ABSTRACT Plasma proteins are increasingly recognized as key regulators of extracellular vesicle (EV) behaviour in circulation. However, von Willebrand factor (vWF)—the largest multimeric glycoprotein in blood and the only blood protein activated by shear—has received little attention in this context. Under elevated shear stress, vWF transitions from a compact globular form to an extended adhesive conformation. Although elevated plasma vWF levels are associated with hypercoagulation in conditions such as malaria, COVID‐19, and cancer, its interactions with EVs or cells under physiological flow have not been systematically investigated. Here, we show that shear‐activated vWF functions as a size‐selective molecular filter that preferentially captures objects smaller than ∼4 µm, including platelets and tumour‐derived EVs, while excluding larger cells. Although intact tumour cells do not directly bind vWF under physiological flow, the coordinated binding of EVs and platelets to extended vWF promotes platelet aggregation, which subsequently traps circulating tumour cells and fosters metastatic dissemination. Our findings reveal a previously unrecognized role of vWF as a shear‐dependent EV‐binding protein that brings EVs and platelets together to enhance coagulation and metastasis. These EV–vWF–platelet aggregates may represent promising biomarkers or therapeutic targets for the prevention of hypercoagulation in cancer and other thrombo‐inflammatory diseases.

Solid tissue-derived extracellular vesicles (ST-EVs) are extracellular vesicles (EVs) separated directly from solid tissues of both vertebrates and invertebrates. ST-EVs provide a physiologically relevant snapshot of tissue-specific molecular dynamics and can be enriched directly in situ, from tissues in their natural state, preserving the native characteristics of ST-EVs. However, their enrichment presents unique technical challenges compared to EVs derived from biofluids or cell culture media. The need for transparent reporting in ST-EV research is crucial to enhance the reproducibility, comparability, and reliability of research findings. The Solid Tissue Task Force, part of the Scientific Reproducibility Subcommittee of International Society for Extracellular Vesicles, aims to recommend reporting parameters and identify outstanding questions related to the pre-analytical and analytical handling of solid tissues, as well as ST-EV separation and characterization. These steps are essential for advancing the understanding of the biological roles of ST-EVs and their potential clinical applications.

Separating and enriching specific extracellular vesicle (EV) subpopulations from the broader EV pool present in tissues and blood is crucial for understanding their role in physiological and pathological conditions. However, high-purity enrichment of specific EV-subpopulations remains challenging due to the lack of suitable techniques. Initial studies have shown that Fluorescence-Activated Cell Sorting (FACS) has great potential for enriching EV subpopulations, despite the technical challenges posed by their small size. Yet, existing protocols have been inconsistent, and proper validation using state-of-the-art sorters has been inadequate. Here, we introduce an EV sorting workflow that overcomes technical challenges and allows for the analysis of EVs from various species, tissue sources and cell culture. We used two fluorescence cell sorters, the BD FACSAria Fusion and the BD FACSDiscover S8, to sort EVs with different fluorescent labels. The successful sorting of EVs was validated using high-sensitivity imaging flow cytometry, transmission electron microscopy, and liquid chromatography tandem mass spectrometry. We defined the optimal parameters for nozzle sizes, flow rates, sample dilutions, and sorting modes, enabling the enrichment of EV populations of interest to nearby 100% purity, including low-frequency EV populations of under 10%, while preserving compatibility with downstream analyses. The workflow presented here provides a powerful tool for both, basic science and translational applications. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64 German Center for Child and Adolescent Health Schilling Foundation T. Von Zastrow Foundation Fielmann Foundation Werner-Otto Stiftung Else Kröner-Fresenius-Stiftung Jung-Stiftung für Wissenschaft und Forschung Studienstiftung des deutschen Volkes

