Background: Recent advances in spatial transcriptomic technologies enable in situ gene expression profiling while preserving spatial context. This capability is particularly important for studying the tumor microenvironment (TME), where diverse and admixed cell populations interact within highly organized spatial niches that influence tumor progression and therapeutic response. However, the limited resolution of early spatial transcriptomic platforms results in each spatial spot capturing transcripts from multiple cell types, making accurate spot deconvolution or annotation a critical yet challenging step in downstream data analysis. The level of complexity will be particularly prominent in heterogeneous samples like the tumor microenvironments where multiple cell types are highly admixed and reliable single-cell reference atlases may usually be unavailable. Methods: In this paper, we developed our method called STEA, which is a novel and accurate reference-independent enrichment-based annotation algorithm for major cell type. Unlike the existing approaches, STEA does not require single-cell RNA sequencing datasets as reference, offering both flexibility and computational efficiency in execution. Results: We performed comprehensive benchmarking using a variety of simulated datasets across different platforms and scenarios and demonstrated the superior accuracy of STEA. Apart from synthetic data, we also evaluated multiple real datasets to further exemplify its practical applicability on both oncology-related and oncology-unrelated data. More importantly, we could confidently demonstrate the high concordance between prediction of STEA and histological classification by experienced pathologist. Conclusion: Our STEA algorithm provides a practical reference-independent framework to complement the cutting-edge spatial transcriptomics in genomics studies, facilitating accurate downstream high-dimensional spatial characterization of cellular and molecular landscapes, reconstruction of tissue architecture as well as cell-cell communication in malignant and non-malignant scenarios. Taken together, our comprehensive evaluation demonstrates the robustness and reliability of STEA, highlighting its potential as a valuable tool for studying complex tissue organization, particularly within heterogeneous TME.

BACKGROUND AND AIMS:The mechanisms underlying metabolic dysfunction-associated steatohepatitis (MASH)-associated hepatocellular carcinoma (HCC) are poorly understood, and effective treatments are lacking. This study explored the translational potential of Resmetirom, a clinically approved thyroid hormone receptor beta (Thrb) agonist, and investigated the mechanistic basis of MASH-associated hepatocarcinogenesis. APPROACH AND RESULTS:Repurposing Resmetirom for MASH-HCC treatment demonstrated significant tumor-suppressive effects across multiple preclinical models. To further investigate its mechanisms, we employed a Western diet plus CCl 4 -induced murine MASH-HCC model, complemented by single-cell RNA sequencing (scRNA-seq) analyses of liver and tumor tissues. These analyses revealed active cell-cell communication within the tumor microenvironment, particularly involving hepatic stellate cells (HSCs) and dysplastic hepatocytes (dys-Heps). These cells showed marked upregulation of midkine (MDK), which in human HCC correlated with shorter relapse-free survival, specifically in non-viral, non-alcohol-associated cases. In mouse models, MDK facilitated M2-like macrophage polarization via interaction with the receptor LRP1, contributing to disease progression from MASH to fibrosis and eventual HCC. Silencing LRP1 in macrophages abolished MDK-driven M2 polarization and increased cytotoxic cytokine secretion, while LRP1-positive macrophages contributed to T cell exhaustion through the CXCL16-CXCR6 axis. MDK expression negatively correlated with Thrb and lipolytic genes, but positively with lipogenesis genes. Resmetirom treatment not only significantly suppressed tumor growth and reduced steatosis but also decreased MDK expression and increased Thrb levels. Combining Resmetirom and an MDK inhibitor (iMDK) synergistically suppressed tumorigenesis in vivo. CONCLUSIONS:Targeting the MDK/LRP1 axis with Resmetirom offers a promising therapeutic strategy for MASH-associated HCC, addressing both metabolic dysfunction and tumor progression.

