Monitoring the Rab27 associated exosome pathway using nanoparticle tracking analysis

Abstract

Exosomes are secreted by many cell types and display multiple biological functions. The ability to both rapidly detect and quantify exosomes in biological samples would assist in the screening of agents that interfere with their release, and which may therefore be of clinical relevance. Nanoparticle tracking analysis, which detects the size and concentration of exosomes, was used to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a known component of the exosome pathway. Inhibition of both Rab27a and Rab27b was observed, resulting in alterations to intracellular CD63+ compartments and the release of fewer exosomes into the culture medium, as determined by nanoparticle tracking analysis and confirmed by immunoblotting and protein quantification. These data show that nanoparticle tracking analysis can be used effectively and rapidly to monitor the disruption of exosome secretion.

Introduction

The term ‘exosome’ was proposed in 1987 to describe vesicles released from multivesicular endosomes in reticulocytes [1]. Later, Raposo and colleagues reported that exosomes secreted from Epstein–Barr virus (EBV)-transformed B lymphocytes bear functional MHC molecules and could present MHC-antigen peptide complexes to specific T cells [2]. Zitvogel and colleagues also discovered that dendritic cells secrete exosomes bearing functional MHC-peptide complexes, which could induce antitumour immune responses in mice in vivo [3]. The later discovery that exosomes can deliver mRNA and microRNA has only served to generate even further interest into the biological functions of exosomes [4]. Modified exosomes have carried siRNA into brain tissue in mice, resulting in specific knock down of the gene BACE1, a therapeutic target in Alzheimer's disease [5]. Furthermore, exosomes encapsulating curcumin or the Stat3 inhibitor JSI124 have been delivered noninvasively to microglia cells via an intranasal route in mice [6]. These studies demonstrate the potential of exosomes in developing novel therapeutic treatments.

Due to the small sizes of exosomes, their analysis and quantification is often problematic. Commonly used techniques include isolation by ultracentrifugation, purification by sucrose cushion or gradients, immunoisolation by antibodies, western blotting, absorption onto latex beads followed by flow cytometry, atomic force microscopy and electron microscopy [7], [8]. Flow cytometry analysis of exosomes has also been reported [9], [10], but is often confounded by the typical lower detection limit of 200–300 nm in most flow cytometers, although newly developed flow cytometers can now achieve better levels of detection.

Recently, nanoparticle tracking analysis (NTA) has been utilised to visualise and measure nanoparticles within the size range of 30 nm to 1000 nm. Particles move rapidly in a liquid sample under Brownian motion and act as point refractors when they pass through a laser beam. Videos can be recorded and a detailed differential particle size distribution graph can be produced using analytical software.

We have previously reported the ability of NTA to detect increases in exosomes released from immune cells [11]. Here, in contrast, we show the use of NTA to monitor decreases in exosome production after using inhibitory RNA technology to suppress the Rab27-associated exosomal pathway in MDA-MB-231 breast cancer cells.