Extracellular vesicles (EVs) act as carriers of biological information from tumors to the bloodstream, enabling the detection of circulating tumor material and tracking of disease progression. This is particularly crucial in glioblastoma, a highly aggressive and heterogeneous tumor that is challenging to monitor. Using imaging flow cytometry (IFCM), we conducted an immunophenotyping analysis of eight glioma-associated antigens and tetraspanins in plasma EVs from 37 newly diagnosed glioblastoma patients (pre- and post-surgery), 11 matched individuals with recurrent glioblastoma, and 22 healthy donors (HD). Tenascin-C (TNC) positive EVs displayed the strongest differences in newly diagnosed and recurrent glioblastoma patients, when compared to non-tumor subjects. Among dual-positive subpopulations, TNC+/CD9+ EVs were the most elevated in newly diagnosed (FC = 7.6, p <0.0001, AUC = 81%) and recurrent patients (FC = 16.5, p <0.0001; AUC = 90%) than HD. In comparison with other CNS tumors (n = 25), this subpopulation was also 34.5-fold higher in glioblastoma than in meningioma cases (p <0.01). Additionally, TNC+/CD9+ EV levels were 3.3-fold elevated in cerebrospinal fluid from glioblastoma patients (n = 6) than controls (p <0.05). Aberrant TNC levels were further observed in glioblastoma EVs from different sources and purified via different methods. Immunohistochemical analysis revealed high levels of TNC in tumor tissues. Spatial transcriptomic analysis indicated a TNC overexpression in malignant cell populations of glioblastoma resections, particularly in cells with mesenchymal-like signatures and chromosomal aberrations. Lastly, we purified TNC+ EVs from plasma of 21 glioblastoma patients by magnetic sorting and detected the oncogenic mutation TERT*C228T by droplet digital PCR. The mutant allele frequency was higher in TNC+ EVs vs TNC-negative EVs (FC = 32, p <0.001), total EVs (FC = 5.3, p <0.001) or cell-free DNA (FC = 5.3, p <0.01). In conclusion, circulating TNC+ EVs may have potential as clinical biomarkers in glioblastoma, and their purification could improve the identification of tumor-specific mutations in liquid biopsies.

Alzheimer’s disease (AD) is the most common form of dementia, characterized by progressive amyloid-β (Aβ) accumulation and neurodegeneration. Extracellular vesicles (EVs) have been implicated in AD pathology, but their relationship to disease progression and the cerebrospinal fluid (CSF) proteome remains poorly understood. In this study, we analysed plasma-derived Aβ42-containing EVs from participants of the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and calculated the ratio between the percentage of Aβ42-positive peripheral EVs and CSF Aβ42 (rAβ42). This ratio was elevated in AD and positively correlated with amyloid pathology as measured by PET. Longitudinal analyses revealed that higher rAβ42 values predicted accelerated hippocampal atrophy and cognitive decline, independent of CSF Aβ42 concentrations. Proteomic profiling of CSF using the SomaScan platform showed that rAβ42 was associated with an inflammatory signature characterized by myeloid activation and type II interferon-related signalling pathways. Together, these findings indicate that Aβ42-carrying EVs reflect an active and inflammatory component of AD pathology and may represent a novel class of biomarkers for disease progression. Understanding the mechanisms that link Aβ42-positive EVs to neuroinflammatory signalling could open new avenues for pathway-specific diagnostics and therapeutic strategies in AD. ### Competing Interest Statement MSW received honoraria from Lilly for educational lectures outside the scope of this manuscript. MK received honoraria from Lilly outside the scope of this work. SG serves on scientific advisory boards for Alzeon, AmyriAD, Advantage, Eisai Canada, Enigma USA, Lilly Canada, Medesis, Lundbeck Foundation, Novo-Nordisk Canada, Okutsa, and TauRx outside the scope of this manuscript. GT has received honoraria as consultant or lecturer from Acandis, Bayer, Boehringer Ingelheim, BMS/Pfizer, Lilly, and TarGED. Corona Foundation Else-Kröner-Fresenius Foundation

Von Willebrand factor (vWF), promoting platelet aggregation in various diseases such as COVID-19, malaria and cancer, is a huge multimeric glycoprotein. This extraordinary size makes vWF a unique shear stress sensing molecule. Below a critical shear stress, vWF is in a globular conformation that prevents platelet binding. Above the critical shear stress, vWF is stretched into platelet accessible fibers. Although previous studies have suggested that leukocytes or cancer cells can bind to vWF fibers, acting forces and the likelihood of cell adhesion has remained largely unexplored. Here, we report that vWF is a size-selective protein that prefers to interact with objects smaller than 4 μm in diameter. Consistently, tumor cell-derived extracellular vesicles (EVs) were able to interact with vWF in parallel to platelets. Although whole tumor cells under flow were unable to bind to vWF per se , binding of EVs and platelets along the vWF fiber promoted platelet aggregation, which in turn entrapped circulating tumor cells. In conclusion, our study highlights the shear-sensitive nature of vWF and its ability to bring EVs and platelets together to enhance coagulation. While EVs-vWF-platelet aggregates may serve as novel biomarkers, their therapeutic disruption may prevent hypercoagulation in disease. ### Competing Interest Statement The authors have declared no competing interest. Mildred Scheel Cancer Career Center HaTriCS4 at University Medical Center Hamburg-Eppendorf German Research Foundation within priority program 2416 CodeChi German Research Foundation, GRK 2873 ‘Tools and Drugs of the Future’ Hamburg Pro Exzellenzia Plus Scholarship Program

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC–MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

