High-Content Imaging and Machine Learning Classify Phenotypical Change in Coronary Artery Endothelial Cells Caused by...

Bisphenol S (BPS) is widely used as a replacement for bisphenol A, yet accumulating evidence suggests that it has comparable endocrine and cardiovascular toxicity. Here, we investigated whether prolonged low-dose BPS exposure induces subtle but classifiable phenotypic alterations in human coronary artery endothelial cells (HCAEC), using an end-to-end experimental and ML pipeline that spans cell culture, high-content imaging, feature extraction, and robust classification. Cells were exposed to 0.1 µM BPS for 96 h and profiled using a cell painting assay and high-content microscopy. Image segmentation yielded ~2500 quantitative features per cell across four compartments-Membrane, Cytoplasm, Ring region (i.e., perinuclear region), and Nucleus-for multiple fluorophores. We systematically compared different classifiers (Random Forest, XGBoost, LASSO logistic regressor) using feature selection (MRMR, ReliefF, LASSO) or transformation-based dimensionality reduction (PCA, autoencoders). Tree-based ensembles robustly handled high-dimensional inputs, with XGBoost combined with ReliefF-selected features achieving the best performance. The most informative descriptors predominantly mapped to mitochondrial and nuclear channels, indicating early alterations in mitochondrial organisation and chromatin-related features. These findings show that chronic low-dose BPS exposure elicits a distinct endothelial phenotype, consistent with early endothelial dysfunction, and demonstrate that integrating high-content imaging with machine learning provides a sensitive, scalable framework for vascular toxicity assessment of environmental contaminants.