Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments

BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles

Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms

Microvesicle-associated AAV vector as a novel gene delivery system

Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery

Detection Of Human c-Myc and EGFR Amplifications in Circulating Extracellular Vesicles in Mouse Tumour Models

Essentially, all cells release extracellular vesicles (EVs) that end up in biofluids, including blood, and the contents of these EVs can provide a window into the status of the cells from which they are released. This is particularly interesting in cancer, since these EVs allow for ‘ex-vivo’ analysis of the properties of the tumours without the need for biopsy. Gene mutations, rearrangements, amplifications, and epigenetic changes in the transcriptome can be monitored in circulating EVs. In this study, we used two human tumour cell lines derived from an epidermoid carcinoma and a medulloblastoma, which had amplification for the epidermal growth factor receptor (EGFR) and c-Myc genes, respectively. Cells were implanted subcutaneously into immunocompromised mice, and levels of gene amplification in both groups of subcutaneous tumours were quantified. We then determined if elevated levels of transcripts for the human EGFR and c-Myc were represented in circulating EVs in tumour-bearing mice. The expression levels of both human EGFR (h-EGFR) and human c-Myc (h-c-Myc) mRNAs in circulating EVs correlated well with their amplified status in the tumours. This data provides further support to the idea that circulating EVs are a potential platform for tumour biomarkers

Markers of inflammation, tissue damage and regeneration in urinary microvesicles from transplanted patients

together with Jan Lötvall and other ISEV board members elected in april 2012, especially