Abstract 6050: Analysis of diverse metabolic pathways using bioluminescent cell-based assays

Abstract

Abstract Cellular energy metabolism is a complex system of many interconnected metabolic pathways that govern both cell health and function. Cancer cells are adept at quickly switching between metabolic pathways to adapt to nutrient limitations in the environment, enhancing their growth advantages while negatively impacting other cells in the tumor microenvironment such as immune cells. To facilitate metabolic characterization of cancer cells, we developed assays for detection of metabolites involved in key pathways, such as glycolysis and glutaminolysis, using a bioluminescent NADH detection technology coupled to specific dehydrogenases. We have now extended these assays to include metabolites involved in glycogen, lipid, ketone body and amino acid metabolism, as well as intermediates in the TCA cycle such as malate, isocitrate, and 2-hydroxyglutarate. A second core bioluminescent hydrogen peroxide detection system was also used to develop an assay for pyruvate. Here we describe the use of these assays to measure metabolite levels in cell lysates and cell culture media collected from cancer cell lines plated in 96- or 384-well plates and in 2D or 3D cultures. Changes in metabolite levels were monitored in response to altered media composition and drug treatments. Levels were also compared between different cell lines. In comparative studies of glioblastoma cell lines, 2-hydroxyglutarate production was higher in cells expressing mutant isocitrate dehydrogenase. By reconfiguring the NADH detection technology to measure cellular enzyme activity rather than metabolite levels, we were able to detect activity of dehydrogenases that participate in the glycolysis pathway, the TCA cycle, and pentose phosphate pathway. Assays for cellular malate dehydrogenase, isocitrate dehydrogenase and other dehydrogenase enzymes were sensitive, requiring ≤ 400 lysed cells per assay. The expanded collection of bioluminescent assays provides a way to quickly characterize the metabolic states and behaviors of cancer cells to help identify pathways that may present therapeutic targets. Citation Format: Donna Leippe, Natasha Karassina, Mike Valley, Kayla Sylvester, Jolanta Vidugiriene. Analysis of diverse metabolic pathways using bioluminescent cell-based assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6050.