Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs.

Yi-Fan Xu,Chinthalapally V Rao,Bethany N Hannafon,Amy Gin,Kritisha Bhandari,Wei-Qun Ding,Xiaohui Xu,Katherine T Morris,Pierre Busson

doi:10.1371/journal.pone.0259563

Abstract

microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC.

Highlights

Introduction miRNA expression is deregulated in pancreatic ductal adenocarcinoma (PDAC) [1, 2] and circulating miRNAs have been explored for the purpose of developing new PDAC biomarkers [3,4,5,6,7]
We have recently reported that miR-1246 and miR-196a are selectively enriched in PDAC cell line-derived exosomes and that plasma exosome miR-196a is a potential biomarker for the detection of early stage PDAC [22]
This is in part due to the fact that extracellular vesicles (EVs) are derived from different cellular sources and present in various sizes [8] and that conventional methods of exosome isolation, namely ultracentrifugation and polymer-based commercial kits, give rise to a mixed population of EVs. miRNA expression among different groups of EVs could significantly differ, which compromises the sensitivity and specificity of circulating exosome miRNAs for PDAC detection

Summary

Introduction

MiRNA expression is deregulated in pancreatic ductal adenocarcinoma (PDAC) [1, 2] and circulating miRNAs have been explored for the purpose of developing new PDAC biomarkers [3,4,5,6,7]. Exosome components collected from biological fluids may serve as biomarkers for PDAC [18,19,20,21,22]. We have recently demonstrated that plasma exosome miR-196a and miR1246 are potential biomarkers for localized PDAC [22]. Results from these previous reports, including ours, indicate that the detection of plasma exosome miRNAs is not sensitive or specific enough to serve as clinically applicable biomarkers for PDAC.

Methods

Results

Discussion

Conclusion