DiscussionThere is an increasing interest in using three-dimensional (3D) spheroids for modelling cancer and tissue biology with the goal of accelerating translation research. The goal of this study was to develop high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compounds treatment and to optimize spheroid assay protocols for higher throughput. Specifically, we optimized spheroid cell culture protocols using low adhesion 96- and 384-well plates for three commonly used cell lines, and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We improved imaging method by using maximum projection algorithm which allows combination of cellular information from multiple slices through 3D object into a single image that allows efficient comparison of spheroids with different phenotypes. Image analysis method provides multi-parametric characterization of single cell and spheroid phenotypes and can be extended to sub-cellular analysis. We report phenotypic readouts of cell scoring, quantification of cell numbers expressing different markers, measurement of cell viability and apoptosis, as well as characterization of spheroid size and shape. We assessed assay performance using established anti-cancer cytostatic and cytotoxic drugs, demonstrated concentration-response effects and measured IC50 values. The 3D spheroid results were compared to 2D cell cultures. Finally, we have screened a library of 119 approved anti-cancer drugs across wide range of concentrations using 116HTC spheroids. The proposed methods can increase throughput and performance of high-content assays for compound screening and fast evaluation of anti-cancer drugs with 3D cell models.