Pancreaticobiliary maljunction is a potential cause of biliary malignancy due to pancreatic enzyme activation from pancreatic juice reflux. Although this reflux phenomenon has been frequently observed in clinical studies, its underlying mechanism has been rarely explored. This study investigated pancreaticobiliary flow in patients with choledochal cysts (CCs) and cholangiocarcinoma, and to explore possible scenarios of how reflux occurs using computational fluid dynamics (CFD). Patient-specific models were first constructed based on two patients with CCs and one with both CCs and cholangiocarcinoma. Particle image velocimetry was used to first visualize the local velocity field and CFD simulations were then performed to visualize and analyze pancreaticobiliary flows (e.g., hepatic bile, gallbladder bile, and pancreatic juice), as well as pancreatic juice reflux. Results showed that for patients with both CCs and cholangiocarcinoma, their digestive function can be impaired compared to those with only CCs, as bile flow movement was reduced by nearly 25%. Bile flow recirculation was more likely to occur in lower region of pancreaticobiliary system in CCs patients with cholangiocarcinoma, implying an increased likelihood of pancreatic enzyme activation due to extended time needed for pancreatic juice to mix with bile in the common channel. Escalating wall shear stress was observed in patients with CCs and cholangiocarcinoma. CFD simulations demonstrated that sphincter of Oddi dysfunction during the bile refilling process can be one of reasons for inducing pancreatic juice reflux. The simulations visualized that concentration of pancreatic juice could reach between 59.2% and 83.8% in common bile duct and cystic duct.

4142 Background: Chemotherapy (chemo) combined with or without PD-(L)1 inhibition is the standard first-line treatment for advanced BTC. After first-line treatment fails, patients (pts) lacking targetable genetic alterations (FGFR2, IDH1, or HER2 etc.) have limited options. For the majority, chemo (FOLFOX as the preferred regimen) is the default but suboptimal option. To date, the therapeutic potential of multi-target ICIs and anti-angiogenesis combination in the post-line regimen of BTC remains unexplored and constitutes a significant clinical demand. Additionally, early preclinical and clinical evidences support the combination blockade of TIGIT, TGF-β and PD-(L)1 plus anti-VEGF therapy in several solid tumors. AK130 is a TIGIT/TGF-β bispecific fusion protein. Ivonescimab, the first approved PD-1/VEGF bispecific antibody, has demonstrated promising efficacy in solid tumors, including BTC. Here, we aim to evaluate the efficacy and safety of AK130 plus ivonescimab in post-line BTC and the primary results from phase 1b is reported. Methods: This was an open-label, multi-center phase 1b/2 study. Pts who had progressed on prior chemo combined with or without a PD-(L)1 inhibitor were enrolled and treated with AK130 and ivonescimab. Phase 1b included dose escalation and expansion parts. In the escalation part, escalating doses of AK130 (10, 30, 45 mg/kg Q3W) were administered using a "3+3+3" design, with ivonescimab at 20 mg/kg Q3W (previously approved dose by NMPA). Dose-limiting toxicities (DLTs) were assessed. Based on safety profile, the expansion proceeded at selected doses, with up to 15 evaluable pts per dose. The primary endpoint of phase 1b was the incidence of AEs and DLTs. The secondary endpoint was ORR. Results: As of Jan 2026, a total of 23 pts was enrolled. 12 pts were enrolled in dose escalation across 3 dose levels (10, 30, 45 mg/kg AK130 Q3W; n = 3, 3, 6). No DLTs were observed, and no maximum tolerated dose (MTD) was established. All pts in the 10 mg/kg and 30 mg/kg groups experienced stable disease (SD). The 45mg/kg group was further expanded to 17 pts, including 15 pts that progressed on prior PD-(L)1 inhibition plus chemo.15 pts from 45mg/kg group were efficacy-evaluable with an ORR of 20.0% (3/15) and a DCR of 73.3% (11/15). 87.5% of the SD pts (7/8) reached shrinkage from baseline. 40.0% pts (6/15) had only one time evaluation and are continuing to be followed. Grade 3 or higher treatment-related adverse events (TRAEs) occurred in 39.1% (9/23) pts. The most common TRAEs were anemia and atopic dermatitis, each reported in 2 pts (8.7%). AK130 45 mg/kg Q3W is planned as the recommended phase 2 dose (RP2D), whose safety and efficacy will be further evaluated in phase 2. Conclusions: AK130 plus ivonescimab demonstrated potential anti-tumor activity with a manageable safety profile as a post-line option for advanced BTC, supporting its further development in this setting. Clinical trial information: NCT06938321 .

