Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay

The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFβ treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity.Author Summary: A common method of evaluating stemness among pluripotent cells or cancer cells is the side population assay, a flow cytometry technique which identifies a subgroup of cells that exhibit differences in dye fluorescence upon blocking of a membrane transporter. A technical limitation of this assay is that it relies on two independent experimental conditions, with and without a transporter inhibitor, preventing evaluation of single cell characteristics that generate population-level shifts in fluorescence. Here, the computational implementation of various forms of cellular heterogeneity allows for ensemble single-cell simulations to be performed in order to assess the underlying properties that give rise to the population-level behavior. We simulated staining in 10,000 kinetic ensembles consisting of 1,000-cell populations with and without inhibitor to determine which cells respond in the assay. We quantitatively establish that a small, responsive subgroup of cells with nonlinear activities associated with transporter number are most likely to recapitulate observed behavior in the side population assay; however, a continuum of phenotypes at different stages of the cell cycle and with a range transporter expression levels will shift fluorescence. We present a new perspective on the phenotype of SP cells at the single-cell level that is determined by biological and experimental kinetic processes, and is not equivalent to a cancer stem cell phenotype.