Spontaneous Cholemia in C57BL/6 Mice Predisposes to Liver Cancer in NASH.

C57BL/6 mice are widely used for metabolic, cancer, and immunologic studies. High-caloric diets induce a heterogeneous phenotype in C57BL/6 mice, with the majority developing obesity and metabolic syndrome, while others remain lean and metabolically healthy. Western diets (WDs), methionine-deficient or choline deficient–based diets are used to study nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH), important risk factors for hepatocellular carcinoma development.2, 3, 4

When conducting metabolic studies with NASH-inducing diets, we found several mice remained lean but developed liver cancer at time points at which previously only NASH had been reported., These mice showed increased markers of biliary damage (serum total bile acids [TBA] and alkaline phosphatase [ALP]). To understand the link between these serum parameters and accelerated liver cancer development, we segregated mice before NASH-diet feeding and followed them up prospectively (Figure 1A and B).

Figure 1

Spontaneous cholemia in C57BL/6 mice upon Western diet feeding. (A) Proposed protocol for screening of cholemic mice. (B) Experimental scheme. (C) Serum TBAs from 6-week-old C57BL/6J Janvier Labs mice. L-TBA (<45 μmol/L) or H-TBA (≥45 μmol/L). (D) Serum TBA before diet feeding. (E) Initial body weight measurement. (F–H) Serum alanine aminotransferase (ALT), ALP, and cholesterol levels. (I) Body weight development. (J) Intraperitoneal glucose tolerance test after 30 weeks of diet. (K and L) Serum ALT and ALP levels. (M) Liver images. (N) Magnetic resonance imaging (MRI) scans of livers after 30 weeks of diet. (O) Liver to body weight ratio (%). (P) Liver tumor incidence. Chow L-TBA, n = 4 mice; WD L-TBA, n = 5 mice; WD H-TBA, n = 5 mice. Data are expressed as means ± SEM. Statistical significance was calculated using either 1-way analysis of variance with the (D-H, J-L– and O) Tukey multiple comparison test or the (P) Fischer exact test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. eMRI, echoMRI; GTT, glucose tolerance test.

Screening male C57BL/6J littermates from Janvier Labs (Le Genest-Saint-Isle, France) at 6 weeks of age identified a heterogeneous serum TBA pattern (Figure 1C). Mice showing serum TBA levels greater than 45 μmol/L (9.1%) were classified as cholemic/high-TBA (H-TBA) (Figure 1C), because complications caused by increased serum TBA only occur at levels greater than 40–45 μmol/L., Cholemia was corroborated in C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME) (14.6%) and Charles River Laboratories (Margate, UK) (23.8%) (Supplementary Figure 1A and B). Importantly, cholemia was not observed in either B6-FVB/N-129 or DBA/2 mice (Supplementary Figure 1C and D).

Next, mice were fed either standard chow or a WD (Figure 1D). Chow feeding caused no accelerated liver cancer phenotype in either low-TBA (L-TBA) or H-TBA mice (data not shown). H-TBA mice lacked any differences in body weight before WD feeding compared with L-TBA mice (Figure 1E). H-TBA mice showed accelerated and increased liver damage (serum alanine aminotransferase, ALT; ALP) upon WD feeding (Figure 1F and G). H-TBA WD-fed mice showed significantly lower levels of serum cholesterol compared with WD L-TBA mice, likely owing to the requirement of cholesterol for bile acid synthesis (Figure 1H). H-TBA, WD-fed mice remained lean throughout the experiment and showed improved glucose homeostasis and serum triglyceride levels (Figure 1I and J, Supplementary Figure 1E–K).

