技术资料

Suspension bath bioprinting,whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing,has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here,we report a systematic investigation of suspension bath properties that support the printing,fusion,and culture of spheroid-based bioinks without added stabilisation. Specifically,agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150 µ m in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however,the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing,fusion,and culture of spheroid-based inks,either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture. View Publication

Antibiotic resistance is a threat to human health,yet recent work highlights how loss of resistance may drive pathogenesis in some bacteria. In two recent studies,we found that β-lactam antibiotics and nutrient stresses faced during infection selected for genetic inactivation of the Pseudomonas aeruginosa antibiotic efflux pump mexEFoprN . Unexpectedly,efflux pump mutations increased P. aeruginosa virulence during infection; however,neither the prevalence of mexEFoprN inactivating mutations in real human infections,nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for virulence enhancing mutations. Using genome sequencing of clinical isolates,we show that mexEFoprN efflux pump inactivating mutations are enriched in P. aeruginosa isolates from cystic fibrosis infections relative to isolates from acute respiratory infections. Combining RNA-seq,metabolomics,genetic approaches,and infection models we show that efflux pump mutants have elevated quorum sensing driven expression of elastase and rhamnolipids which increase P. aeruginosa virulence during acute and chronic infections. Restoration of the efflux pump in a representative respiratory isolate and the notorious cystic fibrosis Liverpool epidemic strain reduced their virulence. These findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity. Subject terms: Antimicrobial resistance,Pathogens,Bacteriology,Molecular evolution View Publication

Proliferating cell nuclear antigen (PCNA),a well-documented anticancer target,is critical for DNA synthesis,replication,and repair. AOH1996,a small-molecule PCNA inhibitor,is currently undergoing clinical trials for the treatment of advanced solid tumors. However,the therapeutic effect of AOH1996 on head and neck squamous cell carcinoma (HNSCC) remains unclear. The effects of AOH1996 on HNSCC biological behaviors and cancer stemness were tested in HNSCC cells and nude mice. The combination treatment of AOH1996 and anti-PD1 was performed in a 4-nitroquinoline N-oxide (4NQO)-induced HNSCC mouse model. RNA sequencing,Western Blotting,immunofluorescence staining,comet assays,and qRT‒PCR were conducted for mechanistic studies. Our results showed that AOH1996 effectively inhibited HNSCC proliferation and invasion both in vitro and in vivo. AOH1996 suppressed HNSCC stemness,development,and metastasis. Moreover,AOH1996 altered the tumor immune microenvironment into an inflamed state with increased CD8 + T-cell infiltration,rendering it a favorable partner for combination therapy with immune checkpoint inhibitors. Mechanistically,AOH1996 induced cellular DNA damage,suppressed cancer stemness through the upregulation of p-TBK1,and promoted the secretion of CD8 + T-cell-recruiting chemokines by stimulating IRF3-mediated transcription. Taken together,our results demonstrated that AOH1996 suppressed tumor growth,eliminated cancer stem cells (CSCs),and synergistically enhanced the efficacy of anti-PD1 immunotherapy in HNSCC. The online version contains supplementary material available at 10.1186/s13287-025-04607-9. View Publication

Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-syn) aggregates throughout the brain,leading to neuronal dysfunction and death. In this study,we used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout screening to identify genes that regulate the entry and accumulation of α-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in α-syn PFFs intracellular accumulation,including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. All confirmed hits affected heparan sulfate (HS),a post-translational modification known to act as a receptor for proteinaceous aggregates including α-syn and tau. Intriguingly,deletion of SLC39A9 and C3orf58 genes,encoding respectively a Golgi-localized exporter of Zn 2+,and the Golgi-localized putative kinase DIPK2A,specifically impaired the uptake of α-syn PFFs,by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains in SLC39A9 -/- and C3orf58 -/- cells indicated major defects in HS homeostasis. Additionally,Golgi accumulation of NDST1,a prime HSPG biosynthetic enzyme,was detected in C3orf58 -/- cells. Interestingly,C3orf58 -/- human iPSC-derived microglia and dopaminergic neurons exhibited a strong reduction in their ability to internalize α-syn PFFs. Altogether,our data identifies new modulators of HSPGs that regulate α-syn PFFs cell surface binding and uptake. Subject terms: Cellular neuroscience,Glycobiology View Publication

