A luminescence-based assay for analyzing energy metabolism in human hepatocellular carcinoma spheroids

Cancer cells often exhibit elevated glycolysis even when oxygen is available, a phenomenon known as the Warburg effect and a potential therapeutic target. Three-dimensional (3D) cell culture systems mimic tumor masses, but whether cancer cells cultured in 3D retain this glycolytic dependence remains unclear. This study examined changes in metabolic pathway dependence using small-molecule inhibitors of glycolysis and mitochondrial ATP synthesis as an indirect approach, and investigated effects of metformin, an emerging antitumor metabolism drug, in a hepatocellular carcinoma cell line. Human hepatocellular carcinoma G2 (HepG2) cells were cultured as hemispherical aggregates. Following metformin pretreatment, small-molecule inhibitors targeting glycolysis, the tricarboxylic acid cycle, and mitochondrial ATP synthesis were applied. After metformin treatment, ATP levels were significantly decreased in the 2D monolayers, whereas ATP levels were unchanged in the 3D hemispheroids. Following the addition of small-molecule inhibitors, glucose dependence of HepG2 monolayers and hemispheroids remained unchanged compared with the vehicle control. In addition, inhibition of mitochondrial ATP synthase did not significantly reduce ATP levels in the 3D configuration. Overall, HepG2 cells in hemispheroids remained glucose-dependent for ATP production, although ATP response to metformin was reduced. Therefore, using 3D spheroids to test anti-Warburg drugs requires careful interpretation, considering different metabolic responses compared with 2D monolayers. Direct flux analysis is needed to further evaluate metabolic pathway dependence.