JHEP Reports

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease, and liver fibrosis is the principal determinant of disease progression and clinical outcome. We recently showed that non-apoptotic caspase-8 in hepatocytes is upregulated in MASH and leads to the induction and secretion of meteorin, which promotes hepatic stellate cell (HSC) activation and liver fibrosis. We address here the major mechanistic question arising from this study, namely how non-apoptotic caspase-8 induces pro-fibrotic meteorin in hepatocytes in MASH. Methods: Mechanistic studies were performed using AML12 hepatocytes; primary mouse and human hepatocytes; and diet-induced mouse models of MASH with hepatocyte-specific caspase-8 deletion. YY1 post-translational modifications were identified by LC-MS/MS and investigated using phosphorylation-defective and phosphomimetic YY1 mutants. Genetic and biochemical approaches were used to interrogate the caspase-8 DED-SRC-GSK3b; pathway, and its relevance was assessed in human MASH liver specimens. Results: In vitro mechanistic studies showed that the amino-terminal death effector domain (DED) of caspase-8 activated a SRC-GSK3b signaling cascade that promoted phosphorylation of YY1 at Ser247, resulting in YY1 nuclear localization and Metrn induction. Disruption of the conserved DED RXDLL motif impaired SRC activation, YY1 phosphorylation, and Metrn expression. Hepatocyte YY1 overexpression increased Metrn expression and fibrosis in wild-type MASH mice but not in hepatocyte-caspase-8-deficient mice. In contrast, phosphomimetic YY1-S247D restored these endpoints even when hepatocyte caspase-8 was lacking. Studies in primary mouse and human hepatocytes and human MASH liver specimens supported conservation of this signaling pathway. Conclusions: These findings identify YY1 Ser247 phosphorylation as a critical molecular event linking non-apoptotic hepatocyte caspase-8 signaling to METRN induction and MASH fibrosis and define a previously unrecognized DED-SRC-GSK3b-YY1 signaling axis with potential therapeutic relevance.

Background & aims: TP53 (p53) coordinates diverse cellular stress response programmes including pro-survival activities, senescence, and cell death. During tissue damage, p53 can shape both the local cellular response to injury, including the fibrotic response, and influence distal organ biology. Fibrosis in the liver is a major driver of hepatocellular carcinoma (HCC) risk within metabolic dysfunction-associated steatohepatitis (MASH). It is also an important determinant of dysfunction in multiple distal tissues including the kidneys, lungs, and heart. Despite significant clinical burden, our understanding of the molecular determinants of fibrotic MASH and its relationship to multiorgan fibrosis remain incomplete. Here, we investigate local and systemic effects of hepatocellular p53 activity and cholesterol during MASH development, with implications for disease prevention. Methods: This study utilised a genetic model of stabilised p53, diet-induced MASH models with varying cholesterol compositions, and an in vitro obesogenic system to investigate p53 activity during liver disease development. Non-invasive imaging and histopathological analyses were employed to monitor p53 activity, MASH, and multiorgan fibrosis in vivo. Complementary approaches, including in vitro human multicomponent liver spheroids, cytokine arrays, and analyses of human MASH transcriptomic and proteomic datasets, were used to examine molecular drivers and patient relevance. Results: Using an inducible mouse model of MDM2 E3 ubiquitin ligase deficiency to stabilise p53, we report that hepatocellular MDM2 E3 loss results in progressive fibrotic damage, robust hepatocellular expression of the p53 target gene CDKN1A/p21 (p21),and induces p21 and fibrosis in the kidneys of male mice in a sex-specific manner. In diet-induced MASH, we observe cholesterol and p53-dependent development of liver fibrosis, high expression of hepatocellular p21, and induction of p21 and fibrosis in the kidneys of male mice-reminiscent of features observed in MDM2 E3-deficient mice. We also observe fibrosis in the lungs and heart of male MASH mice. Both a cholesterol-free obesogenic diet and liver-specific loss of p53 mitigate hepatic fibrosis and systemic induction of p21 and fibrosis. Mechanistically, p53 induces hepatic expression of senescence-associated secretory phenotype (SASP) factors, including GDF15, in vivo. A human multicomponent LiverACE spheroid model showed a concordant trend towards increased GDF15 protein abundance under steatotic stress, while in humans, elevated circulating GDF15 levels in advanced MASH correlate with increased TNFRSF1A and EPHA2, circulating markers linked to kidney injury. Conclusions: Our work identifies undue p53 activity within the liver as a driver of multiorgan fibrosis in a sex-specific manner, affecting male but not female mice. We implicate cholesterol in promoting this pro-fibrotic environment in vivo and highlight circulating factors that could identify at-risk patients for multiorgan fibrosis in MASH.