To date,the effects of endurance exercise training on lymphocyte physiology at the kinome level are largely unknown. Therefore,the present study used a highly sensitive peptide-based kinase activity profiling approach to investigate if the basal activity of tyrosine (Tyr) and serine/threonine (Ser/Thr) kinases of human lymphocytes is affected by the aerobic endurance training status. Results revealed that the activity of various tyrosine kinases of the FGFR family and ZAP70 was increased,whereas the activity of multiple Ser/Thr kinases such as IKK$\alpha$,CaMK4,PKA$\alpha$,PKC$\alpha$+$\delta$ (among others) was decreased in lymphocytes of endurance trained athletes (ET). Moreover,functional associations between several differentially regulated kinases in ET-derived lymphocytes were demonstrated by phylogenetic mapping and network analysis. Especially,Ser/Thr kinases of the AGC-kinase (protein kinase A,G,and C) family represent exercise-sensitive key components within the lymphocytes kinase network that may mediate the long-term effects of endurance training. Furthermore,KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome pathway analysis indicate that Ras as well as intracellular signaling by second messengers were found to be enriched in the ET individuals. Overall,our data suggest that endurance exercise training improves the adaptive immune competence by modulating the activity of multiple protein kinases in human lymphocytes. View Publication

BACKGROUND Lymphotoxin $\beta$ receptor (LT$\beta$R) plays important roles in the development of the immune system and immune response. At the cellular level,ligand-bound LT$\beta$R activates the pro-inflammatory NF-$\kappa$B pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LT$\beta$R and its ligands are promising therapeutic targets. Here,we studied the consequences of perturbed cellular cholesterol content on LT$\beta$R-induced NF-$\kappa$B signaling. METHODS To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228,and endothelial HUVEC cells different treatment regimens with filipin,methyl-$\beta$-cyclodextrin and simvastatin were applied. LT$\beta$R localization was studied by confocal microscopy. The activity of LT$\beta$R-induced NF-$\kappa$B pathway was assessed by measuring the levels of NF-$\kappa$B pathway inhibitor I$\kappa$B$\alpha$ and phosphorylation of RelA transcription factor by Western blotting. The NF-$\kappa$B transcriptional response,production of chemokines and adhesion molecules were examined by qRT-PCR,ELISA,and Western blotting,respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LT$\beta$R with its protein partners were investigated by immunoprecipitation. RESULTS We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-$\beta$-cyclodextrin impaired LT$\beta$R internalization and potentiated LT$\beta$R-dependent activation of the canonical branch of the NF-$\kappa$B pathway. The latter was manifested by enhanced degradation of I$\kappa$B$\alpha$ inhibitor,elevated RelA phosphorylation,substantial increase in the expression of NF-$\kappa$B target genes encoding,among others,cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1,that favored the adhesion of immune cells (NK and T cells,neutrophils) to A549 cells and HUVECs. Mechanistically,we showed that cholesterol depletion stabilized interactions of ligand-stimulated LT$\beta$R with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LT$\beta$R. Thus,drugs modulating cholesterol levels could potentially improve efficacy of LT$\beta$R-based therapies. Video abstract. View Publication

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation,functional assays including drug sensitivity testing,and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+,cytokeratin+,CD44+ phenotype,while UWG01CTC,which was derived from a patient with metastatic neuroendocrine cancer,displays an EpCAM-,weak cytokeratin phenotype,with strong expression of neuroendocrine markers. Further,the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel,well characterised,long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets. View Publication

Scleroderma (SSc) is an autoimmune connective tissue disease characterized by immune dysregulation,vasculopathy,and fibrosis. We have previously demonstrated that low Fli1 expression in SSc fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc,playing key roles in disease pathogenesis. However,the role for immune Fli1 in SSc is not yet clear. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in SSc. Comparison of the expression of Fli1 in monocytes,B- and T-cell fractions of PBMCs isolated from SSc patients and healthy controls (HC),showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from Fli1 flox/flox LysMCre+/+ mice,and found that markers of alternative macrophage activation were increased with Fli1 deletion. Coculture of Fli1-deficient myeloid cells and primary human or mouse fibroblasts resulted in a potent induction of collagen type I,independent of TGF$\beta$ upregulation. We next analyzed global gene expression profile in response to Fli1 downregulation,to gain further insight into the molecular mechanisms of this process and to identify differentially expressed genes in myeloid cells. Of relevance to SSc,the top most upregulated pathways were hallmark IFN-$\gamma$ and IFN-$\alpha$ response. Additionally,several genes previously linked to SSc pathogenesis and fibrosis in general were also induced,including CCL2,CCL7,MMP12,and CXCL10. ANKRD1,a profibrotic transcription co-regulator was the top upregulated gene in our array. Our results show that Fli1-deficient myeloid cells share key features with cells from SSc patients,with higher expression of profibrotic markers and activation of interferon responsive genes,thus suggesting that dysregulation of Fli1 in myeloid cells may contribute to SSc pathogenesis. View Publication

