Intrinsic Tumor Aggressiveness Dictates Hypoxia-Driven Metabolic Programs in Hepatocellular Carcinoma

Hypoxia, a hallmark of hepatocellular carcinoma (HCC), regulates metabolic reprogramming, tumor progression, and therapy resistance. Although hypoxia-induced glycolytic changes are recognized, it remains unclear how intrinsic tumor aggressiveness influences the magnitude and plasticity of metabolic and transcriptional responses to oxygen deprivation. In this study, we investigated the effects of chronic hypoxia (1% O₂ for 48 h) in spheroids generated from two immortalized (HepG2, Hep3B) and two patient-derived HCC cell lines with distinct aggressiveness (HLC19, HLC21). The metabolic activity, energetic status, proliferation, and expression of hypoxia- and metabolism-related genes were assessed, with oxygen levels monitored to validate experimental conditions. It has resulted that immortalized HCC spheroids displayed similar metabolic and transcriptional responses to hypoxia, with enhanced glycolytic activity but limited phenotypic plasticity. Primary HCC spheroids exhibited aggressiveness-dependent differences. Aggressive HLC19 cells showed a pre-established glycolytic phenotype, stable ATP levels, sustained proliferation, and minimal transcriptional remodeling under hypoxia. Less aggressive HLC21 cells relied on the delayed glycolytic activation and induction of hypoxia-responsive genes to maintain viability. Clustering analyses indicated that metabolic strategies, rather than absolute activity, aligned with tumor aggressiveness. These findings suggest that intrinsic tumor aggressiveness shapes hypoxia-driven metabolic programs in HCC and supports the relevance of patient-derived 3D models for studying metabolic adaptation.