Background: Despite the dominance of the Model for End-Stage Liver Disease (MELD) in liver transplantation (LTx) prognosis, its ability to predict early post-transplant survival remains debated. Furthermore, the current critical donor shortage has expanded the use of extended criteria donor (ECD) grafts, which still lack effective prognostic models. To address this, we developed an innovative series of albumin-bilirubin (ALBI)-based multivariable models (MVMs) for predicting perioperative mortality and identifying super high-risk patients. Methods: Among a total of 2,040 recipients included from multicenter, 1,310 recipients were enrolled in the training cohort to develop an MVM for predicting perioperative mortality using logistic regression. Model performance was assessed by receiver operating characteristic (ROC) curve, with the risk threshold defined by decision curve analysis (DCA). External validation was performed in a cohort of 730 patients. For subgroup analyses, the model's discriminatory power, quantified by the area under the ROC curve (AUC), was compared with that of the MELD score. Results: We developed and externally validated two ALBI-based models (ALBI-MVM and ALBI-MVM plus). The ALBI-MVM showed higher predictive power (AUCs: 0.736 and 0.714), identifying a very high-risk group (45.5% mortality; P<0.001) from the ALBI-defined medium/high-risk population. With added variables, performance improved sequentially (AUCs: 0.792 and 0.704), and the ALBI-MVM plus further isolated a super high-risk group (73.3% mortality; P<0.001). Notably, across ECD subgroups-including small-for-size, elderly, and ABO-incompatible (ABOi) grafts-both models showed significantly superior predictive performance over the MELD score. Conclusions: This large multicenter study established and validated ALBI-based MVMs that effectively predicted perioperative mortality and identified super high-risk patients. Especially in major ECD transplantation types, it demonstrated superior predictive performance over traditional systems.

BACKGROUND AND AIMS:Hepatitis B virus (HBV) infection exhibits marked cellular heterogeneity, which conventional poly(A)-based single-cell RNA sequencing fails to resolve this heterogeneity owing to the overwhelming host transcript background. To overcome this, we developed B-BEST (HBV can BE Seen on host Transcriptome), a targeted sc/snRNA-seq approach using custom beads conjugated with HBV-specific probes to simultaneously quantify 5 viral genomic regions (S, X, pgRNA, rcDNA, cccDNA). APPROACH AND RESULTS:We validated B-BEST in HepAD38 cells and integrated it with long-read sequencing, spatial transcriptomics, and in situ hybridization in liver tissues from treatment-naïve patients and antiviral-treated humanized liver-chimeric (Hu-URG) mice. B-BEST revealed significant heterogeneity among HBV-positive hepatocytes. In HBeAg-positive patients, HBV-positive subpopulations enriched for hepatic synthesis/metabolism and mitochondrial function were linked to active viral replication and transcription, with only a mild type I interferon response. Severe inflammation correlated with suppressed HBV replication. Long-read sequencing indicated that integrated HBV transcripts preferentially used host promoters and contributed to HBsAg persistence in HBeAg-negative patients. In Hu-URG mice, entecavir upregulated metabolic pathways, while peginterferon alfa-2b induced broad-spectrum antiviral programs. Notably, clonal expansion of hepatocytes diluted the intrahepatic viral reservoir when viral replication was inhibited, suggesting a proliferative dilution mechanism that may contribute to functional cure. CONCLUSIONS:In summary, our B-BEST platform provides resources for delineating the heterogeneous landscape of HBV infection, identifying host determinants and microenvironmental factors that govern viral replication and persistence, and highlighting hepatocyte proliferation as a potential clearance mechanism for antiviral therapy.

