�����ƽ��

BackgroundThe converging biology between enveloped viruses and extracellular vesicles (EVs) has raised interest in the application of engineered EVs as antiviral therapeutics. Following the recent COVID-19 pandemic, EVs engineered with either the ACE2-receptor or Spike-protein have been proposed as strategy to either decoy SARS-CoV-2, or to compete with its cell entry. For generic use as a platform for future pandemic preparedness, a systematic and quantitative comparison of both strategies is required to assess their limitations and benefits across different variants of concern.MethodsHere we generated EVs decorated with either the ACE2-receptor or the Spike-protein of (Wuhan)-SARS-CoV-2 and used single vesicle imaging for in-depth quantitative characterisation. These vesicles were then systematically tested for anti-viral activity across SARS-CoV-2 variants of concern using both, pseudotype and live virus cellular infection models including primary human bronchial and nasal explants.ResultsSpike-protein EVs or ACE2-EVs recovered from transiently transfected HEK293T cells comprised only a small fraction of the EV secretome (5% or 20%, respectively) and were primarily derived from the plasma membrane rather than multivesicular bodies. Redirecting intracellular trafficking of the Spike protein by mutating its transmembrane or subcellular localisation domains did not increase the yields of Spike-EVs. Both types of vesicles inhibited SARS-CoV-2 (D614G) in a dose dependent manner with kinetics and immunohistochemistry consistent with an inhibition at the initial cell entry stage. ACE2-EVs were more potent than Spike-EVs and at least 500–1000 times more potent than soluble antibodies in a pseudotype model. Surprisingly, ACE2-EVs switched from an inhibitory to an enhancer activity for the Omicron BA.1 variant whereas Spike-EVs retained their activity across all variants of concern.ConclusionsWhile our data show that both types of engineered EVs potently inhibit SARS-CoV, the decoy versus competition strategy may result in diverging outcomes when considering viral evolution into new variants of concern. While Spike-EVs retain their competition for receptor binding even against higher affinity viral Spike mutations, the formation of complexes between ACE2-EVs and the virus may not only result in inhibition by decoy. As EVs are actively internalised by cells themselves, they may shuttle the virus into cells, resulting in a productive alternative cell entry route for variants such as Omicron, that diverge from strict plasma membrane protease cleavage to the use of endosomal proteases for release of their genome.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02223-x.

Electronic cigarettes (e-cigs) have been introduced as a safer alternative to traditional combustible cigarettes and have been growing in popularity. E-cig e-liquids all contain the carrier compounds, vegetable glycerin (VG), propylene glycol (PG), and nicotine, together with different flavors, but the effects of inhalation of these compounds on the airway are not well understood. This study investigates the effects of e-cig exposure on primary human airway epithelial cells grown in air–liquid interface (ALI) cultures, specifically focusing on mucociliary clearance, the lung’s primary host defense mechanism whereby pathogens and particles trapped by mucus are cleared by unidirectional beating by ciliated cells. We developed a microcontroller-based exposure system to reproducibly examine cellular and molecular changes in ALI cultures from e-cig exposure. Here we show heterogeneous, donor-dependent effects of different e-cig flavors on airway epithelial cells. Examining the effects of the unflavored carrier compounds common to all e-cigs, we found that ALI airway cultures exposed to PG:VG (30:70 ratio) with 5% nicotine unflavored e-cigs show a reduction in ciliary beat frequency. Moreover, using transmission electron microscopy, we identified defects in ciliary ultrastructure induced by unflavored e-cigs. Phosphoproteomic analysis uncovered changes in phosphorylation of proteins involved in cadherin and actin binding and the Rho GTPase signaling pathway, which are all involved in cytoskeletal remodeling that may influence ciliary structure and function. Altogether, our findings suggest that exposure to all e-cigs reduces mucociliary clearance.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12931-025-03302-w.

