EVs-delivered OGA disrupts neuronal metabolic flux and glycosylation

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.

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.

Abstract Topic Esophageal Cancer: Adjuvant and Neo-Adjuvant Therapies Background Direct comparisons of health-related quality of life (HRQoL) between neoadjuvant chemoradiotherapy (nCRT) with CROSS regimen and neoadjuvant chemotherapy (nCT) with paclitaxel–cisplatin (TP) for resectable, locally advanced esophageal squamous cell carcinoma (ESCC) are limited. In this randomized controlled trial, we compared the effects of different neoadjuvant regimens on HRQoL (NCT04138212). Methods: In this open-label, randomized controlled trial, patients with resectable, locally advanced ESCC were assigned (1:1) to nCRT or nCT followed by planned esophagectomy. HRQoL was assessed using the EORTC QLQ-C30 and QLQ-OES18 at baseline, mid-treatment, preoperative, pre-discharge, 1-, 3-, and 6-month postoperatively, and transformed to a 0-100 scale. Longitudinal HRQoL was analyzed in the intention-to-treat population using linear mixed-effects models (LMMs with a patient-level random intercept. Prespecified key domains (physical functioning [PF], global health status/quality of life [GHS/QoL], OES18 Dysphagia, and OES18 Pain) were prioritized for primary analyses; other domains were exploratory. Multiplicity was controlled using Holm for overall tests and false discovery rate (FDR) for time-specific comparisons and changes from baseline. A 10-point difference defined the minimal clinically important difference (MCID). Results: Among 456 randomized patients (nCRT: 228; nCT: 228), HRQoL questionnaire completion was 100% at baseline and remained above 75% throughout follow-up. In prespecified key domains, LMMsshowed significant group-by-time interactions for PF, GHS/QoL and OES18 pain (all Holm-adjusted P<0.001), whereas no interaction was observed for OES18 Dysphagia (Holm-adjusted P=0.077). Between-group separation peaked at mid-treatment (FDR-adjusted P<0.05) but did not reach the MCID and was no longer evident from discharge through 6 months. Across both groups, HRQoL temporarily deteriorated during neoadjuvant therapy, was worst at the pre-discharge assessment, and then gradually recovered during postoperative follow-up. By 6 months, HRQoL had largely returned to baseline or improved, although statistically significant changes were generally below the MCID. All other QLQ-C30 and OES18 domains were analyzed exploratorily and showed similar trajectories. Conclusion Collectively, these findings indicate that HRQoL outcomes are clinically comparable between CROSS-regimen nCRT and TP-regimen nCT for resectable, locally advanced ESCC. The short-term treatment-related HRQoL burden did not translate into long-term deficits, and patients' HRQoL returned to baseline by 6 months after surgery.

Gallbladder cancer (GBC), the most prevalent and lethal biliary tract malignancy, lacks effective therapeutic strategies. Solasonine (SS), a bioactive steroidal alkaloid derived from Solanum nigrum L, exhibits antitumor activity in multiple cancers, but its role in GBC and association with ferroptosis remains unknown. The purpose of this study is to investigate the therapeutic effects of SS on GBC and its underlying mechanisms using both in vitro and in vivo models. The efficacy was tested in subcutaneous models. Then, the anti-GBC effects of SS were assessed via CCK-8, colony formation, and EdU assays. Biosafety was evaluated using L-2F7 cells and murine serum biomarkers. RNA sequencing identified key pathways, validated by ChIP-qPCR and dual-luciferase reporter assays. Ferroptosis induction was confirmed by measuring MDA, GSH, ROS, Fe2+, qRT-PCR/Western blot (GPX4/SLC7A11), and rescue experiments. Direct binding of SS to APT2 was demonstrated through molecular docking, CETSA, and SPR. The study employed orthotopic xenograft models, transwell assays, and wound healing assays to investigate the effects of SS on the metastasis of GBC. SS triggered ferroptosis in GBC by elevating MDA, ROS, and Fe2+ while depleting GSH and downregulating GPX4/SLC7A11. Mechanistically, SS directly bound APT2, inhibiting its enzymatic activity and blocking STAT3 depalmitoylation. This prevented p-STAT3 nuclear translocation, suppressing transcription of ferroptosis inhibitors GPX4 and SLC7A11. In vivo, SS suppressed tumor growth by subcutaneous and orthotopic models with no systemic toxicity. Our findings reveal SS as a novel APT2-targeted ferroptosis inducer that inhibits GBC progression via inhibiting the depalmitoylation process of STAT3, providing a safe and effective therapeutic candidate for this intractable malignancy.

