Culture Dimensionality Shapes Drug Sensitivity to Kinase Inhibitor Combinations with Epigenetic or Metabolic Inhibition in a Hepatocellular Carcinoma Model

Background/Objectives: Hepatocellular carcinoma (HCC) remains one of the deadliest malignancies worldwide, and treatment efficacy is often limited by tumor heterogeneity and therapy resistance. Preclinical models, particularly three-dimensional (3D) cell cultures, are increasingly used to better mimic the tumor microenvironment and improve the predictive value of drug testing. This study aimed to compare the effectiveness of different pharmacological treatments for HCC in two-dimensional (2D) monolayer and 3D spheroid HepG2 culture models and to investigate the underlying cell death mechanisms. The multikinase inhibitors sorafenib and regorafenib, the epigenetic histone deacetylase (HDAC) inhibitor panobinostat, and the metabolic phosphoglycerate dehydrogenase (PHGDH) inhibitor NCT-503 were evaluated as single agents and in combination. Results: All tested compounds induced cell death and reduced cellular ATP levels in both 2D and 3D systems. Combination treatments of sorafenib or regorafenib with either panobinostat or NCT-503 generally showed no significant deviation from additivity in cytotoxicity and cellular energy levels, although significant sub-additive deviations were observed for some panobinostat combinations in the ATP analyses. Mechanistically, panobinostat induced apoptotic cell death, as indicated by caspase-3 and PARP cleavage, increased caspase-3/7 activity, and inhibition by the pan-caspase inhibitor zVAD-FMK, whereas NCT-503-mediated cell death occurred largely independently of canonical caspase-mediated apoptosis in 2D cultures. Although similar effects were observed in 3D spheroids, cell death responses were attenuated compared with 2D cultures, whereas reductions in cellular ATP levels remained comparable, indicating a differentiated relationship between ATP depletion and cell death in 2D and 3D cultures. Conclusions: In summary, combining tyrosine kinase inhibition with epigenetic or metabolic targeting agents increased cell death and impaired cellular energy homeostasis more effectively than single-agent treatment in HepG2 cells, with no significant deviation from additivity detected in most combination analyses. Notably, 3D-cultured HepG2 cells showed attenuated cell death responses despite comparable reductions in cellular ATP levels, highlighting differences in drug responses between 2D and 3D culture models. These findings support the use of 3D culture models as a complementary approach to conventional 2D systems for evaluating drug responses, particularly as they reveal an attenuated cell death response compared with 2D cultures.