IntroductionThe prognostic role of STMN1 and its regulatory effects on the tumor microenvironment (TME) in biliary tract cancer (BTC) remain poorly defined. This study aimed to investigate the prognostic value of STMN1 and its predictive capacity for chemotherapy and immunotherapy responses among patients with BTC.MethodsSix independent cohorts encompassing tissue microarray specimens and transcriptional profiling data from BTC patients were included in our analysis. STMN1 protein expression was quantified via immunohistochemistry (IHC) on tissue microarrays.ResultsSTMN1 expression was significantly correlated with inferior overall survival (OS). Multivariate regression further identified STMN1 as an independent adverse prognostic biomarker for OS. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) revealed that elevated STMN1 expression was tightly linked to immune response activation and T-cell infiltration, enabling the formation of an immune-inflamed TME in BTC. IHC validation confirmed positive correlations between STMN1 levels and anti-tumor CD8⁺ T-cell infiltration as well as programmed death-ligand 1 (PD-L1) expression. Patients with low STMN1 expression derived prominent survival benefits from gemcitabine-based adjuvant chemotherapy (ACT), whereas high STMN1 expression predicted superior therapeutic responses to immune checkpoint blockade (ICB) in BTC recipients.DiscussionSTMN1 functions as an independent prognostic biomarker and differential predictive indicator for ACT and ICB efficacy in BTC, likely facilitating the construction of an immune-inflamed TME by promoting tumor immune cell recruitment and infiltration.

Biliary tract cancer (BTC) is typically diagnosed at an advanced stage due to the lack of effective screening tools, resulting in limited therapeutic options and poor survival outcomes. Therefore, there is a critical need for non-invasive strategies that enable early detection and risk stratification. Fragmentomic profiling of cell-free DNA (cfDNA) captures genome-wide fragmentation patterns reflecting tumor-associated chromatin structure and genomic instability, providing a promising approach for non-invasive cancer detection. In this study, we developed a low-pass whole-genome sequencing (WGS)-based framework for BTC detection and postoperative risk assessment. Plasma samples were analyzed to derive three fragmentomic features, including copy number variation, fragment size distribution, and promoter fragmentation entropy, which were integrated into a machine-learning model trained using five-fold cross-validation. The model demonstrated robust performance across independent validation cohorts, outperforming individual fragmentomic features and conventional serum biomarkers, and accurately distinguished BTC from benign biliary diseases. Longitudinal analyses revealed that cfDNA fragmentomic risk scores dynamically tracked disease burden and treatment response. Importantly, postoperative risk scores were independently associated with disease-free survival, highlighting their prognostic value. Collectively, these findings establish a scalable and cost-effective framework for cfDNA-based BTC detection and monitoring using low-pass WGS data. This approach shows strong potential for targeted screening in high-risk populations and for guiding personalized postoperative surveillance and clinical management for BTC patients.

Gallbladder cancer is usually discovered as an incidental finding during or after cholecystectomy for gallstones. Most patients have advanced gallbladder cancer at diagnosis and have lost the opportunity for radical surgery, leading to dismal prognosis. Standard conversion therapy regimen is still in exploration. We reported a patient with unresectable biliary tract cancer (most likely gallbladder cancer) and multiple metastases (TxN2M1) discovered one month after laparoscopic gallstone surgery. The patient received margin-free resection after 9 cycles of immunotherapy (camrelizumab) combined with chemotherapy, followed by adjuvant immunochemotherapy. Pathological complete response was achieved and no disease recurrence occurred 5 months after surgery. Decreased neutrophil count was the only grade 3-4 treatment-related adverse event during the 9 cycles of conversion therapy. This case provides some reference for the conversion therapy of advanced gallbladder cancer or other biliary tract cancers.

Aberrant activation of beta-catenin/TCF is a hallmark of colon cancer. How the functions of nuclear localized beta-catenin are regulated is not fully understood. Here, it was found that FOXN3 (Forkhead box N3) was down-regulated in colon cancer tissues. Forced expression of FOXN3 inhibited the growth, migration and invasion of colon cancer cells, while knocking down the expression of FOXN3 promoted the growth, migration, invasion and metastasis of colon cancer cells. FOXN3 bind to beta-catenin and inhibited beta-catenin/TCF signaling by blocking the interaction between beta-catenin and TCF4. Taken together, these data demonstrated the suppressive roles of FOXN3 in the progression of colon cancer, and indicated that restoring the functions of FOXN3 would be a novel therapeutic strategy for colon cancer.

