New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes
Authors: Gary D Krzyzanowski
M Rohan Fernando
Wayne L Ryan
Chao Jiang
Journal: PLoS ONE (2017)
Discipline: Multidisciplinary
Keywords: cfDNA; exosomes; biomarker discovery; noninvasive diagnostic assays; plasma
Affiliations: Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE, United States of America., Department of Research and Development, CFGenome¬Æ, Omaha, NE, United States of America.

Abstract:  Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive prenatal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K3EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exosomes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma. 

Retracted: No

PubPeer comments:
 A student recently asked me about this paper and how it compares with another paper in the literature by [Jeppesen et al] . Jeppesen, et al, find little DNA associated with EVs. I give my comments below, but let s start with the  TLDR summary: the main takeaway for me from this paper is that polyethylene glycol can precipitate most DNA remaining in plasma pre-cleared with a 16,000 x g centrifugation. Without reflecting on any other papers that report DNA in EVs, this particular paper does not have data that could be used to contradict the conclusions of Jeppesen, et al. My first comment is that the literature on DNA and EVs remains somewhat controversial. I suspect that some of the differences in results are due to donor patient status, where people with more inflammation or more advanced cancer might have more DNA in EVs. However, methodologic differences also have a huge influence on outcomes. Questions I would ask as a reviewer of any EV DNA paper: *How good was the EV separation method, and did the authors truly characterize their EVs (including purity)?* *Were the right comparisons and controls present, like looking at different fractions, not just the supposed EV fraction?* *Were enzymatic treatments and or EV-destructive treatments used to establish the location of DNA (inside or outside EVs)? Were nuclease treatments also complemented with protease treatments? Important: DNA is stable outside the cell mostly as nucleosomes, with 200-bp DNA fragments that are resistant to nuclease unless protein is removed.* *Did the study rule out the possibility that RNA was amplified instead of or in addition to DNA?* Now, comments on the figures and conclusions of this paper. Figure 1: Inconclusive and raises numerous questions. PEG precipitation ( total exosome isolation, a misnomer) is done, then several EV surface markers are probed. A density gradient is also done, and two tetraspanins are shown. However, there is no comparison whatsoever between the outcome of the PEG precipitation and the density gradient. What purity was achieved by the two methods? Where are the negative or depleted markers? What donor is shown in the gradient Westerns? Or is it a different donor from the group in Figure 1A? Why does Figure 1B show only three donors, not four as in 1A? Why are fractions 6-11 shown in Figure 1C, left panel, but only 7-10 in the right panel? Why not show all fractions for both? Which donor and method provided the EM image in Figure 1D? Importantly, most of the particles in the EM resemble lipoprotein particles, not EVs. This seems to be an extremely  dirty prep, consistent with PEG precipitation of lipoprotein particles and few if any EVs. The only thing that looks like an EV (lower right-hand corner) could be some sort of membrane fragment. And since we all inevitably choose our  best EM images, it s likely that the prep contained very few EVs. Figure 2: Inconclusive because key information and controls are missing. Confocal microscopy is used to evaluate dsDNA in EVs based on picogreen reagent. What s missing? Here are a few things. *Controls for A: Dye by itself. Dye plus unprecipitated plasma. Dye plus supernatant remaining after PEG precipitation. Additions for A: Do this for all donors, then quantitate the results.* *B: Controls: dye plus input (as above). Show all fractions, not just the presumed EV-containing fractions. Do it for all donors and quantitate the results.* *C: DNA in blood is fairly DNase-resistant if bound by proteins (as in nucleosomes). Nuclease sensitivity assays for EVs should also include a protease + nuclease condition.* Figure 3: Results are, in my opinion, preliminary and uninterpretable. NTA is not EV-specific. It is not exosome-specific. Most particles in precipitated material from plasma are not EVs. NTA sensitivity declines rapidly for particles smaller than 100 nm in diameter. If the mode is truly a  mode of 39.7 nm, NTA could not establish this. Furthermore, there are almost three times as many  exosomes stained with an unspecified  universal dye as appear in the unstained analysis, and almost two times as many that appear in the picoGreen condition. We might assume that fluorescence mode is more sensitive than scatter; however, if so, we might expect average or mode size to decrease in the fluorescence readings. Instead, reported size increases. Also important is that there s not enough data to make any conclusions. Which donor was this? Which separation method? Why not show all donors and do some statistics? Why not run a few controls, like dye in water, dye in PBS, dye in a whole or depleted plasma fraction? Figure 4: Results suggest that this is nucleosomal DNA, not necessarily EV-associated. Figure 4A,B shows results for just one donor. Figure 4C shows results from two donors. Where are the other donors? Why are they not shown? Note that the DNA size (Figure 4A,B) is the exact size expected for nucleosomal DNA. Figure 5: This is probably the best figure in the paper, since they use a larger n and also examine both input and the supernatant of the precipitated material. One wonders why more donors were not shown in the previous figures. However, the defensible conclusion is not that most DNA in plasma is in EVs, but that almost all DNA in plasma is precipitated by PEG. That is interesting but not surprising. Figure 6: This figure simply doesn t make sense to me. If you ve spun out cells and then spun out larger material with a 16,000 x g spin, where would additional EVs or EV proteins or beta-actin DNA come from during storage? If there are no cells, there would be no new EV biogenesis or cell fragmentation into artifactual  EVs . EVs might be damaged by freeze-thaw and re-form into smaller particles, but this would not create new protein. Maybe there s more aggregation of gunk that can be more easily precipitated by PEG? In any case, I don t think there is enough data to draw any conclusions. Considering the variability in the Western blots, blots from numerous donors would be needed. n is 1 for B and C, and unclear for A and D. It seems like a reviewer must have insisted that a more stringent EV separation be tried (gradients), but that no conclusive experiments were done with this material. This is not as important as the above, but worth noting: The authors spin at 1,600 x g to remove cells, then at 16,000 x g, before moving to EV separation. A 16,000 x g spin is rather fast and not implementable in most clinical labs. Presumably, a spin like this would remove a good deal of cfDNA. The study thus examines a fraction that is very different from the  usual plasma obtained from patients. In conclusion, this study shows that polyethylene glycol can precipitate most DNA remaining in plasma pre-cleared at 16,000 x g. The claim of an internal EV location is not defended by the data presented here. ,