技术资料

Cancer stem cells (CSCs) play crucial roles in tumor metastasis,therapy resistance,and immune evasion. Identifying and understanding the factors that regulate the stemness of tumor cells presents promising opportunities for developing effective therapeutic strategies. In this study on oral squamous cell carcinoma (OSCC),we confirmed the key role of KLF7 in maintaining the stemness of OSCC. Using chromatin immunoprecipitation sequencing and dual-luciferase assays,we identified ITGA2,a membrane receptor,as a key downstream gene regulated by KLF7 in the maintenance of stemness. Tumor sphere formation assays,flow cytometry analyses,and in vivo limiting dilution tumorigenicity evaluations demonstrated that knocking down ITGA2 significantly impaired stemness. Upon binding to its extracellular matrix (ECM) ligand,type I collagen,ITGA2 activates stemness-associated signaling pathways,including PI3K-AKT,MAPK,and Hippo. TC-I 15,a small-molecule inhibitor of the ITGA2-collagen interaction,significantly sensitizes oral squamous cell carcinoma (OSCC) to cisplatin in xenograft models. In summary,we reveal that the KLF7/ITGA2 axis is a crucial modulator of stemness in OSCC. Our findings suggest that ITGA2 is a promising therapeutic target,offering a novel anti-CSC strategy. Subject terms: Cancer stem cells,Cancer therapeutic resistance View Publication

In Alzheimer’s disease,amyloid beta (Aβ) and tau pathology are thought to drive synapse loss. However,there is limited information on how endogenous levels of tau,Aβ and other biomarkers relate to patient characteristics,or how manipulating physiological levels of Aβ impacts synapses in living adult human brain. Using live human brain slice cultures,we report that Aβ 1-40 and tau release levels vary with donor age and brain region,respectively. Release of other biomarkers such as KLK-6,NCAM-1,and Neurogranin vary between brain region,while TDP-43 and NCAM-1 release is impacted by sex. Pharmacological manipulation of Aβ in either direction results in a loss of synaptophysin puncta,with increased physiological Aβ triggering potentially compensatory synaptic transcript changes. In contrast,treatment with Aβ-containing Alzheimer’s disease brain extract results in post-synaptic Aβ uptake and pre-synaptic puncta loss without affecting synaptic transcripts. These data reveal distinct effects of physiological and pathological Aβ on synapses in human brain tissue. Subject terms: Alzheimer's disease,Alzheimer's disease View Publication

The interplay between 3D genomic structure and transposable elements (TE) in regulating cell state-specific gene expression program is largely unknown. Here,we explore the utilization of TE-derived enhancers in naïve and expanded pluripotent states by integrative analysis of genome-wide Hi-C-defined enhancer interactions,H3K27ac HiChIP profiling and CRISPR-guided TE proteomics landscape. We find that short interspersed nuclear elements (SINEs) are the more involved TEs in the active chromatin and 3D genome architecture. In particular,mammalian-wide interspersed repeat (MIR),a SINE family member,is highly associated with naïve-specific genomic interactions compared to the expanded state. Primarily,in the naïve pluripotent state,MIR enhancer is co-opted by ESRRB for naïve-specific gene expression program. This ESRRB and MIR enhancer interaction is crucial for the formation of loops that build a network of enhancers and super-enhancers regulating pluripotency genes. We demonstrate that loss of a ESRRB-bound MIR enhancer impairs self-renewal. We also find that MIR is co-bound by structural protein complex,ESRRB-YY1,in the naïve pluripotent state. Altogether,our study highlights the topological regulation of ESRRB on MIR in the naïve potency state. The online version contains supplementary material available at 10.1186/s13059-025-03577-8. View Publication

Metastasis is one of the leading causes of cancer-related death worldwide. Fascin,a protein that bundles actin filaments to produce protrusions in cancer cells,plays a significant role in the enhancement of cell migration. This protein has been shown that the overexpression of this protein is related to the appearance of different types of cancer,such as colorectal cancer. In this study,we conducted in silico screening of the Enamine library,a compound library with a broad chemical space. Using a ligand-based virtual screening approach based on the pharmacophore model of G2,we identified the predicted inhibitors. First,these compounds were validated by physicochemical analysis. Differential scanning calorimetry (DSF) was used to study the binding between the predicted compounds and fascin protein,followed by an F-actin bundling assay to determine which compounds inhibited the bundling function of fascin. Z1362873773,which exhibited binding to fascin and inhibited F-actin bundling,was further tested in cell cultures to assess its effects on cancer cell viability and migration as well as in organoid models to evaluate potential cytotoxicity. Finally,we established a protocol that can be applied to discover anti-fascin agents from diverse compound libraries. A new molecule has been identified with considerable fascin inhibitory and migration-arresting capacity,which may lead to the development of new therapies to treat cancer. The online version contains supplementary material available at 10.1038/s41598-025-96457-x. Subject terms: Biochemistry,Biophysics,Cancer,Drug discovery,Molecular biology,Virtual drug screening View Publication

