Establishment of a mouse hepatocellular carcinoma tumoroid panel recapitulating inter- and intra- heterogeneity for disease modelling and combinatorial drug discovery

BACKGROUND: Hepatocellular carcinoma (HCC) is characterised by a remarkable molecular heterogeneity and resistance to current therapies. The use of established HCC cell lines has been the gold standard for fundamental and drug screening studies. However, each cell line is a single clone lacking cell heterogeneity, thus limiting the evaluation of anticancer efficacy, with a consequent drop of translatability regarding their clinical effectiveness, notably in relation to HCC complexity. Additionally, 2D monolayer cultures do not reproduce cell-cell and cell-matrix interactions, known to influence biological, molecular, and signalling features of cancer cells and their response to treatments. METHODS: A panel of primary HCC cells and tumoroids was generated from primary tumours of the Alb-R26Met mice. Morphological, molecular, and signalling features were evaluated by histology, imaging, RT-qPCR, and western blots. Proliferation capability and drug efficiency were evaluated through cell viability assays. RESULTS: We report the establishment of eight primary cells from distinct spontaneous HCC of the Alb-R26Met mouse model. By evaluating their morphological, molecular, and signalling pathway characteristics, we illustrate their inter-/intra- tumour heterogeneity. We show their biological features by reporting their distinct proliferation rate and resistance to RTK inhibitors used in the clinic for HCC treatments. Moreover, we document their ability to generate tumoroids, optimizing a protocol for HCC 3D cultures. The robustness of the methodology we established is illustrated by the maintenance of morphological, molecular, and growth features along several passages. Finally, we exemplify the value of this tumoroid panel for anticancer treatment evaluation by assessing the effectiveness of a new combinatorial therapy for HCC that we recently identified. CONCLUSIONS: Outcomes provide a robust methodology for the generation of HCC tumoroids and designate such heterogeneous tumoroid panel as a valuable setting for disease modelling and drug discovery.