Cancer seriously threatens the lives and health of people worldwide, and exosomes seem to play an important role in managing cancer effectively, which has attracted extensive attention from researchers in recent years. This study aimed to scientifically visualize exosomes research in cancer (ERC) through bibliometric analysis, reviewing the past, summarizing the present, and predicting the future, with a view to providing valuable insights for scholars and policy makers. Researches search and data collection from Web of Science Core Collection and clinical trial.gov. Calculations and visualizations were performed using Microsoft Excel, VOSviewer, Bibliometrix R-package, and CiteSpace. As of December 1, 2024, and March 8, 2025, we identified 8,001 ERC-related publications and 107 ERC-related clinical trials, with an increasing trend in annual publications. Our findings supported that China, Nanjing Medical University, and International Journal of Molecular Sciences were the most productive countries, institutions, and journals, respectively. Whiteside, Theresa L. had the most publications, while Théry, C was the most co-cited scholar. In addition, Cancer Research was the most co-cited journal. Spatial and temporal distribution of clinical trials was the same as for publications. High-frequency keywords were "extracellular vesicle," "microRNA" and "biomarker." Additional, "surface functionalization," "plant," "machine learning," "nanomaterials," "promotes metastasis," "engineered exosomes," and "macrophage-derived exosomes" were promising research topics. Our study comprehensively and visually summarized the structure, hotspots, and evolutionary trends of ERC. It would inspire subsequent studies from a macroscopic perspective and provide a basis for rational allocation of resources and identification of collaborations among researchers.

"Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication and valuable biomarkers for various diseases. However, traditional EV isolation and detection methods often struggle with efficiency, scalability, and purity, limiting their clinical utility. Recent advances in microfluidic and paper-based technologies offer innovative solutions that enhance EV isolation and detection by reducing sample volume, accelerating processing times, and integrating multiple analytical steps into compact platforms. These technologies hold significant promise for advancing point-of-care diagnostics, enabling rapid disease detection, personalized treatment monitoring, and better patient outcomes. For example, early detection of cancer biomarkers through EVs can facilitate timely intervention, potentially improving survival rates, while rapid infectious disease diagnostics can support prompt treatment. Despite their potential, challenges such as standardization, scalability, and regulatory hurdles remain. This review discusses recent advancements in microfluidic and paper-based EV diagnostic technologies, their comparative advantages over traditional methods, and their transformative potential in clinical practice."

Exosomes derived from specific cells may be useful for targeted drug delivery, but tracking them in vivo is essential for their clinical application. However, their small size and complex structure challenge the development of exosome-tracking techniques, and traditional labeling methods are limited by weak affinity and potential toxicity. To address these issues, here we developed a novel bio-orthogonal labeling strategy based on phosphatidylinositol derivatives to fluorescently label exosomes from various human and mouse cell types. The different cell-derived exosomes revealed organ-specific distribution patterns and a favorable safety profile. Notably, 4T1 cell-derived exosomes specifically targeted the lungs. When used as drug carriers loaded with anti-inflammatory resveratrol, these exosomes showed significant therapeutic efficacy in mice with acute respiratory distress syndrome (ARDS), effectively reducing inflammatory responses, mitigating pulmonary fibrosis, and restoring lung tissue morphology and function. Our findings provide a novel exosome labeling strategy and an invaluable tool for their in vivo tracking and targeting screening, while exosomes that specifically target the lungs offer a potential therapeutic strategy for organ-specific diseases such as ARDS.

Extracellular vesicles (EVs) are heterogeneous in size, biogenesis, content, and function. Aggressive cancer cells release a distinct, poorly characterized, and particularly large EV subtype, namely large oncosomes (LOs). This study employs an optimized method to improve LO yields and integrates mass spectrometry and RNA sequencing (RNA-seq) to profile their molecular cargo. A consistent set of proteins enriched in LOs is identified across glioma, prostate, and breast cancer cell lines. These proteins are also present as mRNA in LOs from the prostate cancer model and are abundant in plasma LOs from 20 patients with metastasis. Single-LO RNA-seq confirms bulk LO cargo, demonstrating the utility of single-cell technologies for large vesicle analysis. Our patient study provides proof-of-principle evidence that we can use multiomics to delve into EV heterogeneity, biogenesis, and composition. It also suggests that plasma LOs help stratify patients, supporting their potential prognostic value for developing a multi-analyte approach for liquid biopsy.

Studies based on extracellular vesicles (EVs) have been multiplying exponentially for almost two decades, since they were first identified as vectors of cell-cell communication. However, several of these studies display a lack of rigor in EVs characterization and isolation, without discriminating between the different EV populations, thus generating conflicting and unreproducible results. There is therefore a strong need for standardization and guidelines to conduct studies that are rigorous, transparent, reproducible and comply with certain nomenclatures concerning the type of EVs used. The International Society for Extracellular Vesicles (ISEV) published the Minimum Information for Studies of Extracellular Vesicles (MISEV) in 2014, updating it in 2018 and 2023 to reflect different study contexts and technical advancements. The primary objective of this review is to inform future authors about EVs, including their history, nomenclature, and technical recommendations for the for isolation and functionality analysis for conducing EV-based studies according to current standards. Additionally, it aims to inform reviewers about the key parameters required for characterizing EV preparations.

