hepatic carcinoma and immune and resistant

BACKGROUND Hepatocellular carcinoma (HCC) is a prevailing cancer affecting human health. M2 macrophages are essential in mediating immune responses in tumors. This study investigated the action of M2 macrophages in immune escape of HCC. METHODS Mitotic spindle positioning (MISP), IQ motif containing GTPase activating protein 1 (IQGAP1) and programmed cell death-1 (PD-L1) levels in primary HCC/tumor-adjacent tissues were determined by Western blot, followed by correlation analysis. M2 macrophage and CD3+CD8+T cell percentages were estimated by flow cytometry. Hep3B and HepG2 cells were treated with M2 macrophage conditioned medium (M2-CM) and M2 macrophage-derived extracellular vesicles (M2-EVs) and/or co-cultured with CD8+T cells, followed by assessment of cell viability and apoptosis. TNF-α and INF-γ levels were measured by ELISA. MISP and IQGAP1 overexpression plasmids were transfected into HCC cells to explore their role in immune escape. The interactions among MISP, IQGAP1, STAT3, and PD-L1 were analyzed by co-immunoprecipitation. The mechanism of M2-EVs in HCC immune escape was verified in nude mice. RESULTS MISP/IQGAP1/PD-L1 were upregulated in HCC tissues. MISP negatively-correlated with IQGAP1/PD-L1 and IQGAP1 positively-correlated with PD-L1. M2 macrophages were reduced but CD8+T cells were increased in HCC tissues with high MISP expression. M2-CM or M2-EVs inhibited the killing ability of CD8+T cells, increased HCC cell viability, impeded HCC cell apoptosis, induced CD8+T cell apoptosis, downregulated TNF-α and INF-γ, and upregulated PD-L1. M2-EVs facilitated HCC cell immune escape by potentiating IQGAP1 nuclear translocation and activating STAT3 phosphorylation through MISP downregulation. In vivo experiments further verified the action of M2-EVs through MISP. CONCLUSION M2-EVs promote HCC cell immune escape by upregulating PD-L1 through the MISP/IQGAP1/STAT3 axis.

Dysregulated metabolism of immune cells in the tumor microenvironment leads to immune evasion and tumor progression. As a major cell component in the tumor, the metabolic reprogramming of tumor-associated macrophages (TAMs) creates an immunosuppressive microenvironment in hepatocellular carcinoma (HCC). Our study found that sphingolipid (particularly, sphingosine-1-phosphate or S1P) levels are a clinical indicator for prognosis and immunotherapy response in patients with HCC. S1P primarily derived from TAMs, where NIMA-related kinase 2 (NEK2) plays a key role in controlling the activity of serine palmitoyl-CoA transferase, a rate-limiting enzyme in S1P biosynthesis. The S1P produced by NEK2hi TAMs promotes hepatic tumor progression and confers immunotherapy resistance. Targeting S1P synthesis with a NEK2 inhibitor or S1P antagonist disrupted the immunosuppressive function of macrophages, shifted regulatory T cells (Tregs) to TH17 cells, and increased the number and activity of tumor-infiltrating T effectors, thereby enhancing antitumor efficacy in synergy with immune checkpoint blockade therapy.

Durable responses to immune checkpoint blockade (ICB) in hepatocellular carcinoma (HCC) are limited to a minority of patients, yet reliable biomarkers are still lacking. Inflammatory cytokines such as interleukin-8 (IL-8) are associated with HCC progression, and IL-8 is known as the chemoattractant for immunosuppressive myeloid cells. Therefore, we aim to elucidate the ICB resistance mechanisms mediated by the activation of the IL-8/CXCR2 pathway. Single-cell RNA sequencing (scRNA-seq) was performed in advanced HCC patients with baseline and on-treatment biopsy after pembrolizumab in a phase 2 clinical trial cohort. Our data revealed that IL-8 and its receptor, CXCR2, mainly derived from immunosuppressive myeloid-derived suppressor cells (MDSCs). In particular, the high circulating IL-8 level was strongly associated with poor ICB response. This myeloid IL-8/CXCR2 pathway was further elucidated in our ICB-resistant orthotopic mouse model using AZD5069, a clinically available CXCR2 antagonist. Suppression of the IL-8/CXCR2 pathway significantly abrogated MDSC trafficking and immunosuppressive activity, which sensitized the anti-PD-L1 blockade to reduce tumor growth and prolong survival. The association between myeloid IL-8 and ICB therapeutic outcomes also extended to multiple cancer types. Collectively, our study not only suggests a potential non-invasive biomarker for patient stratification and monitoring of ICB response but it also provides a proof of concept for combinational immunotherapy to benefit patients who are non-responsive to ICB monotherapy.

Despite the revolutionary progress in cancer immunotherapy, only a minority of hepatocellular carcinoma (HCC) patients respond to immune checkpoint inhibitors (ICIs). In this study, we found that the oncogenic circular RNA Circ-CDYL in HCC influences the efficacy of immunotherapy and the stemness characteristics of HCC cells by interacting with the hornerin (HRNR) protein. The degraded anti-PD-L1 immunotherapy responses induced by Circ-CDYL and HRNR were confirmed by peripheral blood mononuclear cells (PBMCs) killing assays in HCC cells, patient-derived organoids, and humanized immune system mouse models. Furthermore, Circ-CDYL interference reversed the cytotoxicity and proliferation of CD8+ T cells, resulting in ameliorated immune evasion in tumor spheroids upon anti-PD-L1 treatment. Mechanistically, Circ-CDYL upregulated HRNR expression by stabilizing the HRNR protein through the prevention of its degradation by the E3 ubiquitin ligase synoviolin 1 (SYVN1), which in sequence promoted the phosphorylation of the mTORC1 and p70S6K substrate. The abnormally activated mTORC1-p70S6K signaling increases the stemness of HCC cells and upregulates PD-L1 expression, which may attenuate anti-PD-L1 therapy efficacy via PD-L1+ exosomes. Our study revealed the mechanism by which Circ-CDYL and HRNR regulate the sensitivity of HCC to anti-PD-L1 therapy, and the findings have potential implications for patient stratification and clinical decision-making in HCC immunotherapy.

Background Hepatocellular carcinoma (HCC) is a prevalent malignancy and the third leading cause of cancer-related mortality worldwide. Immune checkpoint inhibitors (ICIs) have emerged as first-line therapies for advanced HCC, substantially improving clinical outcomes. However, resistance to ICIs remains a major therapeutic challenge. Lactylation, a recently identified post-translational modification, has been implicated in tumor progression, although its role in ICIs resistance in HCC remains unclear. Methods Cytotoxicity assays, flow cytometry and orthotopic HCC mouse model were used to evaluate the effects of lactylation in remodeling the immune microenvironment. Chromatin immunoprecipitation sequencing and RNA sequencing were employed to identify lactylation-regulated gene profiles. Programmed cell death-ligand 1 (PD-L1) protein degradation was assayed by cycloheximide-chase analysis and ubiquitination assay. Interactions between major vault protein (MVP) and β-transducin repeat-containing protein (β-TrCP) were analyzed by co-immunoprecipitation experiments. Site-directed mutagenesis and truncation mutants were designed to determine binding sites of MVP-β-TrCP complex. Results Elevated lactylation correlates with poor prognosis and ICIs resistance in patients with HCC. Inhibition of lactylation enhances CD8+ T-cell infiltration and cytokine production. Multiomics analyses identify MVP as a lactylation-regulated factor that suppresses CD8+ T cell-mediated antitumor immunity. Elevated MVP expression is associated with resistance to checkpoint blockade therapy. Mechanistic studies reveal that histone lactylation-induced MVP upregulation stabilizes PD-L1 by preventing β-TrCP-mediated proteasomal degradation. Pharmacological inhibition of lactylation restores ICIs sensitivity in orthotopic HCC mouse models. Conclusions Our findings demonstrate that histone lactylation promotes ICIs resistance via MVP-dependent PD-L1 stabilization. Therefore, targeting lactylation in combination with programmed cell death protein-1/PD-L1 blockade offers a promising strategy to overcome immunotherapy resistance in HCC.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies and epigenetic modifiers play a key role in HCC progression. Histone H3 trimethylation at lysine‐36 (H3K36me3) determines deposition of mRNA de novo N 6‐methyladenosine (m6A) modification. However, it remains largely elusive how long noncoding RNAs (lncRNAs) are selected for proper m6A methylation. The current study provides evidence for L1CAM‐AS1 as a novel H3K36me3‐guided, m6A‐modified lncRNA through integration of genome‐wide H3K36me3 profiles and transcriptome‐wide m6A profiles of HCC cells. The crucial m6A‐modification site in L1CAM‐AS1 exon 3 is recognized by IGF2BP1, leading to increased lncRNA stability. Oncogenic L1CAM‐AS1 shows higher expression in HCC tissues than in normal specimens, and its elevated expression is associated with shorten patient survival. Mechanistically, L1CAM‐AS1 interrupts binding of RAN to the E3 ligase OSTM1, suppresses RAN ubiquitination at Lys152 and Lys167, stabilizes RAN protein, enhances nuclear import of RELA (p65), and activates the NF‐κB signaling, leading to up‐regulated CCL2 expression. L1CAM‐AS1‐induced CCL2 secretion from HCC cells enhances M2 polarization of tumor‐associated macrophages (TAMs). Meanwhile, immunosuppressive M2 macrophages‐released CCL5 augments RELA nuclear import in HCC cells, which in turn activates the NF‐κB signaling. Given the critical role of macrophages in anti‐tumor immunity, inhibition of the L1CAM‐AS1‐RAN axis promotes the efficacy of PD‐1 blockade via TAM reprogramming in HCC mouse models. In conclusion, this study provides novel insights into how epigenetic alternations are involved in antitumor immunity modulation and illustrates promising potentials of L1CAM‐AS1 in immune‐checkpoint inhibitor treatments for HCC.

Summary Immunotherapy has made remarkable achievements in various cancers, but response rates in hepatocellular carcinoma (HCC) remain highly variable. Understanding mechanisms behind this heterogeneity and identifying responsive patients are urgent clinical challenges. In this study, the metagenomic analysis of 65 HCC patients reveals distinct gut microbiota profiles distinguishing responders (Rs) from non-responders (NRs). These findings are further validated through fecal microbiota transplantation (FMT) in mouse models. Notably, Phocaeicola vulgatus (P. vulgatus) is enriched in NRs and diminishes anti-PD-1 efficacy in both syngeneic and orthotopic tumor models. Mechanistically, P. vulgatus suppresses the production of indoleacetic acid (IAA), thereby weakening interferon (IFN)-γ+ and granzyme B (GzmB)+CD8+ T cells and impairing the antitumor immune response. Furthermore, supplementation with IAA restores CD8+ T cell cytotoxicity and counteracts the immune-suppressive effects of P. vulgatus. Our findings establish a causal relationship between P. vulgatus and anti-PD-1 resistance in HCC, highlighting IAA as a potential therapeutic target to enhance immunotherapy outcomes.

Hepatocellular carcinoma (HCC) is a significant global health concern, ranking as the sixth leading cause of cancer-related deaths worldwide. While immune-checkpoint blockade (ICB) has revolutionized cancer treatment, its effectiveness is limited by the immunosuppressive tumor microenvironment (TME) and the diverse etiologies of HCC, resulting in response rates below 30%. We have previously identified key transcriptional and epigenetic regulators that promote immune exclusion and myeloid-mediated immunosuppression underlying ICB resistance in preclinical models (Gut 2018, 2020, 2023, Sci Transl Med 2021). Identifying the central nodes of TME landscapes from ICB-resistant patients is instrumental for developing precise immunotherapy strategies and improving clinical outcomes. We performed single-cell RNA sequencing (scRNA-seq), single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), and spatial transcriptomics to investigate the TME of HCC patients resistant to ICBs (NCT05873244) and treatment naïve-patients as controls. Our multi-omics analysis revealed a subcluster of tumor-associated macrophages (TAMs) that express high levels of lipid-associated genes. In our syngeneic and orthotopic ICB-resistant mouse models, which recapitulate human immune-excluded HCCs, we also found a subset of lipid-associated macrophages (LAMs) is enriched in concordance with the clinical data. Bioinformatics analysis of the open chromatin regions uncovered a transcription factor (TF) that is significantly up-regulated in the TAMs in ICB-resistant HCCs compared to the treatment-naïve tumors. Moreover, chromatin immunoprecipitation-sequencing showed that this TF occupancy in THP-1 cells was enriched in phagocytosis. Our study provides insights into how this TF drives macrophage lipid phagocytosis to promote ICB resistance of HCC. This investigation elucidates the underlying epigenetic and transcriptional changes and identifies potential therapeutic targets for ICB resistance. Our findings may offer valuable insights into the molecular dysregulation of TAM within the immunosuppressive myeloid landscape, forming the basis for developing new strategies against the immunometabolic axis to avert ICB resistance. This study is supported by the General Research Fund (14120621 and 14119023) and the Li Ka Shing Foundation. Xiaohang Long, Stephen Lam Chan, Siyuan Huang, Joaquim Si Long Vong, Xiaoyu Zhan, Yalin Tu, Joseph, JY Sung, Alfred Sze Lok Cheng. Deciphering the regulation and function of lipid associated macrophages in immunotherapy resistance of hepatocellular carcinoma [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 2186.

Gut microbiota has been widely reported to influence anti-PD-1 efficacy in melanoma and lung cancer, in both clinical specimens and animal models. However, the role of gut microbiota in hepatocellular carcinoma (HCC) remains less explored, with limited understanding of the underlying mechanisms. Method: Advanced HCC patients treated with anti-PD-1 with/without anti-CTLA-4 were prospectively enrolled, and categorized into responders and non-responders groups based on their radiological response after 6 months of immunotherapy according to modified Response Evaluation Criteria for Solid Tumors (mRECIST) guideline. The fecal metagenomics and plasma metabolome data were integrated to reveal the profiles of gut microbiota. Meanwhile, fecal microbiota transplantation (FMT), single strain and metabolite intervention animal models, as well as in vitro CD8+T cell culture were employed to study its mechanism of modulating immunotherapy efficacy in HCC. Results: In the metagenomic analysis of 65 patients, the responders and non-responders exhibited distinct gut microbiota profiles, which were replicated in mice via FMT. Lefse analysis revealed a higher abundance of Phocaeicola vulgatus (P. vulgatus) in non-responders compared to responders. Moreover, patients with higher P. vulgatus had a mean progression free survival (PFS) of 11.1 months and overall survival (OS) of 22.9 months, which were significantly shorter than those with lower P. vulgatus (PFS: 30.8 months, p = 0.047; OS: 63.2 months, p = 0.037). Notably, P. vulgatus enriched in non-responders restrained anti-PD-1 efficacy in syngeneic and orthotopic tumor models and inhibited the CD8+T cell cytotoxicity, evidenced by decreased IFN-γ+ and GzmB+ populations. Mechanistically, P. vulgatus reduced gut microbiota-derived indoleacetic acid (IAA), weakening CD8+ T cell effector functions and impairing immune response. Supplementation with IAA restored CD8+ T cell cytotoxicity and counteracted the immune-suppressive effects of P. vulgatus. Conclusion: P. vulgatus impaired anti-PD-1 efficacy in HCC via reducing IAA production, which in turn diminishes the effector functions of CD8+ T cells. This study establishes a causal link between P. vulgatus and anti-PD-1 resistance in HCC, highlighting gut microbiota-derived IAA as a potential therapeutic target. Caining Zhao, Shanshan Li, Thomas Yau, Wenqi Chen, Ren Ji, Xinyuan Guan, Fengming Spring Kong. Phocaeicola vulgatus mediates immunotherapy resistance in hepatocellular carcinoma via reducing indoleacetic acid production [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 2208.

Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related deaths globally. Immune checkpoint blockade (ICB) therapy has shown promise but has yielded limited responses in HCC patients. Combining anti-vascular endothelial growth factor (VEGF) with ICB enhances therapeutic efficacy, however, a deeper understanding of endothelial cell (EC) remodeling is crucial for further improving ICB effectiveness. Our integrated analyses identified a transcription factor-4 (TCF4)-driven transcriptional program, coupled with elevated BRD4 expression, associated with EC remodeling in ICB-resistant HCC. Emerging studies have shown that TCF4 functions depend on BRD4 through recruitment to a specific enhancer region, and BRD4 inhibitors can reduce TCF4 expression. We aim to delineate the microenvironmental cue and transcriptional network that direct EC remodeling for tumor immunosuppression and their role in promoting ICB resistance. Single-cell transcriptomic profiling was performed on samples from a Phase II trial of pembrolizumab (anti-PD-1) in hepatitis B-related HCC patients (NCT03419481). An ICB-resistant HCC mouse model generated via iterative selection was used for validation. The BRD4 inhibitor AZD5153, administered in oral and nanoparticle formulations, was used to suppress BRD4 and TCF4 expression in systemic and endothelial cells. Quantitative immunofluorescence assessed ECs and detected TCF4 and BRD4 levels. scRNA-seq analysis revealed that tumor vessels from ICB non-responders were enriched with disorganized macrovascular-like endothelial cell (MaVEC), which further increased upon ICB. RNA velocity analysis predicted that MaVEC in non-responders likely originate from liver sinusoidal endothelial cell (LEC), indicating LEC-to-MaVEC trans-differentiation during ICB resistance acquisition. TCF4 was identified as the top regulon in MaVEC, with increased activity along the LEC-to-MaVEC trajectory and association with BRD4. Using AZD5153, we observed that TCF4 can be suppressed in ECs, which caused a significant intra-tumoral reversal from MaVEC to LEC phenotypes, resulting in tumor cell apoptosis and regression. Additionally, nanoparticle-delivered EC-targeted AZD5153 achieved similar tumor-suppressive effects at a lower dosage. Our study reveals that the dynamic trans-differentiation from LEC to MaVEC is associated with ICB resistance in HCC, which can be reversed by BRD4 inhibition and possibly mediated by TCF4 transcriptional regulation, targeting their expression in ECs showed improved therapeutic outcomes. Hepatocellular carcinoma, Immune-checkpoint blockade, Endothelial cells, Transcriptional network. Funded by General Research Fund (14118424) & AstraZeneca Pre-clinical Oncology Research Programme & Li Ka Shing Foundation. Baoyi Yin, Shufen Chen, Zhewen Xiong, Patrick Wong, Zhuo Yu, Stephan Chan, Alfred Sze-Lok Cheng. The role of endothelial cell remodeling in driving immunotherapy resistance of hepatocellular carcinoma [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 3852.

Chromatin architecture is frequently altered in cancers, including one of the most prevalent cancers, hepatocellular carcinoma (HCC). Emerging evidence suggests that liquid-liquid phase separation plays a crucial role in shaping chromatin architecture and regulating gene transcription. Immune checkpoint blockade (ICB) therapy has become a new standard of care for HCC. However, the majority of HCC patients exhibit either primary or adaptive resistance to this immunotherapy, highlighting the urgent need for effective combination therapies. This study aims to uncover the differences in chromatin architecture between ICB-sensitive and resistant HCC tumors and dissect the underlying regulatory mechanisms. Hi-C, ATAC-seq, CUT&Tag, and RNA-seq were performed on RIL175 cells from a well-established adaptive ICB-resistant mouse model to generate the multi-omics dataset. To explore the functional role of F1 in ICB resistance of HCC, RIL175-PD1-R cells with F1 knockdown were constructed using the lenti-virus infection. To explore the phase separation ability of F1, F1 protein tagged with mCherry was expressed in E. coli and purified. Droplet formation assay and fluorescence recovery after photobleaching (FRAP) assay were applied. Three-dimensional genomic analysis by Hi-C reveals comparable chromatin interactions between ICB-sensitive and resistant cells at both compartment and topologically associated domain (TAD) levels but an increase in loops within ICB-resistant cells. In accord with this, ATAC-seq results indicate upregulated chromatin accessibility in a subset of chromatin regions in ICB-resistant cells compared to ICB-sensitive cells. Motif analysis identifies F1 as the top transcription factor enriched for the open chromatin regions. CUT&Tag shows that F1 binds to those accessible regions and alters chromatin looping in ICB-resistant cells. Modulating F1 expression in HCC tumor cells significantly increases the therapeutic efficacy of ICB treatment. We also found that F1 undergoes phase separation, which may mediate the changes in chromatin architecture between ICB-sensitive and resistant cells. F1 undergoes phase separation in HCC, particularly in ICB resistant cells. This characteristic of F1 may shape specific three-dimensional chromatin architecture in HCC cells, contributing to their resistance to ICB treatment. Targeting F1 condensates may provide a novel therapeutic strategy to reprogram the genome architecture to improve ICB efficacy for patients with HCC. chromatin architecture, phase separation, hepatocellular carcinoma, immune checkpoint blockade (ICB) therapies This work is supported by NSFC/RGC (460795048) & Li Ka Shing Foundation. Siyun CHEN, Yingnan LIN, Chengpeng Zhong, Yalin TU, Haoran WU, Yiling ZHANG, Jia WANG, Alfred Sze Lok CHENG. Role of transcriptional regulation in chromatin architecture and immunotherapy resistance of hepatocellular carcinoma [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 2751.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality globally. Previous studies have reported that oral cancer overexpression 1 (ORAOV1) is overexpressed in HCC and correlated with poor prognosis, yet its molecular mechanisms remain incompletely understood. In this study, ORAOV1 overexpression was confirmed in HCC tissues via tissue microarray analysis and functionally linked to tumor cell proliferation through a positive correlation with Ki-67 expression in the human HCC cell line MHCC-97L. Bioinformatics analyses using The Cancer Genome Atlas (TCGA) and three Gene Expression Omnibus (GEO) HCC datasets further supported these findings. Multiple mechanisms appear to drive ORAOV1 upregulation, including promoter hypomethylation, amplification of the 11q13 region, and a putative ceRNA network involving AC005332.1, AC012615.1, and hsa-miR-100-5p. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses implicated ORAOV1 in various cellular processes, such as abnormal membrane channel function, extracellular matrix–receptor interactions, IL-17 signaling, and peroxisome proliferator-activated receptor (PPAR) signaling. Co-expression analysis identified significant associations between ORAOV1 and the oncogenes TPCN2 and CCND1. Additionally, ORAOV1 expression correlated with enhanced infiltration of immunosuppressive cells, including regulatory T cells, myeloid-derived suppressor cells, and cancer-associated fibroblasts, as well as upregulation of immune checkpoint markers (PD-1, PD-L1, and CTLA-4). These results indicate that ORAOV1 may modulate the immunosuppressive tumor microenvironment and contribute to resistance against immunotherapy, highlighting its potential as a therapeutic target in HCC.

No abstract available

Immunotherapy resistance conducts the main reason for failure of PD-1-based immune checkpoint inhibitors (ICIs) in patients with hepatocellular carcinoma (HCC). This study aims to clarify the mechanism of nature kill cells (NK) depletion in immunotherapy resistance of HCC. Cancerous /paracancerous tissues and peripheral blood (PB) of 55 HCC patients were collected and grouped according to differentiation degree, FCM, IHC and lymphocyte culture drug intervention experiments were used to determine NK cell depletion degree. Furthermore, a mouse model of HCC in situ was constructed and divided into different groups according to intervention measures of ICIs. Immunofluorescence thermography was used to observe changes in tumor burden. NK cells in cancerous tissues significantly up-regulated TIGIT expression (P < 0.001). Intervention experiments revealed that TIGIT and PD-1 expression decreased gradually with increased PD-1 inhibitor dose in moderately-highly differentiated patients (P < 0.05). Animal experiment showed that tumors proliferation in experimental group was inhibited after PD-1 blockage, WB indicated that ICIs decreased TIGIT and PVRL1 protein expression while increased CD226 and PVRL3 protein expression. We concluded that TIGIT+NK cells competitively bind to PVR with CD226 and promote NK cell depletion. Anti-PD-1 decreases PVRL1 expression through PD-1/PD-L1 pathway, reducing the PVR/TIGIT inhibitory signal pathway, and enhancing function of PVR/CD226 activation signal.

The cGAS‐STING signaling pathway effectively activates antitumor immune responses and holds promise for overcoming drug resistance in hepatocellular carcinoma (HCC) immunotherapy. However, achieving specific activation of this pathway in HCC remains challenging. Here, it is introduced a single‐metal‐doped nanoplatform, ZMRPF, which leverages ferroptosis‐induced mitochondrial DNA (mtDNA) release to stimulate cGAS‐STING‐mediated immune activation. ZMRPF initiates HCC ferroptosis by inducing high levels of lipid reactive oxygen species, leading to mitochondrial stress and the release of endogenous mtDNA. This mtDNA synergistically activates the cGAS‐STING pathway, enhanced by immunoactivating Mn2⁺ ions released from ZMRPF. Concurrently, the tumor antigens released during ferroptosis amplify the activity of antigen‐presenting cells, creating a cascade that links ferroptosis with innate immunity. This cascade drives a robust systemic antitumor immune response, effectively reversing the immunosuppressive microenvironment of HCC. These results demonstrate the ability of ZMRPF to reshape the immune microenvironment of HCC and offer a promising strategy for next‐generation tumor immunotherapy.

The paucity of reliable biomarkers for predicting immunotherapy efficacy in patients with advanced hepatocellular carcinoma (HCC) has emerged as a burgeoning concern with the expanding use of immunotherapy. This study endeavors to delve into the potential peripheral biomarkers capable of prognosticating efficacy in HCC patients who are poised to receive anti-PD-1 monotherapy within the phase III clinical trial, KEYNOTE394. Additionally, we sought to elucidate the underlying molecular mechanisms for resistance to immune checkpoint blockade (ICB) and propose innovative combination immunotherapy strategies for future clinical application. Patient blood samples were collected for single-cell RNA sequencing to evaluate the immune cell signature before receiving ICB therapy. Subsequently, in vitro assays and in vivo murine model experiments were conducted to validate the mechanism that S100A9+CD14+ monocytes play a role in ICB resistance. Our study demonstrates a notable enrichment of S100A9+CD14+ monocytes in the peripheral blood of patients exhibiting suboptimal responses to anti-PD-1 therapy. Moreover, we identified the Mono_S100A9 signature as a predictive biomarker, indicative of reduced efficacy in immunotherapy and decreased survival benefits across various tumor types. Mechanistically, S100A9 activates PD-L1 transcription by directly binding to the CD274 (PD-L1) gene promoter, thereby suppressing T-cell proliferation and cytotoxicity via the PD-1/PD-L1 axis, consequently diminishing the therapeutic effectiveness of subsequent anti-PD-1 treatments. Furthermore, our in vivo studies revealed that inhibiting S100A9 can synergistically enhance the efficacy of anti-PD-1 drugs in the eradication of hepatocellular carcinoma. Our study underscores the significance of S100A9+CD14+ monocytes in predicting inadequate response to ICB treatment and provides insights into the monocyte cell-intrinsic mechanisms of resistance to ICB therapy. We also propose a combined therapeutic approach to enhance ICB efficacy by targeting S100A9.

Introduction: The advent of immune checkpoint blockade (ICB) therapy has revolutionized the treatment approach for hepatocellular carcinoma (HCC). However, the response rate to ICB therapy remains low (below 20%). This limited response rate can be attributed to the considerable heterogeneity observed in HCC cells and the tumor microenvironment (TME). Recent research has highlighted the pivotal role of cancer-associated fibroblasts (CAFs) in orchestrating various components within the TME of HCC. Although the significance of lipid metabolism-associated CAFs in the efficacy of ICB therapy has been demonstrated in HCC murine models [1], the functions and contributions of other CAF subtypes remain largely unknown. Therefore, identifying CAF heterogeneity could advance our comprehension of HCC TME and potentially augment ICB efficacy. Methods: We performed single-cell RNA sequencing on tumor biopsy specimens obtained from patients with advanced HBV-related HCC who had participated in a pembrolizumab (anti-PD1) phase II clinical trial (NCT03419481). Our objective was to examine the dynamic alterations in the HCC TME before and during anti-PD1 treatment (upon 2-cycle). We utilized the Seurat and Cellchat packages for bioinformatics analysis which enabled us to investigate the associations between changes in the proportions of CAF subtypes and the patients' clinical responses, as well as the potential cell-cell interactions involving CAF and other TME components. Results: We identified four CAF subtypes within the TME: cycling, inflammatory, matrix, and vascular CAFs in our anti-PD1 HCC patient cohort. Among the anti-PD1 non-responders, there was a significant increase in the proportions of cycling CAFs (cCAFs) and matrix CAFs (matCAFs) compared to the responders. Notably, matCAFs exhibited a high enrichment of Gene Ontology (GO) Terms related to the extracellular matrix (ECM), whose higher proportion correlated with poor prognosis of patients. Moreover, a distinct collagen pathway signal was found between matCAFs and two immunosuppressive cell types, namely regulatory T cells and M2 macrophages in the non-responders. Discussion and Conclusion: The presence of matCAFs within the HCC tumor may associate with the development of an immunosuppressive TME and unfavorable prognosis for patients. Targeting this specific CAF subtype holds promise as a potential strategy to overcome resistance to ICB therapy. Acknowledgments: The CUHK Strategic Seed Funding for Collaborative Research Scheme and The Li Ka Shing Foundation. References: 1. Zhu, GQ., Tang, Z., Huang, R. et al. CD36+ cancer-associated fibroblasts provide immunosuppressive microenvironment for hepatocellular carcinoma via secretion of macrophage migration inhibitory factor. Cell Discov 9, 25 (2023). Citation Format: Patrick Pak-Chun Wong, Jianquan Cao, Zhewen Xiong, Haoran Wu, Qin Cao, Bonan Chen, Ka-Fai To, Stephen Lam Chan, Wei Kang, Alfred Sze-Lok Cheng. Single-cell landscape of cancer-associated fibroblasts in immunotherapy resistance of hepatocellular carcinoma [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 6524.

