Background The anatomical TNM staging system inadequately reflects the profound survival heterogeneity in gallbladder cancer (GBC), thereby limiting accurate risk stratification and contributing to suboptimal clinical decision-making. To overcome this limitation, we developed a multidimensional and dynamic prognostic framework integrating clinicopathological features, systemic inflammatory markers, and tumor biomarkers, leveraging large-scale multicenter real-world data. Methods A total of 1,354 patients with GBC from 44 medical centers were retrospectively analyzed and randomly assigned to training (n = 947) and validation (n = 407) cohorts. Independent prognostic factors were identified using LASSO and multivariable Cox regression to construct three risk scores: clinicopathological (C-score), blood-based inflammatory (B-score), and tumor marker (T-score). An integrated prognostic model was subsequently developed and evaluated through machine-learning–based benchmarking. Model performance was assessed using time-dependent ROC analysis (6–48 months), calibration curves, and decision curve analysis (DCA). Gene Set Enrichment Analysis (GSEA) was performed to explore the biological relevance of the scoring systems. Results Age, R0 resection status, TNM stage, CA19-9 (log1p), neutrophil-to-lymphocyte ratio (NLR), and lymphocyte-to-CRP ratio (LCR) were identified as independent prognostic factors. The integrated model consistently outperformed individual C-, B-, and T-scores across all follow-up intervals, demonstrating strong discriminative ability with a 12-month AUC of 0.857 in both cohorts. Calibration and decision curve analyses confirmed good model reliability and clinical utility. GSEA revealed distinct molecular associations underlying the three scores, including ECM–receptor interaction, immune-inflammatory signaling (JAK–STAT/NF-κB), and metabolic stress pathways (HIF-1/p53). A web-based dynamic prediction platform was further developed to enable individualized survival estimation. Conclusion This multidimensional framework provides a biologically interpretable and dynamically adaptable tool for prognostic stratification in gallbladder cancer. Implemented through a web-based platform, the model facilitates individualized risk assessment and supports data-driven clinical decision-making.

Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary system and is frequently diagnosed at an advanced stage upon initial presentation. Nonetheless, the introduction of conversion therapy has allowed certain initially inoperable cases of GBC to become candidates for radical resection after comprehensive modalities, including chemotherapy, immunotherapy, and radiotherapy. In the current retrospective analysis, we present two patients with initially unresectable GBC treated at Xiangyang Central Hospital, affiliated with Hubei University of Arts and Science. After receiving a combination of chemotherapy and immunotherapy, substantial tumor regression was observed at both primary and metastatic sites, achieving partial remission according to the Response Evaluation Criteria in Solid Tumors (RECIST). Conversion therapy proved effective, enabling both patients to undergo successful radical resections. Following surgery, the original therapeutic regimen was continued until completion of five cycles. Follow-up extended through November 2025, with no evidence of tumor recurrence observed.

Liver invasion is one of the most frequent events in the progression of gallbladder cancer (GBC). However, the cellular and pathological role of the tumor-liver-interface microenvironment in liver invasion is still enigmatic. Here, we applied single-cell and spatial transcriptomics to systematically investigate the cellular component and gene expression regulation of the microenvironment from the tumor to the liver, specifically the invasive boundary. Our analyses revealed that CXCL9+ macrophage-rich immune cell niches were accumulated in the tumor-liver invasive margin, where 2 subclasses of the CXCL9+ immune cell niches, CXCL9+TRAC+ (CT) and CXCL9+C1QB+ (CC) niches, were identified. CD8+ T cells were recruited by CXCL9+ macrophages through CXCL9-CXCR3 interaction in the CT niche, which was located adjacent to the liver. Moreover, the CC niche was proximal to the tumor core, where tumor cells induced CD8+ T cell exhaustion via LGALS4 expression. In addition, our cohort study showed that high CXCL9 and low LGALS4 in the liver invasion margin demonstrated a favorable prognosis and better responses to anti-PD-1 immunotherapy for patients with gallbladder cancer. Altogether, these findings demonstrate novel cellular and molecular mechanisms underlying liver invasion and offer clinical value for immunotherapies.

