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KRAS-dependent acinar-to-ductal metaplasia (ADM) is a fundamental step in the development of pancreatic ductal adenocarcinoma (PDAC),but the involvement of cell death pathways remains unclear. Here,we show that key regulators of programmed cell death (PCD) become upregulated during KRAS-driven ADM,thereby priming transdifferentiated cells to death. Using transgenic mice and primary cell and organoid cultures,we show that transforming growth factor (TGF)-β-activated kinase 1 (TAK1),a kinase regulating cell survival and inflammatory pathways,prevents the elimination of transdifferentiated cells through receptor-interacting protein kinase 1 (RIPK1)-mediated apoptosis and necroptosis,enabling PDAC development. Accordingly,pharmacological inhibition of TAK1 induces PCD in patient-derived PDAC organoids. Importantly,cell death induction via TAK1 inhibition does not appear to elicit an overt injury-associated inflammatory response. Collectively,these findings suggest that TAK1 supports cellular plasticity by suppressing spontaneous PCD activation during ADM,representing a promising pharmacological target for the prevention and treatment of PDAC. The involvement of cell death pathways in the early stage of pancreatic ductal adenocarcinoma (PDAC) development,especially KRAS-dependent acinar-to-ductal metaplasia (ADM),remains to be investigated. Here,the authors find that TAK1 mediates cell survival during ADM transdifferentiation through suppression of apoptosis and necroptosis,which could be targeted for prevention and treatment of PDAC. View Publication

BackgroundThe trafficking of immune cells between lymphoid organs and circulation depends on gradients of CXCL12 and sphingosine-1-phosphate (S1P),mediated through their cognate receptors C-X-C chemokine receptor type 4 (CXCR4) and S1P receptor type 1 (S1P1). S1P1 facilitates the egress of hematopoietic stem cells and lymphocytes by counteracting CXCR4-mediated retention signals. However,the molecular mechanisms underlying this interplay remain poorly understood. In this study,we uncover CXCR4-S1P1 heteromerization and explore their functional interactions.MethodsBimolecular fluorescence complementation (BiFC) assay,proximity ligation assay (PLA),and quantitative bioluminescence resonance energy transfer (BRET) assay were employed to detect CXCR4-S1P1 heteromerization. Functional properties of the heteromers were assessed using cAMP assay,G protein activation,β-arrestin recruitment,ligand binding,calcium mobilization,and transwell migration assays. S1P1-overexpressing Jurkat T cells were generated via lentiviral transduction,while S1P1-deficient KARPAS299 cells and β-arrestin1/2-deficient HEK293A cells were constructed using the CRISPR/Cas9 system.ResultsCXCR4-S1P1 heteromerization was observed in HEK293A cells overexpressing both receptors. The S1P/S1P1 axis interfered with CXCR4-mediated signaling,while CXCR4 did not affect S1P1-mediated signaling,indicating a unidirectional modulation of CXCR4 by S1P1. CXCL12 binding to CXCR4 remained unchanged in the presence of S1P1,and interference of CXCL12-induced Gαi activation by S1P1 was observed in β-arrestin1/2-deficient cells. BRET analysis revealed that S1P1 interfered with CXCR4-Gαi pre-association and CXCR4 oligomerization,both of which are critical for CXCR4 function. Domain-swapping experiments identified transmembrane domain 3 of S1P1 as essential for this modulation. In Jurkat T cells overexpressing S1P1,CXCR4-mediated signaling and cell migration were diminished,whereas these functions were enhanced in S1P1-deficient KARPAS299 cells. Co-activation of S1P1 attenuated CXCL12-induced migration,while pretreatment with S1P or FTY720-phosphate increased CXCR4-mediated migration by downregulating surface S1P1 in KARPAS299 cells. In primary T cells,PLA confirmed CXCR4-S1P1 heteromerization,and S1P interfered with CXCL12-induced migration.ConclusionsThis study identifies CXCR4-S1P1 heteromers and demonstrates a unidirectional modulation of CXCR4 by S1P1. S1P1 affects CXCR4 function by disrupting its G protein pre-association and oligomerization. These findings underscore the regulatory role of the S1P/S1P1 axis in CXCR4 signaling within the heteromeric context and provide novel insights into the intricate mechanisms governing immune cell trafficking.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02099-x. View Publication