Introduction: Non-alcoholic steatohepatitis (NASH) has emerged as a prominent risk and the most rapidly increasing aetiology of hepatocellular carcinoma (HCC). Despite the effectiveness of immune checkpoint blockade (ICB), like anti-programmed cell death-ligand 1 (anti-PD-L1) antibodies, in a subset of HCC patients, NASH has been shown to impair the anti-tumor immunity in ICB-treated HCC. We aim to elucidate the mechanisms responsible for ICB resistance in NASH-HCC at single-cell resolution. Methods: Orthotopic NASH-HCC mouse models were generated by orthotopically injecting liver cancer cell line into NASH liver induced by methionine-and choline-deficient diet (MCD) or choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Multi-color flow cytometry and single-cell RNA-sequencing (scRNA-seq) were utilized to dissect the liver and tumor immune microenvironment upon anti-PD-L1 treatment. Results: Anti-PD-L1 treatment failed to alleviate tumor burden in NASH mice, indicating that orthotopic NASH-HCC mouse models were resistant to ICB therapy. Compared to the control liver, multi-color flow cytometry demonstrated that CD11b+F4/80+CD206+M2 macrophages accumulated in NASH liver and further increased in liver and tumor upon anti-PD-L1 treatment. Tumor-infiltrating M2 macrophages exhibited the most significant and robust correlation with tumor weight among all immune cell types. Moreover, PD-1 expression on tumor-infiltrating CD8+ T cells positively correlated with tumor weight and intratumoral M2 macrophages. Furthermore, scRNA-seq analysis identified a NASH-specific tumor-infiltrating monocyte subcluster, which may differentiate into an ‘M2-like’ macrophage subtype upon anti-PD-L1 treatment as demonstrated by cell trajectory analysis. Conclusion: Our study provides insights into the significance of monocyte/macrophage reprogramming in ICB resistance in NASH-HCC. Further investigation is conducted to identify the molecular mechanisms during monocyte-macrophage differentiation and explore potential targeted strategies to overcome ICB resistance in NASH-HCC. Acknowledgment: This study is supported by the General Research Fund (14120621 and 14119023), the Collaborative Research Fund (C4045-18W), the Li Ka Shing Foundation and CUHK Strategic Seed Funding for Collaborative Research Scheme. Keywords: hepatocellular carcinoma, single-cell RNA-sequencing, anti-PD-L1, macrophages. Citation Format: Lingyun Zhang, Yiling Zhang, Wenshu Tang, Zhewen Xiong, Xiaoyu Liu, Zhixian Liang, Yuk Wah Tsang, Weiqin Yang, Xiaohang Long, Alfred Sze Lok Cheng. Deciphering the cellular and molecular determinants of immunotherapy resistance in NASH-associated hepatocellular carcinoma by single-cell analysis [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 2662.

Background: Immune checkpoint blockade (ICB) therapies by antibodies have revolutionized the treatment paradigm for a variety of cancers. Although subsets of people exhibit durable responses, resistance and relapse are common in hepatocellular carcinoma (HCC). We therefore aim to delineate the mechanism underlying the failure of ICB therapy in HCC, and more importantly to identify ICB-sensitizing therapeutic approaches. Methods: ICB-resistant orthotopic-grafted murine models were established via a serial selection of HCC cells in ICB-treated mice. Integrated multi-omics analysis (single-cell RNA-seq, single-cell ATAC-seq and ChIP-seq) were applied to decode the mechanisms underlying the ICB resistance and an epigenetic-mediated ICB response. Results: We successfully established the ICB-resistant murine models of HCC, in which tumor intrinsic interferon-gamma (IFNγ) response was most significantly suppressed and characterized by a “cold” tumor microenvironment with decreased lymphocyte activation and intratumoral infiltration. On the other hand, single cell RNA-seq analysis of human HCC biopsies revealed that patients with HDAC1/2/3-high HCC exhibit poor survival upon ICB therapy. Notably, we found that a novel specific HDAC1/2/3 inhibitor CXD101 synergized with PD-(L)1 antibody to induce immunogenic cell death, eradicate tumor and prolong survival in our ICB-resistant mouse models, which was accompanied by enhanced intratumoral infiltration, activation and anti-tumor memory formation of cytotoxic lymphocytes. Mechanistically, CXD101 combined with PD-(L)1 inhibition synergistically reactivated tumor IFNγ pathway via enhancing chromatin accessibility of downstream genes associated with antigen presentation and lymphocytes recruitment. Moreover, the activated immune system induced tumor cell pyroptosis, further igniting antitumor immunity and ICB response. Conclusions: Our findings suggest that corruption of tumor intrinsic IFNγ signaling may confer ICB resistance upon ICB therapy, which can be rectified by class I HDAC inhibitor mediated IFNγ response and tumor cell pyroptosis. Based on these findings, we have commenced a Phase II clinical study of a new epigenetic immunotherapy (CXD101 plus anti-PD-1) for HCC patients resisting anti-PD-(L)1 alone. This project is supported by the CRF (C4045-18W), GRF (14115820), TRS (T11-706/18-N), Li Ka Shing Foundation, CUHK Strategic Seed Funding for Collaborative Research Scheme. We also acknowledge support (funding and study medications) by Celleron Therapeutics. Citation Format: Yalin Tu, Haoran Wu, Chengpeng Zhong, Zhewen Xiong, Jing Wang, Patrick Pak-Chun Wong, Weiqin Yang, Jiahuan Lu, Zhixian Liang, Shufen Chen, Lingyun Zhang, Siyun Chen, Jingying Zhou, Ka-Fai To, Joseph Jao-Yiu Sung, Stephen Lam Chan, David Kerr, Nick La Thangue, Alfred Sze-Lok Cheng. Epigenetic activation of tumoral IFNγresponse and pyroptosis overcomes immunotherapy resistance in hepatocellular carcinoma [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 1424.

Introduction Hepatocellular carcinoma (HCC) remains a leading cause of cancer‐related mortality worldwide, with cellular senescence playing a context‐dependent role in tumor progression and the immunosuppressive microenvironment. This study is aimed at identifying senescence‐related gene signatures through integrated single‐cell and transcriptomic analyses to construct a robust prognostic model for predicting survival and immunotherapy response in HCC patients. Methods We obtained single‐cell RNA sequencing (scRNA‐seq) data from the Gene Expression Omnibus (GEO) database and transcriptomic data from The Cancer Genome Atlas (TCGA). The scRNA‐seq data were processed using the Seurat and Harmony packages for cell clustering and batch correction. Senescence scores were calculated via the AUCell package, and differentially expressed genes were identified using the limma package. Prognostic genes were selected through univariate and LASSO Cox regression (glmnet package) to construct a risk model, which was validated in multiple independent cohorts. Immune infiltration was assessed with single‐sample gene set enrichment analysis (ssGSEA), TIMER, and MCPCounter algorithms, and response to immune checkpoint blockade was predicted using the tumor immune dysfunction and exclusion (TIDE) platform. Experimental validation included qRT‐PCR, Cell Counting Kit‐8 (CCK‐8), wound healing, and Transwell assays in HCC cell lines. Results A total of 80,997 identified cells were allocated to eight clusters, with an evidently higher percentage of natural killer (NK) cells in HCC samples. A higher senescence score was also seen in HCC samples, and poor prognosis was noticed in the patients of high senescence score group. Further, the DEGs were intersected with the genes highly expressed in Population 4 of NK cells to reveal their enrichment in cell cycle and cell division. Further, eight genes (TMEM106C, BSG, COPE, CDCA8, KPNA2, LIG1, UQCRH, and CCT5) with differential expression in HCC were applied to construct the risk model, which could stratify HCC patients into different risks and predict the prognosis. Besides, the high immune infiltration and expression levels of immune checkpoint–relevant genes yet poor immunotherapy response were noticed in HCC patients of high risk. Further validation tests have suggested that the knockdown of CDCA8 repressed the malignant phenotypes of HCC cells. Discussion This integrated analysis establishes a senescence‐related gene signature as a robust tool for prognostic stratification and immunotherapy response prediction in HCC. The model highlights the complex interplay between cellular senescence and the immunosuppressive tumor microenvironment, offering insights for personalized treatment strategies. Furthermore, the identified biomarker CDCA8 represents a promising therapeutic target warranting further investigation. Conclusion These discoveries provide novel evidence on senescence in HCC, which may tailor the pharmacological interventions to improve the clinical management.

Simple Summary Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, and many patients do not respond well to immunotherapy. In this study, we identified a unique group of macrophages—called NFKBIZ+ M0 macrophages—that are enriched in patients non-responsive to anti-PD-1 treatment. These macrophages are activated by hypoxia and release factors such as VEGFA and HBEGF that promote new blood vessel formation and tumor growth. They also produce inflammatory molecules that suppress the immune system, helping the tumor evade immune attack. Further analysis revealed that specific signaling pathways (FOSB–VEGFA and FOS–HBEGF) drive this harmful macrophage behavior. Our findings uncover a new mechanism linking hypoxia, angiogenesis, and immune evasion to treatment resistance in HCC and suggest potential therapeutic targets to improve immunotherapy outcomes.

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The overall response rate to immunotherapy is modest in hepatocellular carcinoma (HCC), and immunotherapy resistance mechanisms are incompletely understood. We report that the E3 ubiquitin ligase Riplet is universally silenced by promoter hypermethylation in HCC. Loss of Riplet modulates fatty acid metabolism to promote terminal exhaustion of CD8 T cells. Riplet loss impedes K48-linked polyubiquitination of fatty acid synthase (FASN), consequently accelerating fatty acid production in HCC. Tumor cell-derived free fatty acids, especially palmitic acid (PA/C16:0), activate STAT3 (signal transducers and activators of transcription 3) by enhancing its palmitoylation in T cells, consequently triggering terminal CD8 T cell exhaustion. HCC cells with Riplet deficiency are resistant to anti-PD-1 therapy, and treatment with an FASN inhibitor overcomes resistance. Our study shows how Riplet can alter lipid metabolism and induce CD8 T cell exhaustion and anti-PD-1 resistance, thus suggesting avenues for combined therapies for treating patients with Riplet-deficient HCC.

Cancer-associated fibroblasts (CAFs) are known to shape the tumor microenvironment, yet the role of extracellular vesicles they produce, particularly migrasomes, in hepatocellular carcinoma (HCC) remains largely unexplored. In this study, we identified a subset of POSTN+ CAFs as the main source of migrasomes in HCC, predominantly located at the tumor–stroma interface. Transcriptomic data from TCGA and ICGC were used to compute a Migrasome_Score via GSVA, revealing strong associations with poor prognosis and immune checkpoint blockade resistance across cancers. Mechanistically, POSTN+ CAF-derived migrasomes enhanced endothelial angiogenic activity by delivering VEGFA and activating the VEGFR2–PI3K–AKT–eNOS pathway, promoting vascular remodeling within tumors. Migrasomes also induced malignant reprogramming of nearby hepatocytes by boosting oxidative phosphorylation and reactive oxygen species (ROS) production, leading to DNA damage and oncogenic transformation—effects reversed by ROS scavenging. Additionally, POSTN+ CAFs and their migrasomes physically hindered immune cell infiltration, forming a barrier that shielded tumor cells from immune surveillance. Together, these findings identify POSTN+ CAF-derived migrasomes as key drivers of HCC progression by promoting angiogenesis, malignant transformation, and immune exclusion. Their strong links to poor prognosis and immunotherapy resistance suggest that they may serve as promising therapeutic targets and prognostic biomarkers in HCC and beyond.

To evaluate the characteristics and management of primary (PR) and acquired resistance (AR) to immune checkpoint inhibitor (ICI) combination treatment in patients with unresectable hepatocellular carcinoma (uHCC). Patients with uHCC who received ICI combination therapy were retrospectively included. Baseline characteristics, pattern of progression, post-resistance management, and prognosis were recorded and analyzed according to resistance type. The primary endpoint was overall survival (OS). A hundred and eighty-eight out of 262 patients (71.8%) developed resistance, including 35.1% with PR and 36.6% with AR. All AR developed within 2 years. Patients with AR had better general conditions, smaller tumors, fewer vessel invasion and better prognosis (median OS [mOS]: 25.8 vs. 8.63 months) than those with PR. Patients with AR were less likely to have multiple lesions and sites exhibiting progression. Occurrence of new lesions led to a decrease in OS in patients with PR but an increase in those with AR. Patients with either PR or AR receiving post-resistance anti-tumor treatment showed improved OS, but patients with AR had significantly longer OS than those with PR. The combination of subsequent local therapy (mOS 25.6 vs. 10.0 months) and changes in the ICI regimen (mOS 27.5 vs. 13.4 months) led to improved OS among patients with PR. AR normally develops within 2 years after the beginning of treatment. Patients with AR have better characteristics in general, less invasive pattens of progression, and better prognosis compared to those with PR. Subsequent local therapy and changes in ICI treatment showed potential to overcome primary resistance.

Increasing evidence indicates that activation of oncogenic pathways contributes to an unfavourable tumour immune microenvironment (TIME), ultimately resulting in resistance to immunotherapy. Here, we aim to identify a critical oncogenic pathway involved in an antigen-expressing c-MYC-lucOSOE/Tp53KO hepatocellular carcinoma (HCC) mouse model that simulates immune response against tumour-associated antigens. Using data-independent acquisition proteomics, we reveal the role of wild-type KRAS in immune escaped mouse HCC tumours, with EGF concurrently activating EGFR/MEK/ERK signalling. Single cell RNA sequencing data analysis reveals that KRAS signalling intrinsically inhibits interferon-mediated MHC-I expression and extrinsically impairs CD8+ T cell activity due to the suppression of CXCL9 through the EGFR/MEK/ERK pathway. We observe KRAS activation in HCC patients who received immune checkpoint inhibitor (ICI) treatments, where it correlates with poor clinical outcomes. Notably, combination therapy with SOS1 inhibitor MRTX0902, Trametinib, and anti-PD-1 antibody effectively increased intratumoural CD8+ T cell infiltration and improved survival. Our study thus reveals that targeting wild-type KRAS signalling in combination with ICIs may serve as an effective treatment strategy for advanced HCC patients. Increasing evidence suggests that activation of oncogenic pathways contributes to an unfavorable tumor microenvironment. Here, the authors show that wild-type KRAS plays a key role in immune evasion in hepatocellular carcinoma by impairing interferon-mediated immunity and promoting resistance to immunotherapy via the EGFR/MEK/ERK pathway.

Immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1) immunotherapy, have emerged as promising treatments for advanced hepatocellular carcinoma (HCC), significantly improving clinical outcomes. However, resistance to ICIs remains a major challenge, and the underlying mechanisms of this resistance are not yet fully understood. This study aimed to investigate the role of S100 calcium-binding protein A9 (S100A9) in mediating resistance to anti-PD-1 therapy. We conducted RNA sequencing (RNA-seq) on tumor samples from anti-PD-1 responders and non-responders in HCC patients. Differential expression analysis identified S100A9 as a potential driver gene of resistance to anti-PD-1 therapy. Subcutaneous tumor models and an orthotopic HCC model established via hydrodynamic transfection were utilized to evaluate the impact of S100A9 on the efficacy of PD-1 therapy. Our findings revealed that S100A9 promotes resistance to anti-PD-1 therapy in HCC. Mechanistically, S100A9 directly interacted with PARP1 and induced its degradation via the ubiquitin-proteasome pathway. This process increased STAT3 phosphorylation at Tyr705, thereby enhancing PD-L1 transcription. Notably, treatment with the S100A9 inhibitor Tasquinimod significantly improved the efficacy of anti-PD-1 therapy in HCC. Our study reveals that S100A9 facilitates immune evasion in HCC by enhancing PARP1 ubiquitination, STAT3 phosphorylation, and PD-L1 expression. Furthermore, combining S100A9 inhibitors with anti-PD-1 antibodies markedly enhances the therapeutic efficacy of ICIs in HCC. These findings highlight S100A9 as a potential therapeutic target for overcoming resistance to immunotherapy in HCC.

Despite the recent success of immune checkpoint inhibitor (ICI) based regimens in advanced stage HCC, clinical outcomes remain poor with a median overall survival (OS) of under two years. There is an unmet need to improve responses to, and durability of, ICI based therapy in HCC. IL-8 is a chemokine promoting the attraction of myeloid cells via receptor CXCR1/2 and has been shown to have numerous pro-tumorigenic roles in the tumor immune microenviroment (TiME). Recent work by others has shown that high plasma IL-8 concentration is associated with inferior outcomes for patients receiving ICI therapy across multiple solid tumors, but its relevance to outcomes in patients with HCC is not well characterized. We prospectively collected whole blood from patients with HCC prior to initiation of standard of care immunotherapy-based regimens at Johns Hopkins (IRB #00267960). Plasma cytokines were profiled via Luminex bead-based immunoassay. To profile the local effects of IL-8 on the TiME, we performed IHC (anti-IL8, anti-CD3, anti-CD8, and anti-CD206) on resected HCC tumor tissue from systemic treatment naive patients (n=13). Finally, to understand the consequences of IL-8 inhibition via CXCR1/2 antagonism, we utilized a syngeneic murine HCC model (RIL-175 in female C57BL/6) treated with medicated SX-682 (anti-CXCR1/2) alone and in combination with intraperitoneal injections of anti-PD1+anti-VEGF. The final clinical cohort included 41 patients with advanced HCC. Patients were predominantly male (82.9%) with Barcelona Clinic Liver Cancer (BCLC) stage C disease (75.6%) who received treatment with atezolizumab + bevacizumab (51.2%). In a multivariable Cox model, baseline plasma IL-8 above or equal to median was associated with inferior progression free survival (PFS; hazard ratio [HR] 3.78, p=0.01) and OS (HR 2.32, p=0.07) after controlling for BCLC stage, albumin-bilirubin (ALBI) index, baseline alpha fetoprotein (AFP), regimen type, and baseline concentration of other cytokines with relevance in HCC (IL-6, IL-10, and VEGF-A). IHC on resected tumor specimens revealed negative correlation between IL-8 staining and CD8 cell density (R=-0.2, p=0.041) and positive correlation with CD206 staining (R=0.24, p=0.05). In our murine model, combination treatment with anti-CXCR1/2, anti-PD1, and anti-VEGF was associated with reduction of tumor volume at day 36 compared to control (p<0.001), anti-CXCR1/2 (p=0.006) and anti-PD1+anti-VEGF (p=0.06). Consistent with prior reports in other solid tumors, high plasma IL-8 concentration was associated with inferior survival on ICI based regimens in HCC. We hypothesize that IL-8 signaling promotes M2 macrophage polarization and in turn results in reduced effector T cell infiltration in the TiME. These results provide rationale for targeting the CXCR1/2 signaling axis in advanced HCC. Jeric Hernandez, Kabeer Munjal, Emma Kartalia, Madelena Brancati, Howard L. Li, James Leatherman, Ervin Griffin, Christopher J. Thoburn, Won Jin Ho, Marina Baretti, Daniel J. Zabransky, Elizabeth M. Jaffee, Mark Yarchoan, Mari Nakazawa. Overcoming ICI resistance with CXCR1/2 antagonism in advanced HCC [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 7456.

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Immunotherapy, notably the immune checkpoint blockade (ICB), has demonstrated significant promise in the management of diverse neoplasms. However, the PD-1 inhibitor has exhibited suboptimal objective response rates and did not achieve the primary endpoints in hepatocellular carcinoma (HCC) patients, primarily due to resistance to ICB fostered by the immunosuppressive tumor microenvironment (TME). To address ICI resistance and minimize adverse effects, we have engineered an innovative tumor-specific nanomedicine for the concurrent administration of aPD-1 and MDK-siRNA. Both in vitro and orthotopic HCC models were employed to investigate and establish the efficacy of the novel tumor-specific nanomedicine in overcoming the immunosuppressive TME. Specifically, the impact of the nanomedicine on the M2 polarization and polyamine metabolism within tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) was delineated. The immunomodulatory and antitumor effects, along with the side effects, of the nanomedicine integrating both aPD-1 and MDK-siRNA were assessed. A dual pH-responsive nanomedicine was successfully fabricated to co-deliver MDK-siRNA and aPD-1. The nanomedicine achieved targeted drug delivery to tumors by engaging with circulating PD-1+ T cells and accompanying their migration into the tumor mass. Additionally, nanomedicine promoted efficient drug release within the acidic TME, deploying aPD-1 for ICI therapy and retaining MDK-siRNA-encapsulated nanomedicine to regulate TAMs and MDSCs synergistically. The synergistic application of MDK-siRNA and aPD-1, coupled with the efficient tumor-targeted drug delivery, potently suppressed M2 polarization and polyamine metabolism in TAMs and MDSCs, thereby overcoming the immunosuppressive TME and leading to significant therapeutic efficacy with minimal side effects in HCC. We have developed an innovative tumor-specific nanocarrier for the co-delivery of aPD-1 and MDK-siRNA. We validated that the synthesized nanomedicine (aPD-1-siRNA@NP) yielded highly effective treatment and minimal side effects in both in vitro and orthotopic HCC models. Our work presents a nanomedicine-based approach for targeted dual-drug delivery, achieving notable efficacy in the treatment of HCC. Aiming at reversing immunosuppressive TME and overcoming ICB resistance for HCC immunotherapy, a dual pH-sensitive nanodrug was successfully developed to facilitate T cell-mediated and tumor-targeted delivery of both aPD-1 and MDK-siRNA. This novel nanodrug achieved remarkable ICB therapeutic efficacy with minimal side effects in HCC. Our efforts offered a nanomedicine strategy for tumor-targeted dual-drug delivery to gain remarkable efficacy in HCC treatment.

Recent approvals of immune checkpoint inhibitor (ICI) based regimens have transformed the clinical landscape of advanced hepatocellular carcinoma (HCC), however, clinical responses are only observed in a minority of patients. Trimethylamine-N-oxide (TMAO) is an amine oxide biosynthesized from trimethylamine (TMA), generated when gut bacteria metabolize dietary choline via the enzyme CutC. While prior studies have shown that TMAO concentrations positively correlate with the risk of several gastrointestinal malignancies, newer studies have demonstrated that TMAO may enhance antitumor immunity in some tumor types. TMAO may therefore play an important role in modulating the tumor immune response, but its significance in HCC is unknown. We prospectively collected whole blood from patients with HCC prior to initiating standard-of-care ICI-based therapy at Johns Hopkins (IRB #00267960). Clinical records were reviewed for demographic characteristics and clinical outcomes. Additionally, we studied the impact of acute and chronic choline supplementation on anti-PDL1 efficacy in a syngeneic murine model of HCC (Hep53.4 in C57BL/6 mice). Baseline choline, TMA, and TMAO plasma concentrations in clinical and preclinical specimens were quantified via liquid chromatography-mass spectrometry (LC-MS) utilizing the SCIEX Triple Quad 6500+ system. The clinical cohort included 42 patients with advanced HCC. Patients were predominantly male (81.0%) with Barcelona Clinic Liver Cancer (BCLC) stage C disease (73.8%) who underwent treatment with atezolizumab + bevacizumab (52.4%) or durvalumab + tremelimumab (31.0%). In a multivariable Cox model, higher plasma TMAO concentration at baseline was associated with inferior progression-free survival (PFS; HR 1.36 per interquartile range [IQR] increase, p=0.032) and overall survival (OS; HR 1.63 per IQR increase, p<0.001) after controlling for BCLC stage, albumin-bilirubin index, alpha fetoprotein (AFP) concentration, and baseline choline and TMA concentrations. In our preclinical model, mice given chronic choline supplementation (1% choline diet for 14 days prior to treatment with anti-PDL1 therapy) had increased plasma TMAO concentrations (p<0.001) and exhibited no response to anti-PDL1 therapy, compared to mice given standard and acute choline (1% choline diet starting concurrently with anti-PDL1 treatment) diets. Our clinical and preclinical results suggest that high plasma TMAO concentration is associated with inferior outcomes on ICI therapies in HCC. These findings contrast with existing preclinical studies that suggest TMAO may potentiate responses to ICI therapy. We hypothesize that chronic TMAO exposure may lead to immune cell exhaustion via promotion of an inflammatory microenvironment and oxidative stress. Studies examining the effect of CutC inhibition are ongoing in our laboratory and may provide further rationale for targeting TMAO in HCC. Mari Nakazawa, Vivian L. Raj, NV Rajeshkumar, Noushin Rastkari, Michael Davis, Elizabeth De Oliveria, Madelena Brancati, Ervin Griffin, Kabeer Munjal, James Leatherman, Sarah Mitchell, Marina Baretti, Won Jin Ho, Mark Yarchoan, Chi V. Dang. Trimethylamine-N-oxide (TMAO) mediates immunotherapy resistance in advanced HCC [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 2222.

Objective We previously reported a characterisation of the hepatocellular carcinoma (HCC) immune contexture and described an immune-specific class. We now aim to further delineate the immunogenomic classification of HCC to incorporate features that explain responses/resistance to immunotherapy. Design We performed RNA and whole-exome sequencing, T-cell receptor (TCR)-sequencing, multiplex immunofluorescence and immunohistochemistry in a novel cohort of 240 HCC patients and validated our results in other cohorts comprising 660 patients. Results Our integrative analysis led to define: (1) the inflamed class of HCC (37%), which includes the previously reported immune subclass (22%) and a new immune-like subclass (15%) with high interferon signalling, cytolytic activity, expression of immune-effector cytokines and a more diverse T-cell repertoire. A 20-gene signature was able to capture ~90% of these tumours and is associated with response to immunotherapy. Proteins identified in liquid biopsies recapitulated the inflamed class with an area under the ROC curve (AUC) of 0.91; (2) The intermediate class, enriched in TP53 mutations (49% vs 29%, p=0.035), and chromosomal losses involving immune-related genes and; (3) the excluded class, enriched in CTNNB1 mutations (93% vs 27%, p<0.001) and PTK2 overexpression due to gene amplification and promoter hypomethylation. CTNNB1 mutations outside the excluded class led to weak activation of the Wnt-βcatenin pathway or occurred in HCCs dominated by high interferon signalling and type I antigen presenting genes. Conclusion We have characterised the immunogenomic contexture of HCC and defined inflamed and non-inflamed tumours. Two distinct CTNNB1 patterns associated with a differential role in immune evasion are described. These features may help predict immune response in HCC.

