Background: 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.Methods: 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.Results: 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.Conclusions: 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.
背景：缺氧是实体肿瘤（包括肝细胞癌）的一个标志性特征，它驱动氧化应激和细胞外基质重塑，促进肿瘤侵袭和转移。由于肿瘤微环境的复杂性，在患者中研究这些机制仍然具有挑战性。 方法：我们使用人肝细胞癌HepG2细胞（ATCC HB-8065）构建了一种无支架三维球体肝细胞癌模型。为表征缺氧驱动的过程，我们采用了一种多参数方法，结合了用于评估代谢活性的MTT实验、用于检测细胞活力和细胞外基质结构的共聚焦显微镜、用于检测细胞凋亡和活性氧的流式细胞术、用于分析基因表达的qRT-PCR以及用于生化谱分析的傅里叶变换红外光谱。 结果：该三维模型表现出进行性的活性氧积累、HIF-1α的稳定化以及向有氧糖酵解的代谢重编程。同时，细胞外基质重塑明显，表现为SPARC和FN1表达增加以及胶原纤维排列改变，反映了侵袭性肿瘤表型。 结论：这种无支架三维肝细胞癌模型重现了肿瘤进展的关键病理生理特征，为研究缺氧-活性氧-细胞外基质关系以及支持靶向治疗策略的临床前评估提供了一个稳健且生理相关的平台。
肿瘤(癌症)患者之家