Phosphorylated nucleosides and their derivatives sit at the center of cellular energetics, signaling, and biosynthesis, yet they are largely invisible to routine reversed-phase metabolomics. Their phosphate groups chelate metal ions, producing severe peak tailing and chromatographic irreproducibility that consign them to the "dark metabolome." We report nucleotidomics, a workflow that recovers this phosphorylated fraction on standard reversed-phase LC-MS. Each sample is split in two; one aliquot is analyzed as-is, the other treated with a nucleoside digestion mix that strips enzyme-accessible phosphate groups, returning phosphorylated forms to their parent nucleosides. Subtracting the untreated signal from the treated one cancels the pre-existing free nucleoside pool common to both aliquots, leaving ∑P: the summed abundance of the phosphorylated forms of each nucleoside. ∑P is therefore an indirect, semi-quantitative readout — it flags phosphate-bearing pools but cannot resolve mono-, di-, and triphosphates. Seven-minute analytical cycles keep total instrument time practical despite the doubled injection count. In two gemcitabine-treated pancreatic ductal adenocarcinoma cell lines, the paired injections returned 1071 putatively annotated metabolites and 23 endogenous phosphorylated species surviving manual curation. The method recapitulated the drug's established pharmacology: extensive phosphorylation of gemcitabine, formation of the deaminated metabolite dFdU and its phosphates, and depletion of the deoxyadenosine and deoxyguanosine phosphate pools, consistent with ribonucleotide reductase inhibition. Untargeted analysis revealed deoxyinosine depletion, likely reflecting reduced deoxyadenosine pools. A high-sensitivity fragmentation protocol with extended ion accumulation (sniperMS2) yielded candidate identifications of trace species: free queuosine phosphates and endogenous 5′-methylthioinosine, to our knowledge not previously measured directly in human cells. ∑P alone gave tentative evidence for GDP-fucose phosphorylation. By rendering the nucleotide constituent of the dark metabolome accessible, nucleotidomics extends routine metabolomic workflows to the cellular nucleotidome without the need for specialized chromatography.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related death. Although molecular stratification and multimodal therapy have improved outcomes in selected patients, overall prognosis is still limited by late diagnosis, heterogeneity, and treatment resistance. Immune checkpoint inhibitors (ICIs) have substantially improved survival outcomes in a subset of patients; however, the overall benefit remains limited, and both primary and acquired resistance are common. Neutrophils, as key effectors of innate immune responses, can be activated by diverse stimuli and release neutrophil extracellular traps (NETs). Growing evidence indicates that neutrophils and NETs contribute to remodeling of the tumor microenvironment (TME) in NSCLC, promoting resistance to ICIs. This review systematically summarizes the biological features, key molecular pathways, and inducing factors of neutrophils and NETs in lung cancer and synthesizes evidence supporting their roles as biomarkers of ICI efficacy and prognosis. We further focus on the mechanisms by which NETs mediate immunosuppression and foster an immune-excluded TME, thereby driving resistance to immunotherapy. In addition, we outline potential therapeutic and combination strategies targeting neutrophils and NETs, providing a theoretical basis for developing optimized immunotherapy approaches for NSCLC that target neutrophils and NETs.

Pancreatic ductal adenocarcinoma (PDAC) continues to rank among the most lethal malignancies, with five-year survival rates stubbornly below 12

Robotic technology has enabled the execution of complex surgical procedures through a fully minimally invasive approach. Among these procedures, robotic pancreatoduodenectomy (R-PD) remains particularly challenging due to its duration and the numerous steps involved. The authors present a standardized approach designed as a reproducible framework potentially applicable to the majority of standard R-PD techniques aimed at enhancing operative workflow. The technique is based on a stepwise model for the resection phase, performed in a counterclockwise sequence that provides a logical and reproducible pathway through the complex anatomy of the pancreatic head. The reconstruction phase follows a second counterclockwise-inspired sequence, relying on standardized anastomotic techniques designed to improve reproducibility and operative flow. Between May 2018 and November 2025, 150 consecutive R-PDs were performed in our General Surgery Unit. The mean console time was 279.44 ± 55.85 min. All the procedures were completed using a fully robotic approach, without conversion to open surgery. The median hospital stay was 12 days (range, 9–23 days). Major complications (Clavien-Dindo ≥3) occurred for 20 patients (13.3

