Early metastasis is the primary factor in the very poor prognosis of pancreatic ductal adenocarcinoma (PDAC), with liver metastasis being the most common form of distant metastasis in PDAC. To investigate the mechanism of PDAC liver metastasis, we found that PDAC cells can promote the formation of pre-metastatic niches (PMNs) through exosomes to facilitate liver metastasis in the early stage. In our study, hepatic stellate cells (HSCs) were treated with PDAC-derived exosomes (PDAC-exo), and the activation of HSCs was detected. A novel transfer RNA-derived fragment, the tRF-GluCTC-0005 was obtained by small RNA sequencing from serum exosomes of PDAC patients. Bioinformatics analysis and RNA pull-down assays revealed the interaction between WDR1 and tRF-GluCTC-0005. A KPC transgenic mouse model and an AAV-mediated sh-WDR1 mouse model were used to detect the mechanism of liver metastasis in vivo. Finally, the dual luciferase reporter assay, protein mutation truncation assay, Co-IP assay, and flow cytometry assay were used to explore the molecular mechanism in HSCs activation and PMNs formation. We found that the tRF-GluCTC-0005 in exosomes binds to the 3’ untranslated region of the mRNA of the WDRl in HSCs and increases mRNA stability. The N-terminals of WDR1 bind to the YAP protein directly, inhibit YAP phosphorylation, and promote the expression of YAP transcription factors. The tRF-GluCTC-0005 in PDAC-exo significantly recruits myeloid-derived suppressor cells (MDSCs) in the liver, creating a PMNs immunosuppressive microenvironment and further advancing liver metastasis from PDAC. Our results suggest that the key of PDAC liver metastasis is the activation of HSCs through upregulation of WDR1 by tRF-GluCTC-0005 in exosomes, which mediates the infiltration of MDSCs to form PMNs.

INTRODUCTION: The ability of carbohydrate antigen 19-9 (CA19-9) to differentiate pancreatic cancer from other benign pancreatic lesions is unsatisfactory. This study explored the diagnostic value of KRAS gene mutations and plasma circulating tumor DNA (ctDNA) in patients with pancreatic cancer. METHODS: The prospective cohort study comprised 149 consecutive patients with solid pancreatic lesions who underwent endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). KRAS subtype mutations were analyzed by digital droplet PCR (ddPCR) in EUS-FNA histopathology tissue samples, and blood samples were sent for plasma ctDNA analysis. The final diagnosis was based on surgical resection pathology or follow-up for at least 2 years. RESULTS: Adding KRAS mutation ddPCR increased the sensitivity and accuracy of EUS-FNA from 71.4% to 91.6% (P < 0.001) and 75.8% to 88.6% (P < 0.001), respectively. By comparison, the sensitivities of circulating biomarkers ctDNA and CA19-9 were only 35.2% and 71.2%. The area under the curve of the receiver operating characteristic curve (AUC) of EUS-FNA and KRAS ddPCR combination was >0.90 for distinguishing pancreatic cancer from benign lesions, whereas the AUC of EUS-FNA and CA19-9 combination was 0.83. The median survival time was significantly shorter in patients with G12D KRAS mutations than that in patients with other mutations (180 vs 240 days, P < 0.001). DISCUSSION: FNA tissue sample KRAS mutation analysis in tissues significantly improves the diagnostic accuracy of cyto/histopathological evaluation in EUS-FNA samples. The combination of EUS-FNA and tissue sample KRAS ddPCR provided a more accurate method for pancreatic cancer diagnosis, superior to the combination of EUS-FNA and CA19-9/ctDNA. G12D KRAS mutations in pancreatic cancer were independently associated with poor overall survival.

Cancer-associated fibroblasts (CAFs) represent a major contributor to tumor growth. Cellular senescence is a state of cell-cycle arrest characterized by a pro-inflammatory phenotype. The potential impact of CAF senescence on tumor progression and the tumor microenvironment (TME) remains to be elucidated. Here, we systematically investigated the relationship between CAF senescence and the TME of pancreatic ductal adenocarcinoma (PDAC) based on multi-omics analysis and functional experiments. CAF senescence promotes tumor progression in vitro and in vivo and contributes to the formation of immunosuppressive TME. A CAF-senescence-related risk score was developed to predict overall survival, immune landscape, and treatment sensitivity in patients with PDAC. Further experiments revealed that plasminogen activator urokinase (PLAU) derived from senescent CAFs (SCAFs) promoted PDAC progression and was involved in immunosuppression. Together, these findings suggested that CAF senescence was correlated with tumor progression, and the CAF-senescence-based machine learning model could potentially predict prognosis in patients with PDAC.

