Journal of extracellular biology最新文献

Therapeutic proteins and peptides have revolutionized modern biomedicine, but their oral delivery is limited by gastrointestinal degradation and barriers. Small extracellular vesicles (sEVs), which are resistant to biochemical degradation and capable of traversing mucus and cellular barriers, hold great promise as next-generation oral delivery vehicles. Oral semaglutide, the first approved oral GLP-1 receptor agonist (GLP-1RA), employs vesicle-mediated transcellular transport, highlighting the potential of sEVs as an effective delivery vehicle. In this study, we demonstrate the successful oral delivery of two GLP-1RAs, semaglutide and previously unexplored tirzepatide, using milk-derived sEVs. Both peptides were efficiently loaded onto sEVs in vitro, and their oral administration effectively reduced blood glucose levels in diabetic db/db mice. Compared with the current SNAC technology, which is limited exclusively to semaglutide, our sEV platform provides broader applicability and versatility for oral peptide drug delivery.

Neural stem cell (NSC)-based therapies offer a promising strategy to promote brain repair by delivering neurotrophic factors, supporting cell replacement, and stimulating endogenous neurogenesis following injury. While numerous studies have highlighted the protective and regenerative potential of NSCs and their extracellular vesicles (EVs), progress toward clinical translation remains hindered by limited molecular characterization of NSC lines and their EV cargo. To address this gap, we characterized two therapeutically relevant human fetal NSC lines, LMNSC01 and LMNSC02, both engineered to express the L-MYC gene, along with their corresponding EVs. LMNSC01 cells primarily differentiated into neurones with limited glial populations, whereas LMNSC02 cells gave rise to all three major neural lineages: neural, glial and oligodendrocyte progenitor cells (OPCs). scRNA-seq revealed distinct transcriptional profiles with minimal overlap between the two LMNSC lines. Using single extracellular vesicle nanoscopy, we observed that both lines released predominantly circular EVs, with LMNSC02-EVs exhibiting higher levels of tetraspanins (CD9, CD63, and CD81) and a larger average diameter than LMNSC01-EVs. Proteomic profiling revealed that LMNSC01-EVs are enriched in proteins involved in cell adhesion, migration, junction formation, and neuronal projection development, while LMNSC02-EVs are enriched in factors related to cytoplasmic translation initiation and biosynthesis. These LMNSC-EVs (collected from undifferentiated LMNSCs) demonstrated neuroprotective effects in a brain organoid model of methotrexate-induced toxicity when added to corresponding LMNSC01- or LMNSC02-derived brain organoids. LMNSC01- and LMNSC02-derived EVs restored neuronal and astrocytic populations but failed to rescue OPCs. These findings demonstrate the therapeutic potential of LMNSC-derived EVs to counter chemotherapy-induced neurotoxicity by preserving neurones and astrocytes, while highlighting the need for repeated or complementary interventions to restore oligodendrocyte populations.

HIV-1 proteins and RNA are incorporated into extracellular vesicles (EVs) via the EV biogenesis machinery. Due to their similar size and content, EVs and HIV-1 particles are hard to separate, and current purification methods often overlook EVs' effects on infectivity. This study co-characterized HIV-1 particles and three EV subtypes to assess their impact on infection. The HIV-infected Raji CD4 DCIR cells' supernatants were harvested 2 and 8 days after infection. The 2-day supernatant was treated with proteinase K to discard viral components outside the EVs. The supernatants were fractionated into three pellets by differential centrifugation: 3K, 17K and 100K. EVs and viral particles were co-characterized for their host and viral contents and the pellets obtained after 8 days post-infection were tested for infectivity. Proteinase K reduced HIV-1 RNA in EVs without affecting p24 concentration. The p24 protein was mostly found in the 17K pellet and HIV-1 RNA was the most abundant in the 100K pellet for both 2- and 8-day productions. Nevertheless, the 3K pellet had the highest infectivity when cells were infected with an equal quantity of virus. Each EV subtype were co-purified with functional virus and uniquely influenced HIV-1 infectivity, underscoring the importance of considering EVs in viral preparations.

