Developing a strain‐dependent susceptibility to hepatocellular carcinoma in a murine model based on histopathological evidence and genetic context

Abstract Hepatocellular carcinoma is a leading cause of cancer‐related mortality worldwide. Smad4 , a well‐established tumor suppressor within the transforming growth factor‐β (TGF‐β) signaling pathway, has an oncogenic potential of its heterozygous loss, still poorly defined, especially across genetically diverse backgrounds. The aim was to investigate whether heterozygous loss of Smad4 increases susceptibility to spontaneous liver tumor development in a genetically diverse murine population. We generated 260 F1(C57BL/6J/ Smad4 +/− X CC) mice, including 131 females and 129 males, by crossing Smad4 +/− males (C57BL/6J background) with females from 14 Collaborative Cross (CC) lines. Mice were maintained and monitored under standardized conditions for up to 80 weeks. Liver tissues were analyzed by gross pathology and histology for evidence of neoplasia. Tumor incidence was calculated per CC line; and Fisher's exact tests were used to evaluate line‐specific susceptibility. Liver tumors developed in Smad4 +/− mice (8/260; 3.08%) background, with no tumors observed in wild‐type littermates. Four CC lines exhibited tumor‐positive, with elevated incidence in CC025 (15.79%, p = 0.0011), CC010 (8.33%, p = 0.0173), and CC005 (6.90%, p = 0.0241). After Bonferroni correction for multiple comparisons, only CC025 remained statistically significant. Histopathology confirmed hepatocellular carcinoma in representative cases. Tumor incidence showed no sex bias and was restricted to specific genotypic contexts, indicating an interaction between heterozygous Smad4 deletion and host genetic background. Partial loss of Smad4 promotes liver carcinogenesis in a strain‐dependent manner, highlighting the importance of genetic modifiers in tumor susceptibility. The use of CC mice provides a valuable platform for uncovering genotype‐specific cancer susceptibilities in preclinical research.