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

## TL;DR

This study shows that endothelial cells respond early to doxorubicin and increase heart cell death, suggesting new ways to test drug toxicity.

## Contribution

The study introduces a machine learning-based method to monitor cardiotoxicity in co-cultures without labels.

## Key findings

- Endothelial cells are more sensitive to doxorubicin than cardiomyocytes or fibroblasts.
- Co-culture with endothelial cells increases doxorubicin-induced apoptosis in cardiomyocytes.
- Inhibiting nitric oxide signaling reduces caspase activity in co-cultures.

## Abstract

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.

•Label-free caspase-3/7 quantification and cardiomyocyte recognition in phase images•Endothelial cells (ECs) are earliest responders to cumulative doxorubicin exposure•Co-culture with ECs accelerates doxorubicin-induced apoptosis in hPSC-cardiomyocytes•Inhibiting NO signaling using L-NAME delays caspase activity in EC-CM co-cultures

Label-free caspase-3/7 quantification and cardiomyocyte recognition in phase images

Endothelial cells (ECs) are earliest responders to cumulative doxorubicin exposure

Co-culture with ECs accelerates doxorubicin-induced apoptosis in hPSC-cardiomyocytes

Inhibiting NO signaling using L-NAME delays caspase activity in EC-CM co-cultures

In this article, Davis and colleagues expose an hPSC-derived multi-cell type cardiac culture to repeated pulses of doxorubicin and use machine learning on phase contrast images to monitor label-free caspase-3/7 dynamics and cardiomyocyte-specific responses. They show that endothelial cells are early responders to doxorubicin and amplify cardiomyocyte apoptosis partly via nitric oxide signaling. This platform enables real-time monitoring of drug-induced cardiotoxicity.