At 32 weeks after starting the diet, the H-TBA, WD-fed mice retained higher liver damage (serum ALT and ALP) and showed liver cancer with 100% penetrance (Figure 1K–P, Supplementary Figure 1L). Thus, H-TBA/cholemic mice show metabolic improvements at the expense of accelerated liver cancer. Histologic analyses showed tumor nodules of different sizes in WD-fed, H-TBA mice, with loss of collagen IV and increased hepatocyte proliferation, indicative of hepatocellular carcinoma (Figure 2A, Supplementary Figure 2A). H-TBA mice showed profound biliary expansion, higher fibrosis, yellow coloration of the serum, and increased serum total bilirubin levels (Figure 2B–F). Although livers of H-TBA mice showed biliary expansion, none of the tumors were of biliary origin (cytokeratin 19 - CK19), while showing positivity for the hepatocyte marker hepatocyte nuclear factor 4 alpha (HNF4A) (Supplementary Figure 2A). In addition, H-TBA mice fed a WD showed significantly higher immune cell infiltrates (Supplementary Figure 2B–F). Notably, the exacerbated liver damage in H-TBA mice was evident at as early as 5 weeks of WD feeding (Supplementary Figure 3A and B).

Figure 2

Cholemic mice show exacerbated biliary damage, liver fibrosis, and up-regulation of proliferative pathways upon Western diet feeding. (A) H&E and collagen IV (COLIV) staining of livers; dashed line indicates the tumor (T) border. Scale bars: 1 mm (low magnification) and 100 μm. (B) Cytokeratin 19 (CK19) and Sirius red staining. Scale bar: 100 μm. (C) Quantification of CK19 and (D) Sirius red–positive area. (E) Serum appearance. (F) Serum total bilirubin (TBIL) levels. (G) Serum TBA profiling. (H and I) Correlation plots. (J) Principal component analysis, (K) gene ontology (GO) analysis, and (L) gene-set enrichment analysis of H-TBA mice compared with L-TBA mice fed a WD. (A–I) Chow L-TBA n = 4; WD L-TBA n = 5; WD H-TBA n = 5 mice. (J–L) WD L-TBA n = 3; WD H-TBA n = 4 mice. Data are expressed as means ± SEM. Statistical significance calculated by 1-way analysis of variance with the (C, D, F, and G) Tukey multiple comparison test or (H and I) linear regression. ∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001. FDR, false discovery rate; MAPK, mitogen-activated protein kinase; NES, normalized enrichment score; NT, non-tumor.

We observed no consistent differences in expression of genes involved in bile acid synthesis or transport between H-TBA and L-TBA mice fed a WD (Supplementary Figure 3C). TBA levels remained strongly increased in H-TBA, WD-fed mice at 32 weeks after starting the diet (Figure 2G), primarily owing to taurine conjugates (taurocholic acid and tauro-β-muricholic acid) and unconjugated cholic acid (Supplementary Figure 3D). Previous reports have shown that taurocholic acid is not only a predictive biomarker of cirrhosis but also drives liver cirrhosis. Here, serum TBA and ALP levels strongly correlated with increased liver fibrosis (Figure 2H and I).

RNA sequencing of liver homogenates from L-TBA and H-TBA mice fed a WD showed a clear separation between groups (Figure 2J). Gene ontology and gene set enrichment analysis indicated increased fibrosis and proliferation as well as activation of the Notch and mitogen-activated protein kinase pathways (Figure 2K and L, Supplementary Figure 3E). Gene set enrichment analysis also showed enrichment for the Yes-associated protein signature in H-TBA mice (Figure 2L), supporting previous findings showing that bile acids activate Yes-associated protein to promote carcinogenesis.

The accelerated liver cancer phenotype in H-TBA cholemic mice was corroborated using an alternate WD with high trans-saturated fats or a choline-deficient high-fat diet (Supplementary Figure 4A–D). In addition, cholemic female mice also developed accelerated liver cancer upon WD feeding (data not shown). Nevertheless, cholemic mice did not show exacerbated liver damage to the well-characterized CCl4-induced model of liver fibrosis (Supplementary Figure 4E–G). This suggests that cholemia-induced accelerated liver damage and cancer may be specific to high-caloric feeding.

Overall, a subset (5%–25%) of all C57BL/6 mice, obtained from different commercial breeders, develop spontaneous cholemia, predisposing them to liver cancer upon high-caloric feeding. The molecular and genetic basis for development of spontaneous cholemia in C57BL/6 mice remains to be investigated. We suggest that future metabolic and liver cancer studies should screen C57BL/6 mice for TBA and exclude cholemic mice to prevent inconsistent or perplexing findings.