Lymph node (LN) metastasis (LNM) in early-stage pancreatic ductal adenocarcinoma (PDAC) predicts systemic dissemination and poor survival, yet its underlying mechanisms remain elusive. In this study, we demonstrated that senescent cancer-associated fibroblasts (senCAF) drive lymphatic remodeling and LNM in early-stage PDAC. Mechanistically, senCAFs increased glucose metabolism and lactate production, which activated lactylation-mediated serine metabolism to protect lymphatic endothelial cells from oxidative stress. Moreover, we discovered CCR4+ regulatory T cells from the draining LNs accumulated around lymphatic vessels, which established an immunosuppressive perilymphatic niche. High-throughput drug screening determined selective clearance of senCAFs via chidamide, attenuated tumor progression, and improved chemoimmunotherapeutic efficacy. We subsequently initiated a clinical trial (chidamide and nab-paclitaxel/gemcitabine plus anti-PD-1/CTLA-4) in patients with metastatic PDAC and reported its preliminary promising results. Collectively, these findings reveal a closed link between cellular senescence and PDAC metastasis, offering the potential senolytic means to improve chemoimmunotherapy efficacy. SIGNIFICANCE:Our findings have revealed a closed link between cellular senescence, metabolic reprograming, and spatial immunosuppressive niche and PDAC metastasis, offering the potential senolytic drugs to improve chemoimmunotherapy efficacy in patients with PDAC.

Background As it is a tumour-associated antigen in epithelial cells, research on claudin18.2 (CLDN18.2) has focused on its role as a therapeutic target in pancreatic cancers and its part in maintaining tight junctions. Objective We elucidate the role of trogocytosis-related CLDN18.2 in CD8 + T cells and pancreatic ductal adenocarcinoma (PDAC) progression. Design We constructed humanised hCD34 + , Trp53 R172H Kras G12D Pdx1-cre (KPC), Cldn18.2 knockout (KO), and patient-derived xenograft/organoid mouse models. Flow cytometry, immunofluorescence, single-cell RNA-sequencing and immunoprecipitation-mass spectrometry (IP-MS) were performed. Results CLDN18.2 + CD8 + T cells indicated poor pancreatic cancer prognosis and immunotherapeutic resistance. CD8 + T cells acquired CLDN18.2 from tumour cells via trogocytosis, inhibiting their activation and cytotoxicity. “Dressed” CLDN18.2 suppressed glucose uptake, glycolysis and cytotoxicity of tumour-infiltrating CD8 + T cells. Mechanically, trogocytosis-related CLDN18.2 induced GSK3β/CK1α-mediated β-catenin phosphorylation, promoting β-catenin ubiquitination and proteasome degradation in CD8 + T cells. CLDN18.2 interacted with β-catenin’s N-terminal domain via its C-terminal domain, further strengthening the interaction between β-catenin and CK1α. Moreover, CLDN18.2 + CD8 + T cells preferentially ‘homed’ to the bone marrow through the CXCL12/CXCR4 axis, skewed haematopoietic stem cell myeloid differentiation and induced systemic immune senescence via IL1α. Notably, preclinical mouse studies showed PC18.1 peptide sensitised immunotherapy and suppressed PDAC progression by disrupting the CLDN18.2/β-catenin interaction in CD8 + T cells. Conclusions Trogocytosis-related CLDN18.2 inhibited the glucose uptake, glycolysis and cytotoxicity of tumour-infiltrating CD8 + T cells by promoting the ubiquitin-proteasomal degradation of β-catenin in PDAC. Therefore, targeting trogocytosis-related CLDN18.2 + CD8 + T cells may be a promising therapeutic strategy to inhibit PDAC progression.