EVs-delivered OGA disrupts neuronal metabolic flux and glycosylation

Background: The feasibility and safety of laparoscopic liver resection (LLR) and open liver resection (OLR) in combined hepatocellular-cholangiocarcinoma (cHCC-CCA) patients remain controversial. This study compared the clinical outcomes of LLR versus OLR for patients with cHCC-CCA. Methods: Clinicopathological features of cHCC-CCA patients who underwent liver resection (LR) between 2010 and 2022 were retrospectively analyzed. Propensity score matching (PSM) was employed to balance intergroup differences. Univariate and multivariate Cox analyses were employed to identify independent predictors of overall survival (OS). Results: Of the 141 cHCC-CCA patients, 78 underwent LLR and 63 underwent OLR. After PSM, the LLR group had lower estimated blood loss (EBL) (200 vs. 300 mL, P=0.004) and shorter postoperative length of stay (LOS) (10.0 vs. 15.0 days, P<0.001). Multifactor Cox regression analyses showed that hepatocellular carcinoma (HCC) as the tumor main ingredient [hazard ratio (HR) =0.323, 95% confidence interval (CI): 0.151-0.693, P=0.004] was an independent protective factor for OS. After PSM, no statistically significant difference in OS was observed between the two groups (60.0 vs. 69.0 months, P=0.54). Conclusions: cHCC-CCA patients undergoing LLR are safe and feasible with lower EBL, and shorter postoperative LOS. No statistically significant difference in long-term OS was observed between LLR and OLR.

INTRODUCTION:Subsegment IVb (S4b) is a common site of direct invasion in gallbladder cancer, and its complete resection is critical to achieve negative surgical margins. However, the portal venous anatomy of S4b is highly complex and exhibits significant interindividual variation, which the conventional Couinaud classification fails to adequately delineate. This study utilized three-dimensional visualization to systematically characterize the anatomical features and branching patterns of the portal veins supplying S4b, with the goal of establishing a practical anatomical classification to inform preoperative planning for radical gallbladder cancer surgery. METHODS:This retrospective analysis utilized thin-slice computed tomography data from 115 patients who underwent abdominal contrast-enhanced computed tomography for a spectrum of clinical indications (e.g., evaluation of focal liver lesions, trauma assessment, or other abdominal pathologies). Three-dimensional reconstruction of the portal venous system was performed using dedicated visualization software. We focused on analyzing the origin, number, course, and common trunk patterns of the internal branches of the left portal vein supplying S4b. RESULTS:S4b was supplied by 1 to 6 portal vein branches, with 2 to 6 branches observed in 87.1% of cases. The overwhelming majority (>90%) of these branches originated from the umbilical portion of the left portal vein. Based on the common trunks supplying both S4a and S4b, the vascular anatomy was classified into four types: type A (no common trunk, 23.4%), type B (one common trunk 53%), type C (two common trunks 20%), and type D (more than two common trunks or a branch derived from the right portal vein 3.4%). Overall, 73% of patients exhibited 1-2 common trunks. The diameter of these common trunks correlated with their number, and their trajectories consistently coursed toward the S4a segment. CONCLUSIONS:The proposed four-type classification systematizes the broad spectrum of S4b portal venous anatomy. This framework facilitates a preoperatively planned, targeted resection in radical gallbladder cancer surgery, which is instrumental in optimizing procedural safety and oncologic outcomes.

