Hepatology

Purpose Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major cause of chronic liver disease and liver-related morbidity worldwide. Although dietary factors may influence MASLD progression, the long-term liver-specific implications of artificially sweetened beverage (ASB) intake remain unclear. We aimed to examine the association between ASB intake and the risk of liver-related adverse events and liver-related death among individuals with MASLD. Methods This prospective cohort study included 50,562 participants with MASLD from the UK Biobank. ASB intake was assessed using 24-hour dietary recalls and categorized as 0, >0-1, and >1 serving/day. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for liver-related adverse events and liver-related death. Restricted cubic spline models were used to assess dose-response patterns, and competing-risk analyses were performed by treating liver-related death as a competing event for liver-related adverse events. Additional substitution, subgroup and sensitivity analyses were conducted to evaluate the robustness of the findings. Results During a median follow-up of 12.8 years, 292 liver-related adverse events and 91 liver-related deaths occurred. Compared with participants reporting no ASB intake, those consuming >1 serving/day had a higher risk of liver-related adverse events in the fully adjusted model (HR 1.40, 95% CI 1.02-1.93; P = 0.039), whereas the association for >0-1 serving/day was not statistically significant (HR 1.26, 95% CI 0.92-1.71; P = 0.149). The risk of liver-related adverse events increased across ASB intake categories (P for trend = 0.023). Restricted cubic spline analysis indicated a positive linear association between ASB intake and liver-related adverse events (P-overall <0.001; P-nonlinearity = 0.72). In competing-risk analysis, the association for >1 serving/day remained consistent after accounting for liver-related death as a competing event (sub-HR 1.40, 95% CI 1.02-1.93; P = 0.038; Gray test P = 0.006). The association was robust in sensitivity analyses. ASB intake was not significantly associated with liver-related death, and beverage substitution analyses showed no significant associations. Conclusion Among individuals with MASLD, high ASB intake, particularly >1 serving/day, was associated with an increased risk of liver-related adverse events, but not liver-related death. This association was consistent across dose-response, competing-risk, and sensitivity analyses, suggesting that high ASB intake may represent a potential dietary risk marker for adverse liver outcomes in MASLD.

HighlightsCERAMIC enables continuous and high-capacity lineage tracing of liver tumor initiation Fatty liver-associated hepatocytes acquire regenerative and premalignant cell states before malignant transformation Lineage reconstruction identifies Hep_Bi-zonal cells as the cellular origin of liver tumor initiation Transcriptional and regulatory programs distinguish tumor-fated hepatocytes from failed-to-transform lineages Peroxisomal metabolism is required for progenitor-state formation and liver tumor initiation Spatial remodeling identifies a macrophage niche associated with tumor-fated hepatocytes Dual ontogenies and functional specialization of lipid-associated macrophages shape the tumor-fated hepatocyte niche Fatty liver disease predisposes to primary liver cancer, yet the lineage routes and niche mechanisms that select rare tumor-fated hepatocytes remain unclear. Here we developed CERAMIC, a high-capacity CRISPR-Cas9 lineage recorder that co-recovers editing scars and transcriptomes from single cells, and applied it to an AKT/NRAS-driven model of MASLD-associated liver tumor initiation. Longitudinal lineage, single-cell and spatial analyses revealed a hierarchical trajectory in which Hep_Bi-zonal cells, rather than Hep_CVlike cells, generated regenerative and neoplastic hepatocyte progenitor states that progressed toward both hepatocellular carcinoma and intrahepatic cholangiocarcinoma lineages. Tumor-fated cells preferentially expanded along a remodeled midlobular-periportal axis and depended on ACOX1-mediated peroxisomal beta-oxidation to withstand lipotoxic and oxidative stress. Spatial and lineage analyses further identified a sequential lipid-associated macrophage niche, in which monocyte-derived LAMs engaged tumor-fated hepatocytes through an LGALS9-P4HB axis, and P4HB inhibition suppressed tumor expansion. These findings define liver tumor initiation as a lineage-restricted process licensed by peroxisomal metabolic adaptation and macrophage-derived niche signals.

