Hepatocellular carcinoma (HCC) is a highly lethal malignancy with obvious heterogeneity features. This study aims to identify circRNAs with key roles in promoting HCC malignancy and stemness properties. Through circRNA profiling comparison in HCC patients and functional screening in HCC cells, circRSU1 emerges as the top candidate. It exhibits a significant higher expression level in tumor tissues compared to adjacent non-tumor liver tissues from HCC patients. Functionally, circRSU1 promotes a spectrum of HCC malignant phenotypes both in vitro and in vivo, including an enrichment of CD24positive cancer stem cell population. Mechanistically, circRSU1 interacts with heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) via two RNA motifs on circRSU1 and two RNA-binding domains on hnRNPA1. This interaction increases hnRNPA1 protein level via reducing its proteasomal degradation. Furthermore, hnRNPA1 enhances HIF-1α protein translation via binding to its internal ribosome entry site (IRES), which subsequently increases the CD24positive cell population. Additionally, circRSU1 further enhances this process not only through increasing the hnRNPA1 protein level, but also through enhancing the interaction of hnRNPA1 with HIF1A IRES, consequently augmenting the CD24positive cell population and the associated malignancy/stemness features of HCC cells. Together, circRSU1 activates the hnRNPA1/HIF-1α/CD24 signaling axis, leading to the increased HCC malignancy and stemness features.

Hepatocellular carcinoma (HCC) exhibits diminished capacity for oxidative utilization of long-chain fatty acids (LCFAs). However, the strategic and mechanistic basis by which HCC cells enact metabolic reprogramming to adapt to impaired LCFAs oxidation and sustain viability remains incompletely defined. Here we report that solute carrier family 27 member 5 (SLC27A5), the specific transporter for LCFAs, is broadly downregulated in HCC cells, resulting in reduced LCFAs uptake. In HCC cells with impaired LCFAs oxidation, diminished LCFAs import caused by SLC27A5 loss does not lead to energy deficiency, but instead prevents lipotoxicity derived from unutilized LCFAs, thereby supporting HCC cell growth. Impaired LCFAs oxidation suppresses peroxisome proliferator-activated receptor alpha (PPAR-α) signaling, which in turn represses SLC27A5 transcription, accounting for the widespread downregulation of SLC27A5 in HCC. Owing to reduced LCFAs uptake, HCC cells with low SLC27A5 rely on the glutamine reductive pathway for fatty acid biosynthesis to maintain total fatty acid levels, rendering these cells highly sensitive to glutaminase inhibition. In conclusion, we demonstrate that SLC27A5 downregulation represents a response to defective LCFAs oxidation in HCC, and reduced LCFAs uptake consequent to low SLC27A5 expression constitutes a survival adaptation that enables HCC to tolerate impaired LCFAs oxidation. Glutaminase inhibitors may serve as a precision therapeutic strategy for HCC characterized by low SLC27A5 expression.

Hepatocellular carcinoma (HCC) is the fastest growing cause of cancer-related mortality and there are limited therapies1. Although endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are implicated in HCC, the involvement of the UPR transducer ATF6α remains unclear2. Here we demonstrate the function of ATF6α as an ER-stress-inducing tumour driver and metabolic master regulator restricting cancer immunosurveillance for HCC, in contrast to its well-characterized role as an adaptive response to ER stress3. ATF6α activation in human HCC is significantly correlated with an aggressive tumour phenotype, characterized by reduced patient survival, enhanced tumour progression and local immunosuppression. Hepatocyte-specific ATF6α activation in mice induced progressive hepatitis with ER stress, immunosuppression and hepatocyte proliferation. Concomitantly, activated ATF6α increased glycolysis and directly repressed the gluconeogenic enzyme FBP1 by binding to gene regulatory elements. Restoring FBP1 expression limited ATF6α-activation-related pathologies. Prolonged ATF6α activation in hepatocytes triggered hepatocarcinogenesis, intratumoural T cell infiltration and nutrient-deprived immune exhaustion. Immune checkpoint blockade (ICB)4 restored immunosurveillance and reduced HCC. Consistently, patients with HCC who achieved a complete response to immunotherapy displayed significantly increased ATF6α activation compared with those with a weaker response. Targeting Atf6 through germline ablation, hepatocyte-specific ablation or therapeutic hepatocyte delivery of antisense oligonucleotides dampened HCC in preclinical liver cancer models. Thus, prolonged ATF6α activation drives ER stress, leading to glycolysis-dependent immunosuppression in liver cancer and sensitizing to ICB. Our findings suggest that persistently activated ATF6α is a tumour driver, a potential stratification marker for ICB response and a therapeutic target for HCC.