Liquid-liquid phase separation (LLPS) enhances oncogenic signaling pathways and advances cancer progression, and has been proposed as a promising cancer biomarker and intervention target. Nevertheless, doubts remain about the prognostic importance of LLPS-related long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC). An LLPS-related lncRNA prognostic signature was generated by drivers and regulators of LLPS, and was validated in external datasets. The underlying genetic changes and functional enrichment of the signature were assessed. The drug sensitivity and response to immunotherapy were predicted in patients categorized as high-risk and low-risk. Clinical samples, phase separation agonist, and dispersant were used to identify lncRNAs with the most significant expression change. Cancer cells with ZNF32-AS2 expression regulation were subjected to colony formation assay, scratch test assay, migration and invasion assay, sorafenib resistance assay, and xenograft tumor model. The signature of LLPS-related hub lncRNAs identified through Weighted Gene Co-Expression Network Analysis showed outstanding performance in training and external validation cohorts consistently, and the molecular characteristics varied between different risk groups. Potential drugs for high-risk individuals were identified, and low-risk individuals demonstrated a more favorable reaction to immunotherapy. ZNF32-AS2 showed the most significant expression change in phase separation agonist and dispersant treatment. ZNF32-AS2 promoted the proliferation, mobility, and sorafenib resistance of liver cancer cells. The LLPS-related lncRNA signature may help assess prognosis and predict treatment efficacy in clinical settings. LLPS-related ZNF32-AS2 promoted the proliferation, mobility, and sorafenib resistance of liver cancer cells, and may be a novel potential biomarker in hepatocellular carcinoma.

Background: Mediastinal cystic lesions account for approximately 15–20% of all mediastinal masses and are difficult to differentiate because of similar imaging manifestation. The aim of this study was to differentiate mediastinum cystic lesions through endoscopic ultrasound‐guided‐fine needle aspiration (EUS‐FNA) and parameters from cyst‐fluid analysis.Methods: Over a period of eight years, 37 patients suspected with mediastinal cystic lesions were assessed. Cyst fluid was collected via EUS‐FNA and further examined using cytological and biochemical techniques. Definitive diagnosis was established based on cytology, surgical pathology, and/or clinical follow‐up.Results: Based on the final pathological reports or long‐term follow‐up, 19 patients were diagnosed with benign cysts, 14 with benign or malignant tumors, 2 with tuberculosis, 1 with an abscess, and 1 with a pancreatic pseudocyst. Computed tomography or magnetic resonance imaging mistakenly distinguished eight cases as solid masses (27.03%), but EUS revealed cystic characteristics. Carcinoembryonic antigen and lactate dehydrogenase (LDH) were evaluated from the cyst fluid obtained by EUS‐FNA. There was no statistically significant difference in carcinoembryonic antigen values between benign and malignant cysts; however the average LDH value in the malignancy group was significantly higher than in the benign group.Conclusion: EUS‐FNA showed great potential for differentiating mediastinal lesions by combining imaging manifestation and cytological examination. The elevated LDH value from cyst fluid chemical analysis could be used as an auxiliary indicator for diagnosing malignancy.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic and immunosuppressive tumor microenvironment (TME), limiting the efficacy of immune checkpoint inhibitors such as anti-programmed cell death 1 (PD-1). Methods: This study aimed to evaluate the therapeutic potential of Y126S, a recombinant IgG1/IgG2 hybrid bispecific antibody (BsAb), in reshaping the immunotherapy-resistant TME in PDAC. Orthotopic PDAC and KPC (Kras ^LSL-G12D/+ ; Trp53 ^LSL-R172H/+ ; Pdx1-Cre) mouse models were established and treated with Y126S, α-connective tissue growth factor (CTGF), α-PD-1, or a combination of α-CTGF and α-PD-1. TME remodeling, antibody distribution, and therapeutic efficacy were assessed using flow cytometry, immunohistochemical/Masson staining, atomic force microscopy, positron emission tomography (PET) imaging, distribution analysis, and other experimental techniques. Results: Here, Y126S was characterized in vitro and its antitumor efficacy was evaluated and validated in orthotopic PDAC mice and KPC mouse models. Notably, Y126S significantly remodeled the TME and demonstrated superior tumor-specific accumulation compared with single α-PD-1 treatment, leading to markedly enhanced antitumor efficacy relative to its parental antibodies or their combination. Mechanistically, Y126S suppressed cancer-associated fibroblasts (CAFs) activation, reduced collagen deposition, and downregulated programmed cell death ligand 1 (PD-L1) expression on CAFs by targeting CTGF and enhanced the anti-PD-1-mediated reinvigoration of cytotoxic CD8 ⁺ T cells, thereby establishing a less desmoplastic and potent tumor-killing microenvironment. Conclusions: Our findings highlight the potential of Y126S as a promising BsAb-based immunotherapy strategy for PDAC by remodeling the desmoplastic and immunosuppressive TME.