Hepatocellular carcinomas (HCC) are genetically heterogeneous cancers frequently characterized by MYC gene amplification or hyperactivating {beta}-catenin (CTNNB1) mutations. Analysis of TCGA transcriptomics revealed that MYC-driven HCC tumors have decreased expression of mtDNA-encoded genes, but increased expression of nuclear-encoded mitochondrial genes. To investigate this apparent discrepancy, we generated MYC- and CTNNB1-driven murine HCCs, all of which displayed aberrant mitochondrial metabolism. Notably, MYC-driven tumors exhibited significant reductions in OXPHOS and TCA cycle activity that correlated with increased ROS levels, as well as elevated mitochondrial turnover through mitochondrial fission and mitophagy. MYC induces the expression of nuclear respiratory factor 1 (NRF1), which regulates DRP1 and other genes to promote receptor-mediated mitophagy. Knocking out DRP1 reduced mitophagy and ROS levels and promoted survival of HCC-bearing mice. These results identify elevated mitochondrial turnover as a potential therapeutic target in MYC-driven HCC. SignificanceHepatocellular carcinoma can arise from multiple oncogenes, making targeted therapy more difficult. Here we show that tumors with MYC amplification lose mitochondrial function via fission and mitophagy upregulation. Targeting mitochondrial quality control results in increased survival suggesting a therapeutic window in MYC-driven HCC.

Background & Aims: Liver transplantation improves outcomes in hepatocellular carcinoma (HCC), yet treatment options for patients with tumor recurrence remain limited to tyrosine kinase inhibitors. Glypican-3 (GPC3)-targeted CAR T cells offer a tumor-directed immune-based therapeutic strategy, but their efficacy may be limited by post-transplant immunosuppression. We developed a CAR T cell platform combining CRISPR/Cas9-mediated FKBP1A disruption to confer resistance to FKBP12-dependent immunosuppressive agents, including tacrolimus, everolimus, and sirolimus, with TRAC knockout to eliminate endogenous T cell receptor expression and reduce alloreactivity. Methods: Human T cells were edited using Cas9 ribonucleoprotein complexes targeting FKBP1A and TRAC, expanded, and transduced with an anti-GPC3 CAR construct. Cytokine production and cytotoxicity were assessed in vitro. Antitumor activity under tacrolimus treatment was evaluated in a Hep G2 xenograft model, and xenoreactivity was assessed in a graft-versus-host disease model. FKBP1A/TRAC double-knockout T cells were enriched using mTOR inhibitor selection combined with CD3-based MACS depletion. PBMCs from liver transplant recipients were used to evaluate feasibility for clinical translation during the early post-transplant period. Results: Tacrolimus suppressed wild-type CAR T cell function but not FKBP1A/TRAC double-knockout CAR T cells, which retained cytokine production, cytotoxicity, and in vivo antitumor activity. Cyclosporine A remained suppressive, enabling its potential use as a pharmacologic control strategy. TRAC disruption reduced xenoreactivity. CD3-based MACS depletion and mTOR inhibition achieved functional double-knockout efficiencies greater than 98%, without compromising cell viability. Functional FKBP1A/TRAC knockout CAR T cells were generated from patient PBMC samples 30 days post-transplant. Conclusions: Dual-edited GPC3 CAR T cells resist tacrolimus-based immunosuppression while limiting alloreactivity, supporting their use for recurrent HCC after liver transplantation. Sequential, high-viability selection in a modular cellular engineering framework enables adaptation to alternative tumor targets and next-generation CAR T cell designs.

Overnutrition-related liver dysfunction and cancer are increasingly prevalent and highly resistant to immunotherapy. While metabolic dysregulation is a hallmark of hepatocellular carcinoma (HCC), how nutrient overload impairs antitumor immunity remains unclear. Here, we show that short-term Western diet (WD) exposure drives near-complete loss of CD8 T cell infiltration and antitumor function in HCC. We identify dietary linoleic acid (LA), the most abundant {omega}-6 fatty acid, as the dominant immunosuppressive driver. Cancer cell-restricted FADS2-mediated desaturation of LA to longer-chain {omega}-6 PUFAs drives their accumulation in the tumor interstitial fluid, suppressing infiltrating CD8 T cells via lipid peroxidation. FADS2 inhibition restores CD8 T cell function and sensitizes WD-driven HCC to PD-1-based immunotherapy. Further, the Parkinsons disease-associated deglycase DJ-1 protects LA-handling proteins from methylglyoxal-mediated glycation, sustaining tumoral immunosuppressive PUFA production. Across multiple independent human MASLD-HCC cohorts, LA metabolic activity correlates with CD8 T cell impairment, immune exclusion, and immunotherapy resistance. Overall, these studies identify a dietary lipid axis as a therapeutically actionable vulnerability in WD-associated HCC.

