3D HepG2 spheroid model exhibits drug-specific response profiles validating their application in therapeutic...

This work establishes a robust and reproducible 96-well plate-based 3D HepG2 tumour spheroid model for drug screening applications. A 7-d culture protocol was optimised to generate HepG2 spheroid model using 96-well ultra-low attachment plates with 5 seeding densities (500, 1000, 2500, 5000 and 10,000 cells/well). Spheroids were evaluated for morphology, viability and proliferation. The optimized model was treated with doxorubicin and sorafenib at their respective IC₅₀ values (2.4 µM and 5.3 µM, determined in HepG2 monolayer). Drug-specific responses were assessed via spheroid growth, viability and expression of apoptotic and drug resistance markers. Spheroids seeded at 500 cells/well exhibited optimal characteristics, including sustained proliferative capacity (up to 30-fold increase relative to day 1) and progressive spheroidal growth (1.85-fold-change in diameter); hence, it was selected for further experiments. Day 4 was identified as the optimal treatment point based on morphology, viability and diameter progression. The 72-h IC₅₀ values were determined to be 2.4 µM for doxorubicin and 5.3 µM for sorafenib; these concentrations were chosen for subsequent treatments on HepG2-derived spheroids. Doxorubicin induced a delayed but potent cytotoxic response, while sorafenib triggered a slower, sustained effect, distinct from its rapid action in monolayer cultures. Furthermore, spheroid diameter did not correlate with viability or proliferation decline, highlighting its limitation as a sole readout. Molecular profiling revealed drug-specific responses in which ABCB1 was upregulated by doxorubicin, while ABCC2 was selectively induced by sorafenib. Sorafenib progressively reduced BAX/BCL2 ratio, suggesting potential resistance development. This study presents an optimized HepG2 spheroid model with validated culture and treatment parameters, capturing drug-specific cellular and molecular responses for therapeutic screening and mechanistic studies in hepatocellular carcinoma.