Figure S2. Cellular compositions of myeloid and T cell subclusters between hepatic metastases and healthy livers.

GSEA results for all ductal subclusters using genesets collected from published articles.

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.

Pancreatic cancer (PC) is a highly lethal disease, and current clinical drugs often have significant side effects. Traditional Chinese medicines, particularly food and medicine homology herbs, present novel opportunities for the development of low-toxicity tumor treatments. It is imperative to further explore the application of these substances in the field of tumor therapy. To investigate the effect of Ci-Gu-Tang (CGT) on PC both in vitro and in vivo and to elucidate its underlying mechanism. In this study, liquid chromatography-mass spectrometry (LC-MS) technology was utilized to validate the constituents of CGT, whereas network pharmacology was employed to further analyze the 37 active ingredients in CGT and their nine targets associated with PC. Protein-protein interaction (PPI) investigations revealed that the three potential key targets are epidermal growth factor receptor (EGFR), AKT serine/threonine kinase 1 (AKT1), and signal transducer and activator of transcription 3 (STAT3). Molecular docking was used to elucidate the binding mode and energy between four active ingredients (beta-sitosterol, stigmasterol, quercetin, and prangenidin) and the above three targets. Subsequent in vitro and in vivo studies further validated its anti-PC efficacy and confirmed the involvement of the EGFR/AKT1/STAT3 signaling pathway. The utilization of patient-derived organoid (PDO) and patient-derived xenograft (PDX) models enhanced the clinical significance of this investigation. The present research utilized network pharmacology, molecular docking, LC-MS, and in vitro and in vivo experiments to examine the effects and mechanism of CGT in the treatment of PC. This study revealed that beta-sitosterol, stigmasterol, quercetin, and prangenidin could be practical components by inhibiting tumorigenesis through the EGFR/AKT1/STAT3 axis. PDO and PDX models also demonstrated the effectiveness of CGT. This study represents a pioneering effort in the use of patient-derived models to investigate the potential of food and medicine homology species in cancer treatment. (c) 2026 The authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Biliary tract cancers (BTCs) comprise biologically heterogeneous subtypes with limited therapeutic options and variable responses to sorafenib. The biological basis underlying this variability remains unclear. In this study, we compared intrahepatic cholangiocarcinoma (iCCA) and gallbladder cancer (GBC) models under defined sorafenib exposure conditions using transcriptomic profiling, Seahorse-based mitochondrial stress assays, pharmacologic combination analysis, and patient-derived organoid models. In representative iCCA models, 1.25 μM sorafenib increased mitochondrial respiration and ATP production, whereas 10 μM sorafenib suppressed this respiratory adaptation. In contrast, representative GBC models exhibited persistent suppression of mitochondrial respiratory function across the tested conditions and did not display a comparable concentration-dependent shift. Integrated transcriptomic and pharmacologic analyses further identified mitochondrial Complex I as a treatment-relevant vulnerability under sorafenib exposure, with stronger combination interactions observed in iCCA models. These findings indicate that iCCA and GBC differ in their metabolic responses to sorafenib and highlight subtype-specific differences in mitochondrial adaptation under treatment stress.

GO functional enrichment analysis of genes downregulated in immunotherapy treated mice.