Gallbladder cancer (GBC) is a highly aggressive malignancy with dismal outcomes. To dissect its molecular characteristics and identify potential therapeutic avenues, we performed proteogenomic characterization of 195 tumors and 135 adjacent non-cancerous gallbladder tissues. Integrative analyses highlighted TP53 and ELF3 mutations as key drivers disrupting signaling and metabolism. ErbB2 amplification, a pivotal genomic event, was associated with reduced canonical PI3K/AKT and RAS/MAPK/ERK signaling yet enhanced proliferative activity. We discovered potential gain-of-function mutations in ErbB2 and ErbB3 predicted to enhance ErbB2-ErbB3 heterodimer activity. ACAT1 and PHGDH were identified as metabolic drivers of GBC liver invasion. Integrated molecular and immune subtyping delineated four distinct multi-omics and immune microenvironment subtypes, each carrying prognostic and therapeutic relevance. Although rare, neuroendocrine GBC was separately characterized, revealing MEIS1 as a potential regulator of neuroendocrine-like features. Together, this study establishes a proteogenomic landscape of GBC, providing biological insights and guiding future translational efforts.

Verification after selective inhibition of OGA or addition of glycosylated substrates

Single cell analysis maker and proteomic PCA analysis

Peripheral nerve invasion (PNI) is an early and decisive step in gallbladder cancer progression that strongly predicts poor postsurgical outcome. The tumor-neuron interactions that drive PNI could represent potential targets and biomarkers to improve treatment of gallbladder cancer. In this study, we demonstrated that gallbladder cancer provoked necroptosis of neurons to enable PNI. Gallbladder cancer cells transferred extracellular vesicles (EV) containing O-GlcNAcase (OGA) to neurons, which activated RIPK1-dependent necroptosis. Mechanistically, EV-derived OGA suppressed RIPK1 glycosylation while enhancing its phosphorylation, thereby activating the RIPK1/RIPK3/MLKL axis to trigger neuronal necroptosis. Subsequent neuronal release of HMGB1 engaged RAGE on gallbladder cancer cells, establishing a loop that accelerated PNI. Moreover, the RAGE antagonist FPS-ZM1 synergized with gemcitabine to suppress tumor progression. Collectively, these findings uncover an EV-mediated cross-talk between gallbladder cancer cells and neurons in which RIPK1-dependent necroptosis and its effector HMGB1 drive PNI, positioning the HMGB1-RAGE axis as a tractable therapeutic target.Significance: Tumor-derived extracellular vesicles trigger neuronal necroptosis that fuels peripheral nerve invasion, creating a tumor-neuron signaling loop that could be leveraged for liquid biopsy and personalized therapy strategies in neurotropic cancers.

Gallbladder cancer (GBC), a lethal malignancy of the biliary tract, is associated with a poor clinical prognosis. Although chemo-immunotherapy combinations demonstrate preliminary efficacy, the molecular determinants of treatment response remain elusive. Emerging evidence implicates aberrant alternative splicing in modulating tumor immunity. Through an in vitro CRISPR/Cas9 screen, we identified SRSF2 as a key RNA-binding protein regulating PD-L1 expression. Intriguingly, SRSF2 does not directly bind PD-L1 mRNA. Multi-omics analyses (mRNA-seq, RIP-seq, and proteomics) revealed that SRSF2 induces exon skipping in hnRNPD, shifting isoform expression from full-length P45 to truncated P40. Functional studies established that P45-but not P40-binds to AU-rich elements in the PD-L1 3'-UTR to promote mRNA degradation. Leveraging this mechanism, we designed splice-switching antisense oligonucleotides (ASOs) that block SRSF2-mediated exon skipping, restoring P45 expression. This intervention effectively reduced PD-L1 levels and potentiated T-cell-mediated cytotoxicity in vitro and in vivo. These findings elucidate a splicing-centric mechanism of immune evasion and highlight the therapeutic potential of splicing modulation in cancer immunotherapy. Proposed model of the SRSF2-hnRNPD-PD-L1 axis in gallbladder cancer (GBC) immune evasion and its therapeutic targeting. Overexpression of SRSF2 drives hnRNPD exon skipping, shifting the isoform balance from the PD-L1-degrading P45 to the truncated P40. This transition stabilizes PD-L1 mRNA and facilitates tumor immune evasion. Conversely, therapeutic intervention with splice-switching ASOs blocks SRSF2-mediated alternative splicing, restores P45 expression, and effectively reactivates T-cell-mediated cytotoxicity against GBC cells.