Background: Metabolic adaptation is a recognized feature of therapeutic resistance in hepatocellular carcinoma (HCC), but it is unclear whether transcriptional states exposed during acquired resistance are restricted to drug adaptation or reflect broader aggressive tumor biology. We tested whether metabolic programs derived from a lenvatinib-resistance model identify a clinically adverse transcriptional state in an independent HCC patient cohort. Methods: The discovery framework was based on GSE186191, comprising parental and acquired lenvatinib-resistant Hep3B and Huh7 cells. A pre-specified 33-gene lipid-source ledger served as a biological anchor, and three discovery-derived programs, MYC Targets V2, mTORC1 Signaling, and Fatty Acid Metabolism, were frozen before patient-level evaluation. In TCGA-LIHC, single-sample enrichment scores for the three programs were population-standardized and summed to generate an integrated metabolic score. Overall survival was assessed by Kaplan-Meier and Cox analyses. Whole-transcriptome differences between high- and low-score tumors were characterized by preranked gene set enrichment analysis (GSEA). Results: The survival cohort comprised 282 patients (118 deaths), with 141 patients in each median-defined score group. High-score patients had shorter overall survival (log-rank P=0.000419). The continuous score was associated with mortality in univariable analysis (HR 1.86, 95% CI 1.33-2.61; P=0.000293) and in the frozen model adjusted for age, sex, and stage indicators (HR 1.93, 95% CI 1.35-2.76; P=0.000350; n=277). In 327 primary tumors, Fatty Acid Metabolism was strongly depleted in high-score tumors (NES -2.06; FDR<0.001). MYC Targets V2 (NES 1.18; FDR=0.232) and mTORC1 Signaling (NES 1.11; FDR=0.229) showed positive directional enrichment without FDR significance. Conclusions: A lenvatinib-resistance-derived transcriptional program is associated with an adverse-survival state in HCC. The strongest patient-level pathway feature is depletion of canonical fatty-acid metabolism, accompanied by directional MYC/mTORC1 features rather than statistically established pathway activation. These findings support a testable model of metabolic identity remodeling but do not establish causality or clinical prediction of lenvatinib response.

Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality, yet its pathogenesis remains incompletely understood. While neural regulation is critical for hepatic homeostasis, how autonomic and neurotrophic signaling is regulated in the liver during ALD progression remains largely unexplored. Here, we performed an integrative analysis of bulk and single-cell transcriptomic datasets of human livers across the spectrum of ALD. We identified stage-dependent remodeling and coordinated expression patterns of autonomic and neurotrophic genes. Autonomic signaling exhibited a consistent decline, with reduced expression of sympathetic (DBH, TH) and the parasympathetic (ChAT) markers. In contrast, pan-neuronal and neuroplasticity-associated genes (UCHL1, TUBB3, STMN2) were upregulated, indicating active neural remodeling. In parallel, factors such as MANF, GDNF, and NRG2, with the receptor NTRK1, were upregulated and positively correlated with clinical indicators of disease severity, inflammation and fibrosis. In contrast, trophic factors including NTF3 and CDNF, and receptors such as GFRA1 and GFRA3, were downregulated and inversely associated with these parameters. Single-cell analyses revealed that neurotrophic factors are predominantly expressed by hepatic stellate cells, supporting their role as resident neuroglia-like cells in the liver. In another independent cohort, we observed a coordinated downregulation of NTF3 and sympathetic nerve markers in alcohol-associated hepatitis (AH), suggesting impaired NTF3-mediated neurotrophic support may contribute to sympathetic dysfunction in AH. Collectively, these findings uncover previously unrecognized neuro-regulatory dimensions in ALD and suggest that dysregulated autonomic and neurotrophic signaling may contribute to disease progression. Conflict of interestThe authors have declared that no conflict of interest exists.

Background: The gut-liver axis is involved in Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD), but the host molecular actors involved in such dialogue have not yet been fully characterized. Objective: We investigated the sex-dependent role of the Constitutive Androstane Receptor (CAR) as a hepatic sensor of microbial signals in MASLD pathophysiology. Design: Male and female Car+/+ and Car-/- mice were fed a western diet (WD) for 4, 14 or 35 weeks. We performed plasma biochemistry, liver histology, bile acid and cholesterol quantification, untargeted multi-omics approach combining liver RNA-sequencing and 16S rRNA sequencing, together with co-housing, fecal microbiota transplantation and analysis of publicly available transcriptomics data in human cohorts. Results: Short-term WD revealed CAR-dependent protection against steatosis, together with perturbations in the transcriptional signatures of multiple transcription factors involved in hepatocyte identity in males. After 14 weeks, CAR deletion increased WD-induced liver damage, inflammation and fibrosis, and disrupted bile acid, cholesterol and gut microbiota homeostasis, in males only. Co-housing and fecal microbiota transplantations revealed that a Car+/+ gut microbiota protected against WD-induced liver damage, while a Car-/- microbiota was reshaped by a Car+/+ host. No CAR-dependent phenotype was observed in females. Finally, in human cohorts, hepatic CAR expression decreased in advanced clinical stages of MASLD. Conclusion: CAR is a sex-dependent protective nuclear receptor against MASLD progression, acting both as a transcriptional regulator of hepatocyte identity and as a mediator of the host-microbiota crosstalk.