BackgroundBiliary atresia (BA) is a severe neonatal liver disease characterized by progressive fibrosis and bile duct obliteration. ObjectiveAlthough immune dysregulation is implicated in the pathogenesis of BA, the specific mechanisms driving bile duct injury remain incompletely understood. This study aimed to characterize tertiary lymphoid structures (TLSs) within extrahepatic biliary remnants (EBRs), identify their cellular mediators, and evaluate the therapeutic potential of targeting IL-21 receptor signaling. DesignWe performed integrated bulk RNA sequencing, single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and flow cytometry on clinical samples from BA patients and non-BA cholestatic controls. TLS maturation was assessed by CD23 immunohistochemistry in EBRs from 148 BA patients and correlated with clinical parameters. Anti-IL-21R antibody treatment was evaluated in a rhesus rotavirus-induced BA mouse model, with treatment initiated on day 4 post-infection. ResultsTLSs were identified in BA EBRs with significantly higher prevalence than in matched liver tissues. Mature TLSs containing CD23 germinal centers were associated with elevated serum matrix metalloproteinase-7, more advanced hepatic fibrosis, and localized autoantibody deposition on injured bile ducts. Single-cell profiling revealed expanded CD4+ T peripheral helper (Tph) cells expressing IL-21 and CXCL13 within TLS-containing EBRs. Tph cells were enriched in peripheral blood of BA patients compared to non-BA cholestatic controls (P = 0.0025), and serum IL-21 was significantly elevated (P < 0.0001). Post-infection IL-21R blockade in the mouse model reduced jaundice incidence, improved weight gain, prevented extrahepatic biliary obstruction, and significantly improved long-term survival. ConclusionTLSs in BA extrahepatic biliary remnants harbor expanded Tph cells associated with IL-21-mediated B cell activation and bile duct injury. IL-21R blockade ameliorated disease in a murine BA model, identifying the IL-21/IL-21R axis as a potential therapeutic target warranting further investigation. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSImmune dysregulation contributes to biliary atresia (BA), with documented lymphocyte infiltration and defective B cell tolerance. However, the cellular mechanisms linking local immune activation to bile duct injury are unclear, and the roles of organized lymphoid structures and specific CD4 T cell subsets in orchestrating local humoral responses have not been characterized. What this study addsThis study demonstrates that mature tertiary lymphoid structures in extrahepatic biliary remnants are associated with disease severity markers and localized bile duct injury in BA. We identify T peripheral helper cells as an expanded IL-21-producing CD4 T cell population within these structures, and show that post-infection IL-21 receptor blockade prevents biliary obstruction and improves survival in a murine BA model. How this study might affect research, practice or policyThese findings identify the IL-21/IL-21R signaling axis as a candidate therapeutic target in BA warranting further preclinical and translational investigation. TLS maturation status in biliary remnants and serum autoantibody levels may serve as potential biomarkers of disease severity, meriting prospective evaluation in clinical cohorts.

Liver regeneration is initiated by rapid vascular changes, yet how blood flow-derived mechanical cues are decoded by liver sinusoidal endothelial cells (LSECs) remains unclear. Here, we found that partial hepatectomy generates temporally distinct mechanical cues in vivo, with a transient rise in shear stress followed by progressive sinusoidal dilation and endothelial stretch. To dissect these forces, we developed a liver regeneration chip that reconstructs sinusoidal architecture and enables independent or coupled manipulation of shear stress and mechanical stretch. Shear-dominant, stretch-dominant, and coupled mechanical modalities induce divergent LSEC regenerative programs involving extracellular matrix remodeling, cell-cycle regulation, cytoskeletal organization, and angiocrine signaling. Mechanistically, force-specific pathways, including Wnt, HIF-1, NF-{kappa}B, and Piezo1-associated signaling, mediate these outputs. Inhibition of these pathways after partial hepatectomy impairs hepatocyte proliferation and survival. These findings reveal that LSECs temporally decode blood flow-derived mechanical forces into distinct regenerative outputs, establishing endothelial mechanotransduction as an upstream regulator of liver regeneration. HIGHLIGHTS{blacksquare} Partial hepatectomy decouples transient shear from progressive stretch in vivo. {blacksquare}A liver regeneration chip recreates structure and distinct mechanical modalities in sinusoids. {blacksquare}Distinct mechanical modalities encode divergent LSEC regenerative programs. {blacksquare}Force-specific LSEC mechanotransduction supports hepatocyte proliferation and survival.

