Tumor-derived serum exosomal microRNA as a novel biomarker for high-grade serous carcinoma

Abstract

Objectives: High-grade serous cancer (HGSC) is diagnosed at an advanced stage due to the lack of a reliable early detection biomarker. Exosomes are microvesicles, 50–100 nm in size, that form when endosomes fuse with cell membranes and are released into the extracellular space. Modern genomic technology allows researchers to isolate and interrogate circulating serum exosomes. We hypothesize that tumor-derived serum exosomes contain microRNAs (miRNAs) that can serve as novel biomarkers for HGSC. Methods: Exosomes were isolated from 500 μL of serum from 15 patients with stage IIIC or IV HGSC and 16 healthy, unaffected controls using the Exoquick kit (System Biosciences, Mountain View, CA). Exosomes were confirmed by size measurement using the NanoSight NS500 (Salisbury, UK) and the presence of common exosomal proteins (HSP70, CD9, CD81, and CD63) through Western blots. miRNAs were extracted from isolated exosomes using the Seramir kit (System Biosciences), and 800 commonly expressed human miRNAs were quantified with the NanoString nCounter platform (NanoString Technologies, Seattle, WA). miRNA expression was validated using Taqman real-time polymerase chain reaction (RT-PCR) with spike-in controls (Life Technologies, Norwalk CT). Two-sided statistical tests were used as appropriate. Results: Expected exosome size was confirmed by Nanosight evaluation, and Western blots confirmed the presence of common exosome-associated proteins: HSP70, CD9, CD81 and CD63. Of the 800 miRNAs measured, 56 were found to have more than twofold higher expression in HGSC samples compared to controls at a strict Bonferroni-corrected P b 0.01. No miRNAs were overexpressed in the control samples. Taqman RT-PCR has validated relative gene expression for select miRNAs. The Figure shows nonsignificant differences in expression for mir-16 and significant differential expression for mir-200a and mir-1283 in the discovery and validation platforms for HGSC cases and unaffected controls. Conclusions: Serum exosomal miRNAs are overexpressed in patients with advanced-stage HGSC compared to healthy controls. These findings suggest that serum exosomal miRNAsmay serve as novel biomarkers for advanced HGSC. Future work will be required to determine if exosomal miRNA expression is sufficient for early detection.