SHR-A1811, an antibody‒drug conjugate consisting of the anti-HER2 antibody trastuzumab conjugated via a cleavable linker to a topoisomerase I inhibitor payload, demonstrated substantial antitumor activity in patients with heavily treated HER2-expressing or mutated advanced solid tumors. The main analysis was reported, and this is a long-term follow-up of the HORIZON-X trial (NCT04446260). This global, multicenter, first-in-human, phase 1 trial enrolled patients aged ≥ 18 years with unresectable, advanced, or metastatic HER2-expressing or mutated solid tumors refractory or intolerant to standard therapies across 38 hospitals. SHR-A1811 was administered intravenously at doses ranging from 1.0 to 8.0 mg/kg every three weeks. The primary endpoints included dose-limiting toxicity, safety, and the recommended phase 2 dose. From September 7, 2020, to June 4, 2024, 396 patients with a median of three prior treatment regimens (IQR 2-5) received SHR-A1811. As of March 12, 2025, the median follow-up was 17.1 months for HER2-positive breast cancer, 10.6 months for HER2-low expressing breast cancer, and 4.3 to 8.2 months in non-breast cancers. The safety profile remained consistent with that of previous reports. Grade 3 or higher treatment-related adverse events occurred in 261 patients (65.9%), and any grade interstitial lung disease was observed in 10 patients (2.5%). The median progression-free survival was 25.0 months (95% CI 17.2-33.6) for HER2-positive breast cancer, 11.0 months (95% CI 8.2-13.8) for HER2-low expressing breast cancer, and 3.5 to 17.2 months for non-breast tumors. This final analysis further confirmed the long-term efficacy and favorable safety profile of SHR-A1811 among heavily prior-treated advanced solid tumors, reinforcing its potential as an effective HER2-targeted therapy.

Enterotoxigenic Bacteroides fragilis (ETBF), which secretes B. fragilis toxin (BFT) and has been associated with colorectal cancer, can be enriched in patients with gall bladder cancer (GBC). Whether and how ETBF contributes to GBC is unclear. Here we confirm, through analysis of patient samples, that ETBF is enriched in GBC tumours, while experiments in mice show that ETBF colonizes the gall bladder. In vitro and in patient-derived organoids, ETBF promoted GBC proliferation. ETBF also promoted tumour growth in a BFT-dependent manner in a mouse GBC allograft model. Mechanistically, the tumorigenic activity of BFT was dependent on the ETBF surface protein, membrane-bound lytic murein transglycosylase D (MltD), which interacts with the host receptor protein, transmembrane serine protease 13 (TMPRSS13). This interaction activated JAK2-STAT3 signalling to promote tumorigenesis. BFT also activated NF-κB signalling, which increased CXCL1 secretion, leading to myeloid-derived suppressor cell recruitment and angiogenesis in the tumour microenvironment. Taken together, these findings uncover mechanisms through which ETBF facilitates GBC development, with potential promise as therapeutic targets to limit GBC progression.

Figure S4. Association between H2AFZ expression and genomic mutations in PDAC.

Cell markers for celltype annotation.

This guideline provides standardized requirements and quality control measures for human gallbladder cancer organoids, supporting their reliable application in research and precision medicine. It aims to promote methodological consistency, facilitate international collaboration, and accelerate translational research and clinical implementation.

Approximately 90% of patients with pancreatic cancer harbor KRAS mutations, predominantly the KRASG12D subtype. HRS-4642, a non-covalent inhibitor targeting KRASG12D, demonstrates potent antitumor efficacy but may ultimately lead to resistance. This study investigates the mechanisms underlying KRASG12D inhibitor resistance and evaluates strategies to enhance treatment sensitivity. Our findings indicate that a glutamine-restricted diet not only reverses KRASG12D inhibitor resistance in pancreatic ductal adenocarcinoma (PDAC) but also achieves remission with prolonging survival. Mechanistically, KRASG12D inhibitor resistance markedly upregulates ANXA1 expression, which, in turn, promotes its binding to the glutamine-related enzyme GOT1 and stabilizes its expression. Additionally, we find that ANXA1 upregulation facilitates mitochondrial localization of GLS1, thereby altering glutamine metabolism. These findings highlight ANXA1-mediated glutamine metabolism as a key driver of KRASG12D inhibitor resistance and support glutamine-restricted diets as a potential therapeutic strategy for KRASG12D mutant PDAC.

Figure S3. Analysis of bulk RNA-seq data on metastatic tumor tissues of anti-PD1 monotherapy, anti-LAG3 monotherapy and anti-PD1 plus anti-LAG3 combined therapy.

