Cell Painting Identifies Phenotypic Clusters Associated with Anticancer Drug Responses in OVCAR-3 Ovarian Cancer Cells

Cell Painting is a high-content imaging approach that enables systematic characterization of cellular responses to chemical perturbations through quantitative morphological profiling. Although this methodology has been widely applied in drug discovery and functional genomics, ovarian cancer models remain comparatively underrepresented in Cell Painting studies. In the present work, we established a Cell Painting workflow for OVCAR-3 ovarian cancer cells and applied it to the phenotypic profiling of 54 anticancer compounds representing diverse mechanisms of action. Cells were exposed to compounds under short- and prolonged-treatment conditions, followed by multiplex fluorescent staining, high-content imaging, feature extraction using CellProfiler, and computational analysis of the resulting morphological profiles. Profiles obtained at the two treatment durations were analyzed separately. For each compound, the treatment condition showing greater within-compound consistency in PCA space was additionally retained to construct a combined dataset. Morphological relationships were explored using Pearson correlation analysis, dimensionality reduction, and unsupervised clustering. Morphological profiles showed reproducible relationships among compounds. Compounds with related mechanisms showed greater morphological similarity than expected by chance, although substantial within-class heterogeneity remained. Statistically significant cluster enrichment was observed for microtubule-targeting agents and topoisomerase inhibitors, whereas other mechanism-related patterns were incomplete. Among the evaluated clustering approaches, Leiden and agglomerative clustering produced similar separation of the Combined dataset, with silhouette scores of 0.486 and 0.482, respectively. Cell Painting identified mechanism-associated but heterogeneous morphological responses in OVCAR-3 cells. The results support the use of this model for exploratory phenotypic profiling while highlighting the influence of exposure conditions and the limited ability of morphology alone to recover annotated molecular mechanisms.