Hospital readmission is used in cirrhosis research as if a return to hospital meant the disease had worsened: it is a trial outcome, a quality indicator and a prediction target. We asked what those readmissions were. Sixty-one adults with alcohol-related cirrhosis, selected because they had been hospitalized more than once, contributed all 326 later admissions to one department. A clinician read each full record and classified it for acute decompensation and for admission mode, blinded to diagnostic codes and structured flags. Fewer than two in five admissions (125, 38.3%) were decompensations. One in three (114) had been booked in advance; none of these was a decompensation, yet they used 29.1% of all bed-days. Among 124 returns within 30 days, planned admissions outnumbered decompensations, 48 to 43. Counting all admissions tracked a patients decompensations only moderately (correlation 0.50) whereas counting urgent admissions tracked them closely (0.93). In crude analysis the sicker patients had fewer admissions, not more, because planned care goes to those well enough to receive it. A liver severity score, computable for 165 admissions, did not separate planned from urgent admissions or decompensations from other stays, even within one patient. Readmission was not the same thing as decompensation.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Background & Aims: Immune-mediated liver injury from immune checkpoint inhibitors (ILICI) is a major immune-related adverse event that limits cancer immunotherapy, yet its tissue-level immunobiology is poorly defined and its management is largely extrapolated from autoimmune hepatitis (AIH). We previously identified a tri-cellular CD8+ T cell-macrophage-hepatocyte injury niche in a murine model of ILICI; here, we tested whether this niche is recapitulated in human disease. Methods: We applied imaging mass cytometry with a 32-marker panel to liver biopsies from patients with ILICI (n = 12), AIH as a disease comparator (n = 14), and healthy controls (n = 2), profiling approximately 297,000 single cells across 144 regions of interest with spatially resolved detection of apoptosis (cleaved caspase-3, cC3) and pyroptosis (cleaved gasdermin D, cGSDMD). Results: We detected histiocyte-rich granulomas in ILICI consisting of macrophages and CD8+ T cells, including activated memory-effector subsets. Permutation-based spatial analysis identified CD8+ T cell-macrophage co-localization as the most frequent significant interaction in ILICI, organizing into integrated innate-adaptive cellular neighborhoods that concentrated cC3- and cGSDMD-positive cells. Descriptively, this contrasted with AIH, in which immune cells and stroma were more spatially compartmentalized. CD8+ T-cell and macrophage densities correlated with Ishak necroinflammation scores, jaundice, and granuloma formation. Conclusions: These findings provide the first single-cell spatial proteomic characterization of human ILICI in situ; they recapitulate the tri-cellular CD8-macrophage-hepatocyte niche we previously defined in a murine model and characterize ILICI as a spatially organized innate-adaptive inflammatory process, nominating myeloid signaling and CD8-macrophage interactions as candidate liver-directed targets to uncouple hepatotoxicity from anti-tumor immunity.

Reliance on invasive liver biopsy endpoints is a major limiting factor in new therapeutic development for metabolic dysfunction-associated steatohepatitis (MASH). While liver fat accumulation is considered a driver of early disease, liver fat reduction is not recognized as a surrogate endpoint by health authorities. Here, we conducted the largest-to-date genome-wide association study (GWAS) of liver fat in 79,642 people with magnetic resonance imaging and developed a polygenic score with liver fat-lowering alleles across 55 genomic loci. In a separate set of 714,886 individuals, a 30% relative liver fat reduction due to the polygenic score was associated with 39% lower odds of liver cirrhosis and 41% lower odds of hepatocellular carcinoma, consistent with a strong causal relationship between lifelong liver fat differences and advanced liver clinical outcomes. A systematic review and meta-analysis of 20 randomized controlled trials including a total of 3,502 MASH patients showed that treatment-induced liver fat reductions were associated with MASH resolution (meta-regression slope in % units of the outcome for each 1% relative reduction in liver fat from baseline, {beta} = -0.70%, p<0.001) and fibrosis improvement ({beta} = -0.24%, p<0.001). These findings demonstrate the profound etiologic impact of liver fat in MASH and provide compelling evidence for liver fat reductions as a reasonably likely surrogate endpoint for future clinical development.

