AbstractMalignant mesothelioma (MM) is a rare cancer caused by exposure to asbestos, this condition continues to represent a significant diagnostic and therapeutic challenge. Due to the long disease latency, non-invasive diagnostic modalities such as breath analysis may enhance MM early detection by identifying the disease specific pattern of volatile organic compounds (VOCs) in exhaled breath. In this study, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) was used to extract VOCs from the headspace of two mesothelioma cell lines: MSTO-211H (biphasic mesothelioma) and NCI-H28 (epithelioid mesothelioma) in addition to MET-5a (non-malignant mesothelial cell line), following exposure to asbestos mineral fibers (actinolite, amosite, crocidolite and chrysotile) and a non-asbestos fiber control (wollastonite). Multivariate statistical analysis was applied to identify VOC-based biomarkers associated with asbestos exposure within the cell lines. Data confirms that exposure to mineral fibers (including the non-asbestos fiber control) induces significant alterations in the levels of as many as 24 VOCs in each cell line. This is the first study to investigate the impact of asbestos mineral fiber exposure on the VOC profile of MM cell lines. This may be relevant to studies involving mesothelioma patients, as these VOCs could also serve as indicators of asbestos exposure in individuals who have been exposed to asbestos.

Efficient isolation and ultrasensitive detection of cancer cell-derived extracellular vesicles (EVs), which carry molecular fingerprints of their parent cells, can enable precise early-stage cancer diagnostics and prognostics. In this study, we developed a label-free impedimetric biosensor using electroactive 3D porous graphene foam (3D-PGF) electrodes to detect CD81 protein and MCF-7 cells-derived EVs in human serum. 1-pyrenebutyric acid N-hydroxysuccinimide ester (PYB-NHS) was non-covalently attached to the 3D-PGF via pi-pi interactions without compromising structural integrity or electrocatalytic performance. Sensor selectivity was achieved by immobilizing anti-CD81 antibodies onto the modified electrode, followed by quenching and blocking with ethanolamine and bovine serum albumin. MCF-7 cells-derived EVs were isolated by ultracentrifugation and characterized using nanoparticle tracking analysis and immunoaffinity plate-based assay. Capture and detection of CD81- and MCF-7-derived EVs were evaluated using electrochemical impedance spectroscopy with a ferro/ferri redox mediator. The sensor demonstrated exceptional sensitivity, with limits of detection of 0.048 fg/mL for CD81 protein and similar to 24 EVs/mL for MCF-7 EVs. Performance was benchmarked against 2D graphene electrodes, highlighting structural and electrochemical advantages. The binder-free 3D-PGF combined with non-covalent functionalization establishes a robust platform for selective, label-free, and ultrasensitive detection of cancer-cell-derived EVs in human serum samples.

ABSTRACT Highly inflammatory mononuclear phagocytes (MNPs) cause tissue damage across the respiratory tract and other organs in response to infections such as SARS‐CoV‐2. Extracellular vesicles (EVs) can metabolically and phenotypically reprogram target cells, suggesting a potential role in COVID‐19 pathology. We hypothesised that EVs drive inflammatory changes in COVID‐19 and investigated their cargo and effects on MNPs. EVs and MNPs were isolated from matched peripheral blood and airways of ventilated patients with and without severe COVID‐19, alongside healthy volunteer blood samples. Characterisation was performed using flow cytometry, microscopy, transcriptomics, PCR, proteomics, mass spectrometry, and bioenergetic profiling. We found that airway EVs from severe COVID‐19 patients carried altered cargo, notably reduced miRNAs and enriched mitochondrial DNA and ATP, a difference absents in the circulation. These DAMP‐rich EVs impaired healthy MNP function, suppressing cytokine production (e.g., IL‐6, IFNα2), and inflammatory programs identified in the monocyte proteomic profile, while also disrupting oxidative phosphorylation—features matching airway MNPs in severe disease. Interaction of COVID‐19 airway EVs with monocytes was diminished and these EVs had altered phosphatidylcholine/ lysophosphatidylcholine content. Our findings reveal a distinct EV cargo that reprograms immune metabolism, identifying a novel immunomodulatory mechanism exploited by SARS‐CoV‐2.