The tumor immune microenvironment (TIME) critically modulates therapeutic responses to immune checkpoint inhibitors (ICIs) in hepatocellular carcinoma (HCC). Relative to the other solid tumors, HCC is characterized by more pronounced intratumoral heterogeneity and has comparatively poorer responsiveness to ICI blockade treatment. Importantly, the correlation of the underlying mechanism of TIME and the molecular underpinnings of immunotherapy resistance in HCC remains elusive. Employing an integrative multi-omics approach, including spatially resolved transcriptomics, single-cell and bulk RNA sequencing, and lipidomics, we delineated the spatiotemporal dynamics and mechanistic basis of resistance to anti-PD-1 therapy in HCC. Our analyses revealed a profoundly immunosuppressive TIME in non-responsive patients, marked by sparse immune cell infiltration within the tumor niche, in contrast to the immune-inflamed TIME observed in the responders. Multidimensional profiling further uncovered dysregulated lipogenesis and aberrant lipid accumulation in tumor cells of the non-responder cases. Notably, tumor-associated macrophages (TAMs), exhausted CD8 T cells, and lipid-enriched tumor cells co-localize at the tumor-immune interface, forming a physical and functional barrier that precludes effective immune cell infiltration into the tumor niche. We also identified the pivotal TAM-tumor cell crosstalk via the ADM-RAMP1-EBP signaling axis, orchestrating lipid metabolism reprogramming and contributing to the attenuated PD-1 therapeutic efficacy. Collectively, these findings provide a comprehensive mechanistic framework for anti-PD1 resistance, unveiling actionable biomarkers and a translational vulnerability to enhance precision therapeutics in HCC.

Background: Hepatitis B virus (HBV) infection is a significant risk factor for hepatocellular carcinoma (HCC). During infection, HBV DNA integrates into the host genome, promoting hepatocarcinogenesis through both gene-dependent and gene-independent mechanisms. It can activate oncogenic gene transcription or produce chimeric and novel proteins that contribute to tumorigenesis. On the other hand, it also compromises genomic stability on a large scale. Furthermore, HBV integration can alter the liver microenvironment, fostering conditions conducive to tumor development. HCC remains one of the most challenging cancers to treat, primarily due to the incomplete understanding of HCC, inadequate diagnostic and prognostic strategies, and limited effective therapeutic options. Summary: Liquid biopsy represents a significant advancement in oncology, offering a noninvasive tool for cancer detection and management. HBV integration detection through liquid biopsy serves as a promising strategy for managing HBV-associated HCC. Importantly, it exhibits preferential patterns that differentiate HCC from chronic hepatitis or cirrhosis in patients, making it a potential biomarker for HCC diagnosis. Moreover, the quantification of HBV-host chimeric reads in the bloodstream can indicate the presence of residual tumor cells post-surgery, serving as a promising biomarker for the screening of HCC recurrence. HBV integration additionally contributes to the production of HBV surface antigen, which is crucial for achieving a functional cure for HBV infection and influences the efficacy of antiviral treatments. Key Messages: HBV integration plays a pivotal role in hepatocarcinogenesis, and its detection via liquid biopsy will greatly enhance the clinical management of HBV-associated HCC. .

Background and Aims:Research on metabolic reprogramming in HCC has increased; however, studies on the metabolism of glycerolipids or interactions between different pathways remain scarce. Enzymes of the glycerol phosphate dehydrogenase (GPD) family, which regulate the glycerol-3-phosphate shuttle, link the metabolic processes of glycolysis and glycerolipids. Therefore, we aimed to understand the role and regulation of GPDs in HCC.Approach and Results:We performed transcriptomic analysis on clinical HCC samples from in-house and public cohorts and detected the upregulation of glycerol-3-phosphate dehydrogenase 1-like (GPD1L) among GPD family genes in HCC. Further analysis showed that high GPD1L expression was associated with more frequent venous invasion and shorter overall survival. Consistent with these clinical findings, GPD1L knockdown suppressed the invasiveness of HCC cells, reduced colony- and sphere-forming abilities, and inhibited stemness gene expression in vitro, while also inhibiting tumor growth and metastasis in vivo. On the other hand, we used mass-spectrometry-based metabolomics to confirm that GPD1L facilitated the biogenesis of the glycerolipid precursor glycerol-3-phosphate (G3P) from dihydroxyacetone phosphate (DHAP). Further untargeted lipidomic analysis revealed that GPD1L supported triacylglycerol synthesis. In addition, our study identified E74-like E-Twenty-Six transcription factor 1 (ELF1) as a direct activator of GPD1L transcription, binding to the GPD1L promoter to boost its transcription while reducing GPD1L expression when ELF1 levels were lowered.Conclusions:GPD1L is overexpressed in human HCCs and is associated with worse clinical outcomes. Aberrant GPD1L expression, driven by ELF1, facilitates conversion of DHAP to G3P to support triacylglycerol synthesis in HCC, promoting tumor growth and metastasis.