This study found that in patients with SLE (n = 5), lethal (let)-7f-5p expression was significantly downregulated in peripheral blood mononuclear cells. Further, high-throughput RNA sequencing was used to mine the differential transcriptome expression in renal tissue exosomes of systemic lupus erythematosus (SLE)-prone mice, and bioinformatics was utilized to analyze non-coding RNAs and coding RNAs in exosomes for their possible roles in SLE. In renal tissues of MRL/lpr SLE-prone mice with exosomes and Pristane-induced SLE mice, we also demonstrated aberrant expression levels of microRNA (miRNA) let-7f-5p. Meanwhile, in the macrophage inflammation model, the expression levels of let-7f-5p were downregulated, that of guanylate binding protein (Gbp2 and Gbp7) were upregulated, and the inflammatory state of macrophages was alleviated following transfection with the let-7f-5p mimic. Co-culturing mesenchymal stem cells with a macrophage model of inflammation resulted in increased let-7f-5p expression and downregulated inflammatory factors, Gbp2 and Gbp7 expression in macrophages. Dual luciferase reporter gene assays confirmed that let-7f-5p directly binds to the 3' UTR of Gbp7 to regulate its expression. Let-7f-5p regulation of the Gbp family is involved in SLE pathogenesis and is a biomarker associated with the inflammatory response with potential clinical applications.

Neuronal extracellular vesicles (microvesicles and exosomes) are emerging secreted vesicular signals that play important roles in the CNS. Currently, little is known about how glutamatergic signalling affects the subcellular localisation of exosome precursor intraluminal vesicles (ILVs), microRNA (miR) packaging into ILVs and in vivo spreading of neuronal EVs. By selectively labelling ILVs and exosomes (but not plasma membrane-derived MVs) with GFP-tagged human CD63 (hCD63-GFP) in cortical neurons, we found that glutamate stimulation significantly redistributes subcellular localisation of hCD63-GFP+ ILVs, especially decreasing its co-localisation with multi-vesicular body (MVB) marker Rab7 while substantially promoting EV secretion. Interestingly, glutamate stimulation only modestly alters EV miR profiles based on small RNA sequencing. Subsequent in vivo cortical neuronal DREADD activation leads to significantly more widespread hCD63-GFP+ area in hCD63-GFPf/+ mice, consistently supporting the stimulatory effect of glutamatergic activation on neuronal EV secretion and spreading. Moreover, in situ localisation of hCD63-GFP+ ILVs and hCD63-GFP+ secreted exosomes from specialised HB9+ and DAT+ neurons were also illustrated in the CNS. Taken together, our results demonstrated that glutamate activity stimulates neuronal exosome secretion and spreading in vitro and in vivo, but only modestly affects miR cargo packaging in neuronal exosomes.

Small extracellular vesicles (small EV) are a conserved means of communication across the domains of life and lately gained more interest in mammalian non-cancerous work as non-cellular, biological therapeutic with encouraging results in recent studies of chronic degenerative diseases. The nucleus pulposus (NP) is the avascular and aneural center of an intervertebral disc (IVD), home to unique niche conditions and affected in IVD degeneration. We investigated autologous and mesenchymal stem cell (MSC) small EVs for their potential to contribute to cell and tissue homeostasis in the NP niche via mass spectrometric proteome and functional enrichment analysis using adult and fetal donors. We compared these findings to published small EV databases and MSC small EV data. We propose several mechanisms associated with NP small EVs: Membrane receptor trafficking to modify signal responses promoting niche homeostasis; Redox and energy homeostasis via metabolic enzymes delivery; Cell homeostasis via proteasome delivery and immunomodulation beyond an association with a serum protein corona. The proteome signature of small EVs generated by NP parent cells is similar to previously published small EV data, yet with a focus on supplementing anaerobic metabolism and redox balance while contributing to the maintenance of an aneural and avascular microniche.

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, both in developed and developing countries. Despite the implementation of various measures in clinical practice that have shown certain curative effects, poor prognosis and irreversible pathological cardiac remodeling continue to limit the therapeutic effect of CVDs. There are still many new mechanisms worth exploring for the regulation of CVDs. Previous studies have highlighted the potential applicability of exosomes in CVDs, and significant research has been conducted in this area. In this review, we summarize the physiological mechanisms of exosomes and the basic research achievements in regulating CVDs via exosomal non-coding RNAs. We also discuss the limitations and prospects of exosome application in CVD treatment.

The therapeutic effect of stem cells is attributed to their direct maturation into somatic cells and their paracrine effects, which influence the extracellular environment. One such component released is extracellular vesicles containing proteins and genetic materials with immunomodulatory functions and facilitating cell-to-cell communication.

The study's main objective was to characterize extracellular vesicles (EVs) from Human Neural Precursor Cells (hNPCs).

Wharton's Jelly mesenchymal stem cells (WJ-MSCs) were isolated by explant technique and characterized by flow cytometry and trilineage differentiation. The hNPCs obtained from neurospheres were produced by seeding WJ-MSCs on a natural functional biopolymer matrix. EVs derived from WJ-MSCs and hNPCs were isolated by precipitation methodology and characterized by flow cytometry, nanoparticle tracking analysis (NTA), scanning electron microscopy (TEM), and proteomic.

hNPCs expressed proteins and genes characteristic of neural precursor cells. The EVs were characterized by flow cytometry and showed varied expression for the markers CD63, CD9, and CD81, indicating different subpopulations based on their origin of formation. NTA and TEM of the EVs exhibited characteristic size, shape, and structural integrity consistent with the criteria established by the International Society for Extracellular Vesicles (ISEV). EV-hNPCs function enrichment analysis of the proteomic results showed that these vesicles presented abundant proteins directly involved in neuronal biological processes such as plasticity, transduction, postsynaptic density, and overall brain development.

