Novel 3D models for β-catenin-driven liver cancers.

BACKGROUND & AIMS: Constitutive activation of β-catenin is a key feature in the pathogenesis of HCC and hepatoblastoma (HB). Activating alterations in the CTNNB1 gene and, to a lesser extent, inactivating alterations in the APC gene are observed in 30-40% of HCC cases and 80-90% of HB cases. For both tumours, therapeutic management remains suboptimal. Therefore, relevant experimental models are needed to improve our understanding and to test new therapeutic approaches. METHODS: Liver organoids (n=17 mice) and tumouroids (n=14 mice) were established from APCΔhep and βcatΔex3 mouse models, which are clinically relevant models for β-catenin-activated HCC and mesenchymal HB. We developed a new methodological approach based on dynamic suspension culture in a rotating bioreactor. Morphological and molecular characteristics, as well as drug responses were evaluated by histology, immunohistochemistry, immunofluorescence, transcriptomics and RT-qPCR. RESULTS: This easy-to-implement methodology allows for the rapid production of large numbers of uniform organoids and tumouroids (30-75 per tumour) with no detectable cell death and hypoxia in their core. The robustness of the approach is also illustrated by the preservation of histological architecture, cell diversity (tumour cells, macrophages, endothelial cells) and gene expression in organoids and tumouroids compared with native liver tissue. Gene set enrichment analysis showed significant enrichment of 10 human HCC/HB tumour signatures in murine tumouroids. Organoids and tumouroids showed expected responsiveness to specific inhibitors, including the β-catenin antagonist WNTinib. CONCLUSIONS: These organoids and tumouroids represent robust in vitro models of β-catenin-driven HCC and mesenchymal HB. They provide a scalable platform for drug screening and may support the development of "à la carte" therapies urgently needed for these indications. Impact and implications; This study addresses the need for representative in vitro models to investigate β-catenin-driven liver cancers. The tumouroids developed here provide robust and reproducible 3D models that closely recapitulate the histological features, cellular diversity, and gene expression profiles of native liver tumours. These models offer a valuable platform for studying tumour biology, evaluating therapeutic candidates, and supporting the development of personalized treatment strategies. They may also contribute to reducing the use of animals in preclinical research.