Objective Metagenomic next-generation sequencing (mNGS) is an effective method for the detection of microorganisms. Early allograft dysfunction (EAD) is a common complication after liver transplantation. The association between early postoperative microorganisms in bile and EAD is unclear, so we evaluated the association of microorganisms and other potential risk factors with EAD. Methods A total of 100 patients who underwent orthotopic liver transplantation with biliary T tube placement in Huashan Hospital Fudan University from March 2021 to July 2022 were studied. Clinical data, the occurrence of EAD, and bile microorganisms’ information detected by mNGS were collected. Results EAD occurred in 22 recipients (22%). Patients with EAD had a longer length of postoperative hospital stay. Bacteroides spp. detected by mNGS in bile was identified as an independent risk factor for EAD. Also, operation time, the MELD score of the recipient, and donor AST level were also independent risk factors for EAD. Conclusion Bacteroides spp. detected by mNGS in bile after liver transplantation was identified as an independent risk factor for EAD, which may reflect the translocation of intestinal flora into the biliary tract and may serve as a potential early warning indicator of poor quality of the donor liver. Recipients with EAD had longer LOS, which may indicate a poor short-term prognosis.

Immune checkpoint blockade has improved the treatment of hepatocellular carcinoma (HCC), yet most patients fail to achieve durable responses to PD-1 blockade. Tumor-associated macrophages are major determinants of immunotherapy resistance, but the tumor-intrinsic epigenetic programs that recruit and polarize macrophages in HCC remain incompletely defined. Here, we identify activating transcription factor 2 (ATF2) as a driver of macrophage-mediated PD-1 resistance in HCC. Integrated single-cell and spatial transcriptomic analyses revealed enrichment of M2-like macrophages in non-responding tumors and identified ATF2 as a candidate tumor-intrinsic regulator associated with this immunosuppressive state. Genetic perturbation of ATF2 in syngeneic HCC models demonstrated that ATF2 promotes tumor growth, M2-like macrophage accumulation, CD8+ T-cell dysfunction, and resistance to PD-1 blockade. Mechanistically, ATF2 enhanced CCL2-dependent macrophage recruitment and polarization by coupling transcriptional activation with enhancer remodeling. ATF2 interacted with KAT3A/CBP to increase global and site-specific histone lactylation, particularly H3K18 lactylation, thereby preserving chromatin accessibility at NF-κB-responsive regulatory elements and sustaining inflammatory and chemokine transcription. ATF2 also directly bound the CCL2 promoter, establishing a dual mechanism for CCL2 activation. Pharmacologic inhibition of ATF2 remodeled the tumor immune microenvironment, reduced CD206+ macrophage infiltration, restored CD8+ T-cell effector function, and sensitized orthotopic HCC tumors to PD-1 blockade without overt toxicity. In patient specimens, elevated ATF2 and H3K18 lactylation correlated with NF-κB target expression, CD206+ macrophage enrichment, reduced CD8+ T-cell infiltration, and non-response to anti-PD-1 therapy. These findings define an ATF2-KAT3A-H3K18la-NF-κB chemokine axis that promotes macrophage-mediated immune evasion and nominate ATF2 as a therapeutic target for overcoming PD-1 resistance in HCC.

The tumor microenvironment (TME) poses a major barrier to effective immunotherapy, yet high-throughput perturbation-mapping approaches to dissect TME spatial complexity and its contextual immune modulators remain lacking. Here, we introduce CRISPR-laser-captured microdissection (LCM) integration mapping of the tumor-immune microenvironment (CLIM-TIME), a scalable platform that integrates CRISPR screening with LCM of metastatic tumors for transcriptomic, deconvolution, and immunofluorescence analyses. CLIM-TIME enables spatially resolved mapping of how tumor suppressor gene (TSG) loss reshapes the TME and modulates immune responses. We identified seven distinct TME subtypes, revealing that DNA repair and Polycomb repressive complex (PRC) TSG loss is linked to immune-infiltrated TMEs sensitive to T cell therapy. In contrast, knockouts of TSGs in the Hippo pathway promoted immune evasion and therapy resistance by fostering myeloid-enriched but T cell-excluded TMEs with elevated extracellular matrix (ECM). Targeting the ECM-crosslinking enzyme LOXL2 effectively remodeled the metastatic TME, enhancing T cell infiltration and improving therapeutic efficacy in lung metastases across multiple cancers.