BACKGROUND AND AIMS:Cholangiocarcinoma (CCA) is an aggressive cancer with rising incidence and mortality worldwide. Chronic liver disease (CLD) is a well-recognized risk factor, but its influence on tumor presentation and clinical outcomes remains unclear. We aimed to compare the clinical course of CCA in patients with and without CLD. METHODS:We retrospectively analyzed 3,743 patients diagnosed with CCA between 2010 and 2024 across international centers. CLD was defined by documented primary sclerosing cholangitis, cirrhosis, viral hepatitis, or other chronic liver disorders; remaining patients were classified as non-CLD. Demographic, clinical, biochemical, treatment, and survival features were compared. RESULTS:Among the CCA cohort, 993 patients had CLD. Compared with non-CLD patients (n=2,750), those with CLD were more frequently male (67% vs. 53%) and younger (median age 63 vs. 66 years). CLD-CCA patients more often presented with intrahepatic tumors (64% vs. 42%), better performance status (ECOG 0: 53% vs. 35%), lower CA19.9 levels (56 vs. 135 U/mL), and earlier-stage disease (localized: 57% vs. 43%; metastatic: 23% vs. 31%). In propensity score-matched analyses, patients with prior CLD were diagnosed at earlier CCA stages than non-CLD controls. Consequently, curative-intent tumor surgery was performed more frequently in CLD patients (60% vs. 48%), resulting into longer median overall survival (mOS 12.2 vs. 11.1 months; HR 0.88, 95%CI 0.80-0.98) and higher 5-year survival (OR 1.70, 95%CI 1.37-2.11), particularly in intrahepatic CCA (mOS: 14.2 vs. 11.1 months; HR 0.77, 95%CI 0.68-0.87; 5-year survival OR 2.19, 95%CI 1.60-3.01). Treatment responses across modalities were comparable between groups. CONCLUSION:Pre-existing CLD is associated with earlier-stage CCA diagnosis and improved survival, supporting the implementation of structured surveillance strategies in high-risk CLD populations. IMPACT AND IMPLICATIONS:This international multicenter study show that pre-existing CLD is associated with earlier-stage CCA diagnosis, likely due to closer clinical surveillance, greater eligibility for curative-intent surgery, and improved survival. Treatment responses were similar regardless of CLD status. These findings support established surveillance in high-risk groups and highlight the need to optimize strategies for selected moderate-to-high risk CLD populations, alongside prospective evaluation of their clinical utility, cost-effectiveness, and potential refinement through more accurate non-invasive biomarkers.