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, exhibits significant cellular and molecular heterogeneity. The advent of single-cell transcriptomic methodologies has significantly disclosed its intra-tumoral cellular diversity to an unprecedented level. Interactions between cells within the tumor microenvironment (TME) are crucial for cancer progression and affect how tumors respond to current therapies. This study aimed to utilize single-cell data to address two main objectives: (1) to dissect the heterogeneous nature of HCC tumors by identifying tumor subclones and their molecular features, key transcription factors, and implicated pathways; and (2) to explore the intricate communication between tumor cells and immune cells, with a focus on the pathways that significantly influence tumor growth and metastasis. The tissue samples of HCC were obtained from nine patients undergoing surgery immediately from the operation theater after resection, with informed consent. Dissociated tumor single cells were subjected to scRNA-seq by 10X Genomics platform. Our analysis identified seven distinct tumor subclones, validating the presence of high tumor heterogeneity. Among these, one subclone, designated as SC6, exhibited particularly distinctive molecular characteristics. Further investigation revealed that NFE2L3 exhibited the highest activity score during the TFs analysis, indicating its significant regulatory role in SC6 cluster, and was intricately associated with pathways involved in extracellular matrix organization and immune regulation. Through cellular communication analysis, we discovered that tumor-associated macrophages (TAMs) and dendritic cells (DCs) exhibited the highest communication intensity with SC6 cluster when acting as the source of ligands. Further investigation revealed critical ligand-receptor pairs, including some integrin family genes functioning as receptors. This interaction suggests that these integrins may engage with ligands secreted by TAMs or DCs within the TME, potentially promoting tumor cell proliferation and migration. Through comprehensive analysis of scRNA-seq data, we elucidated the intricate heterogeneity within HCC, identifying distinct characteristics of various tumor subclones. Our investigation revealed key transcription factors that predominate in specific subclones. Additionally, we uncovered robust intercellular communication between tumor cells and myeloid cells. Qingyang Zhang, Yu-Man Tsui, Vanilla Xin Zhang, Daniel Wai-Hung Ho, Irene Oi-Lin Ng. Single-cell transcriptomic analysis of hepatocellular carcinoma: elucidating intratumoral heterogeneity and immune microenvironment crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6484.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are two distinct types of primary liver cancer (PLC) characterized by a considerable degree of cellular and molecular heterogeneity. We recently developed a web-based cell atlas called LiverSCA with a user-friendly interface, comprehensive functionality, and a complete set of analytical pipelines. In the previous version, LiverSCA includes 40 subjects (35 HCC patients with various etiological risk factors and five healthy controls) with a total cell count of more than 140, 000 cells. It facilitates the exploration of gene expression patterns, cellular composition, and intercellular communication in the liver and PLC tumor microenvironment, aiming to help researchers discover new mechanistic insights into the complex tumor microenvironment of HCC. To further enhance the comprehensiveness of LiverSCA, we have expanded its catalog with additional datasets of different phenotypes/subtypes of liver and PLC, such as intrahepatic cholangiocarcinoma (ICC) and metabolic dysfunction-associated fatty liver disease/steatosis (MASLD/MASH). The current enhanced version of the LiverSCA cell atlas contains six phenotypes (normal, HBV-HCC, HCV-HCC, non-viral HCC, ICC, and MASH), encompassing 63 patients and more than 248, 000 cells. In addition, we have incorporated a comparative visualization method that allows users to simultaneously examine and compare gene expression levels between two different phenotypes. We are committed to the continued development of LiverSCA to narrow the knowledge gap between different research fields and accelerate knowledge transfer in the scientific community. We envision that it will become a valuable resource that supports researchers to conveniently study the cellular and molecular landscape of the liver and PLCs. In addition, as scRNA-seq technology is still rapidly evolving, more features, such as spatial transcriptomics and multimodal omics, will be considered in future updates. Fanhong Zeng, Renwen Long, Tina Suoangbaj, Irene Oi-Lin Ng, Daniel Wai-Hung Ho. LiverSCA 2.0: An enhanced and comprehensive cell atlas of human hepatocellular carcinoma, intrahepatic cholangiocarcinoma and metabolic dysfunction-associated steatotic liver disease/steatohepatitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1088.