Novel therapeutic targets are urgently needed for the aggressive malignancy gallbladder cancer (GBC). G-protein regulated inducer of neurite outgrowth 1 (GPRIN1) is a candidate oncogene, but its function in GBC and its connection to mitochondrial dysregulation remain unknown. In this study, we analyzed clinical samples and demonstrated that GPRIN1 is significantly upregulated in GBC tissues, where its high expression correlates with advanced clinical stage and poor patient prognosis. Functional assays revealed that GPRIN1 is essential for GBC progression, driving cell cycle advancement and maintaining mitochondrial homeostasis. By integrating proteomic and molecular analyses, our study delineates a bimodal and hierarchical regulatory program commanded by GPRIN1 to ensure the robust activation of CDK1. In the nucleus, GPRIN1 functions as a transcriptional co-activator, scaffolding and stabilizing E2F1 to drive CDK1 expression. In parallel, it functions at a post-translational level to directly promote CDK1 activation by physically steering the kinase away from its inhibitor, MYT1, and toward its activator, Cdc25C. This dual-pronged regulation culminates in hyperactivated CDK1, which in turn unleashes a PI3K-Akt signaling cascade to couple relentless cell proliferation with the necessary mitochondrial support. Importantly, genetic or pharmacological disruption of this GPRIN1-CDK1-PI3K/Akt axis completely abrogated tumorigenesis in vitro and in vivo. Taken together, these results reveal GPRIN1 as a master regulator whose dual transcriptional and post-translational control of CDK1 integrates cell cycle progression with mitochondrial homeostasis, suggesting that targeting GPRIN1 may represent a highly specific therapeutic strategy in this lethal malignancy.

e16297 Background: Gallbladder cancer (GBC) is a highly aggressive biliary tract tumor with limited therapeutic options and poor prognosis. Metabolic reprogramming, particularly cholesterol metabolism, has emerged as a critical driver of tumor progression. Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, has been implicated in multiple cancers; however, its role and underlying mechanisms in GBC remain largely unclear. Methods: \We analyzed SQLE expression in human GBC tissues and paired adjacent para-tumor tissues using quantitative PCR, western blotting and immunohistochemistry, and evaluated its association with clinicopathological features and patient survival. Kaplan–Meier survival analysis was performed to determine the prognostic significance of SQLE. Stable SQLE knockdown and overexpression cell lines were established, and functional assays including proliferation, migration, and apoptosis assays were conducted in GBC cell lines, as well as SQLE inhibitor treated groups. Cholesterol quantification, Filipin staining were performed to elucidate the mechanistic role of SQLE in cholesterol-mediated AKT activation. In vivo tumorigenicity and therapeutic response were evaluated using a subcutaneous xenograft model in nude mice. Results: SQLE was significantly upregulated in GBC tissues compared with adjacent para-tumor tissues and was positively correlated with advanced TNM stage. High SQLE expression was associated with significantly shorter overall survival. Functional studies demonstrated that knocking down or inhibiting SQLE markedly suppressed GBC cell proliferation, migration, and invasion, while inducing apoptosis, and overexpressing SQLE had the opposite results. Mechanistically, SQLE inhibition reduced intracellular cholesterol levels and attenuated downstream AKT signaling. In vivo, targeting SQLE significantly inhibited tumor growth in subcutaneous xenograft model in nude mice. Conclusions: Our findings identify SQLE as a critical regulator of cholesterol-dependent AKT activation and a potent oncogenic driver in GBC. Targeting SQLE represents a promising therapeutic strategy and prognostic biomarker for patients with gallbladder cancer.

