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Extracellular Vesicles (EVs) are nanosized particles with significant role in disease pathogenesis and as therapeutic potential. However, the lack of reliable and efficient methods for the characterization, quantification and tracking of EVs, combined with the limitations of detection techniques in differentiating specific EVs subtypes with beneficial properties, makes these process complex and time-consuming. To address this challenge, EVs were engineered using a tricistronic plasmid that encodes fluorescent proteins fused to tetraspanins (eGFP-CD63 and mCherry-CD9), with both fluorophores localized within the luminal space. Double fluorescently labelled small EVs (sEVs) were then produced in a stably transfected HEK293SF-3F6 cell line. The fluorescently labelled sEVs were characterized using a variety of techniques. Protein expression analysis showed that the fused proteins were efficiently produced and incorporated in sEVs, as evidenced by clear fluorescence signal detected. Comparisons of the size distribution and concentration of modified sEVs with controls indicated that sEVs engineering did not affect their biogenesis and morphology. Fluorescently labelled sEVs were then quantified by flow cytometry, allowing to distinguish sEVs from other EVs subtypes or sample particles. The values were then compared to fluorometry measurements, obtaining a linear correlation what enabled a novel sEVs quantification method. The functionality of engineered sEVs was assessed by monitoring their uptake and trafficking in recipient cells, obtaining an efficient internalisation by target cells. Overall, these results demonstrate that the implementation of dual fluorescent methodology is feasible for sEVs characterization, quantification, for in vitro study of EVs interaction with cells, and intercellular communication, as well as a valuable tool in the in vitro development of targeted therapeutic EVs delivery systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Small extracellular vesicles and nanoparticles (sEVPs) are cell-secreted entities with potential as diagnostic biomarkers and therapeutic vehicles. However, significant intrinsic sEVP heterogeneity impedes analysis and understanding of their composition and functions. We employ multidimensional fluorescent labelling on sEVPs, leveraging the robustness of a newly developed membrane probe-conjugated oligoelectrolytes (COEs), and conduct total internal reflection fluorescence (TIRF) microscopy on sEVP arrays. These arrays comprise single sEVPs anchored to a soft material functionalized surface with little bias. We then develop an enhanced algorithm for colocalization analysis of the multiple labels on individual sEVPs and perform deep profiling of particle content. We categorize sEVPs derived from the same cell type into seven distinct subpopulations-some vesicular whereas others non-vesicular, and we demonstrate that sEVPs from four cell types exhibit quantitatively distinguishable subpopulation distributions. Furthermore, we gain insights into specific particle features within each subpopulation, including CD63 counts, relative particle size, relative concentration of cargoes, and correlations among different cargoes. This high-content analysis reveals common cargo sorting features in sEVP subpopulations across different cell types and suggests new statistics within the sEVP inherent heterogeneity that could differentiate sEVPs from two types of cancer cells and two types of normal cells. Collectively, our study presents a robust single-sEVP characterization platform, combining high-content imaging with comprehensive analysis. This platform is poised to advance sEVP-based theranostic assays and facilitate exploration into disease-associated sEVP biogenesis and sEVP-mediated intercellular communication., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Extracellular vesicles (EVs) play crucial roles in cell-cell communication, but the biogenesis of large EVs has remained elusive. Here, we show that the biogenesis of large EVs (>800 nm-2 µm) occurs predominantly through the completion of successful cytokinesis, and the majority of large EVs are midbody remnants (MBRs) with translation activity, and the unique marker MKLP1. Blocking the cell cycle or cytokinesis, genetically or chemically, significantly decreases MBRs and large (800 nm-2 µm), medium (500-800 nm), and small (<300 nm) EVs, suggesting that proliferative cells can also generate all sizes of EVs. The canonical EV markers including CD9, CD63, CD81 localize to the spindle midzone, midbody, and MBRs, suggesting that these markers are not specific for detecting EVs exclusively. Importantly, all commonly used EV isolation methods isolate MBRs, confounding previous EV research. Last, isolated MBRs maintain translation activity regardless of the isolation method. We propose a model for the biogenesis of EVs throughout the cell cycle and suggest that some large EVs are primarily generated from mitotic cells. The discovery of MBRs as a unique class of large, translating EVs has implications for using them as cancer diagnostic markers and for engineering them for therapeutic cargo delivery during mitosis.