Background and objectives Spontaneous bacterial peritonitis (SBP) is a life-threatening complication of cirrhosis with ascites, carrying one- and two-year mortality rates exceeding 70% and 80%, respectively. Fluoroquinolone prophylaxis is the cornerstone of SBP prevention. Real-world longitudinal data on prescribing practices and clinical outcomes from Indian tertiary care centers are sparse. We aimed to evaluate fluoroquinolone prescribing patterns, guideline adherence, and six-month clinical outcomes in SBP patients at a tertiary academic center in North India. Methods This was a pre-specified sub-analysis of a 15-month analytical longitudinal study at AIIMS Rishikesh. Adults (age >/=18 years) admitted with SBP and initiated on fluoroquinolone prophylaxis were enrolled consecutively and followed for six months. Prescribing practices were compared against EASL and AASLD recommendations. The primary outcome was the rate of guideline-directed prescribing. Secondary outcomes included clinical cure at discharge, six-month cure, relapse, regimen modification, adverse drug reactions, and treatment compliance. Categorical variables were compared by Fisher's exact test or chi-squared test (SPSS). Results Forty-eight SBP patients were included (mean age 44.75 +/- 11.94 years; 85.4% male). Guideline-directed fluoroquinolone prophylaxis was prescribed to all patients (100%). Norfloxacin 400 mg once daily was predominant (85.4%), followed by levofloxacin (10.4%) and moxifloxacin (4.2%). Cure at discharge was 85.4%. At six months, 64.6% maintained sustained cure and 22.9% relapsed. Regimen modification occurred in 22.9%, most commonly antimicrobial substitution. Nausea was the only adverse drug reaction (4.8%). Treatment compliance was 73.8%. No patient underwent therapeutic drug monitoring. Conclusions Fluoroquinolone prescribing for SBP prophylaxis at AIIMS Rishikesh was fully concordant with standard guidelines. Despite complete adherence, a relapse rate of 22.9% and frequent regimen modification underscore the limitations of long-term fluoroquinolone prophylaxis, likely reflecting emerging quinolone resistance. Strengthening antimicrobial stewardship is essential to sustain prophylaxis effectiveness in Indian tertiary care settings.

Background & AimsHepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. Here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. MethodsAdult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. ResultsHBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, HBx expression increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative, less differentiated state. ConclusionsHBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. These findings support a model in which HBx promotes a survival-permissive less differentiated state that may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation. Impact and implicationsUnderstanding how HBV promotes hepatocellular carcinoma remains a critical challenge, partly due to the lack of physiologically relevant human derived model systems to study HBx function. Using a differentiated adult human liver organoid system, we show that HBx simultaneously suppresses apoptosis and disrupts hepatocyte identity, providing a mechanistic framework for how HBV may prime hepatocytes for malignant transformation. These findings are particularly relevant for researchers studying HBV pathogenesis and liver cancer, as well as for clinicians aiming to better understand early disease progression. While further validation in more complex multicellular systems is needed, this platform can support the identification of HBx-targeted therapeutic strategies and guide the development of improved adult human derived models for virus-host interaction studies.

Autoimmune hepatitis (AIH) is a chronic progressive liver disease that despite suggestive serum autoantibodies or plasma cell enrichment, remains functionally a diagnosis of exclusion. Whether the broader cellular composition of the liver might enable improved specificity of diagnosis has not been systematically tested. We prospectively recruited patients undergoing a clinically-indicated liver biopsy for suspected AIH and performed single-nucleus RNA sequencing (snRNA-seq) on biopsy tissue to map the cellular landscape of AIH and its diagnostic mimics. Unsupervised clustering on cell-type abundances alone largely separated AIH from non-AIH samples. Among individual populations, a subset of CD8 T-cells marked by high TOX and PD1 expression was the most discriminating feature: its enrichment perfectly distinguished AIH by both snRNA-seq and in situ density (AUC = 1.00), outperforming plasma cell abundance (AUC = 0.83). CD8TOX T-cell enrichment may therefore be the histologic lesion that marks the diagnosis of AIH.

