Targeting cancer stem cells enhances multikinase inhibitor therapy in metabolic dysfunction-associated steatotic liver disease-related hepatocellular carcinoma

Objective: Metabolic dysfunction-associated steatotic liver disease-related hepatocellular carcinoma (MASLD-HCC) is an emerging malignancy with limited therapeutic options. The identity and function of cancer stem cells (CSCs) in MASLD-HCC remain poorly understood. In this study, we characterised CSCs in MASLD-HCC and investigated their contribution to MASLD-HCC tumourigenesis and therapy response. Design: We performed expression profiling in human MASLD-HCC samples (n=29 pairs of tumour and adjacent normal tissues). Advanced in vivo genetic lineage tracing coupled with single-cell RNA sequencing was used to characterise CD133+ CSCs in preclinical models. To establish causality, we developed a hepatocyte-specific CD133-overexpressing mouse model of MASLD-HCC. We identified CD133 protein interactors by mass spectrometry. A novel strategy combining CD133-targeted small interfering RNA (siRNA) nanoparticles with first-line therapy was assessed in clinically relevant MASLD-HCC models. Results: CD133+ CSCs were significantly enriched in human MASLD-HCC tumours and positively correlated with established markers of malignancy. In vivo genetic lineage tracing in mice revealed that CD133+ cells exhibit hallmark CSC properties, including self-renewal, tumour-initiating capacity and multipotent differentiation, as compared with CD133- counterparts. Hepatocyte-specific CD133 overexpression in mice accelerated MASLD-HCC tumourigenesis. Mechanistically, CD133 interacts with myosin heavy chain 9 (MYH9) to stabilise active β-catenin, thereby propagating Wnt/β-catenin signalling that drives CSC phenotypes and tumourigenic potential. Therapeutically, genetic ablation of CD133+ cells or systemic delivery of CD133-siRNA nanoparticles potently sensitised MASLD-HCC to sorafenib and lenvatinib, significantly improving outcomes in MASLD-HCC. Conclusion: This study established CD133+ CSCs as critical mediators through the CD133-MYH9/β-catenin axis in MASLD-HCC. Targeting CD133 enhances multikinase inhibitor efficacy, offering a promising therapeutic strategy for MASLD-HCC.