Introduction Combined vascular endothelial growth factor/programmed death-ligand 1 blockade through atezolizumab/bevacizumab (A/B) is the current standard of care in advanced hepatocellular carcinoma (HCC). A/B substantially improved objective response rates compared with tyrosine kinase inhibitor sorafenib; however, a majority of patients will still not respond to A/B. Strong scientific rationale and emerging clinical data suggest that faecal microbiota transfer (FMT) may improve antitumour immune response on PD-(L)1 blockade. Early trials in melanoma with FMT and reinduction of immune checkpoint blockade (ICI) therapy in patients with anti-PD-1-refractory metastatic melanoma were reported in 2021 and demonstrated reinstatement of response to ICI therapy in many patients. Due to anatomical vicinity and the physiological relevance of the gut-liver axis, we hypothesise HCC to be a particularly attractive cancer entity to further assess a potential benefit of FMT in combination with ICI towards increased antitumour immunity. Additionally, HCC often occurs in patients with liver cirrhosis, where liver function is prognostically relevant. There is evidence that FMT may increase hepatic function and therefore could positively affect outcome in this patient population. Methods and analysis This prospective, multicentre, randomised, placebo-controlled, double-blind phase II clinical trial has been designed to assess immunogenicity and safety of FMT via INTESTIFIX 001 combined with A/B in advanced HCC in comparison to A/B with placebo. Primary endpoints are measured as tumour CD8+ T cell infiltration after 2 cycles of treatment with vancomycin, A/B+INTESTIFIX 001 in comparison to vancomycin-placebo, A/B+INTESTIFIX 001-placebo and safety of the therapeutic combination in advanced HCC. INTESTIFIX 001 is an encapsulated FMT preparation by healthy donors with a high alpha-diversity in their gut microbiome for oral administration, manufactured by the Cologne Microbiota Bank (CMB). Sample size was calculated to achieve a specific expected accuracy for the primary immunological endpoint. 48 subjects will be randomised to reach a goal of 42 usable measurements in the modified intention-to-treat set. Subjects will be randomised in a 2:1 ratio to A/B or placebo (28 A/B, 14 placebo). Ethics and dissemination The study was approved by ethics committee review and the German Federal Ministry of Drugs and Medical Devices. The trial is registered under EU CT no. 2023-506887-15-00. The outcome of the study will be disseminated via peer-reviewed publications and at international conferences. Trial registration number NCT05690048.

Immune checkpoint inhibitors (ICI) benefit some cancer patients but de novo resistance remains poorly understood. Analyzing transcriptional data from two clinical trial cohorts, GO30140 and IMbrave150, we find B cell lymphoma 9 (BCL9), a Wnt/β-catenin co-factor, associated with resistance. We develop a BCL9-targeting peptide, hsBCL9Z96, which suppresses tumor growth in combination with anti-PD-L1 ab in preclinical hepatocellular carcinoma (HCC) mouse models. Multi-omics analyses implicate targeting BCL9 inhibits BMP4 secretion and downregulates CD24 on tumor cells, reprogramming macrophages toward a tumor-suppressive phenotype and promoting macrophage phagocytosis. This in turn rejuvenates T cell immunity via enhanced macrophage-mediated antigen presentation. Our data extend our understanding of how tumor-derived Wnt/β-catenin signaling impedes the innate and adaptive immune responses in the tumor microenvironment and provide preliminary evidence that targeting BCL9 is a promising preclinical strategy to mitigate ICI resistance in HCC. Immune checkpoint inhibitors have shown promise in tumour immunotherapy but resistance has been seen. Here using pre-treatment hepatocellular carcinoma patient biopsies from patients scheduled for immunotherapy, the authors implicate BCL9 and show that a BCL9-targeting peptide promotes anti-tumour immunity in mouse models through targeting macrophages and promoting anti-tumour T cell responses.

Immune checkpoint inhibitors (ICIs) are widely used for treating hepatocellular carcinoma (HCC), yet their efficacy remains limited, with suboptimal response rates. The predictive power of the current biomarker, programmed death ligand‐1 (PD‐L1), is limited by detection variability and glycosylation, underscoring the need for complementary biomarkers to enhance predictive accuracy. In this study, mass spectrometry was employed to identify proteomic alterations in HCC tissues from responders and nonresponders to anti‐programmed cell death‐1 (PD‐1) therapy. Survival analysis established the role of Yin Yang 1 (YY1) in determining ICI efficacy. Coculture models of hepatoma and CD8+ T cells revealed the immunosuppressive function of YY1. Transcriptome sequencing identified polypeptide N‐acetylgalactosaminyltransferase 16 (GALNT16) as a transcriptional target of YY1, and subsequent Western blot and coimmunoprecipitation assays demonstrated that GALNT16 augments PD‐L1 expression. Furthermore, in vivo mouse models demonstrated that YY1 knockdown potentiated the efficacy of anti‐PD‐1 therapy, an effect that was partially reversed by GALNT16 overexpression. Specifically, YY1 upregulates GALNT16, which in turn promotes PD‐L1 glycosylation and stability, leading to diminished CD8+ T cell activity. Thus, GALNT16 knockdown rescued the compromised CD8+ T cell cytotoxicity induced by YY1. Collectively, these results elucidate the YY1/GALNT16/PD‐L1 axis as a pivotal mechanism underlying HCC resistance to ICI therapy. This highlights the therapeutic potential of targeting PD‐L1 glycosylation pathways.

Summary Immune checkpoint inhibitors (ICIs) represent a promising treatment for hepatocellular carcinoma (HCC) due to their capacity for abundant lymphocyte infiltration. However, some patients with HCC respond poorly to ICI therapy due to the presence of various immunosuppressive factors in the tumor microenvironment. Our research reveals that a macrophage-coated tumor cluster (MCTC) signifies a unique spatial structural organization in HCC correlating with diminished recurrence-free survival and overall survival in a total of 572 HCC cases from 3 internal cohorts and 2 independent external validation cohorts. Mechanistically, tumor-derived macrophage-associated lectin Mac-2 binding protein (M2BP) induces MCTC formation and traps immunocompetent cells at the edge of MCTCs to induce intratumoral cytotoxic T cell exclusion and local immune deprivation. Blocking M2BP with a Mac-2 antagonist might provide an effective approach to prevent MCTC formation, enhance T cell infiltration, and thereby improve the efficacy of ICI therapy in HCC.

Background: Although use of immune-checkpoint inhibitors (ICI) has yielded impressive results in cancer patients, resistance to ICI therapies has increasingly been observed, especially in hepatocellular carcinoma (HCC). Materials and Methods: We generated a novel anti-programmed death-ligand 1 (PD-L1) treatment resistant mouse hepatoma cell Hepa1-6 (PD-L1R) by 7-generation of in vivo selection. To dissect tumor cell-intrinsic resistant mechanism, we performed single-cell RNA-sequencing (scRNA-seq) from anti-PD-L1-treated tumors generated from parental or PD-L1R cells. Expression level of target genes was detected by QPCR, western blot and immunohistochemical analyses. Lipid content and fatty acid uptake was examined by Oil Red O and BIODIPY staining. Peroxisome proliferator-activated receptor gamma (PPARγ) promoter methylation level was analyzed by bisulfite pyrosequencing. To investigate the tumor cell-extrinsic factors, we profiled the myeloid and lymphoid lineages by flow cytometry. Results: ScRNA-seq revealed significant gene enrichments in lipid metabolism and PPARγ signaling pathway in PD-L1R tumors, which could be verified in both mRNA and protein levels. Moreover, we observed evident lipid accumulation in the PD-L1R tumors when compared to the parental tumors by Oil Red O staining. Consistently, PD-L1R tumor cells exhibited higher lipid content and enhanced fatty acid uptake in vitro, as demonstrated by BODIPY493/503 and BODIPY-C16 analyses. Of note, palmitic acid could dose-dependently increase Pparg mRNA expression in parental cells to a similar level with the PD-L1R cells, supporting an important role of lipids in inducing PPARγ expression. Pyrosequencing analysis indicated that PD-L1R cells exhibited lower PPARγ methylation level compared with parental cells. Notably, 5-Azacytidine treatment in parental cells significantly reduced the promoter DNA methylation and increased Pparg mRNA expression to similar extent with the PD-L1R tumor cells, thus indicating DNA hypomethylation-driven PPARγ up-regulation. Flow cytometry analysis showed that ICI resistance was associated with lower cytotoxic CD8+ T cells but higher intermediate Th17 cells, myeloid-derived suppressor cells and T regulatory cells in the tumor microenvironment (TME). Conclusions: Our study suggested that lipid exposure may epigenetically up-regulate PPARγ transcription in the development of anti-PD-L1 resistance. Detailed investigation on the crosstalk between aberrant lipid metabolism and immunosuppressive TME is ongoing. Acknowledgment: This study is supported by RGC General Research Fund (14120621 and 14119023), Li Ka Shing Foundation and CUHK Strategic Seed Funding for Collaborative Research Scheme. Citation Format: Zhewen Xiong, Stephen Lam Chan, Jingying Zhou, Xuezhen Zeng, Weiqin Yang, Haoran Wu, Jianquan Cao, Jie-Ting Low, Michael Wing-Yan Chan, Kevin Yuk-Lap Yip, Joseph Jao-Yiu Sung, Alfred Sze-Lok Cheng. Lipid-mediated epigenetic activation of PPAR-gamma signaling promotes immune-checkpoint resistance in hepatocellular carcinoma [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 1426.

Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work. Oncolytic viruses create an inflamed tumour microenvironment allowing T cells to respond to immune checkpoint blockade therapy more efficiently. Authors here show that in a hepatocellular carcinoma model, a dominant anti-viral rather than anti-tumour T cell response is elicited by an oncolytic vesicular stomatitis virus, unless the virus is designed to express tumour antigens, which restores therapeutic benefit.

Immune checkpoint inhibitors (ICI) have improved patient outcomes in hepatocellular carcinoma (HCC); however, most patients do not experience durable benefit. The non-T cell-inflamed tumor microenvironment, characterized by limited CD8+ T-cell infiltration, reduced dendritic cell function, and low interferon-γ-associated gene expression, is associated with a lower likelihood of response to ICI. To nominate new therapeutic targets for overcoming ICI resistance in HCC, we conducted a large-scale multiomic analysis on 900+human specimens (RNA sequencing (RNA-seq), proteomics) and 31 tumor single-cell (sc) RNA-seq samples, with tissue validation through imaging mass cytometry (IMC) and spatial lipidomics by matrix-assisted laser desorption/ionization (MALDI), with experimental investigation by in vitro CD8+ T-cell recruitment and macrophage polarization functional assays using three-dimensional (3D) co-culture models. We discovered 32 oncogenic pathways associated with immune exclusion, with sterol regulatory element binding protein 1 (SREBP1, encoded by SREBF1) as a hub regulator. scRNA-seq analysis showed that SREBP1 signaling is associated with enriched lipid biogenesis pathways in tumor cells, elevated immunosuppressive markers in macrophages, and diminished CD8+ T-cell infiltration. Integration of IMC and MALDI images revealed distinct lipid species differentially abundant in tumor regions with low versus high CD8+ T cell infiltration. Functional studies in a 3D in vitro tumor-immune co-culture system demonstrated that CRISPR-mediated SREBF1 knockout (KO) in HepG2 cells reduced monocyte recruitment, decreased expression of the protumorigenic CD206 marker in macrophages, and enhanced CD8+ T-cell migration compared with wild-type (WT) (p<0.0001). RNA-seq of SREBF1 KO versus WT tumor cells confirmed suppression of lipid biosynthesis genes. Our findings nominate an atlas of tumor-intrinsic drivers of immune exclusion, particularly SREBP1 via pro-tumorigenic macrophage (M2-like) reprogramming. These pathways may represent novel therapeutic targets to enhance antitumor immunity and deserve further study as targeted therapy candidates to enhance ICI in HCC.

Recent evidence has highlighted immune checkpoint inhibitors as among the most promising immunotherapies for various malignancies. However, a significant proportion of HCC patients exhibit poor responses. Lipid metabolic heterogeneity is considered a key driver of cancer progression. However, the role of lipid metabolic reprogramming in HCC immunotherapy resistance remains poorly understood. Herein, we aimed to illuminate the potential relationship between lipid metabolic reprogramming and ICI resistance and provide novel strategies to increase the HCC immunotherapy response. Patients who received PD-1/PD-L1 inhibitors were enrolled. The effect of TACC3 on the tumor microenvironment was validated via single-cell RNA sequencing in HCC-bearing mouse models. Targeted metabolomics was performed to analyze the regulatory role of TACC3 in HCC metabolism. To address HCC immunotherapy resistance, we developed a targeted nucleic acid therapeutic utilizing N-acetylgalactosamine (GalNAc) to conjugate siTACC3. Through clinical cohort analysis, we found that TACC3 was overexpressed in HCC patients with poor response to immunotherapy. Furthermore, we demonstrated that silencing tumor-derived TACC3 optimizes the cytotoxicity of infiltrating CD8+ T lymphocytes. Both in vitro and in vivo assays suggested that TACC3 maintains ACSL4-mediated polyunsaturated fatty acid (PUFA) metabolism in HCC cells. Additionally, TACC3 accelerates ACSL4 expression by interacting with LARP1 and PABPC1, which stabilize ACSL4 mRNA. The results of preclinical models demonstrated the satisfactory efficacy of GalNAc-conjugated siTACC3 combined with PD-1 inhibitor therapy for HCC. In summary, tumor-derived TACC3 impairs the tumor-killing activity of CD8+ T lymphocytes through PUFA metabolism-associated crosstalk. Targeting TACC3 represents a novel and practicable strategy to augment ICI efficacy against HCC.

Abstract Introduction: Immunomodulating effects of Helicobacter pylori (H. pylori) have been shown to inhibit antitumor immunity. Resistance to immune checkpoint inhibitor (ICI)-based therapies is common among patients with hepatocellular carcinoma (HCC). This study aimed to assess the effect of H. pylori on the outcomes of ICI in patients with HCC. Methods: We conducted a multicenter study in patients with HCC across a broad range of treatments. Patients received either ICI-based combination regimens or sorafenib-based therapy. H. pylori serostatus and virulence factors were determined and correlated with overall survival (OS), progression-free survival (PFS), and safety across the treatment modalities. Results: 180 patients with HCC were included; among these, 64 were treated with ICI-based regimen and 116 with sorafenib-based regimen. In patients treated with ICI, median OS was shorter in H. pylori-positive patients (10.9 months in H. pylori-positive vs. 18.3 months; p = 0.0384). H. pylori positivity was associated with a shorter PFS in ICI recipients (3.9 months vs. 6.8 months, p = 0.0499). In patients treated with sorafenib, median OS was not shorter among H. pylori-positive patients (13.4 months in H. pylori-positive vs. 10.6 months; p = 0.3353). Immune-related adverse events and rates of gastrointestinal bleeding were comparable between H. pylori-positive and -negative patients. Conclusion: H. pylori seropositivity was linked to poorer outcomes in patients with HCC treated with ICI. This association was not observed among patients receiving sorafenib-based therapies.

The efficacy of immune checkpoint inhibitors (ICI) in the treatment of hepatocellular carcinoma (HCC) remains limited, highlighting the need for further investigation into the underlying mechanisms. Accumulating evidence indicates that tumor-associated macrophages (TAMs) within the tumor microenvironment (TME) are implicated in immune evasion and treatment resistance. This study aimed to explore the contribution of TAMs in the HCC TME. Our findings reveal the critical involvement of CX3C motif chemokine receptor 1 (CX3CR1)-positive TAMs in inducing T cell exhaustion through interleukin-27 (IL-27) secretion, providing valuable insights into the mechanisms underlying the suboptimal efficacy of anti-PD-1 therapy in HCC. Moreover, we identified prostaglandin E2 (PGE2), released by immune-attacked tumor cells, as a key regulator of CX3CR1+ TAM phenotype transition. To augment the therapeutic response to current anti-PD-1 therapy, we propose an innovative treatment strategy that incorporates targeting CX3CR1+ TAMs in addition to anti-PD-1 therapy. In conclusion, our study contributes to the understanding of TAMs' role in cancer immunotherapy and highlights potential clinical implications for HCC treatment. The combination of targeting CX3CR1+ TAMs with anti-PD-1 therapy holds promise for enhancing the efficacy of immunotherapeutic interventions in HCC patients.

Immune checkpoint inhibitor (ICI) resistance in hepatocellular carcinoma (HCC) poses a major therapeutic challenge. Here we present a Phase 2 trial evaluating stereotactic body radiotherapy (SBRT) combined with sintilimab and bevacizumab biosimilar (PD-1/VEGF blockade) to overcome resistance in ICI-refractory HCC. Twenty-one patients with progressive HCC after ICI therapy receive SBRT followed by sintilimab 200 mg and bevacizumab biosimilar 15 mg/kg every 3 weeks. The primary outcome, objective response rate in non-irradiated lesions is 33.3%, with a disease control rate of 66.7%. Median progression-free survival is 6.2 months, and estimated median overall survival is 24.4 months. SBRT achieves 100% local control, with 33.3% experiencing grade 3 or higher adverse events. Proteomic profiling reveals that responders exhibit lower baseline IFN-γ and elevated IL-6, while post-SBRT increases in IFN-γ, IL-2, and IL-6 correlate with improved outcomes. These results indicate that combination of SBRT in ICI-refractory HCC is effective, well-tolerated, and may be guided by cytokine assessment. Immune checkpoint inhibitor resistance limits treatment options in advanced hepatocellular carcinoma. Here, the authors report a Phase 2 trial of stereotactic body radiotherapy combined with PD-1/VEGF blockade in patients with refractory hepatocellular carcinoma.

e16323 Background: Systemic therapy of hepatocellular carcinoma (HCC) faces several challenges, including tumor heterogeneity and drug resistance. Previous studies have indicated that circulating tumor DNA (ctDNA) may offer valuable insights for predicting the efficacy of systemic therapy in various cancers. Nevertheless, the majority of these studies are predominantly based on mutation detection. Recently, the ctDNA methylation detection has been used for real-time monitoring of tumor dynamic changes and assessment of treatment response. However, its applicability in HCC remains unclear. Methods: For HCC patients undergoing neoadjuvant therapy (immune checkpoint inhibitor [ICI] based), we collected paired plasma samples (n = 54) at baseline (Tb) and prior to surgery (Ts). Serial plasma samples (n = 28) were obtained from HCC patients receiving systemic therapy (ICI based) at baseline (C1) and 3 (C2), 6 (C3), and 9 (C4) weeks following treatment initiation. All samples were tested using the GutSeer panel and the cancer-specific methylation score (CSMS) were calculated. The ctDNA methylation response (CMR) was defined as the difference of CSMS between Tb and Ts greater than 0.01. The concordance between CMR and pathological response (major pathological response [MPR], ≤50% residual viable tumor) and radiological response (assessed by the modified Response Evaluation Criteria in Solid Tumors) was evaluated. Results: We enrolled 27 HCC patients undergoing neoadjuvant therapy. All patients completed at least two courses of ICI and the median treatment-to-surgery interval was 1.7 months. The difference of CSMS (Tb-Ts) was significantly negatively correlated with the proportion of residual viable tumor (R = -0.7, P < 0.001). Among them, 17 patients (63.0%) achieved CMR, of whom 15 (88.2%) were assessed as MPR. In contrast, only 2 (20%) MPR were observed among the remaining 10 patients without CMR. The area under the curve and accuracy of the predictive model based on CSMS were 85.9% and 85.2%, respectively. Additionally, we included 7 HCC patients receiving systemic therapy. Among them, 2 were assessed as partial response with one patient demonstrating a significant reduction in CSMS (~20% decrease from C1 to C4) in advance. As for the 5 patients classified as stable disease or progressive disease, 4 exhibited an early increase in CSMS, including one patient with a rise of ~80% (from C1 to C4). Conclusions: Our study found that ctDNA methylation detection is a promising indicator for monitoring tumor dynamic changes, enabling the prediction of pathological and radiological response in HCC patients during treatment. These discoveries pave the way for the development of effective efficacy surveillance strategies, thereby facilitating individualized management and enhancing the precision of medical interventions.

Introduction: Hepatocellular carcinoma (HCC) harboring CTNNB1 mutations that activate the Wnt/β-catenin pathway demonstrates increased Gd-EOB-DTPA uptake due to overexpressed OATP1B3 (Organic anion transporting polypeptide 1B3) and exhibits immune checkpoint inhibitor (ICI) resistance attributed to an immune-excluded tumor microenvironment (TME) and tumor immune barriers (TIB). This systematic review investigated Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI) for predicting immunotherapy response in HCC. Methods: A systematic search of PubMed, Web of Science, and Cochrane was conducted up to September 10, 2025. Studies linking RER > 0.9 on Gd-EOB-DTPA-enhanced MRI to PFS (RECIST 1.1) in immunotherapy-treated patients were included. Following PRISMA 2020/SWiM guidelines, risk of bias was assessed via QUADAS-2. Meta-analysis was performed using JASP (v0.95.4) via a random-effects model with Restricted Maximum Likelihood (REML) estimation. Heterogeneity was assessed using I² and τ². Notably, the Knapp-Hartung adjustment was applied to calculate 95% confidence interval (CI) to ensure robust inference despite the limited study number. Sources of heterogeneity and robustness were explored using subgroup analyses, meta-regression, and sensitivity analyses. Results: Five studies (n = 253; published 2021–2025) were analyzed. RER ≥ 0.9 was identified as a significant predictor of poor response across diverse ICI regimens, with a pooled HR of 5.79 (95% CI: 1.56–21.50; p = 0.020) and individual estimates ranging from 1.58 to 22.04. However, further analysis indicated that anti-VEGF therapy might mitigate this resistance and partially restore ICI efficacy; the association between high RER and poor survival was not statistically significant in the anti-VEGF cohort (HR = 3.39; 95% CI: 0.39–29.14; p = 0.135). Conclusions: To the best of our knowledge, this is the first systematic review evaluating the predictive utility of Gd-EOB-DTPA-enhanced MRI for HCC immunotherapy. Our findings suggest that an RER ≥ 0.9 serves as a potential non-invasive marker for poor treatment response. Notably, the observation that anti-VEGF combination therapy might mitigate this imaging-defined resistance is hypothesis-generating, underscoring the need for prospective studies to validate optimal strategies for patients with high-RER tumors.

BACKGROUND Hepatocellular carcinoma (HCC) is a highly aggressive cancer with significant intratumoral heterogeneity, likely contributed by the differentiation of cancer stem-like cells (CSCs) into multiple cancer cell lineages, resulting in therapeutic resistance and increased patient mortality. However, there is limited evidence from patient samples regarding the critical role of CSCs in treatment efficacy and patient outcomes. METHODS In this study, through the analysis of single-cell transcriptomic data of HCC patient samples, we identify a rare population of cells with cancer stem-like features. Next, we constructed a 12-gene signature that accurately predicts patient survival. Furthermore, based on the expression levels of the 12-gene signature of CSCs, a CAncer stem-like cell Risk Score (CARS) is computed. RESULTS The results show that patients with a higher CARS exhibit increased resistance to sorafenib but greater sensitivity to immune checkpoint inhibitors (ICI). The identification of CSCs in the single-cell transcriptome also provides a unique opportunity to study the microenvironmental characteristics of CSCs in HCC. HCC samples with higher CARS show a substantial infiltration of B cells, and the glycoprotein signaling of GRN and PSAP is specifically associated with the crosstalk between B cells and CSCs, rather than other tumor cells. CONCLUSION These findings develop CARS of potential prognostic and predictive value in HCC management.

Background Immune checkpoint inhibitors (ICIs) are standard therapy for unresectable HCC, but many patients do not respond. Non-viral HCC, particularly non-alcoholic steatohepatitis (NASH), have been implicated in ICI resistance. Methods We reviewed 288 patients with unresectable HCC who received ICI from 1/2017 to 12/2021. The overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) between patients with viral HCC and non-viral HCC were compared using the full and Child Pugh (CP) class A only cohorts. Results In total, 206 patients (71.5%) had viral HCC (most HCV), and 82 patients had non-viral HCC. Non-viral HCC was associated with worse OS (HR 1.6, 95% CI: 1.1–2.1, P = 0.006) and PFS (HR 1.5, 95% CI: 1.2–2, P = 0.002) in univariate but not multivariate analyses. For the CP class A cohort, non-viral HCC was independently associated with worse OS (HR 1.8, 95% CI: 1.2–2.7, P = 0.005) and PFS (HR 1.9, 95% CI: 1.3–2.7, P < 0.001). Viral HCC and CP class A liver disease was associated with better ORR than non-viral HCC (38% vs. 16%, P = 0.001). Conclusions Following ICI treatment, non-viral HCC correlated with worse OS, PFS, and ORR than viral HCC, particularly in patients with preserved liver function.

Background Programmed cell death protein 1 (PD-1) pathway blockade with immune checkpoint inhibitors (ICIs) is a standard therapy in advanced hepatocellular carcinoma (HCC) nowadays. No strategies to overcome ICI resistance have been described. We aimed to evaluate the use of ipilimumab and anti-PD-1 ICIs (nivolumab or pembrolizumab) combinations in patients with advanced HCC with progression on prior ICIs. Methods Patients with advanced HCC with documented tumor progression on prior ICIs and subsequently received ipilimumab with nivolumab/pembrolizumab were analyzed. Objective response rate (ORR), median duration of response (DOR), time-to-progression (TTP), overall survival (OS), and treatment-related adverse events (TRAEs) were assessed. Results Twenty-five patients were included. The median age was 62 (range: 51–83). About 68% were of Child-Pugh (CP) Grade A and 48% had primary resistance to prior ICI. At median follow-up of 37.7 months, the ORR was 16% with a median DOR of 11.5 months (range: 2.76–30.3). Three patients achieved complete response. The median TTP was 2.96 months (95% CI: 1.61 to 4.31). Median OS was 10.9 months (95% CI: 3.99 to 17.8) and the 1 year, 2 year and 3 year survival rates were 42.4%, 32.3% and 21.6%, respectively. The ORR was 16.7% in primary resistance group and 15.4% in acquired resistance group (p=1.00). All responders were of CP A and Albumin-Bilirubin (ALBI) Grade 1 or 2. CP and ALBI Grades were significantly associated with OS (p=0.006 and p<0.001, respectively). Overall, 52% of patients experienced TRAEs and 12% experienced Grade 3 or above TRAEs. Conclusions Ipilimumab and nivolumab/pembrolizumab can achieve durable antitumor activity and encouraging survival outcomes with acceptable toxicity in patients with advanced HCC who had prior treatment with ICIs.

No abstract available

OBJECTIVE This study aims to investigate the mechanism by which PAARH promotes M2 macrophage polarization and immune evasion of liver cancer cells through VEGF, in order to reveal its role in the progression of liver cancer. METHODS The expressions of PAARH, VEGF, and HIF-1α in liver cancer cells were detected using qRT-PCR and Western blot. Flow cytometry was utilized to analyze the polarization status of macrophages and assess the impact on immune evasion-related markers. The relationship between PAARH and VEGF in macrophage polarization was further explored. Additionally, a tumor-bearing mouse model was established to observe tumor growth. RESULTS The results show that PAARH is upregulated in liver cancer cells, and silencing PAARH significantly inhibits tumor malignancy progression. Under hypoxic conditions, overexpression of PAARH significantly increases VEGF expression, and PAARH regulates M2 macrophage polarization through VEGF. Overexpression of PAARH significantly promotes M2 macrophage polarization, increases levels of PD-L1 and Th2 immune response markers, and enhances cell proliferation, migration, and invasion; it also suppresses M1 macrophage polarization, decreases levels of PD-L2 and Th1 immune response markers, and inhibits cell apoptosis. Silencing VEGF reverses these effects. Silencing PAARH or overexpressing VEGF weakens the malignant phenotype of the cells and immune evasion. Results from the tumor-bearing mouse model indicate that silencing PAARH significantly reduces tumor size and weight, while overexpressing VEGF significantly increases tumor volume and weight. CONCLUSION PAARH enhances the immune evasion capability of liver cancer cells by upregulating VEGF to promote M2 macrophage polarization, suggesting that PAARH may serve as a new therapeutic target for liver cancer.

Background and aims The immunosuppressive tumor microenvironment (TME) plays an essential role in cancer progression and immunotherapy response. Despite the considerable advancements in cancer immunotherapy, the limited response to immune checkpoint blockade (ICB) therapies in patients with hepatocellular carcinoma (HCC) remains a major challenge for its clinical implications. Here, we investigated the molecular basis of the protein O-fucosyltransferase 1 (POFUT1) that drives HCC immune evasion and explored a potential therapeutic strategy for enhancing ICB efficacy. Methods De novo MYC/Trp53−/− liver tumor and the xenograft tumor models were used to evaluate the function of POFUT1 in immune evasion. Biochemical assays were performed to elucidate the underlying mechanism of POFUT1-mediated immune evasion. Results We identified POFUT1 as a crucial promoter of immune evasion in liver cancer. Notably, POFUT1 promoted HCC progression and inhibited T-cell infiltration in the xenograft tumor and de novo MYC/Trp53−/− mouse liver tumor models. Mechanistically, we demonstrated that POFUT1 stabilized programmed death ligand 1 (PD-L1) protein by preventing tripartite motif containing 21-mediated PD-L1 ubiquitination and degradation independently of its protein-O-fucosyltransferase activity. In addition, we further demonstrated that PD-L1 was required for the tumor-promoting and immune evasion effects of POFUT1 in HCC. Importantly, inhibition of POFUT1 could synergize with anti-programmed death receptor 1 therapy by remodeling TME in the xenograft tumor mouse model. Clinically, POFUT1 high expression displayed a lower response rate and worse clinical outcome to ICB therapies. Conclusions Our findings demonstrate that POFUT1 functions as a novel regulator of tumor immune evasion and inhibition of POFUT1 may be a potential therapeutic strategy to enhance the efficacy of immune therapy in HCC.