Early detection of pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge due to its asymptomatic onset and the lack of noninvasive diagnostic tools. Leucine aminopeptidase (LAP) has emerged as a promising biomarker for pancreatic cancer progression, with significantly elevated levels observed in both blood and urine during tumorigenesis. Herein, we report the rational design and synthesis of two LAP-activatable bioluminescent probes, N-LAP-Luc and O-LAP-Luc, for sensitive detection of LAP activity in blood and urine. Compared to N-LAP-Luc, O-LAP-Luc, incorporating a self-immolative linker, exhibits superior sensitivity (LOD = 8.7 mU/L), excellent selectivity, and 13.5-fold higher luminescence signals. Mechanistic studies revealed that this enhancement originates from both lower probe-enzyme binding energy and brighter luciferin substrate released upon activation. In KPC-Luc cells and PDAC mouse models, O-LAP-Luc enabled highly sensitive imaging of endogenous LAP, with significantly prolonged signal persistence and enhanced signal-to-background ratios. Notably, O-LAP-Luc achieved sensitive monitoring of LAP levels in the urine and blood samples from orthotopic PDAC mice, as well as urine specimens from PDAC patients. Furthermore, we established a portable smartphone-based detection platform that allows visual urine testing within 5 min under 365 nm UV light, with quantitative RGB analysis achieving excellent linear correlation (R2 = 0.96). To our knowledge, O-LAP-Luc represents the first bioluminescent probe applied to point-of-care testing of urinary LAP, offering a promising strategy for noninvasive, rapid, and cost-effective diagnosis of PDAC.

Detailed Supplementary Methods and Materials

The labile iron pool in the cell is required for ferroptosis, a form of regulated cell death resulting from excessive lipid peroxidation and membrane damage. Glutathione (GSH) is critical for lipid-peroxide scavenging, and cysteine is the rate-limiting amino acid in GSH synthesis. Cysteine metabolism intricately intertwines with iron metabolism, either directly by participating in assembly of the iron-sulfur cluster or indirectly through the pantothenate pathway and coenzyme A (CoA) synthesis. However, the regulation of iron homeostasis in cystine (Cys2)-deprivation-induced ferroptosis is poorly understood. Here, we show that Cys2 deprivation promotes ferroptosis, at least in part, by activating the iron-starvation response (ISR), and CoA can mitigate ferroptosis by suppressing the ISR. Mechanistically, Cys2 deprivation promotes the oxidation of cytosolic iron-sulfur clusters to activate the ISR; CoA and related small-molecule thiols in the pantothenate pathway suppress the ISR and ferroptosis by preventing the oxidation of iron-sulfur clusters in Cys2-deprived cells. Our findings provide important insight into the regulation of the ISR in Cys2-deprivation-induced ferroptosis, and show that CoA can protect cells from Cys2-deprivation-induced ferroptosis by suppressing the ISR.

Abstract Purpose: Cancer cachexia, characterized by persistent weight loss, skeletal muscle atrophy, and adipose tissue reduction—with progressive skeletal muscle wasting being the predominant manifestation—represents a major cause of mortality in pancreatic ductal adenocarcinoma (PDAC) patients. Cachexia is a common and severe complication in PDAC, significantly impacting patients' quality of life and prognosis. This study investigates the role of the mitochondrial calcium uniporter (MCU) in PDAC-associated cachexia, potentially offering novel interventional strategies for its prevention. Experimental Design: MCU expression was analyzed in PDAC patient tissues and correlated with cachexia incidence. Spontaneous tumor models and orthotopic PDAC models using MCU-overexpressing and control stable cell lines were established to evaluate tumor progression and muscle atrophy. Neutrophil extracellular traps (NETs) formation was detected by H3cit/MPO/DAPI multiplex immunofluorescence. NETs function was examined through anti-Ly6G-mediated neutrophil depletion, DNase I treatment, and Pad4-/- mice. Secretome analysis identified MCU-regulated mechanisms in NETs formation, validated through recombinant protein treatment and receptor inhibition. The role of CCDC25 was investigated using genetic knockout models and AAV9-mediated skeletal muscle-specific silencing. Results: MCU overexpression significantly correlated with cachexia incidence and reduced skeletal muscle mass in PDAC patients. Compared to the MCU-overexpression group, control mice demonstrated attenuated tumor growth and preserved muscle mass independent of tumor burden. MCU-overexpressing PDAC models showed significantly elevated NETs infiltration in skeletal muscle, while NETs clearance prevented muscle atrophy. MCU overexpression induced cellular senescence and senescence-associated secretory phenotype (SASP) secretion (particularly C3 and CXCL1), promoting NETosis through C3aR and CXCR2 signaling. CCDC25 was identified as a critical NET DNA receptor in skeletal muscle, mediating muscle atrophy through RAC1-dependent ROS production. Conclusion: Our study reveals a novel signaling pathway wherein tumor MCU overexpression promotes cellular senescence and SASP secretion, driving NETs formation through C3/CXCL1 signaling. NETs engage CCDC25 receptors on muscle cells, activating the RAC1-ROS signaling pathway that ultimately leads to muscle atrophy. Impact: These findings establish the MCU-NETs-CCDC25 axis as a key mechanism in PDAC-associated cachexia and suggest multiple therapeutic strategies—including MCU inhibition, NETs degradation, and CCDC25 blockade—providing new directions for alleviating cachexia and preserving muscle mass in cancer patients. Citation Format: Xiuchao Wang, Jihui Hao. Mitochondrial calcium uniporter drives cancer cachexia in pancreatic ductal adenocarcinoma through NETosis-mediated skeletal muscle atrophy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3367.