The results indicate that EVs derived from hNPCs maintain key neural precursor characteristics and exhibit marker variability, suggesting distinct subpopulations. Their structural integrity aligns with ISEV standards, supporting their potential as reliable biological entities. The proteomic analysis highlights their role in neuronal functions, reinforcing their applicability in neurodegenerative research and therapeutic strategies.

The EVs were successfully isolated from hNPCs with abundant proteins involved in neuronal processes, making them attractive for acellular therapies to treat neurodegenerative diseases.

Freezing sperm for artificial insemination (AI) has been common for decades, but this method causes damage to sperm, which affects its viability and fertility. Various strategies have been used to treat sperm cryopreservation complications, but their results are still not satisfactory. The latest approach in this field is using extracellular vesicles (EVs). The role of EVs in reproduction, such as spermatogenesis, sperm capacitation, and fertility has been proven. EVs can deliver proteins, lipids, nucleic acids, and other molecules to the sperm for repair. The EVs carry proteins, lipids, nucleic acids, and other molecules, which could be involved in sperm quality, functionality or fertility. The application of EV derived from animal and human cell sources for cryoinjury treatment indicates the improvement of sperm quality after freeze-thawing. In addition, different EV engineering methods regarding various EV cargos could be more influential for cryopreserved sperm treatment because they could provide EV customized content for delivering to cryoinjured sperm, according to their unique needs to enhance viability and fertility. In this review, first, we reminded the sperm cryopreservation complications, and next explained the conventional and modern strategies for overcoming them. Then, we have pointed out the role of EV in sperm development and the following mentioned the study results of using EV from different cell sources in sperm cryoinjuries repair. Also, we suggested several predisposing molecules (including microRNAs and proteins) for EV engineering to treat sperm cryopreservation complications by indirect engineering procedure, including genetic manipulation and incubation with therapeutic molecules, and direct engineering procedure, including electroporation, sonication, incubation, saponin permeabilization, extrusion, CaCl2-heat shock, and freeze/thawing. Finally, we discussed the limitations of EV application and ethical considerations in this context. In the meantime, despite these limitations, we pointed out the promising potential of the EV engineering strategies to reduce infertility rates by helping to overcome sperm cryopreservation challenges.

Extracellular vesicles (EVs) are heterogeneous nanoscale membrane vesicles released by almost all cell types into the circulation. Depending on their biogenesis and cells of origin, EVs show considerable heterogeneity in their biophysical and biomolecular composition and can serve as reflective and dynamic blood biomarkers for personalized medicine. Conventional analytical technologies, however, often lack the compatibility to reveal nanoscale EV features and resolve vesicle heterogeneity. The past decade has since witnessed the development of various nanoplasmonic technologies to empower EV analysis, through bulk and single-vesicle characterization, at an unprecedented scale and resolution. These platforms achieve versatile measurements that are not only size-matched to EV dimensions but can also probe multiplexed biomolecular contents, thereby providing new insights into EV heterogeneity and enabling transformative clinical opportunities. In this review, key characteristics of EVs and their remarkable heterogeneity are introduced. The sensing principles of plasmonic platforms are also discussed, with recent technology developments highlighted to resolve EV heterogeneity, through bulk analyses of EV subpopulations as well as high-resolution single-EV measurements. An outlook is further provided on emerging opportunities, at the interface of biomarker discovery and technology innovation, to develop empowering nanoplasmonic EV platforms for personalized medicine. biosensing; bulk analysis; extracellular vesicles; nanoplasmonics; single-vesicle analysis.

Autophagy and apoptosis are two essential mechanisms regulating cell fate. Although distinct, their signaling pathways are closely interconnected through various crosstalk mechanisms. Lipid rafts are described to act as both physical and functional platforms during the early stages of autophagic and apoptotic processes. Only recently has a role for lipid raft-associated molecules in regulating EV biogenesis and release begun to emerge. In particular, lipids of EV membranes are essential components in conferring stability to these vesicles in different extracellular environments and/or to facilitate binding or uptake into recipient cells. In this review we highlight these aspects, focusing on the role of lipid molecules during apoptosis and secretory autophagy pathways. We describe the molecular machinery that connects autophagy and apoptosis with vesicular trafficking and lipid metabolism during the release of EVs, and how their alterations contribute to the development of various diseases, including autoimmune disorders and cancer. Overall, these findings emphasize the complexity of autophagy/apoptosis crosstalk and its key role in cellular dynamics, supporting the role of lipid rafts as new therapeutic targets.

