Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

ABSTRACTParacrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

<sec><p indent="0mm">Extracellular vesicles (EVs) are released by living cells and play important roles in transporting information between cells. EVs contain physiological and pathological information of the parent cell and can dynamically transmit disease signals. According to different biological pathways, EVs are often classified into apoptotic bodies, microvesicles, exosomes, etc. Due to the size overlap between different vesicles and the inability to determine their origin based on size, accurate differentiation between exosomes and microvesicles is highly challenging. To avoid misunderstandings, the International Society for Extracellular Vesicles (ISEV) recommends using small and large vesicles to distinguish them from each other. </sec><sec> EVs have become a new star in the field of tumor liquid biopsy, due to the high variability of biomarkers available. However, the rapid and accurate separation of specific EVs from complex body fluids or cell culture supernatants is an urgent problem. Many researchers are committed to developing new and efficient methods and technologies for EV separation, including size-based ultracentrifugation, size exclusion chromatography, and ultrafiltration; polymer precipitation method based on hydrophobic interactions; immunoaffinity method based on antigen-antibody affinity; affinity methods based on DNA aptamers, microfluidic methods based on size and immune affinity; and so on. With the development of EV isolation and characterization methods, researchers have developed various EV nucleic acid and protein analysis techniques and conducted clinical trials. In recent years, several products have been commercialized, transforming scientific research into productivity. </sec><sec> DNA, RNA, and proteins are the most commonly used targets for analysis, such as nucleic acid testing during epidemics and influenza, which uses qRT-PCR technology to analyze virus DNA or RNA and tumor marker screening during physical examinations, which analyzes broad-spectrum cancer-related proteins such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigens (CA). EVs are rich in DNA, RNA, and proteins. By analyzing a single component or a combination of multiple components, real-time detection of tumor occurrence, metastasis, and prognosis can be achieved. Nanoscale EVs have played a role in enriching biomarkers, with higher sensitivity than plasma detection. Moreover, the membrane of EVs can effectively prevent RNA and other biomarkers from being degraded by various enzymes present in plasma, further enhancing the sensitivity of EV biomarker detection. Encouraged by the latest research progress, we review the application of EVs in tumor liquid biopsy, introducing the application and progress of EV DNA, RNA, protein, and other biomarkers. </sec><sec> Although EVs have great potential, the road ahead is full of difficulties. To achieve large-scale application of EVs in clinical practice, the following problems must be solved. The first is the separation problem. Currently, there are several methods for separating EVs, but none can efficiently separate EVs or even EV subgroups produced by specific cells without introducing impurities. The second is large-scale production. Currently, the main way to obtain EVs is through the separation of cell culture and culture medium. For adherent cells, the surface area of the culture environment limits the number of EVs produced. The third is standardization. ISEV and the Chinese Society for Extracellular Vesicles (CSEV) have developed guidelines and group standards for the naming, acquisition, isolation, characterization, and reporting of EVs. However, there is still no consensus on how to ensure inter-batch stability, absolute quantification, yield, and quality control of EVs, which makes clinical translation difficult. We look forward to these issues being resolved as soon as possible, taking EV research and application to a new level, and thereby benefiting humanity. </sec>

Liver plays a central role in elimination of circulating extracellular vesicles (EVs), and it also significantly contributes to EV release. However, the involvement of the different liver cell populations remains unknown. Here, we investigated EV uptake and release both in normolipemia and hyperlipidemia. C57BL/6 mice were kept on high fat diet for 20–30 weeks before circulating EV profiles were determined. In addition, control mice were intravenously injected with 99mTc-HYNIC-Duramycin labeled EVs, and an hour later, biodistribution was analyzed by SPECT/CT. In vitro, isolated liver cell types were tested for EV release and uptake with/without prior fatty acid treatment. We detected an elevated circulating EV number after the high fat diet. To clarify the differential involvement of liver cell types, we carried out in vitro experiments. We found an increased release of EVs by primary hepatocytes at concentrations of fatty acids comparable to what is characteristic for hyperlipidemia. When investigating EV biodistribution with 99mTc-labeled EVs, we detected EV accumulation primarily in the liver upon intravenous injection of mice with medium (326.3 ± 19.8 nm) and small EVs (130.5 ± 5.8 nm). In vitro, we found that medium and small EVs were preferentially taken up by Kupffer cells, and liver sinusoidal endothelial cells, respectively. Finally, we demonstrated that in hyperlipidemia, there was a decreased EV uptake both by Kupffer cells and liver sinusoidal endothelial cells. Our data suggest that hyperlipidema increases the release and reduces the uptake of EVs by liver cells. We also provide evidence for a size-dependent differential EV uptake by the different cell types of the liver. The EV radiolabeling protocol using 99mTc-Duramycin may provide a fast and simple labeling approach for SPECT/CT imaging of EVs biodistribution.

