Construction of Human Breast Cancer Organoids and Comet Assay–Based Quantification of Doxorubicin-Induced DNA Damage

Breast cancer is the most common malignancy among women worldwide and exhibits substantial heterogeneity. Doxorubicin (DOX) is widely used in breast cancer treatment, and its primary mechanism of action involves inducing DNA double-strand breaks and triggering cell death. In this protocol, a human BCO model was established to quantitatively assess DOX-induced DNA damage. Primary breast cancer cells were isolated from patient-derived tumor tissues through mechanical and enzymatic dissociation and subsequently cultured in a three-dimensional matrix to generate BCOs. Organoids were then treated with 8 μM DOX for 2 h. DNA damage was evaluated after 2 h of treatment. Following treatment, organoids were dissociated into single cells, embedded in low-melting-point agarose, and electrophoresed under alkaline conditions. Following electrophoresis, DNA migration patterns were visualized as comet structures under fluorescence microscopy and quantitatively analyzed using OpenComet software to determine the percentage of tail DNA (% Tail DNA). The comet assay successfully detected DNA damage in BCOs. Compared to the control group, BCOs exposed to 8 μM DOX for 2 h exhibited elongated comet tails following organoid dissociation and electrophoresis. Quantitative analysis revealed that the percentage of tail DNA was significantly higher in the treated group, indicating the induction of DNA damage. This highly sensitive method supports toxicological and pharmacodynamic studies of DOX and provides a robust technical platform for assessing DNA damage in patient-derived BCO systems.