参考文献

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

The ten‐eleven translocation family of enzymes (TET1/2/3) promotes DNA demethylation and is essential for hematopoiesis. While the roles of TET1 and TET2 are well‐studied in hematopoiesis,the requirement of TET3 in embryonic and adult hematopoiesis is less investigated. In this study,by characterizing embryonic and adult hematopoiesis in Tie2 +/cre ; Tet3 f/f mice,we have established a requirement for TET3 in regulating hematopoietic stem cells (HSCs; CD150 + CD48 – ). We found that loss of TET3 in the fetal liver and adult bone marrow causes a reduction in the percent of long‐term HSCs (LT‐HSCs; CD150 + CD48 – CD34 – ). This was accompanied by reduced colony forming capacity of TET3‐deficient HSCs in vitro and reduced contribution of HSCs after a competitive bone marrow transplantation in vivo. TET3 deficiency increased DNA methylation at several cell cycle regulator genes leading to their down regulation. This is consistent with,and likely underpins,the reduced number of quiescent HSCs in TET3‐deficient bone marrow. These findings uncover a new role for TET3 in HSC homeostasis during embryonic and adult hematopoiesis. 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

Mesenchymal stem cells (MSCs) are promising for cell-based therapies targeting a wide range of diseases. However,challenges in translating MSC-based therapies to clinical applications necessitate standardized protocols following Good Manufacturing Practices (GMP) guidelines. This study aimed at developing GMP-complained protocols for FPMSCs isolation and manipulation,necessary for translational research,by (1) optimize culture of MSCs derived from an infrapatellar fat pad (FPMSC) condition through animal-free media comparison and (2) establish feasibility of MSC isolation,manufacturing and storage under GMP-compliance (GMP-FPMSC). FPMSCs from three different patients were isolated following established protocols and the efficacy of two animal component-free media formulations in the culturing media were evaluated. The impact of different media formulations on cell proliferation,purity,and potency of MSCs was evaluated through doubling time,colony forming unit assay,and percentage of MSCs,respectively. Furthermore,the isolation and expansion of GMP-FPMSCs from four additional donors were optimized and characterized at each stage according to GMP requirements. Viability and sterility were checked using Trypan Blue and Bact/Alert,respectively,while purity and identity were confirmed using Endotoxin,Mycoplasma assays,and Flow Cytometry. The study also included stability assessments post-thaw and viability assessment to determine the shelf-life of the final GMP-FPMSC product. Statistical analyses were conducted using one-way ANOVA with Tukey’s Multiple Comparisons. The study demonstrated that FPMSCs exhibited enhanced proliferation rates when cultured in MSC-Brew GMP Medium compared to standard MSC media. Cells cultured in this media showed lower doubling times across passages,indicating increased proliferation. Additionally,higher colony formation in FPMSCs cultured in MSC-Brew GMP Medium were observed,supporting enhanced potency. Data from our GMP validation,including cells from 4 different donors,showed post-thaw GMP-FPMSC maintained stem cell marker expression and all the specifications required for product release,including > 95% viability (> 70% is required) and sterility,even after extended storage (up to 180 days),demonstrating the reproducibility and potential of GMP-FPMSCs for clinical use as well as the robustness of the isolation and storage protocols. The study underscores the feasibility of FPMSCs for clinical uses under GMP conditions and emphasizes the importance of optimized culture protocols to improve cell proliferation and potency in MSC-based therapies. The online version contains supplementary material available at 10.1186/s12860-025-00539-7. 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

Metabolic reprogramming of amino acids represents a vulnerability in cancer cells,yet the mechanisms underlying serine metabolism in acute myeloid leukemia (AML) and leukemia stem/initiating cells (LSCs/LICs) remain unclear. Here,we identify RNA N 6 -methyladenosine (m 6 A) modification as a key regulator of serine biosynthesis in AML. Using a CRISPR/Cas9 screen,we find that depletion of m 6 A regulators IGF2BP3 or METTL14 sensitizes AML cells to serine and glycine (SG) deprivation. IGF2BP3 recognizies m 6 A on mRNAs of key serine synthesis pathway (SSP) genes (e.g.,ATF4,PHGDH,PSAT1 ),stabilizing these transcripts and sustaining serine production to meet the high metabolic demand of AML cells and LSCs/LICs. IGF2BP3 silencing combined with dietary SG restriction potently inhibits AML in vitro and in vivo,while its deletion spares normal hematopoiesis. Our findings reveal the critical role of m 6 A modification in the serine metabolic vulnerability of AML and highlight the IGF2BP3/m 6 A/SSP axis as a promising therapeutic target. Subject terms: Acute myeloid leukaemia,Cancer metabolism View Publication