Single-Cell Distribution Analysis of AR Levels by High-Throughput Microscopy in Cell Models: Application for Testing...

Cell-to-cell variation of protein expression in genetically homogeneous populations is a common biological trait often neglected during analysis of high-throughput (HT) screens and is rarely used as a metric to characterize chemicals. We have captured single-cell distributions of androgen receptor (AR) nuclear levels after perturbations as a means to evaluate assay reproducibility and characterize a small subset of chemicals. AR, a member of the nuclear receptor family of transcription factors, is the central regulator of male reproduction and is involved in many pathophysiological processes. AR protein levels and nuclear localization often increase following ligand binding, with dihydrotestosterone (DHT) being the natural agonist. HT AR immunofluorescence imaging was used in multiple cell lines to define single-cell nuclear values extracted from thousands of cells per condition treated with DHT or DMSO (control). Analysis of numerous biological replicates led to a quality control metric that takes into account the distribution of single-cell data, and how it changes upon treatments. Dose-response experiments across several cell lines showed a large range of sensitivity to DHT, prompting us to treat selected cell lines with 45 Environmental Protection Agency (EPA)-provided chemicals that include many endocrine-disrupting chemicals (EDCs); data from six of the compounds were then integrated with orthogonal assays. Our comprehensive results indicate that quantitative single-cell distribution analysis of AR protein levels is a valid method to detect the potential androgenic and antiandrogenic actions of environmentally relevant chemicals in a sensitive and reproducible manner.