Wolman disease (WD), the severe infantile form of lysosomal acid lipase deficiency, is a rare metabolic disorder caused by inactivating mutations in the LIPA gene. Although WD is characterized by profound hepatic dysfunction, experimental human systems capable of modelling multicellular liver pathology and supporting therapeutic testing remain limited. Here, we generated an isogenic human model of WD by introducing LIPA loss-of-function mutations into induced pluripotent stem cells and differentiating them into multicellular human liver organoids (HLO). LIPA-deficient HLO preserved hepatic lineage specification while recapitulating key biochemical and cellular features of WD, including loss of LIPA activity, lysosomal expansion, lipid accumulation, and activation of inflammatory and fibrogenic programs. Single-cell RNA sequencing resolved cell-type-specific disease states across hepatocyte-, stromal-, and biliary-like populations, revealing the emergence of a reactive biliary program consistent with ductular reaction, a complex tissue response associated with chronic liver injury. Importantly, this reactive biliary phenotype was supported by targeted gene-expression analysis in WD liver organoids and independently validated in liver tissue from mouse models and WD patients. Isolated LIPA-deficient cholangiocyte organoids failed to reproduce the DR-associated program, indicating that this response depends on multicellular interactions within the hepatic microenvironment rather than on biliary cell-autonomous dysfunction alone. Consistently, hepatocyte-directed AAV-mediated restoration of LIPA expression attenuated metabolic stress, inflammatory and fibrogenic programs, and suppressed ductular reaction both in organoids and in vivo. Together, these findings establish multicellular human liver organoids as a physiologically relevant platform for modelling emergent tissue-level responses in WD and for evaluating therapeutic rescue strategies in a human context.

Background: Pediatric chronic pancreatitis (CP) carries an elevated lifetime risk of pancreatic ductal adenocarcinoma (PDAC), yet the cellular and molecular mechanisms driving disease progression and early neoplastic transformation remain undefined. Methods: We performed single-nucleus RNA sequencing (snRNA-seq) on pancreatic tissue from 15 pediatric CP individuals and 6 healthy controls (HC). Findings were integrated with peripheral blood flow cytometry immunophenotyping of 8 CP and 7 HC individuals and validated by histopathological assessment. Findings: We identified 15 distinct cell populations and profound cellular remodeling in CP, including a 46% reduction in acinar cells and emergence of inflammatory fibroblasts as the dominant stromal population. Acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) populations bearing early PDAC-associated transcriptional signatures were detected in most CP samples. Cell-cell interaction analysis revealed that 68% of CP-specific ligand-receptor interactions converged on ADM and PanIN populations via ECM-integrin and inflammatory pathways. Peripheral blood flow cytometry demonstrated concordant systemic immune activation, including elevated monocyte CCR2 and CD80, increased CD69 on T cells, and upregulated ROR{gamma}t in regulatory T cells. Interpretation: This atlas defines the cellular landscape and intercellular signaling networks underlying pediatric CP, identifying inflammatory fibroblasts and early neoplastic cell states as central features. These findings provide a molecular foundation for understanding cancer risk in pediatric CP and provide a resource to prioritize studies into potential therapeutic targets and biomarkers. Funding: This work was supported by the Network for Pancreatic Organ donors with Diabetes (nPOD) and The Leona M. & Harry B. Helmsley Charitable Trust.

Cholangiocarcinoma (CCA) is a lethal biliary cancer in which chemoresistance is nearly universal, and its tumor immune microenvironment is dominated by immunosuppressive tumor-associated macrophages (TAMs) that exclude cytotoxic CD8+ T cells. How tumor cells sustain this immunosuppressive state during chemotherapy is undefined. Here we demonstrate that therapy-induced senescent-like (Sen-L) cancer cells accumulate after gemcitabine/cisplatin in human and murine CCA, are predominantly cancer cells, and predict shorter survival. Genetic elimination of Sen-L cancer cells reduces tumor burden, lowers TAM abundance, and restores intratumoral CD8+ T cells, establishing them as causal drivers. Growth differentiation factor 15 (GDF-15) is the dominant Sen-L-secreted factor and reprograms macrophages to suppress CD8+ T cells through the non-canonical receptor TGFBR2 and STAT6, and p16-restricted Gdf15 silencing phenocopies Sen-L elimination. Combined with chemotherapy, Sen-L elimination improves survival beyond chemotherapy alone. These findings establish Sen-L cancer cells and their GDF-15 output as causal, targetable drivers of macrophage-mediated immune evasion in CCA. SIGNIFICANCE STATEMENTTherapy-induced senescent-like cancer cells, not stromal cells, are the dominant senescent-like and immunosuppressive population in cholangiocarcinoma, and their elimination restores antitumor immunity. GDF-15 is their dominant secreted effector and engages a non-canonical macrophage receptor, TGFBR2, identifying a cancer-cell-to-macrophage axis and a Sen-L-elimination strategy to restore chemosensitivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/734341v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@7d3fd8org.highwire.dtl.DTLVardef@ea9efborg.highwire.dtl.DTLVardef@16ba16borg.highwire.dtl.DTLVardef@132b821_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. Senescent-like CCA cells promote tumor immunosuppression through TAMs polarization by GDF-15 C_FIG

