exosomesDiscovered nearly 30 years ago, exosomes are now being implicated in one of the most important and promising breakthroughs in medical therapy yet. A quick glance in PubMed reveals over ~3,000 journal references since their discovery in 1983. Not surprisingly, this groundswell of interest has led to the formation of the International Society for Extracellular Vesicles, as well as the American Society for Exosomes and Microvesicles. A recent journal article by Raposo & Stoorvogel published this year in J. Cell Biology presented a remarkable new model for cell-to-cell signaling, one that offers new perspectives on intercellular communication and the possible transference of disease [1].

Image (top) courtesy of http://www.exosome-rna.com.

Exosomes: an overview

Exsomes are small in size, around 150 nm in diameter. They are formed in cellular compartments known as multivesicular endosomes (MVE). MVE were initially believed to only help traffic cellular "garbage" to place where they may be degraded and removed. But about 30 years ago, two different research groups published two independent reports stating that maturing red blood cells literally jettison tiny, ~30-100 nm diameter globules (later dubbed exosomes) out of the cell and into the extracellular matrix [2].

Both teams of researchers cultured immature red blood cells, known as reticulocytes, with a radioactive antibody that bound to the top receptor for transferrin to record this procedure. Every 15 minutes, they would gather subsets of cells and image them using electron microscope. This gave them the option to follow either the receptor or the transferrin as it made its way to the cell surface. They found the transferrin molecule, still bound to its receptor, was sequestered in tiny vesicles that formed inside the bigger MVE. Surprisingly, they demonstrated that some MVEs will fuse together with the cellular membrane and discharge these little vesicles carrying transferrin to the outside the cell.

For the next 15 years, exosomes were all but forgotten until 1996, when Graca Raposo, from the Institut Curie in Paris, France, published her discovery that immune cells such as B lymphocytes also secreted exosomes. She found these vesicles carried membrane-bound molecules vital for an adaptive immune response [3]. Two years later, yet another exosome-secreting cell type was found: the dendritic cell, whose exosomes transport immunologically functional agents that promote induction of anti-tumor response in mice [4]. These results prompted explorations for their clinical use, and formed the bedrock for the hypothesis that exosomes play a large and active role in intercellular communication.

Within the last few years, technical innovations with high-throughput protein analysis have allowed researchers to detail and begin classifying the sort of cargo transported by exosomes (see Figure 1) [5]. Depending on the cell type, exosomes carry and secrete specific sets of proteins that differ from the proteins found in the membrane vesicles released by apoptotic cells, implying that exosomes are secreted only by living cells. Other cells, for example T and B lymphocytes, seem to amplify the number of exosomes secreted when provoked by the binding of a cell-surface receptor.

Recent studies of exosomes purified in vitro have shown that the vesicles may be transduced by other cells, thus transferring any information enclosed in and/or on the exosome. Other exosome-bound molecules can cause the inactivation or even the death of the target cell they fall upon. Some exosomes, for instance, have the Fas ligand, which when bound to a Fas receptor, also referred to as the death receptor, will initiate apoptosis [6].

In 2007, a group directed by Jan Lotvall in Sweden discovered messenger RNA and microRNA inside exosomes [7]. In vitro experiments revealed that the mRNA might be translated into proteins within target cells, providing the very first demonstration of genetic information transfer in humans. This remarkable discovery not only signals a fresh type of intercellular communication, but suggests that exosomes could possibly act similar to viruses, in so far as foreign genetic material is translated to proteins in the cells they "infect." This discovery, and the concomitant development of analysis on microRNAs, has triggered the recent boost in the study of exosomes, the uptick in publications, as well as the packed house of researchers keen to share their latest findings.

Figure 1. Schematic model for exosome fate and cargo sorting.

exosome_cargo_sorting

 

Diagram courtesy of Ramachandran and Palanisamy. NIH.

Cell Talk

Over 2.5 billion years ago, multi-cellular life suddenly appeared on Earth. One explanation for why this formative period in life took so long to evolve is due to the elaborate systems of cell communication required to govern their behaviour in a way that benefits the entire organism. Cells have since developed many sophisticated pathways of communication.

Traditionally, cells communicate across a vast network of connections involving physical cell-cell contact or among surface receptors that can retrieve distant messages from paracrine, synaptic, or endocrine cues [8]. Intracellular signals, such as peptide hormones, growth factors, and neurotransmitters, are often initiated by surface cues that binds to a specific ligand, which then stimulates a cascade of signals that distributes the message internally. Cellular and neural synapses have the unique ability to transport chemical messages between minute gaps in cells. Other certain environmental chemical stimuli, such as chemotaxis, can also elict a cellular response. Any sort of communication not involving direct contact between cells will usually be mediated through the paracrine or endocrine hormone response systems [9].

Exosomes for the 21st Century

Image courtesy of www.systembiosciences.comWhen exosomes were first discovered back in 1983, two different research groups published two independent reports stating that maturing red blood cells literally jettison tiny, ~30-100nm diameter globules out of the cell and into the extracellular matrix. Years later, those globules were termed exosome in order to differentiate this new class of organelle from the endosomal shuttles already known to exist inside cells.