Background Gallstones are one of the most common gastrointestinal conditions closely associated with metabolic dysfunction. The metabolic dysfunction - associated fibrosis - 5 (MAF -5) score has been established as a non - invasive indicator for assessing liver fibrosis in individuals with metabolic abnormalities. However, comprehensive large - scale research examining the correlation between the MAF-5 score and gallstone occurrence remains limited. This study seeks to clarify the link between the MAF-5 score and gallstone prevalence using nationally representative data from the National Health and Nutrition Examination Survey (NHANES). Methods This study examined data from 15,560 NHANES 2017-2020 participants aged 20 years or older, ensuring complete records for MAF-5 scores and gallstone status. Gallstone presence was identified through self-reported physician diagnoses. To assess the relationship between MAF-5 scores and gallstone prevalence, weighted logistic regression models were applied, adjusting for demographic characteristics, lifestyle factors, and health conditions. Subgroup analyses were conducted to evaluate the stability of this association and detect possible interactions. Sensitivity analyses were performed by excluding extreme values ({+/-}3SD) to assess result robustness. Furthermore, MAF-5 scores were divided into quartiles to investigate gallstone prevalence trends, and a restricted cubic spline (RCS) model was employed to visualize response patterns. Results A total of 15,560 participants met the inclusion criteria, with 747 in the gallstone group and 6,367 in the non-gallstone group. MAF-5 scores were significantly higher in the gallstone group (P < 0.001). After adjusting for multiple covariates, each unit increase in MAF-5 score correlated with a 14% higher gallstone prevalence (OR = 1.14, 95% CI: 1.06-1.23). Quartile-based analysis indicated that individuals in the highest MAF-5 quartile had a 2.12-fold higher prevalence of gallstones than those in the lowest quartile (OR = 2.12, 95% CI: 1.13-3.98). RCS analysis confirmed a linear association between MAF-5 scores and gallstone prevalence. Subgroup analyses showed this association remained stable across age, sex, and racial/ethnic groups, with no significant interactions. Sensitivity analyses, excluding extreme values ({+/-}3SD), reinforced the reliability of these findings (OR = 1.15, 95% CI: 1.04-1.28). Conclusion The MAF-5 score is significantly and positively associated with gallstone prevalence, independent of demographic and lifestyle confounders. These findings indicate that the MAF-5 score may be a useful tool for assessing gallstone prevalence in individuals with metabolic dysfunction, offering valuable insights for early screening and targeted health management strategies. Keywords: Gallstones, MAF-5 score, Metabolic dysfunction, NHANES, Liver fibrosis.

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.

The aim of this study was to determine whether the frequency of metabolic dysfunction-associated steatotic liver disease (MASLD) and the severity profile of inflammatory bowel disease (IBD) differ across body mass index (BMI) categories. A retrospective analysis was performed using 400 inpatient medical records from 2023-2024 with verified ulcerative colitis or Crohn's disease and sufficient information on anthropometry, hepatic status, and intestinal disease activity. The cohort comprised 118 patients with normal BMI, 176 with overweight, and 106 with obesity. MASLD status was reconstructed from routine clinical documentation and required hepatic steatosis in the appropriate cardiometabolic context. Ultrasonography was the principal source of evidence for steatosis; laboratory findings, non-invasive indices, and elastography were considered supportive when available. Ulcerative colitis activity was assessed with the Mayo Score and Crohn's disease activity with the Crohn's Disease Activity Index; disease-specific results were harmonized into mild, moderate, and severe categories for pooled comparisons. MASLD was identified in 181 of 400 patients (45.3%). Its frequency increased from 20.3% in the normal-BMI group to 49.4% in the overweight group and 66.0% in the obesity group (chi2=49.29; p<0.001). The largest absolute increase, 29.1 percentage points, occurred between normal BMI and overweight. The proportion of severe IBD rose in the same order, from 6.8% to 14.8% and 32.1% across the three BMI strata (chi2=39.45; p<0.001). In unadjusted pairwise comparisons, the odds of MASLD were higher with overweight than with normal BMI (OR=3.83; 95% CI 2.24-6.55) and higher with obesity than with normal BMI (OR=7.62; 95% CI 4.17-13.91); obesity also differed from overweight (OR=1.99; 95% CI 1.21-3.28). These findings demonstrate an ordered BMI-related pattern in which MASLD becomes more frequent and severe IBD represents a larger share of cases. However, MASLD was still documented in approximately one fifth of patients with normal BMI, indicating that BMI is useful for clinical prioritization but not for excluding steatotic liver disease. Because the study was retrospective, liver assessment reflected routine care, and the effect estimates were not adjusted for treatment exposure, disease duration, or other metabolic covariates, the observed relationships should be interpreted as associations rather than causal effects.

