Abstract P4-07-05: Exosome-mediated trafficking of microRNAs by breast cancer cells

Abstract

Abstract Introduction Cellular communication in the primary tumour micro-environment is known to play a key role in tumour development and progression. Exosomes are membrane-derived nanovesicles that are actively secreted by cells. Exosomes have been implicated in cell-to-cell communication through the transfer of genetic material including messenger RNA (mRNA), and more recently microRNA (miRNA). miRNAs are small non-coding RNAs approximately 22 nucleotides in length. They play an important role in posttranscriptional regulation of gene expression. Recent reports suggest exosome-mediated trafficking of microRNAs between cells. The aim of this study was to identify the panel of exosome-encapsulated microRNAs secreted by breast cancer cells in vitro. Methods Four breast cancer cell lines, MDA-MB-231, BT-20, Sk-Br-3 and T47D were cultured in exosome-depleted media for 48 hours. Exosomes secreted by the cells were isolated through a process of differential centrifugation, microfiltration and ultracentrifugation. The presence of exosomes was first confirmed by Transmission Electron Microscopy (TEM) and Western Blot analysis. Global miRNA array analysis of RNA extracted from exosomes was performed to identify the panel of miRNAs secreted by these cells. miRNA targets of interest were further validated using relative quantification PCR (RQ-PCR). Transfer of Red fluorescent protein (RFP)-labelled exosomes between cell populations was visualized using Confocal microscopy. Results TEM analysis of secreted exosomes revealed vesicular bodies of 40-100nm in size. Immunoblotting confirmed the presence of the exosome-associated protein CD63. MicroRNA array analysis of exosome fractions targeting 2089 miRNAs, revealed secretion of between 324 and 394 miRNAs by the cell lines. The miRNAs appeared to cluster in a biologically relevant fashion. 282 miRNAs were common to exosomes from all 4 cell lines, while a small selection were specific to individual cell lines. Exosome-mediated trafficking of miRNA targets of interest including miR-451a and miR-744-5p was successfully validated using RQ-PCR. Further validated targets included miR-10b, miR-145, miR-492 and miR-498, all of which have established roles in regulation of proliferation and apoptosis in cancer. Confocal microscopy supported visualization of miRNA-enriched exosome release from donor cells and subsequent uptake of the RFP-labelled exosomes by recipient cells. Conclusions A distinct panel of miRNAs are actively and selectively packaged into exosomes and secreted by breast cancer cells. This transfer of functional miRNAs between cells may play an important role in intercellular communication in the primary tumour microenvironment. The data presented also has potentially important implications in the identification of a circulating miRNA signature for breast cancer detection. Citation Format: Doireann P Joyce, Claire L Glynn, James Brown, Emma Holian, Peter Dockery, Michael J Kerin, Roisin M Dwyer. Exosome-mediated trafficking of microRNAs by breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-05.