Extracellular vesicles (EVs) are highly attractive biomarkers for liquid biopsies, a minimally invasive approach for disease detection and monitoring based on the sampling of biofluids. With the aim to foster interaction between EV scientists and clinical experts in laboratory medicine and diagnostics, and thereby support translational EV research, the German Society for Extracellular Vesicles (GSEV) teamed up with INSTAND, the Interdisciplinary Group for Laboratory Medicine (IGLD) and the German Stem Cell Network (GSCN). This year, the annual joint meeting of these societies took place on March 6-7, 2025, at the Kap Europa in Frankfurt. The interdisciplinary gathering featured 117 talks and brought together more than 800 participants from academia and industry to exchange recent advances in the field of laboratory medicine, quality assurance, translational research, and EVs. In this meeting report, we will summarize the key insights and perspectives of the seven sessions, which had been organized by GSEV and which covered a broad range of topics from basic EV biology, their role in different diseases to their use as biomarkers in the clinic.
EV Biology: From Membrane Architecture to Cellular Responses
The question if and how EVs are specifically taken up at target cell membranes to deliver their messages, continues to intrigue EV biologists and was the overarching theme of this session. It began with a presentation by Heiko Herwald from Lund University, who explored the role of EVs for the delivery of inflammatory cargo in sepsis. He showed that EVs from lipopolysaccharide (LPS)-stimulated monocytes can attach to macrophages and transfer membrane and intracellular components, such as TNFα and MyD88, to recipient cells by fusing with the target cell membrane (Papareddy et al, 2024). Notably, MyD88-deficient cells were activated via NF-kB after exposure to these EVs, suggesting that the vesicles are capable of delivering fully functional signalling complexes. Matthias Kneussel from Hamburg University shared his research on protein targeting to EVs and their subsequent interaction with neuronal membranes. Knockdown of the trafficking factor Muskelin/MKLN1 impaired the lysosomal degradation of prion proteins, resulting in their increased packaging into EVs (Heisler et al, 2018). Using primary mouse cortical neuron cultures and knockout models, his group demonstrated that Tspan15 is a core component of EVs released by neurons. By combining live-cell imaging and functional knockout of Tspan15, they showed that EVs lacking Tspan15 exhibited significantly impaired docking to target neurons, while Tspan15-deficiency at the recipient cell membrane had no effect (Stajano et al, 2023). These results identified Tspan15 as a vesicle-intrinsic factor crucial for efficient EV-neuron interaction, independent of target cell expression. Reinhard Lipowsky, a physicist, provided insights into the biophysical aspects of lipid bilayer dynamics. Through molecular dynamics simulations, he identified that small lipid imbalances between the inner and outer leaflets of the bilayer can induce significant shape transitions, such as budding or fusion, driven by lipid asymmetry rather than protein interactions (Lipowsky, 2024). These findings imply that, in some contexts, EV biogenesis and fusion might be primarily governed by lipid-driven processes, independent of protein-mediated pathways. The session concluded with Stefan Momma from Frankfurt University, who presented his novel data on the ABCG2 transporter and its role in stem cell quiescence. He underscored the importance of EV-mediated ceramide export in maintaining the undifferentiated state of stem cells by preventing membrane domain formation. Using pharmacological inhibitors and genetic knockout models, he demonstrated ABCG2 exports ceramide via EVs, as its inhibition led to ceramide accumulation in cells, disrupting membrane microdomains and triggering spontaneous cell differentiation.