Ischemic stroke represents a significant global health challenge, often resulting in death or long-term disability, particularly among the elderly, where advancing age stands as the most unmodifiable risk factor. Arising from the blockage of a brain-feeding artery, the only therapies available to date aim at removing the blood clot to restore cerebral blood flow and rescue neuronal cells from death. The prevailing treatment approach involves thrombolysis by administration of recombinant tissue plasminogen activator (tPA), albeit with a critical time constraint. Timely intervention is imperative, given that delayed thrombolysis increases tPA leakage into the brain parenchyma, causing harmful effects. Strategies to preserve tPA's vascular benefits while shielding brain cells from its toxicity have been explored. Notably, administering neuroserpin (Ns), a brain-specific tPA inhibitor, represents one such approach. Following ischemic stroke, Ns levels rise and correlate with favorable post-stroke outcomes. Studies in rodent models of focal cerebral ischemia have demonstrated the beneficial effects of Ns administration. Ns treatment maintains blood-brain barrier (BBB) integrity, reducing stroke volume. Conversely, Ns-deficient animals exhibit larger stroke injury, increased BBB permeability and enhanced microglia activation. Furthermore, Ns administration extends the therapeutic window for tPA intervention, underscoring its potential in stroke management. Remarkably, our investigation reveals the presence of Ns within extracellular vesicles (EVs), small membrane-surrounded particles released by all cells and critical for intercellular communication. EVs influence disease outcome following stroke through cargo transfer between cells. Clarifying the role of EVs containing NS could open up urgently needed novel therapeutic approaches to improve post-ischemic stroke outcome.

The direct interplay between the immune and nervous systems is now well established. Within the brain, these interactions take place between neurons and resident glial cells, i.e., microglia and astrocytes, or infiltrating immune cells, influenced by systemic factors. A special form of physical cell–cell interactions is the so-called “neuroimmunological (NI) synapse.” There is compelling evidence that the same signaling pathways that regulate inflammatory responses to injury or ischemia also play potent roles in brain development, plasticity, and function. Proper synaptic wiring is as important during development as it is during disease states, as it is necessary for activity-dependent refinement of neuronal circuits. Since the process of forming synaptic connections in the brain is highly dynamic, with constant changes in strength and connectivity, the immune component is perfectly suited for the regulatory task as it is in constant turnover. Many cellular and molecular players in this interaction remain to be uncovered, especially in pathological states. In this review, we discuss and propose possible communication hubs between components of the adaptive and innate immune systems and the synaptic element in ischemic stroke pathology.

Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain-derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV-associated proteins in brain tissue homogenates and cell-derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces 'artificial' proteolytic processing in key BDEV markers, such as Flotillin-1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non-enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non-enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near-physiological setting, thus opening new research approaches.

On April 13-14th, 2023, more than 50 researchers located in Hamburg, mainly working at the University Medical Center Hamburg-Eppendorf (UKE), met to present research projects focusing on extracellular vesicles (EVs). The meeting was supported by State Research Funding (LFF-FV74: Mechanisms of cell communication in Infection; Speaker Maura Dandri, UKE), which aims to develop existing and promising research areas further. This meeting certainly surpassed all expectations: what was first organized as a tentative get-together between a dozen EV scientists, soon revealed the existence of a much broader and strong EV community with more than 50 basic researchers and clinicians currently active in Hamburg. This first UKE-EV meeting created a platform to share experiences, projects, and protocols and mainly discuss future requirements to create a consolidated structure and network in Hamburg, enabling state-of-the-art EV research.

In the last decades, the role of the prion protein (PrP) in neurodegenerative diseases has been intensively investigated, initially in prion diseases of humans (e.g., Creutzfeldt-Jakob disease) and animals (e.g., scrapie in sheep, chronic wasting disease in deer and elk, or "mad cow disease" in cattle). Templated misfolding of physiological cellular prion protein (PrPC) into an aggregation-prone isoform (termed PrP "Scrapie" (PrPSc)), self-replication and spreading of the latter inside the brain and to peripheral tissues, and the associated formation of infectious proteopathic seeds (termed "prions") are among the essential pathogenic mechanisms underlying this group of fatal and transmissible spongiform encephalopathies. Later, key roles of the correctly folded PrPC were identified in more common human brain diseases (such as Alzheimer's disease or Parkinson's disease) associated with the misfolding and/or accumulation of other proteins (such as amyloid-β, tau or α-synuclein, respectively). PrPC has also been linked with neuroprotective and regenerative functions, for instance in hypoxic/ischemic conditions such as stroke. However, despite a mixed "bouquet" of suggested functions, our understanding of pathological and, especially, physiological roles played by PrPC in the brain and beyond is certainly incomplete. Interactions with various other proteins at the cell surface or within intracellular compartments may account for the functional diversity linked with PrPC. Moreover, conserved endogenous proteolytic processing of PrPC generates several defined PrPC fragments, possibly holding intrinsic functions in physiological and pathological conditions, thus making the "true and complete biology" of this protein more complicated to be elucidated. Here, we focus on one of those released PrPC fragments, namely shed PrP (sPrP), generated by a membrane-proximate ADAM10-mediated cleavage event at the cell surface. Similar to other soluble PrPC fragments (such as the N1 fragment representing PrP's released N-terminal tail upon the major α-cleavage event) or experimentally employed recombinant PrP, sPrP is being suggested to act neuroprotective in Alzheimer's disease and other protein misfolding diseases. Several lines of evidence on extracellular PrPC (fragments) suggest that induction of PrPC release could be a future therapeutic option in various brain disorders. Our recent identification of a substrate-specific approach to stimulate the shedding by ADAM10, based on ligands binding to cell surface PrPC, may further set the stage for research into this direction.