BACKGROUND:We report orthotopic combined whole-liver and bilateral-kidney xenotransplantation (xeno-CLKT) from a six-gene-edited pig into a 53-year-old human decedent recipient. METHODS:After simultaneous implantation and reperfusion, graft function was monitored for nearly 5 days using continuous clinical assessments. Host responses were further characterized with single-cell RNA sequencing, proteomics, and metabolomics of blood and graft tissues collected before and after transplantation. FINDINGS:All grafts maintained basic physiological function throughout the observation period, with no evidence of hyperacute rejection. Early post-transplant immune analysis showed expansion of S100A12+ neutrophils, which emerged as central hubs in inferred intercellular communication networks. Systems-level metabolomic profiling indicated that post-transplant metabolic patterns remained positively correlated with the recipient's pre-transplant baseline, with generally higher magnitudes. CONCLUSIONS:This study provides initial evidence for the feasibility of pig-to-human orthotopic whole-liver plus bilateral-kidney transplantation and identifies early immune and metabolic features that may inform perioperative management and future clinical translation. FUNDING:The research was supported by the Guangxi Key Research and Development Program (AB24010059), the Shenzhen Medical Research Fund (SMAF) (B2302008), and the Guangdong Province Guangdong-Shenzhen Joint Key Project (2023B1515120083).

The prognosis for patients diagnosed with hepatocellular carcinoma with bile duct tumor thrombus (HCC-BDTT) remains dismal, and there are presently no universally accepted treatment guidelines to address this complex condition. Long-term outcomes of liver transplantation (LT) for HCC-BDTT patients are unclear, and whether LT is a proper therapeutic option for HCC-BDTT patients remains to be determined. Therefore, we design a clinical trial to evaluate whether LT can improve recurrence-free survival (RFS) and overall survival (OS) in HCC-BDTT patients. This is an open-labeled, single-arm, prospective, multicenter and real-world study aiming to assess the survival outcomes of HCC-BDTT patients in LT. Patients will be enrolled based on histological confirmation of HCC with BDTT. The study is planned to take 4 years, 2 years for enrollment and 2 years for follow-up. We anticipate that LT confers beneficial survival outcomes for HCC-BDTT patients, specifically in terms of the pivotal parameters, such as RFS and quality of life. Upon successful completion of the trial, we will extend our monitoring over a longer follow-up time to accurately estimate important indicators such as OS. We expect that this study provides substantial evidence to refine treatment guidelines through thorough data analysis, ultimately contributing to better patient outcomes and advancing our understanding of the disease.Trial Register: Trial registered at www.clinicaltrials.gov (NCT06928415)

Background/Aims Liver transplantation (LT) following total hepatectomy is a life-saving treatment for hepatocellular carcinoma (HCC). The HCC recurrence after LT hinders the effectiveness of the procedure. The objective of this study is to develop a pre-operative risk stratification model based on a liquid biopsy. Methods We conducted a comprehensive multi-omics study of 260 HCC patients from three centers, including clinical data, low-coverage whole-genome sequencing of cell-free DNA (cfDNA) from plasma, as well as whole-exome, single-nucleus RNA, and spatial transcriptomics from matched tumor and non-tumor tissues. Results We identified cfDNA-derived copy number alteration (CNA) signatures associated with post-transplant recurrence. By integrating cfDNA-derived CNA profiles with single-cell transcriptomic data, we traced recurrence-associated cfDNA to a distinct subpopulation of malignant cells within the primary tumor. These cells were embedded in a pro-metastatic microenvironment of specialized endothelial subtypes and cancer-associated fibroblasts. Notably, most recurrence-associated lesions were detectable in cfDNA prior to liver transplantation (LT). Building on these insights, we developed the ZJU Criteria based on CNA fragments and tumor markers, a pre-LT risk prediction tool that integrates conventional clinical factors with cfDNA-derived CNA signatures, and validated it using internal and independent external cohorts. Conclusion Our findings suggest that post-transplant recurrence commonly originates from advanced subclones that emerge late during tumor evolution. The ZJU Criteria provides an accurate, non-invasive strategy that significantly improves pre-LT risk stratification and clinical decision-making for patients with HCC.

