LuCEMe: An accessible luciferase-based method to functionally assess cellular metabolism across model systems

Metabolic profiling approaches such as extracellular flux analysis, metabolomics, stable isotope tracing and cytometry-based assays have transformed our understanding of cellular metabolism in physiological and pathological contexts. However, most techniques require specialized equipment or reagents, and do not allow differential metabolic analysis in intact multicellular systems. Here, we present LuCEMe, a luciferase-based metabolic phenotyping approach that combines acute metabolic perturbations with quantitative ATP measurements to resolve glycolytic and mitochondrial dependencies and capacities under native culture conditions, including in complex coculture and ex vivo systems without prior cell separation. The assay can also be adapted to increase metabolic resolution and distinguish between glycolytic activity, glucose oxidation and fatty acid/amino acid-driven mitochondrial ATP production. We use LuCEMe to reveal profound metabolic differences between leukemia cells cocultured with macrophages, stromal cells or adipocytes, and demonstrate distinct metabolic rewiring during macrophage polarization, osteogenic and adipogenic differentiation, and T cell activation. Ex vivo LuCEMe analysis identified a particular metabolic adaptation associated with chemotherapy response in acute myeloid leukemia that was not evident from conventional analyses. LuCEMe thus provides an accessible, robust and versatile framework for direct functional metabolic phenotyping across in vitro, ex vivo, and multicellular systems that complements existing metabolic profiling technologies.