The International Society of Extracellular Vesicles (ISEV) is proud to launch a new, open access journal called the Journal of Extracellular Biology (J Ex Bio), covering research on extracellular vesicles (EVs), extracellular particles (EPs), and related biology and applications. With the incredible success of the Journal of Extracellular Vesicles (JEV), the ISEV Journal established in 2012, the Executive Board of ISEV began planning for a second journal in 2018. At the time, it was evident that the EV field was rapidly expanding and that the priority level for acceptance of articles into JEV was increasing. We felt that there was a clear need for another journal in the expanding field of EV research and related areas to fill in the knowledge gap from discoveries of newer types of particles and capture high-quality science and widen the scope of topics covered in this emerging scientific area. In 2019, Kenneth Witwer and Marca Wauben developed a scoping document for what the ISEV Executive Board was calling ‘Journal 2’ at the time. This important document, written in the closing hours of the ISEV 2019 Annual Meeting in Kyoto, Japan, helped frame the development of the Journal of Extracellular Biology. Since then, much work has been undertaken by the ISEV Executive Board (including the Communications and Membership committee led by Susmita Sahoo) to further refine the scope of the journal and work with our publishing partner Wiley, and Talley Management Group to develop a business plan for the journal. So, why the name ‘Extracellular Biology’? The journal is intended to be a vehicle for publishing EV research. Still, we felt there was scope to expand this to other EPs and entities interacting with, or involved in, EV biology. It is, however, increasingly appreciated that other EPs, many of which interact with and co-fractionate with EVs, also contribute to their biological activities. With EVs now established as key players in intercellular communication and other biological functions, they can be harnessed as biomarkers and therapeutics. The overarching vision of J Ex Bio is to provide a broader forum to discuss EVs and their EP cousins: from biogenesis, on to interactions with each other and with other extracellular macromolecules (including but not limited to RNA, lipoproteins and protein complexes), extracellular matrix, and from there to ultimate effects on recipient cells and organs. J Ex Bio will promote interdisciplinary studies investigating the function of EVs and EPs in pathology, normal physiology, interorgan and interorganism communication, their cargo as biomarkers and therapeutics; new technologies and methods to engineer EVs and EPs and their artificial mimics. The topics covered by J Ex Bio are still broadly tied to EVs and could constitute the ‘future core’ of research that the current EV field is constantly exploring and expanding. Additionally, J Ex Bio will allow ISEV to focus on broader themes than those within JEV, yet align with ISEV's mission to ‘advance EV research globally’. Key differences between the scope of J Ex Bio and JEV is that the scope of J Ex Bio is broader and will publish articles where EVs are not the primary focus, but other EPs are. Furthermore, J Ex Bio will also publish some of the products of ISEV Workshops and Taskforces where the topic meets the broader objectives of J Ex Bio. J Ex Bio is now open for submissions. In early 2021, Andrew Hill was appointed Editor in Chief and an editorial board of global leaders in the areas in which J Ex Bio covers has been established. The three Deputy Editors are Juan M. Falcon-Perez (Spain), Minh Le (Singapore) and Mỹ Mahoney (USA), who bring a wealth of scientific and editorial experience. The team of Associate Editors (see https://onlinelibrary.wiley.com/page/journal/27682811/homepage/editorial-board for the list) also provide scientific leadership to the team. We expect that J Ex Bio will become an important vehicle for publishing extracellular vesicle/particle research and contribute to the expanding scientific endeavours in this field, publishing original research articles, short reports, reviews and position papers. Fully open access, the journal will also provide wide reach for published articles. Article submissions can be made at the journal website (https://onlinelibrary.wiley.com/journal/27682811) which is available now.

Extracellular Vesicles: How a Circulating Biomarker Can Double As a Regulator of Blood Pressure.

CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling

Breast cancer (BC) recurrence and metastasis are significant causes of death, mainly due to the emergence of therapy-resistant cells. Secreted factors known as extracellular vesicles (EVs), especially small EVs (SEVs) or exosomes less than 100 nanometers in size, have been implicated in the plasticity associated with drug resistance, BC recurrence, and metastasis. EVs derived from BC cells can transform local or distal non-tumor cells, induce the epithelial-to-mesenchymal transition, and trigger uncontrolled proliferation, angiogenesis, and immune suppression of tumor cells. EVs also mediate resistance to hormone therapy and chemotherapy. Given these oncogenic activities, the production of EVs by BC cells presents an attractive target for inhibition. However, the complexity of EV biogenesis and uptake hampers the effectiveness of most pharmacological inhibitors that target single or specific EV synthetic pathways. Because protein synthesis and sorting are the initial steps in the biogenesis of EVs, we inferred that protein folding is a necessary part of EV synthesis. Previous studies revealed that the protein-folding complex, Chaperonin-Containing TCP-1 (CCT), is upregulated in cancer cells to mitigate the damage from misfolded proteins that accumulate due to increased somatic gene alterations and driver mutations. Using RNA sequencing and optimized methods of EV characterization by flow cytometry and nanoparticle tracking analysis (NTA), we determined that CCT has a role in the production of EVs by BC cells. Inhibition of CCT2, the second subunit of the CCT complex, in BC cells deregulated the gene expression of critical components involved in the early stages of EV biogenesis and decreased the release of CCT2-containing EVs. The exogenous expression of CCT2 in BC cells produced small EVs (SEVs) with increased and selective loading of CCT2 RNA and protein cargo. BC cell culture media, enriched for CCT2-containing SEVs using a 0.1 micromolar filter, increased CCT production and activity in recipient cells. Exogenously expressed CCT2 in BC cells also enriched gene expression associated with extracellular exosomes (GO:0070062). Hence, CCT may be essential for the biogenesis of SEVs with transformative properties and a promising therapeutic target for inhibiting cancer-causing EVs. These studies show that CCT directly regulates components of EV biogenesis in BC cells and advances CCT as an essential driver of BC progression through the production of oncogenic EVs. Citation Format: Annette R. Khaled, Carolyn Dang, Lam Truong, Sydney Laxton, James Velazquez, Shoba Kankipati, Priya Gopalan, SA Litherland. Slamming the breaks on extracellular vesicles that cause breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6584.

Most cells from different organisms release extracellular vesicles (EVs) to the extracellular environment. The term EV is a broad one for all types of vesicles found in body fluids and cell culture in accordance with the recommendation of the International Society for Extracellular Vesicles (ISEV).1 In recent years, Interest in EVs increased because of their function in intercellular communication and their potential use as biomarkers and therapeutics for several diseases.2,3 These EVs contain various DNAs, proteins, mRNAs, and microRNAs (miRNAs) that have potential diagnostic and therapeutic implications and are classified into exosomes, microvesicles, and apoptotic bodies through their biogenesis and secretion mechanisms. Exosomes are 50 to 150-nm vesicles released by living cells. Microvesicles are 100-to 2,000-nm vesicles formed by direct outward budding of the plasma membrane from living cells. Apoptotic bodies are 1-to 4-µm vesicles and released from the plasma membrane as apoptotic cell blebs.4 EVs act as a signaling complex, transfer membrane receptors between cells, deliver proteins to target cells, and modify the receiving cells by horizontal transfer of genetics.5 Many studies have suggested that EVs might be involved in a wide range of biological processes, including immune regulation, inflammation, and tumor development.6 EV have been isolated from different body fluids, such as plasma,7 urine,8 and bronchoalveolar lavage fluid (BALF).9 EVs are highly stable in biological fluids and protected from degradation by their lipid bilayer.4 They play important roles in maintaining homeostasis through intercellular communication in the human airway. Recently, their biological roles draw significant interest in respiratory diseases because their contents, including miRNA, play an important role in the pathogenesis of many respiratory diseases, including lung cancer, interstitial lung disease, chronic obstructive pulmonary disease, and allergic diseases like asthma.10 In the lungs, various types of cells, such as epithelial cells, fibroblasts, endothelial cells, tumor cells, stem cells, and immune cells, can release EVs. Alveolar macrophage-derived EVs can potentially control lung and airway inflammation through intercellular communication. Exposure to various stimuli, such as infection, DNA damage, and smoke exposure, enhances EV secretion and modify EV composition to change the surrounding microenvironment through EV-mediated cell-to-cell communication.11,12,13 Noncancerous cell-derived EVs in the airway show protective functions against injuries, such as tissue recovery and repair, but lung cancer-derived EVs regulate tumor malignancy.14 Exosome secretion is increased during allergic inflammation in the lungs, which may mediate increased intercellular signaling.6 A few studies have reported that EVs may regulate airway inflammation and allergic reactions through their paracrine effects in the lungs.15,16 BALF exosomes from healthy individuals and asthmatic patients exhibit distinct phenotypes and functions. In asthmatics, BALF exosomes may contribute to subclinical inflammation in airway epithelium.17 In 2010, Pegtel et al.17 reported that miRNAs of viral origin are found in EVs secreted by infected cells and that their transfer to non-infected cells led to the regulation of some target genes of these miRNA. It has recently been shown that, EVs derived from Esherichia coli into the bloodstream induced systemic inflammation mimicking sepsis18 and that Staphylococcus aureus-derived EVs are related to the pathogenesis of atopic dermatitis-like inflammation.18,19 In 2013, Kim et al.20 reported the relationship between EVs in indoor dust and neutrophilic pulmonary inflammation. Their study indicated that inhalation of indoor-dust EVs induce both Th1-and Th17-cellresponses and neutrophilic inflammation in the lungs. Furthermore, clinical data suggest that IgG1 sensitization to dust EVs may be related to the clinical manifestation of asthma symptoms in atopic children. Indoor-dust EVs may represent a novel target for the development of diagnostic tool for neutrophilic inflammation-induced airway diseases, such as neutrophilic asthma and COPD. In this current issue of the Allergy, Asthma & Immunology Research, Kim et al.21 reported that serum IgG antibody level to dust EVs in the 3 disease groups and healthy controls had a normal distribution and were significantly higher in patients with non-eosinophilic asthma, COPD or lung cancer than in healthy control subjects. Furthermore, multivariable analysis showed that a high serum anti-dust EV IgG concentration was an independent risk factor for non-eosinophilic asthma, COPD (irrespective of severity), and lung cancer (irrespective of cellular subtypes) after adjustment for age, gender, and cigarette smoking. Although additional prospective studies will be needed to determine whether dust EV exposure has a causal relationship with asthma, COPD, and lung cancer, these findings provide an new insight into the pathogenesis of non-eosinophilic asthma, COPD, and lung cancer, as well as a clue to developing novel diagnostic and/or therapeutic modalities.

