Small extracellular vesicles-shuttled miR-23a-3p from mesenchymal stem cells alleviate renal fibrosis and inflammation by inhibiting KLF3/STAT3 axis in diabetic kidney disease.

Human umbilical cord mesenchymal stem cells-derived small extracellular vesicles (MSC-sEV) provide a pragmatic solution as a cell-free therapy for patients with diabetic kidney disease (DKD). However, the underlying protective mechanisms of MSC-sEV remain largely unknown in DKD. Invivo and in vitro analyses demonstrated that MSC-sEV attenuated renal fibrosis and inflammation of DKD. The underlying mechanism of the MSC-sEV-induced therapeutic effect was explored by high-throughput sequencing, which identified the unique enrichment of a set of miRNAs in MSC-sEV compared with human skin fibroblasts-sEV (HSF-sEV). Vitro experiments demonstrated that the protective potential was primarily attributed to miR-23a-3p, one of the most abundant miRNAs in MSC-sEV. Further, overexpression or knockdown analyses revealed that miR-23a-3p, and its target Krüppel-like factor 3 (KLF3) suppressed the STAT3 signaling pathway in high glucose (HG) induced HK-2 cells were essential for the renal-protective property of MSC-sEV. Moreover, we found that miR-23a-3p was packaged into MSC-sEV by RNA Binding Motif Protein X-Linked (RBMX) and transmitted to HG-induced HK-2 cells. Finally, inhibiting miR-23a-3p could mitigate the protective effects of MSC-sEV in db/db mice. These findings suggest that a systemic administration of sEV derived from MSC, have the capacity to incorporate into kidney where they can exert renal-protective potential against HG-induced injury through delivery of miR-23a-3p.

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

Extracellular vesicles meet mitochondria: Potential roles in regenerative medicine.

Extracellular vesicles (EVs), secreted by most cells, act as natural cell-derived carriers for delivering proteins, nucleic acids, and organelles between cells. Mitochondria are highly dynamic organelles responsible for energy production and cellular physiological processes. Recent evidence has highlighted the pivotal role of EVs in intercellular mitochondrial content transfer, including mitochondrial DNA (mtDNA), proteins, and intact mitochondria. Intriguingly, mitochondria are crucial mediators of EVs release, suggesting an interplay between EVs and mitochondria and their potential implications in physiology and pathology. However, in this expanding field, much remains unknown regarding the function and mechanism of crosstalk between EVs and mitochondria and the transport of mitochondrial EVs. Herein, we shed light on the physiological and pathological functions of EVs and mitochondria, potential mechanisms underlying their interactions, delivery of mitochondria-rich EVs, and their clinical applications in regenerative medicine.

Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma.

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of ß-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

Boron Neutron Capture Therapy-Derived Extracellular Vesicles via DNA Accumulation Boost Antitumor Dendritic Cell Vaccine Efficacy.

Radiated tumor cell-derived extracellular vesicles (RT-EVs) encapsulate abundant DNA fragments from irradiated tumor cells, in addition to acting as integrators of multiple tumor antigens. Accumulating evidence indicates these DNA fragments from damaged cells are involved in downstream immune responses, but most of them are degraded in cells before incorporation into derived RT-EVs, thus the low abundance of DNA fragments limits immune responses of RT-EVs. Here, this study found that different radiations affected fates of DNA fragments in RT-EVs. Boron neutron capture therapy (BNCT) induced DNA accumulation in RT-EVs (BEVs) by causing more DNA breaks and DNA oxidation resisting nuclease degradation. This is attributed to the high-linear energy transfer (LET) properties of alpha particles from the neutron capture reaction of 10B. When being internalized by dendritic cells (DCs), BEVs activated the DNA sensing pathway, resulting in functional enhancements including antigen presentation, migration capacity, and cytokine secretion. After vaccination of the BEVs-educated DCs (BEV@BMDCs), the effector T cells significantly expanded and infiltrated into tumors, suggesting robust anti-tumor immune activation. BEV@BMDCs not only effectively inhibited the primary tumor growth and metastasis formation but also elicited long-term immune memory. In conclusion, a successful DC vaccine is provided as a promising candidate for tumor vaccine.

Acidity and hypoxia of tumor microenvironment, a positive interplay in extracellular vesicle release by tumor cells.

