BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterised by remarkable cellular heterogeneity, which emerges early from the interplay of oncogenic KRAS signalling and inflammatory injury. However, the transcriptional, metabolic and functional properties of these pre-malignant cell states that initiate and drive PDAC progression remain elusive. OBJECTIVE:This study aimed to identify and functionally characterise the critical premalignant cell states that arise from this heterogeneity, to define novel biomarkers and targets for early intervention. DESIGN:Public and in-house scRNA-seq data of pancreatic tumour models were analysed to identify key subpopulations in early cellular heterogeneity. Genetic perturbation in KrasG12D-driven models was performed to assess functional impact. Mechanistic studies used TurboID proximity proteomics, epigenetic profiling and metabolic assays. Clinical relevance was validated in human PDAC cohorts. RESULTS:We identified LY6D as a marker of a distinct, gastric-like cell state that emerges early and persists throughout tumourigenesis. The LY6D+ population exhibits conserved stemness and a unique, pan-stage dependency on oxidative phosphorylation (OXPHOS). Genetic ablation of Ly6d specifically impaired the gastric lineage and delayed tumourigenesis, while its overexpression enhanced tumourigenic and metastatic potential. Mechanistically, the glycosylphosphatidylinositol (GPI)-anchored LY6D protein scaffolds a lipid raft-associated kinase network that drives FOSL1-dependent epigenetic-transcriptional reprogramming. In human PDAC, LY6D+ cells harbour stemness and Epithelial-Mesenchymal Transition (EMT) signatures, and high LY6D expression is an independent prognostic marker of poor survival. CONCLUSION:Our work defines the LY6D+ gastric-like cell state as a key driver linking early pre-malignant heterogeneity to PDAC initiation and progression. LY6D represents a pan-stage therapeutic target and a candidate biomarker for early detection and therapeutic targeting.

Understanding the spatial proteomic landscape of human tumors is essential for dissecting cellular heterogeneity and microenvironmental interactions in cancer biology. Traditional bulk proteomic approaches, however, obscure spatial information and average out signals from distinct cell populations. Here, we present a detailed and reproducible micro-quantitative protocol for spatially resolved proteomic analysis of specific cellular subpopulations isolated from immunohistochemistry (IHC)-labeled formalin-fixed paraffin-embedded (FFPE) tissue sections using laser microdissection (LMD). By combining IHC staining to visually define phenotypically distinct cells within preserved tissue architecture and precise LMD capture, approximately 6000 target cells can be isolated per sample for downstream proteomic quantification. Despite the ultra-low input, optimized lysis and digestion steps ensure consistent peptide recovery and highly reproducible label-free LC-MS/MS data across replicates. Integrating immunohistochemistry staining-guided spatial sampling with ultrasensitive quantitative proteomics, this workflow enables reliable cell-type-specific profiling directly within human tumor tissues. The protocol bridges histopathology and proteomics, offering a practical framework for translational research exploring spatial protein signatures and tumor microenvironmental heterogeneity.

Pancreatic ductal adenocarcinoma (PDAC) relies heavily on glutamine (Gln) utilization to meet its metabolic and biosynthetic needs. How epigenetic regulators contribute to the metabolic flexibility and PDAC's response and adaptation to Gln scarcity in the tumor milieu remains largely unknown. Here, we elucidate that prolonged Gln restriction or treatment with the Gln antagonist, 6-diazo-5-oxo-L-norleucine (DON), leads to growth inhibition and ferroptosis program activation in PDAC. A CRISPR-Cas9 screen identifies an epigenetic regulator, Paxip1, which promotes H3K4me3 upregulation and Hmox1 transcription upon DON treatment. Additionally, ferroptosis-related repressors (e.g., Slc7a11 and Gpx4) are increased as an adaptive response, thereby predisposing PDAC cells to ferroptosis upon Gln deprivation. Moreover, DON sensitizes PDAC cells to GPX4 inhibitor-induced ferroptosis, both in vitro and in patient-derived xenografts (PDXs). Taken together, our findings reveal that targeting Gln dependency confers susceptibility to GPX4-dependent ferroptosis via epigenetic remodeling and provides a combination strategy for PDAC therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy driven by oncogenic KRAS and inflammation-induced cellular heterogeneity, yet the mechanisms underlying tumor-initiating cells (TICs) emergence and maintenance remain unclear. Our study establishes LY6D as a marker of persistent TICs that orchestrate pancreatic cancer progression across all disease stages. Single-cell RNA sequencing of inflammation-driven PDAC models reveals that LY6D+ TICs specifically originate from KRAS-mutant acinar-to-ductal metaplasia (ADM) lesions under inflammatory conditions, maintaining conserved stemness properties and exhibiting a distinctive oxidative phosphorylation (OXPHOS) dependency throughout tumor evolution. Moreover, genetic ablation of Ly6d in Kras G12D pancreata delays tumorigenesis, while forced Ly6d expression enhances tumorigenic potential and metastatic capability. Mechanistically, LY6D-despite lacking intracellular domains-scaffolds lipid raft-associated kinase networks and FOSL1-dependent epigenetic reprogramming to establish a ​stable pro-tumorigenic state. Clinically, LY6D+ cells are enriched in human PDAC and exhibit conserved stemness and epithelial-mesenchymal transition (EMT) properties. Strikingly, LY6D expression levels demonstrate PDAC-restricted prognostic power. Our work defines LY6D as pan-stage TICs marker linking cellular plasticity to PDAC initiation and progression, offering new avenues for early detection and interception of this lethal malignancy. Juanjuan Shi, Yingying Tang, Zhengyan Zhang, Xian Wang, Shixin Meng, Ping Lu, Junyi Xu, Zheng Wang, Yongwei Sun, Jing Xue. LY6D identifies persistent tumor-initiating cells driving pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B119.

