MSR1+ tumor-associated macrophages (TAMs) have been implicated in various malignancies; however, their functional role in Hepatocellular carcinoma (HCC) remains poorly defined. This research seeks to clarify the roles of MSR1+ TAMs in HCC and their influence on the tumor immune microenvironment. Clinical and experimental data indicate that high levels of MSR1+ TAMs correlate with poor prognosis in HCC patients. Transcriptomic analyses and in vitro as well as in vivo functional assays revealed that the immunosuppressive activity of MSR1+ TAMs is closely linked to their secretory profile. MSR1 enhances IL-6 secretion by activating the NF-κB signaling pathway, subsequently facilitating the recruitment of myeloid-derived suppressor cells (MDSCs). This cascade diminishes CD8+ T cell infiltration and effector function, promoting an immunosuppressive tumor microenvironment. In preclinical models, the simultaneous inhibition of MSR1 and PD-L1 markedly reduced tumor growth more effectively than either treatment alone. Our findings demonstrate that MSR1+ TAMs contribute to hepatocellular carcinogenesis through the NF-κB/IL-6 signaling axis by promoting MDSCs accumulation and impairing CD8+ T cell responses. Effectively targeting MSR1+ TAMs can overcome resistance to anti-PD-L1 therapy, offering a promising new immunotherapeutic approach for HCC.

BACKGROUND:Although immunotherapy has revolutionized cancer treatment, hepatocellular carcinoma (HCC) continues to demonstrate limited clinical responses, highlighting the urgent need for novel immunomodulatory strategies. Trained immunity, an emerging paradigm wherein innate immune cells develop a memory-like phenotype through epigenetic and metabolic reprogramming, offers a promising avenue to remodel the immunosuppressive tumor microenvironment. This study investigated whether β-glucan-induced trained immunity could potentiate antitumor immunity against HCC. METHODS:We established orthotopic HCC mouse models to investigate the role of trained immunity induced by whole β-glucan particle (WGP) in the HCC microenvironment, particularly in modulating hepatic apolipoprotein E (APOE)-positive monocytes/macrophages. Transcriptional changes in trained monocytes/macrophages were identified by analyzing single-cell RNA sequencing and bulk RNA-sequencing data from the livers of WGP-treated and control mice. Mechanistic studies were performed using Apoe -/- mice and in situ monocyte/macrophage engineering. Flow cytometry was performed to assess immune cell phenotypes and phagocytosis, while luminescence-based assays were used to evaluate cytotoxic activity. The translational potential was assessed using human monocyte training assays. RESULTS:This study demonstrated that preconditioning with WGP, a trained immunity inducer, increased the accumulation of trained monocytes/macrophages in the liver and suppressed tumor progression in HCC mouse models. Mechanistically, WGP-trained APOE+ monocytes/macrophages exhibited a decrease in lipid accumulation and endoplasmic reticulum stress, thereby enhancing their antitumor function. Genetic deletion of Apoe in monocytes/macrophages abrogated the antitumor effects of WGP, demonstrating that APOE+ monocytes/macrophages are essential mediators of WGP-induced trained immunity. Adoptive transfer of WGP-trained bone marrow-derived macrophages suppressed the growth of HCC in recipient mice. Furthermore, WGP induced trained immunity in human monocytes, leading to enhanced killing of HCC cells. Notably, combination therapy with WGP and anti-programmed death-ligand 1 antibody achieved superior tumor control compared with either monotherapy. CONCLUSIONS:These findings identify a critical role for trained APOE+ monocytes/macrophages in WGP-mediated antitumor immunity in the liver. Harnessing WGP-induced peripheral trained immunity represents a novel therapeutic strategy for HCC.