Background Three-dimensional (3D) cell culture techniques have emerged as a bridge between traditional two-dimensional (2D) cell culture and the complex 3D architecture of living organisms. To overcome the limitations of existing 3D culture methods, we aimed to develop a new 3D culture platform that provides a liquid-matrix interface for cells, enabling both cell-cell and cell-matrix interactions. Methods We generated an inject-embed 3D culture platform with a standardized procedure for operation and quantification. This platform was evaluated using seven cell lines: six liver cancer cell lines including four hepatocellular carcinoma (HCC) lines and two cholangiocarcinoma (CCA) lines, and LX2 hepatic stellate cells representing the liver cancer microenvironment. Cells were cultured in both mono-culture and co-culture setups to assess spheroid and aggregate formation, proliferation, assembly, and their 3D architecture. We also evaluated its utilization in assays of stemness-related gene expression, chemo-resistance, and the tumor malignancy phenotypes. Results In mono-culture, spheroids from all seven cell lines exhibited varying sizes and shapes. In co-culture setup, HCC cells with LX2 predominantly formed mixed LX2-HCC hybrid aggregates, while CCA cells with LX2 formed well-organized CCA-centered/LX2-surrounded aggregates. In both mono-culture and co-culture systems, this inject-embed method supported significant cell proliferation, spheroid/aggregate aggregation, and cell-cell communication. Compared with conventional 2D culture, cells in this 3D system altered gene expression profiles, enhanced stemness-associated gene expression and increased cell resistance to chemotherapeutic agents. Moreover, a known HCC malignancy regulator altered spheroid size and number in this method, demonstrating its suitability for functional studies in cancer field. Conclusions This newly established inject-embed 3D culture method is highly reproducible, effectively promotes spheroid/aggregate growth and assembly, and enables the investigation of cell malignancy features under 3D microenvironment conditions.

Information on antibodies and reagents, including RRIDs

Aberrant Notch signaling has been identified as a key driver of cancer development. Genetic studies in Drosophila showed that the knockout of strawberry notch (sno) mimics the loss of notch. Here, we found that Strawberry Notch 1 (SBNO1) is upregulated in several cancer entities and elucidated the role of SBNO1 in liver cancer development. In hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), SBNO1 protein was significantly increased and localized to the nucleus suggesting its involvement in gene regulation. SBNO1-inhibition reduced cell viability, colony formation and migration and induced distinct expression patterns in HCC and CCA cell lines. However, BioID revealed that SBNO1 similarly modulates gene regulation in HCC and CCA by binding to general transcription factors TAF4 and TAF3. Deletion of Sbno1 in murine liver cancer cells Hep55.1C reduced tumor growth in vivo. In addition, inhibition of Sbno1 significantly reduced liver tumor development in three different mouse models of HCC and CCA. Furthermore, Sbno1-deletion reduced biliary differentiation and angiogenesis in the tumor margin, underscoring the necessity of Sbno1 in Notch-driven CCA formation. Thus, we identified SBNO1 as a transcriptional regulator required for liver cancer development and progression.

Cholangiocarcinoma (CCA) is an aggressive malignancy that originates in the bile ducts and is characterized by late-stage diagnosis and limited treatment options. CCA accounts for approximately 10% to 15% of primary liver tumors. Recent genetic studies have shed new light on this disease, exploring CCA's complexity and finding more effective treatment strategies, particularly based on identifying actionable mutations. Various mouse models for CCA have been established; however, the extent to which these models reflect the complexity of human is not well investigated. Therefore, this study aimed to characterize the available mouse models for CCA studies and compare their characteristics, advantages, and challenges in resemblance to human CCA. We applied tissue-based techniques using classical hematoxylin and eosin, Sirius red, and immunohistochemistry of 16 markers for in-depth characterization of tumor cells, and the tumor microenvironment of 11 different mouse models. Our findings demonstrate that CCAs present with various tumor subtypes, tumor growth patterns, morphologic subtypes, and tumor microenvironment activity. Furthermore, we report here that neoplastic lesions other than CCA, such as hepatocellular carcinoma and nonneoplastic changes in the liver parenchyma (eg, steatosis), occur with significant differences among the investigated models. Nine out of 11 investigated models were suitable for CCA studies as they resemble human CCA features. Overall, our data show that mouse models of CCA represent a valid tool to investigate this deadly disease, but they should be carefully selected, depending on the study's aims and targets in advance.

