons, emitted during radioactive decay, traverse a scintillator plate placed below the cells. This method is used to measure the cell-to-cell heterogeneity in the uptake of tracers such as FDG in cell lines and cultured primary cells. The capacity of the platform to perform multiplexed measurements was demonstrated by measuring differential FDG uptake in single cells subjected to different incubation conditions and expressing different types of glucose transporters. This method opens many new avenues of research in basic cell biology and human disease by capturing the full range of stochastic variations in highly heterogeneous cell populations in a repeatable and high-throughput manner.