Pancreatic ductal adenocarcinoma remains a formidable clinical challenge, with a very poor survival rate. It is highly metastatic and quickly exhibits chemoresistance, both of which are mediated by interactions of the tumor cells with their microenvironment. Extracellular vesicles are key mediators by which tumor cells interact with their microenvironment. Here, we utilized a data-independent acquisition proteomics workflow to characterize the protein cargo of extracellular vesicles isolated from patient-derived primary cells and cell lines of varying aggressiveness. Mapping 1432 unique proteins with high reproducibility, we identified protein signatures that robustly discriminated between short-term and long-term survival phenotypes. While the data set was highly enriched for established exosomal markers, differential expression and PLS-DA modeling revealed that the most significant indicators of short survival (high aggressiveness) were ribosomal proteins. STRING analysis confirmed these as highly connected interactome hubs. These findings suggest that the differential loading of translational machinery into the extracellular space─likely via microvesicles or coisolated nonvesicular particles─contributes to poor prognosis, offering new insights into the systemic secretome of aggressive tumors.

Resistance to therapy is a major driver of the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC), which is projected to become the second leading cause of cancer-related death by 2030. The lack of reliable biomarkers for early detection and the rapid emergence of therapeutic resistance underscore an urgent need for strategies capable of improving patient stratification and overcoming treatment failure. In this context, the receptor tyrosine kinase AXL has gained increasing attention as a mediator of PDAC progression, sustaining tumor cell survival and plasticity while fostering an immunosuppressive, fibrotic microenvironment, that collectively fuels resistance to both cytotoxic and targeted therapies. In this review, we critically evaluate the therapeutic landscape of AXL inhibition in PDAC. We discuss the spectrum of pharmacological approaches under investigation, including small-molecule inhibitors, monoclonal antibodies, and ligand-blocking agents, alongside emerging modalities such as antibody-drug conjugates, bispecific formats, and CAR-T cell platforms, emphasizing both their promise and the persistent barriers of limited tumor penetration and stromal resistance. Furthermore, we highlight the growing interest in AXL as a circulating biomarker, with potential applications in early patient selection, dynamic treatment monitoring, and prediction of resistance. By integrating mechanistic insights with translational advances, this review outlines both the opportunities and the barriers that will shape the future of AXL-directed strategies in PDAC and their potential to address current gaps in therapeutic resistance.