Natural killer (NK) cells are exploited in cellular therapies for cancer. While NK cell therapies are efficient against haematological cancers,it has been difficult to target solid tumours due to low tumour infiltration and a hostile tumour microenvironment. NK‐cell derived extracellular vesicles (NK‐EVs) target and kill cancer cells in vitro and represent an alternative treatment strategy for solid tumours. To exploit their potential,it is necessary to standardize NK‐EV production protocols. Here,we have performed a comparative analysis of EVs from the human NK‐92 cell line cultured in five serum‐free commercial media optimized for growth of human NK cells and one serum‐free medium for growth of lymphocytes. The effect of growing the NK‐92 cells in static cell cultures versus shaking flasks was compared. EVs were purified via ultracentrifugation followed by size‐exclusion chromatography. We found that there were no significant differences in EV yield from NK‐92 cells grown under static or dynamic conditions. However,we found clear differences between the different culture media in terms of EV purity as assessed by the enrichment of the CD63 and CD81 markers in the isolates that translated into their capacity to induce apoptosis of the colon cancer cell line HCT 116. These findings will be instructive for the design of future production protocols for therapeutic NK‐cell derived EVs. View Publication

Low dose methotrexate (LD‐MTX) remains the gold standard in rheumatoid arthritis (RA) therapy. Multiple mechanisms on a variety of immune cells contribute to the anti‐inflammatory effects of LD‐MTX. Inflammatory signaling is deeply implicated in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) fate decisions; raising the question of whether HSPC are also modulated by LD‐MTX. This is the first study to characterize the effects of LD‐MTX on HSPC. CD34 + HSPC were isolated from healthy donors' non‐mobilized peripheral blood. Resting and/or cycling HSPCs were treated with LD‐MTX [dose equivalent to that used in RA patients]. Flow cytometry was performed to assess HSPC viability,cell cycle,surface abundance of reduced folate carrier 1 (RFC1),proliferation,reactive oxygen species (ROS) levels,DNA double‐strand breaks,p38 activation,and CD34 + subpopulations. HSPC clonogenicity was tested in colony‐forming cell assays. Our results indicate that in cycling HSPC,membrane RFC1 is upregulated and,following LD‐MTX treatment,they accumulate more intracellular MTX than resting HSPC. In cycling HSPC,LD‐MTX inhibits HSPC expansion by promoting S‐phase cell‐cycle arrest,increases intracellular HSPC ROS levels and DNA damage,and reduces HSPC viability. Those effects involve the activation of the p38 MAPK pathway and are rescued by folinic acid. The effects of LD‐MTX are more evident in CD34 + CD38High progenitors. In non‐cycling HSPC,LD‐MTX also reduces the proliferative response while preserving their clonogenicity. In summary,HSPC uptake LD‐MTX differentially according to their cycling state. In turn,LD‐MTX results in reduced proliferation and the preservation of HSPC clonogenicity. View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication

Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However,the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs),with unique design features,facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress,using aggregate- and microcarrier-based culture systems. Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling,flow cytometry,and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis,electron microscopy,and Western blotting. Lipidomics provided insights into EV lipid composition,while functional assays assessed the impact of EVs in a D-galactose-induced senescence model. VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size,but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36%) in total lipid content; however,distinct differences were identified in lipid chain lengths and saturation levels,affecting key pathways such as sphingolipid and neurotrophin signaling. Human BVO EVs demonstrated the abilities of reducing oxidative stress and increasing cell proliferation in vitro. Human BVOs differentiated in VWBRs (in particular 40 rpm) produce 2–3 fold higher yield of EVs (per mL) than static control. The bio manufactured EVs from VWBRs have exosomal characteristics and therapeutic cargo,showing functional properties in in vitro assays. This innovative approach establishes VWBRs as a scalable platform for producing functional EVs with defined lipid profiles and therapeutic potential,paving the way for future in vivo studies. The online version contains supplementary material available at 10.1186/s13287-025-04317-2. View Publication