New Methods and Approaches for EV Analysis
In recent years, an even greater heterogeneity among EVs has been revealed than previously anticipated (Menck & Preußer, 2024). Accurate and reliable methods are crucial to isolate, characterize, and quantify specific EV subpopulations and distinguish disease-related from normal EVs. This session therefore focused on single EV analytics and the harmonization of methods, as both are essential for bridging EV research across labs and translating it into the clinic. Erdinc Sezgin from Karolinska Institutet introduced his Single Particle Profiler (SPP), a confocal-based system that can analyse bioparticles, such as EVs, with high resolution (Sych et al, 2025). By tracking fluorescent peaks from labelled particles, it is possible to calculate membrane fluidity, size and relative cargo loading. Using this method, he observed that membrane stiffness varied between EVs and correlated with disease states such as cardiovascular risk. This high-content profiling enables discrimination of disease-associated EV subtypes and could facilitate early diagnosis or risk stratification. Daniel Bachurski, a physician scientist at the University of Cologne, presented "TeLEV", a novel tellurium-based metabolic labelling method for EV proteomes, allowing tracking of EV uptake and proteome transfer at the level of single recipient cells, which is not achievable by conventional labelling methods. By supplementing cells with tellurium-labelled phenylalanine, EVs incorporate the label during protein synthesis. Using healthy PBMCs, he identified preferential EV uptake by monocytes and dendritic cells and observed a disease-specific immune signature following exposure to chronic lymphocytic leukaemia-derived EVs. The neurosurgeon Franz Ricklefs from the University Medical Centre Hamburg-Eppendorf described the standardized collection of liquid biopsy samples from glioblastoma (GBM) patients. Using imaging flow cytometry to immunophenotype plasma-derived EVs, he identified Tenascin-C as a clinical biomarker for GBM and as a tumor antigen allowing to enrich tumor-EVs from the circulation by magnetic activated cell sorting (MACS) (Salviano-Silva et al, 2025). Compared to conventional tissue biopsies, this non-invasive EV-based approach enabled not only longitudinal monitoring but increased the detection sensitivity of relevant tumor mutations. Following the mission on method standardisation, Britta Bettin, a junior post-doc at UMC Amsterdam, highlighted the need for reproducible and comparable concentration measurements of EVs in blood samples using flow cytometry to enable comparison of EV data across different instruments and labs, a prerequisite for clinical validation. Presenting a large interlaboratory comparison involving 25 different flow cytometers, she illustrated that the use of pre-labelled EVs, calibration beads, and protocol harmonization successfully reduced variability in concentration measurements by two thirds. Calibration with standard particles transforms fluorescence and scatter data into the quantitative MESF unit of fluorescence intensity, or nanometres, respectively, facilitating correct gating and differentiation between EVs and cells (Bettin et al, 2023).
From Pathogen Defence to Disease Modulation: The Diverse Functions of EVs in Immunity
EVs are key mediators of communication within the immune system and play a crucial role in inflammation, antigen presentation, and immunity (Buzas, 2023). This session focused on their dual role in host defence against various pathogens and in susceptibility to disease. In the first talk, Pierre-Yves Mantel from the University of Freiburg focused on malaria tropica caused by infection with the parasite Plasmodium falciparum and demonstrated that EVs can suppress resistance to secondary infections: Infected red blood cells release EVs containing abundant miRNAs, including miR-451a, which modulates neutrophil function. The EVs impair reactive oxygen species production, bacterial killing and neutrophil swarming in both primary human neutrophils and the HL-60 neutrophil-like cell line. Pre-incubation of neutrophils with infected EVs led to upregulation of HMOX1 (HO-1), a gene involved in cytoprotective responses and immune tolerance. In vivo, knockout mice lacking miR-451a specifically in red blood cells showed improved neutrophil function and bacterial clearance following a double infection (malaria and salmonella) (Babatunde et al, 2025). Susanne Erdmann from the Max-Planck Institute for Marine Microbiology in Bremen studied EVs in archaeal and marine microbial systems. She showed that EVs from halophilic archaea can bind to and neutralize viruses by acting as decoys, mimicking the host cell surface and absorbing viral particles. Looking at marine microbial EVs using the Tara Oceans dataset, her group revealed that up to 40% of sequences in marine viromes are non-viral, likely derived from EVs (Lücking et al, 2023). In their most recent work, they identified ArvA, a small GTPase, as an essential and conserved driver of EV biogenesis in halophilic archaea. Disruption of ArvA almost completely abolished the release of RNA-containing EVs, directly linking this protein to vesicle formation and cargo selection. This represents a mechanistic parallel to eukaryotic vesicle trafficking and highlights the evolutionary connection of archaeal and eukaryotic EV pathways (Mills et al, 2024). Corissa