Pancreas development is tightly controlled by multilayer mechanisms. Despite years of effort, large gaps remain in understanding how histone modifications coordinate pancreas development. SETD2, a predominant histone methyltransferase of H3K36me3, plays a key role in embryonic stem cell differentiation, whose role in organogenesis remains elusive. Here, by combination of cleavage under targets and tagmentation (CUT&Tag), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and bulk RNA sequencing, we show a dramatic increase in the H3K36me3 level from the secondary transition phase and decipher the related transcriptional alteration. Using single -cell RNA sequencing, we define that pancreatic deletion of Setd2 results in abnormalities in both exocrine and endocrine lineages: hyperproliferative tip progenitor cells lead to abnormal differentiation; Ngn3+ endocrine progenitors decline due to the downregulation of Nkx2.2, leading to insufficient endocrine development. Thus, these data identify SETD2 as a crucial player in embryonic pancreas development, providing a clue to understanding the dysregulation of histone modifications in pancreatic disorders.

PURPOSE:Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge due to its high heterogeneity and aggressiveness. Recognizing the urgency to delineate molecular subtypes, our study focused on the emerging field of lipid metabolism remodeling in PDAC, particularly exploring the prognostic potential and molecular classification associated with fatty acid biosynthesis. METHODS:Gene set variation analysis (GSVA) and single-sample gene set enrichment analysis (ssGSEA) were performed to evaluate the dysregulation of lipid metabolism in PDAC. Univariate cox analysis and the LASSO module were used to build a prognostic risk score signature. The distinction of gene expression in different risk groups was explored by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Weighted Gene Co-expression Network Analysis (WGCNA). The biological function of Acyl-CoA Synthetase Long Chain Family Member 5 (ACSL5), a pivotal gene within 7-hub gene signature panel, was validated through in vitro assays. RESULTS:Our study identified a 7-hub gene signature associated with fatty acid biosynthesis-related genes (FRGs), providing a robust tool for prognosis prediction. The high-FRGs score group displayed a poorer prognosis, decreased immune cell infiltration, and a higher tumor mutation burden. Interestingly, this group exhibited enhanced responsiveness to various compounds according to the Genomics of Drug Sensitivity in Cancer (GDSC) database. Notably, ACSL5 was upregulated in PDAC and essential for tumor progression. CONCLUSION:In conclusion, our research defined two novel fatty acid biosynthesis-based subtypes in PDAC, characterized by distinct transcriptional profiles. These subtypes not only served as prognostic indicator, but also offered valuable insights into their metastatic propensity and therapeutic potential.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of cancer-associated fibroblasts (CAFs) as part of the host response to tumor cells. The origins and functions of transcriptionally diverse CAF populations in PDAC remain poorly understood. Tumor cell-intrinsic genetic mutations and epigenetic dysregulation may reshape the TME; however, their impacts on CAF heterogeneity remain elusive. SETD2, a histone H3K36 trimethyl-transferase, functions as a tumor suppressor. Through single-cell RNA sequencing, we identify a lipid-laden CAF subpopulation marked by ABCA8a in Setd2-deficient pancreatic tumors. Our findings reveal that tumor-intrinsic SETD2 loss unleashes BMP2 signaling via ectopic gain of H3K27Ac, leading to CAFs differentiation toward lipid-rich phenotype. Lipid-laden CAFs then enhance tumor progression by providing lipids for mitochondrial oxidative phosphorylation via ABCA8a transporter. Together, our study links CAF heterogeneity to epigenetic dysregulation in tumor cells, highlighting a previously unappreciated metabolic interaction between CAFs and pancreatic tumor cells.