AbstractColorectal cancer is the second leading cause of cancer-related death in the United States and high-risk individuals face a notably higher likelihood of developing colorectal cancer based on their genetic background. Hence, there is a compelling need for innovative chemopreventive treatments aimed at minimizing colorectal cancer tumorigenesis. Exportin 1 (XPO1; also referred to as CRM1) plays a pivotal role in transporting proteins from the nucleus to the cytoplasm. Various cancers overexpress XPO1, including colorectal cancer, and selective inhibitors of nuclear export compounds, such as eltanexor (KPT-8602), have been developed to target XPO1. Eltanexor demonstrates fewer adverse effects than its precursors and is currently under evaluation in phase I/II clinical trials. This research evaluates eltanexor as a chemopreventive agent for colorectal cancer. Our findings indicate that eltanexor treatment inhibits expression of the common chemoprevention target in colorectal cancer, COX-2. This occurs by eltanexor-dependent reduction of Wnt/β-catenin signaling. Furthermore, XPO1 inhibition leads to forkhead transcription factor O subfamily member 3a nuclear retention, which can modulate β-catenin/TCF transcriptional activity. The in vivo oral treatment of eltanexor to Apcmin/+ mice (a mouse model for familial adenomatosis polyposis) was well tolerated and reduced tumor burden by approximately threefold, along with decreased tumor size. Drug sensitivity assays using organoids from Apcmin/+ mice tumors showed increased sensitivity to eltanexor compared with wild-type organoids. Collectively, these findings highlight XPO1 as a potent target for colorectal cancer chemoprevention.Significance:In this study, we show the XPO1 inhibitor eltanexor acts as an effective colorectal cancer chemopreventive agent both in vivo and in vitro. This occurs by reducing COX-2 expression by modulating the Wnt/β-catenin signaling pathway. Collectively, these findings highlight XPO1 as a potent target for colorectal cancer chemoprevention.

BackgroundHematopoietic Stem and Progenitor Cells (HSPCs) gene therapy has shown significant progress, with commercial approval for at least four distinct haematological disorders, and poised for a rapid expansion in the upcoming years. Despite these advancements, the ex vivo culture of HSPCs continues to present significant challenges. The stress induced by ex vivo culture can negatively impact transplantation outcomes, while the need for exogenous cytokine supplementation contributes to the high costs associated with gene therapy products.MethodsWe developed genetically modified human bone marrow MSCs (GM-MSCs) secreting cytokines such as Stem cell factor (SCF), Thrombopoietin (TPO), FMS-like tyrosine kinase-3-ligand (FLT3L), and Interleukin-3 (IL3), closely resembling bone marrow cellular niche to augment HSPCs culture.ResultsHSPCs proliferate on GM-MSCs akin to standard conditions, devoid of external cytokine supplementation and these HSPCs retain their stem cell characteristics, colony-forming potential, stemness gene signatures, and capacity for long-term multilineage reconstitution in NBSGW mice. We demonstrate that our biomimetic feeder layer supports and alleviates stress associated with Homology Directed Repair (HDR) mediated gene-editing of HSPCs for fetal haemoglobin reactivation for a potential application in β-hemoglobinopathies gene therapy.ConclusionOur GM-MSCs offer a compelling alternative to traditional cytokine supplementation by establishing a biomimetic bone marrow niche that fosters HSPC expansion while maintaining their stemness. These findings underscore the potential of engineered MSCs to revolutionize ex vivo HSPCs culture, ultimately enhancing their therapeutic value for gene therapy applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04474-4.

Hepatocellular carcinoma (HCC) is a highly heterogeneous and immunosuppressive malignancy that frequently exhibits poor responses to immunotherapy, primarily due to immune evasion mediated by myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Despite increasing interest in MDSC-targeted strategies, effective approaches to selectively modulate MDSC function and enhance immunotherapeutic efficacy remain limited. Recent advances in nanotechnology have led to the development of nanodrug delivery systems, particularly for small interfering RNA (siRNA), offering advantages such as protection from degradation and improved delivery specificity. However, traditional liposomal carriers often suffer from low selectivity and widespread biodistribution, increasing the risk of off-target effects. In this study, we designed a pH-responsive lipid nanoparticle (Lip@si-YTHDF2) for the targeted delivery of siRNA against YTHDF2. This approach aimed to suppress MDSC function, inhibit CSC-mediated immune escape, and enhance immunotherapy in HCC. Bioinformatic analyses of GEO and TCGA-LIHC datasets revealed elevated YTHDF2 expression in HCC and its association with poor prognosis. Functional studies in a conditional YTHDF2-knockout mouse model demonstrated that YTHDF2 regulates MDSC activity and promotes tumor progression by stabilizing MYC mRNA through N6-methyladenosine (m6A) modification. Our findings demonstrated that Lip@si-YTHDF2 effectively downregulated MYC expression, diminished the immunosuppressive phenotype of MDSCs, restored T cell-mediated anti-tumor immunity, and significantly inhibited tumor growth in combination with PD-1 checkpoint blockade. This study not only elucidates a novel YTHDF2/m6A/MYC axis in immune evasion but also provides a clinically relevant siRNA delivery platform with promising therapeutic implications for improving HCC immunotherapy.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12951-025-03538-0.