Metastatic hepatocellular carcinoma (HCC) is the most lethal malignancy and lacks effective treatment. FBXL6 is overexpressed in human hepatocellular carcinoma (HCC), but whether this change drives liver tumorigenesis and lung metastasis in vivo remains unknown. In this study, we aimed to identify FBXL6 (F-Box and Leucine Rich Repeat Protein 6) as a key driver of HCC metastasis and to provide a new paradigm for HCC therapy. We found that elevated FBXL6 expression in hepatocytes drove HCC lung metastasis and was a much stronger driver than Kras mutation (KrasG12D/+;Alb-Cre), p53 haploinsufficiency (p53+/-) or Tsc1 loss (Tsc1fl/fl;Alb-Cre). Mechanistically, VRK2 promoted Thr287 phosphorylation of TKT and then recruited FBXL6 to promote TKT ubiquitination and activation. Activated TKT further increased PD-L1 and VRK2 expression via the ROS-mTOR axis, leading to immune evasion and HCC metastasis. Targeting or knockdown of TKT significantly blocked FBXL6-driven immune evasion and HCC metastasis in vitro and in vivo. Notably, the level of active TKT (p-Thr287 TKT) was increased and was positively correlated with the FBXL6 and VRK2 expression levels in HCC patients. Our work provides novel mechanistic insights into FBXL6-driven HCC metastasis and suggests that targeting the TKT-ROS-mTOR-PD-L1/VRK2 axis is a new paradigm for treating patients with metastatic HCC with high FBXL6 expression.

BACKGROUND & AIMS Metabolic syndrome can lead to the clinical manifestation of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) as part of the natural history of NAFLD. Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations. METHODS Here, tumor-normal pairs from 100 subjects diagnosed with NAFLD-HCC were subject to next generation sequencings. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD to NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of underlying CTNNB1 S45P driver mutation. Syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mutand TNFRSF19 in reshaping the tumor microenvironment. RESULTS Mutational process operative in NAFLD-liver inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. ChIP-seq integrated with transcriptome and immune profiling showed for the first time a transcriptional axis of CTNNB1mut/TNFRSF19/repressed senescence-associated secretory phenotype-like (SASP-like) cytokines (including IL6 and CXCL8). This phenomenon could be reverted by Wnt-modulator ICG001. CONCLUSIONS The unique mutational processes in NAFLD-liver and NAFLD-HCC alludes to a "field effect". Whereby, distinct aberrations and shift in driver events reveal a gain-of-function role of CTNNB1 mutations in immune exclusion via TNFRSF19 inhibition of SASP-like features. LAY SUMMARY The increasing prevalence of metabolic syndrome in adult populations poised NAFLD-induced HCC to be the major type of liver cancer of the 21st century. We showed a strong "field effect" in NAFLD-liver from mutational signatures detected and a mechanistic path of activated β-catenin in reshaping the tumor-immune microenvironment.

Histidine metabolism is crucial in role in tumor biology, contributing to tumor progression, immune regulation, and metabolic reprogramming. In hepatocellular carcinoma (HCC), dysregulated histidine metabolism may promote tumor growth and immune evasion, although the specific mechanisms remain poorly understood. Using single-cell RNA sequencing, the expression patterns of histidine metabolism–related genes were evaluated across different cell types in HCC samples. In vivo and in vitro experiments were conducted to validate how histidine treatment affects macrophage and T-cell function. Furthermore, the TCGA database was utilized to construct a prognostic model to identify the key gene BUD23 and to examine its correlation with metabolism and immune infiltration. The proportion of parenchymal cells exhibiting high histidine metabolism was significantly increased, accompanied by a general reduction in immune and stromal cell infiltration. Notably, macrophages and T cells demonstrated impaired antitumor functions. In the high histidine metabolism group, multiple critical cell communication pathways (e.g., MIF, CLEC, MHC II) were downregulated, macrophages shifted toward immunosuppressive subpopulations, T cells exhibited an exhaustion phenotype, and CD8 + T-cell activation was diminished. Further in vivo and in vitro co-culture experiments confirmed that elevated histidine concentrations promoted M2 polarization in macrophages and weakened T-cell cytotoxicity, accelerating tumor proliferation. According to TCGA analyses, BUD23 was upregulated in the high histidine metabolism group and significantly negatively correlated with patient survival and immune cell infiltration. Silencing BUD23 boosted immune cell activation and cytotoxic effects, effectively reversing the immunosuppressive microenvironment. A multivariable Cox regression–based prognostic model indicated unfavorable outcomes in patients with high histidine metabolism. Histidine metabolism drives tumor cell metabolic reprogramming and reshapes the tumor immune microenvironment through intercellular communication, thereby promoting tumor progression. BUD23 shows promise as a biomarker for prognosis and immune response prediction in liver cancer. This study provides new therapeutic targets and theoretical support for liver cancer treatment by targeting histidine metabolism.

Liver metastasis (LM) poses a significant challenge in cancer treatment, with limited available therapeutic options and poor prognosis. Understanding the dynamics of tumor microenvironment (TME) and immune interactions is crucial for developing effective treatments. We find that WNT11 promoted CD8+ T-cell exclusion and suppression, which was correlated with poor prognosis in LM. Mechanistically, WNT11-overexpressing tumor cells directly reduce CD8+ T-cell recruitment and activity by decreasing CXCL10 and CCL4 expression through CAMKII-mediated β-catenin/AFF3 downregulation. WNT11-overexpressing tumor cells promote immunosuppressive macrophage polarization by inducing IL17D expression via the CAMKII/NF-κB pathway, which result in CD8+ T-cell suppression. Moreover, CAMKII inhibition increases the efficacy of anti-PD-1 therapy in mouse model of LM. Serum expression of WNT11 is identified as a potential minimally invasive biomarker in the management of colorectal cancer-LM with immunotherapy. Our findings highlight WNT11/CAMKII axis as a critical regulator of the TME and a promising target for immunotherapy in patients with LM. Activation of the WNT/β-catenin signaling pathway has been associated with immune evasion in several cancer types. Here the authors show that expression of WNT11, a member of the non-canonical WNT signaling pathway, is associated with CD8 + T cell exclusion and resistance to immune checkpoint inhibitors in liver metastasis.

No abstract available

M2-like macrophages promote tumor growth and cancer immune evasion. This study used an in vitro model to investigate how hypoxia and tumor metabolism affect macrophage polarization. Liver cancer cells (HepG2 and VX2) and macrophages (THP1) were cultured under hypoxic (0.1% O2) and normoxic (21% O2) conditions with varying glucose levels (2 g/L or 4.5 g/L). Viability assays and extracellular pH (pHe) measurements were conducted over 96 hours. Macrophages were exposed to the tumor-conditioned medium (TCM) from the cancer cells, and polarization was assessed using arginase and nitrite assays. GC-MS-based metabolic profiling quantified TCM meta-bolites and correlated them with M2 polarization. The results showed that pHe in TCMs decreased more under hypoxia than normoxia (p < 0.0001), independent of glucose levels. The arginase assay showed hypoxia significantly induced the M2 polarization of macrophages (control group: p = 0.0120,0.1%VX2-TCM group: p = 0.0149, 0.1%HepG2-TCM group: p < 0.0001, 0.1%VX2-TCMHG group: p = 0.0001, and 0.1%HepG2-TCMHG group: p < 0.0001). TCMs also induced M2 polarization under normoxic conditions, but the strongest M2 polarization occurred when both tumor cells and macrophages were incubated under hypoxia with high glucose levels. Metabolomics revealed that several metabolites, particularly lactate, were correlated with hypoxia and M2 polarization. Under normoxia, elevated 2-amino-butanoic acid (2A-BA) strongly correlated with M2 polarization. These findings suggest that targeting tumor hypoxia could mitigate immune evasion in liver tumors. Lactate drives acidity in hypoxic tumors, while 2A-BA could be a therapeutic target for overcoming immunosuppression in normoxic conditions.

Background Hepatic inflammation is a common initiator of liver diseases and considered as the primary driver of hepatocellular carcinoma (HCC). However, the precise mechanism of inflammation-induced HCC development and immune evasion remains elusive and requires extensive investigation. This study sought to identify the new target that is involved in inflammation-related liver tumorigenesis. Methods RNA-sequencing (RNA-seq) analysis was performed to identify the differential gene expression signature in primary human hepatocytes treated with or without inflammatory stimulus. A giant E3 ubiquitin protein ligase, HECT domain and RCC1-like domain 2 (HERC2), was identified in the analysis. Prognostic performance in the TCGA validation dataset was illustrated by Kaplan–Meier plot. The functional role of HERC2 in HCC progression was determined by knocking out and over-expressing HERC2 in various HCC cells. The precise molecular mechanism and signaling pathway networks associated with HERC2 in HCC stemness and immune evasion were determined by quantitative real-time PCR, immunofluorescence, western blot, and transcriptomic profiling analyses. To investigate the role of HERC2 in the etiology of HCC in vivo, we applied the chemical carcinogen diethylnitrosamine (DEN) to hepatocyte-specific HERC2-knockout mice. Additionally, the orthotopic transplantation mouse model of HCC was established to determine the effect of HERC2 during HCC development. Results We found that increased HERC2 expression was correlated with poor prognosis in HCC patients. HERC2 enhanced the stemness and PD-L1-mediated immune evasion of HCC cells, which is associated with the activation of signal transducer and activator of transcription 3 (STAT3) pathway during the inflammation-cancer transition. Mechanically, HERC2 coupled with the endoplasmic reticulum (ER)-resident protein tyrosine phosphatase 1B (PTP1B) and limited PTP1B translocation from ER to ER-plasma membrane junction, which ameliorated the inhibitory role of PTP1B in Janus kinase 2 (JAK2) phosphorylation. Furthermore, HERC2 knockout in hepatocytes limited hepatic PD-L1 expression and ameliorated HCC progression in DEN-induced mouse liver carcinogenesis. In contrast, HERC2 overexpression promoted tumor development and progression in the orthotopic transplantation HCC model. Conclusion Our data identified HERC2 functions as a previously unknown modulator of the JAK2/STAT3 pathway, thereby promoting inflammation-induced stemness and immune evasion in HCC.

This study delineates the dual oncogenic and immunomodulatory functions of SLFN5 across a spectrum of human malignancies. By integrating comprehensive multi-omics analyses in breast, liver, and ovarian cancers, we demonstrate that elevated SLFN5 expression is consistently associated with advanced tumor stage, remodeling of the tumor microenvironment toward a more aggressive phenotype, and poorer clinical outcomes. Mechanistically, SLFN5 displays a paradoxical immunoregulatory role: it facilitates the infiltration of immunosuppressive regulatory T cells (Tregs) and polarized macrophages, yet concurrently stimulates key immune checkpoint pathways. Functional enrichment analyses further underscore its central position in coordinating tumor-immune communication. Clinically, SLFN5 expression emerges as a predictive biomarker for heightened sensitivity to epigenetic therapies, including BET and HDAC inhibitors, and shows correlation with established markers of genomic instability, such as elevated tumor mutational burden (TMB) and microsatellite instability (MSI). Collectively, our results establish SLFN5 as a pleiotropic regulator residing at the crossroads of tumor progression and immune evasion. These findings not only provide a molecular rationale for targeting SLFN5 therapeutically but also offer a conceptual framework for designing combination immunotherapy strategies in SLFN5-high malignancies.

No abstract available

Motivating the immune system to target tumour cells plays an increasingly prominent role in the treatment of hepatocellular carcinoma (HCC), but challenges such as low overall response rates persist in current clinical practice. Tumour cell MHC-Class-I (MHC-I) downregulation and antigen loss are typical mechanisms of immune evasion. To this end, a dual-functional RNA-based strategy was conceived for HCC immunotherapy. MHC-I expression on HCC and paratumour tissues from patients was assessed, and the correlations between MHC-I regulators and HCC prognosis were analyzed. Small interfering RNA (siRNA) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) and mRNA encoding tumour antigens were encapsulated in a fluorinated lipid nanoparticle (LNP), which direct nucleic acids primarily to the liver, making it ideal for HCC treatment. Anti-tumour efficacy was investigated in an orthotopic HCC model, with single-cell RNA sequencing used for in-depth analysis of the tumour microenvironment (TME). A marked downregulation of MHC-I expression was observed in HCC tumour cells from a cohort of patients, with this MHC-I suppression correlating with poor prognosis and diminished responsiveness to immunotherapy. Among the various MHC-I regulators, PCSK9 is the only one that shows a significant correlation with the prognosis of HCC patients. Knockdown of PCSK9 inhibited MHC-I degradation and thus increased the efficiency of antigen presentation by up to sixfold compared to untreated tumour cells. The hybrid RNA LNPs (h-LNP) enhanced Th1-mediated immune responses, reinvigorating and expanding anti-tumour immunity within the TME. Following treatment with h-LNPs, the TME showed a pronounced infiltration of CD8+ T cells and NK cells, coupled with a significant reduction in immune-suppressive populations, such as M2-like macrophages, in contrast to the controls. These changes in the immune landscape were accompanied by a marked inhibition of tumour growth in an orthotopic HCC model as well as melanoma, where this dual-functional RNA-regulated system outperformed the control groups. The present study successfully engineered a dual-functional RNA-regulated system that augments tumour cell antigen presentation and reconfigures the immune landscape within the TME, thereby potentiating the anti-tumour efficacy of the mRNA vaccine.

The immune response against cancer is orchestrated by various parameters and site-dependent specificities have been poorly investigated. In our analyses of ten different cancer types, we describe elevated infiltration by regulatory T cells as the most common feature, while other lymphocyte subsets and also expression of immune-regulatory molecules on tumor-infiltrating lymphocytes showed site-specific variation. Multiparametric analyses of these data identified similarities of renal and liver or lung with head and neck cancer. Co-expression of immune-inhibitory ligands on tumor cells was most frequent in colorectal, lung and ovarian cancer. Genes related to antigen presentation were frequently dysregulated in liver and lung cancer. Expression of co-inhibitory molecules on tumor-infiltrating T cells accumulated in advanced stages while T-cell abundance was related to enhanced expression of genes related to antigen presentation. Our results promote evaluation of cancer-specific or even personalized immunotherapeutic combinations to overcome primary or secondary resistance as major limitation of immune-checkpoint inhibition.

Immune checkpoint blockade (ICB) has shown considerable promise for treating various malignancies, but only a subset of cancer patients benefit from immune checkpoint inhibitor therapy because of immune evasion and immune-related adverse events (irAEs). The mechanisms underlying how tumor cells regulate immune cell response remain largely unknown. Here we show that hexokinase domain component 1 (HKDC1) promotes tumor immune evasion in a CD8+ T cell-dependent manner by activating STAT1/PD-L1 in tumor cells. Mechanistically, HKDC1 binds to and presents cytosolic STAT1 to IFNGR1 on the plasma membrane following IFNγ-stimulation by associating with cytoskeleton protein ACTA2, resulting in STAT1 phosphorylation and nuclear translocation. HKDC1 inhibition in combination with anti-PD-1/PD-L1 enhances in vivo T cell antitumor response in liver cancer models in male mice. Clinical sample analysis indicates a correlation among HKDC1 expression, STAT1 phosphorylation, and survival in patients with hepatocellular carcinoma treated with atezolizumab (anti-PD-L1). These findings reveal a role for HKDC1 in regulating immune evasion by coupling cytoskeleton with STAT1 activation, providing a potential combination strategy to enhance antitumor immune responses.

Objective The metabolic characteristics of liver cancer drive considerable hurdles to immune cells function and cancer immunotherapy. However, how metabolic reprograming in the tumour microenvironment impairs the antitumour immune response remains unclear. Design Human samples and multiple murine models were employed to evaluate the correlation between GPR109A and liver cancer progression. GPR109A knockout mice, immune cells depletion and primary cell coculture models were used to determine the regulation of GPR109A on tumour microenvironment and identify the underlying mechanism responsible for the formation of intratumour GPR109A+myeloid cells. Results We demonstrate that glutamine shortage in liver cancer tumour microenvironment drives an immunosuppressive GPR109A+myeloid cells infiltration, leading to the evasion of immune surveillance. Blockade of GPR109A decreases G-MDSCs and M2-like TAMs abundance to trigger the antitumour responses of CD8+ T cells and further improves the immunotherapy efficacy against liver cancer. Mechanistically, tumour cells and tumour-infiltrated myeloid cells compete for glutamine uptake via the transporter SLC1A5 to control antitumour immunity, which disrupts the endoplasmic reticulum (ER) homoeostasis and induces unfolded protein response of myeloid cells to promote GPR109A expression through IRE1α/XBP1 pathway. The restriction of glutamine uptake in liver cancer cells, as well as the blockade of IRE1α/XBP1 signalling or glutamine supplementation, can eliminate the immunosuppressive effects of GPR109A+ myeloid cells and slow down tumour progression. Conclusion Our findings identify the immunometabolic crosstalk between liver cancer cells and myeloid cells facilitates tumour progression via a glutamine metabolism/ER stress/GPR109A axis, suggesting that GPR109A can be exploited as an immunometabolic checkpoint and putative target for cancer treatment.

Tumor immune evasion relies on the crosstalk between tumor cells and adaptive/innate immune cells. Immune checkpoints play critical roles in the crosstalk, and immune checkpoint inhibitors have achieved promising clinical effects. The long non‐coding RNA taurine‐upregulated gene 1 (TUG1) is upregulated in hepatocellular carcinoma (HCC). However, how TUG1 is upregulated and the effects on tumor immune evasion are incompletely understood. Here, METTL3‐mediated m6A modification led to TUG1 upregulation is demonstrated. Knockdown of TUG1 inhibited tumor growth and metastasis, increased the infiltration of CD8+ T cells and M1‐like macrophages in tumors, promoted the activation of CD8+ T cells through PD‐L1, and improved the phagocytosis of macrophages through CD47. Mechanistically, TUG1 regulated PD‐L1 and CD47 expressions by acting as a sponge of miR‐141 and miR‐340, respectively. Meanwhile, TUG1 interacted with YBX1 to facilitate the upregulation of PD‐L1 and CD47 transcriptionally, which ultimately regulated tumor immune evasion. Clinically, TUG1 positively correlated with PD‐L1 and CD47 in HCC tissues. Moreover, the combination of Tug1‐siRNA therapy with a Pdl1 antibody effectively suppressed tumor growth. Therefore, the mechanism of TUG1 in regulating tumor immune evasion is revealed and can inform existing strategies targeting TUG1 for enhancing HCC immune therapy and drug development.

Abstract Background Immune checkpoint blockade, particularly targeting programmed death 1 (PD‐1) and programmed death ligand 1 (PD‐L1), shows promise in treating hepatocellular carcinoma (HCC). However, acquired resistance, especially in patients with ‘hot tumours’, limits sustained benefits. Lysine‐specific demethylase 1 (LSD1) plays a role in converting ‘cold tumours’ to ‘hot tumours’, but its involvement in PD‐1 inhibitor resistance in HCC is unclear. Methods LSD1 and PD‐L1 expression, along with CD8+ T cell infiltration, were assessed using immunohistochemistry in HCC tissues, correlating these markers with patient prognosis. The impact of LSD1 deletion on tumour cell proliferation and CD8+ T cell interactions was examined in vitro. Mouse models were used to study the combined effects of LSD1 inhibition and anti‐PD‐1 therapy on tumour growth and the tumour microenvironment (TME). The clinical relevance of LSD1, CD74 and effector CD8+ T cells was validated in advanced HCC patients treated with PD‐1 blockade. Results LSD1 overexpression in HCC patients correlated with reduced PD‐L1 expression, less CD8+ T cell infiltration and poorer prognosis. LSD1 deletion increased PD‐L1 expression, boosted effector CD8+ T cells in vitro and inhibited tumour growth in vivo. While anti‐PD‐1 monotherapy initially suppressed tumour growth, it led to relapse upon antibody withdrawal. In contrast, combining LSD1 inhibition with anti‐PD‐1 therapy effectively halted tumour growth and prevented relapse, likely through TME remodelling, enhanced CD8+ T cell activity and improved CD74‐mediated antigen presentation. Clinically, low LSD1 expression was associated with better response to anti‐PD‐1 therapy. Conclusion LSD1 deletion reshapes the TME, enhances CD8+ T cell function and prevents acquired resistance to anti‐PD‐1 therapy in HCC. The combination of LSD1 inhibitors and PD‐1 blockade offers a promising strategy for overcoming resistance in advanced HCC. Key points Uncovering the synthetic lethality resulting from LSD1 deletion and PD1 inhibitor co‐administration, evaluating their combined effects on tumour growth and TME remodelling. Elucidating the mechanism underlying the combined therapy of LSD1 deletion with PD1 inhibition for HCC. Exploring the implications of LSD1, CD74 and effector CD8+ T cell expression levels in advanced HCC patients undergoing anti‐PD1 treatment.

BACKGROUND AIMS Atezolizumab plus bevacizumab (Atez/Bev) is the first-line immunotherapy for advanced hepatocellular carcinoma (HCC), yet many patients show primary or acquired resistance. We aimed to identify tumor-intrinsic mechanisms driving immune cold tumor microenvironments (TMEs) and Atez/Bev resistance. APPROACH RESULTS We used a genetically heterogeneous immunocompetent HCC mouse model generated by hydrodynamic injection of a barcoded oncogene library, integrating single-cell RNA sequencing, spatial transcriptomics, and bulk RNA sequencing of human HCC tissues. Retrospective analyses included a multi-institutional registry of 549 Atez/Bev-treated patients and 199 surgically-resected HCC patients. External validation used RNA-seq data from 247 patients registered in the IMbrave150 and GO30140 trials. In mice, Atez/Bev eliminated hot tumors, while pre-existing cold tumors with exhausted effector T cells and Tregs predominated later. Barcode analysis revealed enrichment of NFE2L2 (NRF2) in resistant tumors. NRF2 overexpression suppressed immune infiltration and conferred resistance, which was reversed by NRF2 or COX2 inhibition. In human cohorts, both non-responders and patients with acquired resistance exhibited high NRF2/COX2 expression and immune exclusion, confirmed by spatial profiling. High NRF2 activity predicted shorter progression-free survival (PFS), whereas concomitant COX2 inhibitor use correlated with longer PFS. Plasma prostaglandin E2 (PGE₂) independently predicted poor response and survival. In IMbrave150/GO30140, NRF2 and prostaglandin pathway activation correlated with Atez/Bev resistance but not sorafenib response, validating a treatment-specific mechanism. CONCLUSIONS Tumor-intrinsic activation of the NRF2-COX2-PGE₂ axis drives immune cold TMEs and mediates Atez/Bev resistance in HCC. Targeting this pathway may enhance efficacy, and plasma PGE₂ represents a non-invasive biomarker for stratification.

Most patients with hepatocellular carcinoma (HCC) develop resistance to immune checkpoint blockade (ICB) or STING agonists despite their immune-stimulating activities. Here, we identify increased intratumoral B-cell infiltration as a mediator of acquired resistance. In HCC models with liver fibrosis in male mice, anti-PD-1 ICB or the STING agonist BMS-986301 increase intratumoral B-cell infiltration, circulating IL-10, and TIM-1+ B-cells, promoting tertiary lymphoid structure formation. B-cell depletion combined with ICB or STING agonism improves survival, and STING agonism inhibits distant metastasis. In addition, co-targeting STING and TIM-1 enhances B-cell differentiation and antigen presentation, reduces intratumoral TIM-1+ B-cells, and increases CD86 and MHC class II expression, thereby augmenting CD8+ T-cell-mediated anti-tumor immunity. These findings reveal that B-cells contribute to ICB and STING therapy resistance in HCC, and that B-cell depletion or TIM-1 blockade can overcome acquired resistance to these immunotherapies. B cell subsets expanding during tumor progression have been associated with impaired anti-tumor responses and resistance to immunotherapy. Here the authors report that STING agonism or anti-PD-1 induce intratumoral B cell infiltration, and that depleting B-cells improves response to immunotherapies in preclinical models of hepatocellular carcinoma.

3129 Background: Atezolizumab (atezo; anti-PD-L1) plus bevacizumab (bev; anti-VEGF-A) became a standard treatment for advanced hepatocellular carcinoma (HCC) after demonstrating an overall survival advantage over sorafenib (inhibitor of VEGFR2 & other kinases) in the phase III clinical trial, IMbrave150. However, the mechanisms of primary and acquired resistance to atezo-bev are poorly understood. VEGFR2 + endothelial cells (ECs) are potential cellular targets of bev and may play a key immunomodulatory role in response to atezo-bev. In this study, we utilized single-cell sequencing to identify potential mediators of resistance within EC subsets. Methods: Eight patients with unresectable HCC were enrolled on the INTEGRATE study, treated with atezo-bev, and underwent intensive biospecimen collection (NCT04563338). Serial tumor biopsies were collected and viably cryopreserved including pre-treatment (n=6), 21-28 days after first dose (n=6), and at disease progression (n=2). Single-cell analysis via cellular indexing of transcriptomes and epitopes (CITEseq) has been performed and data from four patients have been analysed to date. Aggregating their nine biopsies, 8,569 hepatocytes (ALB + FABP1 + FGB + ), 29,072 immune cells (CD45 + ), and 8,028 ECs (CD31 + vWF + KDR + ) were annotated. The differential expression of VEGFR2, PD-L1, and other immune checkpoint ligands by tumor vs. immune vs. endothelial cellswere interrogated (Table). Results: VEGFR2 (receptor for VEGF-A) is predominantly expressed by ECs, at high prevalence & intensity. PD-L1 and PD-L2 (ligands of PD-1) are expressed by ECs at low prevalence & intensity. Galectin3 (LAG3 ligand) is widely expressed by hepatocytes, immune cells and ECs; while L-SECtin (LAG3 ligand) is predominantly expressed by ECs but at low prevalence & intensity. ECs had the highest prevalence of galectin9 (TIM3 ligand) expression. Nectin2 (TIGIT ligand) is expressed by both hepatocytes and ECs at high prevalence & intensity. Conclusions: Liver ECs express a broad array of immune checkpoint ligands, which are more frequent than previously anticipated. These EC subsets may potentially drive resistance by contributing to exhaustion of T cell subsets entering the tumor microenvironment. Complete CITEseq, TCR sequencing, and correlative studies from the full cohort are underway. Clinical trial information: NCT04563338 . Hepatocytes Immune cells ECs VEGFR2 (KDR) 0.06% (<0.01) 0.05% (<0.01) 66% (0.8) PD-L1 (CD274) 0.2% (<0.01) 4% (0.03) 2% (0.01) PD-L2 (PDCD1LG2) 0.02% (<0.01) 2% (0.02) 3% (0.02) L-SECtin (CLEC4G) 0% 0.1% (<0.01) 3% (0.05) Galectin-3 (LGALS3) 68% (0.8) 39% (0.5) 43% (0.5) Galectin-9 (LGALS9) 5% (0.04) 28% (0.3) 34% (0.3) PVR (CD155) 14% (0.1) 1% (<0.01) 18% (0.1) Nectin2 (CD112) 60% (0.5) 5% (0.04) 44% (0.4) % = proportion of cells with positive expression. ( ) = normalized mean expression.

