Journal of Extracellular Vesicles最新文献

Extracellular vesicles (EVs) affect the function of cells in living animals. Yet, cell type-specific EV abundances and their distribution in biological fluids are technically challenging to study. Thus, we aimed to develop an in vivo EV reporter system to monitor cell-type-specific EVs, with a focus on adipocyte-derived EVs. While our previously generated EV reporter construct had insufficient sensitivity, we successfully created a sensitive EV reporter using an adeno-associated virus (AAV) vector with Cre-activated expression of human CD63 fused to NanoLuc (CD63-NanoLuc). Moreover, we designed a control AAV construct for monitoring constitutive secretion of NanoLuc (sec-NanoLuc). AAV administration to mice induced adipocyte-specific expression of both reporters. NanoLuc activity was detected in plasma. While sec-NanoLuc was predominantly in plasma and urine, CD63-NanoLuc was highest in adipose tissues (ATs). We challenged mice with a 2-week high-fat diet (HFD), which had minimal effects on body weight and adipogenic markers. Still, the HFD-fed CD63-NanoLuc mice, but not sec-NanoLuc mice, showed significantly higher NanoLuc activity in ATs, lungs, kidneys and urine. Thus, our CD63-NanoLuc and sec-NanoLuc constructs revealed an early effect of HFD on the abundance and distribution of adipocyte-derived EVs and provide a sensitive system for monitoring cell-type-specific EVs in health and disease.

Microinflammation is a key driver of chronic kidney disease (CKD) progression, with interleukin-1β (IL-1β) playing a pivotal role. However, current anti-IL-1β antibody therapies face critical limitations, such as systemic side effects and substantial production costs, which hinder their therapeutic efficacy and clinical translation for CKD intervention. To address this, we developed Bacteroides fragilis-derived outer membrane vesicles (OMVs) encapsulating anti-IL-1β single-chain variable fragment (scFv) and conjugated with kidney-targeting peptides (KKEEE)₃K. Engineered OMV-(KKEEE)₃K-scFv's safety was evaluated in vitro (24, 48 h) and in vivo (2 months). Its anti-inflammatory efficacy was assessed in a high glucose-induced model in vitro, and in various kidney disease mouse models (streptozotocin-induced diabetic nephropathy, lupus nephritis, unilateral ureteral obstruction) in vivo. OMV-(KKEEE)₃K-scFv showed high stability, precisely delivered scFv to proximal renal tubules, had excellent safety, reduced the expression of inflammatory cytokines including tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased cell infiltration, and alleviated renal injury. In conclusion, engineered OMVs effectively deliver anti-IL-1β scFv, mitigating local inflammation and CKD-related renal damage.

Temporomandibular joint osteoarthritis (TMJ-OA) is a progressive degenerative disorder, for which therapeutic interventions remain limited. The disruption of metabolic homeostasis plays a critical role in the pathogenesis and advancement of TMJ-OA. However, it remains unclear whether extracellular vesicles (EVs) as cellular metabolites are correlated with the pathogenesis, treatment and diagnosis of TMJ-OA. In this study, we demonstrated that autologous circulating extracellular vesicles (C-EVs) possessed significant therapeutic potential for TMJ-OA through the targeted removal of senescent chondrocytes. In a randomized clinical trial (ChiCTR2200063153), C-EV administration was found to significantly enhance condylar bone regeneration and alleviate symptoms relative to hyaluronic acid controls, without eliciting any adverse effects. Comparative analysis revealed that joint cavity-derived EVs from TMJ-OA patients (OA-EVs) exhibited structural abnormalities, diminished expression of canonical EV markers, and pro-inflammatory characteristics. In contrast, C-EVs were significantly enriched with functional proteins C1q binding protein (C1QBP). And the level of C1QBP-positive EVs was positively correlated with therapeutic outcomes, thereby establishing C1QBP as a potential predictive biomarker for TMJ-OA. Furthermore, C-EVs reestablished joint homeostasis by regulating the immune microenvironment and tissue regeneration capacity. Mechanistically, C1QBP^high C-EVs upregulated the expression of membrane C1q on senescent chondrocytes, thereby initiating C1q–C1QBP binding, p14ARF translocation to mitochondria, and subsequent cytochrome C/caspase-3-dependent apoptosis. Our findings demonstrate that C-EVs serve a dual therapeutic role by facilitating the clearance of senescent cells via the C1QBP/C1q/p14ARF axis, while promoting tissue regeneration and regulating metabolites homeostasis, offering a novel biological strategy for TMJ-OA treatment.