Melanoma is the most aggressive type of skin cancer with high mortality rates. Early diagnosis is crucial because it significantly improves treatment outcomes, but conventional methods relying on dermoscopy and lesion biopsy have limitations in accuracy during early stages and are invasive. Liquid biopsies offer a minimally invasive alternative, particularly for routine screening. The abundance of cancer cell-driven extracellular vesicles in interstitial fluid can be utilized for point-of-care cancer diagnostics. Here, we developed a stellate silicon microneedle patch, the ExoPatch, coated with Annexin V functionalized hydrogel to isolate melanoma-specific exosomes. The ExoPatch captures exosomes directly from the skin, followed by dissolution of the hydrogel to release the exosomes, which are then detected using a lateral flow immunoassay specific to melanoma markers (MCAM and MCSP). After validating with cell line derived extracellular vesicles and testing with mouse tissue, the ExoPatch isolated 11.5 times more protein from melanoma tissue compared to healthy tissue. Additionally, the ExoPatch effectively distinguished between melanoma and healthy tissues, with its specificity confirmed through Western Blot and electron microscopy analysis. The ExoPatch with melanoma mouse samples produced a 3.5-fold higher signal in the lateral flow immunoassay compared to that of healthy controls. The ExoPatch presents a promising point-of-care diagnostic tool for melanoma, offering significant advantages in terms of rapidness, minimal invasiveness, and ease of use. It has the potential to enhance early detection and routine monitoring in melanoma patients, ultimately improving patient outcomes by reducing the reliance on traditional, invasive biopsies.

Extracellular vesicles are nanoscale vesicles released by a diversity of cells that mediate intercellular communication by transporting an array of biomolecules. They are gaining increasing attention in cancer research due to their ability to carry specific biomarkers. This characteristic makes them potentially useful for highly sensitive, noninvasive diagnostic procedures and more precise prognostic assessments. Consequently, EVs are emerging as a transformative tool in cancer treatment, facilitating early detection and personalized medicine. Despite significant progress, clinical implementation is hindered by challenges in EV isolation, purification, and characterization. However, developing advanced biosensor technologies offers promising solutions to these obstacles. This review highlights recent progress in biosensors for EV detection and analysis, focusing on various sensing modalities including optical, electrochemical, microfluidic, nanomechanical, and biological sensors. We also explore techniques for EV isolation, characterization, and analysis, such as electron microscopy, atomic force microscopy, nanoparticle tracking analysis, and single-particle analysis. Furthermore, the review critically assesses the challenges associated with EV detection and put forward future directions, aiming to usher in a cutting-edge era of precision medicine through advanced, sensor-based, noninvasive early cancer diagnosis by detecting EV-carried biomarkers.

Neurological disorders such as Alzheimer's disease, Parkinson's disease, and stroke pose significant challenges for conventional therapy due to the complexities of the blood-brain barrier (BBB) and the restricted delivery of drugs to the central nervous system. Exosomes, a type of small extracellular vesicle secreted by nearly all cell types, hold substantial promise as delivery vehicles for therapeutic agents in treating these conditions. Notably, stem cell-secreted exosomes have emerged as particularly effective due to their regenerative potential and natural ability to cross the BBB. Similarly, hydrogels have gained recognition as versatile biomaterials capable of supporting sustained release and targeted delivery of therapeutics. The combination of the regenerative properties of stem cell-derived exosomes (SC-Exos) with the structural and functional benefits of hydrogels offers a promising approach for enhancing neurogenesis, modulating neuroinflammation, and facilitating tissue repair. This review explores the origin, structure, and modifications of exosomes as well as the synthesis and incorporation methods of hydrogels in the therapeutic context for debilitating neurological disorders. It highlights recent advancements in using SC-Exos and hydrogels for therapeutic delivery, addressing both current challenges and future applications. Improving our understanding of hydrogels loaded with SC-Exos for cargo transportation and neural tissue regeneration may pave the way for novel therapeutic strategies.

Mitochondria-derived vesicles (MDVs) participate in early cellular defence mechanisms initiated in response to mitochondrial damage. They maintain mitochondrial quality control (MQC) by clearing damaged mitochondrial components, thereby ensuring the normal functioning of cellular processes. This process is crucial for cell survival and health, as mitochondria are the energy factories of cells, and their damage can cause cellular dysfunction and even death. Recent studies have shown that MDVs not only maintain mitochondrial health but also have a significant impact on tumour progression. MDVs selectively encapsulate and transport damaged mitochondrial proteins under oxidative stress and reduce the adverse effects of mitochondrial damage on cells, which may promote the survival and proliferation of tumour cells. Furthermore, it has been indicated that after cells experience mild stress, the number of MDVs significantly increases within 2-6 h, whereas mitophagy, a process of clearing damaged mitochondria, occurs 12-24 h later. This suggests that MDVs play a critical role in the early stress response of cells. Moreover, MDVs also have a significant role in intercellular communication, specifically in the tumour microenvironment. They can carry and transmit various bioactive molecules, such as proteins, nucleic acids, and lipids, which regulate tumour cell's growth, invasion, and metastasis. This intercellular communication may facilitate tumour spread and metastasis, making MDVs a potential therapeutic target. Advances in MDV research have identified novel biomarkers, clarified regulatory mechanisms, and provided evidence for clinical use. These breakthroughs pave the way for novel MDV-targeted therapies, offering improved treatment alternatives for cancer patients. Further research can identify MDVs' role in tumour development and elucidate future cancer treatment horizons.