Embryonic DNA damage resulting from DNA repair deficiencies or exposure to ionizing radiation during early neurogenesis can lead to neurodevelopmental disorders,including microcephaly. This has been linked to an excessive DNA damage response in dorsal neural progenitor cells (NPCs),resulting in p53-dependent apoptosis and premature neuronal differentiation which culminates in depletion of the NPC pool. However,the effect of DNA damage on ventral forebrain NPCs,the origin of interneurons,remains unclear. In this study,we investigated the sequelae of irradiation of mouse fetuses at an early timepoint of forebrain neurogenesis. We focused on the neocortex (NCX) and medial ganglionic eminence (MGE),key regions for developing dorsal and ventral NPCs,respectively. Although both regions showed a typical p53-mediated DNA damage response consisting of cell cycle arrest,DNA repair and apoptosis,NCX cells displayed prolonged cell cycle arrest,while MGE cells exhibited more sustained apoptosis. Moreover,irradiation reduced the migration speed of interneurons in acute living brain slices and MGE explants,the latter indicating a cell-intrinsic component in the defect. RNA sequencing and protein analyses revealed disruptions in actin and microtubule cytoskeletal-related cellular machinery,particularly in MGE cells. Despite massive acute apoptosis and an obvious interneuron migration defect,prenatally irradiated animals did not show increased sensitivity to pentylenetetrazole-induced seizures,nor was there a reduction in cortical interneurons in young adult mice. This suggests a high plasticity of the developing brain to acute insults during early neurogenesis. Overall,our findings indicate that embryonic DNA damage induces region-specific responses,potentially linked to neurodevelopmental disorders.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05643-7. View Publication

Klebsiella pneumoniae (Kp) is responsible for a wide range of infections,including pneumonia,sepsis,and urinary tract infections. However,the treatment options are limited due to the continuous evolution of drug-resistant and hypervirulent variants. It is crucial to investigate the mechanisms behind the high mortality rate of hypervirulent Kp (hvKp) strains to develop new strategies for preventing hvKp from evading the host’s defenses and improving treatment effectiveness for these fatal infections. In this study,we used a hvKp-induced mouse bacteremia model and performed single-cell RNA sequencing to investigate the effects of hvKp infection. Our findings demonstrated that hvKp infection led to a decrease in lymphocytes (lymphopenia),attributed to impaired proliferation and apoptosis. The infiltration of myeloid-derived suppressor cells (MDSCs) in the infected lungs was confirmed to suppress T cell proliferation,leading to lymphopenia. We further identified that hvKp promotes tryptophan metabolism in infected lungs,enhancing the immunosuppressive activity of MDSCs by inducing the production of the enzyme IDO1. Our ex vivo inhibition experiment revealed that L-kynurenine,a product of tryptophan metabolism,inhibits T-cell proliferation and induces T-cell apoptosis,further suppressing T-cell mediated responses against bacteria. Importantly,when we knocked out the Ido1 gene or inhibited IDO1 expression using a specific inhibitor 1-MT in mice,we observed a significant enhancement in T-cell mediated responses against hvKp. These findings highlight the crucial role of MDSCs in hvKp-induced bacteremia and suggest a promising immunotherapeutic approach by inhibiting IDO1 production to combat infectious diseases. Author summaryKlebsiella pneumoniae is a major cause of health care–associated infections worldwide,and infections with hvKp is often associated with cytokine storms and lymphopenia,resulting in higher mortality. However,the underlying mechanisms of lymphopenia during hvKp infection is not fully understood. In this study,we demonstrate a mechanism of immunosuppression of T cells in hvKp infection mouse model by combination of single-cell RNA sequencing,flow cytometry and untargeted metabolomic analysis. We confirm MDSCs can suppress T cell proliferation and induce T cell apoptosis by activating tryptophan metabolism during hvKp infection. Specifically,the downstream metabolite kynurenine,which catalyzes by IDO1 in tryptophan metabolism pathway,serves as the signaling molecule in the interactions between MDSCs and T cells. Our findings provide a new insight in understanding the mechanism of how hvKp manipulate the host immune systems and suggest that pharmacological inhibition of IDO1 could be an effective therapeutic strategy for combating hvKp infection. View Publication

T cells primarily drive the autoimmune destruction of pancreatic beta cells in Type 1 diabetes (T1D). However,the profound yet uncharacterized diversity of the T cell populations in vivo has hindered obtaining a clear picture of the T cell changes that occur longitudinally during T1D onset. This study aimed to identify T cell clonal expansion and distinct transcriptomic signatures associated with T1D progression in Non-Obese Diabetic (NOD) mice. Here we profiled the transcriptome and T cell receptor (TCR) repertoire of T cells at single-cell resolution from longitudinally collected peripheral blood and pancreatic islets of NOD mice using single-cell RNA sequencing technology. We detected disease dependent development of infiltrating CD8 + T cells with altered cytotoxic and inflammatory effector states. In addition,we discovered a high frequency of transcriptionally distinct double negative (DN) T cells that fluctuate throughout T1D pathogenesis. This study identifies potential disease relevant TCR sequences and potential disease biomarkers that can be further characterized through future research. View Publication

