C. Zielke, C.W. Pan, A. Gutierrez, C. Feit, C. Dobson, C. Davidson, B. Sandel, P. Abbyad
Santa Clara University,
Keywords: microfluidics, droplet microfluidics, cancer, single-cell, cell sorting, metabolism
Summary:We present a facile and robust way of separating cells based on elevated glycolytic activity; a biomarker associated to cancer cell malignancy. A hallmark of cancer is aberrant metabolism, which is used routinely in PET scans for cancer diagnostics and monitoring. A key observation of cancer metabolism is aerobic glycolysis (‘Warburg effect’), where cancer cells display high levels of glycolysis even in the presence of oxygen. Glycolysis results in the release of lactate, which is accompanied by secretion of protons, leading to an acidification of the extracellular environment. Elevated glycolysis and high levels of lactate release are linked to cancer aggressivity, promoting metastasis and tumor recurrence. There is increasing evidence that high extracellular acidity is not only a byproduct of the disease, but rather a disease driver. With the connection of glycolysis and malignancy, a label-free technique that selects cells based on glycolysis could have a major impact in oncology. We expand here the use of a technique developed in our lab, SIFT (Sorting by Interfacial Tension), to isolate cancer cell subpopulations based on glycolysis without the additional use of active sorting components or markers. Unlike other label-free microfluidic techniques (inertial, dielectrophoresis, acoustophoresis) that sort based on physical attributes (such as size, density, deformability and dielectric properties), SIFT sorts based on single-cell metabolism. Briefly, single cells are encapsulated into picoliter droplets. After a few minutes of incubation, the pH of droplets containing cells achieve a lower pH. Under specific chemical conditions, the surface tension of droplets is highly dependent on pH. This allows for the sorting of flattened droplets as they expand into a microfabricated rail that is oriented at 45° relative to the direction of flow. Cells displaying high glycolysis can be isolated despite sharing similar physical characteristics as other cells. By altering the flow rates of the external oil on the microfluidic chip, the pH threshold of droplet selection is controlled. This enables the selection of cell subpopulations that have different levels of glycolysis. To demonstrate this principle, we enriched cells that have been exposed to hypoxic conditions or pharmaceuticals. Treatment with the pharmaceutic, 2DG, inhibits glycolysis leading to higher average droplet pH. While 2DG treated cells make up 47 % of cells prior to sorting, selected cells contain exclusively control cells excluding all the 2DG cells. Thus, SIFT can be used to remove cells that have lower glycolytic activity. More importantly, this demonstrates that the technique can be used to isolate and then study a subpopulation of cells that may not be responsive to pharmaceutic treatment targeting glycolysis. Tumor microenvironment, in particular hypoxia, can promote elevated cellular glycolysis. SIFT can preferentially select cells that have been hypoxia treated, that are known to have increased malignancy. As a passive technique, SIFT is easy and inexpensive to implement as it requires no markers, excitation source, detectors, or active sorting components. The technique provides a robust way of separating cells based on glycolytic activity, a biomarker that can track cancer cell malignancy.