The prostate gland is a complex and heterogeneous organ composed of epithelium and stroma. Whilst many studies into prostate cancer focus on epithelium, the stroma is known to play a key role in disease with the emergence of a cancer-associated fibroblasts (CAF) phenotype associated upon disease progression. In this work, we studied the metabolic rewiring of stromal fibroblasts following differentiation to a cancer-associated, myofibroblast-like, phenotype. We determined that CAFs were metabolically more active compared to normal fibroblasts. This corresponded with a heightened lipogenic metabolism, as both reservoir species and building block compounds. Interestingly, lipid metabolism affects mitochondria functioning yet the mechanisms of lipid-mediated functions are unclear. Data showing oxidised fatty acids and glutathione system are elevated in CAFs, compared to normal fibroblasts, strengthens the hypothesis that increased metabolic activity is related to mitochondrial activity. This manuscript describes mechanisms responsible for the altered metabolic flux and shows that prostate cancer-derived extracellular vesicles can increase basal respiration in normal fibroblasts, mirroring that of the disease-like phenotype. This indicates that extracellular vesicles derived from prostate cancer cells may drive an altered oxygen-dependent metabolism associated to mitochondria in CAFs.

Abstract Background Distinguishing benign from malignant pancreaticobiliary disease is challenging because of the absence of reliable biomarkers. Circulating extracellular vesicles (EVs) have emerged as functional mediators between cells. Their cargos, including microRNAs (miRNAs), are increasingly acknowledged as an important source of potential biomarkers. This multicentric, prospective study aimed to establish a diagnostic plasma EV-derived miRNA signature to discriminate pancreatic ductal adenocarcinoma (PDAC) from benign pancreaticobiliary disease. Methods Plasma EVs were isolated using size exclusion chromatography (SEC) and characterised using nanoparticle tracking analysis, electron microscopy and Western blotting. EV-RNAs underwent small RNA sequencing to discover differentially expressed markers for PDAC (n = 10 benign vs. 10 PDAC). Candidate EV-miRNAs were then validated in a cohort of 61 patients (n = 31 benign vs. 30 PDAC) by RT-qPCR. Logistic regression and optimal thresholds (Youden Index) were used to develop an EV-miR-200 family model to detect cancer. This model was tested in an independent cohort of 95 patients (n = 30 benign, 33 PDAC, and 32 cholangiocarcinoma). Results Small RNA sequencing and RT-qPCR showed that EV-miR-200 family members were significantly overexpressed in PDAC vs. benign disease. Combined expression of the EV-miR-200 family showed an AUC of 0.823. In an independent validation cohort, application of this model showed a sensitivity, specificity and AUC of 100%, 88%, and 0.97, respectively, for diagnosing PDAC. Conclusions This is the first study to validate plasma EV-miR-200 members as a clinically-useful diagnostic biomarker for PDAC. Further validation in larger cohorts and clinical trials is essential. These findings also suggest the potential utility in monitoring response and/or recurrence. Graphical Abstract

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) is a feature of many solid tumours and is a key pathogenic driver in the inherited condition Tuberous Sclerosis Complex (TSC). Modulation of the tumour microenvironment by extracellular vesicles (EVs) is known to facilitate the development of various cancers. The role of EVs in modulating the tumour microenvironment and their impact on the development of TSC tumours, however, remains unclear. This study, therefore, focuses on the poorly defined contribution of EVs to tumour growth in TSC. We characterised EVs secreted from TSC2-deficient and TSC2-expressing cells and identified a distinct protein cargo in TSC2-deficient EVs, containing an enrichment of proteins thought to be involved in tumour-supporting signalling pathways. We show EVs from TSC2-deficient cells promote cell viability, proliferation and growth factor secretion from recipient fibroblasts within the tumour microenvironment. Rapalogs (mTORC1 inhibitors) are the current therapy for TSC tumours. Here, we demonstrate a previously unknown intercellular therapeutic effect of rapamycin in altering EV cargo and reducing capacity to promote cell proliferation in the tumour microenvironment. Furthermore, EV cargo proteins have the potential for clinical applications as TSC biomarkers, and we reveal three EV-associated proteins that are elevated in plasma from TSC patients compared to healthy donor plasma.