Background: Connexin 43 (Cx43) plays a crucial role in mediating intracellular communication and facitating the interaction between exosomes and recipient cells. This study investigates whether the activation of cAMP/protein kinase A (PKA) can regulate exosomal Cx43 expression and contribute to the functional recovery following ischemia-reperfusion (I/R) injury., Methods: An intraluminal vascular occlusion was performed on Lewis rats to simulate I/R injury. Concurrently, a PKA activator (8-Bromo-cAMP, 5 mg kg -1 ) or PKA inhibitor (H 89 2HCl, 20 mg kg -1 ) was administered intravenously via the tail vein (n = 10). Exosomes were isolated from cerebrospinal fluid, and the expression of exosomal markers (CD63 and CD81) and Cx43 was analyzed using Western blot. The expression of CD63 and CD81 in astrocytes was measured to assess exosome uptake. Spatial learning and memory capability were evaluated using the Morris water maze test., Results: 8-Bromo-cAMP significantly increased exosome release in cerebrospinal fluid, accompanied by elevated Cx43 expression. Additionally, 8-Bromo-cAMP enhanced exosome uptake by astrocytes, alleviated blood-brain barrier damage and edema, and improved cognitive function., Conclsions: PKA activation enhances exosome production, promotes cognitive function recovery, and attenuates cerebral I/R injury by up-regulating exosomal Cx43 expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Bullous pemphigoid (BP) is a severe autoimmune sub-epidermal bullous disease. Exosomes are small extracellular vesicles secreted by most cell types. The exosomal membrane proteins are implicated in various biological and pathological pathways. This study aims to explore the potential roles of exosomes in BP pathomechanism., Research Design: We collected plasma samples from 30 BP patients and 31 healthy controls. Nanoparticle tracking analysis (NTA) was used to analyze the size and concentration of exosomes. The immunogold labelling experiment and extracellular vesicle (EV) array were performed to detect the content and distribution of exosomes., Results: The exosomes from both the BP and control groups' plasma were successfully extracted. EV Array showed that CD63 and CD9 levels were significantly higher in the BP group than in the control group ( p  < 0.05). Expression levels of the BP180 NC16A and intracellular domain (ICD) were higher in the anti-BP180 positive group versus the controls ( p  < 0.05). The active BP group exhibits higher CD63 and BP180 ICD protein concentrations than the control or inactive BP groups ( p  < 0.05)., Conclusion: BP180 autoantigen fragments were expressed on the exosomal membrane in BP patients. The BP180 ICD and CD63 on exosomes could potentially be novel biomarkers for monitoring disease activity.

The important role of extracellular vesicles, which are considered key mediators of intercellular communication under physiological and pathological conditions, in various cellular processes, including those crucial for mammalian reproduction, has been increasingly studied. Tetraspanins, including CD63, are widely used as markers of extracellular vesicles, but they may also play a role in their biogenesis, cargo selection, cell targeting, and uptake. This study aimed to map the journey of the extracellular vesicle protein tetraspanin CD63 from the testis through the epididymis into mature bull sperm via an approach that included immunohistochemistry (immunofluorescence and immunoperoxidase staining), Western blot analysis, and immunoprecipitation analysis. We described the presence of CD63 in bull testicular and epididymal tissues, extracellular vesicles produced in these organs and spermatozoa during epididymal transit and after ejaculation. In addition, we revealed the nonuniform distribution of potential CD63 partners, such as CD9, integrin αV and syntenin-1, in the sperm head and tail and in extracellular vesicles. These findings contribute to understanding the complex mechanisms underlying sperm maturation and point to the possible involvement of tetraspanins and their associated partners, either as part of extracellular vesicles or sperm membranes, in these processes., Competing Interests: Declarations. Ethics approval and consent to participate: The organs (testes, epididymides) were collected from bulls slaughtered at the local slaughterhouse under Council Directive 98/58/EC, Council Regulation (EC) No. 1099/2009, Regulation (EU) 2016/1012, Slovak National Council No. 39/2007 and Slovak legislation guidelines (directive 432/2012 Z. z.). The experiments were not performed on live animals, therefore ethical approval is not required. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Small extracellular vesicles (sEVs) are important intercellular information transmitters in various biological contexts, but their release processes remain poorly understood. Herein, we describe a high-throughput assay platform, CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening, for identifying key players in sEV release. CIBER screening employs sEVs barcoded with CRISPR-gRNA through the interaction of gRNA and dead Cas9 fused with an sEV marker. Barcode quantification enables the estimation of the sEV amount released from each cell in a massively parallel manner. Barcoding sEVs with different sEV markers in a CRISPR pooled-screening format allows genome-wide exploration of sEV release regulators in a subpopulation-specific manner, successfully identifying previously unknown sEV release regulators and uncovering the exosomal/ectosomal nature of CD63 + /CD9 + sEVs, respectively, as well as the synchronization of CD9 + sEV release with the cell cycle. CIBER should be a valuable tool for detailed studies on the biogenesis, release, and heterogeneity of sEVs., Competing Interests: Competing interests K.K., Y.U., R.K. have filed a patent for the screening system of sEV release regulators (WO2021095842 (published), application by the University of Tokyo). The other authors report no competing interests., (© 2024. The Author(s).)

Naturally occurring cell-derived extracellular vesicles (EVs) have emerged as attractive nanocarriers for drug delivery. However, production of large quantities of EVs for clinical applications in a scalable manner remains a significant challenge. This study investigated at the single cell level how sonoporation, or membrane poration produced by ultrasound-induced microbubble cavitation, impacts EV production using mouse macrophage RAW 264.7 cells stably expressing CD63-GFP as a model system. Real-time fluorescence videomicroscopy detected rapid changes in CD63-GFP, a tetraspanin family member highly enriched in intraluminal vesicles tagged with GFP, to track changes in multivesicular bodies (MVBs), which are the cellular compartments where exosomes originate within the cells. Our results revealed distinct dynamic changes in CD63-GFP intensity and distribution in RAW 264.7 cells in terms of response time and duration depending on whether the cells were directly or indirectly impacted by sonoporation, suggesting reorganization of MVBs in response to direct and indirect mechanisms resulted from the mechanical impact of ultrasound pulse on the cells. Analysis of the supernatant from sonoporation-treated RAW 264.7 cells expressing CD63-GFP demonstrated a delayed and sustained increase in the production of CD63-GFP-positive EVs. These results show the robust and detailed effect of sonoporation and reveal insights into sonoporation-induced EV release useful for guiding the application of sonoporation to enhance large-scale EV production., (© 2024. The Author(s).)

Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike-in recovery, inter- and intra-assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease-free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type-specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Background/aim: Exosomes are extracellular vesicles produced by both normal and cancer cells. Previous research has demonstrated that circulating exosomes derived from cancer cells may create a niche for future metastasis, distant from the primary tumor. In the present report, circulating exosomes were captured and quantified based on exosome-surface proteins in pre- and post-operative serum of breast cancer patients, focusing on the exosome markers CD9 and CD63, as well as HER2, a therapeutic target for breast cancer., Materials and Methods: Eight breast cancer patients were recruited, and their pre- and post-operative serum samples were analyzed for CD63 and CD9; or CD9 and human epidermal growth factor receptor-2 (HER2), double-positive exosomes. An ExoCounter with antibody-conjugated beads was used to capture serum-derived exosomes. Sera from patients with tumors larger than 10 mm were used for analysis. The resected breast cancer was also histopathologically analyzed for the presence of HER2., Results: CD63 and CD9 double-positive serum exosomes and CD9 and HER2 double-positive serum exosomes decreased after surgery in breast-cancer patients whose tumors expressed HER2, as determined by histopathological analysis., Conclusion: Serum exosomes expressing CD9, CD63 and HER2 are candidate biomarkers of tumor burden in HER2-positive breast-cancer patients., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Recently, exosomes that are derived from bone marrow mesenchymal stem cells (BMSCs) have garnered considerable interest due to their significant roles in the processes of bone regeneration and repair. Among the various molecular components present within these exosomes, miR-668-3p has emerged as a pivotal microRNA that may be instrumental in modulating the function and proliferation of osteoblasts, the cells responsible for bone formation. The primary objective of this research was to examine the enhancing effects of BMSC-derived exosomes that are enriched with miR-668-3p on the advancement of osteoblasts in the context of osteonecrosis of the femoral head. Furthermore, the study aimed to analyze how the expression of specific exosomal proteins, namely CD63 and CD9, influences this biological process. To conduct the investigation, BMSCs were isolated from healthy rat models, followed by the extraction of their secreted exosomes. The subsequent phase of the study involved assessing the proliferation and differentiation of osteoblasts by introducing the exosomes enriched with miR-668-3p into an experimental setup representing osteonecrosis of the femoral head. The findings revealed that exosomes derived from BMSCs, which contained miR-668-3p, significantly enhanced the proliferation of osteoblasts as well as the expression of key osteogenic marker genes. Notably, the levels of CD63 and CD9 proteins were markedly increased in the treated groups, indicating that the mechanisms underlying this promotion might involve cell adhesion and the endocytic uptake of exosomes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The lack of circulating biomarkers for tumor monitoring is a major problem in Ewing sarcoma management. The development of methods for accurate tumor monitoring is required, considering the high recurrence rate of drug-resistant Ewing sarcoma. Here, we describe a sensitive analytical technique for tumor monitoring of Ewing sarcoma by detecting circulating extracellular vesicles secreted from Ewing sarcoma cells. Proteomic analysis of Ewing sarcoma cell-derived extracellular vesicles identified 564 proteins prominently observed in extracellular vesicles from three Ewing sarcoma cell lines. Among these, CD99, SLC1A5, and ENO-1 were identified on extracellular vesicles purified from sera of patients with Ewing sarcoma before treatment but not on extracellular vesicles from those after treatment and healthy individuals. Notably, not only Ewing sarcoma-derived extracellular vesicles but also Ewing sarcoma cells demonstrated proteomic expression of CD99 and ENO-1 on their surface membranes. ENO-1 + CD63 + extracellular vesicle detection was reduced after tumor resection while both CD99 + CD63 + and ENO-1 + CD63 + extracellular vesicles were detected in serum from Ewing sarcoma-bearing mice. Finally, the accuracy of liquid biopsy targeting these candidates was assessed using extracellular vesicles from the sera of patients with Ewing sarcoma. Elevated ENO-1 + CD81 + extracellular vesicles in the serum of patients before treatments distinguished patients with Ewing sarcoma from healthy individuals with an area under the curve value of 0.92 (P < 0.001) and reflected the tumor burden in patients with Ewing sarcoma during multidisciplinary treatments. Collectively, circulating ENO-1 + CD81 + extracellular vesicle detection could represent a novel tool for tumor monitoring of Ewing sarcoma., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Extracellular vesicles (EVs), crucial in facilitating the transport of diverse molecular cargoes for intercellular communication, have shown great potential in diagnostics, therapeutics, and drug delivery. The challenge of developing effective preparation methods for EVs is heightened by their intrinsic heterogeneity and complexity. Here, a novel strategy for high EV enrichment is developed by utilizing EV-affinitive-modified cellulose nanofibrils. Specifically, modified cellulose with rich carboxyl groups has outstanding dispersing properties, able to be dispersed into cellulose nanofibrils in solution. These cellulose nanofibrils are utilized as scaffolds for the immobilization of EV-affinitive antibody of CD63 by chemical conjugation. The CD63-modified nanofibrils demonstrate a superior EV capture efficiency of 86.4% compared with other reported methods. The high performance of this system is further validated by the efficient capture of EVs from biological blood plasma, allowing the detection of bioactive markers from EV-derived miRNAs and proteins. The authors envision that these modified cellulose nanofibrils of enhanced capability on EV enrichment will open new avenues in various biomedical applications., (© 2024 The Authors. Small Methods published by Wiley‐VCH GmbH.)

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics.

