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

TY - JOUR

T1 - Exploring cell death mechanisms in spheroid cultures using a novel application of the RIP3-caspase3-assay

AU - Philippi, C I

AU - Hagens, J

AU - Heuer, K M

AU - Schmidt, H C

AU - Schuppert, P

AU - Pagerols Raluy, L

AU - Trochimiuk, M

AU - Li, Z

AU - Bunders, M J

AU - Reinshagen, K

AU - Tomuschat, C

N1 - © 2024. The Author(s).

PY - 2024/7/11

Y1 - 2024/7/11

N2 - 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.

AB - 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.

KW - Receptor-Interacting Protein Serine-Threonine Kinases/metabolism

KW - Humans

KW - Spheroids, Cellular/metabolism

KW - Caspase 3/metabolism

KW - Apoptosis/drug effects

KW - Necroptosis/drug effects

KW - Tumor Necrosis Factor-alpha/metabolism

KW - Cell Death/drug effects

KW - Organoids/metabolism

U2 - 10.1038/s41598-024-66805-4

DO - 10.1038/s41598-024-66805-4

M3 - SCORING: Journal article

C2 - 38992075

VL - 14

SP - 16032

JO - SCI REP-UK

JF - SCI REP-UK

SN - 2045-2322

IS - 1

ER -