3008 Background: Kirsten rat sarcoma (KRAS) G12D is the most prevalent RAS mutation in human cancers, accounting for 10.6-19.2% and 5.8-10.8% of cases in colorectal cancer (CRC) and cholangiocarcinoma (CCA), respectively. Currently, the later-line therapies for metastatic CCA and CRC remain very limited. The KRAS G12D mutation is associated with poor prognosis and confers resistance to conventional chemotherapy. GFH375, a potent, selective, orally bioavailable KRAS G12D inhibitor, has demonstrated antitumor efficacy in pancreatic cancer and non-small cell lung cancer. Here we report the preliminary efficacy results of GFH375 in patients (pts) with advanced KRAS G12D mutant CCA and CRC. Methods: This is a phase I/II study (NCT06500676) evaluating the safety, tolerability, pharmacokinetics and efficacy of GFH375 in pts with advanced solid tumors harboring KRAS G12D mutations. Pts with locally advanced or metastatic CCA and CRC who had failed prior therapies were also enrolled. Tumor assessments were performed every 6 weeks during the first 48 weeks, and every 12 weeks thereafter. All efficacy endpoints, including objective response rate (ORR), disease control rate (DCR) and progression-free survival (PFS) were evaluated according to RECIST v1.1. Circulating tumor DNA (ctDNA) samples were collected at baseline and at the end of treatment for exploratory analyses. Results: As of 31 Oct 2025, twenty CCA and 41 CRC pts were treated with GFH375 once daily (400, 600 or 750 mg). Among the CCA pts (median age: 59.5 yrs; 80.0% male), 17 (85.0%) had metastatic disease at baseline, and the median prior lines of therapy were 2 (range: 1-6). ORR was 35.0% (7/20), and DCR was 95.0% (19/20). Nine out of 12 pts with stable disease (SD) had tumor shrinkage. Median PFS was 6.3 months, and median overall survival (OS) was not reached. Among those CRC pts (median age: 56 yrs; 61.0% male), all had metastatic disease at baseline, and the median prior lines of therapy were 3 (range: 1-6). Among 35 pts who had at least one post-treatment tumor assessment, ORR was 11.4% (4/35), and DCR was 77.1% (27/35). Median PFS was 4.1 months and median OS was not reached. Baseline ctDNA results were available in 19 CCA pts and 36 CRC pts. KRAS G12D mutations were detected in 14 (73.7%) CCA pts and 31 (86.1%) CRC pts. The common co-mutated genes (≥15%) in CCA were TP53, BCL2L11 and APC, while in CRC were TP53, APC, RUNX1, SMAD4 and PIK3CA. The safety profile in the 61 pts was similar to that of the whole population as previously reported. The most common treatment-related adverse events (TRAEs) (≥30%) included diarrhea, nausea, vomiting, aspartate aminotransferase increased, anemia and decreased appetite. Conclusions: GFH375 monotherapy has demonstrated promising antitumor activities in heavily treated pts with CCA and CRC. GFH375 monotherapy in CCA and in combination with other anti-tumor therapies for CRC is under development. Clinical trial information: NCT06500676 .

BACKGROUND: Gallbladder cancer (GBC) is an aggressive malignancy with limited therapeutic options, primarily due to the frequent emergence of resistance to gemcitabine-based chemotherapy. Uncovering molecular mechanisms underlying this resistance is essential for developing more effective treatments. METHODS: Gemcitabine-resistant GBC cell lines were generated and subjected to transcriptomic sequencing to identify resistance-associated genes. A genome-wide CRISPR-Cas9 knockout screen was used to pinpoint key genetic regulators. Functional validation was performed through gene knockdown and overexpression, cell viability and apoptosis assays, colony formation, and DNA damage analysis. A high-throughput virtual screening (HTVS) approach was applied to identify small-molecule inhibitors targeting the E2F8-DNA interaction. The efficacy of selected compounds was tested in vitro and in xenograft mouse models, and further validated using patient-derived organoids (PDOs) established from primary and recurrent gallbladder cancers. RESULTS: The transcription factor E2F8 was identified as a driver of gemcitabine resistance via upregulation of RRM2, a gene involved in DNA repair. Knockdown of E2F8 enhanced sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors in resistant GBC cells by impairing DNA repair. HTVS yielded HIT-4, a small-molecule inhibitor that binds to E2F8 and disrupts its interaction with DNA, leading to reduced RRM2 expression. HIT-4 significantly increased apoptosis and DNA damage when combined with PARP inhibitors. In vivo and in PDO models, HIT-4 and PARP inhibitor co-treatment markedly suppressed tumor growth, extended survival, and showed minimal toxicity. CONCLUSIONS: This study identifies the E2F8-RRM2 axis as a key regulator of gemcitabine resistance in GBC and establishes E2F8 as a druggable target. The novel compound HIT-4, in combination with PARP inhibitors, represents a promising therapeutic strategy to overcome chemoresistance and warrants further clinical investigation.

