Small Extracellular Vesicles from Breast Cancer Cells Induce Cardiotoxicity

Cardiovascular diseases and cancer are leading global causes of morbidity and mortality, necessitating advances in diagnosis and treatment. Doxorubicin (Doxo), a potent chemotherapy drug, causes long-term heart damage due to cardiotoxicity. Small extracellular vesicles (sEVs) carry bioactive molecules-such as proteins, lipids, and nucleic acids-that can modulate gene expression and signaling pathways in recipient cells, including cardiomyocytes. Through the delivery of cytokines, microRNAs, and growth factors, sEVs can influence cell survival, which plays a critical role in the development of cardiotoxicity. This study investigates the role of sEVs derived from breast cancer cells treated or not with Doxo and their potential to induce cardiomyocyte damage, thereby contributing to cardiotoxicity. We isolated sEVs from MCF-7 cells treated or not to Doxo using ultracentrifugation and characterized them through Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), and Western Blotting (WB) for the markers CD63, CD81, and TSG101. We analyzed cytokine profiles using a Multiplex Assay and Cytokine Membrane Array. We exposed Guinea pig cardiomyocytes to different concentrations of sEVs. We assessed their viability (MTT assay), shortening, reactive oxygen species (ROS-DHE dye) production, mitochondrial membrane potential (JC-1 dye), and calcium dynamics (FLUO-4 dye). We performed statistical analyses, including t-tests, ANOVA, Cohen's d, and eta(2) to validate the robustness of the results. Treatment of MCF-7 cells with 0.01 mu M Doxorubicin resulted in increased sEVs production, particularly after 48 h of exposure (similar to 1.79 x 10(8) +/- 2.77 x 10(7) vs. similar to 5.1 x 10(7) +/- 1.28 x 10(7) particles/mL, n = 3, p = 0.0019). These sEVs exhibited protein profiles in the 130-25 kDa range and 93-123 nm sizes. They carried cytokines including TNF-alpha, IL-1 beta, IL-4, IFN-gamma, and IL-10. Exposure of cardiomyocytes to sEVs (0.025 mu g/mL to 2.5 mu g/mL) from both Doxo-treated and untreated cells significantly reduced cardiomyocyte viability, shortened cell length by up to 20%, increased ROS production, and disrupted calcium homeostasis and mitochondrial membrane potential, indicating severe cellular stress and cardiotoxicity. These findings suggest that Doxo enhances sEVs production from breast cancer cells, which plays a key role in cardiotoxicity through their cytokine cargo. The study highlights the potential of these sEVs as biomarkers for early cardiotoxicity detection and as therapeutic targets to mitigate cardiovascular risks in chemotherapy patients. Future research should focus on understanding the mechanisms by which Doxorubicin-induced sEVs contribute to cardiotoxicity and exploring their diagnostic and therapeutic potential to improve patient safety and outcomes in cancer therapy.