BackgroundRecurrent/metastatic head and neck squamous cell carcinoma (R/M-HNSCC) is a severe,frequently lethal condition. Oncogene addiction to epidermal growth factor receptor (EGFR) is a hallmark of HNSCC,but the clinical efficacy of EGFR-targeted therapies remains low. Understanding molecular networks governing EGFR-driven progression is paramount to the exploration of (co)-treatment targets and predictive markers.MethodsWe performed function-based mapping of differentially expressed genes in EGFR-mediated local invasion (fDEGs) using photoconvertible tracers and RNA-sequencing (RNA-seq) in a cellular 3D-model.ResultsUpon alignment with public single-cell RNA-seq (scRNA-seq) datasets and HNSCC-specific regulons,a gene regulatory network of local invasion (invGRN) was inferred from gene expression data,which was overrepresented in budding tumors. InvGRN comprises the central hubs inhibin subunit beta alpha (INHBA) and snail family transcriptional repressor 2 (SNAI2),and druggable fDEGs integrin subunit beta 4 (ITGB4),laminin 5 (LAMB3/LAMC2),and sphingosine kinase 1 (SPHK1). Blockade of INHBA repressed local invasion and was reverted by activin A,laminin 5,and sphingosine-1-phosphate,demonstrating a functional interconnectivity of the invGRN. Epithelial-to-mesenchymal transition (EMT) of malignant cells and the invGRN are induced by newly defined EGFR-activity subtypes with prognostic value that are promoted by amphiregulin (AREG) and epiregulin (EREG). Importantly,co-inhibition of SPHK1 showed synthetic effects on Cetuximab-mediated invasion blockade and high expression of selected fDEGs was associated with response to Cetuximab in patient-derived xenotransplantation (PDX) and R/M-HNSCC patients.ConclusionsWe describe an actionable network of EGFR-mediated local invasion and define druggable effectors with predictive potential regarding the response of R/M-HNSCC to Cetuximab.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02290-1. View Publication

Overweight and obesity are linked to mitochondrial alterations,impaired glucose tolerance and a high risk of type 2 diabetes. Time-restricted eating (TRE) may aid in facilitating weight loss to prevent diabetes. Here,we investigated if TRE in individuals with overweight and prediabetes or obesity affects mitochondrial bioenergetics of peripheral blood mononuclear cells (PBMCs) and platelets using the Seahorse extracellular flux technology. In a 3-month randomized controlled trial,PBMCs/platelets were analyzed from 52 participants before and after a TRE intervention with a 10-h eating window or habitual living. PBMC and platelet respiratory function was evaluated through sequential addition of substrates,uncouplers,and inhibitors in living cells. After 3 months,there were no statistically significant differences in mitochondrial respiration within or between the TRE and control groups. Association analyses between PBMC/platelet respiration and clinical parameters including body mass index and fat mass showed no significant effects. In conclusion,3 months of 10-h TRE does not alter the mitochondrial bioenergetics of PBMCs and platelets in individuals with high risk of type 2 diabetes.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-94652-4. View Publication

ABSTRACTBackgroundDendritic cells (DCs) are known to be crucial in initiating immune responses,but their role in regulating immune checkpoint inhibitor (ICI) efficacy in EGFR mutant NSCLC remains unclear.MethodsPeripheral blood mononuclear cells (PBMCs) were co‐cultured with EGFR mutant cells to evaluate immune scores and DC maturation via high‐throughput sequencing. TIDE scores were used to predict the efficacy of ICI treatment. Gene set enrichment analysis (GSEA) was carried out on DCs to explore the signaling pathway changes underlying the diverse responses to ICIs.ResultsA significant decrease in CD8+ T lymphocytes and cytotoxicity scores was found in EGFR mutant LUAD compared to wild‐type (p < 0.001). Three datasets (GSE135222,GSE126044,and GSE136961) showed that higher DC gene expression was associated with a more favorable response to ICIs (p = 0.028). The CSE241934 dataset showed that the number of conventional DC 1 (cDC1) was higher in the ICI‐sensitive group. The TIDE model suggested that cDC1 was associated with ICIs efficacy. However,GSE32863,GSE75037,and GSE72094 showed no differences in cDC subpopulations between EGFR mutant and wild‐type LUAD. EGFR mutant cells exhibited more suppression in the expression of HLA‐DR,CD40,CD83,and CD86 than the control group. The TIDE model suggested DC maturity was associated with ICI efficacy. GSE241934‐IIT showed that DC maturity was more abundant in the ICI‐sensitive group than that in the resistant group.ConclusionsBoth the number and maturation capacity of DCs are positively correlated with ICI efficacy. The cause of poor ICI efficacy in EGFR mutant LUAD is more likely to be low DC maturity,not number,compared to EGFR wild‐type LUAD. Dendritic cells gene expression signature predicted ICIs efficacy. (A) IRF8 high‐expression group had low TIDE scores,indicating a predicted longer survival period following ICIs treatment. (B) CLEC9A high‐expression group had low TIDE scores,indicating a predicted longer survival period following ICIs treatment. (C–E) The stack diagram showed the cell composition of each sample,GSE135222 (n = 27),GSE126044 (n = 16),GSE136961 (n = 21). Each column in the above figure corresponds to each sample,and a different color indicates the constituent specific gravity of the cells in each sample. The dark blue is the component of DC cells in each sample. (F) The surv_cutpoint value. (G) Kaplan‐Meier survival analysis of the different groups of samples from GEO dataset,patients with high abundance of dendritic cell infiltration had a high therapeutic benefit heel from ICIs. (H)The number of cDC1 in GSE241934‐Real dataset between the sensitive group and resistant group follwing ICIs. (I) The number of cDC2 in GSE241934‐Real dataset between the sensitive group and resistant group follwing ICIs. (J) The number of pDC in GSE241934‐Real dataset between the sensitive group and resistant group follwing ICIs. View Publication