Aims: The association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis., Methods and Results: Wild type (WT), PCSK9 -/- , and LDLR -/- C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR -/- and PCSK9 -/- mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9 -/- mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR -/- mice showing high levels while PCSK9 -/- were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR -/- mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63 + EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63 + EVs were significantly depleted., Conclusions: This research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD., (© 2024. The Author(s).)

Preeclampsia is a hypertensive disorder of pregnancy that can lead to stillbirth and preterm birth if not treated promptly. Currently, the diagnosis of preeclampsia relies on clinical symptoms such as hypertension and proteinuria, along with invasive blood tests. Here, we investigate the role of soluble proteins and exosomes in noninvasive diagnosing preeclampsia non-invasively using maternal urine and urine-derived exosomes. We quantified the levels of particles and the presence of TSG101 and CD63 in urine and urinary exosomes via the biologically intact exosome separation technology (BEST) platform. Then, we obtained higher levels of soluble proteins such as fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) from urine as it was than urinary exosomes. Compared to commercial blood tests, the sensitivity of the sFlt-1/PlGF ratio was found to be 4.0 times higher in urine tests and 1.5 times higher in tests utilizing urine-derived exosomes. Our findings offer promising possibilities for the early and non-invasive identification of high-risk individuals at risk of preeclampsia, allowing for comprehensive preventive management., (© 2024. The Author(s).)

Extracellular vesicles (EVs) are highly heterogeneous, and different EV subpopulations from various origins mediate different biological effects. The separation of different subpopulations of EVs from mixtures is critical but challenging. Epididymosomes are secreted by the epididymal epithelium and play a key role in sperm maturation and function. However, limited access to human epididymal tissue and epididymal fluid has hampered further study of epididymosomes and their potential clinical applications. Here, we established a novel strategy based on flow cytometry sorting to isolate human CD63-positive epididymosomes from ejaculate. We identified CD52, a membrane-located protein expressed exclusively in the epididymis, as the sorting marker for human epididymosomes. Then, CD63-positive epididymosomes were isolated from human semen using a flow cytometry sorting instrument and concentrated. Additionally, we observed that isolated CD63-positive epididymosomes improved sperm function more than other CD63-positive seminal EV subpopulations did, demonstrating the successful isolation of a subpopulation of epididymosomes from human semen and their potential application in the clinic., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Exosomes, emerging as ideal non-invasive biomarkers for disease diagnosis and monitoring, have seldom been explored based on metabolite levels. In this study, we designed and synthesized a pH-responsive phase-transition bifunctional affinity nanopolymer (pH-BiAN) that could efficiently and homogeneously separate exosomes from urine. Specifically, poly-4-vinylpyridine (P4VP) was chosen as the pH-responsive polymer and simultaneously modified with two exosome-affinity components CD63 aptamer and distearoyl phosphoethanolamine (DSPE) through a one-step amide reaction at room temperature. By utilizing two distinct but synergistic affinity mechanisms-the immune affinity between CD63 aptamer and exosomal CD63 proteins, and hydrophobic interactions between the DSPE and the exosomal lipids-pH-BiAN can enable efficient and specific exosome separation. Moreover, during the urine exosome capture procedure, the pH-BiAN outperforms conventional solid exosome separation materials by remaining soluble in the urine sample, significantly enhancing mass transfer and contact efficiency. After exosome capture, pH-BiAN can quickly aggregate and convert to solid upon pH adjustment, allowing for easy centrifugation separation. Afterwards, multiple machine learning models were established by combining liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) untargeted metabolomics for isolated exosomes, and the clinical accuracy of the training and test sets was more than 0.919, which could well distinguish early osteoarthritis patients from healthy people., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Mucin1 (MUC1) protein, involved in cytoprotective and signaling pathways, is abnormally elevated in various cancers, making it a key cancer indicator. Exosomes, which reflect the status of their originating cells, offer potential for cancer diagnosis. Thus, developing a method to detect MUC1-positive exosomes is crucial for the early diagnosis of certain cancers. In this study, we developed a highly sensitive, specific, and simple UV-visible signal amplification method to detect MUC1-positive exosomes using terminal deoxynucleotidyl transferase (TdT). Initially, exosomes were captured on magnetic beads using a CD63 aptamer(apt). The Primer-AuNPs-MUC1 apt complex which we synthesized by low pH loading method was then attached MUC1 proteins on the surface of the exosomes to create a sandwich structure. TdT catalyzed the extension of Biotin-dATP at the 3' end of the primer, introducing multiple biotin sites into the sandwich structure. These sites subsequently bound multiple streptavidin-horseradish peroxidase (streptavidin-HRP), which catalyzed the oxidative color change of the substrate, which can be detected by colorimetric method. This method can detect A549 exosomes in the range of 1.4E+6 to 4.2E+8 particles/mL and shows high specificity for cell lines with different MUC1 expression. Additionally, it successfully distinguished cholangiocarcinoma (CCA) patients (n=11) from healthy individuals (n=7) in clinical serum assays, demonstrating good performance in real sample detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins; however, whether these proteins are uniformly present on each LEL, or if there are cell-type-dependent LEL subpopulations with unique protein compositions is unclear. We employed quantitative, multiplexed DNA-PAINT super-resolution imaging to examine the distribution of seven key LEL proteins (LAMP1, LAMP2, CD63, Cathepsin D, TMEM192, NPC1, and LAMTOR4). While LAMP1, LAMP2, and Cathepsin D were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type-specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts., (© 2024 Bond et al.)