Bone is the most common site of prostate cancer metastasis, leading to significant morbidity, treatment resistance, and mortality. A major challenge in understanding treatment response is the complex, bone metastatic niche. Here, we report the first patient-specific microphysiological system (MPS) to incorporate six primary human stromal cell types found in the metastatic bone niche (mesenchymal stem cells, adipocytes, osteoblasts, osteoclasts, fibroblasts, and macrophages), alongside an endothelial microvessel, and prostate tumor epithelial spheroids in an optimized media that supports their viability and phenotype. We tested two standard of care drugs, darolutamide and docetaxel, in addition to sacituzumab govitecan (SG), currently in clinical trials for prostate cancer, demonstrating that the MPS accurately replicates androgen response sensitivity and captures stromal microenvironment-mediated resistance. This advanced MPS provides a robust platform for investigating the biological mechanisms of treatment response and for identification and testing of therapeutics to advance patient-specific MPS towards personalized clinical-decision making. The authors developed a patient-specific microphysiological system using the LumeNEXT microfluidic platform to more accurately recreate the prostate cancer metastatic bone tumor microenvironment.

Several chemotherapeutic agents act by increasing DNA damage in cancer cells, triggering cell death. However, there is limited understanding of the extent and long-term consequences of collateral DNA damage in normal tissues. To investigate the impact of chemotherapy on mutation burdens and the cell population structure of normal tissue, we sequenced blood cell genomes from 23 individuals aged 3–80 years who were treated with a range of chemotherapy regimens. Substantial additional somatic mutation loads with characteristic mutational signatures were imposed by some chemotherapeutic agents, but the effects were dependent on the drug and blood cell types. Chemotherapy induced premature changes in the cell population structure of normal blood, similar to those caused by normal aging. The results show the long-term biological consequences of cytotoxic agents to which a substantial fraction of the population is exposed as part of disease management, raising mechanistic questions and highlighting opportunities for the mitigation of adverse effects. Mutational signature analysis of blood cells isolated from 23 chemotherapy-exposed samples and 9 nonexposed controls characterizes the effects of various drugs on mutational burden, signature exposure and cell types.

Human iPSC-derived models currently used in eye research usually replicate later events of tissue differentiation rather than early steps involving concurrent development of diverse embryonic cell types. Here we present a multi-timepoint morphological and transcriptomic analysis of a 2D model mirroring the early stages of human whole eye development, self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. SEAM cultures maintained a reproducible growth profile over the standard 28-day differentiation process with quantifiable morphological changes accompanying generation of key ocular cell types over time. Bulk and single-cell RNA-seq analyses at Days 0, 14 and 28 identified dynamic transcriptomic changes indicative of the emerging cell types, including rare stem cell-like populations analogous to those comprising the ciliary marginal zone, transit-amplifying cells, limbal epithelial stem cells, and corneal stromal stem cells. Integrated developmental trajectory analysis highlighted intermediate differentiation states underlying SEAM maturation. Cluster-specific interrogation of eye disease-associated genes demonstrated dynamic temporal patterns and enrichment in relevant developing cell types. These analyses establish a comprehensive baseline of SEAM formation, supporting the potential of the model to facilitate mechanistic studies of genetic variants that may uniquely impact humans, thus improving the success rate in resolving cases presenting with a broad range of developmental eye phenotypes.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06602-9.