A simplified co-culture reveals altered cardiotoxic responses to doxorubicin in hPSC-derived cardiomyocytes in the presence of endothelial cells.

Cardiotoxicity is a significant challenge in cancer therapies, particularly with doxorubicin, a widely used anthracycline. More predictive in vitro models are needed to understand doxorubicin-induced cardiac damage and patient-specific responses. Here, human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs), cardiac fibroblasts (hPSC-cFBs), and endothelial cells (hPSC-ECs) were cultured in mono- or multi-cell-type formats and repeatedly treated with doxorubicin to mimic cumulative clinical exposure. A machine learning-based tool enabled continuous quantification of the early toxicity marker caspase-3/7 and accurately identified hPSC-CMs within mixed cultures. Notably, hPSC-ECs were more sensitive to doxorubicin than hPSC-CMs or hPSC-cFBs, with nitric oxide signaling contributing to the elevated cardiomyocyte toxicity observed in co-culture. These results question the conventional in vitro focus on cardiomyocytes regarding drug-induced cardiac damage, highlighting the interplay among different cardiac cell types in mediating the toxic effects of doxorubicin. Furthermore, the work demonstrates the potential of AI-based tools to provide scalable strategies for assessing drug-induced cardiotoxicity.