Tumour development and progression are driven by intricate interactions among various cell types within the tumour microenvironment (TME). Targeting a single cell type often fails to eradicate cancer, highlighting the need for strategies to co-target multiple cell types. MicroRNA-21-5p, highly abundant in tumour cells and tumour-associated macrophages (TAMs), exerts strong cancer-promoting effects. Epidermal growth factor receptor (EGFR) is overexpressed in various cancer types, and programmed death-ligand 1 (PD-L1) is expressed predominantly by TAMs in multiple cancers. In this study, we developed a dual-targeted engineered milk-derived extracellular vesicles system (7D12/KN035-iEVs), decorated with 7D12 (an EGFR nanobody) and KN035 (a PD-L1 nanobody), to specifically deliver miR-21-5p inhibitors into EGFR⁺ and/or PD-L1⁺ tumour cells and TAMs, thereby inhibiting tumour progression while reprogramming immunosuppressive TME. Notably, this dual-targeting nanomedicine synergistically inhibits tumour growth when combined with immunotherapy and radiotherapy. In summary, this mEV-based nanomedicine represents a promising universal strategy for cancer treatment, offering a versatile platform for targeting multiple components of the TME.

Dendrogenin A (DDA) is a cholesterol-derived antitumour metabolite that promotes the secretion of immunogenic antitumour exosomes (DDA-sEV) enriched in bis(monoacylglycero)phosphate (BMP). BMP is a phospholipid specific to late endosomes and lysosomes, where it plays a crucial role in lipid degradation, regulates the fate of endosomal cholesterol, and contributes to intraluminal vesicle formation. Dysregulation of BMP biosynthesis is associated with multiple diseases. Here, we show that the DDA/LXRβ complex activates the transcription and activity of phospholipase D (PLD) and CLN5, two enzymes involved in BMP biosynthesis. Inhibition of PLD in DDA-treated tumour cells reduces BMP levels in DDA-sEV, impairs their release, and their antitumour immune activity. Blocking BMP on DDA-sEV with a specific antibody abolishes their antitumour reponse, prevents the recruitment of activated dendritic cells (DC) and T cells into tumours, and decreases mouse survival. This blockade also impairs DDA-sEV uptake by immature DC (iDC) and hinders DC maturation and Th1 T cell activation. Notably, neutralising the BMP-presenting receptor on iDC inhibits DDA-sEV uptake and DC maturation. Treatment of iDC with free BMP induces their functional maturation, confirming BMP as a key immune activator. Furthermore, BMP-containing DDA-sEV enhance the efficacy of anti-PD-1 therapy in melanoma. Targeting LXRβ with DDA represents an innovative strategy to stimulate anticancer immunity by increasing BMP levels in tumours and sEV.