Epidermal growth factor receptor (EGFR) mutations are an oncogenic driver in non-small cell lung cancer (NSCLC), but resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a major challenge. Here, we show that mycoplasma infection sustains ERK, AKT, and NF-κB signaling; impairs receptor internalization; and reduces osimertinib sensitivity in EGFR-mutant NSCLC cells. Antibiotic-mediated clearance of Mycoplasma partially restored drug sensitivity, establishing a causal infection-resistance link. Transcriptomic analysis identified converging pathways related to mycoplasma infection and drug resistance, including gene sets enriched for inhibition of receptor internalization and clathrin downregulation at the protein level. Combined inhibition of mTOR and LDHA improved growth suppression in mycoplasma-positive cells. Clinically, mycoplasma infection and higher LDHA expression were associated with poor overall survival of EGFR-TKI-treated patients. These findings establish mycoplasma infection as a causal, non-genetic contributor to EGFR-TKI resistance and support a hierarchical therapeutic strategy combining antibiotic eradication with mTOR and LDHA inhibition.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with a 5-year survival rate of approximately 12%. Treatment options, including surgical resection and chemotherapy, are often ineffective in advanced stages due to late detection. This review examines the role of Erythropoietin-producing human hepatocellular (Eph) receptors in PDAC, emphasizing their potential both as therapeutic targets and biomarkers. Eph receptors, a family of receptor tyrosine kinases, are frequently overexpressed in various cancers and contribute to tumor growth, angiogenesis, and metastasis. Among the molecular drivers implicated in PDAC, EphA2, EphA4, EphA10, and EphB4, have emerged as key players in tumor progression, metastasis, resistance to therapy and poor prognosis. Eph receptor fragments show potential as early detection and prognostic biomarkers, potentially offering higher sensitivity than traditional markers such as CA 19-9. Additionally, emerging therapies targeting these receptors, such as EphA2-directed drug conjugates and small-molecule inhibitors, show promising results in preclinical models. Despite these advancements, their clinical translation remains limited, because of low specificity, poor delivery, and lack of validation in large patient cohorts. This review underscores the significance of Eph receptors in PDAC biology and highlights their dual utility as biomarkers and therapeutic targets, emphasizing the need for in depth investigation to bridge the gap between experimental promise and clinical reality. Harnessing Eph receptor biology holds the potential to improve early diagnosis and open new avenues for precision therapy and better treatment outcomes in one of the deadliest cancers.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterised by profound immune dysfunction and limited response to immunotherapy. Although tertiary lymphoid structures (TLSs) are associated with improved outcomes, most studies focus on their presence or density, providing limited insight into how their organisation contributes to tumour control. Here, we analyse a unique cohort of treatment-naïve PDAC patients enriched for exceptionally rare long-term survivors (LTS, n = 23; >5-year survival) alongside more common short-term survivors (STS, n = 24), enabling direct interrogation of mechanisms underlying durable tumour control. We develop a multi-scale spatial framework integrating multiplex imaging, computational modelling, and transcriptomics to quantify TLS architecture. By modelling TLSs as higher-order immune assemblies, we define their structural states, spatial organisation, assembly rules and tissue context-dependency within the tumour microenvironment. We show that long-term survival is associated with organised, spatially integrated TLSs exhibiting coordinated B and T cell zoning, whereas short-term survival is characterised by disorganised, Treg-enriched, and spatially isolated TLSs, despite similar immune cell abundance. These architectural differences align with transcriptional programmes: LTS tumours display coordinated lymphoid and NF-κB–driven chemokine signalling, while STS tumours exhibit inflammation uncoupled from immune organisation. Together, these findings demonstrate that durable anti-tumour immunity in PDAC is defined by coordinated transcriptional, cellular, and spatial organisation, rather than immune presence alone. This study provides a blueprint for structure-informed strategies to reprogram the tumour microenvironment and improve outcomes in PDAC. Statement of significance Integrating multiplex imaging, computational modelling and transcriptomics in treatment-naïve PDAC enriched for exceptional survivors shows that durable tumour control is associated with organised TLS architecture rather than immune abundance alone, providing a framework for immune-architecture-based biomarkers and therapeutic reprogramming.