PURPOSE:The purpose of this study is to evaluate the efficacy and safety of nab-paclitaxel combined with gemcitabine (AG regimen) as a first-line chemotherapy for patients with unresectable gallbladder cancer (GBC). MATERIALS AND METHODS:A retrospective analysis was conducted on patients diagnosed with GBC via histological examination. Inclusion criteria included locally advanced or metastatic gallbladder cancer deemed unresectable by multidisciplinary team (MDT) consensus; age 18-75 years; ECOG performance status ≤ 2; and AG as first-line chemotherapy. Treatment response was assessed using RECIST 1.1 criteria. Primary endpoints were overall response rate (ORR) and progression-free survival (PFS); secondary endpoints included disease control rate (DCR), overall survival (OS), and adverse events incidence. RESULTS:Thirty-one patients were enrolled. The ORR was 48.4%, and DCR was 90.3%. The median PFS was 8.4 months (95% CI, 5.0-11.8), and the median OS was 20.9 months (95% CI, 16.8-24.9). Among the 11 patients with locally advanced disease, 7 patients (63.6%) successfully converted to a resectable state and underwent surgery. Grade 3/4 treatment-related adverse events occurred in 11 patients (35.5%), predominantly hematologic toxicities, and no treatment-related deaths or treatment discontinuations due to adverse events were observed. CONCLUSION:First-line AG chemotherapy demonstrated promising efficacy and manageable safety for patients with unresectable GBC, with a substantial portion achieving disease control and some converting to resectable status.

Liver metastasis is a frequent and clinically important event across multiple solid tumors, yet a cross-cancer single-cell description of the metastatic immune ecosystem remains incomplete. Here, we integrated single-cell RNA-sequencing data from primary tumors and liver metastases across 12 cancer types and combined cell-state annotation, pseudotime ordering, ligand–receptor inference, and multiplex immunofluorescence as orthogonal tissue-level support. We identified a hepatocyte-like malignant epithelial state enriched in liver metastases, characterized by coexpression of hepatic genes, epithelial markers, oncogenic transcripts such as MYC and KRAS, and inferred copy-number-variation signals. CellChat analysis suggested that this epithelial state may engage RGS1-high and LILRB5-high macrophage states through a CCL3–CCR1-associated signaling axis. Reprocessed tissue imaging supported spatial proximity between TAT+/EPCAM+ epithelial cells and these macrophage populations across several tumor types. In addition, macrophage and T-cell composition differed between primary tumors and liver metastases, and sample-level associations linked RGS1-high macrophages to selected CD4 + T-cell states and LILRB5-high resident-like macrophages to selected CD8 + T-cell states. Overall, this study provides a pancancer descriptive atlas of epithelial–immune programs associated with liver metastasis and nominates candidate cellular associations for future functional testing.

Objective·To systematically evaluate the potential causal associations between plasma protein levels and the risk of gallbladder cancer (GBC) through proteome-wide Mendelian randomization analysis.Methods·GBC genome-wide association study (GWAS) summary data from Japan, Republic of Korea, Finland, and the UK were used for a multi-ethnic meta-analysis to obtain genetic effect estimates for GBC. Summary-level GWAS data of plasma proteomes from two large-scale proteomic studies (deCODE and UKBPPP) were used as exposure variables. A generalized summary-data-based Mendelian randomization (GSMR) analysis was performed to assess the causal associations between each plasma protein level and GBC risk, and significant associations were validated by two-sample Mendelian randomization (TSMR). Significant associations discovered between plasma proteins and GBC risk were also validated using colocalization analysis.Results·GSMR analysis identified 56 plasma proteins significantly causally associated with GBC risk in the deCODE cohort (Padj<0.001), of which 31 proteins were replicated in another independent proteomic cohort (UKBPPP). TSMR further confirmed two proteins with robust causal associations with GBC. Among these two proteins, higher plasma complement receptor 2 (CR2) levels were significantly associated with a reduced risk of GBC (OR=0.258, 95%CI 0.071‒0.935, P=0.039), whereas higher plasma neural cell adhesion molecule 1 (NCAM1) levels were associated with an increased risk of GBC (OR=2.306, 95%CI 1.432‒3.713, P<0.001). The Bayesian colocalization analysis showed strong colocalization between protein quantitative trait loci (pQTL) variants of CR2 and NCAM1 and GBC risk variants.Conclusion·Two potential risk-related biomarkers for GBC, CR2, and NCAM1, were identified in the whole plasma proteome. The elevated CR2 level is linked to a lower risk of GBC, while the elevated NCAM1 level corresponds to a higher risk.