Supplementary Figure Legend from Cancer Exosomes Trigger Fibroblast to Myofibroblast Differentiation

The quick reference card "Storage of urinary

AbstractUrinary extracellular vesicles (uEVs) are enriched with glycosylated proteins which have been extensively studied as putative biomarkers of urological cancers. Here, we characterized the glycosylation and integrin profile of EVs derived from urological cancer cell lines. We used fluorescent europium‐doped nanoparticles coated with lectins and antibodies to identify a biomarker combination consisting of integrin subunit alpha 3 (ITGA3) and fucose. In addition, we used the same cancer cell line‐derived EVs as analytical standards to assess the sensitivity of the ITGA3‐UEA assay. The clinical performance of the ITGA3‐UEA assay was analysed using urine samples of various urological pathologies including diagnostically challenging benign prostatic hyperplasia (BPH), prostate cancer (PCa) and bladder cancer (BlCa). The assay can significantly discriminate BlCa from all other patient groups: PCa (9.2‐fold;p = 0.00038), BPH (5.5‐fold;p = 0.004) and healthy individuals (and 23‐fold;p = 0.0001). Our results demonstrate that aberrantly fucosylated uEVs and integrin ITGA3 can be detected with fucose‐specific lectin UEA in a simple bioaffinity assay for the detection of BlCa directly from unprocessed urine.

AbstractUrine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast‐growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV‐based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Skeletal metastases are the most common form of secondary tumour associated with prostate cancer (PCa). The aberrant function of bone cells neighbouring these tumours leads to the devel-opment of osteoblastic lesions. Communication between PCa cells and bone cells in bone envi-ronments governs both the formation/development of the associated lesion, and growth of the secondary tumour. Using osteoblasts as a model system, we observed that PCa cells and their conditioned medium could stimulate and increase mineralisation and osteoblasts’ differentiation. Secreted factors within PCa-conditioned medium responsible for osteoblastic changes included small extracellular vesicles (sEVs), which were sufficient to drive osteoblastogenesis. Using MiR-seq, we profiled the miRNA content of PCa sEVs, showing that miR-16-5p was highly ex-pressed. MiR-16 was subsequently higher in EV-treated 7F2 cells and a miR-16 mimic could also stimulate mineralisation. Next, using RNA-seq of extracellular vesicle (EV)-treated 7F2 cells, we observed a large degree of gene downregulation and an increased mineralisation. Ingenuity® Pathway Analysis (IPA®) revealed that miR-16-5p (and other miRs) was a likely upstream effec-tor. MiR-16-5p targets in 7F2 cells, possibly involved in osteoblastogenesis, were included for val-idation, namely AXIN2, PLSCR4, ADRB2 and DLL1. We then confirmed the targeting and dow-regulation of these genes by sEV miR-16-5p using luciferase UTR (untranslated region) reporters. Conversely, the overexpression of PLSCR4, ADRB2 and DLL1 lead to decreased osteoblastogene-sis. These results indicate that miR-16 is an inducer of osteoblastogenesis and is transmitted through prostate cancer-derived sEVs. The mechanism is a likely contributor towards the for-mation of osteoblastic lesions in metastatic PCa.