Pancreatic ductal adenocarcinoma (PDAC) is a clinically challenging cancer with a dismal overall prognosis. NSD2 is an H3K36-specific di-methyltransferase that has been reported to play a crucial role in promoting tumorigenesis. Here, the study demonstrates that NSD2 acts as a putative tumor suppressor in Kras-driven pancreatic tumorigenesis. NSD2 restrains the mice from inflammation and Kras-induced ductal metaplasia, while NSD2 loss facilitates pancreatic tumorigenesis. Mechanistically, NSD2-mediated H3K36me2 promotes the expression of IκBα, which inhibits the phosphorylation of p65 and NF-κB nuclear translocation. More importantly, NSD2 interacts with the DNA binding domain of p65, attenuating NF-κB transcriptional activity. Furthermore, inhibition of NF-κB signaling relieves the symptoms of Nsd2-deficient mice and sensitizes Nsd2-null PDAC to gemcitabine. Clinically, NSD2 expression decreased in PDAC patients and negatively correlated to nuclear p65 expression. Together, the study reveals the important tumor suppressor role of NSD2 and multiple mechanisms by which NSD2 suppresses both p65 phosphorylation and downstream transcriptional activity during pancreatic tumorigenesis. This study opens therapeutic opportunities for PDAC patients with NSD2 low/loss by combined treatment with gemcitabine and NF-κBi.

IFNγ signaling is mainly mediated through the activation of the canonical JAK-STAT signaling pathway, transcription factors, and epigenetic modifications. The activation of IFNγ signaling pathway may provide a novel option for tumor immunotherapy, but the outcomes remain controversial. In fact, recent studies suggest that the resistance to IFNγ-dependent immunotherapies is commonly derived from the tumor cell-intrinsic heterogeneity, the molecular mechanism of which remains elusive. Therefore, elucidating the tumor cell-intrinsic heterogeneity in response to IFNγ would be beneficial to improve the efficacy of immunotherapy. Here, we first delineated the epigenetic redistribution and transcriptome alteration in response to IFNγ stimulation, and demonstrated that ectopic gain of H3K4me3 and H3K27Ac at the promoter region mainly contributed to the enhancement of IFNγ-mediated transcriptional activity of interferon-stimulated genes (ISGs). Furthermore, we found that the cellular heterogeneity of PD-L1 expression in response to IFNγ was mainly attributed to cell-intrinsic H3K27me3 levels. Enhancement of H3K27me3 by GSK-J4 limited PD-L1hi tumor growth by salvaging the intratumoral cytotoxicity of CD8+ T cells, which may provide therapeutic strategies to overcome immune escape and resistance to IFNγ-based immunotherapies in pancreatic cancer.

PDF - 922KB, Supplementary Materials and Methods. Supplementary Table S1: Drugs and chemical reagents. Supplementary Table S2: Primers used for Real-time PCR and other sequences. Supplementary Table S3: Antibodies used in this study. Supplementary Figure S1. The expression of ABCG2 was correlated with drug resistance of HCC. Supplementary Figure S2. TM inhibited ABCG2 expression in both HCC and other tumor cell lines. Supplementary Figure S3. Glycosylation inhibitors influenced SP of HCC cells. Supplementary Fig. S4 TM inhibited ABCG2 and p-Akt expression in CD133+/- MHCC-97L cells nude mouse models. Supplementary Figure S5. The anti-tumor effect of chemotherapeutic drugs combined with TM in PLC/PRF/5 cells was evaluated by Western blotting. Supplementary Figure S6. TM enhanced the therapeutic effect of CDDP through Akt-ABCG2 pathway in vivo. Supplementary Figure S6. TM inhibited the glycosylation of EGFR and blocked its downstream signalling. Supplementary Figure S8. The combination treatment effect of sorafenib and TM was evaluated in HCC cell lines.

