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

By B. K. Thakur, Haiying Zhang, A. Becker, Irina R Matei, Yujie Huang, B. Costa-Silva, Yan Zheng, Ayuko Hoshino, H. Brazier, J. Xiang and 12 more. Cell Research, 2014

1,384 citations recorded by the source corpus.

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), mito-chondrial DNA, and oncogene amplifications (i.e., c-myc) 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 transla-tional 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 oligonucle-otides 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 …

Subfields: Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/b80d2d5caa89e11b96682028d166e29452f2424a

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Citations in the corpus

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper (2017, neutral): “treatments prior to vesicle lysis (compared with DNase treatment post-lysis) and read out DNA to demonstrate protection or lack thereof.[60]”
Origins, structures, and functions of circulating DNA in oncology (2016, background): “Recently, it has been demonstrated that exosomes also contain small amounts of DNA and exosomes tumor-derived DNA has been characterized [122]: this seems to exist in two main forms: one double-stranded form external to the microvesicle is bound to the membrane and is of a large size (>2.”
Inflammaging and 'Garb-aging'. (2017, background): “11 DNA but, notably, parts of it such as non-coding sequences, seem to be overrepresented, suggesting an active and selective vesicle compartmentalization of DNA molecules [60-62].”
The biology, function, and biomedical applications of exosomes (2020, positive): “Some studies have suggested that small amounts of DNA can be found in exosomes and that this DNA can be of value in detecting cancer-associated mutations in serum exosomes ( 185 – 188 ).”
Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer (2017, positive): “We and others have previously described the presence of genomic DNA in EVs from cancer-derived cell lines and in patients with pancreatic cancer (13).”
The biology and function of exosomes in cancer. (2016, background): “DNA in exosomes may provide information about cancerspecific mutations (7, 49).”
Innovative approaches for cancer treatment: current perspectives and new challenges (2019, methodology): “In recent years, exosomes detection has been validated as a reliable tool for preclinical practice in different cancer types [64], thanks to the identification of their content: double-stranded DNA (dsDNA) [65, 66], messenger RNA (mRNA), micro RNA (miRNA), long non-coding RNA (lncRNA) [67], proteins”