4131 Background: Biliary Tract Cancer (BTC) is a highly aggressive malignancy with poor survival outcomes, primarily due to the lack of effective early detection methods and late-stage diagnoses. Current diagnostic tools, including imaging and invasive endoscopic procedures, are limited in their sensitivity and specificity for identifying early-stage disease. This study addresses this critical gap by developing a novel, non-invasive approach for BTC detection using circulating cell-free DNA (cfDNA) fragmentomics features. Methods: The study cohort included 163 patients diagnosed with BTC and 165 healthy individuals, divided equally into training and validation cohorts. All participants’ plasma samples were collected for a low-depth whole genome sequencing (WGS) process to extract three key cfDNA fragmentomics features: Copy Number Variation (CNV), Fragment Size Distribution (FSD), and Promoter Fragmentation Entropy (PFE). These features were utilized to develop a machine learning model, which was trained and validated through 5-fold cross-validation. An external cohort of 55 patients with benign diseases and 18 Tis/High-grade cases was used to further evaluate the model robustness. Results: The stacked ensemble model reached an Area Under the Curve (AUC) of 0.96 in the validation cohort, showing excellent performance in identifying BTC from healthy participants. At an 86% training specificity cutoff, sensitivity achieved 90.91% (95% CI: 81.26% - 96.59%) and specificity 87.88% (95% CI: 77.51% - 94.62%). While PFE performed as a strong single feature with an AUC exceeding 0.92. The model demonstrated its effectiveness in early-stage detection, with the sensitivity increasing from 80% in stage I to 95.65% in stage II. The model surpassed traditional biomarkers (AUC > 95% compared to ~75% for CA19-9) and demonstrated consistent performance across subgroups. External validation revealed 89% sensitivity for early lesions and 89% specificity for benign cases, highlighting its potential for non-invasive early detection of BTC. Conclusions: This study demonstrates a reliable and non-invasive strategy for early BTC detection, leveraging cfDNA fragmentomics features and a robust machine learning framework. The model’s high accuracy and reproducibility in both internal and external cohorts highlight its potential for clinical implementation, offering a transformative approach for BTC screening. Early diagnosis enabled by this method may significantly improve patient outcomes and survival rates, marking a major advancement in clinical practice.

PURPOSE:To compare overall survival (OS) and disease-free survival (DFS) in patients with advanced resectable gallbladder carcinoma (GBC) receiving radical resection followed by chemoradiation therapy (S+CRT) or chemotherapy alone (S+CT). METHODS AND MATERIALS:This real-world study included 239 patients (pT2-4, N0-2, M0) treated between February 2017 and November 2021. Inverse probability of treatment weighting (IPTW) was applied to balance baseline variables between S+CRT and S+CT groups. Kaplan-Meier analysis and Cox regression were used to evaluate survival. Predefined subgroups analyses were conducted for 77 patients with incidental GBC and 94 patients with nodal metastasis (pN+). Adverse events and recurrence patterns were recorded for the entire cohort. RESULTS:Median follow-up was 57.4 months. IPTW created 472.6 weighted patients with well-balanced characteristics. S+CRT was associated with higher OS and DFS at 1, 3, and 5 years compared with S+CT (OS, 90.5%, 71.9%, 65.8% vs 81.6%, 56.1%, 50.1%; DFS, 77.8%, 65.1%, 57.7% vs 63.1%, 45.3%, 42.6%). Median OS and DFS were not reached in the S+CRT group. In the incidental GBC subgroup, survival differences were not statistically significant overall, but a landmark analysis beyond 24 months revealed improved OS favoring S+CRT (P = .011). Similarly, in the subgroup with nodal metastasis (pN+), adjuvant CRT was associated with a significant improvement in DFS. CONCLUSIONS:Postoperative CRT significantly improved OS and DFS in balanced patients with stage II-IV GBC. A delayed OS benefit was observed in patients with incidental GBC, whereas patients with nodal metastasis also experienced significantly improved DFS. Although intrahepatic recurrence remained predominant, CRT notably reduced local failures without significantly increasing treatment-related toxicity.

