Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

Abstract

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. We 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.

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles

“…L-EVs and S-EVs were isolated from cell culture media by differential ultracentrifugation followed by density gradient purification (Suppl. Figure 1C), in line with the most recent MISEV2018 guidelines 11 and previous studies that separated large oncosomes from exosomes 7,8,25,42 . Over 99% of the cells from which EVs were isolated were viable (Suppl.…”

“…Finally, immunoblotting of proteins enriched in L-and S-EVs confirmed the nature of the EV preparations ( Figure 2C). L-EVs showed the enrichment of HSPA5 and KRT18 at 1.10-1.15 g/mL, which are enriched in large oncosomes 7,8,25,43 , while S-EVs showed the enrichment of CD81 and TSG101 at 1.10 g/mL, that are typically enriched in exosomes ( Figure 2C), in line with current literature [6][7][8]11,25,43 .…”

“…The isolation of EVs was conducted as previously described with minor modifications 7,8,25 . Cells were grown on 18 x 150 cm 2 -cell culture plates (Corning) until 90% confluence, washed in PBS, and serum-starved for 24 hours before the collection of conditioned cell media.…”

“…Extracellular vesicles (EVs) are lipid-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Several classes of EVs that differ in size, cargo, biogenesis and function have been reported in the last decade [6][7][8][9][10] . However, clearly defined markers that define these classes across different cell lines are currently lacking 11 .…”

New insight into the role of extracellular vesicles in kidney disease

“…While large EVs (L-EV) and small EVs (S-EV) (exosomes) purified from the same cells contained similar amounts of protein, DNA was more abundant in L-EV, despite S-EVs being more numerous. 7 Differences in the abundance levels of the EV miRNAs could discriminate between the three EV populations, apoptotic bodies, microvesicles and exosomes. 8 The varied cargoes of different populations of EVs may suggest their different functional roles.…”