[HTML][HTML] Double-stranded DNA in exosomes: a novel biomarker in cancer detection

BK Thakur, H Zhang, A Becker, I Matei, Y Huang… - Cell research, 2014 - nature.com
BK Thakur, H Zhang, A Becker, I Matei, Y Huang, B Costa-Silva, Y Zheng, A Hoshino…
Cell research, 2014nature.com
Exosomes, small membrane vesicles (30-100 nm) of endocytic origin secreted by most cell
types, contain functional biomolecules, which can be horizontally transferred to recipient
cells [1]. Exosomes bear a specific protein and lipid composition, and carry a select set of
functional mRNAs and microRNAs [2]. Recently, our group has shown that c-Met shed in
exosomes can promote a proangiogenic and prometastatic phenotype in bone marrow-
derived progenitor cells during melanoma progression [3]. In previous research …
Exosomes, small membrane vesicles (30-100 nm) of endocytic origin secreted by most cell types, contain functional biomolecules, which can be horizontally transferred to recipient cells [1]. Exosomes bear a specific protein and lipid composition, and carry a select set of functional mRNAs and microRNAs [2]. Recently, our group has shown that c-Met shed in exosomes can promote a proangiogenic and prometastatic phenotype in bone marrow-derived progenitor cells during melanoma progression [3]. In previous research, retrotransposon RNA transcripts, single-stranded DNA (ssDNA), mitochondrial DNA, and oncogene amplifications (ie, cmyc) have been detected in microvesicles [4-6]. In this report, we provide evidence that tumor-derived exosomes carry double-stranded DNA (dsDNA), as demonstrated through two different approaches, using enzymatic methods (dsDNA-specific shrimp DNase) and physical/structural studies (atomic force microscopy, AFM). Furthermore, we show that exosomal DNA (exoDNA) represents the entire genome and reflects the mutational status of parental tumor cells. We also highlight the translational value of exoDNA in tumor-derived exosomes for its potential usefulness as a circulating biomarker in the early detection of cancer and metastasis. We demonstrate for the first time that the majority of DNA associated with tumor exosomes is double-stranded (Figure 1A-1E). Two types of DNA-specific detection assays were utilized to assess the nature and amount of DNA in three different cancer models including human chronic myeloid leukemia (K-562), human colorectal carcinoma (HCT116), and murine melanoma (B16-F10). One assay is based on the detection of DNA on agarose gels using highly sensitive SYBR Gold nucleic acid staining. The second assay is the QuantiFluor dsDNA Detection System, which uses fluorescent dye that binds specifically to dsDNA (Supplementary information, Data S1). For both assays, we utilized DNases that specifically recognize and digest ssDNA (S1 nuclease) or dsDNA (Shrimp dsDNase). We first verified the specificity of S1 nuclease and dsDNase using purified ssDNA oligonucleotides and Lambda dsDNA as substrates (Supplementary information, Figure S1A). Slight non-specificity of S1 nuclease towards dsDNA was observed. First, to analyze whether DNA is associated with the outer membrane and/or inside exosomes, we extracted DNA from either intact exosomes or exosomes pre-treated with DNases. Remarkably, we observed no change in the pattern of DNA isolated from samples pre-treated with S1 nuclease versus untreated samples (Figure 1A, lane 2 vs lane 5), whereas exosomes pre-treated with dsDNase showed a strong reduction in DNA species greater than 2.5 kb in size and an enrichment of DNA between 100 bp and 2.5 kb (Figure 1A, lane 2 vs lane 8). This indicates that the majority of external exoDNA is dsDNA with a larger size (> 2.5 kb) in comparison to internal exoDNA. Genomic DNA (gDNA) was used as a control for DNase digestion (Figure 1A, lanes 11-13).
To further characterize internal exoDNA, first we eradicated external DNA using dsDNase digestion. Then, isolated internal exoDNA was analyzed using both the S1 nuclease/dsDNase digestion method and the QuantiFluor assay. We observed that S1 nuclease digestion resulted in a reduction of DNA to a much lesser extent than dsDNase digestion (Figure 1A, lane 9 vs lane 10 and Figure 1B-1C). The same pattern was observed in internal exoDNA isolated from exosomes untreated or pre-treated with S1 nuclease (Figure 1A, lane 3 vs lane 4 and lane 6 vs lane 7). Consistently, we observed a strong …
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