A striking characteristic of liver cancer is its extensive heterogeneity, particularly with regard to its varied response to immunotherapy. In this study, we employed multimodal sequencing approaches to explore the various aspects of neoadjuvant nivolumab treatment in liver cancer patients. We used spatially-resolved transcriptomics, single- and bulk-cell transcriptomics, and TCR clonotype analyses to examine the spatiotemporal dynamics of the effects of nivolumab. We observed a significantly higher clonal expansion of T cells in the tumors of patients who responded to the treatment, while lipid accumulation was detected in those of non-responders, likely due to inherent differences in lipid metabolic processes. Furthermore, we found a preferential enrichment of T cells, which was associated with a better drug response. Our results also indicate a functional antagonism between tumor-associated macrophages (TAMs) and CD8 cells and their spatial separation. Notably, we identified a UBASH3B/NR1I2/CEACAM1/HAVCR2 signaling axis, highlighting the intense communication among TAMs, tumor cells, and T-cells that leads to pro-tumorigenic outcomes resulting in poorer nivolumab response. In summary, using integrative multimodal sequencing investigations, combined with the multi-faceted exploration of pre- and post-treatment samples of neoadjuvant nivolumab-treated HCC patients, we identified useful mechanistic determinants of therapeutic response. We also reconstructed the spatiotemporal model that recapitulates the physiological restoration of T cell cytotoxicity by anti-PD1 blockade. Our findings could provide important biomarkers and explain the mechanistic basis differentiating the responders and non-responders.

Single-cell RNA sequencing (scRNA-seq) has revolutionized genomic investigations by enabling the exploration of gene expression heterogeneity at the individual cell level. However, the complexity of scRNA-seq data analysis remains a challenge for many researchers. Here, we present OmniCellX, a browser-based tool designed to simplify and streamline scRNA-seq data analysis while addressing key challenges in accessibility, scalability, and usability. OmniCellX features a Docker-based installation, minimizing technical barriers and ensuring rapid deployment on local machines or clusters. Its dual-mode operation (analysis and visualization) integrates a comprehensive suite of analytical tools for tasks such as preprocessing, dimensionality reduction, clustering, differential expression, functional enrichment, cell–cell communication, and trajectory inference on raw data while enabling alternative interactive and publication-quality visualizations on pre-analyzed data. Supporting multiple input formats and leveraging the memory-efficient data structure for scalability, OmniCellX can efficiently handle datasets spanning millions of cells. The platform emphasizes user flexibility, offering adjustable parameters for real-time fine-tuning, alongside extensive documentation to guide users at even beginner levels. OmniCellX combines an intuitive interface with robust analytical power to perform single-cell data analysis and empower researchers to uncover biological insights with ease. Its scalability and versatility make it a valuable tool for advancing discoveries in cellular heterogeneity and biomedical research.

Background & Aims:HBV integration profiles in the natural history of chronic HBV infection (CHB) have not been well-defined. Hence, we aimed to determine HBV integration profiles across different CHB phases. Methods:We delineated integration profiles from liver biopsies of 55 patients in different CHB phases (3 HBsAg-positive/HBeAg-positive infection; 13 HBsAg-positive/HBeAg-positive hepatitis; 7 HBsAg-positive/HBeAg-negative infection; 12 HBsAg-positive/HBeAg-negative hepatitis; 10 HBsAg seroclearance; 10 occult HBV). Target-capture next-generation sequencing (NovaSeq-6000) was performed, and integrations were characterized on AVID (integrations defined as chimeric fusions in ≥1 soft-clipped reads and ≥2 total reads). Results:HBV integrations were detected in 35 HBsAg-positive (100%), 8 (80%) HBsAg seroclearance, and 7 (70%) occult HBV patients, respectively. There was a stepwise decrease in integration events from HBsAg-positive/HBeAg-positive (median 9.6 [IQR 9.3-10.1] log integrations per liver), HBsAg-positive/HBeAg-negative (8.7 [8.4-9.0] log integrations) and HBsAg-negative patient groups (7.3 [6.8-7.7] log integrations) (p <0.001 for trend). There were no differences in integration frequencies in chronic infection (ALT < the upper limit of normal) and chronic hepatitis (ALT ≥ the upper limit of normal) for either HBeAg-positive or -negative patients (all p >0.05). No significant differences in integration frequencies were noted between HBsAg seroclearance and occult HBV groups (p >0.05). HBV genome integration breakpoints clustered around nucleotide 1800 in all disease phases. Human genome breakpoints were also delineated, and LINC00486 was the most frequently involved human gene in all disease phases. Conclusion:We characterized the HBV DNA integration patterns and genome breakpoints of patients in different CHB disease phases. These findings enhance our understanding of the natural history of CHB. Impact and implications:HBV integration profiles in the natural history of chronic HBV infection have not been well-defined. We utilized next-generation sequencing in a well-characterized cohort of patients with HBV at different disease phases, demonstrating a stepwise decrease in integration events as HBV progressed from HBeAg-positive to HBeAg-negative, and then to HBsAg-negative phases. Human and viral genome breakpoints were also identified. These findings enhance our understanding of the natural history of CHB, and provide insights for antiviral treatment.

