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Acute myeloid leukemia (AML) remains a therapeutic challenge due to drug resistance and relapse. Selinexor,an XPO1 inhibitor,shows limited efficacy as monotherapy,necessitating combination strategies. JQ1,a BET inhibitor targeting MYC,may synergize with Selinexor to enhance antileukemic effects. AML cell lines,primary patient samples,and xenograft models (MLL-AF9,CDX,PDX) were treated with Selinexor and JQ1 alone or combined. Synergy was assessed via viability assays (Compusyn/SynergyFinder),apoptosis (flow cytometry/Western blot),and C-MYC suppression (qPCR/CRISPR). In vivo efficacy was evaluated by tumor burden (flow cytometry) and survival. The combination demonstrated strong synergy (CI < 1,HSA > 10) across AML models,with > 80% inhibition in cell lines and primary samples. Mechanistically,it suppressed C-MYC (protein/mRNA),induced apoptosis (cleaved PARP),and arrested cell cycle. In vivo,the combination reduced leukemic burden in bone marrow,spleen,and liver,extending survival in xenografts. PDX models confirmed efficacy in primary AML cells. Selinexor and JQ1 synergistically target AML by dual C-MYC inhibition,offering a promising strategy to overcome resistance. Further clinical evaluation is warranted. The online version contains supplementary material available at 10.1186/s12967-025-06525-z. View Publication

The airway epithelium provides a first line of defense against pathogens by release of antimicrobial factors and neutrophil-attracting chemokines. Pseudomonas (P.) aeruginosa,a Gram-negative bacterium that expresses flagellin as an important virulence factor,is a common cause of injurious airway inflammation. The aim of our study was to determine the contribution of flagellin to the inflammatory,antimicrobial,and metabolic responses of the airway epithelium to P. aeruginosa . Furthermore,as we previously showed that targeting mTOR limited the glycolytic and inflammatory response induced by flagellin,we assessed the effect of rapamycin on human bronchial epithelial (HBE) cells stimulated with flagellated and non-flagellated P. aeruginosa. Primary pseudostratified HBE cells,cultured on an air-liquid-interface,were treated on the basolateral side with medium,vehicle or rapamycin,exposed on the apical side with flagellated or flagellin-deficient P. aeruginosa,and analyzed for their inflammatory,antimicrobial,and glycolytic responses. Flagellin augmented the P. aeruginosa -induced expression of antimicrobial factors and secretion of chemokines by HBE cells but did not further increase the glycolytic response. Treatment of HBE cells with rapamycin inhibited mTOR activation in general and flagellin-augmented mTOR activation in particular,but did not affect the glycolytic response. Rapamycin,however,diminished the flagellin-augmented inflammatory and antimicrobial response induced by Pseudomonas . These results demonstrate that flagellin is a significant factor that augments the inflammatory and antimicrobial response of human airway epithelial cells upon exposure to P. aeruginosa and suggest that mTOR inhibition by rapamycin in the airway epithelium diminishes these exaggerated responses. View Publication

The development of immunocompetent skin models marks a significant advancement in in vitro methods for detecting skin sensitizers while adhering to the 3R principles,which aim to reduce,refine,and replace animal testing. This study introduces for the first time an advanced immunocompetent skin model constructed entirely from induced pluripotent stem cell (iPSC)-derived cell types,including fibroblasts (iPSC-FB),keratinocytes (iPSC-KC),and fully integrated dendritic cells (iPSC-DC). To evaluate the skin model’s capacity,the model was treated topically with a range of well-characterized skin sensitizers varying in potency. The results indicate that the iPSC-derived immunocompetent skin model successfully replicates the physiological responses of human skin,offering a robust and reliable alternative to animal models for skin sensitization testing,allowing detection of extreme and even weak sensitizers. By addressing critical aspects of immune activation and cytokine signaling,this model provides an ethical,comprehensive tool for regulatory toxicology and dermatological research. View Publication

Neurodevelopmental studies employing animal models encounter challenges due to interspecies differences and ethical concerns. Maturing neurons of human origin,undergoing several developmental stages,present a powerful alternative. In this study,human embryonic stem cell (H9 cell line) was differentiated into neural stem cells and subsequently matured into neurons over 30 days. Ion AmpliSeq™ was used for transcriptomic characterization of human stem cell-derived neurons at multiple time points. Data analysis revealed a progressive increase of markers associated with neuronal development and astrocyte markers,indicating the establishment of a co-culture accommodating both glial and neurons. Transcriptomic and pathway enrichment analysis also revealed a more pronounced GABAergic phenotype in the neurons,signifying their specialization toward this cell type. The findings confirm the robustness of these cells across different passages and demonstrate detailed progression through stages of development. The model is intended for neurodevelopmental applications and can be adapted to investigate how genetic modifications or exposure to chemicals,pharmaceuticals,and other environmental factors influence neurons and glial maturation. View Publication

Directed evolution is a process of mutation and artificial selection to breed biomolecules with new or improved activity. Directed evolution platforms are primarily prokaryotic or yeast-based,and stable mammalian systems have been challenging to establish and apply. To this end,we develop PROTein Evolution Using Selection (PROTEUS),a platform that uses chimeric virus-like vesicles to enable extended mammalian directed evolution campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for directed evolution in mammalian systems. Using PROTEUS,we alter the doxycycline responsiveness of tetracycline-controlled transactivators,generating a more sensitive TetON-4G tool for gene regulation with mammalian-specific adaptations. PROTEUS is also compatible with intracellular nanobody evolution,and we use it to evolve a DNA damage-responsive anti-p53 nanobody. Overall,PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells. Subject terms: Synthetic biology,Synthetic biology,Molecular evolution,Next-generation sequencing View Publication