Metabolic-epigenetic crosstalk accelerates breast cancer (BC) progression; however, the conduit linking glycolytic flux to chromatin remodeling remains incompletely defined. Here, we delineate a kinase-metabolism-epigenetic axis centered on the Mitogen-Activated Protein Kinase Associated Protein 1 (MAPKAP1, also known as SIN1). Single-cell transcriptomic profiling of highly invasive tumors identifies SIN1 as a central node co-enriched with epithelial-mesenchymal transition and proliferation programs. Biochemical mapping and mass spectrometry demonstrate that SIN1 scaffolds TTK to promote phosphorylation of lactate dehydrogenase A (LDHA) at Tyr239 (p-LDHA^239), thereby amplifying glycolysis and lactate production. The resulting lactate accumulation increases histone H3K18 lactylation (H3K18la), which, as shown by ChIP-seq, is enriched at the Solute Carrier Family 2 Member 3 (SLC2A3, also known as GLUT3) promoter, upregulating GLUT3 and enhancing glucose uptake, thus establishing a self-reinforcing SIN1/TTK/LDHA-H3K18la-GLUT3 feed-forward loop. Structure-function analyses using domain truncations, molecular docking, and high-throughput virtual screening nominate LR-90 as a small-molecule inhibitor of the SIN1/TTK/LDHA complex. LR-90 reduces p-LDHA^239, lactate levels, H3K18la occupancy at the GLUT3 promoter, glucose uptake, invasion, and tumor growth, and it synergizes with standard chemotherapy in patient-derived organoids and mouse xenografts. Clinically, elevated SIN1 expression is associated with adverse pathological features and inferior overall survival. Collectively, these findings link SIN1-mediated recruitment of TTK for LDHA phosphorylation to histone lactylation and GLUT3-driven metabolic reprogramming, and suggest that pharmacological disruption of this axis with LR-90, alone or in combination with standard chemotherapy, may offer a therapeutic strategy for high-glycolytic, high-lactate breast cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with poor prognosis due to chemoresistance. Using integrative single-cell RNA sequencing, we identified that the upregulation of mitochondrial calcium uniporter (MCU) may contribute to chemoresistance and stemness maintenance in PDAC. MCU was highly expressed in chemotherapy-resistant PDAC tumors and correlated with enhanced cancer stem cell properties. Mechanistically, MCU-mediated mitochondrial Ca 2+ influx triggered endoplasmic reticulum (ER) stress and the downstream PERK-eIF2α pathway. This cascade activated ATF4 and NRF2, which enhanced the transcriptional regulation of PSAT1 and SLC7A11. These changes promoted de novo glutathione (GSH) synthesis to scavenge reactive oxygen species (ROS) and sustain stemness. Genetic knockdown or pharmacological inhibition of MCU disrupted GSH synthesis, suppressed stemness, and restored sensitivity to nab-paclitaxel plus gemcitabine (AG). High-throughput screening identified MCU inhibitor NB-598, which synergized with AG to inhibit tumor growth in preclinical models. These findings offer a potential novel therapeutic strategy to address chemoresistance in PDAC.

Supplementary Table1 shows the senescence genesets

Supplementary Table 2 shows the patient characteristics in the proof-of-concept clinical trial; Supplementary Table 3 shows treatment-related adverse events in the proof-of-concept clinical trial.

