技术资料

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation,survival and therapy resistance of acute myeloid leukemia (AML) cells. Here,we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA),a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD + . We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis,glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML,we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry,and ROS levels were measured by flow cytometry. Among metabolic enzymes,we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples,while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity,lowers levels of glycolytic intermediates,decreases the cellular NAD + pool,boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead,we found that LDHA is essential to maintain a correct NAD + /NADH ratio in AML cells. Continuous intracellular NAD + supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively,our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD + /NADH balance in support of their abnormal glycolytic activity and biosynthetic demands,which cannot be compensated for by other cellular NAD + recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells. The online version contains supplementary material available at 10.1186/s40170-025-00392-4. View Publication

Bats can host viruses of pandemic concern without developing disease. The mechanisms underlying their exceptional resilience to viral infections are largely unresolved,necessitating the development of physiologically relevant and genetically tractable research models. Here,we developed respiratory and intestinal organoids that recapitulated the cellular diversity of the in vivo epithelium present in Rousettus aegyptiacus,the natural reservoir for the highly pathogenic Marburg virus (MARV). In contrast to human counterparts,bat organoids and mucosal tissue exhibited elevated constitutive expression of innate immune effectors,including type I interferon-ε (IFNε) and IFN-stimulated genes (ISGs). Upon infection with diverse zoonotic viruses,including MARV,bat organoids strongly induced type I and III IFN responses,which conferred robust antiviral protection. Type III IFNλ3 additionally displayed virus-independent self-amplification,acting as an ISG to enhance antiviral immunity. Our organoid platform reveals key features of bat epithelial antiviral immunity that may inform therapeutic strategies for viral disease resilience. Subject terms: Mucosal immunology,Viral infection View Publication

Prenatal alcohol exposure (PAE) affects embryonic development,causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neurodevelopmental disorders and birth defects. To explore the effects of PAE on gastrulation,we used an in vitro model with subchronic moderate (20 mM) and severe (70 mM) ethanol exposures during the differentiation of human embryonic stem cells into germ layer cells. We analyzed genome-wide gene expression (mRNA sequencing),DNA methylation (EPIC Illumina microarrays) and metabolome (non-targeted LC-MS) of the endodermal,mesodermal and ectodermal cells. The largest number of ethanol-induced alterations were observed in endodermal cells,whereas the most prominent changes were in ectodermal cells. Methionine metabolism and genes of the main signaling pathways involved in gastrulation and body patterning were affected by ethanol in all germ layers. Many of the altered genes,including BMP4,FGF8,SIX3 and LHX2,have previously been associated with PAE and phenotypes of FASD,like defects in heart and corpus callosum development as well as holoprosencephaly. Our findings support the early origin of alcohol-induced developmental disorders and strengthen the role of methionine cycle in the etiology of FASD. View Publication

Tumor-initiating cells (TICs) share features and regulatory pathways with normal stem cells,yet how the stem cell niche contributes to tumorigenesis remains unclear. Here,we identify CXCR4 + macrophages as a niche population enriched in normal mammary ducts,where they promote the regenerative activity of basal cells in response to luminal cell-derived CXCL12. CXCL12 triggers AKT-mediated stabilization of β-catenin,which induces Wnt ligands and pro-migratory genes,enabling intraductal macrophage infiltration and supporting regenerative activity of basal cells. Notably,these same CXCR4 + niche macrophages regulate the tumor-initiating activity of various breast cancer subtypes by enhancing TIC survival and tumor-forming capacity,while promoting early immune evasion through regulatory T cell induction. Furthermore,a CXCR4 + niche macrophage gene signature correlates with poor prognosis in human breast cancer. These findings highlight the pivotal role of the CXCL12-CXCR4 axis in orchestrating interactions between niche macrophages,mammary epithelial cells,and immune cells,thereby establishing a supportive niche for both normal tissue regeneration and mammary tumor initiation. Subject terms: Cancer stem cells,Cancer microenvironment,Tumour immunology View Publication

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases,yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage,limited by toxicity from likely excessive signaling. By contrast,mouse Tregs-IL2pa exhibit sustained expansion,long-term survival in immunocompetent mice for over a year,and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype,Treg-specific demethylation,and a diverse TCR repertoire. In vivo,prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly,compared with control Treg,human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability,durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery,Regulatory T cells,Autoimmune diseases,Interleukins View Publication

The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here,we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo,thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies,we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes,resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin,we show resistance of engrafted,multiplex edited human cells in vivo,and a 2-fold enrichment for edited cells in vitro. Together,our results highlight the potential of adenine base editors for improved immune and gene therapies. Subject terms: Haematopoietic stem cells,Bone marrow transplantation,Cell biology View Publication

This study investigates the impact of minimal residual disease (MRD) on relapse in patients with acute myeloid leukemia (AML),focusing on its interaction with immune cells function. A total of 49 AML patients were enrolled in this prospective study and categorized into four groups: MRD − positive with relapse,MRD − positive without relapse,MRD − negative with relapse,and MRD − negative without relapse. Peripheral blood T lymphocyte subpopulations were analyzed using ten-color flow cytometry. CD4 + T cells were co-cultured with leukemia cell lines to assess the impact of CD4 + T cells on leukemia cell proliferation,apoptosis,and cytokine release. In MRD − positive patients,relapsed individuals exhibited significantly higher levels of CD4 + T cells,regulatory T (Treg) cells,and CD4 + CD45RA + naïve T cells compared to non-relapsed patients ( P < 0.0001,P = 0.0016,and P = 0.0066,respectively). Conversely,in MRD − negative patients,relapsed individuals showed a significantly lower percentage of Treg cells ( P = 0.0068). Furthermore,we observed that CD4 + T cells were associated with enhanced leukemia cell proliferation and reduced apoptosis,along with markedly increased IL-10 expression. The available data raise the possibility that CD4 + T cell-derived IL-10 participates in immune microenvironment regulation,a process that may have implications for MRD maintenance and disease recurrence in AML. View Publication

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. View Publication

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 View Publication

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. View Publication

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. View Publication

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 View Publication