Biliary systems are one of the most crucial organs in human physiology, perforating bile secretion and transport. Due to its complex physiological and anatomical structure, the biliary system operates through highly intricate mechanisms involving fluid dynamics, biochemistry. The aim of this research is to develop a biliary biofluid dynamics measurement platform based on Particle Image Velocimetry (PIV) methods for the characterization of fluid mechanical properties of the biliary system. The experimental platform consisting of the PIV unit, the experimental unit, and a high-performance computing unit, is capable to simulate several patho-physiological conditions within the biliary tract and thus provide flow field information in the model. By introducing tracer particles into a biliary fluid, PIV technique is utilized to capture and analyze the motion trajectories of these particles, reconstructing the complex flow patterns within the biliary tract and providing key data for the study of related diseases. This platform is expected to offer new insights and potential utilities for research efforts in biliary fluid dynamics.

Background Gallbladder cancer (GBC) is a rare but highly lethal malignancy with one of the poorest prognoses among cancers of the digestive system cancers. However, current experimental models of GBC face critical limitations, particularly their dependence on immunodeficient hosts, which precludes the investigation of the tumor microenvironment.We aimed to establish a novel syngeneic immunocompetent mouse model that recapitulates human GBC and enables the investigation of tumor-immune interactions. Methods We engineered a murine cell line (mGBC1-ZH) from normal mouse gallbladder organoids expressing Kras and Trp53 (encoding mouse p53 ) mutations. Tumorigenic potential of mGBC1-ZH was evaluated by subcutaneous and orthotopic implantation. RNA-Seq and WES was used to demonstrate its characterization and similarity with human GBC. Immunohistochemistry, CCK8, and transwell assays were used to investigate the role of CXCL5 in GBC. Therapeutic responses to standard first-line chemotherapeutic agents was evaluated in the syngeneic GBC model. Results This model supports both subcutaneous and orthotopic tumor growth in immunocompetent C57BL/6J hosts while preserving hallmark features of human GBC, including biliary epithelial differentiation (CK7+/CK19+), aggressive histopathology, and an immunosuppressive "cold" tumor microenvironment. Genomic characterization revealed recurrent chromosomal instability and copy number alterations mirroring human GBC. Comprehensive transcriptomic profiling revealed profound gene expression changes during the model development, and resembled transcriptional features between mGBC1-ZH and human GBC cell lines and samples. CXCL5 was found to be upregulated in human GBC, and promote tumor cell proliferation, migration, and invasion. Functional validation of the syngeneic GBC model demonstrated therapeutic responsiveness to frontline chemotherapeutics gemcitabine and cisplatin with significant in vivo tumor regression. Conclusions In summary, we establised a novel syngeneic GBC mouse model, overcoming the limitations of traditional models by enabling studies in immunocompetent hosts. This model provides valuable insights into the molecular evolution from normal gallbladder cells to transformed cancer cells and establishes a robust platform for both mechanistic studies and therapeutic development, particularly for immunotherapy approaches. ### Competing Interest Statement The authors have declared no competing interest. * Abbreviations : GBC : Gallbladder cancer; PDXs : patient-derived xenografts; 3D : three-dimensional; EMT : epithelial-mesenchymal transition; TIB : tumor immune barrier; STR : Short Tandem Repeat; WES : whole-exome sequencing; PDAC : pancreatic ductal adenocarcinoma; GEM : gemcitabine; CIS : cisplatin; IC50 : half-maximal inhibitory concentration. the National Natural Science Foundation of China, 32300484, 82373311 Science and Technology Commission of Shanghai Municipality, https://ror.org/03kt66j61, 22ZR1411700, 21JC1401200

The protein sequestosome 1 (p62) can enhance the antioxidant defense of tumor cells against chemotherapeutic agents by increasing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. We focused on exploring whether p62 regulates resistance to 5-fluorouracil (5-Fu) in intrahepatic cholangiocarcinoma (ICC) cells by mediating Nrf2 expression. Higher levels of p62 mRNA and its protein were observed in 5-Fu-resistant ICC samples and cell lines. Knockdown of p62 lowered 5-Fu resistance in 5-Fu-resistant ICC cells, along with a strong inhibition of cell proliferation and a potent promotion of apoptosis in response to 5-Fu stimulation. Importantly, 5-Fu-resistant ICC cells demonstrated a remarkable Nrf2 nuclear translocation, but p62 silencing overtly repressed Nrf2 nuclear translocation under 5-Fu stimulation. Also, p62 overexpression elevated 5-Fu resistance and Nrf2 nuclear translocation in parental ICC cells, but these changes were reversed following Nrf2 silencing. p62 enables ICC cells with 5-Fu resistance by boosting Nrf2 nuclear translocation, suggesting that targeting p62 may resensitize 5-Fu-resistant ICC cells to 5-Fu.

