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Influenza virus particularly affects those with chronic lung conditions such as Chronic Obstructive Pulmonary Disease (COPD). Airway epithelial cells are the first line of defense and primary target of influenza infection and release extracellular vesicles (EVs). EVs can transfer of biological molecules such as microRNAs (miRNAs) that can modulate the immune response to viruses through control of the innate and adaptive immune systems. The aim of this work was to profile the EV miRNAs released from bronchial epithelial cells in response to influenza infection and discover if EV miRNA expression was altered in COPD. Influenza infection of air-liquid interface (ALI) differentiated BCi-NS1.1 epithelial cells were characterized by analyzing the expression of antiviral genes, cell barrier permeability and cell death. EVs were isolated by filtration and size exclusion chromatography from the apical surface wash of ALI cultured bronchial epithelial cells. The EV miRNA cargo was sequenced and reads mapped to miRBase. The BCi sequencing results were further investigated by RT-qPCR and by using healthy and COPD primary epithelial cells. Infection of ALI cultured BCi cells with IAV at 3.6 x 10 6 IU/ml for 24 h led to significant upregulation of anti-viral genes without high levels of cell death. EV release from ALI-cultured BCi cells was confirmed using electron microscopy and detection of known tetraspanin EV markers using western blot and the ExoView R100 platform. Differential expression analyses identified 5 miRNA that had a fold change of >0.6: miR-155-5p, miR-122-5p, miR-378a-3p, miR-7-5p and miR-146a-5p (FDR<0.05). Differences between EV, non-EV and cellular levels of these miRNA were detected. Primary epithelial cell release of EV and their miRNA cargo was similar to that observed for BCi. Intriguingly, miR-155 expression was decreased in EVs derived from COPD patients compared to EVs from control samples. Epithelial EV miRNA release may be a key mechanism in modulating the response to IAV in the lungs. Furthermore, changes in EV miRNA expression may play a dysfunctional role in influenza-induced exacerbations of COPD. However, further work to fully characterize the function of EV miRNA in response to IAV in both health and COPD is required.

Accumulating data have shown that targeting breast cancer stem cells (CSCs) is an auspicious way for anticancer therapies. This study demonstrated that the antirheumatic drug auranofin is a potent CSC inhibitor with anti-CSC action on breast cancer. This research focused on investigating the effect of auranofin on breast cancer and CSCs and its cellular mechanism. Mammosphere formation, colony formation, levels of CD44 high /CD24 low , and aldehyde dehydrogenase 1 expression in the cells were evaluated after auranofin treatment. The anti-CSC properties of auranofin were further examined by gel shift assay and cytokine detection. Auranofin suppressed cell growth, colony formation, migration, and mammosphere formation and triggered apoptosis in breast cancer. Auranofin decreased the CD44 high /CD24 low - and aldehyde dehydrogenase-expressed subpopulations, as well as the Stat3-DNA interaction and phosphorylated Stat3 level. Auranofin also decreased the extracellular levels of interleukin-8 (IL-8) in the mammosphere media. Auranofin suppressed the Stat3/IL-8 signal and killed CSCs; therefore, it may be a potential target for CSCs.

Chromothripsis, the chaotic shattering and repair of chromosomes, is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN), we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%, driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A , a serine threonine kinase, is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo, and represents a druggable axis. Collectively, these findings define chr. 21amp as a prognostic biomarker in BP-MPN, and link chromothripsis to a therapeutic target. Subject terms: Leukaemia, DNA sequencing