Cell-cell interactions in the central nervous system (CNS) are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles (EVs). The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of glial cells in the retina, which forms part of the CNS. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct EVs from highly specialized domains of Müller cells in retinae from adult mice in vivo . We identify VAMP5 as a Müller cell-specific SNARE component that is part of EVs and responsive to ischemia, and we reveal differences between the secretomes of affinity-purified Müller cells and neurons in vitro . Our findings suggest EV-based communication as an important mediator of cellular interactions in the retina. suggested that these cells vesicular release of but the full range of their intercellular communication capacity is unknown. Our findings suggest that Müller cells release EVs from their endfeet facing the vitreous body and their microvilli surrounding photoreceptor segments. We show that Müller cell-derived EVs have a characteristic protein composition that differs substantially from those secreted by neurons.

The prion protein (PrP) is a broadly expressed glycoprotein linked with a multitude of (suggested) biological and pathological implications. Some of these roles seem to be due to constitutively generated proteolytic fragments of the protein. Among them is a soluble PrP form, which is released from the surface of neurons and other cell types by action of the metalloprotease ADAM10 in a process termed ‘shedding’. The latter aspect is the focus of this review, which aims to provide a comprehensive overview on (i) the relevance of proteolytic processing in regulating cellular PrP functions, (ii) currently described involvement of shed PrP in neurodegenerative diseases (including prion diseases and Alzheimer’s disease), (iii) shed PrP’s expected roles in intercellular communication in many more (patho)physiological conditions (such as stroke, cancer or immune responses), (iv) and the need for improved research tools in respective (future) studies. Deeper mechanistic insight into roles played by PrP shedding and its resulting fragment may pave the way for improved diagnostics and future therapeutic approaches in diseases of the brain and beyond.

Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles. The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of radial glial cells in the retina, which forms part of the central nervous system. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct extracellular vesicles from endfeet and microvilli of retinal Müller cells in adult mice in vivo . We identify VAMP5 as a Müller cell-specific SNARE component that is part of extracellular vesicles and responsive to ischemia, and we reveal differences between the secretomes of immunoaffinity-purified Müller cells and neurons in vitro . Our findings suggest extracellular vesicle-based communication as an important mediator of cellular interactions in the retina.

Extracellular vesicles (EVs) are lipid bilayer-enclosed structures that represent newly discovered means for cell-to-cell communication as well as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA, eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 h after experimental stroke in mice and compared them to controls (shams) using nCounter® Nanostring panels, with or without prior RNA isolation. We found that both panels show similar results when comparing upregulated mRNAs in stroke. Notably, the highest upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, thus indicating that regenerative mechanisms already take place at this time-point. The five top overrepresented mRNAs in stroke mice were confirmed by RT-qPCR and, interestingly, found to be full-length. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and predominantly present in small BDEVs (≤ 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation, and that the mRNA cargo of BDEVs indicates a possible participation in inflammatory but also recovery processes after stroke.

The human macula is a highly specialized retinal region with pit‐like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone‐ and rod‐rich retinae from human and mice and identified different expression profiles of cone‐ and rod‐associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell‐derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region‐specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

Extracellular vesicles (EVs) are double membrane structures released by presumably all cell types that transport and deliver lipids, proteins, and genetic material to near or distant recipient cells, thereby affecting their phenotype. The basic knowledge of their functions in healthy and diseased brain is still murky and many questions about their biology are unsolved. In neurological diseases, EVs are regarded as attractive biomarkers and as therapeutic tools due to their ability to cross the blood–brain barrier (BBB). EVs have been successfully isolated from conditioned media of primary brain cells and cerebrospinal fluid (CSF), but protocols allowing for the direct study of pathophysiological events mediated or influenced by EVs isolated from brain have only recently been published. This review aims to give a brief overview of the current knowledge of EVs’ functions in the central nervous system (CNS) and the current protocols to isolate brain-derived EVs (BDEVs) used in different publications. By comparing the proteomic analysis of some of these publications, we also assess the influence of the isolation method on the protein content of BDEVs.