Journal of extracellular biology最新文献

Human milk extracellular vesicles (EVs) are crucial mother-to-baby messengers that transfer biological signals. These EVs are reported to survive digestion and transport across the intestine. The mechanisms of interaction between human milk EVs and the intestinal mucosa, including epithelial uptake remain unclear. Here, we studied the interaction of human milk EVs with the gut barrier components, including intestinal biofluids, enzymes, mucus and epithelium. Additionally, we probed the endocytic mechanisms mediating the EV intestinal uptake. Finally, using proteomic analysis, we determined the existence and identification of proteins enriched in the EV fraction transported across the intestinal epithelium. We show that human milk EVs are largely stable in the biochemical gut barriers and demonstrate high mucus diffusivity. EVs show a high level of epithelial cell uptake (∼70%) and efficient transport across Caco-2 monolayers. Whilst cell uptake of EVs was mediated by multiple routes, none of the pathway-specific inhibitors inhibited their epithelial translocation. Proteomic analysis of EVs transported across Caco-2 monolayers identified 14 enriched EV proteins that may facilitate intestinal transport. These findings significantly expand our understanding of the interactions between human milk EVs and the gut barriers, including their intestinal uptake.

Current state-of-the-art tools for analysing extracellular vesicles (EVs) offer either highly sensitive but unidimensional bulk measurements of EV components, or high-resolution multiparametric single-particle analyses which lack standardization and appropriate reference materials. This limits the accuracy of the assessment of marker abundance and overall marker distribution amongst individual EVs, and finally, the understanding of true EV heterogeneity. In this study, we aimed to define the standardized operating procedures and reference material for fluorescent characterization of EVs with two commonly used EV analytical platforms-nanoparticle tracking analysis (NTA) and nano-flow cytometry (nFCM). We achieved quantitative fluorescence analyses on ZetaView NTA and NanoAnalyzer nFCM instruments, by utilizing yellow-green FluoSpheres (FS) with assigned ERF (equivalent reference fluorophore) values. This standardization technique allowed for fluorescent EV signal to be expressed in ERF units (indicative of bound fluorescent antibodies per EV), thus enabling measurement of target protein marker abundance on individual EVs, and in the whole EV population. The NTA's and nFCM's limits of detection (LoD) were evaluated at 21 and 9 Alexa Fluor 488 (AF488) molecules, respectively. To complement the limited quantification of markers expressed in a few copies per single EV, in-line bulk fluorescence measurements with a plate reader were performed. This provided absolute marker quantification and more insightful analyses of EV heterogeneity and marker stoichiometry. The standardization method outlined in this work unlocks the full analytical potential of NTA and nFCM, enabling cross-platform data comparison. At the same time, it highlights some of the technical challenges and considerations and thus contributes to the ongoing efforts towards the development of EV analytical tools.

Hallal, S. M., Sida, L. A, Tűzesi, Á., Shivalingam, B., Sim, H.-W., Buckland, M. E, Satgunaseelan, L., & Alexander, K. L (2024). Size matters: Biomolecular compositions of small and large extracellular vesicles in the urine of glioblastoma patients. Journal of Extracellular Biology, 3, e70021. https://doi.org/10.1002/jex2.70021

In the originally-published article, author Ágota Tűzesi's name was incorrectly given as Csilla Ágota Tűzesi. This has been corrected in the online version of the article.

We apologize for this error.

Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molecules. They have emerged as potential circulating biomarkers for monitoring responses to cancer therapies. This study aimed to comprehensively characterize plasma-carried EVs in hormone receptor-positive (HR⁺) metastatic breast cancer (MBC) patients treated with first-line CDK4/6 inhibitors (iCDK4/6) combined with endocrine therapy. MBC patients were classified into three groups based on their response to therapy: resistant, intermediate or sensitive. In a prospective cohort, we monitored the concentration of circulating EVs, analyzed their lipid signature and correlated these factors with treatment response. To facilitate the translation of EV research to clinical practice, we established a three-step procedure: (1) EVs were isolated from plasma using semi-automatized size exclusion chromatography (SEC); (2) EV concentration, termed vesiclemia, was determined by drop counting via interferometric light microscopy (ILM); and (3) EV lipid composition was analyzed by mass spectrometry. ILM-based vesiclemia values were highly fluctuating upon iCDK4/6 treatment, while early increase associated with accelerated progression. Of note, vesiclemia remained a steady parameter over a 1-year period in age-matched healthy women. Additionally, analysis of the EV cargo unveiled a distinct sphingolipid profile, characterized by increased levels of ceramides and sphingomyelins in resistant patients within the first 2 months of treatment. Based on 16 sphingolipid species, sensitive and resistant patients were correctly classified with an overall accuracy of 82%. This specific sphingolipid pattern was exclusively discernible within EVs, and not in plasma, highlighting the significance of EVs in the early prediction of individual responses to iCDK4/6 and disease progression. Overall, this study provides insights of the longitudinal characterization of plasma-borne EVs in both a healthy group and HR⁺ MBC patients under iCDK4/6 therapies. Combined vesiclemia and EV sphingolipid profile emphasize the promising potential of EVs as non-invasive biomarkers for monitoring early treatment response.

Spirulina is an edible cyanobacterium that increasingly gaining recognition for it untapped potential in the biomanufacturing of pharmaceuticals. Despite the rapidly accumulating information on extracellular vesicles (EVs) from most other bacteria, nothing is known about Spirulina extracellular vesicles (SPEVs). This study reports the successful isolation, characterization and visualization of SPEVs for the first time and it further investigates the potential therapeutic benefits of SPEVs using a mouse model. SPEVs were isolated using ultracentrifugation and size-exclusion-chromatography. Cryo-Transmission Electron Microscopy revealed pleomorphic outer-membrane-vesicles and outer-inner-membrane-vesicles displaying diverse shapes, sizes and corona densities. To assess short- and long-term immune responses, mice were injected intraperitoneally with SPEVs, which demonstrated a significant increase in neutrophils and M1 macrophages at the injection site, indicating a pro-inflammatory effect induced by SPEVs without clinical signs of toxicity or hypersensitivity. Furthermore, SPEVs demonstrated potent adjuvanticity by enhancing antigen-specific IgG responses in mice by over 100-fold compared to an unadjuvanted model vaccine antigen. Mass-spectrometry identified 54 proteins within SPEVs, including three protein superfamily members linked to the observed pro-inflammatory effects. Our findings highlight the potential of SPEVs as a new class of vaccine adjuvant and warrant additional studies to further characterize the nature of the immune response.

Extracellular vesicles (EVs) are small membrane-bound structures that originate from various cell types and carry molecular cargos to influence the behaviour of recipient cells. The use of EVs as biomarkers for diagnosis and as delivery vehicles for treatment in a wide range of human disease is a rapidly growing field in research and clinical practice. We hypothesized that electric fields (EFs) could influence the release and content of EVs. To examine this hypothesis, we developed a specialized bioreactor enabling cells to thrive in a three-dimensional setting, replicating in-vivo conditions amidst programmable EF environments. We established a three-step EV purification protocol to achieve high-density production of EVs. We also performed mass spectrometry-based proteomics analysis on EV-carrying proteins and used high-resolution nanoparticle flowcytometry for single-vesicle analysis. Findings from this report suggest that electrical stimulation, employing physiologically relevant amplitudes typical in therapeutic deep brain stimulation, influences the release of EVs and their cargo content in a frequency-dependent fashion. This conclusion could carry significant implications for both fundamental biological understanding and medical advancements. First, it raises an intriguing question about how the endogenous electrical activity of neuronal and other cellular assemblies influence the production and composition of EVs. Second, it reveals a novel underlying mechanism of how therapeutic electrical stimulations can modulate EVs and treat human brain disorders. Third, it provides a novel approach to utilize electrical stimulation for generating desired EV cargos in a programmable setting.