Gallbladder cancer (GBC) is a highly aggressive malignancy, and its robust antioxidant defense system frequently leads to profound therapeutic resistance. Targeted disruption of redox homeostasis offers a potential strategy to overcome such resistance. In this study, we developed ICFe@D, a supramolecular nanomedicine co-assembled from iso-liquiritigenin (ISL), Fe³⁺, and chlorin e6 (Ce6) within a DSPE-PEG matrix, to trigger combined ferroptosis and apoptosis. As a tumor microenvironment (TME)-responsive platform, ICFe@D systematically dismantles the antioxidant defenses of GBC cells through a “supply-and-exhaust” mechanism. Specifically, Fe3+-derived Fe2+ and laser-activated Ce6 act as an efficient reactive oxygen species (ROS) generator via Fenton and photodynamic reactions, while ISL severely impairs the intracellular antioxidant capacity by downregulating GPX4 and activating the p62/Keap1/Nrf2/HMOX1 signaling pathway. We evaluated the therapeutic efficacy of this platform in patient-derived organoids (PDOs) and patient-derived organoid xenograft (PDOX) models. ICFe@D demonstrated potent and consistent tumor regression across heterogeneous patient-specific models with minimal systemic toxicity. This study presents a highly translatable and precise nanotherapeutic strategy that effectively sensitizes GBC to ferroptosis, providing a valuable preclinical framework for biliary tract cancer therapy.

PURPOSE:Hepatic metastasis (HM) is the leading cause of death in pancreatic ductal adenocarcinoma (PDAC). However, the underlying cellular and molecular programs remain poorly understood, leading to limited therapeutics for this disease. EXPERIMENTAL DESIGN:In this study, we integrated single-cell RNA sequencing data from paired primary tumors and HMs, along with bulk RNA sequencing and IHC data from hundreds of patients to elucidate metastasis-associated programs. RESULTS:Our analysis identified a metastasis-prone malignant subpopulation, which is associated with a higher risk of HM and a transitional plastic state. This malignant subpopulation represents a poorly differentiated and highly proliferative phenotype, with H2AFZ potentially contributing to this phenomenon. Moreover, the presence of tumor cells in the liver was accompanied by an increased abundance of M2 macrophages, regulatory T cells, and exhausted T cells (Tex) in HMs compared with adjacent tissues, indicative of a shift toward an immunosuppressive environment. Notably, within the tumor environment of HMs, Tex exhibited elevated expression of PDCD1 and LAG3. The combined therapy targeting these two genes effectively inhibited tumor growth in mouse models of metastatic PDAC. CONCLUSIONS:In conclusion, we reveal a metastasis-associated malignant subpopulation and provide a promising therapeutic strategy for metastatic PDAC.

Background:Pancreatic cancer is highly aggressive, with high post-resection recurrence and poor survival. Accurate preoperative assessment of vascular invasion is essential but remains clinically unmet. This study aimed to enable reliable automated assessment of vascular invasion using routine computed tomography (CT). Methods:We developed CRVIA, a clinician-centric artificial intelligence (AI) method that encodes clinical expertise into invasion-omics and emulates clinician's decision-making through causality-enhanced modelling. The model was trained on 1251 cases and validated on internal (1104 cases) and external (3575 cases from five centres) cohorts. Reader studies involving eight radiologists were conducted to evaluate the efficacy of CRVIA's assistance. Primary outcome measures included classification performance, feature distance, and inter-reader agreement. Statistical analyses were conducted using paired t-tests, Mann-Whitney U, Pearson's chi-squared, and permutation tests. Findings:The study cohort comprised 5930 cases from 2062 patients (median age 63.0 years [IQR 56.0-70.0]; 41.1% female). CRVIA achieved AUCs of 0.946 (95% CI 0.929-0.962) internally and 0.943 (95% CI 0.932-0.952) externally, outperforming nine comparison approaches spanning radiomics to foundation models. Performance remained stable across stages, vessel types, and segmentation variations. In reader studies, CRVIA exceeded senior radiologists' accuracy, elevated junior radiologists to expert-level performance, and substantially improved inter-reader agreement. Interpretation:This study presents a reliable, open-source AI tool (code: https://github.com/SJTUBME-QianLab/CRVIA) for accurate, stable, and interpretable vascular invasion assessment in pancreatic cancer, which potentially supports precise treatment, encourages collaborative research, and ultimately benefits patients, especially for resource-limited regions. Funding:National Natural Science Foundation of China and Shanghai Natural Science Foundation.

