Fig 2: HRG protein expression in MASLD/MASH-related HCC patients. IHC analysis of HRG performed on paraffin-embedded human liver specimens from MASLD/MASH-related HCC patients (n = 37, grades G2 to G3). Original magnification as indicated (A). HRG expression was semi-quantitatively scored blinded by a pathologist by means of histological score (H-score) (B). Kaplan–Meier curves of overall survival (C) and recurrence (D) according to HRG peritumoral H-score in MASLD/MASH-related HCC patients (C, D). Statistical analysis was performed using log-rank (Mantel–Cox) test (C, D). Plasma concentration of HRG measured in a cohort of patients (N = 78) with different stages of disease progression including 10 patients with a F0/F1 score, 25 patients with a F3/F4 score (six F3 and 19 F4) and 43 MASLD/MASH patients carrying HCC. Cirrh, cirrhotic liver; F, fibrosis score (E). Kaplan–Meier curves of overall survival according to HRG plasma levels in MASLD/MASH-related HCC patients (F). Statistical analysis was performed using log-rank (Mantel–Cox) test (F). ns, not significant.

Fig 3: HRG deletion impact on HCC angiogenesis. WB analysis for cd105 (A), PECAM1 (B), VEGF-A (C), and VE-cadherin (D) performed in HCCs from wild-type mice (WT) or from HRG knock-out mice (HRG–/–). BIORAD Quantity One software was used to perform the densitometric analysis (data are expressed as fold change relative to the normalized WT expression). Equal loading was evaluated by re-probing membranes for vinculin (A, D), β-actin (B), and GAPDH (C). Statistical differences were assessed by Student’s t-test or Mann–Whitney test for non-parametric values.

Fig 4: Experimental MASLD/MASH-related HCC: the DEN-CDAA murine model. Graphic representation of the rodent model of MASLD-associated hepatocarcinogenesis based on a single injection of diethyl-nitrosamine (DEN) at 2 weeks of age and the subsequent induction of steatohepatitis by the administration of a CDAA diet for 25 weeks (A). Hematoxylin–eosin staining performed on paraffin-embedded HCC tumor masses from wild-type mice (WT) (n = 12) or from HRG knock-out mice (HRG–/–) (n = 11). IHC analysis for HRG performed on paraffin-embedded HCC tumor masses and peri-tumoral tissue from 12 wild-type mice (WT). The bold arrows indicate nuclear pleomorphism. Original magnification as indicated (B, C). HRG expression analyzed by q-PCR in WT mice subjected to CDAA diet for 24 weeks or DEN-CDAA protocol (D). The mRNA values are expressed as fold increase over control values after normalization to the TBP gene expression (D). ns, not significant.

Fig 5: HRG deletion affects the development of experimental MASH-HCC by downregulating inflammatory response and fibrogenesis. Sirius Red staining (A) and IHC analysis for αSMA (B) performed on paraffin-embedded HCC tumor masses and peri-tumoral tissue from 12 wild-type mice (WT) or from 11 HRG knock-out mice (HRG–/–). ImageJ software analysis was performed to evaluate the amount of positivity. The data are means ± SD of 11/12 animals per group. The boxes include the values within the 25th and 75th percentile, whereas the horizontal bars represent the medians. The extremities of the vertical bars (10th–90th percentile) comprise 80% of the values. Statistical differences were assessed by unpaired parametric t-test or Mann–Whitney non-parametric t-test. Original magnification as indicated (A, B). ns, not significant.