PDF file - 7K, Sequences of siRNA fragments used in this study

Figure S1.Flow cytometry analysis of IL-22RA1 expression among PDAC; Figure S2. The correlation analyses between IL-22RA1 and CSCs Figure S3. IL-22RA1hi pancreatic cancer cells have CSCs properties Figure S4. IL-22RA1hi pancreatic cancer cells express higher EMT markers FigureS5. IL-22 promotes stemness of pancreatic cancer cells FigureS6. IL-6 activates STAT3, but unable to promote stemness of PDACs Figure S7. Dot1L is not involved in IL-22 induced stemness of PDACs Table S1. Quantitative real-time PCR and ChIP primers TableS2&3. The co-relationship between IL-22RA1 expression and PDAC patients characteristics

BACKGROUND:Innate immunity and metabolites link to the pathogenesis and severity of acute pancreatitis (AP). However, liver metabolism and its role in immune response and AP progression remain elusive. We investigated the function of liver metabolism in the pathogenesis of AP.METHODS:Circulating ketone body β-hydroxybutyrate (βOHB) levels were determined in AP clinical cohorts and caerulein-induced AP (CER-AP) mouse models receiving seven (Cer*7) or twelve (Cer*12) injection regimens at hourly intervals. Liver transcriptomics and metabolomics were compared between CER-AP (Cer*7) and CER-AP (Cer*12). Inhibition of fatty acid β-oxidation (FAO)-ketogenesis, or supplementation of βOHB was performed in mouse models of AP. The effect and mechanism of βOHB were examined in vitro.FINDINGS:Elevated circulating βOHB was observed in patients with non-severe AP (SAP) but not SAP. These findings were replicated in CER-AP (Cer*7) and CER-AP (Cer*12), which manifested as limited and hyperactive immune responses, respectively. FAO-ketogenesis was activated in CER-AP (Cer*7), while impaired long-chain FAO and mitochondrial function were observed in the liver of CER-AP (Cer*12). Blockage of FAO-ketogenesis (Cpt1a antagonism or Hmgcs2 knockdown) worsened, while supplementation of βOHB or its precursor 1,3-butanediol alleviated the severity of CER-AP. Mechanistically, βOHB had a discernible effect on pancreatic acinar cell damage, instead, it greatly attenuated the activation of pancreatic and systemic proinflammatory macrophages via class I histone deacetylases.INTERPRETATION:Our findings reveal that hepatic ketogenesis is activated as an endogenous protective programme to restrain AP progression, indicating its potential therapeutic value.FUNDING:This work was supported by the National Natural Science Foundation of China, Shanghai Youth Talent Support Programme, and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant.

Owing to the lack of early diagnosis, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal tumours. Because acinar-to-ductal metaplasia (ADM) is a critical process to pancreatic regeneration and PDAC initiation, we applied GSE65146, a dataset composed of transcripts at different time points in wild-type and KrasG12D mutant mice upon pancreatitis induction, to obtain regeneration- and tumour initiation-related genes. By overlapping with genes differentially expressed in human PDAC, we defined the initiation- and progression-related genes, and the most prognostic gene, SULF2, was selected for further verification. By using multiple PDAC genetically engineered murine models (GEMMs), we further verified that the expression of SULF2 was increased at the ADM and PDAC stages. Functionally, SULF2 was able to promote the dedifferentiation of acinar cells as well as the metastatic ability of PDAC. Additionally, our study revealed that SULF2 could enhance TGFβ-SMAD signalling via GDF15. More importantly, serum SULF2 was elevated in patients with PDAC, and in combination with CA19-9, it provided a better method for PDAC diagnosis. Herein, our study screened out key genes for the initiation and progression of PDAC, providing potential indicators for the diagnosis of the disease.

