Exploring cell death mechanisms in spheroid cultures using a novel application of the RIP3-caspase3-assay

• Published in: Scientific reports 2024-07, Vol.14 (1), p.16032-9, Article 16032
• Main Authors: Philippi, C. I., Hagens, J., Heuer, K. M., Schmidt, H. C., Schuppert, P., Pagerols Raluy, L., Trochimiuk, M., Li, Z., Bunders, M. J., Reinshagen, K., Tomuschat, C.
• Format: Article
• Language: English
• Subjects: 631/250/1933; 631/532/2437; 692/308/2171; 692/308/3187; Adaptability; Apoptosis; Apoptosis - drug effects; Apoptosis and necroptosis; Caspase 3 - metabolism; Cell death; Cell Death - drug effects; Cell death mechanisms; Cell differentiation; Epithelial cells; Humanities and Social Sciences; Humans; Monoclonal antibodies; Mortality; multidisciplinary; Necroptosis; Necroptosis - drug effects; Organoids; Organoids - cytology; Organoids - metabolism; Precision medicine; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; RIP3-caspase3 assay; Science; Science (multidisciplinary); Spheroids, Cellular - drug effects; Spheroids, Cellular - metabolism; TNFα -induced stress; Tumor Necrosis Factor-alpha - metabolism; Tumor Necrosis Factor-alpha - pharmacology; Tumor necrosis factor-α
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-66805-4

This study explores the application of the RIP3-caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay’s capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay’s adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research.