Background: Focal liver lesions (FLLs) encompass a wide spectrum of benign and malignant pathologies, and accurate diagnosis is essential for appropriate management. Although advances in imaging have improved lesion characterization, histopathologic assessment remains the diagnostic gold standard for indeterminate lesions. Data on the histopathologic spectrum and diagnostic utility of ultrasound-guided percutaneous liver biopsy (US-PLB) in sub-Saharan Africa (SSA) are limited. This study aimed to characterize the histopathologic findings of US-PLB performed for FLLs at a tertiary referral center in SSA and to identify factors associated with hepatocellular carcinoma (HCC). Methods: We conducted a retrospective observational study of adult patients ([&ge;]18 years) who underwent US-PLB for FLL between January 2021 and December 2024 at Adera Medical and Surgical Center. Patients with indeterminate pathology results, incomplete records, biopsies performed for diffuse liver disease, or lesions classified as LI-RADS 1, 2, or 5 were excluded. Demographic, clinical, laboratory, imaging, histopathologic, and outcome data were extracted from medical records. Descriptive statistics were used to summarize patient characteristics and histopathologic diagnoses. Logistic regression analysis was performed to identify factors associated with HCC. Results: A total of 119 were included in the final analysis. The median age was 56 years (IQR 45-65), and 59.7% were male. No major biopsy-related complications were reported. HCC was the most common histopathologic diagnosis, accounting for 42.9% of cases, followed by secondary metastatic tumors (15.9%) and regenerative nodules (15.9%). Other diagnoses included chronic hepatitis (8.4%), cholangiocarcinoma (5.9%), and hepatic abscess (3.4%). Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections were present in 14.3% and 12.4% of patients, respectively. On multivariate analysis, HBV infection (AOR 7.85, 95% CI 1.45-42.60; p=0.017), HCV infection (AOR 9.03, 95% CI 1.41-57.76; p=0.020), and larger tumor size (AOR 1.27, 95% CI 1.11-1.46; p<0.01) were significantly associated with HCC. Conclusion: Ultrasound-guided percutaneous liver biopsy demonstrated a favorable safety profile for the evaluation of FLL. HCC was the predominant histopathologic diagnosis, reflecting the substantial burden of primary liver cancer in this setting. Chronic viral hepatitis and larger tumor size were significantly associated with HCC. These findings support the continued role of US-PLB in the diagnostic evaluation of indeterminate focal liver lesions and underscores the importance of viral hepatitis prevention, surveillance, and early detection strategies in sub-Saharan Africa.

Liver cancer remains a major global health burden with high mortality and limited treatment response prediction tools. Patient-derived cancer organoids have emerged as promising preclinical models that recapitulate tumor heterogeneity; however, the biological significance of morphological diversity within established organoids remains poorly characterized in hepatocellular carcinoma (HCC). In this exploratory study, we investigated whether distinct organoid growth phenotypes reflect underlying tumor biology and correlate with clinical outcomes. We established liver cancer organoids from resected tumor tissues of 27 patients and analyzed their clinical, histological, and genomic characteristics. Organoids were classified morphologically into cystic and solid types. Whole exome sequencing (WES) was conducted on six matched tumor-organoid pairs to assess genomic fidelity. Associations between organoid establishment, growth characteristics, and clinical parameters were statistically evaluated. Progression-free survival (PFS) was analyzed using the Kaplan-Meier method and univariate Cox proportional hazards regression. Organoids were successfully established in 13 of 27 cases (48.1%). Solid-type organoids were significantly associated with shorter PFS compared to cystic types (HR = 13.91; p = 0.0039, log-rank test). Organoid establishment was more frequent in older patients (>70 years), those with HBV infection, and tumors with positive {beta}-catenin expression. WES analysis demonstrated high concordance in somatic mutation profiles and variant allele frequency distributions between tissues and corresponding organoids. In univariate Cox regression, organoid growth pattern (solid vs. cystic) showed a significant association with PFS within this exploratory cohort (p = 0.0207). Patient-derived liver cancer organoids preserved the genomic and histopathological features of the original tumors. Notably, solid morphology was associated with shorter PFS, suggesting that organoid growth phenotype may serve as a supplementary indicator of tumor biological behavior in HCC. Given the modest cohort size and the absence of multivariate analysis, these preliminary findings should be interpreted with caution and warrant further large-scale validation.

