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.  

Exosome Source

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.  

Exosome Isolation

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

Differential Ultracentrifugation

  • 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

Microfiltration

  • 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

Microfluidic Devices

  • 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.  

References:
1. Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Cur. Protoc. Cell Biol. (2006) Chapter 3: Unit 3.22.
2. Gui Y, Liu H, Lv W, Hu X. Altered microRNA profiles in cerebrospinal fluid exosome in Parkinson disease and Alzheimer disease. Oncotarget 2015;6:37043-37053.
3. Fiandaca MS, Kapogiannis D, Mapstone M, et al. Identification of preclinical Alzheimer’s disease by a profile of pathogenic proteins in neutrally derived blood exosomes: A case-control study. Alzheimers Dement 2015;11:600-607.
4. Jagot F, Davoust N. Is it worth Considering Circulating microRNAs in Multiple Sclerosis? Front Immunol 2016;7:129. doi: 10.3389/fimmu.2016.00129.
5. Zhang ZG, Chopp M. Exosomes in stroke pathogenesis and therapy. J Clin Invest 2016;126:1190-1197. doi: 10.1172/JCI81133.
6. Bruno S, Porta S, Bussolati B. Extracellular vesicles in renal tissue damage and regeneration. Eur J Pharmacol 2016;pii:S0014-2999(16)30427-7. doi: 10.1016/j.ejphar.2016.06.058. 
7. Santucci L, Bruschi M, Candiano G, et al. Urine Proteome Biomarkers in Kidney Diseases. I. Limits, Perspectives, and First Focus on Normal Urine. Biomark Insights 2016;11:41-48. doi:10.4137/BMI.S26229.
8. Junker K, Heinzelmann J, Beckham C, et al. Extracellular Vesicles and Their Role in Urologic Malignancies. Eur Urol 2016;70:323-331. doi: 10.1016/j.eururo.2016.02.046.
9. Cheow ES, Cheng WC, Lee CN, et al. Plasma-derived extracellular vesicles contain predictive biomarkers and potential therapeutic targets for myocardial ischemic injury. Mol Cell Proteomics 2016; pii:mcp.M115.055731. [Epub ahead of print]
10. Kishore R, Garikipati VN, Gumpert A.Tiny Shuttles for Information Transfer: Exosomes in Cardiac Health and Disease. JCardiovasc Transl Res 2016;9:169-175.doi:10.1007 / s12265-016-9682-4.
11. Keller S, Ridinger J, Rupp AK, et al. Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med 2011;9:86.
12. Admyre C, Johansson SM, Rahman Qazi K, et al. Exosomes with Immune Modulatory Features Are Present in Human Breast Milk. J Immunol 2007;179:1969-1978. doi: 10.4049/jimmunol.179.3.1969.
13. Grigor’eva AE, Tamkovich SN, Eremina AV, et al. Exosomes in tears of healthy individuals: Isolation, identification, and characterization. Biochem 2016;10:165-172.
14. Wiggins R, Glatfelter A, Kshirsagar B, Beals T. Lipid microvesicles and their association with procoagulant activity in urine and glomeruli of rabbits with nephrotoxic nephritis. Lab Invest 1987;56:264-272.
15. Harpole M, Davis J, Espina V. Current state of the art for enhancing urine biomarker discovery. Expert Rev Proteomics 2016;13:609-626.
16. Sorrells RB. Synovioanalysis (“liquid biopsy”). J Ark Med Soc 1974;71:59-62.
17.  Momen-Heravi F, Balaj L, Alian S, et al. Current methods for the isolation of extracellular vesicles. Biol Chem 2013;394:1253-1262. doi: 10.1515/hsz-2013-0141.
18. Livshits MA, Khomyakova E, Evtushenko EG, et al. Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Sci Rep 2015;5:17319. doi:10.1038/srep17319.
19. Ghanbari M, Ikram MA, de Looper HW, et al. Genome-wide identification of microRNA-related variants associated with risk of Alzheimer’s disease. Sci Rep 2016;6:28387. doi: 10.1038/srep28387.
20. Zhang T, Shang Q, Lv C, et al. Circulating miR-126 is a potential biomarker to predict the onset of type 2 diabetes mellitus in susceptible individuals. Biochem Biophys Res Commun 2015;463:60-63. doi: 10.1016/j.bbrc.2015.05.017.
21. Farr RJ, Joglekar MK, Hardikar AA Circulating microRNAs in Diabetes Progression: Discovery, Validation and Research Translation. EXS 2015;106:215-244. doi: 10.1007/978-3-0348-0955-9_10.
22. Freres P, Wenric S, Boukerroucha M, et al. Circulating microRNA-based screening tool for breast cancer. Oncotarget 2016;7:5416-5428. doi:  10.18632/oncotarget.6786.
23. Lasser C. Identification and Analysis of Circulating Exosomal microRNA in Human Body Fluids. Methods in Molec Biol 2013;1024:109-128. doi: 10.1007/978-1-62703-453-1_9.