Background: Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are two distinct types of primary liver cancer (PLC) characterized by considerable extents of cellular and molecular heterogeneities. We recently developed a web-based cell atlas called LiverSCA that possesses a user-friendly interface and comprehensive functionalities. It facilitates the exploration of gene expression patterns, cellular compositions, and intercellular communication within the microenvironments of liver and PLC tumors. Methods: To further enhance the documentation of data pinpointing different phenotypes/subtypes of liver and PLC, we extended the catalog of LiverSCA with additional datasets, e.g., ICC and metabolic dysfunction-associated steatotic liver disease/steatosis (MASLD/MASH). Results: The current enhanced version of the LiverSCA cell atlas encompasses six phenotypes (normal, HBV-HCC, HCV-HCC, non-viral HCC, ICC, and MASH), 63 patients, and over 248,000 cells. Furthermore, we have incorporated comparative visualization methods that allow users to simultaneously examine and compare gene expression levels between two different phenotypes. Conclusions: We are committed to the continuous development of LiverSCA and envision that it will serve as a valuable resource to support researchers in convenient investigations into the cellular and molecular landscapes of liver and PLC.

Background Programmed cell death protein-1 (PD-1) blockade has shown promising clinical efficacy in hepatocellular carcinoma (HCC), yet the underlying immunological mechanisms governing response and resistance remain unclear. This study aimed to delineate the temporal and spatial dynamics of T-cell clonotypes and their relationship with pathological response in neoadjuvant PD-1 blockade therapy in HCC. By integrating T-cell receptor (TCR) repertoire analysis with transcriptomic profiling, we sought to elucidate the immune landscape alterations associated with treatment outcomes.Methods We analyzed longitudinal, matched tumor and blood samples from our previous clinical trial (NCT05471674), encompassing 114 specimens, including pre-nivolumab tumor biopsies, post-treatment resected tumors, adjacent non-tumorous livers, and peripheral blood collected before, after, and during follow-up from 19 treatment-naïve HCC. TCR clonality, diversity, and transcriptomic signatures were assessed to characterize immune responses.Results Elevated clonality of intratumoral TCR clonotypes (ITCs) post-treatment correlated with increased pathological tumor necrosis, predominantly driven by the most abundant top 1% ITCs. These dominant clonotypes occupied a significantly larger clonal space and demonstrated increased spatial sharing across tumor, non-tumorous liver, and peripheral blood compartments in nivolumab responders (nivo-Rs). Shared clonotypes were positively associated with tumor necrosis extent and cytolytic activity, suggesting active immune engagement. Post-treatment, shared clonotypes exhibited peripheral expansion and greater intratumoral dominance in nivo-Rs, whereas tumor unique intratumoral TCR clonotypes (ITCs) remained prevalent in non-responders (nivo-NRs). These divergent patterns were linked to higher chemokine expression within the tumor microenvironment of nivo-Rs and impaired human leukocyte antigen (HLA) class II antigen presentation in nivo-NRs, indicating distinct immune landscape configurations influencing therapeutic response.Conclusions Our findings demonstrate that the peripheral infiltration and expansion of high-frequency intratumoral T-cell clonotypes are critical drivers of pathological response to neoadjuvant PD-1 blockade in HCC, highlighting potential immune biomarkers and therapeutic targets to enhance immunotherapy efficacy.