Objective Therapy-induced tumour microenvironment (TME) remodelling poses a major hurdle for cancer cure. As the majority of patients with hepatocellular carcinoma (HCC) exhibits primary or acquired resistance to antiprogrammed cell death (ligand)-1 (anti-PD-[L]1) therapies, we aimed to investigate the mechanisms underlying tumour adaptation to immune-checkpoint targeting. Design Two immunotherapy-resistant HCC models were generated by serial orthotopic implantation of HCC cells through anti-PD-L1-treated syngeneic, immunocompetent mice and interrogated by single-cell RNA sequencing (scRNA-seq), genomic and immune profiling. Key signalling pathway was investigated by lentiviral-mediated knockdown and pharmacological inhibition, and further verified by scRNA-seq analysis of HCC tumour biopsies from a phase II trial of pembrolizumab (NCT03419481). Results Anti-PD-L1-resistant tumours grew >10-fold larger than parental tumours in immunocompetent but not immunocompromised mice without overt genetic changes, which were accompanied by intratumoral accumulation of myeloid-derived suppressor cells (MDSC), cytotoxic to exhausted CD8+ T cell conversion and exclusion. Mechanistically, tumour cell-intrinsic upregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) transcriptionally activated vascular endothelial growth factor-A (VEGF-A) production to drive MDSC expansion and CD8+ T cell dysfunction. A selective PPARγ antagonist triggered an immune suppressive-to-stimulatory TME conversion and resensitised tumours to anti-PD-L1 therapy in orthotopic and spontaneous HCC models. Importantly, 40% (6/15) of patients with HCC resistant to pembrolizumab exhibited tumorous PPARγ induction. Moreover, higher baseline PPARγ expression was associated with poorer survival of anti-PD-(L)1-treated patients in multiple cancer types. Conclusion We uncover an adaptive transcriptional programme by which tumour cells evade immune-checkpoint targeting via PPARγ/VEGF-A-mediated TME immunosuppression, thus providing a strategy for counteracting immunotherapeutic resistance in HCC.

Background: Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancers. Sorafenib remains the globally accepted systemic first-line treatment for advanced HCC although acquired drug-resistance is becoming increasingly common. Hopefully, immune-checkpoint inhibitor therapy has recently revolutionized the treatment for advanced HCC that have poor response to sorafenib, but the durable response rate remains low in most patients for unknown reasons. This fuels a wave of research into the molecular mechanisms of tumor-intrinsic resistance to both chemotherapy and immune checkpoint blockade (ICB), which may provide potential systemic treatment options for advanced HCC patients. Methods and Results: RNA sequencing of sorafenib-resistant HCC cell lines was performed to identify Frizzled-4 (FZD4) as a critical gene for drug resistance that was further validated in animal models. Clinical implication of FZD4 overexpression was studied in HCC patients. Using genetic and pharmacological approaches, we found that FZD4 was sufficient to increase clonogenicity, migration and invasion, and prevent sorafenib-induced apoptosis. In addition, FZD4 could reduce secretion of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), as well as intracellular levels of cytotoxic granzyme A and perforin in CD8+ T cells, indicating significant suppression of CD8+ T cell-mediated antitumor immunity in HCC tumors. In vivo studies also revealed that FZD4 expression promoted tumorigenesis and accelerated HCC cell metastatic lung colonization. Combination therapy of FZD4 inhibitor FzM1 with sorafenib in HCC of MYC/sgp53-induced model showed a tendency to significantly impede tumor growth and improve mice survival. Moreover, gene set enrichment analysis (GSEA) identified the cholesterol homeostasis and fatty acid metabolism signaling signatures as the top activated pathways in FZD4-high HCC cells. This evidence suggests a crucial role of FZD4-involved lipid metabolism linked to the development of therapeutic resistance. Conclusion: Our study demonstrates that FZD4 expression plays a pivotal role in HCC progression and holds the potential for expanding the scope of targeted/ICB therapies to tumors that are currently unresponsive, especially in advanced HCC. The resistance mechanisms will be addressed in further work. Citation Format: Jie Luo, Lanqi Gong, Yuma Yang, Qin Liu, Jiao Huang, Xiaona Fang, Xin-Yuan Guan. Frizzled-4 expression enhances hepatocellular carcinoma progression, sorafenib resistance and suppresses anti-tumor CD8 T cell immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4145.

Background Hepatocellular carcinoma (HCC) is one of the most serious malignancies. The main features of HCC are vascular invasion and drug resistance. Ferroptosis is a novel cell program that is involved in several diseases, such as cancer. Heat shock protein beta 1 (HSPB1) is a major component of heat shock proteins. A recent study showed that HSPB1 could be a new therapeutic target for colorectal cancer with 5-fluorouracil-acquired resistance. However, the functional role of HSPB1 in HCC remains unclear. Aim The aim of this study is to clarify HSPB1 expression in HCC and its potential therapeutic and prognostic value. Methods We collected data on HSPB1 expression levels in HCC and normal liver tissues from The Cancer Genome Atlas and Gene Expression Omnibus databases. We then validated it using immunohistochemistry (IHC). Receiver operating characteristic and Kaplan–Meier survival curves were used to investigate the role of HSPB1 in the prognosis analysis of HCC. Further, we used the online Search Tool for the Retrieval of Interacting Genes/Proteins website, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes to conduct enrichment analysis and identify the predictive signaling pathways. Meanwhile, we used the TIMER and GSVA package of R (v3.6.3) to analyze the association between HSPB1 and immunocyte infiltration. Results Compared to normal tissues, there was differential expression of HSPB1 in pan-cancers. HSPB1 expression was higher in HCC tissues than in normal tissues (p<0.05). There was an evident significant difference between HSPB1 mRNA levels and histologic grade, vascular invasion, and alpha-fetoprotein level (all p values<0.05). Univariate analysis indicated that HCC patients with high HSPB1 levels had shorter overall survival rates than those with low HSPB1 levels (p<0.05). MAPK14, HSPA8, MAPKAPK3, MAPKAPK5, and MAPKAPK2 are essential proteins that interact with HSPB1. There was a significant correlation between HSPB1 expression levels and immune cell infiltration, including CD4+ T cells (r=0.203, p<0.05). Conclusion High HSPB1 expression is closely associated with a worse prognosis in HCC patients, and HSPB1 may be a target of immunotherapy in HCC.

Hepatocellular carcinoma (HCC) is the most common primary liver tumor, often arising in the context of chronic liver disease. Despite recent advances in systemic therapies, including the use of immune checkpoint inhibitors (ICIs), clinical outcomes remain suboptimal, with many patients exhibiting primary or acquired resistance. Accumulating evidence indicates that the dysregulation of epigenetic mechanisms contributes to HCC development, and may also play a crucial role in shaping the tumor immune microenvironment, influencing responses to treatments. In this study, we analyzed the expression profiles of a comprehensive set of epigenetic regulators across publicly available transcriptomic datasets of HCC and non-tumoral liver tissues. Our findings reveal a consistent dysregulation of key epigenetic modifiers, particularly those involved in DNA methylation and histone modification. Furthermore, our analysis underscores the need for a deeper understanding of the epigenetic landscape of HCC, as specific epigenetic patterns are directly associated with disease development, the major mutational, immune, and transcriptional subclasses of HCC, and patient clinical outcomes. Our study provides a foundation for integrating epigenetic biomarkers into patient stratification and therapeutic decision-making. A more comprehensive analysis of epigenetic alterations could pave the way for novel predictive markers and combination strategies that could enhance the efficacy of ICIs in HCC.

BACKGROUND Human hepatocellular carcinoma (HCC) acquired resistance to anti-cancer agents due to the presence of immunosuppressive tumour microenvironment (TME) established by the interaction between tumour cells and immune populations. New treatment targeting the interaction is urgently needed and clinically beneficial to patients with HCC. This study aims to explore the anti-tumour effect of a Traditional Chinese Medicine formula Siwu Decoction (SWD) and its potential mechanism. MATERIALS AND METHODS The chemical profile of SWD was determined by high-performance liquid chromatography coupled with mass spectrometry. In vitro and in vivo effects of SWD in regressing HCC were assessed. The role of myeloid-derived suppressor cells (MDSCs) in mediating SWD-induced HCC inhibition was determined by adoptive transfer assay. The regulation of SWD-induced interaction between HCC cells and MDSCs was also confirmed both in vitro and in vivo. RESULTS SWD dose-dependent inhibited the HCC growth and lung metastasis in an orthotopic growth tumour in mice, without significant toxicity and adverse side effect. SWD induced necroptosis in HCC cells, but did not directly inhibit in vitro culture of MDSCs, instead, SWD-treated HCC cell culture supernatant suppressed MDSCs by inducing its cell apoptosis. The necroptotic response of HCC cells can also suppress the MDSCs population in the TME without reducing circulating MDSCs infiltration into the tumours. Adoptive transfer of MDSCs recovered tumour growth and lung metastasis of HCC in SWD-treated mice. In HCC cells, SWD induced a necroptotic response, and blockade of necroptotic response in HCC cells recovered the MDSCs population in vitro and in vivo, and restored tumour growth and lung metastasis in SWD-treated mice. A combination of SWD improves the anti-HCC efficacy of sorafenib without inducing adverse side effects. Albiflorin, the effective compound of SWD, its anti-HCC manner has been verified to be consistent with that of SWD. CONCLUSION Our study observed for the first time that SWD can suppress HCC by regulating MDSCs through necroptosis of tumour cells in the TME. The main effective compound of SWD, albiflorin can be a potential adjuvant therapy in the clinical management of human HCC.

Background & Aims Sora is the first-line drug for advanced hepatocellular carcinoma (HCC). However, acquired resistance to Sora treatment largely hinders its therapeutic efficacy, and the mechanisms underlying Sora resistance remain poorly understood. Here, we revealed a new mechanism by which Sora promotes the differentiation of regulatory T (Treg) cells to suppress the immune response in the HCC tumor microenvironment (TME) and induce Sora resistance. Methods Human liver tissues were obtained from HCC patients. Female C57BL/6J, OT-II, and Foxp3GFP mice were also used. Flow cytometry was used to analyze immune cells in TME. Flow cytometry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay were performed to evaluate Treg cell differentiation. Immunoblotting was conducted to identify relevant proteins. Mouse and human tumor tissues were evaluated via multiplex immunofluorescence staining. Sora-treated HCC tissues and Sora-treated Treg cells were subjected to RNA sequencing analysis. Tumor models were generated and treated with Sora, Sora combined with an anti-CD25 antibody, or Sora combined with the Foxo1 inhibitor AS1842856. Results First, we found through bioinformatic analysis that Sora suppresses the immune response in HCC. Furthermore, Sora increased the Treg cell population to promote the formation of an immunosuppressive TME in HCC. In vitro, Sora promoted Treg cell differentiation and increased the immunosuppressive activity of Treg cells. Activating VEGF and AKT abolished the effect of Sora on Treg cell differentiation, whereas inhibiting Foxo1 compromised Sora-induced Treg cell differentiation, indicating that the induction of Treg cells by Sora is dependent on the VEGFR/AKT/Foxo1 pathway. Finally, Treg inactivation by an anti-CD25 antibody or the Foxo1 inhibitor AS1842856 in combination with Sora showed greater efficacy in the treatment of HCC. Conclusions Sora induced Treg cell differentiation by inhibiting VEGFR/AKT signaling and activating Foxo1, thus suppressing the immune response and reducing Sora efficacy. Treg inactivation might be a promising strategy to alleviate the immunosuppressive TME and overcome Sora resistance.

No abstract available

CD276, is a fatal recently discovered immune checkpoint proteins of B7 family. Due to the not clearly uncovered signal pathways that involved in the expression of CD276 in tumors, few strategies were discovered to regulate CD276. Here, we newly discovered that abnormal tumor mitochondrial activation played a vital important role in raising CD276 expression through targeting AMPK/mTOR signal pathway. Then, it was also revealed that clinical usable lipid-lowering drugs with mitochondria oxidative phosphorylation (OXPHOS) and glycolysis inhibiting capacity, like fenofibric acid (FFA), exhibited desired programmed death ligand-1 (PD-L1) and CD276 co-suppression capacity. To better deliver FFA to tumor mitochondria, IR-FFA was synthesized by linking the mitochondria-targeting heptamethylene cyanine IR-68 with FFA, followed by self-assembly with albumin (Alb) to create IR-FFA@Alb nanoparticles. By doing so, the dosage needed for IR-FFA@Alb to depress CD276 and PD-L1 expression was 100 times lower than free FFA. Then, IR-FFA@Alb monotherapy effectively inhibited tumor growth both in vitro and in vivo. Moreover, the combination therapy of IR-FFA@Alb nanoparticles and radiotherapy (RT) effectively avoid the frequently occurred immune tolerance phenomenon of RT by co-depression CD276 and PD-L1. These results altogether showed the possibility of using lipid-lowering drugs as multi-functional immune checkpoint inhibitors to sensitize tumor therapy.

No abstract available

Abstract Background/Aim: STAT3 is involved in the progression of several cancers, and has been proposed as target for therapy. Indeed, the multitargeted tyrosine kinase inhibitor drug regorafenib, which indirectly inhibits STAT3, can significantly enhance the effects of anti-programmed death receptor (PD)-1 therapy in hepatocellular carcinoma (HCC) models. Here, we studied the impact of a direct STAT3 inhibitor on the tumor microenvironment and PD-1 blockade efficacy in HCC models. Materials and Methods: Orthotopic mouse models of HCC (RIL-175 and HCA-1 grafts in syngeneic mice) were used to test the efficacy of the selective STAT3 inhibitor STX-0119 alone or combined with anti-PD-1 antibodies. We evaluated the effects of therapy on tumor vasculature and the immune microenvironment using immunofluorescence (IF), cell viability assay and quantitative real-time (qRT)-PCR in tumor tissues. Results: Combining anti-PD-1 antibodies with a STX-0119 failed to show a growth delay or survival benefit compared to each agent alone or control in any of the HCC models. Interestingly, evaluation of intratumoral CD8+ T cell infiltration by IF showed a significant increase after one-week treatment with STX-0119 (p=0.034). However, STX-0119 treatment simultaneously promoted increased immunosuppression in the tumor microenvironment by increasing the proportion of Tregs, tissue hypoxia and α-SMA activated cancer-associated fibroblasts (CAFs) measured by IF. Consistent with these findings, we found increased immature tumor vessels by IF and VEGF, Tgf-β and Vash2 expression by qPCR. Conclusion: Pharmacologic STAT3 inhibition could significantly enhance CD8+ T cell infiltration in HCC but also significantly alter the immunosuppression and vascular abnormalization in the tumor microenvironment.

Background and Aims: BRCA1 (BRCA1 DNA repair associated) and PALB2 (partner and localizer of BRCA2) interact with each other to promote homologous recombination and DNA double‐strand breaks repair. The disruption of this interaction has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. Approach and Results: We demonstrated that mice with disrupted BRCA1‐PALB2 interaction were more susceptible to HCC than wild‐type mice. HCC tumors arising from these mice showed plenty of T‐lymphocyte infiltration and a better response to programmed cell death 1 (PD‐1) antibody treatment. Mechanistically, disruption of the BRCA1‐PALB2 interaction causes persistent high level of DNA damage in HCC cells, leading to activation of the cyclic GMP‐AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway in both malignant hepatocytes and M1 macrophages in the tumor microenvironment. The activated cGAS‐STING pathway induces programmed cell death 1 ligand 1 expression via the STING–interferon regulatory factor 3 (IRF3)–signal transducer and activator of transcription 1 pathway, causing immunosuppression to facilitate tumorigenesis and tumor progression. Meanwhile, M1 macrophages with an activated cGAS‐STING pathway could recruit T lymphocytes through the STING‐IRF3 pathway, leading to T‐lymphocyte infiltration in tumors. After normalizing immune responses by PD‐1 antibody treatment, the infiltrating T lymphocytes attack tumor cells rapidly and effectively. Conclusions: This study reveals that persistent DNA damage caused by a defective BRCA pathway induces tumor immunosuppression and T‐lymphocyte infiltration in HCC through the cGAS‐STING pathway, providing insight into tumor immune microenvironment remodeling that may help improve HCC response to PD‐1 antibody treatment.

While checkpoint immunotherapy effectively mobilizes T‐cell responses against tumors, its success in hepatocellular carcinoma (HCC) is frequently undermined by immunosuppressive myeloid cells within the tumor microenvironment. This study investigates the role of nuclear protein 1 (NUPR1), a gene prominently expressed in tumor‐associated macrophages (TAMs), in mediating this suppression and influencing immunotherapy outcomes. Through comprehensive analysis of single‐cell RNA sequencing (scRNA‐seq) datasets and functional assays in vitro and in vivo, NUPR1 is identified as a critical regulator within TAMs. The upregulation of NUPR1 is associated with enhanced M2 macrophage polarization and increased expression of immune checkpoints PD‐L1 and SIRPA, resulting in CD8+ T cell exhaustion and a diminished response to immunotherapy. Mechanistically, NUPR1 inhibits the ERK and JNK signaling pathways, thereby creating an immunosuppressive milieu conducive to tumor progression. Additionally, tumor‐derived lactate is shown to upregulate NUPR1 expression in macrophages via histone lactylation, perpetuating a feedback loop that intensifies immune suppression. Pharmacological targeting of NUPR1 reverses M2 polarization, curtails tumor growth, and augments the efficacy of PD‐1 blockade in preclinical models, positioning NUPR1 as both a potential biomarker for immunotherapy responsiveness and a therapeutic target to boost immunotherapy efficacy in HCC.

Background Exosomes play an important role in the tumor microenvironment (TME) and the mechanisms of tumor immune escape in hepatocellular carcinoma (HCC). It is known that immunosuppressive genes, involved in the processes of tumor immunosuppression, are associated with cancer progression. This study aimed to explore the prognostic values of exosome-related immunosuppression genes (ERIGs) in HCC. Methods The RNA-seq transcriptome data of 374 HCC patients were obtained from the Cancer Genome Atlas (TCGA) database. The TCGA cohort was randomly divided into the training cohort and validation cohort in a 1:1 ratio. WGCNA analysis and Pearson correlation analysis were used to identify ERIGs. The Lasso regression method was used to construct a 5-ERIG signature. The prognostic value of our signature was examined in the First Affiliated Hospital of Wenzhou Medical University (FAHWMU) cohort. Results Univariate Cox regression analysis was used to screen prognostic ERIGs. Subsequently, these prognostic ERIGs were included in Lasso regression analyses to identify 5 key ERIGs (ASAP1, IARS1, GTF3C2, TPD5L2 and SLC52A2) and construct a 5-ERIG signature. The patients in the low-risk group had better prognosis than those in the high-risk group. Univariate and multivariate cox regression revealed that risk score was an independent prognostic risk factor of HCC. Gene set enrichment analysis (GSEA) showed that this signature was highly associated with TME-related pathways. Subsequent analyses revealed the potential role of the signature in regulating the TME in HCC. In addition, a lower immunotherapy score was found in patients with high risk-score. Of note, this signature was confirmed to have a good performance in predicting HCC prognosis in the FAHWMU cohort. Moreover, knockdown of 5 ERIGs of this signature contributed to the suppression the Hep3B cell proliferation. Conclusions We generated a novel prognostic 5-ERIG signature to accurately predict the prognosis of patients with HCC, and this signature may serve as an indicator of immunotherapy for HCC. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-10463-0.

Disorders of polyamine metabolism may contribute to the development of hepatocellular carcinoma (HCC), but the precise mechanism remains unknown. This study reports that spermine synthase (SMS), an enzyme involved in polyamine biosynthesis, is overexpressed in HCC and not associated with hepatitis virus infection in HCC patients. The results of analyzing the clinical data of HCC patients showed that SMS level as a categorical dependent variable was related to clinicopathological features of poor prognosis. Furthermore, the Kaplan-Meier survival analysis and ROC curve indicated that increased SMS level is associated with poor survival rate in HCC and may be a potential biomarker to discriminate HCC tissues. However, SMS overexpression limited the therapeutic effect of immune checkpoint blockade (ICB), which seemed to be related to the immunosuppressive effect of the HCC immune microenvironment formed by higher mRNA transcript levels of immune checkpoints and higher infiltration levels of immunosuppressive cells. In samples with high and low SMS expression, functional enrichment analysis of the differentially expressed genes (DEGs) showed that SMS may be linked to the occurrence and development of HCC by affecting a variety of immune-related pathways, such as Intestinal immune network for IgA production, Fc gamma R-mediated phagocytosis, Antigen processing and presentation, Th1 and Th2 cell differentiation. Subsequently, analysis of the co-expression network of SMS in the liver hepatocellular carcinoma (LIHC) cohort revealed that SMS has a broad impact on multiple important immune- and metabolic-related processes in HCC. In summary, SMS is a promising biomarker to differentiate the prognosis, immune characteristics, and holds promise as a potential target for ICB therapy to improve HCC.

Cancer immunotherapy has demonstrated significant efficacy in various tumors, but its effectiveness in treating Hepatocellular Carcinoma (HCC) remains limited. Therefore, there is an urgent need to identify a new immunotherapy target and develop corresponding intervention strategies. Bioinformatics analysis has revealed that growth differentiation factor 15 (GDF15) is highly expressed in HCC and is closely related to poor prognosis of HCC patients. The previous study revealed that GDF15 can promote immunosuppression in the tumor microenvironment. Therefore, knocking out GDF15 through gene editing could potentially reverse the suppressive tumor immune microenvironment permanently. To deliver the CRISPR/Cas9 system specifically to HCC, nanocapsules (SNC) coated with HCC targeting peptides (SP94) on their surface is utilized. These nanocapsules incorporate disulfide bonds (SNCSS) that release their contents in the tumor microenvironment characterized by high levels of glutathione (GSH). In vivo, the SNCSS target HCC cells, exert a marked inhibitory effect on HCC progression, and promote HCC immunotherapy. Mechanistically, CyTOF analysis showed favorable changes in the immune microenvironment of HCC, immunocytes with killer function increased and immunocytes with inhibitive function decreased. These findings highlight the potential of the CRISPR‐Cas9 gene editing system in modulating the immune microenvironment and improving the effectiveness of existing immunotherapy approaches for HCC.

ETHNOPHARMACOLOGICAL RELEVANCE Jianpi-Huayu Decoction (JHD), derived from the traditional Sijunzi decoction, is a traditional Chinese medicine compound that has been clinically proven to have anti-hepatocellular carcinoma (HCC) effects. According to our years of clinical application, JHD showed promising potential in enhancing the therapeutic effect of immune checkpoint inhibitors. However, despite its clinical promise, the immunoregulatory mechanisms by which JHD influences the tumor microenvironment (TME) remain largely unexplored. AIM OF THE STUDY This study aimed to investigate how JHD modulates immune components within the TME and to elucidate its underlying mechanisms of immunosuppression in the context of HCC. MATERIALS AND METHODS JHD was prepared as a spray-dried powder using a double decoction process and applied in both in vivo and in vitro experiments. Subcutaneous and orthotopic HCC models in mice were used to evaluate JHD's effects on immune modulation and tumor suppression. Transcriptomic sequencing, qRT-PCR, immunohistochemistry, and western blotting were employed to assess immune-related pathways. In vitro, the effects of JHD on HCC cell proliferation and molecular mechanisms were analyzed using co-culture systems, MTT and LDH assays, ELISA, qRT-PCR, and western blotting. RESULTS Our results showed that JHD could inhibit the growth of liver cancer in immunocompetent mice, and enhanced immune function may be one of the mechanisms of JHD anti-liver cancer effect by comparative study with immunodeficient mice. Sequencing analysis further found its regulatory effect on DAP12 pathway. The results of bioinformatics analysis of HCC immunity reveal that increased infiltration of immunosuppressive tumor-associated macrophages (TAMs) and decreased natural killer (NK) cells activity are hallmark features of HCC. Notably, TREM1 is significantly upregulated during HCC progression and has emerged as a key regulator of the tumor immune microenvironment. Silencing TREM1 reduced the immunosuppressive phenotype of TAMs, enhanced NK cell-mediated cytotoxicity, and improved the antitumor immune response. JHD exerted its effects by modulating the TREM1/DAP12 signaling axis, thereby diminishing TAMs immunosuppression and promoting NK cell activity. Furthermore, JHD synergistically enhanced the therapeutic efficacy of anti-PD-1 antibodies in vivo, with a favorable safety profile. CONCLUSIONS TAMs and NK cells play a pivotal role in driving immunosuppression in HCC. By targeting TREM1/DAP12 pathway , JHD improves immunosuppression TME and augments the antitumor efficacy of PD-1 inhibitors.

Dysregulation of the aldehyde dehydrogenase (ALDH) family has been implicated in various pathological conditions, including cancer. However, a systematic evaluation of ALDH alterations and their therapeutic relevance in hepatocellular carcinoma (HCC) remains lacking. Herein, we found that 15 of 19 ALDHs were transcriptionally dysregulated in HCC tissues compared to normal liver tissues. A four gene signature, including ALDH2, ALDH5A1, ALDH6A1, and ALDH8A1, robustly predicted prognosis and defined a high-risk subgroup exhibiting immunosuppressive features like regulatory T cell (Tregs) infiltration. Single-cell profiling revealed selective overexpression of tumor necrosis factor receptor superfamily member 18 (TNFRSF18) on Tregs, upregulated in high-risk HCC patients. We identified ALDH2 as a tumor suppressor in HCC, with three novel phosphorylation sites mediated by protein kinase C zeta that enhanced enzymatic activity. Mechanistically, ALDH2 suppressed Tregs differentiation by inhibiting β-catenin/TGF-β1 signaling in HCC. Collectively, our integrated multi-omics analysis defines an ALDH-Tregs-TNFRSF18 axis that contributes to HCC pathogenesis and represents potential therapeutic targets for this aggressive malignancy.

Transmembrane domain-containing 7 (CMTM7) is a protein located at the plasma membrane. It plays a role in regulating the development and immune microenvironment of tumor cells. However, the impact of CMTM7 on hepatocellular carcinoma (HCC) is not well understood. To better understand the role of CMTM7 in HCC, the correlations of CMTM7 with clinical characteristics, patient prognosis, chronic inflammation, and immune cell infiltration were analyzed using tissue microarray slides, sequencing datasets and various analysis tools (Web). The bulk sequencing analysis indicated that elevated expression of CMTM7 appears to promote chronic inflammation, immunosuppression, M2 macrophage infiltration, a diminished response to cancer immunotherapy, and an unfavorable clinical prognosis in patients with hepatocellular carcinoma (HCC). Further investigation through single-cell RNA sequencing and multiple fluorescence staining demonstrated that CMTM7 serves as a molecular marker for M2 macrophages and is associated with T cell exhaustion as well as highly plastic stem-like characteristics. We propose that CMTM7 may represent a novel immune checkpoint for HCC patients experiencing suboptimal therapeutic outcomes. Utilizing the Connectivity Map and AutoDock Vina, we predicted two potential compounds targeting CMTM7—fasudil and arachidonyltrifluoromethane—as promising therapeutic candidates. Collectively, these findings suggest that CMTM7-positive macrophages play significant roles in establishing an immunosuppressive tumor microenvironment while promoting highly plastic and stem-like traits in HCC cells, ultimately contributing to poor prognostic outcomes.

Myeloid cells are abundant and plastic immune cell subsets in the liver, to which pro-tumorigenic, inflammatory and immunosuppressive roles have been assigned in the course of tumorigenesis. Yet several aspects underlying their dynamic alterations in hepatocellular carcinoma (HCC) progression remain elusive, including the impact of distinct genetic mutations in shaping a cancer-permissive tumor microenvironment (TME). Here, we generated somatic HCC mouse models bearing clinically-relevant oncogenic driver combinations and subsequent pathway activation that faithfully recapitulated different human HCC subclasses. We identified cancer genetics’ specific and stage-dependent alterations of the liver TME associated with distinct histopathological and malignant HCC features. These models ranged from T cell-rich, more indolent HCC to aggressive tumors exhibiting heightened myeloid cell infiltration. Interestingly, MAPK-activated, NrasG12D-driven tumors presented a mixed phenotype of prominent inflammation and immunosuppression in a T cell-excluded TME, contrasting with NrasG12V HCC, enriched in adaptive immune cells. Mechanistically, we identified a NrasG12D cancer cell-driven, MEK-ERK1/2-SP1-dependent GM-CSF secretion enabling the accumulation of immunosuppressive and proinflammatory monocyte-derived Ly6Clow cells. GM-CSF blockade curbed the accumulation of this myeloid cell subset, reduced inflammation, induced cancer cell death and prolonged animal survival. Furthermore, the anti-tumor effect of GM-CSF neutralization synergized with the clinically-approved inhibition of the vascular endothelial growth factor (VEGF) to inhibit HCC outgrowth. These findings underscore the striking alterations of the myeloid TME consequential to MAPK pathway activation intensity and the potential of GM-CSF inhibition as a myeloid-centric therapy tailored to subsets of HCC patients.

Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated.

Immunosuppression in a tumor microenvironment is associated with enhanced tumor progression. Natural killer group 2 (NKG2) family proteins, including inhibitory receptors and activators, can be used as attractive targets for immunotherapy of immune checkpoint inhibition. We further explore the expression level prognostic value of NKG2A and NKG2D in hepatitis B virus‐related hepatocellular carcinoma (HBV‐HCC).

No abstract available

Angiogenesis and immunosuppression have been described as closely related processes that can occur in parallel. As an inhibitor of matrix metalloproteinase, whether the level of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in hepatocellular carcinoma (HCC) reflects a link between angiogenesis and immunosuppression is still unknown. We analyzed RNA expression, immune infiltration and survival of HCC from The Cancer Genome Atlas databases. Immune scores and stromal scores were calculated based on the ESTIMATE algorithm to quantify the immune and stromal components in HCC. The association between RECK and clinicopathological features was further investigated by immunohistochemistry on tissue microarray. We found that the prognosis of patients with high RECK expression was significantly better than that of patients with low RECK expression. High RECK expression was associated with high ESTIMATE Score, recruitment of more tumor-infiltrating lymphocytes, low tumor purity, and high PD-L1 expression. In addition, positive RECK expression was associated with a lower incidence of vascular invasion and recurrence, a lower level of alpha fetoprotein (AFP) and microvessel density and a better tumor differentiation. Multivariate analyses revealed that reduced RECK expression was an independent prognostic factor for recurrence and poor prognosis. In conclusion, high RECK expression reflects an immunogenic and hypovascularity status in HCC. RECK is a promising prognostic marker for survival of HCC and may act as a complementary indicator for patients to receive anti-angiogenic therapy or immunotherapy.

Portal vein tumor thrombosis (PVTT) is a significant risk factor for metastasis in hepatocellular carcinoma (HCC) patients and is therefore associated with poor prognosis. The presence of PVTT frequently accompanies substantial hypoxia within the tumor microenvironment, which is suggested to accelerate tumor metastasis, but it is unclear how this occurs. Recent evidence has shown that the hypoxia-inducible factor HIF-1α induces epithelial-to-mesenchymal transition (EMT) in tumor cells to facilitate metastasis. In this study, we investigated whether hypoxia-induced EMT in cancer cells also affects immune cells in the tumor microenvironment to promote immunosuppression. We found that hypoxia-induced EMT increased the expression of the CCL20 cytokine in hepatoma cells. Furthermore, coculture of monocyte-derived macrophages with hypoxic hepatoma cells revealed that the expression of indoleamine 2, 3-dioxygenase (IDO) was induced in monocyte-derived macrophages in a CCL20-dependent manner. In turn, these IDO-expressing monocyte-derived macrophages suppressed T-cell proliferation and promoted the expansion of immunosuppressive regulatory T cells. Moreover, high CCL20 expression in HCC specimens was associated with PVTT and poor patient survival. Collectively, our findings suggest that the HIF-1α/CCL20/IDO axis in hepatocellular carcinoma is important for accelerating tumor metastasis through both the induction of EMT and the establishment of an immunosuppressive tumor microenvironment, warranting further investigation into the therapeutic effects of blocking specific nodes of this signaling network.

Background While recent clinical trials of combination immunotherapies for hepatocellular carcinoma (HCC) have shown promising clinical efficacy and survival improvements breakthroughs, there is still much room for further improvement. A key limiting factor for HCC immunotherapy is the intrinsic immunosuppression within the liver microenvironment, resulting in suboptimal priming of tumor-specific CD8 cytotoxic T cells and thus immune evasion by the tumor. Hence, identifying new key molecular pathways suppressing T-cell responses within the liver is critical for the rational design of more effective combination immunotherapies for HCC. Methods We identified the 5-HT2A serotonin receptor as a potential target for HCC immunotherapy in a chemical screening approach and validated that targeting 5-HT2A signaling could be a viable approach for HCC immunotherapy via in vitro and in vivo studies. Results Disruption of 5-HT2A signaling using either a selective antagonist small molecule, ketanserin, or by knockout of its coding gene Htr2a augments the cytotoxic effector phenotype of mouse CD8 T cells activated in vitro with immunosuppressive liver non-parenchymal cells. Ketanserin treatment of in vitro activated human CD8 T cells also increased expression of the cytotoxic effector molecules granzyme B and perforin. Abrogation of 5-HT2A signaling was associated with increased expression of cytotoxicity-related genes such as granzyme B and reduced expression of transcription factors downstream of MAP kinase signaling. In vivo, systemic ketanserin treatment significantly prolonged survival of HCC tumor-bearing mice and was non-inferior to α-programmed death ligand 1 (PD-L1)+α-vascular endothelial growth factor A (VEGFA) combination antibody treatment. Combining ketanserin with αPD-L1+αVEGFA antibodies also significantly prolonged survival relative to control-treated mice while preserving the occurrence of complete tumor regression observed with αPD-L1+αVEGFA treatment alone. Conclusions Together, our data describe a role for 5-HT2A as a negative regulator of the cytotoxic effector phenotype in CD8 T cells and highlight the therapeutic potential of targeting 5-HT2A for HCC immunotherapy.

ABSTRACT Interfering with tumor metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Featuring a rapid proliferation rate and exacerbated glycolysis, hepatocellular carcinoma (HCC) creates a highly hypoxic microenvironment with excessive production of lactic and carbonic acids. These metabolic conditions promote disease aggressiveness and cancer-related immunosuppression. The pH regulatory molecules work as a bridge between tumor cells and their surrounding milieu. Herein, we show that the pH regulatory molecules CAIX, CAXII and V-ATPase are overexpressed in the HCC microenvironment and that interfering with their pathways exerts antitumor activity. Importantly, the V-ATPase complex was expressed by M2-like tumor-associated macrophages. Blocking ex vivo V-ATPase activity established a less immune-suppressive tumor microenvironment and reversed the mesenchymal features of HCC. Thus, targeting the unique cross-talk between tumor cells and the tumor microenvironment played by pH regulatory molecules holds promise as a strategy to control HCC progression and to reduce the immunosuppressive pressure mediated by the hypoxic/acidic metabolism, particularly considering the potential combination of this strategy with emerging immune checkpoint-based immunotherapies.

BACKGROUND AIMS Hepatocellular carcinoma (HCC) remains a leading cause of cancer mortality in China. Anti-angiogenic drugs (AADs) plus immune checkpoint blockade (ICB) combination therapy shows considerable promise for HCC; however, its efficacy is hampered by immunosuppression within the tumor microenvironment. High endothelial venules (HEVs) facilitate lymphocyte migration and tumor infiltration. The aim was to study the formation, functional mechanisms, and clinical relevance of HEVs in the treatment of HCC with combination therapy. APPROACH RESULTS Single-nucleus RNA sequencing, flow cytometry, and immunohistochemistry revealed increased HEV expression and higher CD3⁺ T-cell infiltration in HCC tissue after combination AAD and ICB therapy. Multiplex immunohistochemistry and spatial analysis demonstrated that CCR7⁺CD8⁺ T cells were spatially associated with HEVs. Pseudotime analysis of human T cells and treatment of Hepa1-6 orthotopic liver tumor mouse models with CCR7+CD8+ T-cell transfusions were used to show that CCR7+CD8+ T cells can differentiate into cytotoxic effector T cells. The same models demonstrated that combination therapy activated VEGFC and non-canonical NF-κB pathways, promoting HEV formation. Kaplan-Meier analysis revealed that high HEV density correlated with improved clinical response and prolonged survival. CONCLUSIONS HEVs are pivotal in modulating immune activity within the HCC tumor microenvironment. Targeting the VEGFC-NF-κB (non- canonical)-HEV axis could be a promising therapeutic strategy to enhance antitumor immunity and improve outcomes in patients with HCC who are receiving combination AAD plus ICB therapy.

Hypoxia, immune evasion, and metabolic reprogramming are key features of hepatocellular carcinoma (HCC) that limit the efficacy of therapies. Members of the solute carrier (SLC) family regulate metabolite homeostasis within tumor cells to maintain tumor survival under stressed conditions like hypoxia. Investigating the SLC family members could offer insights into the hypoxic microenvironment of HCC and potentially help identify improved therapeutic strategies. Here, we identified SLC27A2 as a hypoxia-responsive gene frequently downregulated in HCC, correlating with increased aggressiveness and adverse outcomes. HCC cells with low SLC27A2 expression exhibited enhanced hypoxia tolerance and resistance to hypoxia-related therapies, such as TACE and anti-angiogenic agents. Mechanistically, SLC27A2 downregulation led to reduced long-chain fatty acid (LCFA) intake, preventing LCFA accumulation under hypoxic conditions and mitigating cytotoxicity caused by lipid peroxidation. SLC27A2-deficient HCC cells were sensitive to glutaminase and fatty acid synthase inhibitors. The LCFAs in SLC27A2-deficient HCC were taken up by tumor-associated macrophages (TAMs), activating PPAR-γ transcriptional activity and promoting the enrichment of SPP1+ TAMs. Although this process led to immunosuppression, it presented an opportunity for applying anti-PD-1 therapy. In conclusion, the redistribution of LCFAs between HCC cells and TAMs, mediated by low SLC27A2 expression in cancer cells, supports adaptation to stress, promoting tumor progression and immunosuppression. The redistribution of LCFAs introduces molecular vulnerabilities and specific therapeutic opportunities for HCC.

Background Targeted therapies directed at tumor immune checkpoint, like programmed death-ligand (PD-L)1/programmed death (PD)-1, have shown remarkable progress. Nevertheless, treatment efficacy in hepatocellular carcinoma (HCC) is notably compromised due to the intricate immune microenvironment. Exploring alternative checkpoints beyond PD-L1/PD-1, including those not located on the cell surface, may improve our understanding of their roles in areas like diagnostic potential and immune tolerance in HCC. Aims To explore the roles of serum exosomal CD155 (exo-CD155) in HCC. Study Design Experimental study. Methods We separated and analyzed serum exosomes from HCC patients. We quantified the concentrations of serum soluble CD155 (sCD155) and serum exo-CD155, and examined their association with disease progression, hepatitis B surface antigen (HBsAg) presence, and the concentrations of α-fetoprotein fraction L3 (AFP-L3) or alpha-fetoprotein (AFP). Additionally, we assessed the diagnostic effect through the receiver operating characteristic (ROC) curve, and the immune suppressive effect on natural killer (NK) cells of exo-CD155. Results This study reveal elevated exo-CD155 levels in all HCC patients, with a significant increase in early-stage patients, exhibiting normal AFP/AFP-L3 or HBsAg-positive status. Exo-CD155 is linked to the progression of HCC and shows significant diagnostic effectiveness for the disease. Furthermore, the incubation of NK-92MI with exosomes derived from HCC patients leads to a substantial reduction in immune function, which can be partially counteracted with an antibody that blocks T cell immune receptor immunoglobulin and ITIM domains, (TIGIT)-blocking antibody. Conclusion These results disclose exo-CD155 shows promise for serving as a biomarker for HCC, especially in early-stage patients or those with normal AFP/AFP-L3 levels. Moreover, serum exosomes from HCC patients suppress NK cell immune functions through the TIGIT/CD155 pathway, contributing to immune tolerance in HCC.

The collagen β (1-O) glycosyltransferase 25 domain 1 (GLT25D1), a crucial collagen-modifying enzyme (CME), plays a pivotal role in multiple pathophysiological processes. However, its prognostic and biological roles in hepatocellular carcinoma (HCC) have not been reported. CME-related genes (CMEGs) were obtained from the Molecular Signatures Database (MSigDB), differentially expressed CMEGs (DECMEGs) and prognostic ones were identified. GLT25D1 expression was determined at the mRNA and protein levels in multiple datasets and in our HCC cohort. Its prognostic performance was evaluated and the immune microenvironment was investigated. The effects of GLT25D1 on tumorigenesis were further explored via in vitro and in vivo experiments. Four potential prognosis-associated DECMEGs, including GLT25D1, were identified. GLT25D1 was noticeably up-regulated in HCC tissues and significantly associated with advanced tumor grade and stage. Enrichment analysis revealed that GLT25D1 could participate in regulating immune responses and various carcinogenic processes. HCC patients with high GLT25D1 expression had decreased CD8+ T cells and increased M0 macrophages, leading to an immunosuppressive microenvironment. Our in vivo and in vitro experiments confirmed the increased GLT25D1 expression, and GLT25D1 knockdown impaired the HCC malignant phenotypes. Our results showed that GLT25D1 could be a carcinogenic indicator reflecting poor prognosis and might serve as a potential risk biomarker for HCC patients.

No abstract available

The treatment of hepatocellular carcinoma (HCC) is particularly challenging due to the inherent tumoral heterogeneity and easy resistance towards chemotherapy and immunotherapy. Arsenic trioxide (ATO) has emerged as a cytotoxic agent effective for treating solid tumors, including advanced HCC. However, its effectiveness in HCC treatment remains limited, and the underlying mechanisms are still uncertain. Therefore, this study aimed to characterize the effects and mechanisms of ATO in HCC. By evaluating the susceptibilities of human and murine HCC cell lines to ATO treatment, we discovered that HCC cells exhibited a range of sensitivity to ATO treatment, highlighting their inherent heterogeneity. A gene signature comprising 265 genes was identified to distinguish ATO-sensitive from ATO-insensitive cells. According to this signature, HCC patients have also been classified and exhibited differential features of ATO response. Our results showed that ATO treatment induced reactive oxygen species (ROS) accumulation and the activation of multiple cell death modalities, including necroptosis and ferroptosis, in ATO-sensitive HCC cells. Meanwhile, elevated tumoral immunogenicity was also observed in ATO-sensitive HCC cells. Similar effects were not observed in ATO-insensitive cells. We reported that ATO treatment induced mitochondrial injury and mtDNA release into the cytoplasm in ATO-sensitive HCC tumors. This subsequently activated the cGAS-STING-IFN axis, facilitating CD8+ T cell infiltration and activation. However, we found that the IFN pathway also induced tumoral PD-L1 expression, potentially antagonizing ATO-mediated immune attack. Additional anti-PD1 therapy promoted the anti-tumor response of ATO in ATO-sensitive HCC tumors. In summary, our data indicate that heterogeneous ATO responses exist in HCC tumors, and ATO treatment significantly induces immunogenic cell death (ICD) and activates the tumor-derived mtDNA-STING-IFN axis. These findings may offer a new perspective on the clinical treatment of HCC and warrant further study.

Background and aims Berberine is one of the most promising clinically tested natural alkaloids, and immunotherapy using natural killer (NK) cells is a potentially effective treatment for hepatocellular carcinoma (HCC). This study aims to elucidate the effect of berberine on the anti-HCC effect of NK92-MI cells. Materials and methods Human HCC SMMC-7721 and Hep3B cells were co-incubated with NK92-MI cells, berberine (60 or 80 μmol/L), or their combination for 36 h. To evaluate the killing effect of NK92-MI cells on HCC cells, the release of lactate dehydrogenase (LDH) in HCC cells was measured. The anti-tumor effects of berberine, NK92-MI cells, and their combinations were evaluated by MTS, EdU, Tunel, and Western blot assays. A male BALB/c nude mouse subcutaneous tumor model was used to investigate the anti-HCC effect of berberine and NK92-MI cells in vivo. Results The LDH assay showed that berberine enhanced the cytotoxicity of NK92-MI cells on HCC cells. The combination of berberine and NK92-MI cells demonstrated more obvious anti-HCC effect than did the berberine or NK92-MI single treatment in inhibiting cell proliferation and inducing apoptosis both in vitro and in vivo. Mechanistically, the expression of programmed cell death-ligand 1 (PD-L1) in HCC cells was upregulated after co-culture with NK-92MI cells. PD-L1 expression was knocked down, thereby inhibiting the proliferation and promoting apoptosis of HCC cells, and inhibited by berberine that blocked the secretion of interferon gamma (IFN-γ), thereby enhancing the anti-tumor effect of NK92-MI cells. Conclusions Current data show that the immunomodulatory role of berberine is to enhance the cytotoxic effect of NK92-MI cells and inhibit tumor immune escape by reducing the expression of PD-L1. Our study provides a rationale for the clinical application of berberine in combination with NK cells for the treatment of HCC.

Evidence recently showed that pleiotropic cytokine interferon-gamma (IFN-γ) in the tumor microenvironment (TME) plays a positive role in hepatocellular carcinoma (HCC) progression through the regulation of liver cancer stem cells (LCSCs) in HCC. The present study explored the role and potential mechanism of mitochondrial programmed cell death-ligand 1 (PD-L1) and its regulation of ferroptosis in modulating the cancer stemness of LCSCs. It was shown that mimicking TME IFN-γ exposure increased the LCSCs ratio and cancer stemness phenotypes in HCC cells. IFN-γ exposure inhibited sorafenib (Sora)-induced ferroptosis by enhancing glutathione peroxidase 4 (GPX4) expression as well reactive oxygen species (ROS) and lipid peroxidation (LPO) generation in LCSCs. Furthermore, IFN-γ exposure upregulated PD-L1 expression and its mitochondrial translocation, inducing dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and correlating with glycolytic metabolism reprogramming in LCSCs. The genetic intervention of PD-L1 promoted ferroptosis-dependent antitumor effects of Sora, reduced glycolytic metabolism reprogramming, and inhibited cancer stemness of HCC in vitro and in vivo. Our results revealed a novel mechanism that IFN-γ exposure-induced mitochondrial translocation of PD-L1 enhances glycolytic reprogramming to mediate the GPX4-dependent ferroptosis resistance and cancer stemness in LCSCs. This study provided new insights into the role of mitochondrial PD-L1- Drp1-GPX4 signal axis in regulating IFN-γ exposure-associated cancer stemness in LCSCs and verified that PD-L1-targeted intervention in combination with Sora might achieve promising synergistic anti-HCC effects.

The PD-L1/PD-1 axis is a classic immunotherapy target. However, anti-PD-L1/PD-1 therapy alone can not achieve satisfactory results in solid tumors, especially liver cancer. Among the several factors involved in tumor anti-PD-L1/PD-1 treatment resistance, tumor-associated macrophages (TAMs) have attracted attention because of their immunosuppressive ability. TAMs with a macrophage receptor with a collagenous structure (MARCO) are a macrophage subset group with strong immunosuppressive abilities. Clinical specimens and animal experiments revealed a negative correlation between MARCO + TAMs and patient prognosis with liver cancer. Transcriptional data and in vitro and in vivo experiments revealed that MARCO + TAM immunosuppressive ability was related to secretion. MARCO suppressed IFN-β secretion from TAMs, reducing antigen presentation molecule expression, infiltration, and CD8+T cell dysfunction, thus producing an immunosuppressive microenvironment in liver cancer. MARCO can promote dying tumor cell clearance by macrophages, reducing tumor-derived cGAMP and ATP accumulation in the tumor microenvironment and inhibiting sting-IFN-β pathway activation mediated by P2X7R in MARCO+TAMs. Animal experiments revealed that the MARCO and PD-L1 monoclonal antibody combination could significantly inhibit liver cancer growth. Conclusively, targeting MARCO+TAMs can significantly improve anti-PD-L1 resistance in liver cancer, making it a potential novel immune target for liver cancer therapy.

Hepatocellular carcinoma (HCC) emerges from chronic inflammation, to which activation of hepatic stellate cells (HSCs) contributes by shaping a pro-tumorigenic microenvironment. Key to this process is p62, whose inactivation leads to enhanced hepatocarcinogenesis. Here, we show that p62 activates the interferon (IFN) cascade by promoting STING ubiquitination by tripartite motif protein 32 (TRIM32) in HSCs. p62, binding neighbor of BRCA1 gene 1 (NBR1) and STING, triggers the IFN cascade by displacing NBR1, which normally prevents the interaction of TRIM32 with STING and its subsequent activation. Furthermore, NBR1 also antagonizes STING by promoting its trafficking to the endosome-lysosomal compartment for degradation independent of autophagy. Of functional relevance, NBR1 deletion completely reverts the tumor-promoting function of p62-deficient HSCs by rescuing the inhibited STING-IFN pathway, thus enhancing anti-tumor responses mediated by CD8+ T cells. Therefore, NBR1 emerges as a synthetic vulnerability of p62 deficiency in HSCs by promoting the STING/IFN pathway, which boosts anti-tumor CD8+ T cell responses to restrain HCC progression.

No abstract available

BACKGROUND Hepatocellular carcinoma (HCC) is a common cancer that is fatal and has a dismal prognosis. Obovatol (Ob), a novel lignan derived from the leaf and stem bark of Magnolia obovata Thunb, has exhibited anti-tumor effect on diverse tumors. However, its effect and mechanisms on HCC remain to be further explored. METHODS Huh7 and Hep3B cells, as well as BALB/c nude mice were used to determine the function and mechanisms of Ob on growth, invasion and immune escape by cell counting kit-8, transwell, enzyme-linked immunosorbent assay (ELISA) and western blot experiments. RESULTS Ob reduced the cell viability of Huh7 and Hep3B cells, with a IC50 value of 57.41 µM and 62.86 µM, respectively. Ob declined the invasion ability, the protein expression of N-cadherin and the concentrations of IL-10 and TGF-β, whereas increased the E-cadherin expression and the contents of IFN-γ and IL-2 in Hep3B and Huh7 cells. Mechanically, Ob decreased the protein level of p-JAK/JAK, p-STAT3/STAT3 and PD-L1, which was partly restored with the treatment of RO8191, an activator of JAK/STAT3 axis. The effect of Ob on the cell viability, the invasion ability, the protein level of N-cadherin and E-cadherin, and the concentrations of IL-10, TGF-β, IFN-γ and IL-2 in both Hep3B and Huh7 cells was reversed with the management of RO8191. In vivo, Ob reduced tumor volume and weight, the level of N-cadherin, PD-L1, p-JAK/JAK, and p-STAT3/STAT3, with an elevated expression of E-cadherin and IFN-γ. CONCLUSION Ob downregulated the JAK/STST3/PD-L1 pathway to attenuate the growth, invasion and immune escape of HCC.

Immunotherapy using anti-programmed death 1 ligand 1 (PD-L1) antibodies has shown clinical efficacy against hepatocellular carcinoma (HCC) and is recognized as the first-line treatment for unresectable HCC. PD-L1 expression is affected by various cytokines produced by immune cells in the tumor microenvironment; however, there is limited information about the effects of cytokine interactions on PD-L1 expression. In this study, we examined how cytokines induce PD-L1 expression in HCC cells. Both interferon gamma (IFN-γ) and interleukin 1 beta (IL-1β) induced PD-L1 expression, and the two cytokines enhanced PD-L1 expression in combination compared to that when administered alone. The Janus kinase/signal transducer and activator of transcription signaling pathway activated by IFN-γ is the major pathway of PD-L1 expression. The increase in interferon regulatory factor 1 expression and IFN-γ receptor expression induced by IL-1β was associated with the synergistic effect of IFN-γ and IL-1β on PD-L1 expression. These findings strongly indicate that IFN-γ and IL-1β affect the mechanism underlying immune resistance in HCC cells.

Hepatocellular carcinoma (HCC) is one of the most fatal tumours worldwide and has a high recurrence rate. Nevertheless, the mechanism of HCC genesis remains partly unexplored, while the efficiency of HCC treatments remains limited. The present study analysed the expression of nuclear receptor subfamily 4 group A member 1 (NR4A1) in tumour‐infiltrating natural killer (NK) cells derived from both human patients with HCC and tumour‐bearing mouse models, as well as the features of NR4A1high and NR4A1low NK cells. In addition, knockout of NR4A1 by CRISPR/Cas9 and adoptive transfer experiments were applied to verify the function of NR4A1 in both tumour‐infiltrating NK cells and anti‐PD‐1 therapy. The present study found that NR4A1 was significantly highly expressed in tumour‐infiltrating NK cells, which mediated the dysfunction of tumour‐infiltrating NK cells by regulating the IFN‐γ/p‐STAT1/IRF1 signalling pathway. Knockout of NR4A1 in NK cells not only restored the antitumour function of NK cells but also enhanced the efficacy of anti‐PD‐1 therapy. The present findings suggest a regulatory role of NR4A1 in the immune progress of NK cells against HCC, which may provide a new direction for immunotherapies of HCC.

Immunotherapies such as immune checkpoint blockade have achieved remarkable success in treating cancer. Unfortunately, response rates have been limited in multiple cancers including hepatocellular carcinoma (HCC). The critical function of epigenetics in tumor immune evasion and anti-tumor immunity supports harnessing epigenetic regulators as a potential strategy to enhance the efficacy of immunotherapy. Here, we discovered a tumor-promoting function of FTSJ3, an RNA 2'-O-methyltransferase, in HCC by suppressing anti-tumor immune responses. FTSJ3 was upregulated in hepatocellular carcinoma, and high FTSJ3 expression correlated with reduced patient survival. Deletion of FTSJ3 blocked HCC growth and induced robust anti-tumor immune responses. Mechanistically, FTSJ3 suppressed double-stranded RNA (dsRNA)-induced IFNβ signaling in a 2'-O-methyltransferase manner. Deletion of RNA sensors in HCC cells or systemic knockout of type I IFN receptor IFNAR in mice rescued the in vivo tumor growth defect caused by FTSJ3 deficiency, indicating that FTSJ3 deletion suppresses tumor growth by activating the RNA sensor-mediated type I IFN pathway. Furthermore, FTSJ3 deletion significantly enhanced the efficacy of PD-1 immune checkpoint blockade. The combination of FTSJ3 deficiency and anti-PD-1 antibody treatment effectively eradicated tumors and increased the survival time. In conclusion, this study reveals an epigenetic mechanism of tumor immune evasion and, importantly, suggests FTSJ3-targeting therapies as potential approach to overcome immunotherapy resistance in HCC patients.

Background Genomic screening uncovered interferon-gamma (IFNγ) pathway defects in tumours refractory to immune checkpoint blockade (ICB). However, its non-mutational regulation and reversibility for therapeutic development remain less understood. Objective We aimed to identify ICB resistance-associated druggable histone deacetylases (HDACs) and develop a readily translatable combination approach for patients with hepatocellular carcinoma (HCC). Design We correlated the prognostic outcomes of HCC patients from a pembrolizumab trial (NCT03419481) with tumourous cell expressions of all HDAC isoforms by single-cell RNA sequencing. We investigated the therapeutic efficacy and mechanism of action of selective HDAC inhibition in 4 ICB-resistant orthotopic and spontaneous models using immune profiling, single-cell multiomics and chromatin immunoprecipitation-sequencing and verified by genetic modulations and co-culture systems. Results HCC patients showing higher HDAC1/2/3 expressions exhibited deficient IFNγ signalling and poorer survival on ICB therapy. Transient treatment of a selective class-I HDAC inhibitor CXD101 resensitised HDAC1/2/3high tumours to ICB therapies, resulting in CD8+T cell-dependent antitumour and memory T cell responses. Mechanistically, CXD101 synergised with ICB to stimulate STAT1-driven antitumour immunity through enhanced chromatin accessibility and H3K27 hyperacetylation of IFNγ-responsive genes. Intratumoural recruitment of IFNγ+GZMB+cytotoxic lymphocytes further promoted cleavage of CXD101-induced Gasdermin E (GSDME) to trigger pyroptosis in a STAT1-dependent manner. Notably, deletion of GSDME mimicked STAT1 knockout in abolishing the antitumour efficacy and survival benefit of CXD101-ICB combination therapy by thwarting both pyroptotic and IFNγ responses. Conclusion Our immunoepigenetic strategy harnesses IFNγ-mediated network to augment the cancer-immunity cycle, revealing a self-reinforcing STAT1-GSDME pyroptotic circuitry as the mechanistic basis for an ongoing phase-II trial to tackle ICB resistance (NCT05873244).