Abstract Neoadjuvant therapy (NAT) is becoming an essential component in the management of digestive system malignancies, yet whether the microbiome modulates NAT efficacy remains unclear. Here, we profile the intratumoral microbiome of patients with digestive system cancers following NAT and identify Sphingomonas as markedly enriched in pancreatic ductal adenocarcinoma (PDAC) tumors from responders. Sphingomonas was likewise detected in gastric and colorectal cancers with favourable NAT responses. Using mouse models, we show that Sphingomonas stably colonizes pancreatic, gastric, and colorectal tumors and augments NAT efficacy through secretion of sphingosine. Mechanistically, Sphingomonas-derived sphingosine binds lipid raft structures on the plasma membrane and is preferentially internalized by immune cells, where it selectively activates the ceramide-sphingomyelin synthetic pathway rather than the Sphingosine-1-Phosphate (S1P) pathway. This process amplifies lipid raft organization, promotes immune synapse formation, and enhances immune-mediated tumor cell killing. We further develop a lipid-raft-targeted sphingosine delivery system that boosts immune activation and sensitizes tumors to NAT. Together, these findings uncover a previously unrecognized microbial determinant of NAT responsiveness in digestive system cancers and provide a conceptual and translational framework for microbiome-based patient stratification and therapeutic sensitization. Citation Format: Bin Wang, Xiuchao Wang, Jihui Hao. Sphingomonas-driven sphingosine signalling enhances neoadjuvant therapy responses in digestive system cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4896.

In this phase 2 study (NCT05047991), patients with unresectable metastatic pancreatic adenocarcinoma were randomized to receive NALIRIFOX (liposomal irinotecan, 5-FU, leucovorin, and oxaliplatin) or gemcitabine plus nab-paclitaxel. The primary endpoint was progression free survival (PFS). Secondary endpoints included other efficacy outcomes (overall survival, objective response rate, disease control rate, and duration of response), as well as safety, pharmacokinetic parameters, and evaluation of the relationship between UGT1A1*6 and UGT1A1*28 polymorphisms and safety. A total of 117 patients were enrolled and randomly assigned to NALIRIFOX (n = 78) or gemcitabine plus nab-paclitaxel (n = 39). At a median follow-up of 18.7 months (interquartile range [IQR], 7.5–22.1) for NALIRIFOX and 12.1 months (IQR: 6.4–14.8) for the gemcitabine plus nab-paclitaxel, median PFS was 7.6 months (95

2540 Background: T-cell exhaustion and poor persistence limit tumor infiltrated lymphocytes (TILs) efficacy in solid tumors. GK01 is an autologous tumor-reactive T-cell product enriched for stem cell memory T cells (TSCM: CD45RA + CD62L + ) and diverse T-cell receptor (TCR) clonotypes to promote durable engraftment. We conducted GUARDIAN-01 (NCT06954558), a phase I study of GK01 plus IL-2 in advanced solid tumors. Methods: This single-arm, open-label study enrolled patients with advanced solid tumors (ECOG PS 0-1; measurable disease per RECIST v1.1). Patients received standard lymphodepletion, GK01 (5×10 9 -1×10 11 cells per manufacturing yield), and IL-2 (300,000 IU/kg IV q12h, up to 5 days). Repeat infusion was permitted at investigator discretion based on clinical benefit. Primary endpoint was safety; secondary endpoints included objective response rate (ORR), disease control rate (DCR), and cellular kinetics via absolute lymphocyte count (ALC) and TCR sequencing. Results: From March 2025 to January 2026, 6 patients were enrolled (median age 53.5 years; median 2 prior lines; tumor types included gastric n = 3, pancreatic n = 1, penile SCC n = 1, and melanoma n = 1). Manufacturing succeeded in all patients; median time from tissue procurement to infusion was 28 days (range 25-39). Infused GK01 exhibited median TSCM frequency of 71% (range 42-92%) and demonstrated robust expansion and stemness properties. Upon tumor challenge, these T cells secreted IFN-γ at a median of 1,632 pg/mL (range, 35 - 4,886). Median dose was 2.8×10 10 cells (range 1.4×10 10 -8.8×10 10 ); 2 patients received repeat infusion. The median total IL-2 dose administered was 4 (range 1-5), with the first dose administered approximately 6 hours after GK01 infusion. No dose-limiting toxicities (DLTs) occurred. G3/4 adverse events were exclusively hematologic (neutropenia, thrombocytopenia, leukopenia, lymphopenia in all patients), attributable to lymphodepletion. Chills, fever, and erythroderma occurred in all patients but G1-2, resolving within 2 weeks. At median follow-up of 169 days (range 80-297), ORR was 66.7% (4/6 PR; 2/6 SD), and DCR was 100%. The median peak ALC reached at 11.3×10 9 /L (range 4.9-22.7) at days 7-9 post-infusion, remained elevated at 3.1×10 9 /L (range 2.0-6.4) at 1 month. Among patients with available peripheral-blood samples (n = 4), product-derived TCR clonotypes comprised 94% of the circulating repertoire at day 7 and 92% at 2 months. Conclusions: In this first-in-human study, GK01 plus IL-2 demonstrated a favorable safety profile with no DLTs and manageable toxicity. The TSCM-enriched product achieved a 67% ORR and 100% DCR, with product-derived clonotypes persisting at > 90% of the T-cell repertoire at 2 months. These findings validate the stemness-enriched T-cell platform and support expansion cohorts in selected solid tumor indications. Clinical trial information: NCT06954558 .