Cerebral cavernous malformations (CCMs) are clusters of thin-walled enlarged blood vessels in the central nervous system that are prone to recurrent hemorrhage and can occur in both sporadic and familial forms. The familial form results from loss-of-function variants in the CCM1 , CCM2 , or CCM3 gene. Despite a better understanding of CCM pathogenesis in recent years, it is still unclear why CCM3 mutations often lead to a more aggressive phenotype than CCM1 or CCM2 variants. By combining high-throughput differentiation of blood vessel organoids from human induced pluripotent stem cells (hiPSCs) with a CCM1 , CCM2 , or CCM3 knockout, single-cell RNA sequencing, and high-content imaging, we uncovered both shared and distinct functions of the CCM proteins. While there was a significant overlap of differentially expressed genes in fibroblasts across all three knockout conditions, inactivation of CCM1 , CCM2 , or CCM3 also led to specific gene expression patterns in neuronal, mesenchymal, and endothelial cell populations, respectively. Taking advantage of the different fluorescent labels of the hiPSCs, we could also visualize the abnormal expansion of CCM1 and CCM3 knockout cells when differentiated together with wild-type cells into mosaic blood vessel organoids. In contrast, CCM2 knockout cells showed even reduced proliferation. These observations may help to explain the less severe clinical course in individuals with a pathogenic variant in CCM2 and to decode the molecular and cellular heterogeneity in CCM disease. Finally, the excellent scalability of blood vessel organoid differentiation in a 96-well format further supports their use in high-throughput drug discovery and other biomedical research studies. The online version contains supplementary material available at 10.1007/s10456-025-09985-5.

Anti-interleukin (IL)-4Rα monoclonal antibodies (mAb) improve lung function and decrease the number of exacerbations in patients with COPD type (T)2 inflammation. However, the involvement of early innate immune responses underlying these treatment effects is not well known. We sought to understand the effect and mechanisms of IL-4Rα mAb treatment on bronchial epithelial cells (BECs) from COPD patients under T2 inflammatory conditions with and without rhinoviral infection. Primary BECs from healthy and COPD patients were grown at an air–liquid interface and stimulated with IL-4 or IL-13 cytokines in the presence of IL-4Rα mAb. Cells were infected with human rhinovirus 1B and collected 24 h after infection. Antiviral mediators ( i.e. , interferons (IFNs) and pattern recognition receptors (PRRs)), as well as chemokine and alarmin expression, were measured by reverse transcriptase quantitative PCR and ELISA. Treatment with IL-4Rα mAb (100 nM) inhibited the eotaxin-3 (CCL26) gene after IL-4/IL-13 induction (p<0.05) in COPD BECs. However, no significant changes in rhinovirus-induced IFN-β, PRRs or thymic stromal lymphopoietin gene responses were observed with IL-4/IL-13 stimulation and IL-4Rα mAb treatment. A significant increase in mucin 5AC gene expression was observed with both IL-4 and IL-13 stimulation, but it was not reduced with IL-4Rα treatment in BECs. Inhibition of IL-4Rα reduced CCL26 levels without affecting antiviral immune responses in BECs from COPD patients. Inhibition of IL-4Rα reduced IL-4/IL-13 signalling without broadly suppressing the immune system, which might suggest that inhibition of the IL-4Rα pathways may prevent COPD exacerbations through reduction of eosinophil chemotaxis.

Human airways contain specialized rare epithelial cells including CFTR-rich ionocytes that regulate airway surface physiology and chemosensory tuft cells that produce asthma-associated inflammatory mediators. Here, using a lung cell atlas of 311,748 single cell RNA-Seq profiles, we identify 687 ionocytes (0.45%). In contrast to prior reports claiming a lack of ionocytes in the small airways, we demonstrate that ionocytes are present in small and large airways in similar proportions. Surprisingly, we find only 3 mature tuft cells (0.002%), and demonstrate that previously annotated tuft-like cells are instead highly replicative progenitor cells. These tuft-ionocyte progenitor (TIP) cells produce ionocytes as a default lineage. However, Type 2 and Type 17 cytokines divert TIP cell lineage in vitro, resulting in the production of mature tuft cells at the expense of ionocyte differentiation. Our dataset thus provides an updated understanding of airway rare cell composition, and further suggests that clinically relevant cytokines may skew the composition of disease-relevant rare cells. Subject terms: Interleukins, Systems analysis, Differentiation, Sequencing