Visser, a PhD student at Leibniz-HKI, studied neutrophil-derived EVs in the context of infections with Aspergillus fumigatus. Her work showed that infection-induced EVs differ in their proteomic cargo and show antifungal activity, as evidenced by mitochondrial fragmentation of Aspergillus hyphae and reduced growth (Shopova et al, 2020). Using a novel imaging approach, a direct interaction between EVs and fungal structures was visualized (Visser et al, 2024). Immune cells also play an important role in fibrosis (Huang et al, 2020). In this regard, Mareike Lehmann from the Philipps University in Marburg focused on how EVs modulate lung ageing and fibrotic disease, focusing on idiopathic pulmonary fibrosis. She highlighted that senescent cells, which accumulate with age, release a senescence-associated secretome, much of which is packaged in EVs. These senescence-EVs were particularly enriched in pro-inflammatory and extracellular matrix-modifying factors. On a functional level, EVs isolated from aged mice or fibrotic lungs impaired the regenerative capacity of alveolar epithelial cells in organoid cultures and precision-cut lung slices. Notably, SFRP1 was identified as a key pro-fibrotic mediator on fibroblast-derived EVs, and loss of SFRP1 abrogated the fibrogenic effects of these vesicles in vivo (Burgy et al, 2024). Together, all of these studies highlight the multifaceted roles of EVs in immune regulation and underscore the potential for harnessing EVs in therapeutic approaches of infectious and inflammatory diseases.
Crossing Barriers: EVs in Brain Development and Neurological Diseases
The role of EVs in the neurological system continues to intrigue the field. In particular, the question if, and how, EVs can cross the blood-brain-barrier and target specific cell populations in the brain remains a matter of discussion that was a main focus of this session. Stefan Liebner from Goethe-University Frankfurt Medical School provided a closer insight into the biology of the blood-brain-barrier (BBB) which varies considerably by species and site, and likely changes over time. Using genetic mouse models, Liebner’s team demonstrated that endothelial-specific activation of β-catenin signalling converted the typically leaky vasculature of the subfornical organ into a more restrictive, BBB-like phenotype. This tightening of the vessel barrier was shown to impact neuronal activation in response to water deprivation, illustrating a direct link between vascular properties and brain function (Benz et al, 2019). He proposed that these leaky regions might serve as hotspots for EV exchange between blood and brain, especially under pathological conditions. As an example, he showed that BEAS-2B lung epithelial cells treated with particulate matter (PM2.5) secrete EVs that induced alterations in the tight junctions of mouse brain microvascular endothelial cells, consequently reducing their transepithelial barrier function. Silvia Cappello from LMU Munich demonstrated the importance of EV-mediated communication during brain development. She focused on how radial glial precursors, neurons and astrocytes are involved in EV-mediated communication during brain development. Proteomic analysis of EVs isolated from cerebral organoids at different developmental stages revealed dynamic and cell type-specific cargo patterns (Forero et al, 2024). Using super-resolution microscopy, her team found that RFP-labelled EVs from the different cell populations were preferentially taken up by specific recipient cell populations and differed in their subcellular distribution: While progenitors internalised EVs into the nucleus, neurons retained them docked on the membrane and astrocytes in the cytoplasm. Moving to the role of EVs in neurological disorders, David Otaegui, head of the neuroscience department at the Biogipuzkoa Health Research Institute in Spain, presented his research on EVs as biomarkers in multiple sclerosis (MS). Using the ExoView technology, he showed that the tetraspanin profile of EVs is distinct between serum and cerebrospinal fluid (CSF). L1CAM was suggested as a marker for neuronal-derived EVs in CSF, albeit with low specificity, which differed between MS disease stages, but not comparing healthy controls and MS patients (Bravo-Miana et al, 2024). Analyses of plasma EVs revealed a variety of circular RNAs with disease- and stage-specific expression patterns. Treatment with fingolimod altered both the abundance and miRNA content of plasma EVs, indicating therapy-related changes in EV cargo. In addition, bacterial RNA was detected in plasma-derived EVs, suggesting a possible influence of the microbiome on neuroinflammation, although the biological significance of this finding remains to be clarified. Berta Puig from the University Medical Center Hamburg-Eppendorf studied EVs in a mouse stroke model. Based on the observation that astrocyte markers were increased in the penumbra, the area around the stroke, she characterized EVs derived from astrocytes originating from the ischemic penumbra area by immunoprecipitation and proteomic analyses. The results revealed an enrichment of synaptic proteins 24 h post-stroke, possibly reflecting the attempt of astrocytes to rescue neuronal damage. She identified IL-17 as an important mediator of post-ischemic brain damage, which is released by astrocytes and induces a detrimental recruitment of neutrophils. IL-17 blockade reduced infarct size.
