Single-cell morphological profiling reveals insights into cell death

Analysis at the single-cell level is a powerful approach to study biological processes and responses to perturbations. However, its application in morphological profiling with phenomics remains underexplored. Here, we use the Cell Painting assay to investigate morphological effects of 53 small molecule compounds, associated with six distinct programmed cell death mechanisms, across six concentrations in MCF7 cells. To compare single-cell and aggregated analysis strategies, we conduct both supervised and unsupervised evaluations aimed at identifying features linked to programmed cell death. We apply an energy distance as a metric to quantify morphological perturbation strength, enabling efficient filtering. Among three tested feature extraction methods, self-supervised DINO embeddings applied to single-cell data captured high-resolution morphological patterns. Focused analyses of apoptosis-inducing compounds revealed biological heterogeneity attributable to specific molecular targets and concentration-dependent effects, which were not apparent in aggregated profiles. In contrast, multi-class classification models for the six programmed cell death mechanisms trained on single-cell features achieved F1 scores of 79.86%, while models trained on aggregated features reached F1 scores of up to 89.97%. Our results highlight the advantages of single-cell data for unsupervised exploration and show that aggregated representations yield more robust and accurate performance in supervised models.