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.

Inflammatory cell infiltration within the tumor microenvironment compromises the basement membrane' integrity, contributing to chemotherapy resistance and distant metastasis. This study investigates the interplay between inflammatory responses and basement membrane-related genes (BMIRGs) in lung adenocarcinoma (LUAD). With datasets from the TCGA and GEO databases, the BMIRG risk-score model was developed based on univariate Cox regression and a LASSO-Cox regression model. Nine genes were identified as comprising the BMIRG signature. Using this signature, we developed the predictive models, including BMIRG risk scores and nomogram and Cox regression models. Stratifying patients into high-and low-risk groups based on BMIRG risk scores revealed a more favorable prognosis in the latter group. Additionally, the signature exhibited correlations with drug sensitivity, immunotherapy response, infiltration of multiple immune cells and inflammatory factors in microenvironment. The role of one BMIRG signature, CCL20, was further validated using in intro experiments and tissue samples from Chinese patients with LUAD. Functional experiments revealed that inhibiting CCL20 attenuated cell migration in both A549 and H1299 cells. Immunohistochemistry and Western blot analyses demonstrated that CCL20 protein levels were significantly elevated in LUAD tissues compared to adjacent non-tumor tissues and also associated with tumor the TNM stage. High CCL20 expression was also linked to shorter overall survival. In conclusion, the BMIRG signature is a robust predictor of survival and offers a valuable clinical tool for LUAD management. Moreover, CCL20 emerges as a potential diagnostic and therapeutic target, given its high expression in LUAD patients and association poorer prognosis.

BACKGROUND:Telomere homeostasis serves as a key regulatory mechanism linking aging and cancer. While telomere attrition imposes a proliferative barrier by inducing cellular senescence, abnormal telomere elongation circumvents this constraint, thereby granting malignant cells unlimited replicative capacity. This study systematically explores the causal relationship between telomere length and cancer risk, with the goal of elucidating the molecular pathways involved in telomere-driven tumorigenesis. METHOD:Mendelian randomization (MR) was employed to establish a causal link between telomere length and pan-cancer susceptibility. Multiple MR models were employed to ensure the robustness of the results. Telomere-associated genes were identified through SNPense analysis, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Key gene regulatory networks were identified and visualized using the MCODE and cytoHubba algorithms. The expression profile of TERT across diverse cancer types was analyzed using TCGA datasets, and its diagnostic potential was evaluated via receiver operating characteristic (ROC) curve analysis. Correlations between TERT expression and immune cell infiltration were further explored. RESULTS:Longer telomere length was significantly associated with an increased risk of 33 cancer types (IVW OR = 1.27-1.41, all P < 0.001). A total of 143 telomere-related genes were identified, with functional enrichment highlighting their involvement in genome integrity and telomere maintenance. TERT emerged as the most influential hub gene (MCC score = 169). Transcriptomic analyses from TCGA demonstrated widespread TERT overexpression in 16 cancers (e.g., CHOL, LIHC, LUAD), a finding corroborated by RT-qPCR validation. TERT exhibited high diagnostic performance (AUC > 0.85 in 16 cancers, peaking at AUC = 0.97 in LUSC). Immune infiltration analysis revealed a positive correlation with Th2 cells (r = 0.42) but negative correlations with dendritic cells (r = - 0.38) and macrophages (r = - 0.31). CONCLUSION:This study proposes a comprehensive framework linking telomere length regulation to cancer progression through the TERT axis. Telomere dysfunction contributes to tumorigenesis via two key mechanisms: promoting genomic instability and altering the immune microenvironment. These findings offer new insights into telomere-driven oncogenesis and lay a conceptual foundation for precision diagnostics and targeted therapies in oncology.

