Single-cell-kinetics approach to discover functionally distinct subpopulations within phenotypically uniform populations of cells.

Phenotypically uniform cell populations may contain subpopulations with different activities of enzymes, membrane transporters, and other functional units which can be characterized kinetically. Here we propose the first approach to the discovery of such subpopulations; we term it single-cell approach to discover subpopulations or SCADS for short. SCADS combines microscopy, single-cell kinetic analysis, and population/cluster analysis to discover a functionally distinct subpopulation of cells. In this proof-of-principle work, we used SCADS to search for subpopulations with distinct kinetic patterns of membrane transport in bulk tumor cells (BTCs) and tumor-initiating cells (TICs). We used two classical Michaelis parameters, Vmax and KM, to kinetically characterize the rate of transport. We found that the BTC population was homogeneous with respect to membrane transport. When analyzing TICs, we discovered three main functionally distinct subpopulations: (i) cells with a high rate of transport (high Vmax), (ii) cells with high-affinity transporters (low KM), and (iii) cells with activity and affinity similar to those in BTCs (low Vmax and high KM).