Fluorescent protein-based pH biosensors enable the tracking of pH changes during protein trafficking and, in particular, exocytosis. The recent development of chemogenetic reporters combining synthetic fluorophores with self-labeling protein tags offers a versatile alternative to fluorescent proteins that combines the diversity of chemical probes and indicators with the selectivity of the genetic encoding. However, this hybrid protein labeling strategy does not avoid common drawbacks of organic fluorophores such as the risk of off-target signal due to unbound molecules. Here, we describe a novel fluorogenic and chemogenetic pH sensor based on a cell-permeable molecular pH indicator called pHluo-Halo-1 , whose fluorescence can be locally activated in cells by reaction with HaloTag, ensuring excellent signal selectivity in wash-free imaging experiments. pHluo-Halo-1 was selected out of a series of four fluorogenic molecular rotor structures based on protein chromophore analogues. It displays good pH sensitivity with a p K a of 6.3 well-suited to monitor pH variations during exocytosis and an excellent labeling selectivity in cells. It was applied to follow the secretion of CD63-HaloTag fusion proteins using TIRF microscopy. We anticipate that this strategy based on the combination of a tunable and chemically accessible fluorogenic probe with a well-established protein tag will open new possibilities for the development of versatile alternatives to fluorescent proteins for elucidating the dynamics and regulatory mechanisms of proteins in living cells.

The formation of a lytic immunological synapse (IS) is crucial for cytotoxic lymphocytes to accurately target and effectively eliminate malignant cells. While significant attention has been focused on the lymphocyte side of the IS, particularly its role as a secretory domain for lytic granules, the cancer cell side of the IS has remained relatively underexplored. Recent findings have revealed that cancer cells can rapidly polarize their actin cytoskeleton toward the IS upon interaction with natural killer (NK) cells, thereby evading NK cell-mediated cytotoxicity. In this Brief Research Report, we present preliminary findings suggesting that actin cytoskeleton remodeling at the cancer cell side of the IS is associated with the targeted secretion of small extracellular vesicles towards the interacting NK cell. We observed that multivesicular bodies (MVBs) preferentially accumulate in the synaptic region in cancer cells exhibiting synaptic accumulation of F-actin, compared to those lacking actin cytoskeleton remodeling. Extracellular immunofluorescence staining revealed increased surface exposure of CD63 at the cancer cell side of the IS, suggestive of the fusion of MVBs with the plasma membrane. This hypothesis was supported by a pH-sensitive probe demonstrating dynamic trafficking of CD63 to the extracellular region of the IS. Collectively, our data support the notion that cancer cells can engage in targeted secretion of extracellular vesicles in response to NK cell attack, underscoring the need for further research into the potential role of this process in facilitating cancer cell immune evasion., Competing Interests: The authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Biolato, Filali, Pereira Fernandes, Moreau, Mgrditchian, Hoffmann and Thomas.)

Exosomes, extracellular vesicles (EVs) with an average size of 50-150 nm, transfer various biomolecules and exchange signaling molecules between cells in a paracrine manner. Molecular investigations have revealed that EVs can reflect real-time metabolic changes in normal- and cancer-origin cells and thus harbor valid diagnostic biomarkers. Despite these advantages, the detection of low concentrations of cancer cell EVs in biological fluids is still a great challenge. Here, a new electrochemical Exosensor based on platinum-perovskite is developed for the direct detection of EVs using a biotinylated monoclonal CD63 antibody as a capture element. The label-free method exhibited higher sensitivity with a lower limit of quantification of 2000 EVs/µL with a dynamic linear range (LDR) of 2000 to 14,000 EVs/μL compared with other available methods. To enhance the selectivity of detection, EVs were simultaneously sandwiched between secondary antibodies of PSA (prostate-specific antigen), as an FDA-approved prostate cancer biomarker. Data indicated that this Exosensor can distinguish normal and cancer EVs in samples from healthy individuals and prostate cancer patients. Taken together, this technology offers a unique approach to label-free quantification of EVs and cancer detection in the early stages., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The objective of the present study was to compare principal cell-specific aquaporin-2 (AQP2) abundances in urinary extracellular vesicles (uEVs) on the first postoperative day in deceased-donor kidney transplant recipients without and with acute kidney injury. We measured uEV markers (CD9 and CD63) and the abundances of proximal tubular sodium-glucose transporter 2, distal tubular sodium/chloride cotransporter, and principal cell-specific aquaporin-2 using Western blotting of urine. uEV-AQP2 levels were normalized to living donor controls. The validation cohort consisted of 82 deceased-donor kidney transplant recipients who had a median age of 50 years (IQR 43 to 57 years). A total of 32% of recipients had acute kidney injury. The median uEV-AQP2 was significantly higher in recipients with acute kidney injury compared to immediate allograft function (2.05; IQR 0.87 to 2.83; vs. 0.81; IQR 0.44 to 1.78; p < 0.01). The Youden index indicated a uEV-AQP2 threshold of 2.00. Stratifying uEV-AQP2 into quartiles showed that recipients with higher uEV-AQP2 levels had higher rates of acute kidney injury (Cochran-Armitage, p = 0.001). The discovery cohort showed elevated CD9, CD63, and uEV-AQP2 levels in urine from recipients with acute kidney injury compared to immediate allograft function. We were able to quantify the damage of principal cells after kidney transplant to predict acute kidney injury using uEV-AQP2.