To investigate the biological function and the underlying mechanism of CD39 in cholangiocarcinoma. Quantitative reverse transcription PCR (RT-qPCR), western blot (WB), and immunohistochemistry staining was used to evaluate the expression level of CD39 in cholangiocarcinoma. Kaplan–Meier and Cox hazard ratio regression analyses were implicated to evaluate the prognostic significance of CD39. Cell counting kit-8 (CCK-8) was carried out to evaluate the proliferative capacity, while transwell assay was used to detect the migration and invasion ability. In addition, B-NDG mice were used for the in vivo assay. The potential protein binding with the CD39 was identified through co-immunoprecipitation. CD39 was aberrantly expressed in the tumor tissue and cholangiocarcinoma cell lines. CD39 was identified as an independent poor prognostic factor in cholangiocarcinoma. In addition, in vitro and in vivo data indicated that the knockdown of CD39 could suppress the proliferation, migration, and invasion ability. The opposite results were observed when CD39 was overexpressed. Mechanistically, CD39 could bind with Annexin A2 (ANXA2), which influences the phosphorylation level of ANXA2 at the Tyr24 site, thereby promoting the activation of PI3K/AKT signaling, which resulted in the biological change in cholangiocarcinoma. CD39 was identified as an independent prognostic factor of poor overall survival for patients with cholangiocarcinoma. In terms of the biological role of CD39, our data indicated that CD39 promoted the progression and metastasis of cholangiocarcinoma through binding with ANXA2, and through activating the PI3K/AKT signaling. In brief, CD39 is a potential prognostic factor and therapeutical target for cholangiocarcinoma.

Gallbladder cancer (GBC) is a biliary tract cancer with a poor prognosis. Consistent evidence suggests that fasting has extensive antitumor effects in various cancers and influences levels of poly (rC)-binding protein 2 (PCBP2). However, whether fasting and PCBP2 are involved in GBC remains unknown. We assessed the expression of PCBP2 in GBC tumor tissues and cells. Knockdown and overexpression of PCBP2, combined with in vitro and in vivo assays using fasting mimic medium or diets, were conducted to provide functional significance. The effect of PCBP2 on glycolysis was assessed by glucose uptake, lactate production, oxygen consumption rate, and limiting glycolytic-associated enzymes (PDK1, PKM2, and HK-2). We found that fasting could inhibit glycolysis and cell migration/invasion in GBC cells and that fasting mimic diets could significantly inhibit GBC cell proliferation in a mouse xenograft model. PBCP2 was upregulated in GBC tumor tissues and cells. Moreover, PCBP2 is a key downstream target of fasting, and fasting decreases PCBP2 expression in GBC cells. PCBP2 knockdown inhibits GBC cell proliferation, migration/invasion, and glycolysis, whereas PCBP2 overexpression has the opposite effect. Through co-immunoprecipitation, we identified a physical connection between PCBP2 and the angiopoietin-like protein ANGPTL4. PCBP2 can negatively regulate the expression of ANGPTL4. Hence, fasting inhibits cell proliferation, migration/invasion, and glycolysis through PCBP2/ANGPTL4 signaling. We conclude that PCBP2 is a target of fasting and is involved in cell migration/invasion and glycolysis through the negative regulation of ANGPTL4 in GBC. PCBP2 represents a potential therapeutic target for GBC.