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide despite recent therapeutic advances, driven in part by its marked etiological and molecular heterogeneity and the lack of broadly effective therapeutic targets. Identifying conserved tumor dependencies shared across distinct etiological backgrounds may provide new opportunities for targeted therapy. Here, we developed an integrative computational framework to systematically integrate transcriptomic, functional genomics, and clinical datasets for the identification and prioritization of candidate tumor dependency genes in liver cancer. We reanalyzed transcriptomic data from murine models of liver cancer driven by genotoxic (DEN), oncogenic (c-Myc), and inflammatory (lymphotoxin) stimuli, identifying more than 380 genes consistently upregulated across all tumor models. Functional enrichment analysis revealed a strong overrepresentation of cell cycle-related pathways and liver cancer signatures. Integration with DepMap dependency datasets identified 26 genes with strong dependency scores. Candidate genes were further prioritized by comparing their expression across models of liver regeneration, chronic liver injury, and liver cancer. Analysis of the TCGA-LIHC cohort confirmed significant overexpression of all 26 genes in human HCC, with high expression associated with poor patient survival. Together, these findings establish an integrative framework for identifying conserved tumor dependencies, providing a prioritized set of proliferation-associated genes for functional evaluation as therapeutic targets in HCC.

Background and aimsAlcohol-associated hepatitis (AH) is characterized by excessive inflammation and blunted antiviral interferon (IFN) responses. We hypothesized that specific gut microbiome-derived metabolites could selectively enhance interferon signaling while limiting NF-{kappa}B mediated inflammation, thereby restoring immune balance in AH. Our goal is to identify microbiome-derived metabolites that differentially regulate the NF-{kappa}B and IFN signaling pathways. Methods and resultsWe used human monocytic THP1-Dual cells, which secrete reporters for NF-{kappa}B and IFN signaling, to model innate immune responses and screened a library of 152 gut microbiome-derived metabolites. From the metabolite screen, 4-hydroxyphenylacetic acid (4-HPAA) emerged as a unique immunomodulator: in LPS-challenged cells, 4-HPAA selectively increased IFN signaling with minimal NF-{kappa}B activation. 4-HPAA was evaluated in vivo using a NIAAA-model, with 4-HPAA supplementation (0.4mg/ml) added to the diet. In the NIAAA-model, dietary 4-HPAA did not induce liver injury and was associated with enhanced interferon-stimulated gene expression. Simultaneously, 4-HPAA reduced pro-inflammatory markers such as Il1{beta}, Ly6g and F4/80 compared to the group exposed to ethanol alone. Metabolomic profiling of mouse cecal contents revealed 4-HPAA supplementation counteracted ethanols metabolic effects, selectively reducing triglyceride-associated lipids that had accumulated with ethanol feeding. Conclusions4-HPAA enhances interferon signaling and antiviral gene induction while dampening NF-{kappa}B-driven inflammation in the presence of LPS, both in vitro and in vivo. In an acute-on-chronic alcohol injury model, 4-HPAA attenuated hepatic inflammation, reduced immune cell recruitment, and activated antioxidant defenses, reflecting a shift toward a more hepatoprotective effect. 4-HPAA treatment was associated with reduced pro-inflammatory markers and modest attenuation of ethanol-induced liver injury. Additionally, 4-HPAA reversed ethanol-induced lipid-dysregulation, particularly triglyceride accumulation, highlighting its metabolic benefit in alcohol-fed mice. In conclusion, 4-HPAA rebalances immune and metabolic pathways by enhancing IFN signaling, suppressing NF-{kappa}B inflammation, and reversing alcohol-induced hepatic injury and lipid accumulation.