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein,we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore,the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFN$\gamma$ production,also Ca2+- and CaM-dependent,was instead not reduced in HNSCC T cells,which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity,but not IFN$\gamma$ production,and reduced their chemotaxis in the presence of adenosine,thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally,1-EBIO enhanced INF$\gamma$ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore,they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers. View Publication

Preclinical in vitro models provide an essential tool to study cancer cell biology as well as aid in translational research,including drug target identification and drug discovery efforts. For any model to be clinically relevant,it needs to recapitulate the biology and cell heterogeneity of the primary tumor. We recently developed and described a conditional reprogramming (CR) cell technology that addresses many of these needs and avoids the deficiencies of most current cancer cell lines,which are usually clonal in origin. Here,we used the CR cell method to generate a collection of patient-derived cell cultures from non-small cell lung cancers (NSCLC). Whole exome sequencing and copy number variations are used for the first time to address the capability of CR cells to keep their tumor-derived heterogeneity. Our results indicated that these primary cultures largely maintained the molecular characteristics of the original tumors. Using a mutant-allele tumor heterogeneity (MATH) score,we showed that CR cells are able to keep and maintain most of the intra-tumoral heterogeneity,suggesting oligoclonality of these cultures. CR cultures therefore represent a pre-clinical lung cancer model for future basic and translational studies. View Publication

Toll-like receptor 8 (TLR-8) plays a role in the pathogenesis of autoimmune disorders and associated gastrointestinal symptoms that reduce quality of life of patients. Dietary interventions are becoming more accepted as mean to manage onset,progression,and treatment of a broad spectrum of inflammatory conditions. In this study,we assessed the impact of N-glycans derived from bovine lactoferrin (bLF) on the inhibition of TLR-8 activation. We investigated the effects of N-glycans in their native form,as well as in its partially demannosylated and partially desialylated form,on HEK293 cells expressing TLR-8,and in human monocyte-derived dendritic cells (MoDCs). We found that in HEK293 cells,N-glycans strongly inhibited the ssRNA40 induced TLR-8 activation but to a lesser extent the R848 induced TLR-8 activation. The impact was compared with a pharmaceutical agent,i.e.,chloroquine (CQN),that is clinically applied to antagonize endosomal TLR- activation. Inhibitory effects of the N-glycans were not influenced by the partially demannosylated or partially desialylated N-glycans. As the difference in charge of the N-glycans did not influence the inhibition capacity of TLR-8,it is possible that the inhibition mediated by the N-glycans is a result of a direct interaction with the receptor rather than a result of pH changes in the endosome. The inhibition of TLR-8 in MoDCs resulted in a significant decrease of IL-6 when cells were treated with the unmodified (0.5-fold,p {\textless} 0.0001),partially demannosylated (0.3-fold,p {\textless} 0.0001) and partially desialylated (0.4-fold,p {\textless} 0.0001) N-glycans. Furthermore,the partially demannosylated and partially desialylated N-glycans showed stronger inhibition of IL-6 production compared with the native N-glycans. This provides evidence that glycan composition plays a role in the immunomodulatory activity of the isolated N-glycans from bLF on MoDCs. Compared to CQN,the N-glycans are specific inhibitors of TLR-8 activation and of IL-6 production in MoDCs. Our findings demonstrate that isolated N-glycans from bLF have attenuating effects on TLR-8 induced immune activation in HEK293 cells and human MoDCs. The inhibitory capacity of N-glycans isolated from bLF onTLR-8 activation may become a food-based strategy to manage autoimmune,infections or other inflammatory disorders. View Publication

Glioblastoma (GBM) is the most common and aggressive malignant tumor in adult brain. Even with the current standard therapy including surgical resection followed by postoperative radiotherapy and chemotherapy with temozolomide (Temo),GBM patients still have a poor median survival. Reprogramming of tumor cells into non-malignant cells might be a promising therapeutic strategy for malignant tumors,including GBM. Based on previous studies using small molecules to reprogram astrocytes into neuronal cells,here we further identified a FTT cocktail of three commonly used drugs (Fasudil,Tranilast,and Temo) to reprogram patient-derived GBM cells,either cultured in serum containing or serum-free medium,into neuronal like cells. FTT-treated GBM cells displayed a neuronal like morphology,expressed neuronal genes,exhibited neuronal electrophysiological properties,and showed attenuated malignancy. More importantly,FTT cocktail more significantly suppressed tumor growth and prolonged survival in GBM patient derived xenograft than Temo alone. Our study provided preclinical evidence that the neuronal reprogramming drug cocktail might be a promising strategy to improve the existing treatment for GBM. View Publication