The complex and continuously evolving features of the tumor microenvironment, varying between tumor histotypes, are characterized by the presence of host cells and tumor cells embedded in a milieu shaped by hypoxia and low pH, resulting from the frequent imbalance between vascularity and tumor cell proliferation. These microenvironmental metabolic stressors play a crucial role in remodeling host cells and tumor cells, contributing to the stimulation of cancer cell heterogeneity, clonal evolution, and multidrug resistance, ultimately leading to progression and metastasis. The extracellular vesicles (EVs), membrane-enclosed structures released into the extracellular milieu by tumor/host cells, are now recognized as critical drivers in the complex intercellular communication between tumor cells and the local cellular components in a hypoxic/acidic microenvironment. Understanding the intricate molecular mechanisms governing the interactions between tumor and host cells within a hypoxic and acidic microenvironment, triggered by the release of EVs, could pave the way for innovative strategies to disrupt the complex interplay of cancer cells with their microenvironment. This approach may contribute to the development of an efficient and safe therapeutic strategy to combat cancer progression. Therefore, we review the major findings on the release of EVs in a hypoxic/acidic tumor microenvironment to appreciate their role in tumor progression toward metastatic disease.

Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics.

Aberrant regulation of serine metabolism drives extracellular vesicle release and cancer progression.

Cancer cells secrete extracellular vesicles (EVs) to regulate cells in the tumor microenvironment to benefit their own growth and survive in the patient's body. Although emerging evidence has demonstrated the molecular mechanisms of EV release, regulating cancer-specific EV secretion remains challenging. In this study, we applied a microRNA library to reveal the universal mechanisms of EV secretion from cancer cells. Here, we identified miR-891b and its direct target gene, phosphoserine aminotransferase 1 (PSAT1), which promotes EV secretion through the serine-ceramide synthesis pathway. Inhibition of PSAT1 affected EV secretion in multiple types of cancer, suggesting that the miR-891b/PSAT1 axis shares a common mechanism of EV secretion from cancer cells. Interestingly, aberrant PSAT1 expression also regulated cancer metastasis via EV secretion. Our data link the PSAT1-controlled EV secretion mechanism and cancer metastasis and show the potential of this mechanism as a therapeutic target in multiple types of cancer.

Increased levels of thymidine kinase 1 in malignant cell-derived extracellular vesicles.

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

Exosomes: the next frontier in vaccine development and delivery.

Exosomes are small disk-shaped extracellular vesicles (EVs) that are naturally released into the environment by different types of cells. Exosomes range from 30-150 nm in size and contain complex RNA and proteins. They are widely found in body fluids such as blood, saliva, urine and breast milk and participate in cell communication by functioning as cell messengers. Almost all cell types can transmit information and exchange substances through the production and release of exosomes to regulate proliferation, differentiation, apoptosis, the immune response, inflammation, and other biological functions. Because exosomes exist widely in various body fluids, they are easy to obtain and detect and have the potential for use in disease diagnosis and prognosis detection. Exosomes can be genetically fused with targeted proteins, enhancing their biocompatibility and immunogenicity. Therefore, exosomes are the preferred vector tools for vaccines. In this review, we describe the characteristics of exosomes and discuss their unique and ambiguous functions in the immune microenvironment after infection. In this regard, we explored the ability of exosomes to carry immunogenic virus antigens and to establish adaptive immune responses. Exosomes can provide an interesting platform for antigen presentation and since vaccines are a powerful method for the prevention of infectious diseases, we further review the advantages and disadvantages of the use of exosomes in vaccine preparation. Overall, exosomes are emerging as a promising avenue for vaccine development.

Enhancing circulatory myokines and extracellular vesicle uptake with targeted exercise in patients with prostate cancer (the MYEX trial): a single-group crossover study.