Chronic spontaneous urticaria (CSU) is associated with skin mast cell activation, and its triggering mechanisms are not completely elucidated. Evidence suggests an autoimmune component of CSU. Our aim was to assess the usefulness of an autoimmune mast cell activation test (aiMAT) for diagnosing and differentiating CSU into different subtypes. We enrolled 43 patients with active, uncontrolled CSU before starting treatment with omalizumab and 15 controls. Patients were evaluated based on omalizumab response. aiMATs were performed using non-IgE-sensitized (NS) or myeloma IgE-sensitized (S) LAD2 cells, which were then stimulated with CSU/control sera (25 µL and 10 µL). The expression of CD63 was assessed with flow cytometry. CD63 response on NS-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera ( p = 0.0007) and with 10 µL CSU/control sera ( p = 0.0001). The ROC curve analysis demonstrated an area under the curve (AUC) of 0.82. The cutoff for autoimmune-non-IgE-sensitized-MAT was 40.3% CD63+ LAD2, which resulted in 73.3% sensitivity and 81.4% specificity. CD63 response on S-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera ( p = 0.03). The ROC curve analysis demonstrated an AUC of 0.66. The cutoff for the autoimmune-myeloma IgE-sensitized-MAT was 58.4% CD63+ cells, which resulted in 62.8% sensitivity and 66.7% specificity. Overall, 36 out of 43 (84%) patients responded to omalizumab, and 7 (16%) were nonresponders. We found no differences between LAD2 CD63 response and response to omalizumab. In conclusion, aiMAT could represent a new diagnostic tool in CSU. Additional studies are needed to evaluate the potential benefits during omalizumab therapy.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts., (© 2024 Robinson et al.)

Purpose: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis., Methods: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis., Results: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases., Conclusion: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

As the role of exosomes in physiological and pathological processes has been properly perceived, harvesting them and their internal components is critical for subsequent applications. This study is a debut of intermittent lysis, which has been integrated into a simple and easy-to-operate procedure on a single paper-based device to extract exosomal nucleic acid biomarkers for downstream analysis. Exosomes from biological samples were captured by anti-CD63-modified papers before being intermittently lysed by high-temperature, short-time treatment with double-distilled water to release their internal components. Exosomal nucleic acids were finally adsorbed by sol-gel silica for downstream analysis. Empirical trials not only revealed that sporadically dropping 95 °C ddH 2 O onto the anti-CD63-modified papers every 5 min for 6 times optimized the exosomal nucleic acids extracted by the anti-CD63 paper but also verified that the whole deployed procedure is applicable for point-of-care testing (POCT) in low-resource areas and for both in vitro (culture media) and in vivo (plasma and chronic lesion) samples. Importantly, downstream analysis of exosomal miR-21 extracted by the paper-based procedure integrated with this novel technique discovered that the content of exosomal miR-21 in chronic lesions related to their stages and the levels of exosomal carcinoembryonic antigen originated from colorectal cancer cells correlated to their exosomal miR-21., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs., Objective: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L)., Methods: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry., Results: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation., Conclusions: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Exosomes, extracellular vesicles secreted by cells, play a crucial role in intercellular communication by transferring information from source cells to recipient cells. These vesicles carry important biomarkers, including nucleic acids and proteins, which provide valuable insights into the parent cells' status. As a result, exosomes have emerged as noninvasive indicators for the early diagnosis of cancer. Colorimetric biosensors have garnered significant attention due to their cost-effectiveness, simplicity, rapid response, and reproducibility. In this study, we employ sporopollenin microcapsules (SP), a natural biopolymer material derived from pollen, as a substrate for gold nanoparticles (AuNPs). By modifying the SP-Au complex with CD63 aptamers, we develop a label-free colorimetric biosensor for exosome detection. In the absence of exosomes, the SP-Au complex catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), resulting in a color change from colorless to blue. However, the addition of exosomes inhibits the catalytic activity of the SP-Au complex due to coverage of exosomes on AuNPs. This colorimetric biosensor exhibits high sensitivity and selectivity for exosome detection, with a detection limit of 10 particles/μL and a wide linear range of 10 - 10 8 particles/μL. Additionally, the SP-Au biosensor demonstrates remarkable resistance to serum protein adsorption and excellent catalytic stability even in harsh environments, making it highly suitable for clinical diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and poses a huge challenge to global healthcare. Early and accurate detection of amyloid-β (1-42) (Aβ42), a key biomarker of AD, is crucial for effective diagnosis and intervention of AD. Specific or overexpressed proteins on extracellular vesicles (EVs) describe a close correlation with the occurrence and development of diseases. EVs are a very promising non-invasive biomarker for the diagnosis of AD and other diseases. As a sensitive, simple and rapid analytical method, fluorescence resonance energy transfer (FRET) has been widely applied in the detection of EVs. Herein, we developed a dual labelling strategy for simultaneously detecting EV membrane proteins of Aβ42 and CD63 based on FRET pair consisting of Au nanoclusters (AuNCs) and polydopamine nanospheres (PDANSs). The constructed nanoprobe, termed EV MPFAP assay, could specifically measure the Aβ42 and CD63 on EVs with excellent sensitivity, high specificity and satisfactory accuracy. The limit of detection of EV MPFAP assay was 1.4 × 10 3 particles mL -1 and the linear range was from 10 4 to 10 8 particles mL -1 . EV MPFAP assay was successfully used to analyze plasma EVs to distinguish AD and healthy mice. We expect that EV MPFAP assay can be routinely applied for early diagnosis and development-monitoring of AD, thus facilitating the fight against AD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflect physiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work, we present the advantages of an immunoaffinity-based method for uEVs' isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples.

