Liver histology

Baseline liver biopsy and terminal samples (both from the left lateral lobe) were fixed overnight in 4% paraformaldehyde. Liver tissue was paraffin-embedded and sectioned (3 µm thickness). Sections were stained with hematoxylin–eosin (HE), picro-sirius rRed (PSR, Sigma-Aldrich, Broendby, Denmark), anti-galectin-3 (cat. 125402, Biolegend, San Diego, CA), alpha-smooth muscle actin (α-SMA, cat. ab124964, Abcam, Cambridge, UK), anti-type I collagen (Col1a1, cat. 1310-01, Southern Biotech, Birmingham, AL), anti-Ki67 (Cat#14-5698-82, eBioscience, San Diego, CA) or anti cytokeratin-19 (CK19, cat#10712–1-AP, Proteintech, Rosemont, IL) using standard procedures^87,97. An automated deep learning-based digital imaging analysis pipeline (Gubra Histopathological Objective Scoring Technology, GHOST) was applied to obtain more accurate and objective method for assessment of histopathological scores²⁰, using the clinical NAFLD Activity Scoring (NAS) and fibrosis staging system according to NASH Clinical Research Network (CRN) scoring system as outlined by Kleiner et al.³⁹ In addition, deep learning-based image analysis was applied to histopathological scoring variables for quantifying whole-section number of lipid-laden hepatocytes (% of hepatocytes with lipids droplets), number of inflammatory foci (foci per mm²), hepatocyte ballooning index (cells/mm²) as well as proportionate (%) area of perisinusoidal and periportal fibrosis, respectively. Additionally, quantitative histomorphometry was performed using a digital imaging software (Visiomorph®, Visiopharm, Hørsholm, Denmark) for the determination of whole-section liver fat (HE-staining), fibrosis (PSR, Col1a1), inflammation (galectin-3), hepatic stellate cell (HSC) activation (α-SMA), cell proliferation (Ki67) and progenitor cell/cholangiocyte activation (CK19), expressed relative (%) to total sectional area. The total number and individual size (mm²) of macroscopic surface tumors per animal was quantified. Histological classification of hepatic tumors was performed by an expert clinical histopathologist, using DIO-NASH-HCC mice fed the GAN for ≥ 68 weeks. For reticulin staining, slides were immersed in potassium permanganate solution, followed by sulfuric acid, oxalic acid, ferric ammonium sulfate solution, silver nitrate solution, formaldehyde solution, gold chloride solution, and sodium thiosulfate solution (#1.00251, Sigma-Aldrich, St. Louis, MO). Sections were rinsed with distilled water before immersion in each solution and dehydrated in graded ethanol and xylene before cover slipping. Glutamine synthetase staining was used to support differentation between normal liver architecture with pericentral staining and neoplastic liver with diffuse or no staining. HCCs were subsequently evaluated according to the WHO three-tiered grading system (G1-G3) based on cytological features and differentiation³⁰.