PurposeQuantitative real-time PCR (qRT-PCR) represents a robust methodology to investigate alterations in gene expression patterns during tumorigenesis. The quantification of target gene expression is conventionally standardized through normalization against a stably expressed reference gene. However,the expression profile of a specific reference gene can exhibit variability across different tissue types and diverse physiological conditions. This study aimed to identify a suitable reference gene from a pool of ten potential candidates for the comparison of gene expression profiles between six human breast cell lines,comprising both normal breast (MCF-12A) and breast cancer cells (MCF-7,BT-474,SK-BR-3,MDA-MB-468,MDA-MB-231).MethodsFour different mathematical approaches were used to calculate the stability of reference gene expression (comparative ΔCt method,NormFinder,coefficient of variation and RefFinder).ResultsStability analysis identified ACTB as a suitable reference gene across all cell lines. As we are specifically interested in studying metabolic adaptation of breast cancer,we applied the same approach to identify a suitable reference gene also after maintaining the cell lines in L-arginine-deficient medium for up to 72 h. The stability ranking of reference genes fluctuated after L-arginine was depleted.ConclusionIn the context of investigating specific cell lines under certain conditions,we propose the identification of reference genes that exhibit optimal stability and suitability. View Publication

The persistence of latent HIV-1 proviruses in CD4+ T cells is a major obstacle to curing HIV. The “shock and kill” strategy involves reversing latency with latency-reversing agents (LRAs) and selectively inducing cell death in infected cells. However,current LRAs have shown limited efficacy in eliminating the ex vivo HIV reservoir and thus failed in clinical study. In this study,we repurposed PZ703b,a pro-apoptotic protein degrader initially developed for anti-leukemia therapy,to target HIV eradication. PZ703b induced the degradation of Bcl-2 and Bcl-xL,activating the non-canonical NF-kB pathway and caspases cascade,resulting in latency reversal and the selective apoptosis of infected cells. The treatment of ex vivo CD4+ T cells from ART-suppressed HIV-1 patients led to approximately a 50% reduction in the replication-competent reservoir. While this result does not reach the threshold required for a complete cure,it demonstrates the potential of a dual degrader of Bcl-2/Bcl-xL in reversing HIV latency and inducing selective cell death. Our study provides a proof-of-concept for using dual degraders of Bcl-2/Bcl-xL as a novel category of LRAs in therapeutic strategies aimed at reducing HIV reservoirs. This approach may pave the way for the further exploration of targeted interventions to eliminate the HIV-inducible reservoir. View Publication

CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases,but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1,NTSR2,and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable,target specific,non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-91153-2. View Publication

Tractable,patient-relevant models are needed to investigate cancer progression and heterogeneity. Here,we report an alternative in vitro model of lung squamous cell carcinoma (LUSC) using primary human bronchial epithelial cells (hBECs) from three healthy donors. The co-operation of ubiquitous alterations (TP53 and CDKN2A loss) and components of commonly deregulated pathways including squamous differentiation (SOX2),PI3K signalling (PTEN) and the oxidative stress response (KEAP1) is investigated by generating hBECs harbouring cumulative alterations. Our analyses confirms that SOX2-overexpression initiates early preinvasive LUSC stages,and co-operation with the oxidative stress response and PI3K pathways to drive more aggressive phenotypes,with expansion of cells expressing LUSC biomarkers and invasive properties. This cooperation is consistent with the classical LUSC subtype. Importantly,we connect pathway dysregulation with gene expression changes associated with cell-intrinsic processes and immunomodulation. Our approach constitutes a powerful system to model LUSC and unravel genotype-phenotype causations of clinical relevance. Patient-relevant in vitro models remain crucial to study lung cancer progression and heterogeneity. Here,the authors develop an in vitro model of lung squamous cell carcinoma using primary human bronchial epithelial cells from healthy donors,revealing signalling pathways that are critical for early tumour development and invasion. View Publication