Extracellular vesicles (EVs) from blood plasma are promising biomarkers, as they carry surface markers indicative of their cell of origin. Size-exclusion chromatography (SEC) is commonly employed for EV enrichment, but the choice of pore size and plasma volume can significantly impact the yield, purity, and composition of isolated EVs. In this study, we systematically compared Izon SEC columns with pore sizes of 35 and 70 nm, using either 500 µL plasma (qEVoriginal, “small” column) or 10 mL plasma (qEV10, “large” column). Due to limited material obtained from small columns, fractions had to be pooled for downstream analyses, precluding detailed characterization of individual fractions. In contrast, the larger columns provided sufficient material to analyse each fraction separately, across multiple platforms, including nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), single-EV flow cytometry, MACSPlex surface protein array, immunoblotting, and LC-MS/MS. These analyses consistently identified fractions 1–3 as “EV-rich,” characterized by enrichment of EV markers and reduced levels of abundant plasma proteins. Moreover, a comparison of pore sizes demonstrated that the 70 nm column yielded a higher EV recovery with improved purity compared to the 35 nm column, including a greater abundance of immune cell-derived markers. Together, these findings established that the large 70 nm SEC columns are optimal for isolating EV-rich fractions from plasma, maximizing both EV yield and purity, while minimizing non-EV contaminants.

The tumour microenvironment (TME) constitution is decisive for cancer outcome and is manifested in diffusion-weighted (DW) magnetic resonance imaging (MRI). We hypothesized that the TME metabolic state is reflected by mitochondrial DNA (mtDNA) secreted in extracellular vesicles (EVs) and examined whether plasma EV-mtDNA variants may divulge MRI-assessed TME attributes of rectal cancer aggressiveness. On the diagnostic MRI scans from 60 rectal cancer patients, the apparent diffusion coefficient (ADC) was calculated on DW images (n = 29), and tumour volume (n = 57) and extramural vascular invasion (EMVI; all patients) were determined on anatomical images. Plasma EVs (all patients) were isolated by size exclusion chromatography and verified for EV features. The EV-mtDNA was sequenced along with mtDNA in whole blood (WB; normal tissue) to calculate the EV/WB-mtDNA total variant number (TVN) and heteroplasmic variant number (HVN)—as a proxy for TME intracellular mtDNA variants expelled in EVs. Low EV/WB-mtDNA TVN and HVN, indicative of hampered clearance of mutated mtDNA via EVs, were associated with low ADC (high TME cell density; p = 0.018, p = 0.005) and a large tumour volume (p = 0.002, p = 0.003). Likewise, low EV/WB-mtDNA TVN and HVN were associated with positive EMVI (tumour infiltration in blood vessels; p = 0.002, p = 0.003) and histologic ypN stage 1–2 (lymph nodes with tumour cells surviving radiotherapy; p = 0.002, p = 0.005), both indicators of high tumour aggressiveness. High cellular density may hamper the clearance of pathogenic tumour mtDNA variants by EVs and thus promote rectal cancer aggressiveness.

Trial Registration: ClinicalTrials.gov: NCT01816607. Registered 22 March 2013, https://clinicaltrials.gov/ct2/show/NCT01816607

Wang, Q., R. Jing, N. Cao, et al. 2025. “ Generalizability of Enzyme-Based Isolation Approach for Extracellular Vesicles From Traditional Medicinal Plants.” Journal of Extracellular Biology 4, no. 10: e70090. https://doi.org/10.1002/jex2.70090.

In the originally published article, the incorrect grant number was given for the grant from the Tianjin Belt and Road Project. The correct grant number is given below.

Incorrect

18JCQNJC79400

Correct

24PTLYHZ00330

We apologize for this error.

