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Lung cancer is the leading cause of cancer-related deaths among men and women in the world, accounting for the 25% of cancer mortality. Early diagnosis is an unmet clinical issue. In this work, we focused to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the pleural lavage, a proximal fluid in lung cancer patients, as a source of potential biomarkers. We isolated EVs by ultracentrifuge method from 25 control pleural fluids and 21 pleural lavages from lung cancer patients. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 288 out of the 754 miRNAs that were contained in the OpenArray. The differential expression analysis yielded a list of 14 miRNAs that were significantly dysregulated (adj. p-value < 0.05 and logFC lower or higher than 3). Using Machine Learning approach we discovered the lung cancer diagnostic biomarkers; miRNA-1-3p, miRNA-144-5p and miRNA-150-5p were found to be the best by accuracy. Accordance with our finding, these miRNAs have been related to cancer processes in previous studies. This results opens the avenue to the use of EV-associated miRNA of pleural fluids and lavages as an untapped source of biomarkers, and specifically, identifies miRNA-1-3p, miRNA-144-5p and miRNA 150-5p as promising biomarkers of lung cancer diagnosis.

Background Mesenchymal stem cells (MSCs)-based and MSCs-derived extracellular vesicle (EV) therapies, have garnered significant attention as potential treatments for severe Coronavirus disease 19 (Covid-19), leading to evaluation in numerous clinical trials. This interest stems from the observed ability of MSCs, and their EVs, to mitigate the hyper-inflammatory state that characterizes severe cases of the disease. However, despite the promising preclinical and early clinical results, the precise mechanisms of action remain unclear, hindering therapy optimization. Methods This study investigated the response of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) to a diverse panel of sera collected from multiple hospitalized Covid-19 patients, aiming to recapitulate in vitro the spectrum of inflammatory conditions MSCs encounter in vivo. Results Exposure to Covid-19 serum activated WJ-MSCs, increasing proliferation and RNA/protein synthesis. Transcriptomic analysis revealed an early and significant downregulation of the inflammation-related long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 . This downregulation of NEAT1 and MALAT1 , triggered by Covid-19 serum, suggests a potential mechanism by which WJ-MSCs may limit excessive inflammation. Importantly, reducing MALAT1 (and to a lesser extent NEAT1 ) expression in WJ-MSCs enhanced the therapeutic potential of MSC-derived EVs. This suggests that targeting pro-inflammatory lncRNAs in parental cells could be a promising therapeutic strategy for managing Covid-19 and other inflammatory lung diseases.

Background Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide. Current systematic methods for diagnosing have inherent limitations so development of a minimally-invasive diagnosis, based on the identification of sensitive biomarkers in liquid biopsies could therefore facilitate screening among population at risk. Methods In this study, we aim to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the peritoneal lavage as a source of potential miRNA diagnostic biomarkers. We isolated EVs by ultracentrifugation from 25 ascitic fluids and 25 peritoneal lavages from non-cancer and CRC patients, respectively. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 371 miRNAs. Results 210 miRNAs were significantly dysregulated (adjusted p value < 0.05 and abs(logFC) ≥ 1). The top-10 miRNAs, which had the AUC value higher than 0.95, were miRNA-199b-5p, miRNA-150-5p, miRNA-29c-5p, miRNA-218-5p, miRNA-99a-3p, miRNA-383-5p, miRNA-199a-3p, miRNA-193a-5p, miRNA-10b-5p and miRNA-181c-5p. Conclusions This finding opens the avenue to the use of EV-associated miRNA of peritoneal lavages as an untapped source of biomarkers for CRC.

Endometrial cancer (EC) is the sixth most common cancer in women worldwide and is responsible for more than 89,000 deaths every year. Mortality is associated with presence of poor prognostic factors at diagnosis, i.e., diagnosis at an advanced stage, with a high grade and/or an aggressive histology. Development of novel approaches that would permit us to improve the clinical management of EC patients is an unmet need. In this study, we investigate a novel approach to identify highly sensitive and specific biomarkers of EC using extracellular vesicles (EVs) isolated from the peritoneal lavage of EC patients. EVs of peritoneal lavages of 25 EC patients were isolated and their miRNA content was compared with miRNAs of EVs isolated from the ascitic fluid of 25 control patients. Expression of the EV-associated miRNAs was measured using the Taqman OpenArray technology that allowed us to detect 371 miRNAs. The analysis showed that 114 miRNAs were significantly dysregulated in EC patients, among which eight miRNAs, miRNA-383-5p, miRNA-10b-5p, miRNA-34c-3p, miRNA-449b-5p, miRNA-34c-5p, miRNA-200b-3p, miRNA-2110, and miRNA-34b-3p, demonstrated a classification performance at area under the receiver operating characteristic curve (AUC) values above 0.9. This finding opens an avenue for the use of EV-associated miRNAs of peritoneal lavages as an untapped source of biomarkers for EC.

