Microvesicles in body fluids are a heterogenous group of cell-released vesicles composed of exosomes, microparticles and apoptotic membrane blebs as its main representatives. They are mostly composed of proteins and lipids but also contain nucleic acids. In the present report we demonstrate that a recently discovered population of membrane vesicles termed exosomes, carry genetic information that can be used for diagnostic purposes. We demonstrate that i) esRNA of sufficient quantity can be extracted from body fluid exosomes, that ii) the genetic information is protected from degradation in exosomes, and that iii) in selected examples the esRNA can be used for the determination of SNPs in transcripts as well as for the detection of specific transcripts. We propose that the analysis of esRNA could provide new insights into the transcriptome of the body for example during disease or pregnancy.
For prenatal diagnostics fetal cells are often obtained by invasive procedures like amnioscentesis or chorion villus sampling. These methods constitute a risk of fetal misscarriage and injury and are therefore only offered to women with/at high-risk pregnancies. One of the most promising approaches is the use of cell-free nucleic acids in sera. Cell-free fetal DNA (cff DNA) was first discovered in 1997 in maternal plasma and serum of pregnant women and offers an excellent posibility as starting material for non-invasive prenatal diagnosis [26
]. The majority of cell free DNA is of maternal origin, only 3-6% of circulating cell-free DNA is of fetal origin [26
]. This limits further analysis of cff DNA to fetal targets differing from the maternal ones. Additionally, cell-free fetal DNA and RNA have been isolated from other body fluids e.g. maternal plasma [27
], amniotic fluid [28
], and cerebrospinal fluid [29
]. Although not tested at that time, it is quite likely that these nucleic acids are associated with microvesicles which could explain their relative stability in the nuclease-rich environment of body fluids. The enrichment of fetal derived exosomes by marker proteins is a big challenge and would allow the discrimination between maternal and fetal cell-free nucleic acids.
Microparticles, i.e. exosomes are also present in serum, pleural effusions and ascites of cancer patients [9
]. As stated above, these exosomes most likely represent a mixture derived from various cell types. Recently, we have shown that exosomes derived from the tumor can be distinguished from normal cell exosomes by marker expression [30
]. Exosomes in the ascites derived from ovarian cancer carried the marker set EpCAM, CD24 and CD9 that appear to exist on a common exosome type [30
]. In the present study we used for the analysis of amniotic fluid, urine and saliva exosomes other exosomal marker proteins such as Annexin-1, CD24, HSP-70 or ADAM10. It should be pointed out that at presence there is no evidence that these markers are shared by all exosomes.
An important feature is that, just like cells, exosomes can be isolated by antibodies and MACS procedures. Thus, mAb to membrane proteins overexpressed in tumors such as CD24 or EpCAM can be used to enrich tumor derived exosomes [30
]. This technique is not only limited to the body fluid surrounding the tumor, as exosomes can become detectable in the serum and therefore allows minimal invasive collection methods [15
]. The miRNA profiling of ovarian malignant ascites derived exosomes revealed unique expression signatures derived from the tumor [31
]. Exosomes from glioblastoma patients expressed esRNA for a truncated and oncogenic form of the epidermal growth factor receptor, known as EGFRvIII that can be transferred via exosomes to neighbouring cells [32
]. Thus, it is possible that exosomes derived from the tumor can serve as messengers (for their diagnosis) and mediators of tumor progression [33
Although knowledge about the secretion from MVBs and the requirements for protein sorting into exosomes is growing, it is presently not known how genetic information is recruited into exosomes. An important question is whether the esRNA and miRNA content of exosomes is representative for the cell of origin. Valadi et al showed that microarray assessments of esRNA from mouse and human mast cell lines revealed the presence of mRNA from approximately 1,300 genes, many of which were not present in the cytoplasm of the donor cell [21
]. Another study reported that miRNA from ovarian tumor cells and exosomes from the same patients were positive for 218 of 467 mature miRNAs analyzed. The levels of only 8 specific microRNAs were similar between cellular and exosomal miRNAs [31
]. Further studies are needed to address this important question.