Deciphering Disease using Exosome Biomarkers
Exosomes are small (<150 nm), circulating, membrane-bound vesicles, pinched via endocytosis from the membranes of cells and released via exocytosis following membrane fusion of multivesicular bodies1. Despite their diminutive size, exosomes contain a wealth of information about the health of their cells and organs of origin, making them perfectly suited to diagnostic applications as biomarkers of disease. When formed, exosomes encapsulate a small sampling of key cellular components, such as plasma membrane (including surface proteins), cytosol, DNAs, RNAs (including mRNAs and microRNAs) and proteins. Isolating exosomes and analyzing their constituent parts can reveal modifications common to disease states, enabling earlier detection and less invasive diagnoses.
Minimally invasive methods for disease characterization and diagnosis are already commonplace for some diseases in which sample accessibility is not limited, such as blood cancers and skin disorders, but exosome biomarkers are an essential tool for developing minimally invasive methods for studying the natural history of diseases that strike more privileged and difficult-to-sample compartments, like the brain and spinal cord2-5, kidneys6-8 and heart and vessels9, 10.
Exosomes can be isolated from nearly every fluid in the body, but–for optimal diagnostic or prognostic value–it’s best to select a fluid in direct contact with the organ(s) of interest. For disease-state analysis throughout the body, blood is a reasonable first choice, as it is the fluid that contacts every organ system. Other biological fluids that have successfully been interrogated for their exosomal content include amniotic fluid11, breast milk12, saliva11, tears13 and urine14, 15. These so-called liquid biopsies, a term first used in the scientific literature in 197416, enable the non-invasive surveillance of organ system function through the composition of the fluids surrounding them, including their exosome content.
The purity of exosome preparations is of paramount importance for reliable and repeatable analyses, so special attention should be paid to maintaining the purity of exosome-containing fluids. When obtaining your fluid sample, be careful to avoid the accidental contamination of your pure fluid compartment with other biological fluids. This form of contamination is less of a concern when drawing blood, where blood is the target fluid, but when drawing fluid from other compartments–like the subarachnoid space of the spinal column or the synovial joint of the knee–special precautions should be taken to avoid contaminating your sample with blood from the needle stick. Even when non-invasively collecting fluid–for instance, via a clean catch of voided urine–best practices for avoiding sample contamination should be followed.
There are currently several commonly used methods for isolating exosomes from liquid biopsies, including differential ultracentrifugation, density gradient centrifugation, microfiltration, antibody-coated magnetic beads and microfluidic devices, with differential ultracentrifugation being considered the gold standard.17 As differential ultracentrifugation is the most widely used method for exosome isolation, special note should be made of the importance of using the correct protocol with the correct rotor to ensure the reproducibility and reliability of results.18
Comparison of Exosome Isolation Methods
- Benefits: Gold stardard method, easily adapted to account for sample viscosity
- Disadvantages: Long spin times, cannot separate virus from exosome, protein co-precipitation
Density Gradient Centrifugation
- Benefits: Less unwanted co-precipitation, specialized methods enable separation from virus
- Disadvantages: Nonspecific precipitation of same-size species
- Benefits: Improved speed
- Disadvantages: Matrix/membrane interaction, co-purification of proteins, sub-optimal recovery
Antibody-Coated Magnetic Beads
- Benefits: Highly specific isolation of a population of exosomes
- Disadvantages: Not suitable for large volume samples, exosomes may not elute intact from beads
- Benefits: Lower cost, less sample required
- Disadvantages: Variable efficiencies across available protocols
Whichever exosome isolation method you choose, there is an art to the science of exosome isolation, in that more is not always better, and the best guide is good judgment and previous experience. Using overly stringent size cutoffs or antigenicity requirements can be as detrimental as using overly broad size cutoffs or antigenicity requirements. The key to keep in mind is that sample purity and integrity are of the highest priority. Maintaining a pure exosome sample requires vigilance at all stages of the exosome isolation procedure to prevent sample loss and to maintain the reliability of the data.
As is the case with any membrane-bound structure, from exosome to animal cell, rough or improper handling may lead to lysis, and unintentional lysis of your exosomes before you’re ready is the primary route to sample loss. To that end, be mindful of performing each step of your protocol at the recommended temperature and in the recommended buffer, while keeping your sample away from detergents and matrix metalloproteases.
Mishandling your samples, particularly during washes, can lead to the loss of your entire sample. Always mark your tubes to reflect the specific step of your protocol–this can help prevent discarding a supernatant that contains valuable exosomes or tossing a tube that has a pellet worth preserving. This is the most common error made in any protocol where there are variable steps that include washes and supernatants. To be forewarned is to be forearmed, but so is being measured, aware and methodical.
Profiling Disease with Exosomes
To diagnose or track a disease’s progress using exosome biomarkers, one must first develop a baseline of a healthy or non-diseased exosomal profile. This can involve a population study, to evaluate the gamut of possible variations of a healthy profile, a longitudinal study of a single patient’s exosomal profile, before and after disease onset (which requires either extreme patience or fortuitous coincidence), or–and this option will interest those without access to an endless supply of non-needle-averse patients–a comparison between exosomes produced by a “normal” cell line and a “diseased” cell line.
One of the more common exosomal cargos to be used in the diagnosis and prognosis of disease is microRNAs (miRNAs), as their presence or absence may be used as a biomarker to directly predict disease risk19, onset20, progression21 or remission22. Isolating miRNAs from exosomal fractions has been standardized23 and is now a commonly used method for enriching for disease-specific miRNAs from across the body or within a specific organ system.
Deciphering disease states with exosome biomarkers, whether in a cell line or across a human population, is a robust technique that is shedding light on the major predictive value of these circulating messengers; messengers who, in recent history, were written off as packaged waste-disposal units. There is clearly still much to be gleaned from these tiny messengers.
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