Impeding hypoxia-driven tumor progression in hepatocellular carcinoma: clinical and pre-clinical analyses using 2D and 3D in vitro models

BACKGROUND: Hypoxia is a hallmark of the hepatocellular carcinoma (HCC) microenvironment, promoting tumor progression, therapy resistance, and poor prognosis. Central mediators of the hypoxic response are the hypoxia-inducible factors (HIFs), particularly HIF-1α, whose functional relevance in clinically representative models remains incompletely understood. METHODS: In this study, we performed an in-depth characterization and functional analysis of HIF-1α by generating HIF1A knockout (KO) models in two-dimensional (2D) and three-dimensional (3D) HCC culture systems, including tumor spheroids and fibrotic-like collagen-fibrin hydrogels, to better recapitulate the complexity of the tumor microenvironment (TME). RESULTS: Analyses of publicly available transcriptomic datasets revealed that HIF1A was significantly upregulated in tumor tissues and associated with higher grade, stage, and poor survival. In contrast, EPAS1 was downregulated and correlated with improved outcomes. Functional silencing and KO experiments confirmed that HIF-1α promoted tumor cell survival, invasion, and adaptation to hypoxia, while HIF-2α played only a limited role. HIF1A deletion impaired the expression of downstream targets such as VEGF and BNIP3 and altered ABCB1 levels. Importantly, HIF-1α loss markedly reduced viability and structural integrity in 3D cultures, highlighting the added value of using physiologically relevant models to uncover microenvironment-driven phenotypes. CONCLUSIONS: Altogether, our results identify HIF-1α as a central regulator of hypoxia-mediated tumor behavior in HCC and provide a strong rationale for its therapeutic targeting to disrupt tumor adaptation and improve patient outcomes. Moreover, these findings underscore the relevance of integrating both advanced 3D and complex 2D culture systems to better capture the structural, biochemical, and mechanical features of the TME.