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

Abstract

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 (i.e., c-myc) have been detected in microvesicles 4 , 5 , 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). Figure 1 Identification and characterization of exoDNA and its potential use as a diagnostic tool. (A) Equal amounts of DNA extracted from K-562 exosomes, which were untreated (Set a), pre-treated with S1 nuclease (Set b) or dsDNase (Set c), were digested with either S1 nuclease (lanes 3, 6 and 9) or dsDNase (lanes 4, 7 and 10). Digestion of gDNA (Set d) with S1 nuclease (lane 12) or dsDNase (lane 13) serves as controls. (B , C) Analysis of internal exoDNA isolated from HCT116 (B) and B16-F10 (C) after removal of external exoDNA as in A . The results are representative of 2-3 experiments performed independently. (D) AFM image of exoDNA. ExoDNA was extracted from K-562 cells and absorbed on a mica surface in the presence of 5 mM Mg 2+ . Scale bar, 500 nm. (E) Internal exoDNA was extracted from exosomes secreted by different types of cancer cell lines, including melanoma (B16-F10), pancreatic cancer (Pan02, Pan02 H3, PANC-1, AsPC-1, BXPC-3 and HPAF-II), breast cancer (MDA-MB-231 and MDA-MB-4175), lung cancer (H1650 and H1975), and leukemia (NB-4), and digested with dsDNase. Abundance of dsDNA inside the exosomes, before and after digestion with dsDNase, was expressed as “nanogram of DNA per microgram of exo-Protein”. (F) Circular view of the readings of fragments along each chromosome in the whole-genome sequencing analysis of exoDNA isolated from murine melanoma B16-F10 cell-derived exosomes. (G) ExoDNA represents gDNA shown by comparative genomic hybridization array analysis of B16-F10 exoDNA vs gDNA. (H - J) Mutational analysis of exoDNA. BRAF(V600E) mutation in exoDNA isolated from either cultured melanoma cell lines (H) or circulating exoDNA isolated from SK-MEL-28 melanoma-bearing mice (J) was detected by AS-PCR analysis. gDNA isolated from SK-MEL-28 and SK-MEL-103 cells serves as a positive and negative control, respectively, for V600E mutation (WT (V) and mutant (E) alleles). Arrow indicates the size of expected PCR products. AS-P