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Abstract Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV‐based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell‐free plasma or serum. Because available one‐step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2‐step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV‐ and tumour‐biomarkers in pure FPLC‐derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases‐associated biomarkers in pure EVs, supporting the feasibility of using single‐vesicle analysis for liquid biopsy.

Previous work on murine models and humans demonstrated global as well as tissue-specific molecular ageing trajectories of RNAs. Extracellular vesicles (EVs) are membrane vesicles mediating the horizontal transfer of genetic information between different tissues. We sequenced small regulatory RNAs (sncRNAs) in two mouse plasma fractions at five time points across the lifespan from 2–18 months: (1) sncRNAs that are free-circulating (fc-RNA) and (2) sncRNAs bound outside or inside EVs (EV-RNA). Different sncRNA classes exhibit unique ageing patterns that vary between the fcRNA and EV-RNA fractions. While tRNAs showed the highest correlation with ageing in both fractions, rRNAs exhibited inverse correlation trajectories between the EV- and fc-fractions. For miRNAs, the EV-RNA fraction was exceptionally strongly associated with ageing, especially the miR-29 family in adipose tissues. Sequencing of sncRNAs and coding genes in fat tissue of an independent cohort of aged mice up to 27 months highlighted the pivotal role of miR-29a-3p and miR-29b-3p in ageing-related gene regulation that we validated in a third cohort by RT-qPCR.

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.

Abstract Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor‐dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre‐analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood‐EV), a tool to record and report information about pre‐analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre‐analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence‐based optimization of pre‐analytical protocols through meta‐analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter‐laboratory comparative EV studies, and (iv) improve the peer review process.

The functional role of airway basal cells has not been comprehensively studied in idiopathic pulmonary fibrosis (IPF). Here, the authors show that airway basal cells of IPF patients display a distinct phenotype, are profibrotic if transplanted to mice and that fibrosis can be ameliorated by Src iinhibitors.

Summary: The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Extracellular vesicles (EVs) are promising agents for liquid biopsy—a non-invasive approach for the diagnosis of cancer and evaluation of therapy response. However, EV potential is limited by the lack of sufficiently sensitive, time-, and cost-efficient methods for their registration. This research aimed at developing a highly sensitive and easy-to-use immunochromatographic tool based on magnetic nanoparticles for EV quantification. The tool is demonstrated by detection of EVs isolated from cell culture supernatants and various body fluids using characteristic biomarkers, CD9 and CD81, and a tumor-associated marker—epithelial cell adhesion molecules. The detection limit of 3.7 × 105 EV/µL is one to two orders better than the most sensitive traditional lateral flow system and commercial ELISA kits. The detection specificity is ensured by an isotype control line on the test strip. The tool’s advantages are due to the spatial quantification of EV-bound magnetic nanolabels within the strip volume by an original electronic technique. The inexpensive tool, promising for liquid biopsy in daily clinical routines, can be extended to other relevant biomarkers.