Abstract 5585: Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening

Abstract

Abstract Extracellular vesicles (EV) are a promising biomarker source and present in several biological fluids such as plasma (8.0 x 1011 particles/mL, 121 ± 2.3 nm in diameter) and urine (1.1 x 1011 particles/mL, 152 ± 11.2 nm in diameter). In order to streamline marker screening and validation in translational studies, we developed and optimized high throughput filter-based EV capture, poly(A)+ RNA isolation and gene expression assay protocols for RT-qPCR and RNA-seq. First, we confirmed that plasma and urinary EV mRNA profiles (12 to 15 genes by RT-qPCR) are stable up to 1 week at 4°C, and up to 5 repeats of freeze-thaw cycles, and urinary EV was stable up to 10 months at -80°C, indicating EV mRNA could be a useful biomarker source. Although EV mRNA is less abundant (550 pg mRNA from 1 mL plasma) compared to tissue mRNA or EV non-coding RNA, the optimized protocol allows quantification of about 9,000 genes in average by RNA-seq from 2 mL frozen plasma (N=3) with high reproducibility among the replicates (Spearman correlation: 0.9086 to 0.9326) and between the commercial EV kits (Spearman correlation: 0.9207 to 0.9683). About 77% of the 1000 most abundant genes in plasma EV are expressed ubiquitously throughout various organs but some are expressed specifically in blood (12%), spleen (4.4%), esophagus mucosa (1.2%), etc. according to the Genotype-Tissue Expression (GTEx) database. In urinary EV, other than ubiquitously expressed genes (88%), the genes specific to liver (1.5%), bladder (1%), brain (1%), etc. are present. These data suggest EV mRNA are released from various organs and circulating in biological fluids. The protocol was further applied to biomarker discovery for bladder cancer, and differential gene expression analysis of EV mRNA was conducted using bladder cancer (N=4), cancer remission (N=2), and healthy/disease control urine samples (N=4). Ingenuity pathway analysis indicated that cancer-related molecular and cellular functions are activated in bladder cancer urinary EV but not in remission, while immune-related functions are activated both in cancer and remission. These data indicate that EV mRNA is a promising biomarker source for cancer biomarker screening, and could be used in translational studies. Citation Format: Taku Murakami, Cindy Yamamoto, Mieko Ogura, Melanie Oakes, Hiroshi Harada. Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5585.