Cells across biological kingdoms release extracellular vesicles (EVs) as a means of communication with other cells, be their friends or foes. This is indeed true for the intracellular malaria parasite Plasmodium falciparum (Pf), which utilizes EVs to transport bioactive molecules to various human host systems. Yet, the study of this mode of communication in malaria research is currently constrained due to limitations in high-resolution tools and the absence of commercial antibodies. Here, we demonstrate the power of an advanced spectral flow cytometry approach to robustly detect secreted EVs, isolated from Pf-infected red blood cells. By labeling both EV membrane lipids and the DNA cargo within (non-antibody staining approach), we were able to detect a subpopulation of parasitic-derived EVs enriched in DNA. Furthermore, we could quantitatively measure the DNA-carrying EVs isolated from two distinct blood stages of the parasite: rings and trophozoites. Our findings showcase the potential of spectral flow cytometry to monitor dynamic changes in nucleic acid cargo within pathogenic EVs.

Over the past few decades, extensive basic, translational and clinical research has been devoted to deciphering the physiological and pathogenic roles of extracellular vesicles (EVs) in the nervous system. The presence of brain cell-derived EVs in the blood, carrying diverse cargoes, has enabled the development of predictive, diagnostic, prognostic, disease-monitoring and treatment-response biomarkers for various neurological disorders. In this Review, we consider how EV biomarkers can bring us closer to understanding the complex pathogenesis of neurological disorders such as Alzheimer disease, Parkinson disease, stroke, traumatic brain injury, amyotrophic lateral sclerosis and multiple sclerosis. We describe how translational research on EVs might unfold bidirectionally, proceeding from basic to clinical studies but also in the opposite direction, with biomarker findings in the clinic leading to novel hypotheses that can be tested in the laboratory. We demonstrate the potential value of EVs across all stages of the therapeutic development pipeline, from identifying therapeutic targets to the use of EVs as reporters in model systems and biomarkers in clinical research. Finally, we discuss how the cargo and physicochemical properties of naturally occurring and custom-engineered EVs can be leveraged as novel treatments and vehicles for drug delivery, potentially revolutionizing neurotherapeutics.

Cell communication is crucial for coordinating physiological functions in multicellular organisms, with exosomes playing a significant role. Exosomes mediate intercellular communication by transporting proteins, lipids, and nucleic acids between cells. These small, membrane-bound vesicles, derived from the endosomal pathway, are integral to various biological processes, including signal transmission and cellular behavior modulation. Recent advances highlight the potential of exosomes, especially dendritic cell-derived exosomes (DEXs), for diagnostic and therapeutic applications, particularly in cancer immunotherapy. DEXs are distinguished by their ability to present antigens and stimulate immune responses more effectively than exosomes from other cell types. They carry a cargo rich in immunostimulatory molecules and MHC-peptide complexes, which facilitate robust T-cell activation and enhance tumor-specific immune responses. The unique properties of DEXs, such as their ability to cross biological barriers and resist tumor-induced immunosuppression, position them as promising candidates for therapeutic applications. Here, I review the reports on the bidirectional interaction between dendritic cells and T cells through exosomes and their role in medicine.

Small extracellular vesicle (sEV)-based therapies have gained widespread interest, but challenges persist to ensure standardization and high-scale production. Implementing upstream processes in a chemically defined media in stirred-tank bioreactors (STBr) is mandatory to closely control the cell environment, and to scale-up production, but it remains a significant challenge for anchorage-dependent cells.

We used a human β cell line, grown as monolayer or in suspension as spheroid in stirred systems. We assessed the consequences of culturing these cells in 3D with, or without fetal bovine serum in a chemically defined medium, for cell growth, viability and metabolism. We next explored how different scale-up strategies might influence cell and spheroid formation in spinner flask, with the aim to transfer the process in instrumented Ambr®250 STBr. Lastly, we analyzed and characterized sEV production in monolayer, spinner flask and STBr.

Generation of spheroids in a chemically defined medium allowed the culture of highly viable cells in suspension in stirred systems. Spheroid size depended on the system's volumetric power input (P/V), and maintaining this parameter constant during scale-up proved to be the optimal strategy for standardizing the process. However, transferring the spinner flask (SpF) process to the Ambr®250 STBr at constant P/V modified spheroid size, due to important geometric differences and impeller design. Compared to a monolayer reference process, sEV yield decreased two-fold in SpF, but increased two-fold in STBr. Additionally, a lower expression of the CD63 tetraspanin was observed in sEV produced in both stirred systems, suggesting a reduced release of exosomes compared to ectosomes. This study addresses the main issues encountered in spheroid culture scale-up in stirred systems, rather conducive for the production of ectosomes.

Intercellular communication is an essential hallmark of multicellular organisms for their development and adult tissue homeostasis. Over the past two decades, attention has been focused on communication mechanisms based on various membrane structures, as illustrated by the burst of scientific literature in the field of extracellular vesicles (EVs). These lipid bilayer-bound nano- or microparticles, as vehicle-like devices, act as regulators in various biological and physiological processes. When EVs are internalized by recipient cells, their membrane and cytoplasmic cargoes can interfere with cellular activities, affecting pathways that regulate cell proliferation, differentiation, and migration. In cancer, EVs can transfer oncogenic factors, stimulate neo-angiogenesis and immunosuppression, reprogram stromal cells, and confer drug resistance traits, thereby remodeling the surrounding microenvironment. Although the mechanisms underlying EV biogenesis and uptake are now better understood, little is known about the spatiotemporal mechanism(s) of their actions after internalization. In this respect, we have shown that a fraction of endocytosed EVs reaches the nuclear compartment via the VOR (VAP-A-ORP3-Rab7) complex-mediated docking of late endosomes to the outer nuclear membrane in the nucleoplasmic reticulum, positioning and facilitating the transfer of EV cargoes into the nucleoplasm via nuclear pores. Here, we highlight the EV heterogeneity, the cellular pathways governing EV release and uptake by donor and recipient cells, respectively, and focus on a novel intracellular pathway leading to the nuclear transfer of EV cargoes. We will discuss how to intercept it, which could open up new avenues for clinical applications in which EVs and other small extracellular particles (e.g., retroviruses) are implicated.