Drug resistance and relapse are still major challenges in acute myeloid leukemia (AML) because of the inability to effectively eradicate leukemia stem cells (LSCs). Senescence induction combined with immune killing may offer promising strategies for LSC eradication. However, whether and how drug-resistant LSCs retain stemness via senescence and immune regulation remains unknown. The immunoproteasome subunit PSMB10 expression levels were analyzed by single-cell RNA-seq data, along with the bioinformatics analysis of publicly available AML datasets, and quantified using RT-qPCR and flow cytometry (FCM) analysis on clinical samples from AML patients. The cellular senescence was evaluated by the assays of cell proliferation, cell cycle, senescence-associated β-galactosidase activity, and senescence-associated secretory phenotype factors. In vitro T-cell killing assay was played to determine immune escape reprogramming of AML cells. FCM was conducted to estimate intracellular drug concentration and cellular apoptosis rates. Human AML xenografts and PSMB10 knockout syngeneic mouse bone marrow transplantation models were utilized to investigate the function of PSMB10. Various techniques were employed for mechanism studies, including Lentivirus transduction or siRNA transfection, western blotting, co-immunoprecipitation assays, luciferase reporter assays, polysome profiling assays, quantitative proteomics, etc. PSMB10 mRNA was significantly upregulated in the surviving nonsenescent LSCs, exhibiting a 13-fold increase compared to senescent LSCs following chemotherapy. The specific high expression of PSMB10 in post-chemotherapy nonsenescent LSCs predicts a poor AML prognosis. The genetic inactivation of PSMB10 resulted in increased senescence and cytotoxic T lymphocyte (CTL) killing, as well as increased intracellular drug concentrations and drug-induced cellular senescence in different types of human AML cells, which also impeded human and murine leukemia initiation and stemness maintenance in vivo with a 19-fold decrease in the frequency of human LSCs and a 7.6-fold decrease of drug-resistant mouse LSCs, while normal hematopoietic cells remained unaffected. Mechanistically, the downregulation of PSMB10 boosted SLC22A16-mediated drug endocytosis and further induced chemotherapy drug-mediated senescence through the RPL6/RPS6-MDM2-P21 pathway in AML cells. Additionally, downregulating PSMB10 also impeded MHC-I protein degradation-induced escape of CTL killing. PSMB10 is a key candidate molecular target for eradicating drug-resistant LSCs via senescence and immune regulation. The online version contains supplementary material available at 10.1186/s13046-025-03420-9.

Despite the major roles of choroid plexus epithelial cells (CPECs) in brain homeostasis and repair, their developmental lineage and diversity remain undefined. In simplified differentiations from human pluripotent stem cells, derived CPECs (dCPECs) display canonical properties and dynamic motile multiciliated phenotypes that interact with Aβ uptake. Single dCPEC transcriptomes over time correlate well with human organoid and fetal CPECs, while pseudotemporal and cell cycle analyses highlight the direct CPEC origin from neuroepithelial cells. In addition, time series analyses define metabolic (type 1) and ciliogenic dCPECs (type 2) at early timepoints, followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contract. These temporal patterns are then confirmed in independent derivations and mapped to prenatal stages using human tissues. In addition to defining the prenatal lineage of human CPECs, these findings suggest dynamic models of ChP support for the developing human brain. Subject terms: Differentiation, Neural stem cells, Functional clustering, Cell fate and cell lineage