Chronic hepatitis B virus (HBV) infection contributes to hepatocellular carcinoma by disrupting host transcription, cell-cycle control, and apoptotic signaling. Isochlorogenic acid A (ICAA), a natural compound with antiviral and hepatoprotective properties, was previously shown to inhibit HBV replication by interfering with multiple steps of the viral life cycle. Because chronic HBV often reflects an imbalance between proliferation and cell death, we investigated how ICAA affects gene expression related to these processes in the presence or absence of HBV. We performed transcriptome analysis using RNA sequencing (RNA-seq) in HepAD38 cells (a HepG2-derived stable HBV-expressing line) and HepG2 control cells (HBV-negative) treated with ICAA or DMSO. HBV caused major differences in gene expression in HepAD38 cells compared with HBV-negative HepG2 cells. Principal component analysis showed that ICAA significantly altered HBV-dependent expression patterns, resulting in 189 differentially expressed genes (DEGs) that were regulated in opposite directions by both HBV and ICAA. Functional enrichment analysis highlighted pathways in viral carcinogenesis, apoptosis, MAPK signaling, and p53 signaling. Annexin V/propidium iodide assays showed apoptotic cells in both treated and untreated HepAD38 cultures, with only minor pattern changes. Mechanistically, in untreated HBV-positive cells caspase-9 cleavage failed to activate PARP, suggesting that induction of intrinsic apoptosis is followed by blocked execution. In contrast, ICAA inhibits caspase-9 cleavage in a dose-dependent manner, while activating PARP. Consistent with this, ICAA treatment increased apoptotic DNA fragmentation in HepAD38, reflecting the proapoptotic potential of ICAA under these conditions facilitating the elimination of HBV-positive cells by apoptosis. These findings highlight the potential therapeutic relevance of this compound in processes associated with HBV pathogenesis, together with its antiviral effect. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/733975v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@38d107org.highwire.dtl.DTLVardef@235a13org.highwire.dtl.DTLVardef@ee988aorg.highwire.dtl.DTLVardef@60cb13_HPS_FORMAT_FIGEXP M_FIG C_FIG

Summary Background Metronidazole (MET) resistance in Helicobacter pylori (H. pylori) exceeds 50-60% globally, yet MET-containing bismuth quadruple therapy (BQT) achieves &gt90% eradication in MET-resistant infections. We hypothesise this discordance stems from a structural limitation of two-fold dilution: a pharmacometric grey zone between the 128 and 256 &microg/mL breakpoints where treatable isolates are systematically misclassified as high-level resistance. Methods In a real-world cohort of 4610 treatment-na&iumlve children (2019-2024), checkerboard assays determined the bismuth-MET synergy factor (SF). Population PK/PD modelling simulated gastric MET exposure (AUC<sub&gt0-24</sub&gt/MIC &ge 70) at 1 g, 1&middot5 g and 2 g daily doses, incorporated bismuth SF, and estimated MIC coverage against the European Committee on Antimicrobial Susceptibility Testing (EUCAST) population distribution. Findings were validated against published adult trials. Findings 56&middot4% (485/860) of isolates were MET-resistant (MIC &gt 8 &microg/mL). At [&le;] 1 g/day MET, BQT offered no advantage over triple therapy for resistant infections. At 1&middot5 g/day, the synergy threshold was surpassed, achieving &gt90% eradication in MET-resistant infections, with efficacy independent of baseline susceptibility status. PK/PD modelling showed 1&middot5 g/day MET plus bismuth (median SF = 8) extended coverage to ~180 &microg/mL. To align modelled coverage with the &gt90% eradication rate, the dominant subpopulation of nominally high-resistant isolates must have a geometric mean MIC well below 256 &microg/mL (centred near 150 &microg/mL), consistent with a mixture of two log-normal subpopulations and confirming systematic upward misclassification by two-fold dilution in the grey zone. Interpretation This grey zone is a structural blind spot of two-fold dilution, not random measurement error. The &plusmn1-dilution reproducibility limit renders the interval inherently unresolvable. We propose a three-tier strategy: omit routine MET susceptibility testing for treatment-na&iumlve patients on optimised BQT; reserve categorical testing for BQT failures; and explore genomic/metabolomic biomarkers beyond MIC paradigms in this enriched population. Funding None. Keywords: Helicobacter pylori; Metronidazole resistance; Bismuth quadruple therapy; Antimicrobial susceptibility testing; Pharmacokinetic/pharmacodynamic modelling; Pharmacometric grey zone

