Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics.

The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT; six preservation and five non-preservation) and three blood processing intervals (BPI; 1, 8 and 72 h) on defined performance metrics (n = 9).

Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles.

Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments.

Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions.

Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation.

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine.

Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system.

Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization.

Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have been less thoroughly investigated most probably due to the methodological challenges in separating BEVs from their matrix and host-derived eukaryotic extracellular vesicles (EEVs) such as exosomes and microvesicles.

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles.

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids.

The generation and use of recombinant extracellular vesicles as biological reference material.

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.