Precision cancer medicine with small tyrosine kinase inhibitors (TKIs) directed toward oncogenic drivers, are important treatment regimens for solid tumours. The epidermal growth factor receptor (EGFR)-TKI osimertinib is a preferred therapy for patients with non-small cell lung cancer (NSCLC) driven by activating mutations in EGFR, unfortunately responses are heterogeneous. This calls for non-invasive methods to predict or monitor treatment response, for example, via biomarker analyses in blood. To reveal such putative biomarkers, we analysed the proteome of extracellular vesicles (EVs) from osimertinib resistant or responsive NSCLC cells in vitro and from EVs isolated from serum samples of NSCLC patients treated with osimertinib in second line within the phase II clinical trial TREM. The protein cargo of the EVs was analysed by mass spectrometry (MS) and proximity extension assay (PEA). Western blotting, ELISA and single vesicle analysis was performed to validate and further confirm the expression of certain proteins. MS profiling of the NSCLC cells and their released EVs revealed a protein signature associated with osimertinib refractoriness. Among them were CSPG4, HSPG2, MCAM, L1CAM, TAGLN, THBS1 and TNC. GO-pathway analysis related several of these proteins to the focal adhesion and proteoglycan in cancer pathways. Some of these proteins, including CSPG4, which when suppressed by transient siRNA transfection in NSCLC cells resulted in reduced cell viability, were expressed also in EVs from serum of the NSCLC patients. Moreover, PEA profiling of the serum-isolated EVs revealed signatures associated with immune cells, best response and/or progression-free survival, including PD-L1, CD73/NT5E, FR-alpha/FOLR1, LAMP3, FASLG1 and ANXA1. In summary, we demonstrate that protein profiling of EVs in relation to osimertinib refractoriness has the potential to identify possible biomarkers that can indicate osimertinib treatment resistance, for example, CSPG4, HSPG2, TAGLN, TNC, THBS1, ANXA1 and CD73/NT5E. Studies in expanded cohorts should be conducted to further validate these putative osimertinib biomarkers.

The intestine plays a crucial role in regulating metabolism and immunity, with functional decline occurring during injury and ageing. Stimulating the neogenesis of intestinal stem cells (ISCs) by activating the WNT/β-catenin signalling pathway represents a promising approach for intestinal tissue regeneration and injury repair. However, effective oral delivery of functional WNT signalling agonists to the gut remains challenging. Herein, we report a potent WNT/β-catenin signalling-inducing small extracellular vesicles (sEV) that can be administered orally and present remarkable therapeutic efficacy. We demonstrate that active R-spondin1 (RSPO1) protein can be loaded onto the surface of sEV via heparan sulfate proteoglycans. Notably, sEV-delivered RSPO1 (evRSPO1) effectively induces WNT/β-catenin signalling-inducing activity, enhances ISCs proliferation, and supports intestinal organoid growth in vitro. Importantly, oral administration of evRSPO1 activates the WNT/β-catenin signalling pathway in the cryptic stem cell niche, thereby accelerating tissue repair and regeneration in a radiation-induced intestinal injury model. Furthermore, evRSPO1 treatment induces ISCs proliferation and reverses the intestinal senescence phenotype in aged mice. Collectively, this study establishes evRSPO1 as a potential first-in-class, orally deliverable therapeutic that overcomes biological barriers to activate ISCs, enabling efficient intestinal tissue repair and rejuvenation.

Extracellular vesicle (EV) secretion is an important, though not fully understood, intercellular communication process. Lipid metabolism has been shown to regulate EV activity, though the impact of specific lipid classes is unclear. Through analysis of small EVs (sEVs), we observe aberrant increases in sEV release within genetic models of cholesterol biosynthesis disorders, where cellular cholesterol is diminished. Inhibition of cholesterol synthesis at multiple synthetic steps mimics genetic models in terms of cholesterol reduction and sEVs secreted. Further analyses of sEVs from cholesterol-depleted cells revealed structural deficits and altered surface marker expression, though these sEVs were also more easily internalized by recipient cells. Transmission electron microscopy of cells with impaired cholesterol biosynthesis demonstrated multivesicular and multilamellar structures potentially associated with autophagic defects. We further found autophagic vesicles being redirected towards late endosomes at the expense of autophagolysosomes. These findings were subsequently validated in cellular models of head and neck cancer, where cholesterol depletion induced sEV release and promoted late endosome-autophagosome fusion. Through CRISPR-mediated inhibition of autophagosome formation, we mechanistically determined that release of sEVs after cholesterol depletion is autophagy dependent. We conclude that cholesterol imbalance initiates autophagosome-dependent secretion of sEVs, which may have pathological relevance in diseases of cholesterol disequilibrium.