Abstract Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non‐invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell‐derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor‐matched pairs of adjacent‐normal versus disease‐associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease‐discriminating mRNA. A set of transcripts including Caveolin‐1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression‐free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV‐associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in‐silico model was produced, showcasing the superiority of this multi‐modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

Communication between diseased cells and the microenvironment is a complex yet crucial element in progression of varied pathological processes. Recent studies in cancer highlight an important role for small extracellular nanovesicles secreted by cancer cells as modulators of cancer-associated stroma, leading to enhanced angiogenesis and metastatic priming. The intrinsic factors regulating extracellular nanovesicle biogenesis and secretion are therefore relevant in studies of nano-communication in the cancer milieu. We generated prostate cancer cells bearing stable knockdown of several candidate vesicle regulating factors and examined the impact on cell health, vesicle secretion and on communication with fibroblastic stromal cells. We highlight that RAB11B and RAB35 regulate phenotypically distinct nanovesicle populations, each accounting for only around 20% of the total. Depleting RAB35, but not RAB11B leaves a remaining population of vesicles whose phenotype is insufficient for driving fibroblast to myofibroblast differentiation, leading to attenuated motile behaviours in 3D in vitro models. Co-implantation of tumour cells with stromal fibroblasts in xenografts similarly showed that RAB11B knockdown had little effect on growth rates in vivo. In contrast, significant attenuation in growth, and attenuation of myofibroblasts at the tumour site was evident when using RAB35-knockdown cells. The study concludes that a RAB35 regulated nanovesicle sub-population is particularly important for communication between cancer and stromal cells, and is required for generating a tumour-supportive microenvironment.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points. ARTICLE HISTORY Received 20 September 2018 Accepted 25 September 2018

The Journal of Cancer Metastasis and Treatment is an open access journal focused on cancer metastasis and treatment, including the occurrence, development, progression, metastasis, and treatment of oncologic disease. It covers basic, translational and clinical research related to cancer cell biology, genomics, precision medicine, oncology internal medicine, radiotherapy and radiology, obstetrics and gynecology, pediatrics, surgery, hematology, neurooncology, etc.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

ABSTRACT Exosomes are a distinct population of extracellular vesicles of endocytic origin with a protein repertoire similar to the parent cell. Although tumour‐derived exosomes harbour immunosuppressive characteristics, they also carry tumour antigens and thus potentially contribute to immune activation. The aim of this study was to examine the impact of prostate cancer exosomes on tumour antigen cross‐presentation. DU145 cells, transduced with shRNA to knockdown Rab27a (DU145 KD ) that inhibits exosome secretion, triggered significantly stronger tumour‐antigen‐specific T cell responses when loaded onto dendritic cells (DC) than control DU145 cells. Enhanced T cell response was prevented by adding purified exogenous DU145 exosomes to DU145 KD cells, demonstrating that the dominant effect of tumour exosomes is immunosuppression and not antigen delivery. CD8 + T cell responses were impaired via exosomal regulation of DC function; exosomes triggered the expression of CD73, an ecto‐5‐nucleotidase responsible for AMP to adenosine hydrolysis, on DC. CD73 induction on DC that constitutively express CD39 resulted in an ATP‐dependent inhibition of TNFα‐ and IL‐12‐production. We identified exosomal prostaglandin E 2 (PGE 2 ) as a potential driver of CD73 induction, as inhibition of PGE 2 receptors significantly reduced exosome‐dependent CD73 induction. The results reveal a hitherto unknown suppression of DC function via exosomal PGE 2 , adding a new element to tumour exosome–immune cell cross‐talk. Abbreviations : AMP: adenosine monophosphate; ATP: adenosine triphosphate; BLCL: B lymphoblastoid cell line; CME: exosomes enriched from cell line conditioned media; DC: dendritic cell; DMSO: dimethyl‐sulfoxide; DU145 C : DU145 cells with irrelevant knockdown control; DU145 KD : DU145 cells with Rab27a knockdown; ELISA: enzyme‐linked immunosorbent assay; FBS: fetal bovine serum; GM‐CSF: granulocyte‐monocyte colony stimulating factor; HLA: human lymphocyte antigen; IL: interleukin; LPS: lipopolysaccharide; mfi: mean fluorescence intensity; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffer solution; PGE 2 : prostaglandin E 2 ; TRF: time‐resolved fluorescence.