022 | Physiologic remodelling in 3D hepatocellular carcinoma models: temporal evolution from day 0 to day 9

Serena Munaò, Alessandra Armeli, Desirèe Bonfiglio, Giovanna Calabrese | Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences ChiBioFarAm, University of Messina, Italy.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and remains a major cause of cancer‑related mortality worldwide. Its poor prognosis is driven by late diagnosis, high recurrence rates, and limited responsiveness to systemic therapies. The marked cellular heterogeneity of HCC, together with dynamic remodelling of the tumor microenvironment (TME), contributes to therapeutic resistance and aggressive clinical behaviour. Conventional two‑dimensional (2D) culture systems fail to reproduce these structural and biochemical complexities, underscoring the need for physiologically relevant in vitro models. In this study, we established a three‑dimensional (3D) HCC model using the hanging‑drop technique to investigate tumor evolution across defined time points. Spheroids generated at different seeding densities (400–1400 cells/drop) were analysed at Days 0, 3, 6, and 9 to assess morphological, functional, and molecular progression. Over time, HCC spheroids exhibited a pronounced increase in malignant phenotype, characterized by enhanced proliferative capacity, increased structural compactness, and features consistent with invasive behaviour. Live/Dead staining confirmed high viability throughout the time course, while proliferation assays demonstrated a progressive rise in metabolic and mitotic activity. Molecular profiling via qRT‑PCR revealed a coordinated, time‑dependent activation of multiple oncogenic genes. Stemness and proliferation markers (SOX9, STK5) were upregulated, while EMT progression was reflected by decreased CDH1 and increased TGFβ1. Osteogenic and metastatic mimicry genes (BGLAP, RUNX2) improved sharply, indicating enhanced phenotypic plasticity. Inflammatory and stress‑response mediators (IL6, TNFα, COX2, iNOS, NFκB) were strongly induced, alongside increased expression of cell‑cycle regulators (CDC25A, CDKN1A) and reduced TP53. ECM‑remodelling and invasion‑associated genes (SPARC, SPP1, MMP2, MMP9) were markedly upregulated, highlighting progressive acquisition of invasive traits. Protein–protein interaction (PPI) analysis further revealed a highly coordinated molecular background within the 3D spheroid environment. Central regulatory hubs such as TP53, RUNX2, and SPP1 anchored interactions across key biological domains, including inflammation (TNFα, IL6), matrix remodelling (SPARC, MMP2), osteogenic signalling (BGLAP, RUNX2, SPP1), and cell‑cycle control (CDC25A, CDKN1A), suggesting dynamic crosstalk between tumor‑driven pathways and stromal or differentiation signals. Overall, these findings underscore the utility of this 3D model in capturing the molecular complexity of HCC, providing a robust and biomimetic platform for the investigation of oncogenic pathways and the preclinical screening of novel therapeutic strategies.