Background & Aims: Fatigue is a central symptom of chronic liver disease (CLD), substantially impacting health-related quality of life (HRQoL). This study aimed to further understand CLD symptomatology, including fatigue, and its impact on HRQoL from a patient perspective. Methods: Abbott Global Assessment of Patients unmet needs (aGAP) was a multinational, cross-sectional survey in adults with compensated CLD in China, India and Mexico, conducted between July and November 2024. Adult participants who self-reported that they had physician-diagnosed CLD and were experiencing fatigue completed a quantitative survey to assess symptom burden and included three HRQoL patient-reported outcome (PRO) questionnaires (Patient-Reported Outcomes Measurement Information System [PROMIS]-29+2, Work Productivity and Activity Impairment - Specific Health Problem version 2.0 [WPAI: SHP], Multidimensional Fatigue Inventory [MFI]). Results: Overall, 505 participants (China: 200; Mexico: 105; India: 200) completed the study. Participants reported that their CLD-related fatigue sometimes, often or always affected their self-esteem/confidence (45.1%) and ability to maintain or acquire new employment (38.6%). Most participants reported moderate (51.3%) or serious (26.9%) fatigue, with 33.5% experiencing fatigue every day or almost every day. Many participants felt their social life was negatively impacted by their fatigue (47.3%) and that there were related financial difficulties (53.9%). Use of validated PRO tools demonstrated severe fatigue (MFI: overall mean [SD] 13.9 [3.4] general fatigue and 13.4 [3.6] physical fatigue) as well as substantial levels of work and activity impairment (WPAI: SHP overall mean [SD] 53.0 [26.4]) and high levels of anxiety, pain interference, depression and sleep interference (PROMIS T-scores [&ge;]54). Conclusions: Fatigue has a substantial impact on HRQoL among adults with CLD across several countries, highlighting a global unmet need for targeted interventions to effectively identify and manage the condition.

Background. Activation of de novo lipogenesis (DNL) is a major hallmark of steatotic liver disease (SLD), a highly prevalent group of diseases ranging from a harmless steatosis to more severe steatohepatitis, cirrhosis, and hepatocellular carcinoma. We interrogated herein the early alterations in liver proteome, transcriptome, lipidome, inflammation, fibrosis, and oxidative status in a mouse model of progressive SLD induced by constitutive activation of PI3K-AKT signaling, the major inducer of DNL. Methods. Male, 8 weeks-old mice bearing phosphatase and tensin homolog (Pten) deletion and therefore constitutive activation of PI3K-AKT in hepatocytes (albumin-Cre) and littermate controls were evaluated for liver steatosis, injury, lipid peroxidation, metabolism, proteome, lipidome and transcriptome either upon feeding with a chow or high-fat diets supplemented or not with linoleic acid or after treatment with vehicle or acetyl-CoA carboxylase (ACC) inhibitor (ND-630, 20 mg/kg/day, i.p.) Results. Mice bearing Pten deletion and therefore constitutive activation of PI3K-AKT in hepatocytes display steatohepatitis characterized by enhanced DNL, severe steatosis, hepatocyte ballooning, inflammation, fibrosis and hepatomegaly, which, unexpectedly, was associated with reduced lipid peroxidation, increased GSH content and enhanced rates of triacylglycerol turnover, fatty acid {beta}-oxidation, tricarboxylic acid cycle flux, and mitochondrial respiration, altogether promoting a robust lipidome remodeling mainly defined by enrichment of DNL-derived fatty acids in detriment of a broad depletion of essential fatty acids (linoleic and -linolenic acids) and cardiolipins. Dietary restoration of liver linoleic acid content mildly attenuated liver inflammation, without however affecting lipid peroxidation and improving steatosis and fibrosis, whereas pharmacological acetyl-CoA carboxylase (ACC) and DNL inhibition markedly reduced steatosis, inflammation and fibrosis, despite further lowering hepatic linoleic acid content. Conclusions. Collectively, these findings support a harmful role of essential fatty acids depletion fomenting liver inflammation and identify DNL as a major culprit of steatohepatitis induced by constitutive PI3K-AKT activation.