OBJECTIVE:Gallbladder cancer (GBC) is a rare gastrointestinal malignancy with a global 5-year survival rate of less than 5%. Early diagnosis is challenging owing to the lack of specific clinical symptoms. Additionally, the high heterogeneity of gallbladder tumors limits the clinical utility of unimodal deep-learning methods for GBC diagnosis. This study aimed to develop a novel multimodal deep-learning model to facilitate the preoperative diagnosis of GBC in more patients. METHODS:We conducted a retrospective multicenter study using contrast-enhanced arterial phase computed tomography (CT) images and laboratory examination data from 300 patients (150 GBC cases and 150 non-GBC cases) extracted from electronic medical records of two Grade A tertiary hospitals in Shanghai between 2018 and 2020. A novel two-stage multimodal diagnostic model (GBC-DiagNet) was developed: the first stage achieved coarse segmentation of the gallbladder region using a position-constrained 3D Attention U-Net (improved by combined sampling) to avoid over-segmentation; the second stage realized GBC detection via an adaptive feature fusion strategy, which optimizes the weighted integration of handcrafted radiomic, deep radiomic and laboratory examination features to enhance diagnostic performance. RESULTS:On the independent test set, the model achieved an accuracy of 0.933 (95% confidence interval [95% CI]: 0.927-0.94), specificity of 0.912 (95% CI: 0.904-0.922), sensitivity of 0.962 (95% CI: 0.937-0.986), precision of 0.893 (95% CI: 0.875-0.911), an F1-score of 0.926 (95% CI: 0.919-0.932) and AUC (area under the curve) of 0.9706 (95% CI: 0.961-0.981). Compared with the optimal unimodal model, our model improved accuracy, sensitivity, and F1-score by 14.28%, 16.76%, and 16.85%, respectively. Furthermore, compared to state-of-the-art deep-learning architectures (ResNet, DenseNet, MobileNet, ConvNeXt, ViT), our model exhibited absolute improvements of 7.68% in accuracy, 8.03% in F1-score, and 0.0059 in AUC. CONCLUSION:The proposed multimodal model integrating contrast-enhanced CT and laboratory data achieves stable and clinically meaningful diagnostic performance for gallbladder cancer, supporting its utility as an artificial intelligence-assisted tool for preoperative noninvasive diagnosis.