Summary Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor of hepatocellular carcinoma (HCC). However, the mechanism and target therapy of NAFLD-HCC are still unclear. Here, we identify that the N6-methyladenosine (m6A) methyltransferase METTL3 promotes NAFLD-HCC. Hepatocyte-specific Mettl3 knockin exacerbated NAFLD-HCC formation, while Mettl3 knockout exerted the opposite effect in mice. Single-cell RNA sequencing revealed that METTL3 suppressed antitumor immune response by reducing granzyme B (GZMB+) and interferon gamma-positive (IFN-γ+) CD8+ T cell infiltration, thereby facilitating immune escape. Mechanistically, METTL3 mediates sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) mRNA m6A to promote its translation, leading to the activation of cholesterol biosynthesis. This enhanced secretion of cholesterol and cholesteryl esters that impair CD8+ T cell function in the tumor microenvironment. Targeting METTL3 by single-guide RNA, nanoparticle small interfering RNA (siRNA), or pharmacological inhibitor (STM2457) in combination with anti-programmed cell death protein 1 (PD-1) synergized to reinvigorate cytotoxic CD8+ T cells and mediate tumor regression. Together, METTL3 is a therapeutic target in NAFLD-HCC, especially in conjunction with immune checkpoint blockade (ICB) therapy.

No abstract available

BACKGROUND Liver cancer, particularly hepatocellular carcinoma (HCC), is characterized by a high mortality rate, attributed primarily to the establishment of an immunosuppressive microenvironment. Within this context, we aimed to elucidate the pivotal role of eukaryotic elongation factor 2 kinase (eEF2K) in orchestrating the infiltration and activation of natural killer (NK) cells within the HCC tumor microenvironment. By shedding light on the immunomodulatory mechanisms at play, our findings should clarify HCC pathogenesis and help identify potential therapeutic intervention venues. METHODS We performed a comprehensive bioinformatics analysis to determine the functions of eEF2K in the context of HCC. We initially used paired tumor and adjacent normal tissue samples from patients with HCC to measure eEF2K expression and its correlation with prognosis. Subsequently, we enrolled a cohort of patients with HCC undergoing immunotherapy to examine the ability of eEF2K to predict treatment efficacy. To delve deeper into the mechanistic aspects, we established an eEF2K-knockout cell line using CRISPR/Cas9 gene editing. This step was crucial for verifying activation of the cGAS-STING pathway and the subsequent secretion of cytokines. To further elucidate the role of eEF2K in NK cell function, we applied siRNA-based techniques to effectively suppress eEF2K expression in vitro. For in vivo validation, we developed a tumor-bearing mouse model that enabled us to compare the infiltration and activation of NK cells within the tumor microenvironment following various treatment strategies. RESULTS We detected elevated eEF2K expression within HCC tissues, and this was correlated with an unfavorable prognosis (30.84 vs. 20.99 months, P = 0.033). In addition, co-culturing eEF2K-knockout HepG2 cells with dendritic cells led to activation of the cGAS-STING pathway and a subsequent increase in the secretion of IL-2 and CXCL9. Moreover, inhibiting eEF2K resulted in notable NK cell proliferation along with apoptosis reduction. Remarkably, after combining NH125 and PD-1 treatments, we found a significant increase in NK cell infiltration within HCC tumors in our murine model. Our flow cytometry analysis revealed reduced NKG2A expression and elevated NKG2D expression and secretion of granzyme B, TNF-α, and IFN-γ in NK cells. Immunohistochemical examination confirmed no evidence of damage to vital organs in the mice treated with the combination therapy. Additionally, we noted higher levels of glutathione peroxidase and lipid peroxidation in the peripheral blood serum of the treated mice. CONCLUSION Targeted eEF2K blockade may result in cGAS-STING pathway activation, leading to enhanced infiltration and activity of NK cells within HCC tumors. The synergistic effect achieved by combining an eEF2K inhibitor with PD-1 antibody therapy represents a novel and promising approach for the treatment of HCC.

Background Indoleamine-2,3-dioxygenase 1 (IDO1) is responsible for tumor immune escape by regulating T cell-associated immune responses and promoting the activation of immunosuppressive. Given the vital role of IDO1 in immune response, further investigation on the regulation of IDO1 in tumors is needed. Methods Herein, we used ELISA kit to detect the interferon-gamma (IFN-γ), Tryptophan (Trp), and kynurenic acid (Kyn) levels; western blot, Flow cytometry, and immunofluorescence assays detected the expression of the proteins; Molecular docking assay, SPR assay and Cellular Thermal Shift Assay (CETSA) were used to detect the interaction between IDO1 and Abrine; nano live label-free system was used to detect the phagocytosis activity; tumor xenografts animal experiments were used to explore the anti-tumor effect of Abrine; flow cytometry detected the immune cells changes. Results The important immune and inflammatory response cytokine interferon-gamma (IFN-γ) up-regulated the IDO1 expression in cancer cells through the methylation of 6-methyladenosine (m6A) m6A modification of RNA, metabolism of Trp into Kyn, and JAK1/STAT1 signaling pathway, which could be inhibited by IDO1 inhibitor Abrine. CD47 is IFN-γ-stimulated genes (ISGs) and prevents the phagocytosis of macrophages, leading to the cancer immune escape, and this effect could be inhibited by Abrine both in vivo and in vitro. The PD-1/PD-L1 axis is an important immune checkpoint in regulating immune response, overexpression of PD-1 or PD-L1 promotes immune suppression, while in this study Abrine could inhibit the expression of PD-L1 in cancer cells or tumor tissue. The combination treatment of Abrine and anti-PD-1 antibody has a synergistic effect on suppressing the tumor growth through up-regulating CD4+ or CD8+ T cells, down-regulating the Foxp3+ Treg cells, and inhibiting the expression of IDO1, CD47, and PD-L1. Conclusion Overall, this study reveals that Abrine as an IDO1 inhibitor has an inhibition effect on immune escape and has a synergistic effect with the anti-PD-1 antibody on the treatment of HCC.

Background Hepatocellular carcinoma (HCC) is one of the most common malignancies with a high lethality rate. ZMIZ2 is a transcriptional co-activator implicated in various human diseases. However, the role and molecular mechanism of ZMIZ2 in HCC remains to be elucidated. Methods The expression and prognostic value of ZMIZ2 in HCC was excavated from public databases and explored by bioinformatic analysis. Then the expression of ZMIZ2 and related genes was further validated by quantitative RT-PCR, western blotting, and immunohistochemistry. Loss and gain-of-function experiments were performed in vitro and in vivo to investigate the function of ZMIZ2 in HCC. In addition, transcriptome sequencing and immunoprecipitation was conducted to explore the potential molecular mechanisms of ZMIZ2. Results ZMIZ2 was highly expressed in HCC and associated with poor prognosis. Silencing ZMIZ2 significantly inhibited HCC cell proliferation, cell cycle process, migration, and invasion in vitro, and also inhibited the progression of HCC in vivo. Additionally, ZMIZ2 expression was correlated with immune cell infiltration in HCC samples. Somatic mutation analysis showed that ZMIZ2 and TP53 mutations jointly affected the progression of HCC. Mechanistically, ZMIZ2 interacted with LEF1 to regulate malignant progression of HCC by activating the Wnt/β-catenin pathway. Conclusion ZMIZ2 was overexpressed in HCC and associated with poor prognosis. The overexpression of ZMIZ2 was corelated with malignant phenotype, and it facilitated HCC progression via LEF1-mediated activation of the Wnt/β-catenin pathway. Furthermore, ZMIZ2 could be served as a prognostic biomarker and a new therapeutic target for HCC.

Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, effective therapeutic strategy. Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, antiviral, and antitumor effects. Although IFN-γ is a promising antitumor agent, its application is limited by resistance in tumor cells. A20 is a zinc-finger protein that was initially identified as a gene product induced by tumor necrosis factor α in human umbilical vein endothelial cells. In this study, we found that silencing of A20 combined with IFN-γ significantly represses cell viability, and induces apoptosis and cell-cycle arrest in HCC cells. By investigating mechanisms implicated in A20 and IFN-γ-mediated signaling pathways, we revealed that the phosphoinositide 3-kinase/Akt signaling pathway and antiapoptotic B-cell lymphoma 2 proteins were repressed. Moreover, we also found that phosphorylation of STAT1 and STAT3 was significantly enhanced after the downregulation of A20 in combination with treatment of IFN-γ. Inhibitor of STAT1 but not STAT3 could block the antitumor effect of IFN-γ. Therefore, targeting A20 enhances the cytotoxicity of IFN-γ against HCC cells and may present a promising therapeutic strategy for HCC.

Background This is a phase II clinical trial to investigate the immunotherapeutic effect of Curcumin, Piperine, and Taurine (CPT) combination in hepatocellular carcinoma (HCC). Methods Twenty-six HCC patients aged (50–80 years) were recruited for administration of a daily dose of 5 g of curcumin, 50 mg of piperine, and 500 mg of taurine divided into three doses for successive 3 months. The three components (CPT) were prepared in one capsule. Patients were assessed after each month (cycle) for the plasma levels of CD4, CD8, CD25, Interleukins-2 (IL-2), IL-6, IL-12, Interferon-gamma (IFN- γ), Lactate dehydrogenase (LDH), and Vascular endothelial growth factor (VEGF), FOXP3 mRNA, and miRNA 21 . Results There was a significant increase in the plasma levels of CD4 and CD8, while a significant decrease in the CD25 level after the second and third cycles compared to the baseline level [P < 0.001 for both]. Also, there was a significant increase in the plasma levels of IL-2, IL-12, and IFN-γ [ P = 0.001, P = 0.006, and P = 0.029; respectively], while there was a significant decrease in IL-6, VEGF-α, LDH, and Alpha-fetoprotein (AFP) after CPT administration compared to the baseline levels [P < 0.001, P < 0.001, P = 0.020, and P = 0.004; respectively]. The expression level of miRNA-21 was significantly decreased after CPT administration compared to the baseline level [5.5±0.88, 4.1±0.78, 3±0.75, and 2.5±0.76; respectively, P<0.001]. Though there was a noticeable decrease in the FOXP3 expression after each cycle, however, it didn’t reach a significant level [5.3±0.8, 4.2±0.76, 3.2±0.67, and 2.5±0.79; respectively, P=0.184]. Conclusion CPT could exhibit a potential immune-stimulating effect in HCC patients. The current trial had been registered at the National Hepatology and Tropical Medicine Research Institute (NHTMRI), with a registration number of NHTMRI-IRB 2-21 on 5th January 2021.

Simple Summary We studied the relationship between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in 749 hepatocellular carcinoma (HCC) patients from 5 cohorts using a DNA repair pathway score. We show that the DNA repair pathway was enhanced by the stepwise carcinogenic process of HCC, notably in grade 3 compared to grade 1 or 2 HCC. DNA repair high HCC was associated with worse survival, elevated intratumor heterogeneity, and mutation load, but not with the fraction of immune cell infiltration nor cytolytic activity. The expression of proliferation- and other cancer aggressiveness-related gene sets was also increased. Interestingly, these features were more pronounced in low-grade compared to high-grade HCC. In conclusion, the DNA repair score may be used to understand the role of DNA repair pathways in patient prognosis and treatment sensitivity and be used to improve patient outcome. To our knowledge, this is the first study using DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC. Abstract Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related deaths worldwide. In this study, a total of 749 HCC patients from 5 cohorts were studied to examine the relationships between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in HCC, utilizing a DNA repair pathway score. Our findings suggest that the DNA repair pathway was not only enhanced by the stepwise carcinogenic process of HCC, but also significantly enhanced in grade 3 HCC compared with grade 1 and 2 tumors. DNA repair high HCC was associated with worse survival, elevated intratumor heterogeneity, and mutation load, but not with the fraction of immune cell infiltration nor immune response. HCC tumors with a DNA repair high score enriched the cell proliferation- and other cancer aggressiveness-related gene sets. Interestingly, these features were more pronounced in grade 1 and 2 HCC compared to grade 3 HCC. To our knowledge, this is the first study to use DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC. The DNA repair score may be used to better understand and predict prognosis in HCC.

Di(2-ethylhexyl) phthalate (DEHP), as an endocrine disruptor, is often used as a plasticizer in various polyvinyl chloride plastic products and medical consumables. Epidemiological studies have shown that long-term large intake of DEHP may be a risk factor for liver dysfunction. Long-term exposure to DEHP is associated with liver disease and aggravates the progression of chronic liver injury. However, the effects of DEHP on hepatocellular carcinoma (HCC) are rarely studied. In this study, we sought to determine the effects of DEHP on HCC induced by carbon tetrachloride combined with diethylnitrosamine, and further study its molecular mechanism. It was found that DEHP exposure significantly promotes tumor immune escape and activates signaling pathways involved in related protein expression of tumor immune escape, including PD-L1, JAK2, and STAT3. In addition, the trends observed in the HepG2 cells assay are consistent with vivo conditions. In summary, DEHP may play a tumor-promoting role in HCC mice and IFN-γ stimulated HepG2 cells, which may be related to the JAK2/STAT3 signaling pathway.

Background Steatotic liver disease-related hepatocellular carcinoma (SLD-HCC), a rising global challenge, is characterised by unique tumour microenvironment (TME) adaptations. Objective This study investigates the immune microenvironment and interactions driving immunosuppression and potential resistance to immunotherapy in SLD-HCC. Design We employed single-cell transcriptomics, cytometry by time-of-flight (CyTOF) and two independent spatial transcriptomics platforms to study the TME of 22 SLD-HCC and 31 non-SLD-HCC cases. Findings were further validated using multiplex immunohistochemistry in an independent cohort of 103 patients, an HCC model and an immunotherapy-treated patient cohort to evaluate clinical relevance. Results Our findings revealed significant alterations in immune and lipid metabolism pathways, particularly in regulatory T cells (Tregs) and cancer-associated fibroblasts (CAFs), suggesting distinct cellular adaptations to a high-fat TME and general immunosuppression. CyTOF revealed a cold, immunosuppressive TME with reduced CD8+ T cells and increased Tregs. Spatial transcriptomics further highlighted distinct Treg–CAF clusters localised at tumour margins, suggesting a spatially organised immunosuppressive niche. Mechanistically, tumour necrosis factor superfamily member 14 (TNFSF14)-tumour necrosis factor receptor superfamily member 14 (TNFRSF14)-mediated Treg–CAF interaction was identified as a critical driver of immunotherapy resistance in SLD-HCC. Blocking TNFRSF14 in an HCC model fed with a high-fat diet resulted in reduced Tregs, increased active CD8+ and memory CD4+ T cells, and a synergistic effect with anti-programmed cell death protein 1 therapy to enhance antitumour immunity and overcome immunotherapy resistance in SLD-HCC. Conclusion This study uncovers critical immune and metabolic adaptations in SLD-HCC, identifying TNFSF14-TNFRSF14 signalling as a key driver of immunotherapy resistance. Targeting this signalling axis enhances antitumour immunity and improves immunotherapy efficacy, offering a promising therapeutic strategy for SLD-HCC.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide. Unfortunately, the inherently immunosuppressive tumor microenvironment (TME) severely limits the anti-tumor efficacy of conventional therapies. Immunogenic cell death (ICD) is a rare immunostimulatory form of cell death and can activate adaptive immune responses as a way to remodel the immunosuppressive tumor microenvironment. In this study, we found that bufadienolide-based nanocrystalline formulations (HBE NCs) can reshape the tumor microenvironment by inducing an ICD, thereby achieving superior anti-tumor efficacy. HBE NCs could effectively induce Hepa1-6 cells to release damage-associated molecular patterns, such as CRT as an "eat me" signal, HMGB1 as a "danger" signal and ATP as a "find me" signal to an active immune system. HBE NCs improved the maturation of dendritic cells, enhanced the infiltration of CD4+T and CD8+T cells, depleted the number of immunosuppressive MDSCs and Treg cells, and successfully reconstructed immunosuppressed "cold" tumors into immune-activated "hot" tumors in HCC mice. Notably, dying Hepa1-6 cells treated with HBE NCs can be used as an in vivo "inactivated vaccine" to effectively enhance the ability of mice to resist the challenge of a live tumor. Our study demonstrates a promising strategy for remodeling the immunosuppressive TME of liver cancer.

No abstract available

N6-methyladenosine (m6A) is important in regulating mRNA stability, splicing, and translation, and it also contributes to tumor development. However, there is still limited understanding of the comprehensive effects of m6A modification patterns on the tumor immune microenvironment, metabolism, and drug resistance in hepatocellular carcinoma (HCC). In this study, we utilized unsupervised clustering based on the expression of 23 m6A regulators to identify m6A clusters. We identified differential m6A modification patterns and characterized m6A-gene-cluster A, which exhibited poorer survival rates, a higher abundance of Treg cells, and increased expression of TGFβ in the tumor microenvironment (TME). Additionally, m6A-gene-cluster A demonstrated higher levels of glycolysis activity, cholesterol metabolism, and fatty acid biosynthesis. We also found that the m6A score was associated with prognosis and drug resistance. Patients with a low m6A score experienced worse prognoses, which were linked to an abundance of Treg cells, upregulation of TGFβ, and increased metabolic activity. HCC patients with a higher m6A score showed improved prognosis following sorafenib treatment and immunotherapy. In conclusion, we reveals the association between m6A modification patterns and the tumor immune microenvironment, metabolism, and drug resistance in HCC. Furthermore, the m6A score holds potential as a predictive factor for the efficacy of targeted therapy and immunotherapy in HCC.

No abstract available

BACKGROUND & AIMS Regulatory T cell (Treg) depletion increases antitumor immunity. However, severe autoimmunity can occur following systemic loss of Tregs, which could be avoided by selectively depleting intratumoral Tregs. Herein, we aimed to investigate the role of tumor-infiltrating CCR4+ Tregs in hepatocellular carcinoma (HCC) and to provide a potential target strategy for immunotherapy. METHODS CCR4+ Tregs were analyzed by flow cytometry in murine models and clinical samples. The function of tumor-infiltrating and induced CCR4+ Tregs was interrogated by genetic and epigenetic approaches. To block CCR4+ Treg chemotaxis, we developed an N-terminus recombinant protein of CCR4 (N-CCR4-Fc) as a neutralizing pseudo-receptor that effectively bound to its ligand CCL22. The efficacy of CCR4 antagonism as an immunotherapeutic agent was evaluated by tumor weights, growth kinetics and survival curves. RESULTS CCR4+ Tregs were the predominant type of Tregs recruited to hepatitis B-associated HCC (HBV+ HCC), correlating with sorafenib resistance and HBV load titers. Compared with CCR4- Tregs, CCR4+ Tregs exhibited increased IL-10 and IL-35 expression, and enhanced functionality in suppressing CD8+ T cells. CCR4+ Tregs also displayed PD-1+TCF1+ stem-like properties. ATAC-seq data revealed substantial chromatin remodeling between tumor-infiltrating Tregs (TIL-Tregs) and induced Tregs, suggesting that long-term chromatin reprogramming accounted for the acquisition of enhanced immunosuppressive stem-like specificity by CCR4+ TIL-Tregs. Treatment with a CCR4 antagonist or N-CCR4-Fc blocked intratumoral Treg accumulation, overcame sorafenib resistance, and sensitized tumors to PD-1 checkpoint blockade. CONCLUSIONS Intratumoral stem-like CCR4+ Tregs orchestrated immunosuppressive resource cells in the tumor microenvironment. CCR4 could be targeted to enhance antitumor immunity by specifically blocking infiltration of Tregs into the tumor microenvironment and inhibiting maintenance of the TIL-Treg pool. LAY SUMMARY Targeting regulatory T cells is a promising approach in cancer immunotherapy; however, severe autoimmunity can occur following systemic regulatory T cell loss. This could be avoided by selectively depleting intratumoral regulatory T cells. Herein, targeting intratumoral stem-like CCR4+ regulatory T cells helped to overcome sorafenib resistance and sensitize tumors to immune checkpoint blockade in mouse models of liver cancer. This approach could have wide clinical applicability.

Background and Aims Transcriptome sequencing revealed high expression of DDR2 in oxaliplatin-resistant hepatocellular carcinoma (HCC). This study aimed to explore the role of DDR2 in oxaliplatin resistance and immune evasion in HCC. Methods Oxaliplatin-resistant HCC cell lines were established. The interaction between DDR2 and STAT3 was investigated, along with the mechanisms involved in DDR2/STAT3-mediated PD-L1 upregulation and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) accumulation both in vitro and in vivo. Results DDR2 was found to induce the phosphorylation of STAT3, leading to its nuclear translocation. Conversely, the activation of STAT3 enhanced DDR2 expression. A positive feedback loop involving DDR2/STAT3 was identified in oxaliplatin-resistant HCC, which was associated with PD-L1 upregulation and PMN-MDSCs accumulation. Knockdown of DDR2 and STAT3 sensitized oxaliplatin-resistant HCC cells to oxaliplatin and resulted in decreased PMN-MDSCs and increased CD8+ T cells in the tumor microenvironment. Enzyme-linked immunosorbent array and MDSC transwell migration assays indicated that oxaliplatin-resistant HCC cells recruited PMN-MDSCs through CCL20. Dual luciferase reporter assays demonstrated that STAT3 can directly enhance the transcription of PD-L1 and CCL20. Furthermore, treatment with a PD-L1 antibody in combination with CCL20 blockade had significant antitumor effects on oxaliplatin-resistant HCC. Conclusions Our findings revealed a positive feedback mechanism involving DDR2 and STAT3 that mediates the immunosuppressive microenvironment and promotes oxaliplatin resistance and immune evasion via PD-L1 upregulation and PMN-MDSC recruitment. Targeting the DDR2/STAT3 pathway may be a promising therapeutic strategy to overcome immune escape and chemoresistance in HCC.

While HCC is an inflammation-associated cancer, CRLM develops on permissive healthy liver microenvironment. To evaluate the immune aspects of these two different environments, peripheral blood-(PB), peritumoral-(PT) and tumoral tissues-(TT) from HCC and CRLM patients were evaluated. 40 HCC and 34 CRLM were enrolled and freshly TT, PT and PB were collected at the surgery. PB-, PT- and TT-derived CD4+CD25+ Tregs, M/PMN-MDSC and PB-derived CD4+CD25− T-effector cells (Teffs) were isolated and characterized. Tregs’ function was also evaluated in the presence of the CXCR4 inhibitor, peptide-R29, AMD3100 or anti-PD1. RNA was extracted from PB/PT/TT tissues and tested for FOXP3, CXCL12, CXCR4, CCL5, IL-15, CXCL5, Arg-1, N-cad, Vim, CXCL8, TGFβ and VEGF-A expression. In HCC/CRLM-PB, higher number of functional Tregs, CD4+CD25hiFOXP3+ was detected, although PB-HCC Tregs exert a more suppressive function as compared to CRLM Tregs. In HCC/CRLM-TT, Tregs were highly represented with activated/ENTPD-1+Tregs prevalent in HCC. As compared to CRLM, HCC overexpressed CXCR4 and N-cadherin/vimentin in a contest rich in arginase and CCL5. Monocytic MDSCs were highly represented in HCC/CRLM, while high polymorphonuclear MDSCs were detected only in HCC. Interestingly, the function of CXCR4-PB-Tregs was impaired in HCC/CRLM by the CXCR4 inhibitor R29. In HCC and CRLM, peripheral blood, peritumoral and tumoral tissues Tregs are highly represented and functional. Nevertheless, HCC displays a more immunosuppressive TME due to Tregs, MDSCs, intrinsic tumor features (CXCR4, CCL5, arginase) and the contest in which it develops. As CXCR4 is overexpressed in HCC/CRLM tumor/TME cells, CXCR4 inhibitors may be considered for double hit therapy in liver cancer patients.

Background Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death globally, characterized by high heterogeneity and drug resistance, which significantly impacts clinical outcomes. The tumor microenvironment (TME) plays a critical role in HCC initiation and progression, with immune cell infiltration and immune checkpoint expression closely linked to tumor prognosis. N-glycosylation of proteins modulates immune responses within the TME. ALG3, a key N-glycosylation enzyme, is involved in protein glycosylation. Although ALG3 expression has been studied in various tumors, its role in regulating the immune microenvironment and its prognostic significance in HCC remain unclear. Methods This study comprehensively evaluates ALG3 expression in HCC and its relationship with the immune microenvironment using various techniques. First, bioinformatics analysis of HCC-related data from the TCGA database was performed to investigate ALG3 expression patterns in tumor tissues and its correlation with clinical features. Multiplex immunohistochemistry (mIHC) was then used to validate ALG3 expression in HCC tissue samples and examine its relationship with immune cell infiltration. Additionally, cell experiments and 3D human organoid-based culture models were employed to further assess the role of ALG3 in the HCC immune microenvironment. Results The results showed significant overexpression of ALG3 in HCC tissues, with high expression correlating significantly with poor tumor prognosis. Further analysis revealed that high ALG3 expression was associated with reduced infiltration of CD8+ T cells and CD68+ macrophages in both tumor and stromal areas, while positively correlating with increased infiltration of FOXP3+ regulatory T cells (Tregs). Notably, ALG3 expression levels were also positively correlated with PD-L1 expression in HCC tissues. Conclusions ALG3 may serve as a potential prognostic biomarker and an immunotherapy target in HCC.

Immune checkpoint inhibitors (ICIs) combined with anti‐angiogenic agents manifest improved survival in advanced hepatocellular carcinoma (HCC), but responses remain heterogeneous. Although high PIVKA‐II expression correlates with advanced disease stage, early recurrence, shorter survival, and may predict resistance to anti‐PD‐1 plus lenvatinib therapy, the tumor microenvironment (TME) and resistance mechanisms in HCC with high PIVKA‐II expression remain unclear. Clinical data from 156 resected HCC patients and 104 patients treated with anti‐PD‐1 plus lenvatinib are analyzed to correlate PIVKA‐II expression with clinical features and outcomes. Single‐cell RNA sequencing (scRNA‐seq) is performed on tumors from 15 untreated and 7 treated patients. Mechanistic findings are validated in vitro and in vivo. High PIVKA‐II expression is associated with advanced disease stage, increased microvascular invasion (MVI), early recurrence, and poor response to therapy. ScRNA‐seq revealed an immunosuppressive TME enriched with regulatory T cells (Tregs), exhausted CD8⁺ T cells, and SPP1⁺ tumor‐associated macrophages (TAMs). Mechanistically, tumors with high PIVKA‐II expression upregulated NQO1, which stabilized p65 by inhibiting ubiquitination, activating the NF‐κB/CXCL12 axis, and recruiting Tregs. This pathway mediated therapeutic resistance. Plerixafor, a CXCL12 inhibitor, disrupted this axis and significantly enhanced anti‐tumor efficacy when combined with anti‐PD‐1 plus lenvatinib in vivo. PIVKA‐II is a potentially effective biomarker for predicting resistance to anti‐PD‐1 plus lenvatinib therapy. Its high expression denotes an immunosuppressive TME. Targeting the NQO1/CXCL12/Tregs axis with Plerixafor may restore sensitivity and improve outcomes.

No abstract available

BACKGROUND The tumor microenvironment (TME) plays a crucial role in the limited efficacy of existing treatments for hepatocellular carcinoma (HCC), with tumor-associated endothelial cells (TECs) serving as fundamental TME components that substantially influence tumor progression and treatment efficacy. However, the precise roles and mechanisms of TECs in HCC remain inadequately understood. METHODS We employed a multi-omics profiling strategy to investigate the single-cell and spatiotemporal evolution of TECs within the microenvironment of HCC tumors showcasing varied responses to immunotherapy. Through an analysis of a clinical cohort of HCC patients, we explored the correlation between TEC subpopulations and immunotherapy outcomes. The influence of TEC subsets on the immune microenvironment was confirmed through comprehensive in vitro and in vivo studies. To further explore the mechanisms of distinct TEC subpopulations in microenvironmental modulation and their impact on immunotherapy, we utilized TEC subset-specific knockout mouse models as well as humanized mouse models. RESULTS In this research, we identified a new subset of CXCL12+ TECs that exert a crucial role in immune suppression within the HCC TME. Functionally, CXCL12+ TECs impede the differentiation of CD8+ naïve T cells into CD8+ cytotoxic T cells by secreting CXCL12. Furthermore, they attract myeloid-derived suppressor cells (MDSCs). A bispecific antibody was developed to target both CXCL12 and PD1 specifically, showing significant promise in bolstering anti-tumor immune responses and advancing HCC therapy. CONCLUSIONS CXCL12+ TECs are pivotal in mediating immunosuppression within HCC microenvironment and targeting CXCL12+ TECs presents a promising approach to augment the efficacy of immunotherapies in HCC patients. IMPACT AND IMPLICATION This investigation reveals a pivotal mechanism in the HCC TME, where CXCL12+ TECs emerge as crucial modulators of immune suppression. The discovery of CXCL12+ TECs as inhibitors of CD8+ naïve T cell activation and recruiters of MDSCs significantly advances our grasp of the dynamic between HCC and immune regulation. Moreover, the development and application of a bispecific antibody precisely targeting CXCL12 and PD1 has proven to enhance immune responses in a humanized mouse HCC model. This finding underscores a promising therapeutic direction for HCC, offering the potential to amplify the impact of current immunotherapies.

