Detection of mitochondria-targeting anticancer agents in tumor organoids

Mitochondrial reprogramming is a hallmark of cancer, enabling tumor cells to adapt to hostile microenvironments and resist therapy. While some cancers suppress oxidative phosphorylation, others remain highly dependent on mitochondrial respiration, making mitochondria an attractive therapeutic target. This study presents a robust workflow for evaluating mitochondrial function and drug-induced mitochondrial toxicity in cancer organoids using the Agilent Seahorse XF Flex Analyzer and XF Flex Organoid Microplate. Colon cancer cell line-derived organoids embedded in Matrigel were used to evaluate the mitochondrial inhibitory effects of metformin, a well-characterized metabolic modulator. The Seahorse XF 3D Mito Stress Test and XF Mito Tox Assay enabled real-time measurement of oxygen consumption rate (OCR) and quantification of mitochondrial toxicity via the Mito Tox Index (MTI) - a unitless metric that differentiates between inhibition and uncoupling. The workflow incorporates optimized organoid seeding, image-based normalization, and reproducible metabolic profiling using the XF Flex Organoid Microplate. Results demonstrated dose-dependent mitochondrial inhibition by metformin and revealed differential susceptibility between 2D monolayer and 3D organoid cultures. These findings validate the compatibility of XF technology for organoid-based metabolic profiling in preclinical cancer research and underscore the utility of the MTI for cross-model potency comparisons of mitochondrial-targeting agents. This approach supports the development of new approach methodologies (NAMs) aligned with FDA initiatives to reduce animal testing and improve clinical translatability. The workflow is readily adaptable to patient-derived organoid (PDO) models, offering a scalable platform for screening mitochondrial-targeting therapies in oncology.