EVs as Modulators of the Tumor Microenvironment and Biomarkers in Cancer
EVs have evolved as central mediators of communication between cancer cells and their surrounding microenvironment. Understanding the specific mechanisms of EV-induced stroma cell reprogramming and establishing them as reliable biomarkers, or therapeutic targets, in cancer, remain central focus areas of EV research in Germany (cf. results of the GSEV study 2025 detailed in this issue). In chronic lymphocytic leukemia, Jérôme Paggetti from the Luxembourg Institute of Health highlighted the role of tumor-derived EVs in creating a favourable immune microenvironment. Packed with checkpoint ligands such as PD-L1 and immunosuppressive miRNAs, EVs isolated directly from CLL tissue suppressed T cell activation as well as the expression of granzyme B and perforin (Gargiulo et al, 2023). When CLL-EV secretion was abrogated by Rab27a/b knockdown, EVs were no longer able to suppress T cell function, thus slowing disease progression. When EVs were reintroduced, this effect was restored. In line with these results, Viviane Ponath, a post-doc at Philipps-University Marburg, confirmed an immunomodulatory role of EVs in the tumour microenvironment. She observed that BAG6, a ligand for NKG2DL, is loaded onto EVs and stimulated NK cell tumor killing activity. Pancreatic ductal adenocarcinoma patients with low BAG6 expression had a poor overall survival. In line, knocking out BAG6 in mice resulted in faster tumour growth and reduced the infiltration of T cells in the tumor microenvironment. Mechanistically, the knockout of BAG6 was associated with the release of IL33-containing tumor-EVs that induced mast cell activation and promoted tumor growth (Alashkar Alhamwe et al, 2024). Shifting towards the influence of tumor-EVs on neighbouring cancer cells, Kendra Maaß from the German Cancer Research Centre presented her research on pediatric ependymomas, which often harbour mesenchymal traits associated with poor prognosis. Using proteomics and high-content imaging, she showed that CD44-high mesenchymal tumors secrete EVs enriched in mesenchymal markers, which promote epithelial-to-mesenchymal transition (EMT). Inhibition of EV release with different inhibitors (e.g. GW4869, fluoxetine) reverted the EMT phenotype and reduced tumour cell invasion in vitro, while slowing tumor growth in mouse xenografts and prolonging survival in vivo. Combining the EV inhibitor fluoxetine with radiotherapy had an additional therapeutic benefit and might be a promising strategy for the treatment of aggressive brain tumors. Zoltan Takats from Imperial College London focused on lipidomics of EVs as a non-invasive diagnostic tool for breast and ovarian cancer. He showed that cancer cell lines and their EVs differ in the lipid profile, with triglycerides and phosphatidylglycerol being enriched in cells, and sphingolipids, in particular ceramides, in EVs. In cell line models, EVs were correctly classified to cancerous or non-cancerous as well as to the respective subtype based on their lipid profile. In a pilot study in breast cancer patients, his team identified consistent lipid signatures that distinguish cancerous from healthy states and tumour subtypes (Dorado et al, 2024). Together, these studies highlight not only the functional diversity of EVs within the tumor microenvironment but also emphasize the urgent need for standardization and validation of EV-based biomarkers to enable their translation into clinical applications.