BACKGROUND AND AIMS:The development of hepatocellular carcinoma (HCC) is intricately linked to metabolic processes and immune evasion strategies. As an emerging metabolic vulnerability in HCC, the detailed molecular mechanisms of sterol O-acyltransferase 1 (SOAT1) and its role in immune regulation remain unclear. This study aimed to elucidate the mechanism involved and evaluate the potential of SOAT1 as a therapeutic target. APPROACH AND RESULTS:We explored the role of SOAT1 using genetical and pharmaceutical inhibition in cell lines, patient-derived organoids, and mouse models. Co-culture systems, flow cytometry, and immunohistochemistry were employed to assess tumor-immune interactions. Multi-omics were performed to elucidate the underlying molecular mechanisms. The efficacy of inhibiting SOAT1 alone and in combination with anti-PD1 therapy in vivo was tested. SOAT1 was significantly upregulated in HCC tumors and was associated with increased tumorigenicity and immune-evasive characteristics. SOAT1 deficiency disrupted lipid homeostasis, leading to the accumulation of saturated fatty acids, reactive oxygen species, and endoplasmic reticulum stress, followed by NF-κB activation. This signaling triggered the production of pro-inflammatory cytokines, adhesion molecules, and the recruitment of CD11c + antigen-presenting cells and cytotoxic CD8 + T cells into tumors. Moreover, SOAT1 knockout reduced tumor burden, and the combination of SOAT1 inhibition with PD-1 blockade exhibited synergistic antitumor effects. CONCLUSIONS:SOAT1 functions as both a metabolic vulnerability and an immune regulator in HCC. Its inhibition disrupts tumor-promoting metabolic processes while enhancing immune activation, presenting it as a promising therapeutic target. Combining SOAT1 inhibition with PD-1 blockade holds potential for improving outcomes in HCC immunotherapy.

Germline mutations of the deubiquitinase BRCA1-associated protein 1 (BAP1) lead to the “BAP1 cancer syndrome” characterized by development of cancers. However, the role of BAP1 in hepatocellular carcinoma (HCC) is unclear. We found that BAP1 was upregulated at mRNA level in human HCCs and significantly correlated with a more aggressive tumour behaviour. Intriguingly, we observed cytoplasmic but no or minimal nuclear BAP1 in human HCC samples by immunohistochemistry. We observed that, while BAP1 protein was found mainly in the cytoplasm and less in the nuclei of HCC cell lines, BAP1 expression was predominantly nuclear in HepG2 cells, by cell fractionation and immunofluorescence analyses. Functionally, in the orthotopic liver injection mouse model, silencing the BAP1 predominant nuclear expression of HepG2 cells promoted intrahepatic tumor metastasis, with more frequent tumor microsatellite formation and venous invasion. With transcriptomic profiling, we identified RHOJ amongst the downregulated targets in HepG2 cells upon BAP1 knockdown. Subsequent overexpression of RHOJ suppressed cell migration in HCC cells, suggesting that BAP1 might upregulate RHOJ resulting in reduced cell migratory ability of HCC cells. Furthermore, we identified two transcription factors, CTCF and NRF1, which activated BAP1 transcription by binding to BAP1 promoter region. On the other hand, we uncovered that O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) physically bound to BAP1 in the nucleus, resulting in diminished stability of the nuclear BAP1. Intriguingly, OGT transcription was upregulated and was also under the control of CTCF and NRF1 in human HCC, acting as a negative regulator of BAP1. To summarize, this study uncovered the underlying mechanisms of the regulation of BAP1 and that loss of the nuclear localization of BAP1 protein contributed to enhanced cell migration in vitro and more aggressive tumor behavior in human HCCs.

The evident concordance between tissue and liquid biopsies in hepatocellular carcinoma (HCC), a prevalent and lethal cancer worldwide, has positioned liquid biopsy as a valuable non-invasive tool for the clinical management of HCC. Among its analytes, circulating cell-free DNA (cfDNA) has recently gained significant attention as a key biomarker for HCC. This review provides an overview of recent advancements in the use of cfDNA for the detection and diagnosis, treatment decision-making, and recurrence surveillance of HCC. The various merits of cfDNA underscore its strong potential for clinical integration in HCC. However, there is also an emerging imperative that arises from the varying cfDNA-related methodologies, which demonstrate disparate outcomes across studies, emphasizing the importance of systematic evaluation and standardization to ensure consistent and equitable patient care.