Malignant melanoma has one of the lowest 5-year survival rates of any cancer and is characterised by its high invasiveness and metastatic potential, with especially poor outcomes in patients who develop brain metastases. Crosstalk between melanoma cells and cells of the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), is a central driver of disease progression. While the role of melanoma-derived small extracellular vesicles (sEVs) in reprogramming stromal cells has been well documented, the reciprocal effects of CAF-derived sEVs remain less clear. Here, using an in vitro model of melanoma CAFs, we show that CAF sEVs alter melanoma cells and fibroblasts to promote oncogenic traits and remodel endothelial cells, including brain microvascular cells, in ways consistent with early pre-metastatic niche (PMN) changes. Multi-omics cargo profiling revealed significant differential expression of proteins and RNAs linked to extracellular matrix organisation, vascular remodelling, and patient outcomes, with functional validation identifying THBS1 as an EV cargo that restrains endothelial sprouting while potentially promoting barrier destabilisation. Together, these findings suggest that CAF-derived sEVs contribute to local and distal PMN remodelling, highlight their potential as therapeutic targets, and identify EV cargoes with promise as circulating biomarkers in melanoma.

Extracellular vesicles (EVs) have gained increasing attention in recent years due to their pivotal role in both health and disease. Emerging from both eukaryotic and prokaryotic cells, EVs serve as essential mediators of intercellular communication, exceeding the simplistic interactions observed with individual molecules. In this comprehensive review, we will focus on both Bacterial Extracellular Vesicles (BEV) and on Host derived Extracellular Vesicles (HEV) and highlight mechanistic principles, as well as their transformation into diagnostic and therapeutic tools. We will start with a short introduction into the biogenesis and principal properties of BEV and HEV. We will then focus on the composition of BEV and introduce OMICs-based studies that helped to unravel their complex constitution. As both BEV and HEV interact with different epithelial and endothelial barriers and shape their properties, we will highlight mechanistic principles for both EV types. Starting from the intestinal system, where we will look at BEV and how these BEV overcome the intestinal barrier to change distant organs and the patient's immune system. We will then visit other endothelial and epithelial sites of the human body and summarize how HEV shapes these barriers and how HEV can overcome these barriers. We will then turn towards diagnostic and therapeutic approaches. As both BEV and HEV are currently suggested as diagnostic markers and are being investigated as potential therapeutic agents. Lastly, we will discuss current challenges and provide an outlook on the future in the field. This review seeks to raise awareness for both bacterial and host-derived EVs, highlighting that they present two sides of the same coin.

Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbe-derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remains unclear. This study investigated the role of OMVs and OMV-associated small RNA (sRNA) species from pathogenic and commensal Bacteroides fragilis (ETBF and NTBF, respectively) in eliciting different immune responses from intestinal epithelial cells. To distinguish the differences in the sRNA profiles of the two strains and their OMVs, RNA-seq, qRT-PCR, and northern blotting were conducted to identify enrichment of discrete sRNA species in OMVs, which were also differentially expressed between the two strains. Specifically, coding and non-coding RNAs were enriched in OMVs from NTBF and ETBF, with BF9343_RS22680 and BF9343_RS17870 being significantly enriched in ETBF OMVs compared to NTBF. To understand the effects of OMVs on pattern recognition receptors, reporter cells of Toll-like receptor (TLR) activation were treated with OMVs, demonstrating activation of TLRs 2, 3, and 7. Treatment of Caco-2 and HT29-MTX cells with OMVs demonstrated increased expression of IL-8. Surprisingly, we discovered that degradation of RNase-accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL-8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV-associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications.

Small extracellular vesicles (sEVs), ranging from 30 to 200 nm in diameter, are a subset of extracellular vesicles that play an essential role in intercellular communication by transporting bioactive molecules between cells. Due to their diverse cargo and origins, sEVs form a highly heterogeneous population. Previous studies have suggested that recipient cells uptake specific sEVs to ensure accurate signalling, but direct experimental evidence on how cells effectively communicate in such a complex and noisy environment has been lacking. In this study, we provide direct experimental evidence by developing a method to retrieve and analyze internalized sEVs from the intracellular membrane compartments of recipient cells. Using this sEV retrieval method, we isolated sEV subpopulations from two types of breast cancer cells after incubation with sEVs derived from mixed cell cultures. We then assessed their functional roles by evaluating the phenotypic responses of cells treated with these retrieved sEV subpopulations. Our results reveal apparent differences in their functional impacts, indicating that cells employ a function-based mechanism to selectively uptake sEVs. This finding advances our understanding of how cells enhance communication efficiency via sEVs. Further investigation into this phenomenon could offer deeper insights into intercellular communication and its role in health and disease.