Toll-interacting protein (Tollip) suppresses excessive pro-inflammatory signaling,but its function in airway epithelial responses to IL-13,a key mediator in allergic diseases,remains unclear. This study investigates Tollip knockdown (TKD) effects in primary human airway epithelial cells using single-cell RNA sequencing,providing the first single-cell analysis of TKD and the first exploring its interaction with IL-13. IL-13 treatment upregulated key genes,including SPDEF,MUC5AC,POSTN,ALOX15,and CCL26,confirming IL-13’s effects and validating our methods. IL-13 reduced TNF-α signaling and epithelial-mesenchymal transition in certain cell types,suggesting a dual role in promoting type 2 inflammation while suppressing Th1-driven inflammation. Tollip deficiency alone significantly amplified TNF-α signaling and inflammatory pathways in goblet,club,and suprabasal cells. Comparisons between TKDIL13 vs IL13 and TKD vs CTR revealed that IL-13 does not substantially alter Tollip deficiency response in most cell types,reinforcing findings in TKD vs CTR. Tollip deficiency alters the response to IL-13 in a cell-type-specific manner,strongly downregulating TNF-α signaling in goblet cells but only weakly in basal and club cells. Tollip deficiency enhances IL-13’s suppression of Th1 inflammatory responses in goblet cells. These novel insights in Tollip-IL-13 interactions offer potential therapeutic targets for asthma and related diseases. The online version contains supplementary material available at 10.1186/s13104-025-07255-7. View Publication

Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome,resulting in inconsistent findings across studies. Identifying a core set of genes consistently involved in COPD pathogenesis,independent of patient variability,is essential. We integrated lung tissue sequencing data from patients with COPD across two centers. We used weighted gene co-expression network analysis and machine learning to identify 13 potential pathogenic genes common to both centers. Additionally,a gene-based model was constructed to distinguish COPD at the molecular level and validated in independent cohorts. Gene expression in specific cell types was analyzed,and Mendelian randomization was used to confirm associations between candidate genes and lung function/COPD. Preliminary in vitro functional validation was performed on prioritized core candidate genes. Tissue inhibitor of metalloproteinase 4 (TIMP4) was identified as a key pathogenic gene and validated in COPD cohorts. Further analysis using single-cell sequencing from mice and patients with COPD revealed that TIMP4 is involved in ciliated cells. In primary human airway epithelial cells cultured at the air-liquid interface,TIMP4 overexpression reduced ciliated cell numbers. We developed a 13-gene model for distinguishing COPD at the molecular level and identified TIMP4 as a potential hub pathogenic gene. This finding provides insights into shared disease mechanisms and positions TIMP4 as a promising therapeutic target for further investigation. The online version contains supplementary material available at 10.1186/s12931-025-03238-1. View Publication

Development and lineage choice are driven by interconnected transcriptional,epigenetic and metabolic changes. Specific metabolites,such as α-ketoglutarate (αKG),function as signalling molecules affecting the activity of chromatin-modifying enzymes. However,how metabolism coordinates cell-state changes,especially in human pre-implantation development,remains unclear. Here we uncover that inducing naive human embryonic stem cells towards the trophectoderm lineage results in considerable metabolic rewiring,characterized by αKG accumulation. Elevated αKG levels potentiate the capacity of naive embryonic stem cells to specify towards the trophectoderm lineage. Moreover,increased αKG levels promote blastoid polarization and trophectoderm maturation. αKG supplementation does not affect global histone methylation levels; rather,it decreases acetyl-CoA availability,reduces histone acetyltransferase activity and weakens the pluripotency network. We propose that metabolism functions as a positive feedback loop aiding in trophectoderm fate induction and maturation,highlighting that global metabolic rewiring can promote specificity in cell fate decisions through intricate regulation of signalling and chromatin. Subject terms: Embryonic stem cells,Embryology View Publication

SLC26A4 is the second most common cause of hereditary hearing loss worldwide. This gene predominantly harbors pathogenic variants,including splice,nonsense,and missense. Although missense variants are relatively common,their specific effects on protein function remain unclear. Consequently,there is an urgent need to establish an in vitro system to investigate how these variants impact SLC26A4 protein function. Genetic testing was conducted to determine the specific types of underlying genetic variants in patients. Following this,we employed plasmid transfection to evaluate the effects of the variants on both protein expression levels and the protein's subcellular localization. Thereafter,we transformed peripheral blood mononuclear cells (PBMCs) from the proband into induced pluripotent stem cells (iPSCs) through Sendai virus‐mediated transduction. Genetic testing revealed that the proband carried compound heterozygous variants: SLC26A4 c.919‐2A > G and c.317C > A. The c.317C > A variant markedly decreased the expression levels of SLC26A4 mRNA and its encoded protein. Additionally,it led to the protein's accumulation in the cytoplasm as aggregates. We successfully reprogrammed peripheral blood mononuclear cells from the proband into induced pluripotent stem cells (iPSCs) and verified that these iPSCs retained their pluripotency,differentiation potential,and genetic integrity. These results provide important insights into the mechanisms by which SLC26A4 gene variants lead to hearing loss. View Publication