Extracellular vesicle-associated repetitive element DNAs as candidate osteosarcoma biomarkers

Abstract

Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. Despite that high-risk factors have been identified, no test for early detection is available. This study aimed to identify circulating nucleic acid sequences associated with serum extracellular vesicle (EV) preparations at the time of OS diagnosis, as a step towards an OS early detection assay. Sequencing of small nucleic acids extracted from serum EV preparations revealed increased representation of diverse repetitive element sequences in OS patient versus control sera. Analysis of a validation cohort using qPCR of PEG-precipitated EV preparations revealed the over-representation of HSATI, HSATII, LINE1-P1, and Charlie 3 at the DNA but not RNA level, with receiver operating characteristic (ROC) area under the curve (AUC) ≥ 0.90. HSATI and HSATII DNAs co-purified with EVs prepared by precipitation and size exclusion chromatography but not by exosome immunocapture, indicative of packaging in a non-exosomal complex. The consistent over-representation of EV-associated repetitive element DNA sequences suggests their potential utility as biomarkers for OS and perhaps other cancers.

Extracellular Vesicles: A New Source of Biomarkers in Pediatric Solid Tumors? A Systematic Review

“…Cambier et al analyzed repetitive DNA and RNA elements present in EVs isolated from serum from patients and healthy controls ( 74 ). In this report, different EV isolation and purification approaches were used: ExoQuick in the discovery cohort and PEG precipitation, SEC and immunoaffinity capture in different subgroups within the validation cohort.…”

“…However, the samples isolated by PEG precipitation and immunoaffinity were also analyzed by ExoView. This visualization technique depends on immunocapture of EVs to a microarray chip by different EV-enriched surface proteins ( 74 ). In the discovery cohort sequencing of RNA and DNA resulted in identification of 4 repetitive elements upregulated in serum from patients with osteosarcoma, in comparison to healthy controls.…”

DNA in extracellular vesicles: from evolution to its current application in health and disease

“…mtDNA has been found in EVs enriched from the plasma of atrial fibrillation [ 36 ] and chronic heart failure patients [ 37 ]. Accumulating observations also imply that EV-DNA includes DNA from retrotransposable elements and satellite repeat DNA [ 31 , 38 ]. In particular, the recent finding of Cambier et al (2021) confirmed that repetitive element DNAs, such as HSATI, HSATII, LINE1-P1, and Charlie 3, were associated with EVs derived from the serum of osteosarcoma patients but not with CD9+ or CD81+ exosomes [ 38 ].…”

Extracellular Vesicle Derived From Mesenchymal Stem Cells Have Bidirectional Effects on the Development of Lung Cancer

“…In addition to the above methods, precipitation of EVs by using polyethylene glycol (PEG) is also a good option. Centrifugation followed by mixing with an equal volume of freshly prepared PEG solution can provide EVs of sufficient purity ( 37 ). At present, differential centrifugation is still the most commonly used separation method because of its simplicity and economy ( 38 ).…”