Neuroendocrine neoplasms (NEN) are thought to originate from diffuse neuroendocrine networks and therefore most frequently arise in the gastrointestinal tract and lungs. The liver is a frequent site of metastasis of NEN but also the existence of primary hepatic NEN has been proposed. Due to the impact on disease management, it is urgently required to discriminate the origin of hepatic NEN metastases and to identify clinically relevant subgroups. Using a comprehensive set of NEN (N = 212) from two independent cohorts, we show that the DNA methylation profiles of NEN of distinct anatomical localizations differ significantly and primary tumor-metastasis pairs cluster together, enabling the identification of the tumor origin. Furthermore, the subgroup of hepatic NEN without clinically detectable primary tumor, thus classified as primary hepatic NEN, does not form a distinct cluster by DNA methylation analysis but colocalizes with various subgroups of extrahepatic NEN. Organ-specific subtyping of NEN delineates a foregut-like epigenetic profile for hepatic NEN with unknown primary. We propose a classifier with high prediction accuracy for each of the different organ sites. In conclusion, our results demonstrate that DNA methylation profiling enables precise prediction of NEN origin and suggests that a substantial proportion of presumed primary hepatic NEN may in fact represent misclassified secondary hepatic NEN of unknown primary.

Chronic inflammation across tissues is associated with an increased risk of developing cancer[1][1]–[3][2]. While potentially oncogenic somatic mutations have been demonstrated to persist and expand in healthy organs[4][3]–[6][4], what triggers a subset of cells harbouring deleterious mutations to transition into a neoplasm or an aggressive adenoma with poor prognosis[7][5],[8][6] is not well-understood. Unlike normal, healthy cells, benign cells harbouring mutations perceive inflammation in chronic disease differently, potentiating the progression from physiological inflammation to tumorigenesis[9][7]. Here, we reveal that a subset of epithelial cells with mutations are poised to transition from pre-neoplastic state to early neoplasm, through rewiring of epithelial IL-1β responses and inflammatory macrophage recruitment. We characterise this process by leveraging a mouse model of biliary tract cancer (cholangiocarcinoma), in which deleterious mutations are introduced to tumour suppressor genes in common cancer pathways ( Trp53 and Pten ), and by quantifying differences in cell states and corresponding gene expression dependencies in the absence or presence of liver inflammation. Critically, we find that targeting the epithelial-derived signals of tissue-wide inflammation (namely COX2) is insufficient to limit tumorigenesis; rather, targeting the reactivation of oncogene-induced developmental signals, such as NOTCH, prevents this pre-neoplastic to neoplastic transition, demonstrating that oncofoetal switching is a pharmacologically-tractable target in patients with a high risk of developing cancers on the background of inflammation. ### Competing Interest Statement The authors have declared no competing interest. Cancer Research UK, PRCBTP-May24/100001, C52499/A27948, CTRQQR-2021\100006 Chief Scientist Office, EPD/22/12 PSC Support, EJPROJ23 Carnegie Trust for the Universities of Scotland, https://ror.org/00dfrzy25, RIG012508 UK Research and Innovation, EP/Y028546/1, MR/Z506199/1 [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-6 [5]: #ref-7 [6]: #ref-8 [7]: #ref-9

1Laboratory of Experimental Hepatology and Drug Targeting (HEVEPHARM), University of Salamanca, National Institute for the Study of Liver and Gastrointestinal Diseases (CIBEREHD), IBSAL, Salamanca, Spain 2Institute of Pathology, University Hospital Heidelberg, Heidelberg University, Heidelberg, Germany 3Department of Surgery, Erasmus MC Transplantation Institute, University Medical Center Rotterdam, Rotterdam, Netherlands Abbreviations: CAF, cancer associated fibroblast; CK, cytokeratin; FFPE, formalin-fixed paraffin-embedded; HAIC, hepatic arterial infusion chemotherapy; HBV; hepatitis B virus; iCCA, intrahepatic cholangiocarcinoma; ICIs, immune checkpoint inhibitors; TLS, tertiary lymphoid structures; TME, tumor microenvironment. Correspondence Rocio I.R. Macias, Campus M. Unamuno, Edificio Departamental, Lab. B-17 University of Salamanca, 37007, Salamanca, Spain. Email: [email protected]