MethodsThe NOZ human gallbladder cancer cell line and GelMA hydrogel bioink were used to construct a 3D gallbladder cancer model via extrusion-based 3D bioprinting, aiming to simulate the tumor microenvironment. The morphology, viability, and proliferation of cells in the 3D constructs were assessed using microscopy, live/dead cell staining, and the cell counting kit-8 (CCK-8) assay. Following RNA extraction, RNA sequencing was performed on the Illumina NovaSeqTM 6000 platform. Differential gene expression analysis was conducted using DESeq2, and the results for selected genes were validated by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). Drug sensitivity was evaluated by determining the half-maximal inhibitory concentration (IC50) through dose-response experiments with gemcitabine (GEM), cisplatin (DDP), nab-paclitaxel and 5-fluorouracil (5-FU). Statistical analyses were performed using GraphPad Prism 9.0 and R software (version 4.4.0), and P<0.05 was considered statistically significant.ResultsThe 3D model showed stable lattice structures with high cell viability and spheroid formation, accompanied by significantly enhanced proliferation compared with 2D cultures (P<0.001). Transcriptome analysis revealed 617 differentially expressed genes (235 upregulated, 382 downregulated), enriched in cell cycle regulation, cytokine signaling, and extracellular matrix remodeling. RTFQ-PCR confirmed consistency with RNA-seq results. Drug response assays demonstrated higher IC50 in 3D models versus 2D for all agents tested, indicating reduced chemosensitivity and stronger resemblance to clinical resistance.ConclusionThis study established a 3D bioprinted gallbladder cancer model. Compared with 2D culture, the 3D model showed lower sensitivity and higher IC50 to GEM, DDP, nab-paclitaxel, and 5-FU, consistent with drug-tolerant phenotypes of clinical solid tumors. Therefore, the 3D model is superior to the 2D model in recapitulating clinically relevant drug resistance and tolerance and can be utilized for investigating drug resistance mechanisms and for screening/validating candidate drugs or combination regimens.Background and purposeGallbladder cancer is a highly aggressive gastrointestinal malignancy with a 5-year survival rate of less than 10%. Conventional two-dimensional (2D) cell cultures poorly mimic the tumor microenvironment, limiting translational drug screening. Three-dimensional (3D) bioprinting enables the construction of biomimetic tumor models with controllable structure and function. This study aimed to establish a 3D bioprinted gallbladder cancer model and compare its biological and pharmacological features with 2D cultures.

ETHNOPHARMACOLOGICAL RELEVANCE:As a traditional Chinese medicine, Wu-Mei-Wan (WMW) has shown promise as a second-line treatment for gallbladder cancer, but its mechanism remains to be explored. AIM OF THE STUDY:To determine the specific mechanism of WMW active ingredients (Palmatine et al.) inducing ferroptosis in gallbladder cancer (GBC) and its synergistic potential with gemcitabine. MATERIALS AND METHODS:Subcutaneous tumor in nude mice was used to analyze the combined effect of gemcitabine. The effective components into blood were identified by HPLC. Combined with proteomics, network pharmacology and bioinformatics analysis, the effective components and targets of WMW promoting ferroptosis in GBC were identified in vitro and in vivo. The anticancer effects of WMW on different GBC cell lines were evaluated by CCK-8 assay, colony formation and EdU staining. A variety of molecular biology experiments were used to explore the mechanism. RESULTS:WMW treatment enhanced the sensitivity of GBC to gemcitabine, which induced ferroptosis and effectively inhibited the malignant phenotype of GBC. Network pharmacology and blood component identification identified the key components of WMW inhibiting GBC. Palmitine and other components were identified as active components into the blood. Proteomics and molecular docking validation further revealed the STAT3-centered regulatory network in GBC cells. Molecular experiments have shown that WMW induced ferroptosis by negatively regulating downstream molecules of p-STAT3 transcription of GPX4, ACSL4, HIF1α, and FTH1. CONCLUSIONS:WMW induces ferroptosis in GBC through the p-STAT3 axis and enhances sensitivity to gemcitabine, suggesting the potential of WMW as a second-line therapeutic for GBC.

Helicobacter pylori (H. pylori) manipulates the host immune system to establish a persistent colonization, posing a serious threat to human health, but the mechanisms remain poorly understood. Here we integrate single-cell RNA sequencing and TCR profiling for analyzing 187,192 cells from 11 H. pylori-negative and 12 H. pylori-positive individuals to describe the human gastric ecosystem reprogrammed by H. pylori infection, as manifested by impaired antigen presentation and phagocytosis function. We further delineate a monocyte-to-C1QC+ macrophage differentiation trajectory driven by H. pylori infection, while T cell responses exhibit broad functional impairment and hyporesponsiveness with restricted clonal expansion capacity. We also identify an HLA-DRs- and CTLA4-expressing T cell population residing in H. pylori-inhabited stomach that potentially contribute to immune evasion. Together, our findings provide single-cell resolution information into the immunosuppressive microenvironment shaped by H. pylori infection, offering critical insights for developing novel therapeutic approaches to eliminate this globally prevalent pathogen.