Cancer-derived extracellular vesicles (EVs) are membrane-enclosed structures of highly variable size. EVs contain a myriad of substances (proteins, lipid, RNA, DNA) that provide a reservoir of circulating molecules, thus offering a good source of biomarkers. Researchers from Cedars-Sinai Medical Center demonstrate here that large EVs (L-EV) (large oncosomes) isolated from prostate cancer (PCa) cells and patient plasma are an EV population that is enriched in chromosomal DNA, including large fragments up to 2 million base pair long. While L-EVs and small EVs (S-EV) (exosomes) isolated from the same cells contained similar amounts of protein, the DNA was more abundant in L-EV, despite S-EVs being more numerous. Consistent with in vitro observations, the abundance of DNA in L-EV obtained from PCa patient plasma was variable but frequently high. Conversely, negligible amounts of DNA were present in the S-EVs from the same patients. Controlled experimental conditions, with spike-ins of L-EVs and S-EVs from cancer cells in human plasma from healthy subjects, showed that circulating DNA is almost exclusively enclosed in L-EVs. Whole genome sequencing revealed that the DNA in L-EVs reflects genetic aberrations of the cell of origin, including copy number variations of genes frequently altered in metastatic PCa (i.e. MYC, AKT1, PTK2, KLF10 and PTEN). These results demonstrate that L-EV-derived DNA reflects the genomic make-up of the tumour of origin. They also support the conclusion that L-EVs are the fraction of plasma EVs with DNA content that should be interrogated for tumour-derived genomic alterations.
The DNA contained in L-EVs is of high molecular weight and mirrors
the genetic make-up of their cells of origin
(a) PC3 L-EV and S-EV DNA were extracted in agarose plugs by incubation in lysis buffer for 24, 48 or 72 h, and high molecular weight DNA was resolved by pulse field gel electrophoresis (PFGE), which revealed that L-EVs contain large DNA fragments (100 kbp–2 Mbp). (b) Representative fluorescence intensity profiles of the gel lanes in G (48 h) containing L-EV DNA, S-EV DNA and cell DNA. (c) PC3 EV DNA was extracted in agarose plugs or using a commercially available kit (DNeasy Blood and Tissue kit, QIAGEN). Lower molecular weight DNA was resolved by PFGE, showing that EVs, and L-EVs in particular, contain mostly high molecular weight DNA, which is fragmented when it is extracted using a commercial kit. (d) DNA from PC3 cells and PC3-derived L-EVs was analysed by whole genome sequencing (WGS), which showed that somatic copy number variations (SCNV) detected in PC3 cells were accurately reflected in PC3-derived L-EVs. The profiles demonstrate copy number gains (above two copies, red) and losses (below two copies, blue) across all chromosomes. SCNVs were identified using read depth-based algorithm BIC-seq.; bin size 0.5 Mbp. The density of the reads was normalized using chromosome 2, which resulted to be the least aberrant chromosome. SCNV summary track at the bottom was obtained by Integrative Genomic Viewer (IGV) of 150 human mCRPC genomes and shows that SCNV detected in PC3 cell line largely overlaps with SCNV detected in mCRPC patients. Copy number gains are marked in red and losses are marked in blue. (e) CIRCOS circular visualization of the indicated genomic rearrangements detected in both PC3 cells and PC3-derived L-EVs.