Supplementary Figure S2. H22 orthotopic HCC immunotherapy model

BACKGROUND & AIMS:Endothelial cell (EC) damage is an initiating event in acute cellular rejection after liver transplantation (LT). However, the origin and characteristics of post-transplant neonatal ECs remain controversial. We aimed to uncover the mechanisms underlying EC-T cell interactions after transplantation and to develop an EC-targeted strategy to alleviate transplant rejection. METHODS:Leveraging single-cell RNA sequencing from 13 human and 4 murine liver allografts, we mapped the functional atlas of ECs. Allogeneic orthotopic LT in CAG;R26-tdTomato and Cd34-CreERT2;R26-tdTomato mice confirmed the cellular origin of ECs. We used CellChat, multiplex immunohistochemistry, and in vitro co-culture models to investigate the mechanism of EC-T cell interactions. Using a platelet-based bio-delivery system, we achieved targeted delivery of a CXCL12 monoclonal antibody (αCXCL12). RESULTS:ECs in transplanted livers exhibited a dual origin, being derived from both donor and recipient cells. Recipient-derived ECs were characterized by high CD34 expression, high stemness, and pro-inflammatory characteristics. Using genetic lineage-tracing mice combined with an orthotopic LT model, we found that recipient CD34+ cell-derived ECs peaked at 2 weeks post-LT and declined by 4 weeks, consistent with the temporal pattern of rejection. We further identified that CD34+ ECs recruit and potentiate Th1 and cytotoxic CD8+ T cells via the CXCL12-CXCR4 axis and co-stimulatory molecules. In turn, cytotoxic CD8+ T cells induced pyroptosis of CD34+ cell-derived ECs through the Caspase1-GSDMD pathway. Platelets loaded with αCXCL12 specifically targeted ECs in the transplanted liver, reducing T-cell infiltration and mitigating rejection. CONCLUSIONS:We identified a population of recipient-derived CD34+ ECs that exacerbates acute rejection by activating T cells through the CXCL12-CXCR4 axis. We further developed a platelet-based delivery strategy that precisely targets EC-derived CXCL12 and effectively prevents acute cellular rejection. IMPACT AND IMPLICATIONS:We integrated single-cell transcriptomic data from human and murine liver allografts to delineate the dual cellular origins and functional atlas of endothelial cells. Using lineage-tracing mice in an allogeneic orthotopic liver transplantation model, we tracked the fate of CD34+ cells and demonstrated the contribution of recipient-derived CD34-lineage endothelial cells to liver allograft angiogenesis. These CD34-lineage endothelial cells recruited T cells through the CXCL12-CXCR4 axis and activated them via co-stimulatory molecules. Furthermore, a platelet-based biological delivery strategy targeting CXCL12 in CD34-lineage endothelial cells alleviated T cell-mediated rejection. This study provides an endothelial cell-centered perspective and proposes a potential novel immunosuppressive strategy for liver transplantation.

Supplementary Fig S6. PGE2 released by tumor cells under immune attack promotes CX3CR1 upregulation on macrophages

Supplementary Figure S4. The effect of different immunotherapy combinations on tumor infiltrating NK/NKT cells

Antiplatelet therapy is a well-established risk factor for gastrointestinal bleeding, yet hemobilia induced by these agents remains rarely reported. Acute cholangitis secondary to choledocholithiasis in patients on antiplatelet therapy presents significant clinical challenges, as the medication can precipitate hemobilia, subsequently exacerbating the cholangitis. Here, we report a 73-year-old female with a history of multiple biliary interventions and long-term aspirin use, exemplifying this clinical challenge. Although elective endoscopic retrograde cholangiopancreatography (ERCP) was scheduled following a 1-week aspirin discontinuation, the sudden onset of severe abdominal pain necessitated emergency intervention. ERCP revealed hemobilia with clots obstructing the major duodenal papilla. Therapeutic ERCP successfully evacuated the clots, extracted the stones, and placed a nasobiliary drain. The patient stabilized without requiring angiography or surgery. This case highlights that abrupt changes in abdominal pain patterns in calculous cholangitis patients receiving antiplatelet therapy should raise suspicion for hemobilia, underscoring the need for timely and effective clinical intervention.

Supplementary Figure S3. The effect of CX3CR1 neutralizing antibody on macrophages