A novel immunoassay for ApoB-100, the main protein component of lipoproteins, enables the development of methods to enrich extracellular vesicles from human plasma while depleting both lipoproteins and free proteins.

Understanding communication mechanisms between unicellular parasites is crucial in the development of novel therapies, as they rely on diverse modes of communication (like extracellular vesicle release, cytoneme, and filopodia formation) to regulate their behavior and survival.

Neuroblastoma (NB) is a pediatric tumor of neural crest origin accounting for 7% of cancers and 15% of cancer deaths. Solid malignant tumors commonly contain hypoxic areas that stimulate release of extracellular vesicles (EVs), lipid bound vesicles secreted by cells into the extracellular space. In our study we demonstrated that EVs from hypoxic NB cells promote pro-metastatic features in vitro and in an in vivo zebrafish model. We characterized the microRNA cargo of EVs from NB cells and compared their expression in hypoxia vs. normoxia, finding that miR-210-3p was the most upregulated. By using both in vivo and in vitro models we demonstrated that EVs released by hypoxic NB cells induced changes that favor a malignant phenotype in normoxic NB cells by transferring miR-210-3p. We showed that EVs isolated from cells transfected with miR-210-3p-mimic and cultured in normoxic condition, increased the migration ability and invasiveness of NB cells like EVs from hypoxic NB cells. Cells treated with EVs isolated from cells transfected with miR-210-3p-inhibitor and cultured in hypoxic condition were unable to stimulate migration and invasion as efficiently as EVs derived from control transfected cells, despite cells were cultured in hypoxic conditions. We then focused on the effects of EVs in metastatic niches formation in vivo. Intravascular injection within the zebrafish embryo allows a non-invasive visualization of EVs dispersion, uptake, and interactions with host cells. We first demonstrated that EVs released under hypoxic conditions promote angiogenesis and are more easily internalized by endothelial cells than those purified from normoxic cells. Furthermore, we proved via microscopy imaging and cell sorting that the group injected with hypoxic EVs had higher numbers of macrophages. We then focused on the region of the embryo called caudal hematopoietic tissue (CHT) as a potential metastatic site. After EVs injection, we evaluated through qPCR the expression of mmp9, required for HSPC (hematopoietic stem and progenitor cells) egress from the CHT, and cxcl8 that enhances HSPC colonization in the CHT. Results highlighted that the expression of these genes increased after hypoxic EVs injection. We showed that hypoxic EVs induce i. angiogenesis in vivo, required for tumor and metastasis growth, and ii. increased number of macrophages, a major contributor to the tumor microenvironment which produce growth factors and chemokines promoting tumor angiogenesis. Our data demonstrate that hypoxic EVs can modify the behavior of recipient cells in the CHT, as a potential metastatic site, to prepare a ‘fertile soil’ for cancer cells colonization. In conclusion, our findings suggest that EVs released by hypoxic NB cells induce changes that favor a malignant phenotype in normoxic NB cells by transferring miR-210-3p both in vitro and in vivo. Citation Format: Anna Fietta, Pina Fusco, Giuseppe Germano, Elisa Cimetta. Hypoxic extracellular vesicles induce a pro-metastatic phenotype in neuroblastoma: from the delivery of miR-210-3p to the preconditioning of a metastatic niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1320.

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge.

Targeting Extracellular Vesicle Secretion in Combination with Venetoclax Synergistically Induces Apoptosis in FLT3-ITD+ AML