BACKGROUND: Circular RNA (circRNA) is considered to be an important regulator of human diseases, including atherosclerosis (AS). However, the role of circ_ARHGAP32 in AS formation needs further confirmation. OBJECTIVE: To explore the role of circ_ARHGAP32 in AS formation. METHODS: Oxidized low density lipoprotein (ox-LDL) was used to treat vascular smooth muscle cells (VSMCs) to mimic AS cell models in vitro. The expression of circ_ARHGAP32, microRNA (miR)-665, and fibroblast growth factor 2 (FGF2) was analyzed by quantitative real-time PCR. VSMCs function was measured by EdU assay, cell counting kit 8 assay and transwell assay. Protein expression was determined using western blot analysis. Dual-luciferase reporter assay and RNA pull-down assay were performed to verify RNA interaction. RESULTS: Circ_ARHGAP32 was highly expressed in AS patients and ox-LDL-induced VSMCs. Knockdown of circ_ARHGAP32 repressed ox-LDL-induced proliferation and migration in VSMCs. Circ_ARHGAP32 sponged miR-665 to positively regulate FGF2. MiR-665 inhibitor reversed the regulation of sh-circ_ARHGAP32 on ox-LDL-induced VSMCs proliferation and migration. MiR-665 also had a suppressive effect on the proliferation and migration of ox-LDL-induced VSMCs, and this effect could be reversed by FGF2 overexpression. CONCLUSIONS: Circ_ARHGAP32 might be a potential target for AS treatment, which promoted ox-LDL-induced VSMCs proliferation and migration by regulating miR-665/FGF2 network.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy driven by genetic mutations and/or epigenetic dysregulation. Gemcitabine chemotherapy is the first-line regimen for pancreatic cancer but has limited efficacy. Our previous study revealed the role of SETD2-H3K36me3 loss in the initiation and metastasis of PDAC, but little is known about its role in tumor metabolism. Here, we found that SETD2-deficient PDAC enhanced glycolysis addiction via upregulation of glucose transporter 1 (GLUT1) to meet its large demand for glucose in progression. Moreover, SETD2 deficiency impaired nucleoside synthesis by directly downregulating the transcriptional level of transketolase (TKT) in the pentose phosphate pathway. The metabolic changes confer SETD2-deficient PDAC cells with increased sensitivity to gemcitabine under glycolysis restriction conditions. Collectively, our study provides mechanistic insights into how SETD2 deficiency reprograms glycolytic metabolism to compensate for insufficient nucleoside synthesis, suggesting that glycolysis restriction combined with gemcitabine might be a potential therapeutic strategy for PDAC patients with SETD2 deficiency.

Genetic and epigenetic alterations play central roles in shaping the immunosuppressive tumor microenvironment (TME) to evade immune surveillance. The previous study shows that SETD2-H3K36me3 loss promotes KRAS-induced pancreatic tumorigenesis. However, little is known about its role in remodeling the TME and immune evasion. Here, it is shown that SETD2 deficiency can reprogram neutrophils to an immunosuppressive phenotype, thereby promoting immune escape during pancreatic tumor progression. By comprehensive profiling of the intratumoral immune cells, neutrophils are identified as the subset with the most significant changes upon Setd2 loss. Setd2-deficient pancreatic tumor cells directly enhance neutrophil recruitment and reprogramming, thereby inhibiting the cytotoxicity of CD8+ T cells to foster tumor progression. Mechanistically, it is revealed that Setd2-H3K36me3 loss leads to ectopic gain of H3K27me3 to downregulate Cxadr expression, which boosts the PI3K-AKT pathway and excessive expression of CXCL1 and GM-CSF, thereby promoting neutrophil recruitment and reprogramming toward an immunosuppressive phenotype. The study provides mechanistic insights into how tumor cell-intrinsic Setd2 deficiency strengthens the immune escape during pancreatic tumorigenesis, which may offer potential therapeutic implications for pancreatic cancer patients with SETD2 deficiency.

Perineural invasion (PNI) is a common feature of pancreatic ductal adenocarcinoma (PDAC) and is one of the important causes of local recurrence in resected pancreatic cancer, but the molecular mechanism remains largely unexplored. Here, we used immunohistochemistry staining to determine the expression of CD74. Then the in vivo PNI model, in vitro neuroplasticity assay, cell proliferation assay, wound healing and Transwell-based invasion assay were performed to examine the function of CD74 in pancreatic cancer cell lines. ChIP assay and Luciferase reporter assay were used to illustrate the mechanism underlying CD74 induced GDNF expression. We confirmed that the expression level of CD74 was an independent predictor of PNI and poor prognosis for PDAC. Moreover, we found that upregulation of CD74 on PDAC enhanced its migration and invasive capabilities and potentiated the secretion of neurotrophic factor GDNF to promote the neuroplasticity. Mechanistically, CD74 promoted GDNF production via the AKT/EGR-1/GDNF axis in PDAC. Taken together, our findings suggest a supportive role of CD74 in the PNI of PDAC, and deepen our understanding of how cancer cells promote neuroplasticity in the microenvironment of PDAC.