Recurrence after tumor resection and liver transplantation remains a major clinical challenge in hepatocellular carcinoma (HCC). We examined tumor samples from HCC liver transplant patients to identify drivers of disease recurrence. Integration of proteomic profiling of human HCC samples with or without recurrence and T cell killing assays linked the cholesterol esterification enzyme SOAT1 to immune evasion and cancer recurrence. Genetic or pharmacological inhibition of SOAT1 sensitized liver cancer cells to CD8+ T cell-mediated immunosurveillance, anti-programmed cell death 1 (anti-PD-1) therapy, and chimeric antigen receptor (CAR)-T cell therapy and increased intratumoral CD8+ T cell infiltration. Mechanistically, the inhibition of SOAT1-mediated cholesterol esterification disrupted intratumoral cholesterol and lipid metabolism and reduced unsaturated fatty acids and prostaglandin E2 production; this impaired the antioxidant capacity and metabolic resilience of cancer cells under immune attack. Accordingly, SOAT1 inhibition enhanced immunotherapy efficacy in obesity-associated tumors and under immunosuppressant-induced conditions. Thus, cholesterol esterification supports redox fitness and cancer cell resilience upon immune attack, suggesting a strategy to prevent recurrence and improve immunotherapy outcomes.

Hepatic ischemia/reperfusion injury (IRI) commonly complicates liver transplantation (LT). However, the precise mechanisms underlying hepatic IRI remain incompletely understood. We acquired single-cell RNA sequencing (scRNA-seq) and transcriptome RNA sequencing data of LT patients from the GEO database. Employing scRNA-seq, we delved into the interplay between non-immune and immune cells in hepatic IRI, pinpointing genes exhibiting altered expression patterns. Integrating insights gleaned from scRNA-seq and transcriptome RNA sequencing datasets, we deepened our comprehension of cellular interactions and underlying mechanisms in hepatic IRI. Additionally, we conducted preliminary validation of identified gene expression alterations using immunofluorescence techniques. Using scRNA-seq, we detected significant changes in the populations of liver sinusoidal endothelial cells (LSECs) and monocytes after hepatic ischemia-reperfusion injury (IRI). By integrating scRNA-seq with bulk transcriptome RNA sequencing data, we identified key genes with dysregulated expression in LSECs (ICAM1, SOCS3, NFKBIZ, JUND, TNFRSF12A and HSPA6) and monocytes (SOCS3, JUND, FPR2 and NR4A2). Our analysis of cell communication indicated that the ANXA1-FPR2 axis might be a pivotal signature in mediating interactions between LSECs and monocytes. We then established a mouse model for IRI, and further analyses using flow cytometry and immunofluorescence showed a significant increase in monocyte proportion post-IR (p < 0.01). Consistently, Western Blot also revealed significant upregulation of ANXA1 and FPR2 (p < 0.01). Our study elucidated the cellular interactions and signalling pathways following IRI. The interplay between LSECs and monocytes likely triggers a cascade of events, promoting monocyte infiltration and amplifying inflammatory responses, thus worsening the deleterious effects of IRI.