Metastatic progression requires cancer cells to adapt to the unique constraints of distant organ microenvironments, yet the mechanisms that drive organ-specific adaptations remain poorly understood. Here, we show that the liver actively rewrites metastatic cancer cell identity, driving tumor cells toward a hepatobiliary progenitor-like state. Through integrated transcriptomic, proteomic, metabolomic, and epigenomic analyses of patient-derived rapid-autopsy samples and experimental models, we identify this state as selectively enriched in liver metastases. It is characterized by co-activation of hepatic and biliary/progenitor regulators HNF4A and SOX9 and is observed across multiple epithelial cancers, indicating a conserved response to the hepatic niche. Mechanistically, hepatocyte-derived TGF-{beta} and hypoxia converge to activate a HIF-1-ACLY axis, increasing nuclear acetyl-coenzyme A availability and histone acetylation at hepatic lineage regulatory elements to drive hepatobiliary reprogramming. This coordinated niche-response program can be captured transcriptionally and is associated with inferior overall survival. Disruption of this pathway suppresses hepatic reprogramming and impairs liver metastatic fitness. These findings identify the liver as an active determinant of metastatic cell fate, linking microenvironmental signaling to metabolic and chromatin remodeling programs that enable lineage plasticity. More broadly, they reveal organ-specific reprogramming as a fundamental principle of metastasis and a therapeutic vulnerability in liver metastases.

Pancreatic ductal adenocarcinoma (PDAC) is dependent on autophagy for growth. Chloroquine/Hydroxychloroquine (CQ/HCQ), the sole FDA-approved autophagy inhibitors, have shown limited clinical efficacy as cancer therapies. To identify approaches to improve PDAC response to CQ, we performed a CQ-anchored, CRISPR-Cas9 mediated loss-of-function screen. We identified that the loss of genes encoding proteins upstream in the autophagy pathway enhanced CQ-mediated growth suppression. This indicated that simultaneous targeting of two distinct nodes of the same pathway, vertical inhibition, may be a more effective strategy than single node inhibition. We demonstrated that genetic loss or pharmacological inhibition of VPS34, a protein necessary for autophagosome nucleation, sensitized PDAC cells to inhibitors of the terminal stage of the autophagy pathway, including CQ and an inhibitor of PIKfyve. We extended this concept to the initiation complex and demonstrated that ULK1/2 inhibition synergized with CQ and PIKfyve inhibition to impair PDAC cell growth and increase apoptosis. Anticipating mechanisms of resistance to vertical autophagy inhibition, we performed reverse-phase protein array profiling and identified that vertical inhibition of the autophagy pathway resulted in enhanced activation of the PI3K-AKT-mTORC1 signaling pathway. Increased mTORC1 signaling resulted in heightened sensitivity to bi-steric mTORC1 inhibition in both cell line and organoid models of PDAC. This study identifies novel anti-autophagy inhibitor combinations that may improve the clinical efficacy of autophagy inhibition for PDAC treatment. IMPLICATIONSVertical inhibition of the autophagy pathway reduces pancreatic cancer cell growth, increases apoptosis, and enhances sensitivity to mTORC1 inhibition; thereby representing a novel therapeutic strategy for autophagy-driven pancreatic cancer.

The human liver is composed of a heterogeneous mix of cell types. How these distinct populations contribute individually and collectively to liver function remains poorly understood. Although single-cell technologies have advanced our understanding of liver biology, individual studies have often been limited by small donor cohorts and inconsistent cell type annotations. Integrating multiple datasets can overcome these challenges and better capture biological variability. We present the Human Liver Cell Atlas (HLiCA), an integrated reference of non-disease liver cells assembled from eight datasets across six research centers, encompassing more than 525,000 cells from 110 donors. Developed in collaboration with the Human Cell Atlas Liver Bionetwork, the HLiCA incorporates expert-curated cell annotations refined through community feedback and dedicated cell type annotation meetings. The HLiCA classifies cells into six lineages and expands the cell type resolution to include 47 distinct cell types. Starting from raw sequencing reads, we realigned all data and performed rigorous benchmarking to ensure robust integration across technical and biological variables. Genetic ancestry was inferred for all samples to evaluate the range of ancestral backgrounds represented in the atlas. The expanded cell type annotation enabled identification of previously unrecognized liver cell types, including NRXN1+ stromal cells. Their presence was validated using spatial transcriptomics, which localized NRXN1+ stromal cells to periportal regions. With the number of donors included in the HLiCA we were able to examine cell type specific associations with demographic covariates. In hepatocytes, drug metabolism genes showed differential expression between sexes, and in cholangiocytes, mucus-production genes varied with age. As the largest and most genetically diverse human liver cell atlas to date, the HLiCA provides a comprehensive, well-annotated reference for the field, annotated by expert consensus. This resource will enable deeper interrogation of liver cellular diversity, architecture, and function in the healthy human liver and serve as a reference to understand changes that occur with disease.