BACKGROUND AND AIMS:HCC is an aggressive cancer with a poor clinical outcome. Understanding the mechanisms that drive tumor initiation is important for improving treatment strategy. This study aimed to identify functional cell membrane proteins that promote HCC tumor initiation. APPROACH AND RESULTS:Tailor-made siRNA library screening was performed for all membrane protein-encoding genes that are upregulated in human HCC (n = 134), with sphere formation as a surrogate readout for tumor initiation. Upon confirmation of membranous localization by immunofluorescence and tumor initiation ability by limiting dilution assay in vivo, LanC-like protein-1 (LANCL1) was selected for further characterization. LANCL1 suppressed intracellular reactive oxygen species (ROS) and promoted tumorigenicity both in vitro and in vivo. Mechanistically, with mass spectrometry, FAM49B was identified as a downstream binding partner of LANCL1. LANCL1 stabilized FAM49B by blocking the interaction of FAM49B with the specific E3 ubiquitin ligase TRIM21, thus protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppressed the Rac1-NADPH oxidase-driven ROS production, but this suppression of ROS was independent of the glutathione transferase function of LANCL1. Clinically, HCCs with high co-expression of LANCL1 and FAM49B were associated with more advanced tumor stage, poorer overall survival, and disease-free survival. In addition, anti-LANCL1 antibodies targeting the extracellular N-terminal domain were able to suppress the self-renewal ability, as demonstrated by the sphere formation ability of HCC cells. CONCLUSIONS:Our data showed that LANCL1 is a cell surface protein and a key contributor to HCC initiation. Targeting the LANCL1-FAM49B-Rac1-NADPH oxidase-ROS signaling axis may be a promising therapeutic strategy for HCC.

ObjectiveFat mass and obesity-associated protein (FTO), an eraser ofN6-methyadenosine (m6A), plays oncogenic roles in various cancers. However, its role in hepatocellular carcinoma (HCC) is unclear. Furthermore, small extracellular vesicles (sEVs, or exosomes) are critical mediators of tumourigenesis and metastasis, but the relationship between FTO-mediated m6A modification and sEVs in HCC is unknown.DesignThe functions and mechanisms of FTO and glycoprotein non-metastatic melanoma protein B (GPNMB) in HCC progression were investigated in vitro and in vivo. Neutralising antibody of syndecan-4 (SDC4) was used to assess the significance of sEV-GPNMB. FTO inhibitor CS2 was used to examine the effects on anti-PD-1 and sorafenib treatment.ResultsFTO expression was upregulated in patient HCC tumours. Functionally, FTO promoted HCC cell proliferation, migration and invasion in vitro, and tumour growth and metastasis in vivo. FTO knockdown enhanced the activation and recruitment of tumour-infiltrating CD8+T cells. Furthermore, we identified GPNMB to be a downstream target of FTO, which reduced the m6A abundance of GPNMB, hence, stabilising it from degradation by YTHN6-methyladenosine RNA binding protein F2. Of note, GPNMB was packaged into sEVs derived from HCC cells and bound to the surface receptor SDC4 of CD8+T cells, resulting in the inhibition of CD8+T cell activation. A potential FTO inhibitor, CS2, suppresses the oncogenic functions of HCC cells and enhances the sensitivity of anti-PD-1 and sorafenib treatment.ConclusionTargeting the FTO/m6A/GPNMB axis could significantly suppress tumour growth and metastasis, and enhance immune activation, highlighting the potential of targeting FTO signalling with effective inhibitors for HCC therapy.

Abstract Background: Hepatocellular carcinoma (HCC) is prevalent globally and the third leading cause of cancer-related mortality. N-myc downstream regulated 1 (NDRG1) was found to be tumor promoting in some cancers while suppressive in others. The expression of NDRG1 is induced by stress signals such as DNA damage and hypoxia. In HCC, it has been found to promote proliferation and metastasis. Contradictory results, however, were also suggested on the functions of NDRG1. Methods: Our own HCC cohort and multiple publicly available databases were examined for NDRG1 expression and clinicopathologic correlation. NDRG1 expression under hypoxic conditions was investigated. Various functional assays were performed for NDRG1-knockdown HCC cell lines Huh-7 and PLC/PRF/5. Results: With RT-qPCR analysis on our 102 pairs of patients’ HCC tumor and corresponding non-tumorous liver samples, we found NDRG1 to be upregulated in HCC tumors. Similarly, RNA-sequencing analysis comparing 41 pairs of samples confirmed NDRG1 enrichment in HCC tumors. The upregulation was significantly and positively correlated with the absence of tumor encapsulation and cellular differentiation. These were further supported by TCGA and ICGC databases, in which upregulation of NDRG1 was significantly correlated with poor HCC patient overall survival. Interestingly, in the TCGA database, only 17% of the HCC cases were inferred to have copy number amplification of NDRG1 gene while NDRG1 mutations were rarely found. As hypoxia is a major tumor microenvironmental factor in HCC, we investigated NDRG1’s expression level in HCC cells under hypoxic or normoxic conditions and found significant increase in NDRG1 expression under hypoxic environment. We further found that NDRG1 knockdown inhibited cell proliferation, self-renewal ability, and migration. RT-qPCR analysis found lower LGR5 and SOX2 expression upon NDRG1 knockdown. This is in line with the positive correlation between NDRG1 and various cancer stemness-related gene expression, including LGR5, CD24, MYC, SOX2, and NOTCH1, in TCGA database. Lastly, in the resected HCC tumors on which spatial transcriptomics using Visium 10X and single-cell RNA-sequencing using Chromium 10X were performed, we found NDRG1 enrichment in both tumors cells and cancer associated fibroblasts (CAFs). Conclusion: NDRG1 was found to be significantly upregulated in HCC cells in our in-house cohort and publicly available databases. NDRG1 consistently promotes various oncogenic features of HCC cells, including proliferation, self-renewal ability, and migration. In addition, NDRG1 was found to be enriched in both the HCC cells and CAFs. Citation Format: Jingyi Lu, Lu Tian, Yu-Man Tsui, Xia Wong, Qingyang Zhang, Daniel Wai-Hung Ho, Irene Oi-Lin Ng. The roles of NDRG1 in HCC progression and its clinical significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6982.

