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Jun 15, 2013 (Vol. 33, No. 12)

Exploring Exosomal RNA

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    Uptake by HeLa cells of exosomes labeled with SYTO® RNASelect™ stain: A FLoid® Cell Imaging station was used. Red: Alexa Fluor® 594 phalloidin (allows for visualization of cell membrane); Blue: DAPI (cell nucleus); Green: SYTO® RNASelect™ stain (allows for localization of exosomal RNA). [Alexander Vlassov/Life Technologies]

    Tiny, subcellular membrane-bound vesicles called exosomes are periodically released by a wide variety of cell types, carrying along with them proteins, microRNA (miRNA), and mRNA fragments representative of their cells of origin.

    It is thought that exosomes may shuttle information from cell to cell. 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.

    Exosomes can be easily isolated from bodily fluids by ultracentrifugation and filtration, among other approaches, offering an attractive potential alternative to invasive biopsies for diagnostic and prognostic purposes.

    While representative of the cell of origin, exosome cargo can over- or under-represent the molecular content of the originating cell. Consequently, it is believed that a selective packaging mechanism is involved in the loading of exosomes, though such a mechanism has not yet been demonstrated.

    During the “International Society of Extracellular Vesicles” (ISEV) meeting held in Boston this April, GEN spoke with several scientists for whom sequencing the RNA content of exosomes is a key aspect of their work.

    Novartis’ Maja M. Janas, Ph.D., is working to identify miRNAs commonly enriched or reduced in exosomes across eight different liver cancer cell lines as part of a larger effort to investigate the mechanism of selective miRNA sorting into exosomes.

    Dr. Janas, a postdoctoral fellow at the Biologics Center, Novartis Institute for BioMedical Research, explained that she is looking for common RNA motifs, secondary structures, and proteins that bind these miRNAs. From there, she is working to identify miRNAs that are selectively enriched or reduced in exosomes from metastatic liver cancer cell lines, relative to those in nonmetastatic liver cancer cell lines.

    To date, she has evaluated two liver cancer cell lines—one metastatic, one not. Dr. Janas said that deep sequencing of precursor and mature miRNA using the Illumina HiSeq platform revealed miR-21 was the most highly enriched miRNA in exosomes from the metastatic cell line, and that miR-378, miR-30a, miR-10a, and let-7i were found exclusively in the metastatic exosomes. Among those miRNAs observed exclusively in exosomes from the nonmetastatic cell line were miR-192, miR-191, miR-27b, and miR-101.

    Dr. Janas intends to use these selectively exported miRNAs as reporters in an RNAi screening assay for factors regulating the exosomal miRNA pathway using a liver cancer donor and receptor cell-line system, she said. Interestingly, sequencing revealed more precursor miRNAs than mature miRNAs in exosome cargo, Dr. Janas reported, adding that she intends to investigate why.

    Meanwhile, scientists at Life Technologies are profiling the RNA in exosomes released by HeLa cells.

    In an talk discussing his team’s approach, Alexander “Sasha” Vlassov, Ph.D., senior staff scientists and manager, R&D, Life Technologies, noted that deep sequencing of RNA cargo, using either the Ion Torrent™ Personal Genome Machine or Ion Proton™ platform, has revealed that this content is primarily short RNA of 20–200 nucleotides in length, including miRNA, tRNA, and many short noncoding RNAs. The cargo also includes some full-length mRNA and ribosomal RNA, though little or no DNA.

    Dr. Vlassov emphasized that exosomes are not the only entities containing extracellular circulating RNA. A significant fraction of circulating miRNA, for instance, is bound to proteins rather than encapsulated in exosomes, he said. On average, each exosome contains only 1 to 10 RNA molecules, he noted, assuming an average length of 100 nucleotides. However, taking into account that exosomes are present in very high numbers in body fluids (typically >109 per mL), as a population they are capable of inducing significant biological effects.

    Speaking with GEN, Dr. Vlassov said the exosome field is “very exciting,” and that, given its potential for use in diagnostics and therapeutics for cancer and other diseases, Life Technologies is in the process of developing a complete arsenal of tools for exosome and microvesicle research.

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    Antigen-presenting cells internalize seminal exosomes. Blood-derived dendritic cells (DC) or vaginal Langerhans cells (LC) were exposed to red DiI-labeled seminal exosomes (SE) for 1 hr and stained for MHC-II (green) and the nucleus (blue). LCs isolated from ex vivo vaginal tissue conjugated to unstained T cells (TC). [Lucia Vojtech]

    Over at the University of Washington, Lucia Vojtech, Ph.D., and her colleagues are investigating why sexual transmission of HIV seems particularly resistant to vaccine-based prevention. Their hypothesis was that exosomes in human semen may carry a factor—or factors—that reduce the immune responsiveness of the genital mucosa.

    It has previously been shown that factors in semen do directly influence early events in viral transmission and the immune response in the genital mucosa, but the exact components involved are not well understood.

    Dr. Vojtech has found that exosomes are present in huge numbers in human semen, with individual ejaculates from six different donors containing, on average, 22 trillion exosomes. She also found that these exosomes rapidly enter antigen-presenting cells of the genital mucosa.

    Further experiments showed that these exosomes could deliver functional miRNA to recipient cells. Deep sequencing using an Illumina HiSeq 2000 system revealed that the exosomes contained many different types of small RNAs, including miRNA, Y-RNA, piwi-RNA, and tRNA.

    At least 162 miRNAs were present in all six samples, and), immunoregulatory activities have already reported or predicted for the most abundant miRNAs the researchers observed (including let-7b and miR-375). Dr. Vojtech noted that the miRNA content of the exosomes appeared highly selective, pointing to a selective packaging mechanism suggested by many in the field.

Readers' Comments

Posted 01/31/2014 by Mark

WOW...OK, I do not know if these papers are the result of fraud or misconduct as you suggest but I guess that it is good that people know that the method does not work so they do not burn their money, tough situation though.

Posted 10/23/2013 by anonymous

Thank you, the content and the work cited in this article is excellent. Clearly exosome biology is very important and maybe one day treatments will go into the clinic. It is a pity that the field has lost some credibility because all the fuss in the media about the use of exosomes to deliver siRNAs across the blood brain barrier that finished in fiasco. I mean has anybody being able to replicate the results Alvarez-Erviti 2011 or El Andaloussi 2012? Both works are from the same people and I know several top groups that have tried to replicate their results unsuccessfully. This is like the RVG siRNA of Kumar 2007, I cannot believe that Nature does not retract these papers and make so many people lose their time. I guess that they also make money from the advertisements and do not care that citations come from reviews and discussions rather than from other groups replicating their results and using exosomes to deliver siRNAs across the blood brain barrier or in other case what are they waiting for?

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