EVs in Liquid Biopsies: Insights from DNA, RNA, and Protein Profiling
Despite the promise of EVs as minimally invasive biomarkers for cancer detection and therapy monitoring, several challenges currently hinder their clinical translation, including the often low sensitivity and specificity of EV biomarker assays, as well as the unresolved question of which biofluid is most suitable for certain tumor subtypes. This joint session together with the IGLD addressed these issues and presented several examples illustrating how EV biomarkers identified in vitro can be translated to clinical diagnostics. Basant Thakur, group leader at the University Hospital Essen, reviewed his work on the detection of double-stranded DNA in EVs, which can recapitulate the mutation status of the parent tumour. However, he found that only a fraction of EVs carry DNA, making bulk measurements insensitive. While sequencing of EV DNA provides a high fidelity match to the tumour genome, allowing identification of cancer type and detection of driver mutations (e.g. BCR-ABL in K562 cells), clinical translation to patient plasma samples remains challenging due to low sensitivity. Following up on the detection of nucleic acids in EVs, PhD student Christian Grätz from the Technical University of Munich described an in vitro biomarker discovery pipeline using the anaplastic thyroid cancer cell line Cal62 treated with vandetanib, a multi-kinase inhibitor used to treat thyroid cancers. Cellular transcriptomics and proteomics revealed dose-dependent responses, which were used to define 21 biomarker candidates, among which eight were detected in cell-free RNA. Four showed consistent and significant expression changes (NDRG1, ISG upregulated; FOXN1, TPX2 downregulated). Principal component and regression analysis confirmed that the biomarker signature discriminates treated from control samples, with strong classification performance at higher drug doses. Continuing the analysis of EV-associated RNA, Marie-Nicole Theodoraki, clinician scientist at Technical University of Munich, presented a comprehensive study of EVs as diagnostic and prognostic biomarkers in head and neck squamous cell carcinoma, comparing plasma and saliva as biofluids. Her group showed that EVs from patients differ in protein (e.g., PD-L1, CD16, CD44v3) and RNA profiles from healthy donors. Tumor-exclusive miRNAs were identified and correlated with disease stage, HPV status and recurrence. Saliva-derived EVs showed higher tumour specificity, particularly in PD L1 and CD44v3 expression, suggesting that saliva is more informative for tumor diagnostics in head and neck cancer. Junior group leader and current GSEV president Kerstin Menck from the University of Münster focused particularly on large EVs (lEVs) as a source of predictive biomarkers in non-small cell lung cancer. Using flow cytometry, her group identified six lEV-associated markers (PD-L1, EMMPRIN, EGFR, MUC1, ROR1, ROR2) to be significantly elevated in patients compared to controls. Surprisingly, PD-L1 was more highly expressed on lEVs than on small EVs. Importantly, patients with high PD-L1+ lEVs at baseline responded better to immunotherapy, especially those with negative or low PD-L1 expression in tissue biopsies, identifying PD-L1+ lEVs as a novel predictive biomarker for this patient subgroup, which outperformed tissue biopsies as current gold standard (Schöne et al, 2024). Overall, these presentations underscore both the potential and the current limitations of EV-based biomarkers, particularly regarding assay sensitivity, specificity, and the optimal choice of biofluid for different cancer subtypes. Addressing these challenges will be essential for successful clinical implementation.