Oxysterols (OS) are emerging as active drivers of cancer, shaping aggressive, immunosuppressive tumour niches rather than acting as passive cholesterol byproducts. Here, we developed and applied an advanced LC-MS strategy for the comprehensive qualitative and quantitative profiling of the total (free and esterified) OS amount in healthy human pancreatic epithelial nestin-expressing (HPNE) cells and pancreatic ductal adenocarcinoma (PANC-1) cells. Both cell types were cultured under normoxic and hypoxic conditions to elucidate the impact of oxygen availability on OS metabolism. OS separation was achieved using two complementary stationary phases, i.e., cyanopropyl (ES-CN) and pentafluorophenyl (F5), providing orthogonal chromatographic selectivity. OS identification relied on retention matching with analytical standards across both columns and was further confirmed by the evaluation of diagnostic in-source fragmentation patterns. This multidimensional analytical framework ensured the unambiguous screening of up to 14 OS in cells and their extracellular vesicles (EVs). The tumour cell phenotype, together with hypoxic conditions, was associated with a marked accumulation of ring-oxidized OS, including 3β,5α,6β-cholestantriol, 7-ketocholesterol, and 7-hydroxycholesterol epimers, suggesting either a metabolic rewiring or an increased intracellular oxidative environment. In contrast, side-chain OS showed distinct dependencies on cell type and oxygen availability: 24(S)-hydroxycholesterol was reduced in tumour cells while the amount of 26(R/S)-hydroxycholesterol was increased when both cell types were grown in normoxic conditions. EVs exhibited a unique OS signature dominated by 25-hydroxycholesterol and 5β,6β-epoxycholesterol. This study proposes a robust, orthogonal LC-MS workflow for OS profiling and provides new insights into the regulation of OS levels in pancreatic cancer, paving the way for future fundamental biochemical investigations.

Abstract Cancer is the second leading cause of death worldwide and causes nearly 9.6 million deaths each year according to the WHO. Although medical technology and screening programs continue to improve, delayed diagnosis remains a major challenge. Artificial intelligence (AI) offers a promising approach for cancer detection and risk prediction. This study developed an AI-based platform that integrates gene panel data, traditional risk factors, and healthcare professional assessments to identify individuals at high cancer risk. The system applied machine learning models including random forest, support vector machines, convolutional neural networks (CNN), deep learning algorithms, and Natural Language Processing to analyze complex associations between genetic variants and clinical risk factors. The platform used Python programming with Django, MySQL, ChartJS, JSCharting, HTML, CSS, and JavaScript frameworks. Developers created independent front-end and back-end modules and managed version control with Git. Researchers evaluated the platform in 514,506 cases across 39 multicenter units. The deep learning engine analyzed medical data and risk factors using CNN, support vector machines, and random forest classifiers. The platform also integrated ECG and mammography analysis with general health screening to provide comprehensive risk assessment. Two CNN models supported colon cancer analysis with different fully connected layers for classification of colon adenocarcinoma and benign colonic tissue (https://smartcancer.ir/). Random Forest-based models estimated short-term 5-year risk and long-term lifetime risk. The platform integrated Electronic Health Records (EHR) with blockchain technology to improve security, privacy, and data integrity. The system also generated family trees to support genetic counselling. The platform recommended the next clinical step based on low-risk or high-risk status and stored previous records for specialist follow-up. This study presents a novel framework that combines multiple screening modalities with AI-driven digital analysis for identification of high-risk individuals. Integration of federated learning, blockchain security, and AI-based diagnostics can improve cancer screening, support clinical decision-making, reduce cancer risk, and optimize healthcare resources. Citation Format: Aida Yavari Kondori, Ahmadreza Tavasouli, Mona Maftouh, Ghazaleh Pourali, Zahra Yousefli, Soodabeh Shahidsales, Ali Alamdaran, Masoud Pezeshkirad, Marjaneh Farazestanian, Elisa Giovannetti, Amir Avan, Hamid Naderi. Smart Health Screening in Identification of Individuals at High Cancer Risk Using Artificial Intelligence [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A036.