Exosomes were initially thought of as mere taxis of cellular garbage. But as new research emerged, it became apparant these MVE "garbage trucks" are actually powerful mediators of gene transfer between cells. Furthermore, exosomes are endowed with an effective messaging system, in addition to having the machinery to alter the function and physiology of neighboring cells. Numerous publications have already described the associations between specific markers inside an exosome with several types of cancer, neurodegenerative diseases, inflammation, and cardiovascular disease [10]. This marks the first time researchers have shown that in humans, genetic information can be shuttled from one cell to another. This ability for exosomes to influence genetic transcription at remote cells offers a potential fingerprint as to their cellular origins, a deep insight with tremendous potential to influence a patient care [11].

In the near future, exosomes will have a greater use in diagnostic arrays that are more sensitive and accurate, which can lead to a faster diagnosis and medical intervention. Researchers have even surmised that artificially made exosomes could be suited to deliver drugs that target and kill whatever cell is harming the body [12].

Exosomes have also been found to be in abundance in all body fluids including blood, saliva, urine, and breast milk [13]. In fact, we have not yet located a single cell in which does not excrete exosomes. One cell can send a bundle of molecules, and it can be received by another cell to help that cell regulate.

protein_and_rna_transfer

 

The previous images come courtesy of www.systembiosciences.com

Some common exosome proteins to look for are:

RNA Transport from the Nucleus Can Be Regulated

In mammals, approximately one-twentieth of the total mass of RNA synthesized ever leaves the cell [14]. RNA that has been incompletely processed or is damaged, in any way, is degraded inside the nucleus as part of the cells quality control system. This process is carried out by cellular exosomes.

Research published in 2007 in Nature Cell Biology, reported that exosomes from human mast cells contained mRNA from approximately 1,300 genes [15]. In vitro translation proved that these RNA fragments inside exosomes are still functional. Further investigation revealed that the RNA transported inside an exosome also contains mRNA and microRNA which were not present in the cytoplasm of the donor cell [16]. Valadis publication, in particular, sparked intense conversation about this new type of cellular communication. This is especially true for molecular biologists researching the biology of cancer because these gene fragments can be analyzed to determine a cancer diagnosis or the stage of progression. Since it's original publication date, Valadis research article has been cited over 1,300 times, making it as much an influential a paper as it is an amazing numerical coincidence!

The field of exosome research is predicted to continue to grow and evolve. 20-30 years from now, exosomes will be used for many targeted therapies and diagnostic applications.

If you would like more information about exosomal RNA and other related industry news, you can find that here. If you wish to participate in conversations about exosomes, check out ExosomesTalk curated by scientists at Life Technologies.

Next time, we'll travel inside an exosome and discover how their structures influence their function. Part 3 we will discuss the challenges in exosome research, as well as a product review of exosome products and diagnostic applications.

 

SOURCES

[1,2] Protein and RNA transfer by extracellular vesicles. Raposo & Stoorvegel. Published by Rockefeller University Press.

[3] G. Raposo et al., "B lymphocytes secrete antigen-presenting vesicles," J Exp Med, 183:1161-72, 1996.

[4] L. Zitvogel et al., "Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes," Nat Med,4:594-600, 1998.

[5] S. Mathivanan, R.J. Simpson, "ExoCarta: A compendium of exosomal proteins and RNA," Proteomics, 9:4997-5000, 2009.

[6] Activated T Cell Exosomes Promote Tumor Invasion via Fas Signaling Pathway. by Cai, Yang, Yu, Yu, Jiang, Wang, Yang, Wang, Cao, Wang. The Journal of Immunology. June 15, 2012. Vol. 188 no. 12 5954-5961. <http://www.jimmunol.org/content/188/12/5954.full>.

[7,15,16] Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. by Valadi, Ekstrom, Bossios, Sjostrand, Lee, Lotvall. PubMed, June 2007. <http://www.ncbi.nlm.nih.gov/pubmed/17486113>, <http://www.nature.com/ncb/journal/v9/n6/full/ncb1596.html>

[8,9,12] Exosomes: Tiny Packets of RNAs with Major Significance and Potential. Jerry Zon. <http://zon.trilinkbiotech.com/2013/09/23/exosomes-tiny-packets-rnas-major-significance-potential/>. Sept. 23, 2014. [Sept. 20, 2014].

[10] MicroRNAs as modulators and biomarkers of inflammatory and neuropathic pain conditions. Andersen, Duroux, Gazerani. Neurobiology of Disease. Vol. 71. Nov. 2014. Pages 159-168. <http://www.sciencedirect.com/science/article/pii/S0969996114002320>.

[11] Horizontal Transfer of RNAs: Exosomes as mediators of intercellular communication. by Ramachandran and Palanisamy. NIH. Oct 19, 2011. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3263325/>.

[13] Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. by Li, Zeringer, Barta, Shageman, Cheng, and Vlassov. PubMed, Sept 2014. <http://www.ncbi.nlm.nih.gov/pubmed/24009880>.

[14] Molecular Biology of The Cell. by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter. 4th Edition. Published by Garland Science, 2002. Pages 442-443.