We developed a cell atlas named LiverSCA on human liver cancer single-cell RNA sequencing data. It has a user-friendly web interface and comprehensive functionalities aiming to help researchers to make easy access to cellular and molecular landscapes of the tumor microenvironment in liver cancer. LiverSCA includes a complete analytical pipeline that allow mechanistic exploration on a wide variety of functionalities, such as cell clustering, cell annotation, identification of differentially expressed genes, functional enrichment analysis, analysis of cellular crosstalk, and pseudo-time trajectory analysis. Notably, our intuitive web interface allows users, particularly wet-lab researchers, to easily explore and undertake data discovery, without the need to handle any of the raw data.

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a common malignancy worldwide. About half of all new liver cancers worldwide each year occur in China due to a high prevalence of hepatitis B virus (HBV) infection. HBV DNA integrates into the human genome, disrupting the endogenous tumor suppressors/regulatory genes or enhancing the activity of proto-oncogenes. It would be useful to examine the different NGS-based databases to provide a more unbiased and comprehensive survey of HBV integration. We aimed to take advantage of publicly available datasets of different regional cohorts to determine the disparity landscapes of integration events among sample cohorts, tissue types, chromosomal positions, individual host and viral genes, and genic locations. By comparing HCC tumor with non-tumorous liver, landscape of HBV integration was delineated at gene-independent and gene-dependent manners. Moreover, we performed mechanistic investigations on how HBV-TERT integration led to TERT activation and derived a score to predict patients’ prognostication according to their clonal disparity landscape of HBV integration. We revealed a global geographical disparity of HBV integration that the landscape of HBV integration between HCC and non-tumorous liver varied in regional cohorts, suggesting the different degrees of clonal enrichment. In the Mainland China, Hong Kong and Mongolia cohorts, HCC tumors had higher numbers of events than the non-tumorous livers, whereas in the French and US cohorts, the reverse was true. Moreover, most HBV integrations were positionally enriched at the telomeres and centromeres, and this highlighted the novel co-involvement of HBV integration at these regions, which likely introduces greater genomic instability. Furthermore, we constructed a large meta-cohort of multiple ethnicities to refine the landscape of HBV integration. This enabled the identification of events at key HCC-related genes (e.g., TERT, KMT2B, and CCNA2) and gene families (e.g., cyclin, cadherin, and contactin). As TERT is the most frequently integrated gene by HBV, we further investigated the mechanistic modulation of TERT transcription activation by combining and analyzing our in-house sample cohort with a previous report and luciferase reporter assays. This revealed the positive concurrent influence by both the orientation and relative distance of HBV integration (closer to transcription start site) from the TERT gene. Additionally, we observed clonal disparity of HBV integration among patients and the higher level of the clonal disparity score correlated with poor prognostication of HCC patients. Our study uncovered the different levels of clonal enrichment of HBV integration and identified mechanistic insights and prognostic biomarker. This strengthens our understanding of HBV-associated hepatocarcinogenesis. Citation Format: Xueying Lyu, Karen Man-Fong Sze, Joyce Man-Fong Lee, Abdullah Husain, Irene Oi-Lin Ng, Daniel Wai-Hung Ho. Disparity landscapes of HBV integration in hepatocellular carcinoma: Mechanistic characterization and functional implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1481.