BACKGROUND:Neutrophils are increasingly recognised as immunosuppressive drivers of hepatocellular carcinoma (HCC), yet their persistence in the oxidative, lipid-rich tumour microenvironment remains poorly understood. OBJECTIVE:To elucidate the metabolic and molecular programmes that enable tumour-associated neutrophils (TANs) to resist ferroptosis and sustain immunosuppression in HCC. DESIGN:We employed human HCC samples, multiple murine HCC models, transcriptomic and lipidomic profiling, genetic loss-of-function systems and therapeutic interventions. Ferroptosis sensitivity, lipid metabolic rewiring and immunological consequences of TANs were systematically evaluated across models and validated in patient datasets and biospecimens. RESULTS:TANs in human HCC and mouse models exhibit pronounced lipid accumulation and oxidative stress compared with peripheral neutrophils. Multi-omic profiling revealed that TANs are enriched for lipid-binding gene programmes and undergo rewiring towards sphingolipid and unsaturated fatty acid metabolism. We identified triggering receptor expressed on myeloid cells 2 (TREM2) as a key lipid-sensing receptor selectively expressed in TANs. Functional deletion of TREM2 reprogrammed the tumour immune microenvironment, restoring CD8+ T cell activity and suppressing HCC progression. Mechanistically, tumour-derived sphingosine-1-phosphate (S1P) activates TREM2, triggering nuclear factor erythroid 2-related factor 2 (NRF2)-mediated transcription of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), thereby promoting ferroptosis resistance. TREM2 expression is transcriptionally induced by granulocyte-macrophage colony-stimulating factor-signal transducer and activator of transcription 3 (GM-CSF-STAT3) signalling. Genetic deletion of TREM2, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout of sphingosine kinase 1/2 (SPHK1/2) in tumour cells, or pharmacological inhibition of S1P synthesis disrupts this protective lipid-immune circuit, sensitises TANs to ferroptosis and restricts tumour growth. Therapeutically, a peptide-based TREM2 inhibitor reprogrammes TANs, restores CD8+ T cell function and enhances anti-programmed cell death protein 1 (PD-1) immunotherapy efficacy. Clinically, TREM2+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in HCC tumours, correlate with SPHK1/2 expression and T cell dysfunction and associate with poor patient prognosis. CONCLUSION:Our study uncovers the S1P-TREM2-NRF2 axis as a critical metabolic-immune circuit that preserves neutrophil survival and immunosuppressive function in HCC. Targeting this lipid-dependent ferroptosis resistance pathway offers a promising therapeutic strategy to overcome immunotherapy resistance in liver cancer.

While transarterial chemoembolization (TACE) is widely used as a bridging therapy for hepatocellular carcinoma (HCC) patients awaiting liver transplantation (LT), the prognostic value of TACE-induced tumor necrosis and the mechanisms driving post-TACE recurrence remain poorly understood. In a cohort of 265 HCC patients beyond the Milan criteria undergoing LT, we stratified them by pre-transplant TACE response: extensive necrosis (EN, n = 74), limited necrosis (LN, n = 79), and non-TACE (n = 112). Strikingly, patients with LN exhibited significantly higher post-LT recurrence rates than both the EN (P = 0.007) and non-TACE (P = 0.026) groups. RNA-sequencing of matched HCC specimens revealed significant up-regulation of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) in TACE-treated residual tumors compared to untreated tumors. Clinically, high LGR5 expression (n = 60) correlated with reduced recurrence-free survival compared to low expression (n =147, P = 0.015) and was an independent predictor of tumor recurrence (HR = 1.89). Mechanistically, isolated LGR5high cells demonstrated more malignant biological properties than LGR5low cells. Hypoxia-inducible factor 1α (HIF-1α) greatly up-regulated the expression of LGR5 in cell lines and patient-derived organoids under hypoxic stress. This HIF-1α-driven LGR5 induction markedly enhanced the invasive capacity and epithelial–mesenchymal transition of LGR5high cells, effects that were abrogated by LGR5 knockdown. Furthermore, LGR5 activated the MAPK signaling pathway under hypoxia, and its silencing suppressed this activation. Our findings establish the extent of TACE-induced necrosis as a critical prognostic marker and unveil the novel HIF-1α/LGR5/MAPK axis as a key driver of metastatic progression in residual HCC, positioning LGR5 as a promising therapeutic target to prevent post-TACE recurrence.

