New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

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.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017

“…It clearly established now that the DNA is double stranded and can also be in the single-stranded form. In plasma, most genomic DNA was associated to EVs, and genetic profiling (of EVs) identified tumour-related mutations such as KRAS in pancreatic cancer patients [12]. Importantly, regarding tumour heterogeneity the mutational profile of biopsies from distinct sites within a patient can differ.…”

A High-Throughput Protocol for Isolating Cell-Free Circulating Tumor DNA from Peripheral Blood

“…cfDNA fragment lengths can vary, with typical cfDNA profiles showing an approximately 165bp chromatosome (145 nucleosome+20bp linker histone H1) and 330bp dimer as the dominant DNA species where tumor-specific mutations are routinely observed [4,18]. cfDNA derived from PB or plasma had similar size profiles (Figure 2A & B), which encouraged us to proceed with library construction, target capture and sequencing experiments.…”

Noninvasive prenatal detection of fetal trisomy and single gene disease by shotgun sequencing of placenta originated exosome DNA: a proof-of-concept validation

“…Thus, exoDNA may be an important contributor to plasma cfDNA. As a supporting evidence, Rohan Fernando et al recently exploited droplet digital PCR and confocal microscopy to show that exosomes in human blood of non-pregnant donors contained double strand DNA, which accounts for the main proportion of plasma cfDNA [27]. Give the similarity between exoDNA and cfDNA, it is reasonable to speculate that exoDNA may have great clinical value in detecting fetal disease as to the application of cfDNA in NIPT.…”