An image-based approach to visualize ADC internalization and cytotoxicity in patient-derived organoids

Antibody-drug conjugates (ADCs) represent an emerging class of cancer therapies designed to address the limitations of traditional chemotherapy, particularly the toxicity that arises from healthy cells being exposed to cytotoxic agents. ADCs combine a target-specific monoclonal antibody with a cytotoxic payload, enabling selective delivery to cancer cells and significantly reducing adverse effects. HUB Patient-Derived Organoids (PDOs) are advanced 3D in vitro models derived from adult stem cells that faithfully replicate the architecture, genetics, and physiology of original patient tissues. By preserving patient-specific genetic and phenotypic traits, including surface marker expression, HUB has established a comprehensive and well-characterized biobank of tumor-derived PDOs. This biobank provides a powerful platform for translational research, drug discovery, and the development of targeted therapies. In this study, we present an organoid-based, image-based internalization assay that complements standard Cell Titer-Glo viability assays by enabling direct visualization of ADC internalization. This assay facilitates a deeper exploration of the mechanisms of action of ADCs. We pre-selected tumor-derived organoids based on HER2 expression, which was assessed using flow cytometry. After treating the organoids with FDA-approved HER2-targeting ADCs, we utilized the internalization assay to support the viability data and to define key parameters such as the timing of action, the effect of the payload, and whether cell death resulted from ADC internalization. Overall, these results reinforce the value of patient-derived organoids as a physiologically relevant preclinical platform for ADC development. They also highlight the suitability of the described image-based assay for investigating critical stages of ADC mechanisms, including internalization and payload activity.