Multivesicular bodies (MVBs) are vesicles of endosomal origin containing intraluminal vesicles, which upon fusion with plasma membrane, secrete exosomes. They play a significant role in the physiology and pathology of type-2 diabetes (T2D) due to disrupted intercellular communication. The role of MVBs and their influence on insulin secretory granules (ISGs) of β-cells or their characterization is yet to be uncovered. In our study, we compared MVBs to largely well-characterized ISGs in β-cells. This study compares the density, localization, and exocytosis of CD63+ compartments (CD63+c) with NPY labelled ISGs (NISGs) in β-cells. For this, tetraspanin CD63 was exploited to majorly label MVBs in β-cells. These labels preserve the structural integrity of labelled compartments and mostly do not localize with other endo-lysosomal compartments. This study showed that the β-cells have a significantly higher density of NISGs than CD63+c. CD63+c and NISGs are spatially localized apart within β-cells. The proteins that localize with CD63+c are different from the ones that localize with NISGs. Exocytosis of NISGs occurs at the periphery of the β-cells and takes significantly less time when compared to the release of CD63+c, which is non-peripheral and takes a longer duration. Mechanistically, the availability of CD63+c for exocytosis was assessed and found that an equilibrium is maintained between docking and undocking states at the plasma membrane. Although there are a high number of short-term residing, visiting CD63+c at the plasma membrane, the availability of CD63+c for exocytosis is maintained due to docking and undocking states. Further, a significant reduction in the density of NISGs and CD63+c was observed in β-cells isolated from T2D donors compared to healthy counterparts. Studying the effect of MVBs on insulin secretion in physiological and T2D conditions has huge potential. This study provides a strong basis to open new avenues for such future studies.

Urinary extracellular vesicles (uEVs) are a promising substrate for discovering new biomarkers. In order to investigate the origin of uEVs and the cargo they carry, some types of downstream analysis of uEVs may require concentration and enrichment as well as removal of contaminating substances. Co-isolation of the abundant urinary protein uromodulin with uEVs can be a problem, and may interfere with some techniques, in particular with proteomic analysis tools. Methods of separating out uromodulin and its removal have also not been standardized. This review highlights aspects of uromodulin structure that makes it recalcitrant to separation from uEVs, summarizes frequently used techniques for uEV enrichment and how they affect uromodulin separation, and specific methods for uromodulin removal during preparation of uEVs. The necessity of uromodulin removal for various study endpoints is also examined.

The promise of urinary extracellular vesicles (uEVs) in biomarker discovery is emerging. However, the characteristics and compositions of different uEV subpopulations across normal physiological and pathological states require rigorous explication. We recently reported proteomic signatures of small (s)-uEVs (<200 nm membranous nanoparticles) and described putative biomarkers corresponding to the diagnosis, tumour burden and recurrence of the lethal adult primary brain tumour, glioblastoma. Here, we comprehensively characterise uEV populations with significantly different mean and mode particle sizes obtained by differential centrifugation at 100,000 × g (100K-uEVs; smaller) and 17,000 × g (17K-uEVs; larger) using Fourier-transform infrared spectroscopy and quantitative data-independent acquisition mass spectrometry. We show distinct differences in protein and lipid content, prominent protein secondary structures, and proteome distributions between uEV populations that can distinguish glioblastoma patients from healthy controls and correspond to clinically relevant tumour changes (i.e., recurrence and treatment resistance). Among the key findings is a putative seven-protein biomarker panel associated with 17K-uEVs that could distinguish all glioblastoma patients from healthy controls and accurately classify 98.2% of glioblastoma samples. These novel, significant findings demonstrate that both uEV populations offer individual and combined biomarker potential. Further research is warranted to elucidate the complete diagnostic, prognostic, and predictive capabilities of often-neglected 17K-uEV populations.

Exosomes are cell-derived nanovesicles that play a crucial role in intercellular communication, presenting promising potential as biomarkers and therapeutic agents. Bovine milk exosomes (MK-Exo) show production scalability and cost-effectiveness, offering distinct advantages over cell-derived exosomes. However, exosome storage and transportation are challenging owing to their unstable nature, necessitating preservation at ultralow temperatures. Research findings suggest that freeze-drying could provide a viable solution; however, different sources of exosomes may require specific protocols. In this study, we aimed to successfully isolate high-purity MK-Exo and develop a specialized freeze-drying and lyophilization method for improved long-term preservation of MK-Exo. Specifically, the stability of the lyophilized MK-Exo was evaluated using storage stability tests. Notably, lyophilized MK-Exo remained stable for at least 3 months under high temperature of 50°C and for at least 24 months under low temperatures of 2°C–8°C, preserving their physicochemical properties and biological activity. Conclusively, these findings provide a potential solution for ambient-temperature transportation of MK-Exo, facilitating their industrial-scale production.