Lysine acetylation affects hepatocellular carcinoma (HCC) malignancy through multiple pathways. Evidence from our laboratory and other groups indicates that long chain acyl CoA synthetase 4 (ACSL4) is a crucial oncoprotein in HCC. However, the precise mechanisms underlying the post-translational regulation of ACSL4 acetylation remains unknown. Here, we report a previously unknown mechanism of ACSL4 regulation involving acetylation at the lysine 49 (K49) site by the acetyltransferase MOF. ACSL4 acetylation hindered its degradation via the ubiquitin-proteasome pathway. Using mass spectrometry and subsequent verification, we demonstrated that TRIM21 is an E3 ubiquitin ligase responsible for ACSL4 proteasomal degradation. Further mechanistic studies revealed that MOF-mediated ACSL4-K49 acetylation counteracted the TRIM21-mediated degradation of ACSL4. Functionally, we demonstrated that acetylated ACSL4 promotes lipid accumulation and HCC progression, both in vitro and in vivo. Clinically, ACSL4-K49 acetylation is frequently increased in HCC samples, and elevated ACSL4-K49 acetylation is associated with poor prognosis in patients with HCC. Together, these findings unveil a novel regulatory mechanism of ACSL4, highlighting its pivotal role in modulating HCC progression.

Nifuroxazide (NFX), an antibacterial agent, also exhibits notable antitumor effects by inhibiting STAT3 signaling, which is often aberrantly activated and linked to chemoresistance in tumors such as hepatocellular carcinoma (HCC). Combining NFX with chemotherapy may enhance therapeutic efficacy, but its poor solubility limits oral bioavailability. To address this, we developed a biomimetic delivery system by loading NFX into murine macrophage-like RAW264.7 cells (Mφ-NFX). This strategy aims to improve drug delivery, enhance antitumor effects, with the potential to circumvent or overcome drug resistance. We evaluated the efficacy of Mφ-NFX alone and in combination with Oxaliplatin in vitro and in preclinical HCC models. Macrophages effectively carried and delivered NFX to tumor cells and tissues without significant toxicity to the carriers. Additionally, NFX promoted macrophage polarization toward the M1 phenotype within the tumor microenvironment. Mφ-NFX significantly inhibited tumor growth and increased the M1/M2 macrophage ratio. Co-treatment with Mφ-NFX and Oxaliplatin demonstrated enhanced tumor suppression and modulation of the tumor microenvironment. Mechanistically, NFX and Oxaliplatin acted synergistically via inhibition of the AKT/β-catenin pathway. In conclusion, this macrophage-based NFX delivery platform offers improved antitumor activity and sensitization to chemotherapy, presenting a promising strategy for HCC treatment.

[This corrects the article DOI: 10.3389/fimmu.2026.1822904.].

Immunosuppressive therapy following organ transplantation is essential for ensuring long-term graft survival but leaves patients vulnerable to complications such as infection, malignancy, and severe side effects. Immunosuppressants currently in use are typically hydrophobic with low oral bioavailability and must be taken indefinitely to maintain immune tolerance. Furthermore, these drugs lack tissue repair capacity, which limits their effectiveness in transplantation. To address this clinical gap, an immunomodulatory hydrogel (iGEL) was developed, integrating prodrug engineering with inflammation-restricted pharmacokinetics. A binary pharmacology-loaded iGEL spontaneously formed upon subcutaneous implantation using a dual-syringe system. Acting as a tissue-adhesive depot, iGEL released key antirejection and tissue-regenerative agents, enabling localized immunomodulation in response to rejection-induced inflammation. In mouse major histocompatibility complex-mismatched allo-transplant models, iGEL suppressed T-cell activity while promoting vascular reconstruction and regulating local cytokine profiles to remodel immune-regenerative dynamics. For both immunocompetent and metabolically compromised hosts, iGEL effectively restored the functionality of skin allografts and markedly extended survival. The study introduces a locally syringeable and adaptive hydrogel depot that harnesses pathological cues to mediate complementary immune regulation and tissue repair, ensuring long-term graft survival without systemic immunosuppression.

Tumor recurrence after liver transplantation (LT) for hepatocellular carcinoma (HCC) is the most significant obstacle impacting the prognosis of patients. Managing the clinical application of immunosuppressive agents is difficult, owing to their limited therapeutic range and notable intrapatient variability (IPV). In this study, we explored the association between the IPV of different immunosuppressants and LT outcomes according to HCC. The study retrospectively analyzed 242 patients with HCC undergoing LT between January 2015 and December 2020. We initially stratified the administration of sirolimus into two groups to observe its impact on post-transplant prognosis. Subsequently, we investigated the influence of high and low immunosuppressant IPV on patient outcomes within each group. Overall survival in patients was significantly increased with sirolimus compared with patients solely receiving tacrolimus (P < 0.05), especially among patients beyond the Hangzhou criteria (P < 0.0001). High IPV was defined as a coefficient of variance > 30