Physiopathological Features in a Three-Dimensional In Vitro Model of Hepatocellular Carcinoma: Hypoxia-Driven Oxidative Stress and ECM Remodeling

<i>Background</i>: Hypoxia is a hallmark of solid tumors, including hepatocellular carcinoma (HCC), where it drives oxidative stress and extracellular matrix (ECM) remodeling, promoting tumor invasion and metastasis. Investigating these mechanisms in patients remains challenging due to the complexity of the tumor microenvironment. <i>Methods</i>: We developed a scaffold-free three-dimensional (3D) spheroid model of HCC using human hepatocellular carcinoma HepG2 cells (ATCC HB-8065). To characterize hypoxia-driven processes, a multiparametric approach combining MTT assays for metabolic activity, confocal microscopy for viability and ECM organization, flow cytometry for apoptosis and ROS detection, qRT-PCR for gene expression, and FTIR spectroscopy for biochemical profiling were performed. <i>Results</i>: The 3D model exhibited progressive ROS accumulation, stabilization of HIF-1α, and metabolic reprogramming toward aerobic glycolysis. In parallel, ECM remodeling was evident, with increased expression of SPARC and FN1 and collagen fiber alignment, reflecting an invasive tumor phenotype. <i>Conclusions</i>: This scaffold-free 3D HCC model recapitulates key physiopathological features of tumor progression, providing a robust and physiologically relevant platform to investigate the hypoxia-ROS-ECM relationship and to support preclinical evaluation of targeted therapeutic strategies.