Biliary tract cancer (BTC) is becoming more common worldwide, with geographic differences in incidence and risk factors. In Europe, BTC may be associated with primary sclerosing cholangitis, lithiasis, and liver cirrhosis, but is more frequently observed as a sporadic disease. BTC increasingly affects patients under 60 years, resulting in a significant social and economic burden. Early diagnosis remains challenging due to vague symptoms in 50% of patients with BTC, and lack of specific biomarkers, resulting in late presentation and poor prognosis. The identification of patients at increased risk and reliable biomarkers require collaborative efforts to make faster progress. This Series paper highlights the disparities in access to diagnostic tools and multidisciplinary care in Europe, particularly in economically disadvantaged regions, while identifying priority areas for improvement. Addressing these inequities requires harmonised guidelines, accelerated pathways to curative treatments, and improved awareness among healthcare professionals and the public. Multidisciplinary teams (MDTs) are crucial for the diagnosis of BTC and for improving patient outcomes, yet inconsistencies exist in their implementation not only between different countries, but also between different centres within a country. Collaboration and standardisation of diagnostic and treatment protocols across Europe are essential to effectively address the management of patients with BTC. Copyright (c) 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Introduction and Objectives Cholangiocarcinoma (CCA) incidence and mortality are rising globally. Chronic liver diseases (CLD) are recognized risk factors. This study aimed to compare the clinical presentation and outcomes of CCA in patients with and without CLD, using data from the International CCA Registry. Patients and Methods The international CCA Registry is a multicenter observational study enrolling cases from 54 centers across Latin America, Europe, and Asia (2010–2024). Results Among 3,693 patients enrolled, 916 had CLD and 2,777 did not. Common CLD conditions were fatty liver disease, cirrhosis, viral hepatitis, and primary sclerosing cholangitis. Compared to non-CLD patients, those with CLD were more often male (69% vs. 53%), younger at diagnosis (63 vs. 66 years), and had higher rates of metabolic risk factors, alcohol use, and smoking. Intrahepatic CCA was more frequent in CLD patients (64% vs. 43%), whereas distal CCA was more common in non-CLD cases (20% vs. 9%). CLD patients had better performance status (ECOG 0: 53% vs. 35%), lower CA19-9 levels (59.0 vs. 134.5 U/mL), and more localized disease (56% vs. 48%). Curative-intent surgery was more frequent in the CLD group (59% vs. 48%), translating into longer median overall survival (12.3 vs. 11.0 months) and higher 5-year survival (OR = 1.67; p < 0.001). The benefit was especially evident in intrahepatic CCA. Treatment responses were comparable between groups. Conclusions CCA is diagnosed at earlier stages in individuals with CLD, likely due to certain clinical surveillance, leading to better prognosis. Prospective validation and standardized surveillance protocols are warrant.

Roles of liver-specific genes (LSGs) in tumor initiation and progression are rarely explored in hepatocellular carcinoma (HCC). Here we show that LSGs are generally downregulated in HCC tumor tissues compared to non-HCC liver tissues, and low-LSG HCCs show poor prognosis and the activated c-Myc pathway. Among the c-Myc- and patient prognosis-associated LSGs, PGRMC1 significantly blocks c-Myc-induced orthotopic HCC formation. The role of PGRMC1 depends on its localization to the endoplasmic reticulum (ER) membrane, where PGRMC1 interacts with PERK through their ER luminal domains. This interaction in turn activates PERK in an ER stress-independent manner, which phosphorylates eIF2α and consequently inhibits c-Myc protein translation. In HCC patients, PGRMC1 level is significantly reduced in tumor tissues and negatively associated with the c-Myc signature. Patients with low-PGRMC1 in their tumors have poor prognosis. Collectively, deregulated LSGs in HCC are associated with the c-Myc pathway activation and PGRMC1 blocks c-Myc-induced hepatic carcinogenesis through promoting ER stress-independent PERK activation. The roles of liver-specific genes in hepatocellular carcinoma (HCC) remain to be explored. Here the authors report that PGRMC1, localized to the endoplasmic reticulum (ER) membrane, functions as a tumor suppressor in HCC by inhibiting c-Myc protein translation via activation of PERK independent of ER stress.