Objective: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles., Results: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies., (© 2024. The Author(s).)

Secretory granule (SG) fusion is an intermediate step in SG biogenesis. However, the precise mechanism of this process is not completely understood. We show that Golgi-derived mast cell (MC) SGs enlarge through a mechanism that is dependent on phosphoinositide (PI) remodeling and fusion with LC3 + late endosomes (amphisomes), which serve as hubs for the fusion of multiple individual SGs. Amphisome formation is regulated by the tyrosine phosphatase PTPN9, while the subsequent SG fusion event is additionally regulated by the tetraspanin protein CD63 and by PI4K. We also demonstrate that fusion with amphisomes imparts to SGs their capacity of regulated release of exosomes. Finally, we show that conversion of PI(3,4,5)P 3 to PI(4,5)P 2 and the subsequent recruitment of dynamin stimulate SG fission. Our data unveil a key role for lipid-regulated interactions with the endocytic and autophagic systems in controlling the size and number of SGs and their capacity to release exosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the regulatory effect of Diwu Yanggan (DWYG) Decoction on lysoglycerophospholipids (Lyso-GPLs) in circulating exosomes in a mouse model of nonalcoholic fatty liver disease (NAFLD)., Methods: Circulating exosomes isolated from mouse serum by size exclusion chromatography were morphologically characterized using transmission electron microscope and examined for surface markers CD9, CD63 and TSG101 using Western blotting. Twenty-four male Kunming mice were randomized into 3 groups for normal feeding (control, n =8) or high-fat diet feeding for 1 week to induce NAFLD, after which the latter mice were given DWYG decoction (treatment group, n =8) or normal saline (model group, n =8) by gavage for 4 weeks. After the last treatment, blood samples were collected from the mice for testing serum TC, HDL-C, LDL-C, ALT and AST levels and isolating circulating exosomes. Using multivariate statistical analysis based on targeted metabolomics strategy, the potential biomarkers for Lyso-GPLs in the exosomes were screened., Results: The isolated exosomes about 100 nm in size had a typical saucer-like structure with distinct double-layer membranes and a mean particle size of 137.5 nm and expressed the specific surface marker proteins CD9, CD63 and TSG101. The mouse models of NAFLD had significantly increased serum levels of TC, HDL-C, LDL-C and AST and lowered serum ALT level. A total of 43 Lyso-GPLs with significant reduction after DWYG Decoction treatment were identified in NAFLD mice., Conclusion: DWYG Decoction can regulate Lyso-GPLs in circulating exosomes in NAFLD mice, which provides a new clue for studying the therapeutic mechanism of DWYG Decoction for liver disease.

Exosomes have significant functions in intercellular communication, as well as in tumor migration and invasion. Nevertheless, the precise identification of exosomes poses a significant obstacle due to their low abundance in biofluids and potential disruption caused by free protein molecules, such as CD63 protein. In this study, we have developed a signal amplification method for precise detection of exosomes using a proximity ligation hybridization triggered structure-switching approach. The method involves the dual-recognition of exosomes by two probes: an aptamer probe that recognizes the exosomal surface protein CD63 (L1 probe), and a cholesterol probe that targets the biolipid layer of the exosomes (L2 probe). Based on the dual-recognition of exosomes, we have successfully developed an accurate and sensitive approach that integrates the proximity ligation hybridization technique with a structure-switching based signal cycle. This approach allows for the simultaneous analysis of two biomarkers, enabling both quantification and tracing of exosomes without the need for enzymes. Eventually, the proposed method exhibits a wide detection range of 5 orders of magnitude and a low limit of detection of 36 particles per μL, making it suitable for a wide range of applications in the fields of biological science, biomedical engineering, and personalized medicine.

Extracellular vesicles such as exosomes are now recognized as key players in intercellular communication. Their role is influenced by the specific repertoires of proteins and lipids, which are enriched when they are generated as intraluminal vesicles (ILVs) in multivesicular endosomes. Here we report that a key component of small extracellular vesicles, the tetraspanin CD63, sorts cholesterol to ILVs, generating a pool that can be mobilized by the NPC1/2 complex, and exported via exosomes to recipient cells. In the absence of CD63, cholesterol is retrieved from the endosomes by actin-dependent vesicular transport, placing CD63 and cholesterol at the centre of a balance between inward and outward budding of endomembranes. These results establish CD63 as a lipid-sorting mechanism within endosomes, and show that ILVs and exosomes are alternative providers of cholesterol., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles ( sm HIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Extracellular vesicles (EVs) are small cellular organelles involved in intracellular signaling and cell-to-cell interactions. Recent studies suggested that exosomes may have potential applications in the diagnosis and treatment of cancer and neurodegenerative diseases. In this study, extracellular vesicles of the human nonsmall cell lung cancer cell line H1299 and the unlabeled antiCD63 antibody were imaged using a new label-free terahertz chemical microscopy (TCM) technique to detect changes in the terahertz wave amplitude. To verify the high specificity of the protein biomarkers and the sensitivity of the biosensor surface, we also confirmed the selective binding of the antibody to the antigen, bovine serum albumin, and cancer cells. We also performed real-time measurements of the interaction between EVs from the H1299 cell and the antiCD63 antibody, which showed that the amount of change in the terahertz intensity increased with increasing concentration and the time to saturation decreased. Finally, to reuse the used biosensors (sensing plates), plasma-oxygen cleaning was used, and the activity of the biosensor surface was confirmed by terahertz microscopy and atomic force microscopy and was found to be reusable after less than 3 min of cleaning. Consequently, terahertz chemical microscopy was able to detect the presence or absence of antigen-antibody binding and its reaction rate and binding strength.