Alzheimer's disease (AD) is one of the most common progressive neurodegenerative diseases leading to impairments in memory, orientation, and behavior. However, significant work is still needed to fully understand the progression of such disease and develop novel therapeutic agents for AD prevention and treatment. Small extracellular vesicles (sEVs) have received attention in recent years due to their potential therapeutic effects on AD. The aim of this study was to determine the potential effect of sEVs in an in vitro model of AD. sEVs were isolated from human Wharton's jelly mesenchymal stem cells (MSCs) by asymmetric depth filtration, a method developed recently by us. AD was modeled in vitro using cells obtained from the hippocampi of newborn 5xFAD transgenic mice carrying mutations involved in familial AD. After isolation, sEVs underwent detailed characterization that included scanning electron microscopy, nanoparticle tracking analysis, confocal microscopy, Western blotting, and Luminex assay. When added to 5xFAD hippocampal cells, sEVs were nontoxic, colocalized with neurons and astrocytes, decreased the level of Aβ peptide, and increased the synaptic density. These results support the possibility that sEVs can improve brain cell function during aging, decrease the risk of AD, and potentially be used for AD therapeutics.

The exchange of molecular information across kingdoms is crucial for the survival of both plants and their pathogens. Recent research has identified that plants transfer their small RNAs and microRNAs into fungal pathogens to suppress infection. However, whether and how plants send defense proteins into pathogens remains unknown. Here, we report that rice (Oryza sativa) plants package defense proteins into extracellular vesicles (EVs) and deliver them to the fungal pathogen Rhizoctonia solani. These EVs, enriched with host defense proteins, are internalized by the fungal cells. Reducing the transfer of host defense proteins via EVs results in increased disease susceptibility. Furthermore, the overexpression of host defense proteins in either rice plants or the fungal cells reduced the infection. Therefore, plants use EVs to send defense proteins into fungal pathogens, thereby combating infection. This mechanism represents a form of protein exchange between plants and pathogens, which contributes to reducing crop diseases.

The current medical need to respond to different diseases has sparked great interest in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) due to their great regenerative potential and as drug carriers by playing a critical role in cell-cell communication. However, due to their heterogeneity, there is no standardized universal method for their identification and characterization, which limits their clinical application. This study, following the recommendations and methodologies proposed by MISEV2023 for the characterization of EVs, shows for the first time a detailed morphological, protein, and biochemical comparison between EVs derived from three different MSCs sources (placenta, endometrium, and dental pulp). The information obtained from the different applied assays suggests that there are substantial differences between one EVs source and another. It also offers valuable insights that provide the guidelines to ease their profiling and therefore improve their selection, in order to speed up their use and clinical application; additionally, the knowledge obtained from each characterization test could facilitate new researchers in the field to choose a specific cell source to obtain EVs and select the appropriate methods that provide the necessary information according to their requirements.

Small extracellular vesicles (sEVs), which carry lipids, proteins and RNAs from their parent cells, serve as biomarkers for specific cell types and biological states. These vesicles, including exosomes and microvesicles, facilitate intercellular communication by transferring cellular components between cells. Current methods, such as ultracentrifugation and Tim-4 affinity method, yield high-purity sEVs. However, despite their small size, purified sEVs remain heterogeneous due to their varied intracellular origins. In this technical note, we used high-speed atomic force microscopy (HS-AFM) in conjunction with exosome markers (IgGCD63 and IgGCD81) to explore the intracellular origins of sEVs at single-sEV resolution. Our results first revealed the nanotopology of HEK293T-derived sEVs under physiological conditions. Larger sEVs (diameter > 100 nm) exhibited greater height fluctuations compared to smaller sEVs (diameter ≤ 100 nm). Next, we found that mouse-origin IgGCD63, and rabbit-origin IgGcontrol and IgGCD81, exhibited the iconic 'Y' conformation, and similar structural dynamics properties. Last, exosome marker antibodies predominantly co-localised with sEVd ≤ 100 nm but not with sEVd > 100 nm, demonstrating the CD63-CD81-enriched sEV and CD63-CD81-depleted sEV subpopulations. In summary, we demonstrate that nanoscopic profiling of surface exosome markers on sEVs using HS-AFM is feasible for characterising distinct sEV subpopulations in a heterogeneous sEV mixture.

Neurological diseases present a wide range of conditions, intricate diagnosis and treatment processes, and complex prognostic considerations. Therefore, research focusing on the diagnosis and treatment of these diseases is crucial. Exosomal miRNAs are small RNA molecules enclosed in membrane vesicles, released by cells and known to play roles in the development of various neurological disorders. They also serve as specific biomarkers for these conditions. Drawing on extensive research on exosomal miRNAs in diseases like stroke, Alzheimer's, epilepsy, Parkinson's, and neuroregeneration, this paper provides a comprehensive review of the relationship between exosomal miRNAs and neurological diseases. We strive to offer current and detailed theoretical understandings to help with the diagnosis and treatment of these disorders.

