Single-cell-kinetics approach to compare multidrug resistance-associated membrane transport in subpopulations of cells.

Multidrug resistance (MDR) driven by active efflux of drugs from the cells is one of the major obstacles in chemotherapies. Understanding the nature of MDR and designing more efficient chemotherapies requires the comparison of the efflux rate between different subpopulations of cells. Here we propose a single-cell-kinetics approach for such a comparison. In essence, the entire cell population is loaded with a suitable fluorescent substrate for MDR-associated membrane transporters. The kinetics of substrate efflux from individual cells is followed by time-lapse fluorescence microscopy and analyzed at the single-cell level. Microscopy is also used to assign cells to different subpopulations based on differences in morphology or level of staining by molecular probes. The kinetic parameters obtained for individual cells are then averaged for different cell subpopulations and the mean values of these parameters are finally compared between subpopulations. To test our single-cell-kinetics approach, we studied MDR-related efflux for two subpopulations of cultured breast cancer cells: cells in 2N and 4N phases of the cell cycle. The assignment of cells to 2N and 4N subpopulations was done by fluorescent DNA staining after the completion of efflux. By using the single-cell-kinetics approach, we were able to prove for the first time that the rates of MDR-related efflux differ in 2N and 4N phases of the cell cycle. We foresee that this approach will be an important tool in studies of MDR and in designing combination chemotherapies.