INTRODUCTION: Physical activity is associated with improved disease progression and cancer-specific survival in patients with prostate cancer (PCa). However, the mechanisms underlying these associations remain unclear, while the relative impact of exercise modes is unknown. This study aims to examine the differential impact of exercise mode on tumour-suppressive skeletal muscle-associated systemic molecules as well as their delivery mechanism. This study will compare the effects of the two main exercise modes, aerobic and resistance, on (1) circulatory myokine levels, (2) skeletal muscle-induced extracellular vesicle abundance and cargo contents, and (3) uptake of extracellular vesicles (EVs) in PCa cells in patients with localised or advanced PCa. METHODS: A single-group cross-over design will be used for patients at opposite ends of the disease spectrum. A total of 32 patients (localised PCa, n = 16; metastatic castrate-resistant PCa, n = 16) will be recruited while capitalising on two ongoing studies. Ethics amendment has been approved for two ongoing trials to share data, implement the acute exercise sessions, and collect additional blood samples from patients. The patients will undertake two exercise sessions (aerobic only and resistance only) in random order one week apart. Blood will be collected before, after, and 30 min post-exercise. Circulating/EV-contained myokine levels (irisin, IL-6, IL-15, FGF-21, and SPARC) and plasma skeletal muscle-induced EVs will be measured using ELISA and flow cytometry. PCa cell line growth with or without collected plasma will be examined using PCa cell lines (LNCaP, DU-145, and PC-3), while evaluating cellular uptake of EVs. Ethics amendments have been approved for two capitalising studies to share data, implement acute exercise sessions and collect additional samples from the patients. DISCUSSION: If findings show a differential impact of exercise mode on the establishment of an anti-cancer systemic environment, this will provide fundamental knowledge for developing targeted exercise prescriptions for patients with PCa across different disease stages. Findings will be reported in peer-reviewed publications and scientific conferences, in addition to working with national support groups to translate findings for the broader community. TRIAL REGISTRATION: The registration for the two capitalising studies are NCT02730338 and ACTRN12618000225213.

Plant-derived extracellular nanovesicles: a promising biomedical approach for effective targeting of triple negative breast cancer cells.

Introduction: Triple negative breast cancer (TNBC), a highly aggressive subtype accounting for 15-20% of all breast cancer cases, faces limited treatment options often accompanied by severe side effects. In recent years, natural extracellular nanovesicles derived from plants have emerged as promising candidates for cancer therapy, given their safety profile marked by non-immunogenicity and absence of inflammatory responses. Nevertheless, the potential anti-cancer effects of Citrus limon L.-derived extracellular nanovesicles (CLENs) for breast cancer treatment is still unexplored. Methods: In this study, we investigated the anti-cancer effects of CLENs on two TNBC cell lines (4T1 and HCC-1806 cells) under growth conditions in 2D and 3D culture environments. The cellular uptake efficiency of CLENs and their internalization mechanism were evaluated in both cells using confocal microscopy. Thereafter, we assessed the effect of different concentrations of CLENs on cell viability over time using a dual approach of Calcein-AM PI live-dead assay and CellTiter-Glo bioluminescence assay. We also examined the influence of CLENs on the migratory and evasion abilities of TNBC cells through wound healing and 3D Matrigel drop evasion assays. Furthermore, Western blot analysis was employed to investigate the effects of CLENs on the phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal- regulated kinase (ERK) expression. Results: We found that CLENs were internalized by the cells via endocytosis, leading to decreased cell viability, in a dose- and time-dependent manner. Additionally, the migration and evasion abilities of TNBC cells were significantly inhibited under exposed to 40 and 80 µg/mL CLENs. Furthermore, down-regulated expression levels of phosphorylated phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK), suggesting that the inhibition of cancer cell proliferation, migration, and evasion is driven by the inhibition of the PI3K/AKT and MAPK/ERK signaling pathways. Discussion: Overall, our results demonstrate the anti-tumor efficiency of CLENs against TNBC cells, highlighting their potential as promising natural anti-cancer agents for clinical applications in cancer treatment.

Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

Advances in preparation and engineering of plant-derived extracellular vesicles for nutrition intervention.

Plant-derived extracellular vesicles (PLEVs), as a type of naturally occurring lipid bilayer membrane structure, represent an emerging delivery vehicle with immense potential due to their ability to encapsulate hydrophobic and hydrophilic compounds, shield them from external environmental stresses, control release, exhibit biocompatibility, and demonstrate biodegradability. This comprehensive review analyzes engineering preparation strategies for natural vesicles, focusing on PLEVs and their purification and surface engineering. Furthermore, it encompasses the latest advancements in utilizing PLEVs to transport active components, serving as a nanotherapeutic system. The prospects and potential development of PLEVs are also discussed. It is anticipated that this work will not only address existing knowledge gaps concerning PLEVs but also provide valuable guidance for researchers in the fields of food science and biomedical studies, stimulating novel breakthroughs in plant-based therapeutic options.