Cancer multidrug resistance (MDR) is one of the main challenges for cancer treatment efficacy. MDR is a phenomenon by which tumor cells become resistant to several unrelated drugs. Some studies have previously described the important role of extracellular vesicles (EVs) in the dissemination of a MDR phenotype. EVs’ cargo may include different players of MDR, such as microRNAS and drug-efflux pumps, which may be transferred from donor MDR cells to recipient drug-sensitive counterparts. The present work aimed to: (i) compare the ability of drug-sensitive and their MDR counterpart cells to release and capture EVs and (ii) study and relate those differences with possible distinct fate of the endocytic pathway in these counterpart cells. Our results showed that MDR cells released more EVs than their drug-sensitive counterparts and also that the drug-sensitive cells captured more EVs than their MDR counterparts. This difference in the release and capture of EVs may be associated with differences in the endocytic pathway between drug-sensitive and MDR cells. Importantly, manipulation of the recycling pathway influenced the response of drug-sensitive cells to doxorubicin treatment.

mTOR complex 1 signalling regulates polyamine metabolism and thereby promotes tumorigenesis, through regulation of the stability of a key enzyme, AMD1. Cell proliferation in prostate cancer The mTOR pathway regulates multiple metabolic pathways in cancers but knowledge of how oncogenic signals regulate polyamine production, which is important in spreading cancer cells, is limited. Arkaitz Carracedo and colleagues add to the existing knowledge. Using a mixture of metabolic analysis techniques, they show that mTOR signalling regulates polyamine metabolism and thereby promotes tumorigenesis through regulation of the stability of a key enzyme, AMD1. They validated this pathway in mouse and human cancer specimens and found that ADM1 is upregulated in prostate cancer specimens with activated mTORC1. This adds to the complexity of cancer cell metabolism and offers new therapeutic avenues. Activation of the PTEN–PI3K–mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation 1 , 2 . Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model 3 and human biopsies 4 of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S -adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S -adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus 5 exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Several etiologies result in chronic liver diseases including chronic hepatitis C virus infection (HCV). Despite its high incidence and the severe economic and medical consequences, liver disease is still commonly overlooked due to the lack of efficient non-invasive diagnostic methods. While several techniques have been tested for the detection of fibrosis, the available biomarkers still present severe limitations that preclude their use in clinical diagnostics. Liver diseases have also been the subject of metabolomic analysis. Here, we demonstrate the suitability of 1H NMR spectroscopy for characterizing the metabolism of liver fibrosis induced by HCV. Serum samples from HCV patients without fibrosis or with liver cirrhosis were analyzed by NMR spectroscopy and the results were submitted to multivariate and univariate statistical analysis. PLS-DA test was able to discriminate between advanced fibrotic and non-fibrotic patients and several metabolites were found to be up or downregulated in patients with cirrhosis. The suitability of the most significantly regulated metabolites was validated by ROC analysis. Our study reveals that choline, acetoacetate and low-density lipoproteins are the most informative biomarkers for predicting cirrhosis in HCV patients. Our results demonstrate that statistical analysis of 1H-NMR spectra is able to distinguish between fibrotic and non-fibrotic patients suffering from HCV, representing a novel diagnostic application for NMR spectroscopy.

Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex molecular cargo comprising several populations with unique roles in biological processes. These vesicles are closely associated with specific physiological features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in pathophysiological processes by actively triggering genetic or metabolic responses. However, the heterogeneity of their structure and composition hinders their application in medical diagnosis and therapies. This diversity makes it difficult to establish their exact physiological roles, and the functions and composition of different EV (sub)populations. Ensemble averaging approaches currently employed for EV characterization, such as western blotting or ‘omics’ technologies, tend to obscure rather than reveal these heterogeneities. Recent developments in single-vesicle analysis have made it possible to overcome these limitations and have facilitated the development of practical clinical applications. In this review, we discuss the benefits and challenges inherent to the current methods for the analysis of single vesicles and review the contribution of these approaches to the understanding of EV biology. We describe the contributions of these recent technological advances to the characterization and phenotyping of EVs, examination of the role of EVs in cell-to-cell communication pathways and the identification and validation of EVs as disease biomarkers. Finally, we discuss the potential of innovative single-vesicle imaging and analysis methodologies using microfluidic devices, which promise to deliver rapid and effective basic and practical applications for minimally invasive prognosis systems. Understanding the heterogeneity of extracellular vesicles is crucial for unraveling their functions. This review describes the benefits, challenges and contributions of the state-of-the art methods used in single-vesicle analysis.