Tumor-associated macrophages (TAMs) are key promoters of inflammatory breast cancer (IBC),the most aggressive form of breast cancer. The receptor tyrosine kinase AXL is highly expressed in various cancer types,including IBC,but its role in TAMs remains unexplored. We examined the effects of AXL inhibitor TP-0903 on tumor growth and tumor microenvironment (TME) component M2 macrophages (CD206 + ) in IBC and triple-negative breast cancer mouse models using flow cytometry and immunohistochemical staining. Additionally,we knocked out AXL expression in human THP-1 monocytes and evaluated the effect of AXL signaling on immunosuppressive M2 macrophage polarization and IBC cell growth and migration. We then investigated the underlying mechanisms through RNA sequencing analysis. Last,we performed CIBERSORT deconvolution to analyze the association between AXL expression and tumor-infiltrating immune cell types in tumor samples from the Inflammatory Breast Cancer International Consortium. We found that inhibiting the AXL pathway significantly reduced IBC tumor growth and decreased CD206 + macrophage populations within tumors. Mechanistically,our in vitro data showed that AXL promoted M2 macrophage polarization and enhanced the secretion of immunosuppressive chemokines,including CCL20,CCL26,and epiregulin,via the transcription factor STAT6 and thereby accelerated IBC cell growth and migration. RNA sequencing analysis further indicated that AXL signaling in immunosuppressive M2 macrophages regulated the expression of molecules and cytokines,contributing to an immunosuppressive TME in IBC. Moreover,high AXL expression was correlated with larger populations of immunosuppressive immune cells but smaller populations of immunoactive immune cells in tissues from patients with IBC. AXL signaling promotes IBC growth by inducing M2 macrophage polarization and driving the secretion of immunosuppressive molecules and cytokines via STAT6 signaling,thereby contributing to an immunosuppressive TME. Collectively,these findings highlight the potential of targeting AXL signaling as a novel therapeutic approach for IBC that warrants further investigation in clinical trials. The online version contains supplementary material available at 10.1186/s13058-025-02015-8. View Publication

Transition from the manual processes that are performed during the initial research and development (R&D) stage to automated processes for later and commercial stage cell therapy manufacturing can be challenging. It often requires significant effort,time,and costs – which hinders the therapy’s access to the clinic. To ease this transition,we have developed a novel and flexible manufacturing platform,Bioreactor with Expandable Culture Area (BECA),that aims to support both R&D and manufacturing to accelerate cell therapies from bench to bedside. This report introduces two models in this manufacturing platform: BECA-S for manual small-scale operation at R&D phase and BECA-Auto for functionally closed and automated scaled-out operation at manufacturing phase. We employed these two models to streamline transition of the T cell culture process from manual to automated and reported insignificant differences in the culture outcome between the two. Our work represents the first detailed development and demonstration of a standalone cell manufacturing platform that facilitates a seamless transition between manual and automated processing for autologous T cell therapy manufacturing. View Publication

Mounting evidence indicates that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exosomes) combine the advantages of hucMSC pluripotency with their nanoscale dimensions,enhancing their clinical potential through prolonged circulation half-life. Despite these promising characteristics,research on their immunological toxicity remains insufficient. This study focuses on the impact of hucMSC-exosomes on the general toxicity and immunopathological indicators. When mice received tail vein injections of 6 × 10 10 hucMSC-exosomes particles,we observed no significant changes in body weight,feed intake,blood composition,organ indices,or histopathological findings throughout the 14 days observation period. Similarly,blood levels of immunoglobulins,cytokines,and lymphocyte subpopulations remained stable. The hucMSC-exosomes produced no detectable negative effects on immune organs including the thymus,spleen,and bone marrow. These findings indicate that intravenous administration of 6 × 10 10 particles of hucMSC-exosomes appears relatively safe at the murine level. This assessment of safety and immunological impact following intravenous hucMSC-exosomes infusion offers experimental support for potential clinical applications and future analyses in this field. View Publication

Microglia perform key homeostatic functions to protect the central nervous system (CNS). However,in many brain disorders their protective functions are abrogated,contributing to disease progression. Therefore,studies of microglial function are critical to developing treatments for brain disorders. Different in vitro microglia models have been established,including primary human and rodent cells,induced pluripotent stem cell (iPSC)-derived models,and immortalised cell lines. However,a direct comparative analysis of the phenotypic and functional characteristics of these models has not been undertaken. Accurate modelling of human microglia in vitro is critical for ensuring the translatability of results from the bench to the brain. Therefore,our study aimed to characterise and compare commonly utilised in vitro microglia models. We assessed four established microglia models: primary human microglia,human iPSC-derived microglia,the human microglial clone 3 (HMC3) cell line,and primary mouse microglia,with primary human brain pericytes acting as a negative control. Primary human microglia,iPSC-derived microglia,and mouse microglia stained positive for myeloid-cell markers (Iba1,CD45 and PU.1),while HMC3 cells only stained positive for mural-cell markers (PDGFRβ and NG2). Distinct secretomes were observed in all cell models in response to inflammatory treatment,with iPSC-derived microglia showing the most significant inflammatory secretions. Notably,nitric oxide was only secreted by mouse microglia. Although all cell types exhibited phagocytic capacity,primary human microglia and iPSC-derived microglia displayed significantly higher levels of phagocytosis. Overall,comparative analysis revealed notable differences between human microglia,iPSC-derived microglia,HMC3 cells and mouse microglia. Such differences should be considered when using these models to study human brain diseases. Experimental findings obtained from mouse models or cell lines should ultimately be cross validated to ensure the translatability of results to the human condition. View Publication

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

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

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