AIMS:Post-transplant diabetes mellitus (PTDM) is a well-known phenomenon; however, there is currently no consensus regarding early postoperative hyperglycemia in liver transplant recipients. METHODS:This prospective observational study utilized continuous glucose monitoring (CGM) for approximately one week post-liver transplantation to assess hyperglycemia and its association with infections during hospitalization. RESULTS:Data from 95 patients (37 with diabetes, 58 without diabetes) revealed a 100 % incidence of early hyperglycemia. Patients in the group with diabetes exhibited significantly lower time in range (TIR), higher mean glucose levels, high blood glucose index (HBGI), maximal glucose levels, and mean daily differences compared to the group without diabetes (all P < 0.05). Infections, including pneumonia, intra-abdominal infection, and bloodstream infection, occurred in 30.53 % of patients during the early post-transplant period. In patients with diabetes, TIR was an independent protective factor against early infection (OR 0.865, 95 % CI 0.765-0.970), while higher mean glucose level (OR 1.526, 95 % CI 1.050-2.218) and time above range (TAR, OR 1.105, 95 % CI 1.022-1.196) were independent risk factors. CONCLUSIONS:The study highlights the high prevalence of early postoperative hyperglycemia in liver transplant recipients and suggests that reduced TIR and increased TAR may elevate infection risks, particularly in patients with preoperative diabetes.

BackgroundLiver transplantations (LTs) with extended criteria have produced surgical results comparable to those obtained with traditional standards. However, it is not sufficient to predict hepatocellular carcinoma (HCC) recurrence after LT according to morphological criteria alone. The present study aimed to construct a nomogram for predicting HCC recurrence after LT using extended selection criteria.MethodsRetrospective data on patients with HCC, including pathology, serological markers and follow-up data, were collected from January 2015 to April 2020 at Huashan Hospital, Fudan University, Shanghai, China. Logistic least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analyses were performed to identify and construct the prognostic nomogram. Receiver operating characteristic (ROC) curves, Kaplan-Meier curves, decision curve analyses (DCAs), calibration diagrams, net reclassification indices (NRIs) and integrated discrimination improvement (IDI) values were used to assess the prognostic capacity of the nomogram.ResultsA total of 301 patients with HCC who underwent LT were enrolled in the study. The nomogram was constructed, and the ROC curve showed good performance in predicting survival in both the development set (2/3) and the validation set (1/3) (the area under the curve reached 0.748 and 0.716, respectively). According to the median value of the risk score, the patients were categorized into the high- and low-risk groups, which had significantly different recurrence-free survival (RFS) rates (P < 0.01). Compared with the Milan criteria and University of California San Francisco (UCSF) criteria, DCA revealed that the new nomogram model had the best net benefit in predicting 1-, 3- and 5-year RFS. The nomogram performed well for calibration, NRI and IDI improvement.ConclusionsThe nomogram, based on the Milan criteria and serological markers, showed good accuracy in predicting the recurrence of HCC after LT using extended selection criteria.

Introduction With the continuous advancement of surgical technique,com-bined vascular resection has become increasingly common dur-ing complex surgical procedures.In such cases,ensuring the safe and effective reconstruction of blood vessels after resection is of paramount importance.When direct vascular reconstruction is not feasible,the application of vascular grafts becomes necessary to re-store vascular continuity and function.Commonly employed vascu-lar grafts in clinical practice include allogeneic graft vessels(AGVs),autologous vessels,and artificial vessels.Among these,AGVs offer distinct advantages particularly in its complex structures and sat-isfying histocompatibility,making it a valuable option for vascular reconstruction.