[This corrects the article DOI: 10.7150/jca.46351.].

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

Gallbladder cancer (GBC) is highly aggressive and has poor prognosis, with most patients only diagnosed at an advanced stage. Furthermore, treatment options are limited, and their effect is unsatisfactory. Bromodomain-containing protein (BRD) is an epigenetic regulator that plays a carcinogenic role in several tumors, including squamous cell lung cancer, acute myeloid leukemia, synovial sarcoma, and malignant rhabdomyosarcoma. However, the expression, biological function, and molecular mechanisms of action of BRD9 in GBC are still unknown. Kaplan-Meier analysis, qRT-PCR, and analysis of clinical features were used to assess the clinical significance of BRD9 in GBC. Cell Counting Kit-8 and colony formation assays were performed to determine the effects of BRD9 on cell growth. The functional role of BRD9 in GBC was explored using qRT-PCR, western blotting, siRNA, and CHIP-qPCR. mRNA sequencing was performed to explore the underlying mechanisms of BRD9, and a nude mouse model of GBC was established to explore the anti-tumor effects of the BRD9 inhibitor I-BRD9 in vivo. BRD9 expression was elevated in GBC tissues compared with adjacent non-tumor tissues, and high BRD9 expression was associated with poor prognosis in patients with GBC. BRD9 knockdown by siRNA significantly decreased cell growth. Targeting BRD9 with I-BRD9 inhibited the proliferation of GBC cells without significant toxic effects. Additionally, I-BRD9 treatment suppressed CST1 expression in GBC cell lines, thereby inhibiting the PI3K-AKT pathway. The transcription factor FOXP1 was found to interact with BRD9 to regulate CST1 expression. Collectively, these results suggest that BRD9 may be a promising biomarker and therapeutic target for GBC.

Background The effect of neoadjuvant chemotherapy (NACT) in gallbladder cancer (GBC) patients remains controversial. The aim of this study was to assess the impact of NACT on overall survival (OS) and cancer specific survival (CSS) in patients with localized or locoregionally advanced GBC, and to explore possible protective predictors for prognosis. Methods Data for patients with localized or locoregionally advanced GBC (i.e., categories cTx-cT4, cN0-2, and cM0) from 2004 to 2020 were collected from the Surveillance, Epidemiology, and End Results (SEER) database. Patients in the NACT and non-NACT groups were propensity score matched (PSM) 1:3, and the Kaplan-Meier method and log-rank test were performed to analyze the impact of NACT on OS and CSS. Univariable and multivariable Cox regression models were applied to identify the possible prognostic factors. Subgroup analysis was conducted to identify patients who would benefit from NACT. Results Of the 2676 cases included, 78 NACT and 234 non-NACT patients remained after PSM. In localized or locoregionally advanced GBC patients, the median OS of the NACT and non-NACT was 31 and 16 months (log-rank P < 0.01), and the median CSS of NACT and non-NACT was 32 and 17 months (log-rank P < 0.01), respectively. Longer median OS (31 vs 17 months, log-rank P < 0.01) and CSS (32 vs 20 months, log-rank P < 0.01) was associated with NACT compared with surgery alone. Multivariable Cox regression analysis showed that NACT, stage, and surgery type were prognostic factors for OS and CSS in GBC patients. Subgroup analysis revealed that the survival hazard ratios (HRs) of NACT vs non-NACT for localized or locoregionally advanced GBC patients were significant in most subgroups. Conclusions NACT may provide therapeutic benefits for localized or locoregionally advanced GBC patients, especially for those with advanced stage, node-positive, poorly differentiated or undifferentiated disease. NACT combined with radical surgery was associated with a survival advantage. Therefore, NACT combined with surgery may provide a better treatment option for resectable GBC patients.