Immune interactions and regulation with CD39+ extracellular vesicles from platelet concentrates.

Introduction: CD39 plays an important role in the immunoregulation and inhibition of effector cells. It is expressed on immune cells, including Tregs, and on extracellular vesicles (EVs) budding from the plasma membrane. Platelet transfusion may induce alloimmunization against HLA-I antigens, leading to refractoriness to platelet transfusion with severe consequences for patients. Tregs may play a key role in determining whether alloimmunization occurs in patients with hematologic disorders. We hypothesized that CD39+ EVs might play an immunoregulatory role, particularly in the context of platelet transfusions in patients with hematologic disorders. Such alloimmunization leads to the production of alloantibodies and is sensitive to the regulatory action of CD39. Methods: We characterized CD39+ EVs in platelet concentrates by flow cytometry. The absolute numbers and cellular origins of CD39+ EVs were evaluated. We also performed functional tests to evaluate interactions with immune cells and their functions. Results: We found that CD39+ EVs from platelet concentrates had an inhibitory phenotype that could be transferred to the immune cells with which they interacted: CD4+ and CD8+ T lymphocytes (TLs), dendritic cells, monocytes, and B lymphocytes (BLs). Moreover, the concentration of CD39+ EVs in platelet concentrates varied and was very high in 10% of concentrates. The number of these EVs present was determinant for EV-cell interactions. Finally, functional interactions were observed with BLs, CD4+ TLs and CD39+ EVs for immunoglobulin production and lymphoproliferation, with potential implications for the immunological management of patients.

Exosomes in Bone Cancer: Unveiling their Vital Role in Diagnosis, Prognosis, and Therapeutic Advancements.

Bone cancer among adolescents and children exhibits varying survival outcomes based on disease state. While localized bone cancer cases have a survival rate exceeding 70%, metastatic, refractory, and recurrent forms are associated with significantly poorer prognoses. Initially believed to be mere vehicles for cellular waste disposal, exosomes are now recognized as extracellular vesicles facilitating intercellular communication. These vesicles influence cellular behaviors by transporting various biomolecules, such as proteins, DNA, RNA, and lipids, among cells. The role of exosomes in regulating the progression of bone cancer is increasingly evident, impacting critical processes like tumorigenesis, proliferation, metastasis, angiogenesis, immune evasion, and drug resistance. Current research underscores the substantial potential of exosomes in promoting the progression and development of bone cancer. This review delves into the complex process of exosome biogenesis, the variety of cell-derived exosome sources, and their applications in drug delivery and therapeutics. It also examines ongoing clinical trials focused on exosome cargo levels and discusses the challenges and future directions in exosome research. Unlike costly and invasive traditional diagnostic methods, exosomal biomarkers offer a non-invasive, cost-effective, and readily accessible routine screening through simple fluid collection that aims to inspire researchers to investigate the potential of exosomes for cancer theragnostic. Through comprehensive exploration of these areas, the review seeks to enhance understanding and foster innovative solutions to cancer biology in the near future.

Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.

Acute neuroinflammation promotes a metabolic shift that alters extracellular vesicle cargo in the mouse brain cortex.

Neuroinflammation is initiated through microglial activation and cytokine release which can be induced through lipopolysaccharide treatment (LPS) leading to a transcriptional cascade culminating in the differential expression of target proteins. These differentially expressed proteins can then be packaged into extracellular vesicles (EVs), a form of cellular communication, further propagating the neuroinflammatory response over long distances. Despite this, the EV proteome in the brain, following LPS treatment, has not been investigated. Brain tissue and brain derived EVs (BDEVs) isolated from the cortex of LPS-treated mice underwent thorough characterisation to meet the minimal information for studies of extracellular vesicles guidelines before undergoing mass spectrometry analysis to identify the differentially expressed proteins. Fourteen differentially expressed proteins were identified in the LPS brain tissue samples compared to the controls and 57 were identified in the BDEVs isolated from the LPS treated mice compared to the controls. This included proteins associated with the initiation of the inflammatory response, epigenetic regulation, and metabolism. These results allude to a potential link between small EV cargo and early inflammatory signalling.