Signature genes of M1 and M2 macrophages derived from PMID: 33545035.

Gallbladder cancer (GBC) is characterized by a bile acids (BAs)-rich microenvironment that imposes significant endoplasmic reticulum (ER) stress. While the transcription factor AP-2α (TFAP2A) is upregulated in GBC, its functional role in this specific stress context remains unknown. This study investigates how TFAP2A enables GBC cell adaptation and survival under BAs stress. TFAP2A expression was analyzed in the tissues of patients with GBC via RNA sequencing, immunohistochemistry, and western blot. In vitro functional assays and patient derived organoid models were used to assess TFAP2A’s role in GBC. Interaction partners were identified through astral data-independent acquisition (DIA) proteomics and confirmed by coimmunoprecipitation. Protein stability was evaluated using cycloheximide chase and ubiquitination assays. In vivo validation was performed using cell-derived and patient derived xenograft models. Golgi integrity and ER stress markers were analyzed by immunofluorescence and western blot. TFAP2A was highly expressed in GBC tissues and shows cytoplasmic accumulation correlating with BA levels. Under BAs stress, TFAP2A translocated to the cytoplasm, where it interacted with the Golgi protein GOLIM4 and stabilized it by inhibiting its ubiquitin-mediated degradation. This TFAP2A/GOLIM4 maintained Golgi structure and alleviated ER stress, as evidenced by downregulation of proapoptotic CHOP and upregulation of GRP78/BiP. Consequently, it promoted GBC cell survival, proliferation, and tumor growth. Knockdown of either TFAP2A or GOLIM4 suppressed malignant phenotypes and increased ER stress-induced apoptosis, both in vitro and in vivo. This study identified a novel, nontranscriptional function of TFAP2A in response to BAs stress. By stabilizing GOLIM4 and enhancing secretory capacity, the TFAP2A/GOLIM4 axis represented a UPR-independent adaptive pathway that confers a survival advantage to GBC cells. Targeting this interaction may offer a new therapeutic strategy for this aggressive malignancy.

Signature genes of all ductal subclusters.

Background: Gastric signet-ring cell carcinoma (GSRCC) is an aggressive gastric cancer subtype with abundant mucin production and high metastatic propensity. However, scarcity of specific biomarkers has impeded clinical diagnosis and mechanistic research. This study systematically compares GSRCC and gastric adenocarcinoma (AC) to identify biomarkers and elucidate molecular basis of GSRCC’s aggressive behavior. Methods: We performed single-cell RNA sequencing (scRNA-seq) on surgically resected primary GC tissues, validating our findings using public datasets and functional experiments. Results: We identified expansion of a mucin-secreting epithelial subcluster (Mucous_muc5ac) in GSRCC, characterized by high MUC5AC, TFF1, and other prognosis-associated genes. Within this population, a MUCL3+ subpopulation (Cluster 1) spatially corresponded with classic signet-ring morphology, validating MUCL3 as a specific marker for these cells. Multi-omics analysis revealed that MUCL3+ signet-ring cells exhibit genomic instability, dedifferentiation, and enrichment of TNF-α/NF-κB, TGF-β/EMT, and hypoxia pathways, with elevated metastasis/angiogenesis gene scores and high TFF1 expression. Functional validation confirmed that TFF1 was associated with increased gastric cancer cell migration. Conclusions: Our study characterizes the MUCL3+ signet-ring cell subpopulation, highlighting the diagnostic utility of MUCL3 and suggesting TFF1 as a candidate for further investigation. These findings establish a foundation for advancing precision diagnosis and mechanistic understanding of GSRCC.