Anoikis-Related Genes (ARGs) lead to the organism manifesting resistance to anoikis and are associated with unfavorable prognostic outcomes across various malignancies.Therefore, it is crucial to identify the pivotal target genes related to anoikis in HCC .We found that ARGs were significantly correlated with prognosis and immune responses in HCC. The core gene, SPP1, notably promoted anoikis resistance and metastasis in HCC through both in vivo and in vitro studies. The PI3K-Akt-mTOR pathway played a critical role in anoikis suppression within HCC contexts. Our research unveiled SPP1’s role in enhancing PKCα phosphorylation, which in turn activated the PI3K-Akt-mTOR cascade. Additionally, SPP1 was identified as a key regulator of MDSCs and Tregs migration, directly affecting their immunosuppressive capabilities.These findings indicate that in HCC, SPP1 promoted anoikis resistance and facilitated immune evasion by modulating MDSCs and Tregs.

Background Programmed cell death-1 (PD-1) immune checkpoint inhibitors are not effective in treating all patients with hepatocellular carcinoma (HCC), and regulatory T cells (Tregs) may determine the resistance to anti-PD-1 therapy. Methods Patients were divided into two groups based on the clinical efficacy of anti-PD-1 therapy. Flow cytometry was used to determine the phenotype of CD4+, CD8+, and Tregs in peripheral blood mononuclear cells (PBMCs). CD4+CD45RA+T cells were sorted to analyze Treg differentiation and function. Results No significant differences were found between resistant and sensitive patients in the percentage of CD4+ T cells and Tregs in PBMCs or the differentiation and function of induced Tregs (iTregs). However, iTregs from resistant patients presented higher monocarboxylate transporter (MCT) expression. Lactate induced more iTregs and improved OXPHOS levels in the resistant group. MCT1 and MCT2 were highly expressed in tumor-infiltrating Tregs, and patients with higher MCT1 expression had worse clinical outcomes. Combinatorial therapy with MCT antibody and anti-PD-1 therapy effectively inhibited tumor growth. Conclusion MCT and its downstream lactate signal in Tregs can confer anti-PD-1 resistance and may be a marker of poor prognosis in HCC.

Indoleamine 2,3-dioxygenase (IDO) has been described as a major mechanism of immunosuppression in tumors, though the mechanisms of this are poorly understood. Here, we find that expression of IDO by tumor cells results in aggressive tumor growth and resistance to T-cell-targeting immunotherapies. We demonstrate that IDO orchestrates local and systemic immunosuppressive effects through recruitment and activation of myeloid-derived suppressor cells (MDSCs), through a mechanism dependent on regulatory T cells (Tregs). Supporting these findings, we find that IDO expression in human melanoma tumors is strongly associated with MDSC infiltration. Treatment with a selective IDO inhibitor in vivo reversed tumor-associated immunosuppression by decreasing numbers of tumor-infiltrating MDSCs and Tregs and abolishing their suppressive function. These findings establish an important link between IDO and multiple immunosuppressive mechanisms active in the tumor microenvironment, providing a strong rationale for therapeutic targeting of IDO as one of the central regulators of immune suppression.

Background Immune checkpoint blockade (ICB) monotherapy provides poor survival benefit in hepatocellular carcinoma (HCC) due to ICB resistance caused by immunosuppressive tumor microenvironment (TME) and drug discontinuation resulting from immune-related side effects. Thus, novel strategies that can simultaneously reshape immunosuppressive TME and ameliorate side effects are urgently needed. Methods Both in vitro and orthotopic HCC models were used to explore and demonstrate the new role of a conventional, clinically used drug, tadalafil (TA), in conquering immunosuppressive TME. In detail, the effect of TA on M2 polarization and polyamine metabolism in tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) was identified. After making clear the aforementioned immune regulatory effect of TA, we introduced a nanomedicine-based strategy of tumor-targeted drug delivery to make better use of TA to reverse immunosuppressive TME and overcome ICB resistance for HCC immunotherapy. A dual pH-sensitive nanodrug simultaneously carrying both TA and programmed cell death receptor 1 antibody (aPD-1) was developed, and its ability for tumor-targeted drug delivery and TME-responsive drug release was evaluated in an orthotopic HCC model. Finally, the immune regulatory effect, antitumor therapeutic effect, as well as side effects of our nanodrug combining both TA and aPD-1 were analyzed. Results TA exerted a new role in conquering immunosuppressive TME by inhibiting M2 polarization and polyamine metabolism in TAMs and MDSCs. A dual pH-sensitive nanodrug was successfully synthesized to simultaneously carry both TA and aPD-1. On one hand, the nanodrug realized tumor-targeted drug delivery by binding to circulating programmed cell death receptor 1-positive T cells and following their infiltration into tumor. On the other hand, the nanodrug facilitated efficient intratumoral drug release in acidic TME, releasing aPD-1 for ICB and leaving TA-encapsulated nanodrug to dually regulate TAMs and MDSCs. By virtue of the combined application of TA and aPD-1, as well as the efficient tumor-targeted drug delivery, our nanodrug effectively inhibited M2 polarization and polyamine metabolism in TAMs and MDSCs to conquer immunosuppressive TME, which contributed to remarkable ICB therapeutic efficacy with minimal side effects in HCC. Conclusions Our novel tumor-targeted nanodrug expands the application of TA in tumor therapy and holds great potential to break the logjam of ICB-based HCC immunotherapy.

Summary Although ACVR2A mutations are prevalent in non-viral hepatocellular carcinomas (HCCs), the underlying mechanism remains unelucidated. Our molecular investigation reveals that ACVR2A impairment induces hyperglycolysis through the inactivation of the SMAD signaling pathway. Using syngeneic transplantation models and human clinical samples, we clarify that ACVR2A-deficient HCC cells produce and secrete lactate via the upregulation of lactate dehydrogenase A (LDHA) and monocarboxylate transporter 4 (MCT4) expression levels, which promotes regulatory T (Treg) cell accumulation and then acquires resistance to immune checkpoint inhibitors. Remarkably, genetic knockdown and pharmacological inhibition of MCT4 ameliorate the high-lactate milieu in ACVR2A-deficient HCC, resulting in the suppression of intratumoral Treg cell recruitment and the restoration of the sensitivity to PD-1 blockade. These findings furnish compelling evidence that lactate attenuates anti-tumor immunity and that therapeutics targeting this pathway present a promising strategy for mitigating immunotherapy resistance in ACVR2A-deficient HCC.

Despite recently approved immunotherapy combinations, Hepatocellular carcinoma (HCC) remains among the most therapeutically intractable cancers with a 5-year survival rate of only 18%. Underlying chronic liver disease due to hepatitis B/C infection, alcohol abuse, hemochromatosis, or nonalcoholic steatohepatitis promotes HCC development. Current therapeutic strategies include surgery, radiotherapy and multikinase inhibitors alone or with immune-checkpoint blockade (ICB). In particular, the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) are known drivers of HCC through promotion of tumor growth, survival, tissue invasion, and angiogenesis. Previously, our collaborators showed that the limited clinical benefit of MET kinase inhibition was partially a result of induced upregulation PD-L1 leading to local T cell suppression. We hypothesized that combining MET inhibition with blockade of the PD-1 immune checkpoint would overcome the limitations of each individual approach and act synergistically to promote HCC tumor regression. We first validated that both MET selective (Capmatinib and Tivantinib) and non-selective (Cabozantinib) inhibitors all induced PD-L1 on HCC cell lines in vitro. Next, we compared Capmatinib and Cabozantinib alone and with PD-1 ICB in vivo in the ICB-sensitive Hepa1-6 and HCA-1 models of HCC. In HCA-1, statistically significant benefit of the MET inhibitor and αPD-1 combination was evident in limitation of tumor growth and extension of survival. In each case, the Capmatinib combination trended toward better outcomes with PD-1 blockade. Mechanistically, the Capmatinib and αPD-1 combination decreased tumor Treg cells while promoting increased accumulation of activated, non-exhausted, proliferating CD8 T cells. Central memory CD8 T cell frequencies were increased by the combination during the effector phase, and combination treated animals were better protected against subsequent rechallenge than those cured by αPD-1 alone. In the myeloid compartment, M1 macrophage frequencies were increased while PMN-MDSC both decreased in frequency and in Arginase 1 levels. Finally, we studied a serially-passaged, αPD-1 refractory DEN HCC model. Despite lack of efficacy of either component therapy, the MET inhibitor and αPD-1 combination significantly extended survival and inhibited tumor growth. As in the αPD-1 sensitive setting, Capmatinib appeared to offer the most therapeutic benefit with αPD-1. Ongoing studies are focused on characterizing changes mediated by the combination therapy which confer aPD-1 sensitivity to the otherwise refractory DEN HCC tumor microenvironment. Molecular studies are focused on understanding the superior ICB potentiating capacity of Type I MET inhibitors versus the other classes. Given the clinical availability of numerous MET and PDL-1 inhibitors, we hope to inform near-term optimization of MET and αPD-1 clinical trial design for HCC. Citation Format: Ricardo A. de Azevedo, Broderick Turner, Priyamvada Jayaprakash, Krithikaa Rajkumar Bhanu, Anupallavi Srinivasamani, Brittany Morrow, Michelle Winkler, Shweta Mahendra Hedge, Arthur Liu, Ravaen Slay, Michael A. Curran. Proposing the best MET inhibitor to improve anti-PD-1 efficacy in HCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5745.

BACKGROUND Hepatocellular carcinoma (HCC) is the most common liver cancer. Exploring non-apoptotic regulated cell death (RCD) offers a strategy to overcome drug resistance. This study investigates a risk model based on non-apoptotic RCD-related genes to predict clinical outcomes and guide immunotherapy. METHODS We identified genes associated with non-apoptotic RCD in HCC through weighted gene co-expression network analysis (WGCNA) and differential analysis. We then employed non-negative matrix factorization (NMF) clustering to categorize HCC into molecular subtypes related to non-apoptotic RCD and identified differentially expressed genes (DEGs) among these subtypes. We developed a prognostic model utilizing Cox regression and LASSO analysis, stratifying patients into specific risk groups and validating the model's prognostic significance. We subsequently analyzed immune functions and tumor mutation burden (TMB). Finally, we identified potential drugs and evaluated drug sensitivity specific to HCC. RESULTS We identified four non-apoptotic RCD genes and classified patients into three subtypes. We observed significant differences in immune characteristics and prognostic outcomes among these groups. Six DEGs emerged as key indicators for risk assessment, leading to a prognostic model. High-risk patients face poorer survival rates and increased mortality. Independent prognostic analyses confirm that these models can effectively predict patient outcomes. Notably, in high-risk patients, immune-related functions appear suppressed, facilitating tumor immune evasion. CONCLUSION We developed a risk model focused on non-apoptotic RCD genes. This model accurately predicts the prognosis for HCC patients. It may also offer new insights for clinical decisions and immunotherapy.

Hepatocellular carcinoma (HCC) is one of the most common malignancies globally with poor prognosis. Cancer-associated fibroblasts (CAFs) play multiple functions in the regulation of tumorigenesis, metastasis and therapeutic resistance of cancer. The current study aimed to explore the role of CAFs-related genes in the prognosis and immunotherapy response in HCC. CAFs-related genes were identified by using single-cell RNA-sequencing analysis. Least absolute shrinkage and selection operator (LASSO) analysis was conducted to develop a CAFs-related prognostic signature (FRPS) in TCGA dataset and verified in ICGC, GSE14520 and GSE76427 cohorts. Several tools, including Tumor Immune Dysfunction and Exclusion (TIDE) score, immunophenoscore, and Tumor Mutation Burden (TMB) score were used to evaluate the value of FRPS in predicting immunotherapy benefits. The FRPS constructed based on 10 genes (RGS5, CNN3, PALLD, FLNA, KLHL23, MYC, NDRG2, SERPINE1, CD151 CALU) served as an independent risk factor and showed stable and powerful performance in predicting the overall survival rate of HCC patients with an AUCs of 0. 734, 0.727, and 0.717 in 2-, 3-, and 4-year ROC curve in TCGA cohort. Low risk score indicated a higher abundance of CD8+ T cells and NK, and lower abundance of Treg. Moreover, HCC patients with low risk score had a higher PD1&CTLA4 immunophenoscore, higher TMB score, and lower TIDE score. Moreover, high risk score indicated a lower IC50 value of 5-fluorouracil, camptothecin, cisplatin, docetaxel, gemcitabine, paclitaxel, afatinib, crizotinib, dasatinib, erlotinib, erlotinib, gefitinib, lapatinib, and osimertinib in HCC. Our study develops a novel FRPS HCC. The FRPS acts as a risk factor for the prognosis of HCC patients and it can predict the immunotherapy benefits of HCC patients.

BACKGROUND Genetic alterations in oncogenic pathways are critical for cancer initiation, development, and treatment resistance. However, studies are limited regarding pathways correlated with prognosis, sorafenib, and transcatheter arterial chemoembolization (TACE) in hepatocellular carcinoma (HCC). METHODS In this study, 1928 patients from 11 independent datasets and a clinical in-house cohort were screened to explore the relationships among canonical pathway alterations in HCC patients. The molecular mechanisms, biological functions, immune landscape, and clinical outcomes among three heterogeneous phenotypes were further explored. RESULTS We charted the detailed landscape of pathway alterations in the TCGA-LIHC cohort, screened three pivotal pathways (p53, PI3K, and WNT), identified co-occurrence patterns and mutual exclusively, and stratified patients into three altered-pathway dominant phenotypes (ADPs). P53|PI3K ADP characterized by genomic instability (e.g., highest TMB, FGA, FGG, and FGL) indicated an unfavorable prognosis. While, patients in WNT ADP suggested a median prognosis, enhanced immune activation, and sensitivity to PD-L1 therapy. Remarkably, sorafenib and TACE exhibited efficacy for patients in WNT ADP and low frequent alteration phenotype (LFP). Additionally, ADP could work independently of common clinical traits (e.g., AJCC stage) and previous molecular classifications (e.g., iCluster, serum biomarkers). CONCLUSIONS ADP provides a new perspective for identifying patients at high risk of recurrence and could optimize precision treatment to improve the clinical outcomes in HCC.

Next-generation sequencing has provided information on actionable targets and biomarkers of response in oncology. In hepatocellular carcinoma (HCC), Wnt/CTNNB1 mutations characterize the immune-excluded class (cold tumors) and might represent the biomarkers predicting resistance to immune checkpoint inhibitors. Large-scale validation of these data is needed to customize immunotherapy in advanced HCC. See related article by Harding et al., p. 2116

PURPOSE The establishment of a preclinical model of the abscopal effect on HCC and evaluation on whether the hypofractionated RT multi-tumor Hepa1-6 mouse HCC model could be used to suppress non-radiated tumor mass would be implemented in this study. METHODS AND MATERIALS Hepa1-6 mouse liver cancer cell lines were used to form tumors and immunogenicity was analyzed using ELISpot and immune cell labeled antibody. Interferonβ (IFNβ) expression was confirmed through PCR. RESULTS After investigation, the intratumoral transcription of type Ⅰ IFN increased by 2-fold. The anti-tumor immune response to Hepa 1-6 cells induced by radiation was increased. Moreover, the influx of activated CD8+ T cell was increased in non-irradiated tumors. The number of dendritic cells and activation status were evaluated by flow cytometry on the second day after irradiation. The flow cytometry presented significantly increased DC population expressing the CD11c molecule in tumor draining lymph nodes. Furthermore, as the irradiation leads to adaptation of immune resistance of tumor cells against RT, we explored to elucidate potent tool to overcome the resistance and confirmed the capability of PD-L1 antibody to survive the late RT resistance. CONCLUSIONS The immunological mechanism of the abscopal effect was revealed and the application of PD-L1 inhibitor successfully performed as a breakthrough to the late RT resistance in Hepa1-6 tumor model.

Introduction Adrenomedullin (ADM), a multifunctional peptide, has been implicated in various inflammatory and autoimmune diseases. However, its role in cancer, particularly in NSCLC, remained under-explored. This called for a pan-cancer analysis of ADM, investigating its expression, genomic alterations, prognostic value, immune associations, and relations with drug sensitivity to provide insights into its potential as a therapeutic target and biomarker. Methods ADM expression data from normal and tumor tissues was retrieved and analyzed through HPA and Timer 2.0 online platforms. Genetic alterations, copy number variations (CNVs), and methylation patterns were analyzed using cBioPortal and GSCA platforms. The data for survival analysis was extracted from TCGA and GEO database and analyzed through GEPIA and PrognoScan online platforms. ADM’s correlations with immune checkpoint genes, immune cell infiltration, MSI, and TMB were evaluated using data from Timer and TCGA via R. Drug sensitivity analysis was performed with GDSC and CTRP databases, supported by network visualizations. IHC staining was conducted on LUAD patients’ samples to assess ADM’s relationship with EGFR-TKI resistance and immune microenvironment. Results ADM was widely expressed across normal tissues, with high levels in adipose tissue, endocrine organs, digestive and reproductive systems. Pan-cancer analysis revealed that ADM expression was upregulated in multiple cancer types, including CESC, ESCA, GBM, HNSC, KICH, KIRC, LUSC, PCPG, THCA, and UCEC, and correlated with advanced pathological stages in THCA, KIRP, and HNSC. Furthermore, high ADM expression was significantly linked to poor prognosis in patients with LGG, LUAD, MESO, THYM, LIHC, HNSC, GBM, KICH, KIRP, CESC, PAAD, and STAD, while its negative influence on OS and RFS was validated in LUAD. In addition, ADM exhibited genetic alterations, including amplification and deep deletion across multiple cancer types. Strong and consistent positive correlations were witnessed between ADM and several immune checkpoint genes, including CD274 (PD-L1), CD276, TNFRSF18, TNFSF9, and PVR in pan-cancer analysis, indicating its role in the development of suppressive immune microenvironment and T cell exhaustion. Besides, ADM showed significant correlations with immune cell infiltration, and TMB/MSI, highlighting its role in immune regulation and its potential as a predictive biomarker for immunotherapy. Significantly, ADM expression was correlated with multiple drug sensitivity, particularly chemotherapy and tyrosine kinase inhibitors (TKIs) therapy. Moreover, positive correlations between its expression and EGFR-TKI resistance, CD8+ T cell infiltration and tumor proportion score (TPS) in LUAD were validated in patients’ samples, emphasizing its potential in guiding personalized therapy. Discussion This pan-cancer analysis revealed ADM’s pivotal role in progression, immune modulation, and therapeutic response, especially in LUAD. ADM held promise as a prognostic biomarker and a potential therapeutic target in immune modulation and resistance management. Future research should focus on experimental validation and elucidation of ADM-mediated pathways, which might provide novel insights into cancer biology and improve clinical outcomes

Background The high metastasis rate is one of the main reasons for the poor prognosis of patients with hepatocellular carcinoma (HCC). Coagulation factor Xa (FXa) and its receptor proteinase-activated receptor-2 (PAR-2) proven to promote tumor metastasis in other forms of cancer. Here, we explore the role and mechanism of FXa in the regulation of resistance of anoikis and immune escape of HCC. Methods In vitro and in vivo experiments were conducted to explore the role of FXa in HCC metastasis and its potential mechanism. The effects of FXa inhibitor rivaroxaban on HCC immunotherapy were evaluated using intrahepatic metastasis animal models and clinical trial (No. ChiCTR20000040540). We investigated the potential of FXa inhibition as a treatment for HCC. Results FXa was highly expressed in HCC and promoted metastasis by activating PAR-2. Mechanistically, FXa-activated PAR-2 endows HCC cells with the ability of anoikis resistance to survive in the circulating blood by inhibiting the extrinsic apoptosis pathway. Furthermore, suspension stimulation-induced phosphorylation of STAT2, which promotes programmed death-ligand 1 (PD-L1) transcription and inhibits the antitumor effects of immune cells by inhibiting the infiltration of CD8+T cells in tumors and the levels of secreted cytokines. In vivo inhibition of FXa with rivaroxaban reduced HCC metastasis by decreasing PD-L1 expression and exhausting tumor-infiltrating lymphocytes. Notably, the combination of rivaroxaban and anti-programmed death-1 monoclonal antibody (anti-PD-1) programmed Death-1 monoclonal antibody (anti-PD-1) induced synergistic antitumor effects in animal models. Most importantly, rivaroxaban improved the objective response rate of patients with HCC to immune checkpoint inhibitors and prolonged overall survival time. Conclusions FXa-activated PAR-2 promotes anoikis resistance and immune escape in HCC, suggesting the potential for combining coagulation inhibitors and PD-1/PD-L1 immune checkpoint blockade to enhance the therapeutic efficacy of HCC.

Hepatocellular carcinoma (HCC) is a highly invasive malignant tumor. Tumor-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) play important roles in the invasive process and immune escape of HCC. To overcome these barriers, this work has designed a biomimetic dual-engine nanodisruptor (ZHL@CM) by encapsulating losartan (LST) and Radix Hedysari polysaccharide (HPS) within a zeolitic imidazolate framework (ZIF) and camouflaging the construct with macrophage membranes. In the acidic tumor microenvironment, ZHL@CM rapidly releases LST, which penetrates the dense ECM and reprograms CAFs to suppress collagen synthesis at the source. Simultaneously, Zn2+ released from ZIF directly degrades peritumoral ECM components, collectively alleviating matrix stiffness and dismantling the physical barrier. These effects facilitate the deep penetration of macromolecular agents and immune effector cells into tumor tissues. The liberated HPS efficiently infiltrates the tumor parenchyma and reprograms TAM2 toward a proinflammatory phenotype by activating the TLR4 signaling cascade. Beyond local remodeling, ZHL@CM elicits robust systemic immune activation, markedly suppressing metastatic dissemination. This work offers a mechanistically informed strategy for synergistic immunomodulation in HCC, providing a versatile platform for overcoming stromal and immune resistance in solid tumors.

Radiotherapy plays an increasingly crucial role in the treatment of hepatocellular carcinoma (HCC). However, resistance to radiotherapy remains a significant obstacle to achieving optimal treatment outcomes. Our objective is to elucidate the mechanisms underlying radiotherapy resistance. Through proteomic sequencing of radiotherapy-resistant cell lines and patient-derived xenograft tissues from HCC patients, we identified that Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) is upregulated in both radiotherapy-resistant cell lines and tissues. After radiotherapy treatment, DNAPK phosphorylates TACC3 at 315 threonine, leading to enhanced protein stability of TACC3. TACC3 facilitated the proliferative capacity and radiotherapy resistance of HCC cells by promoting the interaction between XRCC5 and XRCC6 through specific residues within its coiled-coil domain, namely ILE736, ASN742 and GLU773. This interaction facilitates DNA damage repair via the non-homologous end joining pathway in response to radiation, thereby contributing to the radiotherapy resistance in HCC cells. Furthermore, TACC3 increases the production of IL-4 and IL-10 within HCC cells, inducing the differentiation of M0 macrophages to M2 macrophages within the immune microenvironment, leading to the suppression of CD8+T cell cytotoxic functions and creating an immunosuppressive microenvironment in HCC. Targeting TACC3 with inhibitor KHS101 significantly inhibit the proliferation of HCC and improve the immune microenvironment of HCC.

The extracellular matrix (ECM) plays a pivotal role in the initiation and progression of hepatocellular carcinoma (HCC) by facilitating the proliferation of HCC cells and enabling resistance to Anoikis. ECM also provide structural support that aids in the invasion of HCC cells, thereby influencing the tumor microenvironment. Due to genetic variations and molecular heterogeneity, significant challenges exist in the treatment of HCC, particularly with immunotherapy, which frequently leads to immune tolerance and suboptimal immune responses. Therefore, there is an urgent need for a multi-omics-based classification system for HCC that clarifies the molecular mechanisms underlying the establishment of immune phenotypes and Anoikis resistance in HCC cells. In this study, we employed advanced clustering algorithms to analyze and integrate multi-omics data from HCC patients, with the objective of identifying key genes that possess prognostic potential associated with the Anoikis resistance phenotype. This methodology resulted in the development of a consensus machine learning-driven signature (CMLS), which demonstrates robust predictive capabilities by examining variations in epigenetics, transcription, and immune metabolism, as well as their effects on the core differential gene, plasminogen (PLG). The integrated multi-omics approach has identified PLG as a critical node within the gene regulatory network associated with Anoikis resistance and immunometabolic phenotypes. As an independent risk factor for poor prognosis in patients with HCC, PLG facilitates Anoikis resistance and enhances the migration of HCC cells. This study provides novel insights into the molecular subtypes of HCC through the application of robust clustering algorithms based on multi-omics data. The constructed CMLS serves as a valuable tool for early prognostic prediction and for screening potential drug candidates that may enhance the efficacy of immunotherapy, thereby establishing a foundation for personalized treatment strategies in HCC. Our data underscore the pivotal role of PLG in the development of Anoikis resistance and the immunometabolic phenotype in HCC cells. Furthermore, we present compelling experimental evidence that PLG functions as a significant tumor promoter, suggesting its potential as a target for the formulation of tailored therapeutic strategies for HCC.

The membrane‐associated RING‐CH (MARCH) family, a member of the E3 ubiquitin ligases, has been confirmed by a growing number of studies to be associated with immune function and has been highlighted as a potential immunotherapy target. In our research, hepatocellular carcinoma (HCC) patients were divided into C1 and C2 MARCH ligase-related patterns by the non-negative matrix factorization (NMF) algorithm. Multiple analyses revealed that the MARCH ligase-related cluster was related to prognosis, clinicopathological characteristics, and the tumor immune microenvironment (TIME). Next, the signature (risk score) of the MARCH prognosis was constructed, including eight genes associated with the MARCH ligase (CYP2C9, G6PD, SLC1A5, SPP1, ANXA10, CDC20, PON1, and FTCD). The risk score showed accuracy and stability. We found that the correlations between risk score and TIME, tumor mutation burden (TMB), prognosis, and clinicopathological characteristics were significant. Additionally, the risk score also had important guiding significance for HCC treatment, including chemotherapy, immunotherapy, and transarterial chemoembolization (TACE).

Simple Summary Drugs that help the immune system attack cancer, known as immunotherapy, benefit only a small fraction of patients with cancer that has spread distant from the primary organ. Thus, markers that indicate immunotherapy failure are needed. Overall changes to DNA structure that lead to the gain or loss of extra copies of genes are known as copy number alteration (CNA) burden. In this study, we obtained data on CNA burden, clinical information, and overall tumor mutations, known as tumor mutational burden (TMB), for 1661 patients. These patients were all treated with immunotherapy and had their cancer specimens analyzed by an approved clinical test called MSK-IMPACT. We report that CNA burden could predict those patients who do not benefit from immunotherapy. Additionally, tumors with high CNA but low TMB could identify those with the worst survival. This work can better match patients to their most effective treatment of immunotherapy or other therapies. Abstract Immune checkpoint blockade (ICB) benefits only a subset of advanced cancer patients, and predictive biomarkers for immunotherapy response are needed. Recently, copy number alteration (CNA) burden has been proposed to predict ICB resistance. We assessed this finding using the publicly accessible data for 1661 ICB-treated patients whose tumors were profiled by MSK-IMPACT, an approved targeted assay in clinical care. We tested the hypothesis that the continuous increase in CNA burden is associated with poor overall survival following ICB. In addition, we hypothesized that the combinatorial biomarkers of tumor mutational burden (TMB) and CNA burden would better stratify patients for immune status and ICB response. Of the 1661 cases, 79% (n = 1307) were treated with anti PD-1/PD-L1 and the remaining 21% (n = 354) with anti CTLA-4 or the combination of both. In a multivariate analysis, increase in CNA burden was associated with poor overall survival [HR = 1.52, 95% CI (1.01–2.30), p = 0.04]. The combination of biomarkers TMB and CNA burden stratified patients into four clinically distinct subsets among which “LowTMB/HighCNA” showed the worst survival (p < 0.0001). The four patient subsets had unique CNA profiles and enriched pathways, which could predict transcriptional and phenotypic effects related to immune signaling and CD8+ T-cell abundance in the tumor microenvironment. CNA burden was associated with poor overall survival in patients receiving ICB and could improve patient stratification when incorporated with TMB. These findings may guide patient selection for immunotherapy or alternative strategies.