Immunotherapy, particularly effective in tumors with a high mutational burden, is very often administered in combination with chemotherapy. Several tumor types with a high mutational rate include melanoma and non-small cell lung cancer (NSCLC), which are particularly sensitive to immunotherapy. For NSCLC, conventional platinum-based doublet chemotherapy has been extended with drugs targeting signaling pathways (such as the epidermal growth factor receptor) and immune checkpoint inhibitors (ICI) directed against PD-1 and PD-L1. This review highlights the potential role of the membrane antigens CD73 and CD39 in enhancing the efficacy of combined immuno-chemotherapy. These ecto-nucleotidases catalyze the degradation of extracellular ATP to AMP and subsequently to adenosine (Ado), a potent immunosuppressive metabolite that acts through adenosine receptors. Consequently, CD73 and CD39 function as key downregulators of immunogenic signaling. Both CD73 and CD39 are highly expressed not only on tumor cells but also on immune and endothelial cells within the tumor microenvironment. Conventional chemotherapy may further upregulate their expression, contributing to drug resistance and impaired immune responses. To counteract these effects, inhibitors of CD73 and CD39, both monoclonal antibodies and small molecules, are currently under clinical evaluation, with early results indicating potential therapeutic benefit. Although this evidence supports the involvement of CD73 and CD39 in modulating responses to immunotherapy, particularly in combination with chemotherapy, the precise mechanisms underlying these interactions remain unclear. Elucidating these pathways will be critical for optimizing treatment strategies and improving clinical outcomes in malignancies such as NSCLC. This review highlights the critical role of these pathways in optimizing treatment strategies and improving clinical outcomes in malignancies such as NSCLC.

Textbook outcome (TO) is an “all-or-none” composite quality metric increasingly used to benchmark oncologic surgery, but its relationship with functional recovery after rectal cancer resection remains unclear. We evaluated TO after robotic rectal resection with primary anastomosis, identified predictors of TO achievement, and assessed associations with oncologic and urogenital outcomes. We retrospectively analyzed consecutive patients undergoing robotic rectal cancer resection with primary anastomosis between January 2015 and December 2025 in a high-volume robotic general surgery unit. Pelvic magnetic resonance imaging pelvimetry was reviewed preoperatively. Validated urinary and sexual function questionnaires were administered preoperatively and at 1, 6, and 12 months postoperatively. Disease-free survival (DFS) was assessed in stage II–III patients. Among 115 patients, 67 (58.2

Background: The bile microbiome has been shown to be associated with the development of pancreatic ductal adenocarcinoma (PDAC). However, the utility of bile fluid as a potential source of microbial biomarkers remains unknown. We aimed to characterize the bile microbial composition in PDAC compared to benign and malignant pancreatico–biliary disease, as well as correlate our findings with the pancreatic intratumoral and neighboring adjacent tissue (NAT) microbiome. Methods: Prospective matched pancreatic tumor, NAT, and bile samples were obtained from 54 patients who underwent surgery for a head of pancreas mass at Royal Surrey NHS Hospital Trust. Full-length 16S rRNA (V1-V9) gene sequencing was performed on the Oxford Nanopore MinION™ platform. The cohort consisted of 30 PDAC, 14 biliary tract cancers, and 10 benign cases. Results: We identified biliary microbial biomarkers Streptococcus (false discovery rate [FDR] = 0.0047) , Klebsiella (FDR = 0.0095) , Enterobacter (FDR = 9.68 × 10 −7 ), and Veillonella (FDR = 0.0140) that were found both in the bile and tumor in patients having surgery for PDAC. These bacterial genera were significantly more abundant in PDAC tumors compared to matched NAT and benign disease. We detected a negligible number of microbial reads in the NAT samples. Our microbial signature was highly predictive of PDAC within tissue (AUC = 0.9233) and bile (AUC = 0.8101). Positive bile cultures in the PDAC cohort increased the risk of deep-seated surgical site infections (SSIs), delayed gastric emptying, and post-operative pancreatic fistula. Biliary stenting did not affect microbial composition, and the abundance of specific genera significantly correlated with overall survival and disease-free survival in PDAC. Conclusion: We have shown that the normal pancreas is a relatively sterile organ, whilst the PDAC tumor and bile are colonized with specific genera. In fact, 71% of the tumor microbiome is shared with the bile microbiome in PDAC, suggesting that bile may serve as a potential future diagnostic and prognostic microbial biomarker as well as a surrogate readout of the tumor microbiome. The function of these microbes during pancreatic tumorigenesis remains to be determined. Furthermore, intra-operative biliary culture may serve as a low-cost predictor of post-operative outcomes.

Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate due to late-stage diagnosis. Although SUIT2 sublines differ in metastatic and drug-resistant behavior, the mechanisms behind these differences are unclear. Fluid shear stress can regulate tumor cell migration, and the mechanosensitive factor yes-associated protein (YAP) promotes PDAC progression. However, the impact of shear stress on YAP signaling in PDAC cells remains unknown. This study aims to better understand how fluid shear stress influences the behavior of PDAC cells and to elucidate the mechanisms by which shear stress regulates their metastatic potential, in order to support the development of more effective therapeutic strategies. Using the BioFlux-Shear-Flow-System, we observed that under static conditions, the more metastatic SUIT2-007 cells displayed greater migratory capacity than the less metastatic SUIT2-028 cells. However, exposure to fluid shear stress significantly enhanced the migration capacity of SUIT2-028 cells. Consequently, the migratory capacities of the two cell lines became comparable under shear stress conditions. RNA-sequencing revealed differences in yes-associated protein (YAP) activation between SUIT2-028 and SUIT2-007. Functional assays using a YAP activation inhibitor and scratch assay demonstrated that inhibition of YAP activity reduced migration in both tumor cell lines and abolished the differences in their migratory capacities. Our findings highlight YAP as a mechanosensitive regulator of pancreatic cancer cell migration under shear stress, offering insights into how biomechanical forces can drive metastatic capacity.

Energy metabolism plays a crucial role in determining the aggressiveness of cancer. In this study, we assessed the impact of drug-induced modulation on the expression and prognostic significance of crucial factors involved in glycolytic metabolism: lactate dehydrogenase A (LDH-A) and glucose transporter type 1 (GLUT-1). In patient samples diagnosed with pleural Malignant Mesothelioma (MM), expression levels of LDH-A and GLUT-1 were studied both at baseline and after platinum-based-chemotherapy. High GLUT-1 and LDH-A levels were associated with shorter survival, and chemotherapy increased GLUT-1 expression, further correlating with poor prognosis. Utilizing LDH-A (NHI-2) and GLUT-1 (PGL14) inhibitors, we examined their effects on migration and apoptosis in immortalized (H2052, H2452) and primary (STO, MESO-II) MM cells. PGL14 and NHI-2 decreased migration, increased reactive oxygen species (ROS) and apoptosis rates. Inhibitors, both single and in combination, disintegrated the MM spheroids, while the bioluminescence from spheroid-forming cells decreased from 1.3 × 105 in the control group to 9.7 × 104 and 7.1 × 104 [RLU/s] after NHI-2 and PGL14/NHI-2 treatment, respectively. Overexpression and chemotherapy-induced modulation of LDH-A and GLUT-1 correlated with poor MM prognosis. Combined inhibition of these two metabolic determinants impeded MM cell migration, stimulated ROS production and apoptosis, and affected spheroids' growth, offering promise for new treatment development.

Pancreatoduodenectomy (PD) remains a technically demanding procedure with substantial postoperative morbidity. The robotic PD (R-PD) may offer advantages over open PD (O–PD), but its role is still under scrutiny. This study compared R-PD and O–PD within a mature, high-volume single-institution experience. From October 2008 to May 2024, 500 PDs were performed at our institution (388 O-PDs and 112 R-PDs since January 2018). The 112 R-PD cases were 1:1 case-matched with 112 O–PD cases to ensure comparability in terms of patient-related surgical risk (sex, age, BMI, ASA score), disease-related factors (histological diagnosis, and T stage) and surgeon-related expertise. Univariable and multivariable logistic regression analyses were performed to identify predictors of clinically relevant postoperative pancreatic fistula (POPF) and delayed gastric emptying (DGE). Mean operative time was comparable between groups (389 vs 399 min, p = 0.315), with no conversions to open surgery for R-PD. Clinically relevant POPF occurred in 5.4