The ε4 isoform of apolipoprotein E (ApoE) is the most significant genetic risk factor for Alzheimer’s disease. Glial cells are the main source of ApoE in the brain, and in microglia, the ε4 isoform of ApoE has been shown to impair mitochondrial metabolism and the uptake of lipids and Aβ42. However, whether the ε4 isoform alters autophagy or lysosomal activity in microglia in basal and inflammatory conditions is unknown. Altogether, microglia-like cells (iMGs) from eight APOE 3/3 and six APOE 4/4 human induced pluripotent stem cell (iPSC) lines were used in this study. The responses of iMGs to Aβ42, LPS and IFNγ were studied by metabolomics, proteomics, and functional assays. Here, we demonstrate that iMGs with the APOE 4/4 genotype exhibit reduced basal pinocytosis levels compared to APOE 3/3 iMGs. Inflammatory stimulation with a combination of LPS and IFNγ or Aβ42 induced PI3K/AKT/mTORC signaling pathway, increased pinocytosis, and blocked autophagic flux, leading to the accumulation of sequestosome 1 (p62) in both APOE 4/4 and APOE 3/3 iMGs. Exposure to Aβ42 furthermore caused lysosomal membrane permeabilization, which was significantly stronger in APOE 4/4 iMGs and positively correlated with the secretion of the proinflammatory chemokine IL-8. Metabolomics analysis indicated a dysregulation in amino acid metabolism, primarily L-glutamine, in APOE 4/4 iMGs. Overall, our results suggest that inflammation-induced metabolic reprogramming places lysosomes under substantial stress. Lysosomal stress is more detrimental in APOE 4/4 microglia, which exhibit endo-lysosomal defects. The online version contains supplementary material available at 10.1186/s12974-025-03470-y.

Testicular somatic cells play an important role in supporting spermatogenesis. Leydig cells (LCs) and peritubular myoid cells (PTMs) originate from a common progenitor population and show similar expression signatures in adulthood, making it difficult to distinguish and isolate the two in vitro. In this study, new surface markers for identifying adult LCs (ALCs) and PTMs were discovered by reanalyzing testicular single-cell dataset. Differential expressions of ITGA9 and NGFR were confirmed through immunofluorescence staining of human testes. A novel Fluorescence activated Cell Sorting (FACS) protocol is established for the isolation of ALCs and PTMs based on the two markers. Long-term culture of both cells were performed and their characteristics were characterized and explored. ITGA9+ /NGFR + cells were positive for markers of PTMs (SMA, CNN1) and negative for markers of ALCs (HSD3B, STAR), and were able to form tubular and spheroid structures in vitro. In contrast, ITGA9-/NGFR + cells were positive for ALC markers and negative for PTM markers, and showed a capacity of testosterone production in vitro. Also, both cells were negative for Sertoli cell marker SOX9. When the two cells were cultured, they can expand for more than 15 passages. Our study established a novel and efficient method for identifying and isolating human ALCs and PTMs, which provides a great potential for researches of the two cell types in human. The online version contains supplementary material available at 10.1186/s12958-025-01389-w.

Dynamic regulation of metabolic activities in astrocytes is critical to meeting the demands of other brain cells. During neuronal stress, lipids are transferred from neurons to astrocytes, where they are stored in lipid droplets (LDs). However, it is not clear whether and how neuron-derived lipids trigger metabolic adaptation in astrocytes. Here, we uncover an endolysosomal function that mediates neuron-astrocyte transcellular lipid signaling. We identify Tweety homolog 1 (TTYH1) as an astrocyte-enriched endolysosomal protein that facilitates autophagic flux and LD degradation. Astrocyte-specific deletion of mouse Ttyh1 and loss of its Drosophila ortholog lead to brain accumulation of neutral lipids. Computational and experimental evidence suggests that TTYH1 mediates endolysosomal clearance of ceramide 1-phosphate (C1P), a sphingolipid that dampens autophagic flux and LD breakdown in mouse and human astrocytes. Furthermore, neuronal C1P secretion induced by inflammatory cytokine interleukin-1β causes TTYH1-dependent autophagic flux and LD adaptations in astrocytes. These findings reveal a neuron-initiated signaling paradigm that culminates in the regulation of catabolic activities in astrocytes. Subject terms: Organelles, Glial biology, Lipid signalling

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here, we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45, CD56, CD14, and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells, as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis, which also revealed colocalization of lymphoid markers with carbonic anhydrase 9, a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells by tumor cells. Finally, we show evidence of chromosomal DNA being transferred from immune cells to tumor cells through physical contact. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell, resulting in a fusion phenotype that expresses both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally, further studies into trogocytosis and other mechanisms of contact-mediated cellular transfer will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other cancers.