Metabolic Liabilities Of Human Colon Carcinoma Spheroids Are Different Compared To Standard 2d Cultures

The search for metabolic liabilities in tumor cells is typically performed in standard 2D cultures. This approach lacks the 3D context and heterogeneity of tumor tissue in vivo, which has significant impact on their metabolic behavior. One solution to modeling complex tumor cell interactions is to use cultured tumor spheroids. To assess bioenergetic function in individual spheroids, a modified, 96-well plate for the Seahorse Bioscience XFe96 Extracellular Flux Analyzer was created. Here, we demonstrate the capability of measuring mitochondrial oxygen consumption – a measure or mitochondrial respiration – and extracellular acidification – a measure of glycolytic flux – in individual HCT116 colon carcinoma spheroids. Spheroids were cultured using the hanging drop method for 4 days. Mitochondrial function was determined using the Cell Mito Stress Test which profiles 6 specific indices of mitochondrial activity. Cells grown in spheroids had a markedly increased spare respiratory capacity compared to cells in 2D culture. These data are consistent with a shift from the highly proliferative nature of 2D culture to a more physiological model where proliferation and differentiation is more balanced. We hypothesize that viewing metabolism holistically allows for a more complete view of cellular bioenergetic function and potential liabilities. To understand the metabolic potential of cells in the context of their basal metabolism, two new metrics for describing mitochondrial function were established. By stimulating maximal oxygen consumption by combined exposure to oligomycin and FCCP, and maximal extracellular acidification rate (ECAR) using rotenone, antimycin A, and oligomycin, the potential to switch metabolic pathways was determined. Metabolic Capacity Index was identified using the maximal responses in each direction, and Metabolic Flexibility Index was calculated as the angle of the line to identify cellular preference for OXPHOS or glycolysis. Interestingly, plastic-adherent HCT116 cells only exhibited potential to increase glycolysis, while HCT116 spheroids had a more balanced response. These data are consistent with the finding that spheroids have increased spare capacity. Implications of this work for future studies in cancer biology will be discussed. Citation Format: Brian P. Dranka, Pamela Swain, George W. Rogers, Ajit S. Divakaruni, Andy Neilson, David A. Ferrick. Metabolic liabilities of human colon carcinoma spheroids are different compared to standard 2D cultures. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B07. doi:10.1158/1538-7445.CHTME14-B07