Background The incidence of various types of cancers, including leukemia, is on the rise and many challenges in both drug resistance and complications related to chemotherapy appeared. Recently, the development and application of extracellular vesicles (EV) such as exosomes in the management of cancers, especially leukemia, holds great significance. In this article, we extracted exosomes from NALM6 cells and assessed their regulatory effects on proliferation and apoptosis in mesenchymal stem cells (MSCs). Method and result We first verified the exosomes using various techniques, including flow cytometry, transient electron microscopy, dynamic light scattering (DLS), and BCA protein assay. Then MTT analysis and flowcytometry (apoptosis and cell cycle assay) besides gene expressions were employed to determine the state of MSC proliferations. The results indicated that exosome-specific pan markers like CD9, CD63, and CD81 were present. Through DLS, we found out that the mean size of the exosomes was 89.68 nm. The protein content was determined to be 956.292 µg/ml. Analysis of MTT, flow cytometry (cell cycle and apoptosis assay), and RT-qPCR showed that in the dose of 50 µg/ml the proliferation of MSCs was increased significantly (p-value < 0.05). Conclusion All these data showed that exosomes use several signaling pathways to increase the MSCs' proliferation and drug resistance, ultimately leading to high mortalities and morbidities of acute lymphoblastic leukemia., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Tumor-associated macrophages (TAMs) constitute a substantial part of human hepatocellular carcinoma (HCC). The present study was devised to explore TAM diversity and their roles in HCC progression., Methods: Through the integration of multiple 10 × single-cell transcriptomic data derived from HCC samples and the use of consensus nonnegative matrix factorization (an unsupervised clustering algorithm), TAM molecular subtypes and expression programs were evaluated in detail. The roles played by these TAM subtypes in HCC were further probed through pseudotime, enrichment, and intercellular communication analyses. Lastly, vitro experiments were performed to validate the relationship between CD63, which is an inflammatory TAM expression program marker, and tumor cell lines., Results: We found that the inflammatory expression program in TAMs had a more obvious interaction with HCC cells, and CD63, as a marker gene of the inflammatory expression program, was associated with poor prognosis of HCC patients. Both bulk RNA-seq and vitro experiments confirmed that higher TAM CD63 expression was associated with the growth of HCC cells as well as their epithelial-mesenchymal transition, metastasis, invasion, and the reprogramming of lipid metabolism., Conclusions: These analyses revealed that the TAM inflammatory expression program in HCC is closely associated with malignant tumor cells, with the hub gene CD63 thus representing an ideal target for therapeutic intervention in this cancer type., (© 2024. The Author(s).)

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/μL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers., (© 2024. The Author(s).)

Extracellular vesicles (EVs) originating from cancer cells incorporate various critical biomolecules that can aid in early cancer diagnosis. However, the rapid analysis of these micro vesicles remains challenging due to their nano-scale size and overlapping dimensions, hindering sufficient capture in terms of quantity and purity. In this study, an acoustofluidic device was developed to enhance the yield of immune-captured EVs. The channel of the device was modified with degradable gelatin nanoparticles (∼220 nm) to increase the surface roughness, and subsequently treated with CD63 antibodies. The acoustic-induced streaming would prolong the rotation time of the EVs in the targeted continuous flow area, improving their aggregation towards the surrounding pillars and subsequent capture by the specific CD63 antibodies. Consequently, the capture efficiency of the device was improved when the signal was on, as evidenced by enhanced fluorescence intensity in the main channel. It is demonstrated that the acoustofluidic device could enhance the immune capture of EVs through acoustic mixing, showcasing great potential in the rapid and fast detection of EVs in liquid biopsy applications.

Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 10 7 particles per μL, while the measurable range spanned from 6.50 × 10 7 to 1.30 × 10 9 particles per μL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) have rapidly received Food and Drug Administration (FDA) approval as a new type of therapy for patients with advanced hormone receptor-positive breast cancer. However, with the widespread application of CDK4/6i, drug resistance has become a new challenge for clinical practice and has greatly limited the treatment effect. Here, the whole microenvironment landscape of ER + breast cancer tumors was revealed through single-cell RNA sequencing, and a specific subset of cancer-associated fibroblasts (CD63 + CAFs) was identified as highly enriched in CDK4/6i resistant tumor tissues. Then, we found that CD63 + CAFs can distinctly promote resistance to CDK4/6i in breast cancer cells and tumor xenografts. In addition, it was discovered that miR-20 is markedly enriched in the CD63 + CAFs-derived exosomes, which are used to communicate with ER + breast cancer cells, leading to CDK4/6i resistance. Furthermore, exosomal miR-20 could directly target the RB1 mRNA 3'UTR and negatively regulate RB1 expression to decrease CDK4/6i sensitivity in breast cancer cells. Most importantly, we designed and synthesized cRGD-miR-20 sponge nanoparticles and found that they can enhance the therapeutic effect of CDK4/6i in breast cancer. In summary, our findings reveal that CD63 + CAFs can promote CDK4/6i resistance via exosomal miR-20, which induces the downregulation of RB1 in breast cancer cells, and suggest that CD63 + CAFs may be a novel therapeutic target to enhance CDK4/6i sensitivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)