Exosomes and circulating vesicles as biomarkers

 

Almost all biological cells secrete various chemicals and compounds that include proteins, lipids, and genetic material.  These chemicals move from place to place and communicate with each other and their surrounding environment in three ways:  by direct cell-to-cell contact, transfer of secreted molecules, or intercellular transfer of extracellular vesicles (EVs).  Scientists and physicians with emerging interests in EVs and their role in intercellular communication are studying the clinical applications of EVs as both collectors of cellular debris and messengers for recipient cells.   Read More

Extracellular vesicles

An extracellular vesicle (EV) is the generalized name to describe these “vehicles” that transport important information between cells. These “vehicles” can originate from multivesicular endosomes (membrane-bound compartments found inside cells that contain a nucleus ) or they can be shed from the plasma or cell membrane. They can also be found in a variety of biological fluids such as blood, urine, saliva and the media of cultured cells. The field uses a variety of different names to classify these vesicles, with size of the vesicle being one determinant of the nomenclature used. Exosomes are often the smallest of these vesicles, with a size range of 70 – 200 nanometers. Microvesicles (MVs) is used for larger particles, but it has yet to be determined if these two subgroups are functionally different. Most scientists simply refer to exosomes and MVs, collectively, as EVs. Circulating vesicles are most likely comprised of both exosomes and MVs although the current technology is just beginning to provide the capability to discriminate between exosomes and MVs. This is important because although the mechanisms of exosomes and MVs may be the same, their formation and release into the body are likely to be different.

Scientists believe that exosomes are actively secreted by many, if not all, living cells.  Exosomes carry important chemicals and compounds that are delivered to other cells although the signal that turns on the command to “secrete” remains unclear.  Exosomes are known to alter function and physiology including tissue repair, neural communication, and the transfer of pathogenic proteins.  Scientists also know that exosomes can be “captured” by other cells, which then “hijack” the information enclosed in or on the exosomes.  These more recent discoveries have sparked the interests of investigators to see if there are any additional physiological systems and functions that exosomes might affect.

EV isolation challenges

EVs are small, which can make them difficult to isolate from blood and tissue samples.  Traditional techniques to isolate EVs include time-intensive, high speed centrifugation followed by immunoblotting or ELISA assays.  Although there are several commercially available kits that promote “easy” isolation of EVs, the isolation procedure in some of these kits fails to differentiate among the different sizes of EVs and other aggregates.  And, these traditional assays require large volume samples and can suffer from insufficient purity that is required by today’s complex molecular analyses.

Exosomes-work flow2

Work flow for EV isolation and characterization

Selective capture of EVs using microfluidics

MGH investigators have begun to test a new microfluidics device for the selective capture of EVs.  In this new technology developed by other MGH investigators, the surface of the microfluidic chip has been coated with the antibody anti-EGFR, which has a high affinity to bind to cancer and other types of important cells.  From a tiny sample of blood serum or plasma, the chip then captures either cells or EVs by affinity binding.  This technique provides a low cost method to obtain pure EVs due to its high throughput and gentle manipulation of the cells passing through the microfluidic chambers.

EVs and cancer cells

The MGH investigator group is building upon its success in developing and using novel microfluidic chip-based devices to isolate very small and rare cells, including extremely rare (1 in 10 billion cells) circulating tumor cells (CTCs) from the blood of cancer patients.

Exosomes-EVs isolated on chip

Microfluidic chip capture of ‘wild type EGFR EVs (green) and EGFRvIII (red) from glioblastoma cells

Since the mid 1990s,scientists have reported that exosomes are released from many different types of human cells in both healthy and disease states.  Based on their expertise in exploring blood-based biomarkers like CTCs, the MGH investigators are very interested in the complementary benefits from the CTCs and these tumor-secreted EVs both in the diagnosis and prognosis of cancer.

Cancer cells have been shown to release EVs in abundance and EVs can be measured in the patient’s blood.  This provides an excellent opportunity to develop novel biomarkers.  EVs are found at a significantly higher frequency in blood in comparison to CTCs and are far less delicate.  Conveniently, EVs can withstand the rigorous freeze-thaw conditions of sample collection and processing.

Furthermore, these cancer-related EVs have been implicated in promoting tumor progression by manipulating the surrounding microenvironment.  Researchers have hypothesized that EVs shed from the tumor membranes transport ribonucleic acid (RNA) and proteins that may promote tumor growth.

Studying EVs in glioblastoma

EVs are present in the blood serum of patients with glioblastoma, a fast-growing, Grade IV cancerous brain tumor, which affects both older adults and children.  Glioblastoma accounts for about 15% of all brain tumors and patients usually have a poor prognosis, with an expected survival less than 15 months following the diagnosis.

Two MGH investigator groups are collaborating in a project to purify MVs from the serum of patients with glioblastoma and use their RNA content as potential biomarkers.  The investigators are particularly interested in studying the gene expression of epithelial growth factor receptor (EGFR).  Mutations leading to the overexpression or upregulation of EGFR are associated with several types of cancer, including glioblastoma and are believed to contribute to the uncontrolled cell division or rapid growth of this deadly brain tumor.  A specific mutation known as EGFRvIII is often observed in glioblastoma and mutations of EGFR and its related compounds are present in about 30% of all epithelial cancers.

Relevant Publications

Skog J, Würdinger T, van Rijn S, Meijer DH, Gainche L, Curry Jr WT, Carter BS, Krichevsky AM, Breakefield XO. Gliobastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008 Dec;10(12):1470-6. PubMed PMID: 19011622; PubMed Central PMCID: PMC3423894

Tickner JA, Urquhart AJ, Stephenson SA, Richard DJ, O’Byrne KJ. Functions and therapeutic roles of exosomes in cancer. Front Oncol. 2014 May 27;4:127. PubMed PMID: 24904836; PubMed Central PMCID: PMC4034415

Chen C, Skog J, Hsu CH, Lessard RT, Balaj L, Wurdinger T, Carter BS, Breakefield XO, Toner M, Irimia D. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip. 2010 Feb 21;10(4):505-11. PubMed PMID: 20126692l PubMed Central PMCID: PMC3136803

Contact

Shannon Stott, PhD Xandra Breakefield, PhD Brian Nahed, MD
sstott@mgh.harvard.edu xbreakefield@mgh.harvard.edu bvnahed@partners.org
617-643-2658 617-726-5728 617-724-5805

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Grace McDonald-SmithExosomes and circulating vesicles as biomarkers