The therapeutic potential of extracellular vesicles (EVs) in reducing oral inflammation is thoroughly examined in this review, with an emphasis on gingivitis, periodontitis, and oral mucositis. It explains the complex relationship between microbial dysbiosis and host immune responses in the aetiology of oral inflammation. Pathophysiological mechanisms of periodontitis are examined, emphasising the roles played by periodontal pathogens and inflammatory mediators in the disease's chronic course and systemic effects. Preclinical research is providing new evidence that EVs originating from various cellular sources control immune cell dynamics towards a pro-healing phenotype, promote tissue regeneration, and have immunomodulatory qualities. EV-based therapies appear to be a promising new therapeutic technique with potential benefits over traditional methods for the treatment of oral inflammatory illnesses by specifically altering inflammatory signalling pathways. This review highlights the potential of EVs to improve patient outcomes in oral health and emphasises the need for additional clinical research to clarify the therapeutic efficacy and underlying mechanisms of EVs in periodontal therapy.

Exosomes play pleiotropic tumor-promoting functions and are secreted by fusion of multivesicular bodies (MVBs) with the plasma membrane. However, MVBs are also directed to lysosomes for degradation, and the mechanism controlling different fates of MVBs remains elusive. Here, we show that the pro-tumor protein WDR4 enhances exosome secretion from mouse and human cancer cells through degrading the endosomal sorting complex required for transport (ESCRT)-associated Bro1-family protein PTPN23. Mechanistically, PTPN23 and ALIX compete for binding to syntenin, thereby directing MVBs toward degradation and secretion, respectively. ALIX, but not PTPN23, recruits actin-capping proteins CAPZA1/CAPZB to prevent branched filamentous actin (F-actin) accumulation around MVBs, thus enabling MVBs trafficking to the cell periphery for secretion. Functionally, WDR4/ALIX-dependent exosomes load a set of pro-tumor proteins through LAMP2A, thereby potentiating metastasis and immune evasion in mice. Our study highlights a previously unappreciated coupling between the biogenesis mechanism and the fate decision of MVBs and its importance in determining exosomal cargos, which have a profound impact on tumor progression.

Circulating tumor cells (CTCs) are pivotal in cancer progression, and in vitro CTC models are crucial for understanding their biological mechanisms. This study focused on the characterization of extracellular vesicles (EVs) from CTC lines derived from a patient with metastatic colorectal cancer (mCRC) at different stages of progression who progressed despite therapy (thus mirroring the clonal evolution of cancer).

Morphological and size analyses revealed variations among EVs derived from different CTC lines. Compared with the Vesiclepedia database, proteomic profiling of these EVs revealed enrichment of proteins related to stemness, endosomal biogenesis, and mCRC prognosis. Integrin family proteins were significantly enriched in EVs from CTC lines derived after therapy failure. The role of these EVs in cancer progression was analyzed by assessing their in vivo distribution, particularly in the liver, lungs, kidneys, and bones. EVs accumulate significantly in the liver, followed by the lungs, kidneys and femurs.

This study is a pioneering effort in highlighting therapy progression-associated changes in EVs from mCRC patients via an in vitro CTC model. The results offer insights into the role of metastasis initiator CTC-derived EVs in cancer spread, suggesting their utility for studying cancer tissue distribution mechanisms. However, these findings must be confirmed and extended to patients with mCRC. This work underscores the potential of CTC-derived EVs as tools for understanding cancer dissemination.

It is known that the transformation of liver cancer-mediated fibroblasts into cancer-related fibroblasts (CAFs) is beneficial to the development of liver cancer. However, the specific mechanism is still unclear.

Human hepatocarcinoma (HepG2) cells were treated with short hairpin RNA (shRNA) of platelet-derived growth factor-D (shPDGF-D) vector, and the exosomes secreted by the cells were separated using ultracentrifugation and identified by using nanoparticle tracking analysis, transmission electron microscope, and western blot analysis. Exosomes were co-cultured with mouse primary fibroblasts, and then the activity, proliferation, cell cycle, migration, epithelial-mesenchymal transition- (EMT-) and CAF marker-related protein expression levels of fibroblasts were determined by cell counting kit-8 (CCK-8), immunofluorescence, flow cytometry, wound healing, real-time reverse transcription-PCR, and western blotting assays, respectively. Co-cultured fibroblasts were mixed with HepG2 cells and injected subcutaneously into mice to construct animal models. The size and weight of xenograft tumor and the expression of epithelial-mesenchymal transition- (EMT-), angiogenesis- and CAFs marker-related proteins were detected.

The exosomes inhibited the proliferation, migration, EMT, and induced cell cycle arrest, as well as decreased the expression of α-SMA, FAP, MMP-9, and VEGF in fibroblasts. In vivo, sh-PDGF-D inhibited tumor growth, reduced the expressions of CD31, vimentin, α-SMA, FAP, MMP9, and VEGF, and promoted the expression of E-cadherin.

Exosomes derived from HepG2 cells transfected with shPDGF-D prevent normal fibroblasts from transforming into CAFs, thus inhibiting angiogenesis and EMT of liver cancer.

