Protocols for identification and assessment of bioenergetic differences in diverse pediatric cancer specimens

Abstract Metabolism is frequently altered in cancer (e.g. the Warburg effect, the tendency to overuse glycolysis at the expense of oxidative phosphorylation) and both treatment (e.g. chemotherapy) and nutrition can have the potential to differentially influence metabolism in cancer and healthy tissues. Monitoring metabolic dysregulation in cancer specimens and upon treatments is technically challenging particularly for rare, diverse and hardly metabolically characterized tumors such as those derived of pediatric origin, but may have important clinical applications for improved cancer treatment. Here, we defined a set of detailed protocols to examine functional bioenergetics in cells, organoids, organotypic tissue slices and primary samples from biopsies of childhood cancer patients using Seahorse Cell Analyzers. We provided seven application notes as examples to illustrate the possibility of monitoring metabolic heterogeneity and drug responses in 2D and 3D patient samples. We also provided several potential causes and solutions for troubleshooting to improve data consistency and reliability.