This study aims to identify branched-chain amino acid (BCAA) plasma metabolites and gene signatures that enhance prognostic assessments in non-small cell lung cancer (NSCLC) patients receiving immunotherapy. Plasma metabolites were measured using untargeted UPLC-MS/MS (n = 94 and 40), with lymphocyte subset tests on 72 patients. BCAA-related subtypes were identified in NSCLC datasets (n = 274, 176, and 196). A prognostic risk model was developed and validated in NSCLC (n = 16, 27, 24, and 339), melanoma (n = 25), and pan-cancer ICIs cohorts (n = 330 and 81). Immune cell infiltration and prognostic signatures were validated using mIF (n = 21 in CHCAMS), scRNA-seq (n = 8 and 21), and spatial transcriptomics (n = 2 and 6). Cell and animal experiments involving HMGCS1 were conducted in a lung cancer model. Additionally, based on our previous findings that B cells with higher malignancy exhibited enhanced cholesterol homeostasis pathways in diffuse large B-cell lymphoma (DLBCL), we further analyzed the prognostic value of HMGCS1 using our spatial transcriptomics (n = 10) and immunohistochemistry (IHC, n = 39) in DLBCL. Our plasma metabolite analysis showed higher L-leucine levels were associated with better prognosis and had higher T cell counts and CD4+ T cell counts (P < 0.05). In GEO datasets, four NSCLC subtypes were identified, showing distinct prognostic outcomes and tumor microenvironment. Five BCAA-related genes (ACAT2, ALDH2, HMGCS1, MLYCD, and PPM1 K) formed a prognostic risk model for NSCLC, validated through Kaplan–Meier and ROC curve analyses in ICI cohorts (P < 0.05). HMGCS1 was an independent prognostic value in ICI cohorts and was negatively correlated with CD8+ T cell infiltration, while positively correlating with tumor severity, cholesterol homeostasis, and BCAA degradation across multiple platforms, including GEO datasets, our mIF cohort, public scRNA-seq, and spatial transcriptomics (P < 0.05). And our cell and animal function experiments found HMGCS1 overexpression promotes metabolic pathways and accelerates tumor growth, whereas HMGCS1 knockdown suppresses tumor progression in a mouse model treated with PD-1 monoclonal antibody (P < 0.05). In DLBCL, high HMGCS1 expression was associated with shorter overall survival, enriched in B cells and relapsed patients, correlated with cholesterol homeostasis and amino acid degradation pathways, and its prognostic value was further validated at the protein level by our IHC cohort (P < 0.05). This study identifies a BCAA-related plasma metabolites and gene signature as effective prognostic markers for NSCLC patients receiving immunotherapy, with HMGCS1 as a key prognostic factor influencing tumor progression and immune response.

A pH/thermo-responsive nanoframeworks has been engineered using ZIF-8 decorated with the chitosan (C)-poly(N-isopropylacrylamide) (C-PNIPAM) for the co-delivery of oxaliplatin (OXP) and cabazitaxel (CTX) in liver cancer cells. The chitosan PNIPAM nanoparticles (CPNPs) exhibited particle diameters of 205 nm and a surface charge of + 28 mV. The CTX and OXP loading contents in the CPNPs were 12.10

Recurrence after liver cancer resection and transplantation remains a critical clinical challenge, driven by immune evasion. However, effective immune-sensitizing targets for preventing or managing relapse remain limited. In this study, we integrated differential proteomic profiling of human liver cancer samples with or without subsequent recurrence and T cell killing assays to uncover a pivotal role for the cholesterol esterification enzyme SOAT1 in immune evasion and cancer recurrence. Genetic knockout or pharmacological inhibition of SOAT1 markedly sensitized both mouse and human cancer cells to immune surveillance and T cell-mediated killing. Mechanistically, blocking cholesterol esterification disrupted excessive cholesterol biosynthesis, impairing cancer cells’ antioxidant capacity and metabolic resilience under immune attack. Importantly, SOAT1 inhibition effectively suppressed tumor immune escape and improved the efficacy of anti-PD1 and CAR-T cell therapies, even in immunocompromised conditions. These findings highlight cholesterol esterification as a key driver of cancer cell redox metabolism resilience under immunosurveillance and position SOAT1 as a promising target to prevent cancer relapse and enhance immunotherapy outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Chimeric antigen receptor (CAR) T-cell therapy for malignant tumors has reached a crucial stage, with recent studies underscoring the role of T-cell exhaustion in determining the efficacy of CAR-T therapy. This trailblazing discovery has opened new avenues to augment the potency of CAR-T therapy. Basic leucine zipper ATF-like transcription factor (BATF) is indispensable in alleviating T-cell exhaustion and is pivotal in the early stages of CD8(+) T-cell differentiation. In cooperation with other transcription factors, it plays a key role in the differentiation and maturation processes of exhausted T cells. A deeper comprehension of BATF's mechanisms in T-cell biology may yield novel insights into amplifying the efficacy of CAR-T therapy.