Hepatic stellate cells promote structural and functional remodeling of three-dimensional hepatocellular carcinoma coculture spheroids

BACKGROUND: Hepatic stellate cells (HSCs) are major regulators of the hepatocellular carcinoma (HCC) tumor microenvironment, contributing to extracellular matrix remodeling, tumor progression, and therapeutic resistance. Although three-dimensional (3D) coculture spheroid models better represent tumor-stroma interactions than conventional monocultures, the impact of HSCs on spheroid architecture and function remains incompletely characterized. This study investigated how HSCs influence the structural and functional properties of a 3D HCC model. METHODS AND RESULTS: Spheroids were produced using HepG2 cells, LX-2 cells (HSCs), or a 1:1 coculture system (CCS) and characterized over 96 h. Morphometric analysis demonstrated that CCS spheroids exhibited reduced area and perimeter together with increased circularity and normalized solidity, indicating enhanced structural organization and compaction. Scanning electron microscopy (SEM) confirmed distinct ultrastructural organization among the spheroid models. Flow cytometry revealed fewer membrane-compromised cells in CCS spheroids than in HepG2 spheroids. Although the percentage of active caspase-3-positive cells was unchanged, active caspase-3 fluorescence intensity was lower in both HepG2-like and LX-2-like populations. CCS spheroids showed higher collagen type I alpha-1-associated fluorescence, predominantly in the α-SMA-positive compartment, consistent with a stromal collagen-producing phenotype. Biochemical analyses revealed distinct extracellular lactate profiles. Extracellular LDH activity was lower in CCS spheroids than in HepG2 spheroids, although normalized LDH release did not differ between these groups. Functionally, CCS spheroids displayed reduced sorafenib sensitivity, with an IC30 approximately 3-fold higher than HepG2 monoculture. CONCLUSIONS: LX-2 incorporation was associated with greater spheroid organization, increased collagen type I-related expression, altered cell-death and extracellular metabolic profiles, and reduced sorafenib sensitivity, supporting the value of HepG2/LX-2 coculture spheroids for studying selected tumor-stromal interactions.