Targets, pitfalls and reference materials for liquid biopsy tests in cancer diagnostics

Assessment of cell free DNA (cfDNA) and RNA (cfRNA), circulating tumor cells (CTC) and extracellular vesicles (EV) in blood or other bodily fluids can enable early cancer detection, tumor dynamics assessment, minimal residual disease detection and therapy monitoring. However, few liquid biopsy tests progress towards clinical application because results are often discordant and challenging to reproduce. Reproducibility can be enhanced by the development and implementation of standard operating procedures and reference materials to identify and correct for pre-analytical variables. In this review we elaborate on the technological considerations, pre -analytical variables and the use and availability of reference materials for the assessment of liquid biopsy targets in blood and highlight initiatives towards the standardization of liquid biopsy testing

A supporting ecosystem to mature extracellular vesicles into clinical application

Research into extracellular vesicles (EV) has yielded important biological insights and raised the prospect of developing novel diagnostics and therapeutics for a wide range of pathologies. As with other emerging and transformative fields in research, it will require a broad, supportive base for EV research to mature and to develop clinical application. Here, we identify several focus areas to further improve reproducibility and reliability specifically for EV research and make recommendations for minimal experimental guidelines, transparency tools, reference materials, validation, identification of contaminants, data sharing, coaching through education, and funding opportunities

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. Here, we present a step-by-step procedure to separate and characterize BEVs from human body fluids. BEVs are separated through the orthogonal implementation of ultrafiltration, size-exclusion chromatography (SEC) and density-gradient centrifugation. Size separates BEVs from bacteria, flagella and cell debris in stool; and blood cells, high density lipoproteins (HDLs) and soluble proteins in blood. Density separates BEVs from fibers, protein aggregates and EEVs in stool; and low-density lipoproteins (LDLs), very-low-density lipoproteins (VLDLs), chylomicrons, protein aggregates and EEVs in blood. The procedure is label free, maintains the integrity of BEVs and ensures reproducibility through the use of automated liquid handlers. Post-separation BEVs are characterized using orthogonal biochemical endotoxin and Toll-like receptor-based reporter assays in combination with proteomics, electron microscopy and nanoparticle tracking analysis (NTA) to evaluate BEV quality, abundance, structure and molecular cargo. Separation and characterization of BEVs from body fluids can be done within 72 h, is compatible with EEV analysis and can be readily adopted by researchers experienced in basic molecular biology and extracellular vesicle analysis. We anticipate that this protocol will expand our knowledge on the biological heterogeneity, molecular cargo and function of BEVs in human body fluids and steer the development of laboratory research tools and clinical diagnostic kits. Bacterial extracellular vesicles (BEVs) in human body fluids are analyzed using ultrafiltration, size-exclusion chromatography and density-gradient centrifugation to separate the BEVs, followed by post-separation characterization with orthogonal biochemical methods

Exosomes released from breast cancer carcinomas stimulate cell movement

For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis

Preparation of multi-omics grade extracellular vesicles by density-based fractionation of urine

The multidimensional cargo of extracellular vesicles (EV) released in urine is a reflection of the pathophysiological processes occurring within their cells and tissues of origin in the urogenital system. Here, we describe a step-by-step protocol for density-based separation of urinary EV with high specificity and repeatability. The implementation of integrative omics allows the study of the molecular complexity of highly purified urinary EV, supporting the identification of EV-specific functions and biomarkers