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Exosomes, Deep Sequencing, and Biomarker Potential

At the 2013 annual International Society for Extracellular Vesicles (ISEV) (http://www.isevmeeting.org/) conference held in Boston, April 17-20, Michiel Pegtel, Ph.D., Immunologist in the Pathology Department at the VU University Medical Center in Amsterdam, The Netherlands, gave an oral presentation describing how comprehensive deep sequencing analysis revealed non-random small RNA incorporation into tumor exosomes and discussed the biomarker potential of these results. Exosomes (image) are tiny subcellular membrane-bound vesicles (30-150 nm in diameter) that are released by a wide variety of normal cell types and cancer cells, and that can carry membrane and cellular proteins, as well as microRNA (miRNA), and various other types of RNA, including mRNA fragments, representative of the cell of origin. It is thought that exosomes may serve the purpose of shuttling information from one cell to another. For instance, it has been shown that exosomes can carry material from cancer cells that acts to suppress the immune system and stimulate angiogenesis, thus encouraging cancer growth. In talking with BioQuick, Dr. Pegtel emphasized the key role of deep sequencing in his work. β€œIn contrast to closed profiling technologies such as microarray and RT-PCR arrays, deep sequencing has literallyy opened our eyes on the complexity of the human transcriptome. Our previous work using individual quantitative RT-PCR could demonstrate that human B cells infected with the tumor virus Epstein Barr (EBV) secrete exosomes that contain fully mature and functional virus-encoded miRNAs. At the ISEV meeting, Nobel laureate Dr. Phillip Sharp stressed the importance of quantitation in RNA research. I could not agree more as this applies directly to research concerning transfer of functional RNA via exosomes. In our virus model, we could show that transfer of relatively small copy numbers of miRNAs from an EBV-infected B cell into an uninfected recipient dendritic cell could directly influence gene regulation. Quantitation of RNA copy numbers in exosomes is important, but perhaps it is less important how many are in one or a thousand exosomes. More important is what is actually transferred from one cell to another. Because technology has progressed, our laboratory is now using the unprecedented power of deep sequencing with the Illumina HiSeq 2000 and 100 paired-end protocol on both (cancer) cells and secreted exosomes. With this approach and sophisticated bioinformatics in collaboration with Dr Michael Hackenberg from the Univeristiy of Granada (Spain) some interesting discoveries could be made. Besides the presence of nearly all small RNA families (tRNAs, snoRNAs, Y-RNAs, miRNAs, etc.), it turned out that many smaller fragments of these RNAs are preferentially sorted or excluded from secretion by exosomes indicating an underlying biology. Perhaps more interesting is that these small RNA fragments do not adhere to the general rules for Dicer-dependent miRNA processing. Finally, it appears that these alternatively processed small RNAs are more abundant in cancer cells and unequally distributed between cells and exosomes. Because such exosomes are present in nearly all biofluids--urine, blood, breast milk, saliva etc., they are considered very powerfull biomarkers. Thus, deep sequencing has revolutionized not only what we know about RNAs inside cells, but also what is secreted into the extracellular milieu that can be used for diagnostics. Because of the quantitative nature of deep sequencing technology, we have gained additional insights into the potential mechanisms that control secretion of known and newly discovered RNAs via exosomes. These findings eventually will tell us more about cell-cell communication in healthy individuals and what is disturbed in disease. Decoding the messages that are sent out by, for instance, cancerous cells will hopefully improve and simplify diagnosis such that we will all benefit from this exciting work.” [ISEV conference web site] [ISEV conference blog]