Liver metastasis is common in patients with gallbladder cancer (GBC), imposing a significant challenge in clinical management and serving as a poor prognostic indicator. However, the mechanisms underlying liver metastasis remain largely unknown. Here, we report a crucial role of tyrosine aminotransferase (TAT) in liver metastasis of GBC. TAT is frequently up-regulated in GBC tissues. Increased TAT expression is associated with frequent liver metastasis and poor prognosis of GBC patients. Overexpression of TAT promotes GBC cell migration and invasion in vitro, as well as liver metastasis in vivo. TAT knockdown has the opposite effects. Intriguingly, TAT promotes liver metastasis of GBC by potentiating cardiolipin-dependent mitophagy. Mechanistically, TAT directly binds to cardiolipin and leads to cardiolipin externalization and subsequent mitophagy. Moreover, TRIM21 (Tripartite Motif Containing 21), an E3 ubiquitin ligase, interacts with TAT. The histine residues 336 and 338 at TRIM21 are essential for this binding. TRIM21 preferentially adds the lysine 63 (K63)-linked ubiquitin chains on TAT principally at K136. TRIM21-mediated TAT ubiquitination impairs its dimerization and mitochondrial location, subsequently inhibiting tumor invasion and migration of GBC cells. Therefore, our study identifies TAT as a novel driver of GBC liver metastasis, emphasizing its potential as a therapeutic target.

BackgroundIn the era of immunotherapy, neoadjuvant immunochemotherapy (NAIC) for the treatment of locally advanced esophageal squamous cell carcinoma (ESCC) is used clinically but lacks of high-level clinical evidence. This study aimed to compare the safety and long-term efficacy of NAIC followed by minimally invasive esophagectomy (MIE) with those of neoadjuvant chemotherapy (NAC) followed by MIE.MethodsA prospective, single-center, open-label, randomized phase III clinical trial was conducted at Henan Cancer Hospital, Zhengzhou, China. Patients were randomly assigned to receive either neoadjuvant toripalimab (240 mg) plus paclitaxel (175 mg/m2) + cisplatin (75 mg/m2) (toripalimab group) or paclitaxel + cisplatin alone (chemotherapy group) every 3 weeks for 2 cycles. After surgery, the toripalimab group received toripalimab (240 mg every 3 weeks for up to 6 months). The primary endpoint was event-free survival (EFS). The pathological complete response (pCR) and overall survival (OS) were key secondary endpoints. Adverse events (AEs) and quality of life were also assessed.ResultsBetween May 15, 2020 and August 13, 2021, 252 ESCC patients ranging from T1N1-3M0 to T2-3N0-3M0 were enrolled for interim analysis, with 127 in the toripalimab group and 125 in the chemotherapy group. The 1-year EFS rate was 77.9% in the toripalimab group compared to 64.3% in the chemotherapy group (hazard ratio [HR] = 0.62; 95% confidence interval [CI] = 0.39 to 1.00; P = 0.05). The 1-year OS rates were 94.1% and 83.0% in the toripalimab and chemotherapy groups, respectively (HR = 0.48; 95% CI = 0.24 to 0.97; P = 0.037). The patients in the toripalimab group had a higher pCR rate (18.6% vs. 4.6%; P = 0.001). The rates of postoperative Clavien-Dindo grade IIIb or higher morbidity were 9.8% in the toripalimab group and 6.8% in the chemotherapy group, with no significant difference observed (P = 0.460). The rates of grade 3 or 4 treatment-related AEs did not differ between the two groups (12.5% versus 12.4%).ConclusionsThe interim results of this ongoing trial showed that in resectable ESCC, the addition of perioperative toripalimab to NAC is safe, may improve OS and might change the standard treatment in the future.