Alpha-1 antitrypsin deficiency (AATD) caused by the Z variant leads to hepatic accumulation of misfolded AAT polymers and liver disease. Although proteotoxic stress is well established, its impact on lipid metabolism, mitochondrial function, and organelle homeostasis remains incompletely understood. The effects of Z-AAT accumulation were investigated in Z-HepG2 cells and 3D patient-derived ZZ hepatic organoids through protein aggregation, lipid storage, mitochondrial structure and function, peroxisomal dynamics, and comprehensive transcriptomic and proteomic analyses. Z-AAT expression led to intracellular polymer accumulation and reduced secretion, together with lipid accumulation, mitochondrial structural abnormalities, increased mitochondrial number but impaired respiratory capacity. Metabolic profiling revealed reduced oxidative phosphorylation and partial reliance on glucose metabolism. Peroxisomes displayed increased mass, consistent with altered lipid handling. Multi-omics analysis demonstrated widespread transcriptional and proteomic reprogramming related to protein synthesis, lipid metabolism, and mitochondrial function. Proteomic analysis confirmed proteotoxic stress-induced mitochondrial dysfunction, impaired lipid handling, and activation of stress response, inflammatory and vesicular trafficking pathways. Importantly, lipid supplementation elicited adaptive mitochondrial transcriptional responses in control cells, whereas Z-HepG2 cells showed a blunted response to lipid challenge. In conclusion, Z-AAT accumulation disrupts hepatic lipid processing and impaired mitochondrial and peroxisomal homeostasis, producing diminished metabolic flexibility likely contributing to AATD-associated liver disease.

BackgroundFontan-associated liver disease (FALD) is a universal complication of the Fontan palliation characterized by chronic congestion and progressive hepatic fibrosis. Current diagnostics rely on invasive biopsies or non-specific biochemical and imaging biomarkers that fail to capture early fibrogenesis, creating a critical need for non-invasive biomarkers to stratify disease severity. MethodsWe utilized a translational ovine Fontan model (n = 19) to investigate circulating serum extracellular vesicles (sEVs) as reporters of hepatic pathology. Longitudinal serum samples paired with liver elastography were collected, and sEVs were subjected to multi-omic profiling including small RNA sequencing and proteomics. Regularized regression was used to identify transcriptomic predictors, which were integrated with time post-surgery into an ordinal logistic regression framework to construct the Fontan EV Score (FES). Model performance was evaluated on a held-out test cohort and benchmarked against established serological fibrosis indices. To validate the biological relevance of the FES panel, TGF-{beta}-treated human liver organoids were generated and scored miRNA expression was assessed. ResultsThe sEV proteome exhibited robust separation by surgical physiology, while the small RNA cargo was primarily stratified by fibrotic status. Bioinformatic analysis confirmed a high hepatic origin for these transcripts and identified enrichment of inflammatory pathways including Toll-like receptor and Interleukin-17 cascades in fibrotic subjects. The FES, incorporating time post-surgery and eleven small RNA biomarkers, demonstrated high predictive accuracy in the independent testing cohort with an AUC of 0.876 for moderate and 0.963 for severe fibrosis, substantially outperforming APRI (AUC = 0.618) and FIB-4 (AUC = 0.731). In TGF-{beta}-treated human liver organoids, several scoring miRNAs, including miR-125a-5p and miR-193b-5p, were directionally responsive to profibrotic stimulation. ConclusionsCirculating sEVs carry a liver-associated molecular cargo that can be leveraged for the non-invasive prediction of FALD severity. The FES provides a biologically validated scoring system that substantially outperforms existing serological indices and offers a new avenue for early detection and risk stratification of FALD Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LIFontan-associated liver disease (FALD) is a nearly universal consequence of the Fontan circulation, driven by chronic venous hypertension and reduced cardiac output. C_LIO_LICurrent surveillance tools, including transaminases, composite serological indices (APRI, FIB-4), and elastography, have limited sensitivity and specificity for detecting and staging hepatic fibrosis in the Fontan population. C_LIO_LICirculating small extracellular vesicles (sEVs) carry tissue-derived molecular cargo and have shown diagnostic potential in other liver diseases, but their utility in FALD has not been explored. C_LI What New Information Does This Article Contribute?O_LIMulti-omic profiling of circulating sEVs in a translational ovine Fontan model reveals that the small RNA cargo is stratified by fibrotic status and enriched for inflammatory pathways associated with hepatic stellate cell activation. C_LIO_LIThe Fontan EV Score (FES), integrating time post-surgery with eleven circulating small RNA biomarkers, predicts FALD severity with substantially greater accuracy than APRI and FIB-4. C_LIO_LITGF-{beta}-treated human liver organoids confirm that several FES-associated miRNAs are directly responsive to profibrotic stimulation, providing biological validation independent of Fontan hemodynamics. C_LI This study demonstrates that circulating sEVs function as non-invasive reporters of hepatic fibrogenesis in the Fontan circulation and introduces the first EV-based scoring system for FALD risk stratification. The FES achieved an AUC of 0.876 for moderate and 0.963 for severe fibrosis in an independent test cohort, outperforming established serological indices that were originally developed for viral hepatitis but which perform poorly in congestive hepatopathy. By combining molecular biomarker discovery with in vitro functional validation, this work establishes a foundation for developing targeted, non-invasive diagnostics to guide surveillance and clinical decision-making in the growing Fontan patient population.

