Abstract 3994: Comparing ATP-based and image-based viability readouts for assessing treatment responses in patient-derived organoids

High-throughput viability determination often relies on quantifying intracellular Adenosine TriPhosphate (ATP) levels. While this offers a robust option for assessing the efficacy of various compounds, it poses significant challenges when assessing compounds that interfere with metabolic or differentiation pathways, which affect intracellular ATP levels. HUB Organoid Technology is built on the discovery that adult stem cells proliferate and organize into three-dimensional organotypic structures when embedded into an extracellular matrix. Patient-Derived Organoids (PDOs) are generated from normal and malignant tissues and stored as high-quality biobanks that yield highly reproducible results. HUB Organoids® recapitulate complex characteristics of the parental tissue, including molecular heterogeneity and morphological and functional traits. Notably, PDOs can differentiate when subjected to the right conditions. This study compared ATP-based and image-based viability readouts to determine the best fit for quantifying treatment response in a panel of PDOs exposed to drugs with different modes of action. Herein, we show that an ATP-based viability readout fails to detect a viability shift in organoids treated with compounds known to modulate differentiation and metabolism, which instead becomes apparent when using an image-based readout. On the other hand, both readouts are equally effective at detecting changes in organoid viability following treatment with Oxaliplatin and 5-FU, which affect DNA damage and replication, respectively. Our results reveal the importance of consulting an expert organoid service provider who has an in-depth understanding of organoid biology and who can tailor your study based on your compound's expected mode of action, thus increasing your program´s chance of success. Citation Format: Claudia Beaurivage, Tao Yang, Anna Schepers, Mayke Doorn, Sumeyra Mucuk, Merel Derksen, Sylvia F. Boj. Comparing ATP-based and image-based viability readouts for assessing treatment responses in patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3994.