Cholangiocarcinoma (CCA) is a highly lethal epithelial malignancy that can arise at any site within the biliary tract. Our findings revealed that FUN14 domain-containing protein 1 (FUNDC1) expression was significantly elevated in CCA samples compared to matched peritumoral tissues. Survival analysis indicated that patients with high FUNDC1 expression had shorter overall survival. Mitochondrial membrane potential (MMP) alterations were observed. Specifically, knockdown of FUNDC1 induced MMP disruption in CCA cells. Additionally, we detected damaged mitochondria and increased production of reactive oxygen species (ROS), which were further exacerbated by FUNDC1 knockdown in CCA cells. Further analysis revealed upregulation of Gpx4 and SLC7A11, along with downregulation of NCOA4 in CCA patients. These changes were accompanied by increased glutathione (GSH) levels and decreased malondialdehyde (MDA) and C11-BODIPY levels in CCA cells, effects that were nullified by FUNDC1 knockdown in vivo. We also observed increased expression of Ras-related C3 botulinum toxin substrate 1 (RAC1), a member of the Rho GTPase family, in human CCA samples. Immunoprecipitation analysis demonstrated that an extracellular segment of FUNDC1 (amino acids 96-133) mediated its interaction with RAC1. Furthermore, while FUNDC1 knockdown effectively induces ferroptosis, RAC1 knockdown does not. Notably, this induction is abrogated upon overexpression of FUNDC1. In vivo experiments also confirmed that knockdown of FUNDC1 and RAC1 significantly reduced tumor volume. These findings suggest that RAC1 is involved in FUNDC1-induced malignant transformation in CCA. Overall, our data indicate that FUNDC1 promotes CCA progression through mitochondrial function-dependent ferroptosis, highlighting a promising target for the development of FUNDC1-based therapies for CCA.

ICC is a malignant tumor that originates from the intrahepatic bile ducts with insidious symptoms and a poor prognosis. Early diagnosis methods and therapeutic targets are urgently needed for ICC. We utilized a comprehensive set of analytical techniques to elucidate the role and mechanisms of DCDC2 in ICC. Our study included protein microarrays, transcriptome analysis, functional assays, immunofluorescence, dual-luciferase reporter assays, as well as xenograft models and humanized PBMC models. Our study demonstrates that elevated levels of anti-DCDC2 autoantibodies in the serum of ICC patients indicate its potential utility as a diagnostic biomarker. Comprehensive in vitro and in vivo analyses reveal that DCDC2 promotes ICC proliferation, metastasis, and immune evasion. Mechanistically, DCDC2 stabilizes ENO1, resulting in enhanced AKT phosphorylation and increased expression of FGL1. Notably, elevated FGL1 levels significantly impair CD8+ T cell functionality via the FGL1-LAG3 axis. Our findings position anti-DCDC2 autoantibody as a promising diagnostic biomarker for ICC, associated with poor prognostic outcomes, and elucidate its critical role in tumor growth and immune evasion through its interaction with ENO1.

Urothelial carcinoma (UC) is a highly malignant disease with significant public health implications. Despite advancements in oncology, early diagnosis and effective prognostic tools remain limited. This study aimed to develop a machine learning model using complete blood count (CBC) data to predict clinical outcomes in UC patients. A retrospective, two-center cohort study was conducted, analyzing 23 CBC variables from 477 UC patients at Xuhui Hospital of Fudan University (discovery cohort) and 297 UC patients from Putuo People's Hospital of Tongji University (validation cohort). CBC data were collected before treatment and three months posttreatment, with overall survival (OS) as the primary endpoint. Nine machine learning models were developed in the discovery cohort and validated independently. Feature selection identified a logistic regression (LR) model incorporating white blood cell (WBC) count and lymphocyte percentage (LYMPH%) as the optimal predictor. The model achieved high performance, with an area under the ROC curve (AUC) of 0.93 (95 %CI: 0.90-0.97), area under the precision-recall curve (AUPRC) of 0.94 (95 %CI: 0.89-0.99), positive predictive value (PPV) of 0.87 (95 %CI: 0.75-0.98), negative predictive value (NPV) of 0.82 (95 %CI: 0.78-0.87), accuracy of 0.83 (95 %CI: 0.80-0.88), and F1 score of 0.82 (95 %CI: 0.79-0.86) in the discovery cohort, and comparable results in the validation cohort (AUC 0.88 [95 %CI: 0.84-0.93], AUPRC 0.81 [95 %CI: 0.75-0.86], PPV 0.77 [95 %CI: 0.71-0.84], NPV 0.89 [95 %CI: 0.84-0.95], accuracy 0.84 [95 %CI: 0.80-0.89], and F1 score 0.80 [95 %CI: 0.74-0.87]). Decision curve analysis demonstrated consistent net benefits, while Kaplan-Meier analysis indicated significantly shorter OS in the "predict worse outcomes" subgroup. Posttreatment, WBC counts increased and LYMPH% decreased in deceased patients, whereas survivors showed the opposite trends (P < 0.05). These findings suggest that a simple, cost-effective CBC-based machine learning model can effectively predict UC prognosis, aiding clinical decision-making.