Advances and Challenges in EV Therapeutics
EVs have rapidly gained attention as promising therapeutic agents due to their natural ability to transport biomolecules between cells, influence cellular behaviour, and traverse biological barriers. Their low immunogenicity and toxicity as well as high biocompatibility and stability make them very attractive tools for targeted therapy (Akbar, 2021). The current advances in EV engineering for therapeutics were introduced by the keynote talk of André Görgens from the Karolinska Institutet in Stockholm in the joint session of GSEV with the IGLD and INSTAND. He presented a new bioengineering method using multiplex bead-based EV flow cytometry to sort EV domains loaded with the protein of interest. EVs can be functionalized with an antibody-binding moiety specific for Fc domains, allowing decorating them with selected monoclonal antibodies to specifically target them to cancer tissue. The approach has already been successfully applied to melanoma mouse models, in which Fc-EVs decorated with the PD-L1 antibody Atezolizumab accumulated in the tumor tissue and, upon loading with the chemotherapeutic drug doxorubicin, suppressed tumor growth and prolonged animal survival (Wiklander et al, 2024). Translating EVs as therapeutics into the clinic demands more than data, it requires consensus on production, identity, and function. This topic was discussed in the session on EV therapy, which was held together with the GSCN. Augustas Pivoriunas, Head of the Department of Stem Cell Biology at the State Research Institute Centre for Innovative Medicine in Vilnius, presented his method for scaling up mesenchymal stem cell (MSC) -derived EV production for Parkinson's disease therapy. Using bioreactor systems and Good Manufacturing Practice (GMP) -grade protocols, his group increased EV yield and maintained product quality. Isolated EVs reduced α-synuclein aggregation and supported the survival of dopaminergic neurons in vitro. In vivo, treated mice showed improved motor protein function, underlining the clinical potential of the isolated EVs. Eva Rohde, Head of the Department of Transfusion Medicine at the University Hospital Salzburg, then took a critical look at the regulatory challenges, but also the opportunities, of bringing EV-based therapeutics to the clinic. Challenges include ensuring GMP compliance, product consistency and developing potency assays to measure biological activity. She noted problems with poor reporting and lack of efficacy data in many clinical studies on EV-based therapeutics. Early regulatory engagement is key for successful translation. Karen Bieback from the University Medicine Mannheim raised the question of whether MSC-derived EVs are indeed the primary therapeutic agents or whether the effects arise from the full secretome of the cells. Despite widespread attribution of therapeutic effects to MSC-EVs in various organs, her group observed significant equipment- and operator-dependent technical variability and was unable to replicate the immunosuppressive or pro-angiogenic effects using EVs alone. In their hands, conditioned medium and protein-rich fractions harboured most of the activity, raising the question of whether the EV fraction is indeed the true therapeutic agent, or whether we should rethink our product definitions. This question was critically followed up in a panel discussion with the three speakers, who were supported by Halvard Bönig, Bernd Giebel and Daniel Besser. While no consensus was achieved on the question, all panellists agreed that there is an urgent need for standardized assays with robust controls, more reproducible in vivo models, and dose-response data.
With 28 EV-related talks spanning fundamental science, technological advances, therapeutic development, and diagnostics, the joint IGLD-INSTAND-GSEV meeting 2025 has highlighted EVs not only as functional cargo carriers fulfilling important tasks in health and disease, but also as versatile diagnostic and therapeutic tools. It has become clear that the field is moving from bulk to single-EV analysis. Rigorous reporting as well as standardized workflows are no longer optional; they are a prerequisite for reproducible science. Clinical EV research must prioritize biofluid selection, cell-of-origin tracking, and regulatory alignment in order to foster translation. The close collaboration between basic scientists, clinicians, and industry partners will be key to overcoming remaining hurdles and ensuring that EV research continues to deliver impactful solutions for patient care in the coming years.
Acknowledgements
We thank all chairs of the meeting for taking and providing notes. MD is a member of CiM-IMPRS, the joint graduate school of the Cells-in-Motion Interfaculty Center of the University of Münster and the International Max Planck Research School-Molecular Biomedicine in Münster. This work was funded by the Wilhelm Sander-Stiftung (project 2022.139.1) and the Interdisciplinary Center for Clinical Research (IZKF) Münster (project Hai4/007/25).