Tumor-associated macrophages (TAMs) are pivotal in the immunosuppressive tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC). The efficacy of targeting the CSF-1/CSF-1R axis in PDAC remains uncertain. Using single-cell RNA sequencing on specimens from patients treated with Surufatinib plus chemotherapy, we identified a distinct subset of damage-associated macrophages (DAMs) characterized by high GPR34 expression. In Gpr34ΔLyz2 mouse models and in vitro co-cultures, GPR34+ macrophages responded to tissue damage by releasing lysophosphatidylserine (LysoPS), which enhanced CXCL16 secretion and efferocytosis. This efferocytosis promoted MHC-I degradation via the macrophage lysosomal pathway, leading to CD8+ T cell exhaustion. Combining a GPR34 antagonist with chemotherapy and surufatinib significantly enhanced anti-tumor responses in preclinical models. These findings identify GPR34 as a promising immune therapeutic target.

This document provides detailed descriptions of patient recruitment procedures for the case-control cohort, methods for genomic feature extraction, model construction and training, statistical analyses, feature importance assessment, and both in silico and experimental evaluations of the limit of detection. It also includes the design of the prospective screening cohort and comprehensive clinical information on the pancreatic cancer–related cases identified therein.

This file contains supplementary figures illustrating the development and validation of the cfDNA-based pancreatic cancer detection model, including prediction score distributions across clinical subgroups, feature importance analyses, assessments of the limit of detection, and estimated screening benefits. It also includes representative medical imaging and pathological results of the pancreatic cancer related-cases identified in the prospective cohort.

Supplementary Table 4 shows primers and probes used in experiments.

This file includes supplementary tables summarizing cohort demographics, in silico and empirical estimates of the limit of detection (LOD), model performance across clinical and demographic subgroups, results from the non-pancreatic cancer disease cohort and prospective screening cohort, and simulated clinical impact in hypothetical populations.

Pancreatic cancer remains a highly lethal malignancy due to late-stage diagnosis and limited therapeutic options. This study presents the development and validation of a noninvasive circulating cell-free DNA (cfDNA)-based model for early pancreatic cancer detection. In a case-control study comprising 232 patients with pancreatic cancer and 235 healthy controls, the model demonstrated high diagnostic accuracy (AUC = 0.9799 in training; 0.9622 in validation). A prospective cohort study involving 1,926 individuals with diabetes and obesity established risk factors for pancreatic cancer and further assessed its clinical applicability. The model detected 75% of pancreatic cancer cases, including all stage 0 patients, with a lead time of up to 298 days, significantly outperforming CA19-9. Additionally, it demonstrates potential for distinguishing high-risk from low-risk pancreatic cysts, thereby facilitating more precise risk stratification. This study highlights the potential of cfDNA-based screening as a scalable, noninvasive tool for early pancreatic cancer detection, warranting further large-scale clinical validation to enhance patient outcomes. SIGNIFICANCE:This study develops a cfDNA-based model for early pancreatic cancer detection, demonstrating high accuracy and prospective clinical validation. By enabling presymptomatic identification and risk stratification, this noninvasive approach enhances early intervention and improves outcomes, supporting potential clinical applicability and representing a meaningful step toward improving pancreatic cancer screening and management. See related commentary by Tsui and Lo, p. 10.