Background: To date, no precision oncology approach has been established for HCC. Despite the diverse underlying causes, HCC development exhibits a uniform pathophysiology characterised by chronic hyper-proliferation, resulting from hepatocyte apoptosis and compensatory liver regeneration. This chronic hyper-proliferative pressure, termed regeneration stress, drives genomic instability during HCC onset, yet its therapeutic potential remains poorly explored. This study aimed to identify targetable vulnerabilities tied to regeneration stress and establish clinically applicable markers for treatment stratification. Methods: Weighted gene co-expression network analysis (WGCNA) was applied on external bulk RNA-seq datasets to define a LIVer REgeneration Stress Signature (LIVRESS). The signature was functionally validated using HCC patient-derived organoids (HCC-Org), and vulnerabilities were mapped using mid-throughput drug screening, single-molecule and single-cell assays, and multi-omic integration. Results: High LIVRESS scores, characterised by enrichment in replication, mitotic and DNA damage repair pathways, identified a subset of HCC patients with aggressive disease and poorer survival across aetiologies. HCC-Org with high LIVRESS scores displayed exquisite sensitivity to multiple inhibitors of the checkpoint kinase ATR. Although HCC-Org models exhibited a baseline reduction in replication fork speed, sensitivity to ATR inhibitor (ATRi) was decoupled from replication fork dynamics and rather linked to intrinsic mitotic instability. ATR inhibition triggers mitotic failure and apoptosis in LIVRESSHigh HCC-Org. This killing effect was significantly potentiated by combining ATRi with PARPi or WEE1i. Multi-omic integration identified KPNA2 as a surrogate biomarker of ATRi sensitivity. Conclusion: Our findings demonstrate that a subset of HCC-Org, characterised by high liver regeneration-associated stress, is vulnerable to ATRi-based therapies. By focusing on a comprehensive regenerative stress model, we establish a framework to stratify HCC patients and implement biomarker-driven, ATR-based therapies for HCC patients with advanced disease. Impact and implications: Regeneration stress is a key factor that drives genomic instability in HCC, providing a basis for the LIVRESS to identify patients dependent on ATR-mediated checkpoints. These findings reveal a conceptual shift for researchers and trialists: ATRi efficacy is decoupled from replication fork dynamics and instead leverages mitotic fragility. Practically, the LIVRESS and its IHC surrogate marker (KPNA2) offer a scalable roadmap for physicians to improve patient stratification in ATRi-based precision oncology trials. While requiring prospective validation, these results pave the way toward biomarker-driven therapies for advanced HCC.

DNA lesions impede RNA polymerase II, yet the transcriptional consequences of that stress are less understood than the lesions themselves. We reanalysed total RNA and matched whole genomes from 438 liver lesions across four divergent mouse backgrounds, one of them the separate species M. caroli, 428 of them induced by a single dose of diethylnitrosamine (DEN) and 10 arising spontaneously, together with untreated liver and, critically, histologically normal liver from treated, tumour-bearing animals (paired normal liver). Three post-transcriptional changes accompany transformation in every background. Canonical splicing efficiency falls by 3.6 to 4.8 percentage points, and in genes that acquire them, cancer-specific junctions account for a median of 20-25% of junctions and arise at weaker splice signals. Output shifts toward circular RNA: a five-method consensus pipeline identifies 56,418 recurrent backsplice junctions, 9,718 of them detected in all four backgrounds, with ~2-fold enrichment in circRNA abundance and an expanded repertoire of distal configurations. Adenosine-to-inosine editing sites fall 2.2- to 3.8-fold, most steeply at intronic sites. Exposure changes not only these outputs but their coordination. In untreated C57BL/6J liver, splicing efficiency, mean relative backsplice span and editing co-vary positively (pairwise {rho} +0.78 to +0.87). That coordination is lost in exposed tissue, and paired normal liver shows the same loss as tumour. Pooling conditions obscures the effect because opposing estimates cancel. In C57BL/6J, paired normal liver already shows tumour-like splicing, circRNA abundance and editing roughly eight months after one exposure, while backsplice architecture has begun to reorganise. These samples carry a median of 337 callable somatic single-nucleotide variants against 78,843 in tumours and show no elevation of Mki67 or cell-cycle programmes. The post-transcriptional phenotype is therefore a durable property of damaged tissue established before morphological transformation.