P39 Blood cell-surface-bound exosomes as a new source of tumor-specific microRNA

BackgroundMicrovesicles release by neoplastic cells were found in blood and carrying a large number of tumor distinctive molecules, like miRNA/mRNA, proteins. Considering extensive surface of blood cells we wonder if exosomesare circulated exclusively in plasma or could be associated with surface of blood cells.Materials and MethodsMicrovesicles circulating in blood of healthy women (HW) and breast cancer patients (BCP) were studied. Blood was fractionated into plasma and cellular fractions; in order to release cell surface bound material, blood cells were treated as it was described for cell-surface-bound (CSB) cirDNA (Tamkovich, 2005).Exosomes were pelleted at 100,000g from 0.1μm filtered supernatants obtained by plasma and CSB eluates centrifugation at 17,000g, resuspended in PBS and stored in aliquots at −80°C. Size distribution of the microparticles was characterized by TEM (Jeol, Japan), anti CD-63, CD-24, CD-9 antibodies (BD Biosciences, USA) were used as exosome markers, protein concentration was measured by NanoOrange Protein Quantitation kit (Molecular Probes, USA), total RNA and miRNA were isolated by mirVana kit, 28S rRNA were quantified by RT-qPCR after reverse transcription with 6-mer random primers, miRNA using TaqMan Small RNA Assays kit (Applied Biosystems, USA). DNA was isolated using “DNA Isolation Kit” (BioSilicaLtd, Russia) and measured by TaqMan multiplex real-time PCR for LINE1 and α-satellite repeats (Bryzgunova, 2011). Results: TEM with immunogold labeling demonstrate presence of exosomes in 30–100nm membrane-wrapped particles isolated from both plasma and CSB eluates. TEM, NanoOrange and 28S rRNA RT-qPCR data demonstrate that CSB exosomes constitutes 2/3 of total blood exosomes. Exosomes ranged 50–70nm prevail in blood of BCP, whereas 30–50nm exosomes in blood of HW. Exosomal DNA is less than 0.3% of cell-free blood DNA. RNA integrity and specific quantity checked by Bioanalyzer (Agilent Technologies, USA) do not differ in cell-free and CSB exosomes. Preliminary data demonstrate overpresentation of cancer-specific miRNA (miR-103, miR-191, miR-195) in exosomes bound with erythrocyte’s as compared with exosomes bound with leukocytes or circulating in plasma.ConclusionExosomal DNA obviously do not have any diagnostic value in contrast to RNA. CSB exosomes represent valuable source of material for small-invasive cancer diagnostics