Pulmonary lymphangioleiomyomatosis (LAM) is metastatic sarcoma but mechanisms of LAM metastasis are unknown. Extracellular vesicles (EV) regulate cancer metastasis but their roles in LAM have not yet been thoroughly investigated. Here, we report the discovery of distinct LAM-EV subtypes derived from primary tumor or metastasizing LAM cells that promote LAM metastasis through ITGα6/β1-c-Src-FAK signaling, triggered by shuttling ATP synthesis to cell pseudopodia or the activation of integrin adhesion complex, respectively. This signaling leads to increased LAM cell migration, invasiveness, and stemness and regulates metastable (hybrid) phenotypes that are all pivotal for metastasis. Mouse models corroborate in vitro data by demonstrating a significant increase in metastatic burden upon the exposure to EV through distinct mechanisms involving either lung resident fibroblasts or metalloproteinases' activation that are EV subtype dependent. The clinical relevance of these findings is underscored by increased EV biogenies in LAM patients and the enrichment of these EV cargo with lung tropic integrins and metalloproteinases. These findings establish EV as novel therapeutic target in LAM, warranting the future clinical studies.

Liver cancer is a significant global health concern due to its poor prognosis, often resulting from late-stage diagnosis and limited treatment options. While non-invasive methods such as ultrasound, blood tests (like AFP and PIVKA-II), CT scans, and MRIs are commonly employed in liver cancer diagnosis, they can occasionally be limited in sensitivity or associated with high costs. This has heightened the demand for innovative, non-invasive biomarkers that enable early and accurate diagnosis, leading to increased interest in the potential of exosomes. Exosomes are small vesicles released by cells and have the potential to serve as biomarkers for liver cancer. They contain a variety of biomolecules, including nucleic acids, proteins, and lipids, which can offer important information about cell health and disease progression. Developing fast, accurate, sensitive, and reliable techniques for detecting exosomes is essential. Biosensors, analytical tools for biological samples, have emerged as powerful instruments for analyzing exosomes. This review focuses on recent advancements in biosensor technology for exosome detection and explores future perspectives. The goal is to promote the development of innovative biosensor-based methods for detecting exosomes to enable earlier diagnosis and better clinical management of liver cancer.

Oxidative stress, a source of genotoxic damage, is often the underlying mechanism in many functional cell disorders. Extracellular vesicles (EVs) have been shown to be key regulators of cellular processes and may be involved in maintaining cellular redox balance. Herein, we aimed to develop a method to assess the effects of EVs on DNA oxidation using porcine seminal plasma extracellular vesicles (sEVs).

The technique was set using a cell-free plasmid DNA to avoid the bias generated by the uptake of sEVs by sperm cells, employing increasing concentrations of hydrogen peroxide (H2O2) that generate DNA single-strand breaks (SSBs). Because SSBs contain a free 3'-OH end that allow the extension through quantitative PCR, such extension -and therefore the SYBR intensity- showed to be proportional to the amount of SSB. In the next stage, H2O2 was co-incubated with two size-differentiated subpopulations (small and large) of permeabilized and non-permeabilized sEVs to assess whether the intravesicular content (IC) or the surface of sEVs protects the DNA from oxidative damage. Results obtained showed that the surface of small sEVs reduced the incidence of DNA SSBs (P < 0.05), whereas that of large sEVs had no impact on the generation of SSBs (P > 0.05). The IC showed no protective effect against DNA oxidation (P > 0.05).

Our results suggest that the surface of small sEVs, including the peripheral corona layer, may exert a protective function against alterations that are originated by oxidative mechanisms. In addition, our in vitro study opens path to detect, localize and quantify the protective effects against oxidation of extracellular vesicles derived from different fluids, elucidating their function in physiopathological states.

Small extracellular vesicles (sEVs) play crucial roles in intercellular communication. However, the internalization of individual sEVs by recipient cells has not been directly observed. Here, we examined these mechanisms using state-of-the-art imaging techniques. Single-molecule imaging shows that tumor-derived sEVs can be classified into several subtypes. Simultaneous single-sEV particle tracking and observation of super-resolution movies of membrane invaginations in living cells reveal that all sEV subtypes are internalized via clathrin-independent endocytosis mediated by galectin-3 and lysosome-associated membrane protein-2C, while some subtypes that recruited raft markers are internalized through caveolae. Integrin β1 and talin-1 accumulate in recipient cell plasma membranes beneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggers Ca2+ mobilization induced by the activation of Src family kinases and phospholipase Cγ. Subsequent Ca2+-induced activation of calcineurin-dynamin promotes sEV internalization, leading to the recycling pathway. Thus, we clarified the detailed mechanisms of sEV internalization driven by paracrine adhesion signaling.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplantion (HSCT) with lentiviral (LV)-based gene therapy has great potential to treat MLD. Achieving the optimal balance between high enzyme production for therapeutic efficacy and maintaining a low vector copy number (VCN) is crucial. Insufficient enzyme levels can lead to the progression of motor symptoms, undermining treatment goals. Conversely, elevated VCN increases the risk of genotoxicity, which poses safety concerns, and contributes to higher production costs, making the therapy less accessible. Striking this balance is essential to maximize clinical benefit while minimizing risks and costs. To address this need, we increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human hematopoietic stem cells (HSCs) with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4× more ARSA activity than the currently US and European Union (EU)-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa-knockout (KO) MLD mice transplanted with Arsa-KO bone marrow (BM) cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching wild-type (WT) mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating MLD patients.