Abstract 3590: Investigation of exosome-encapsulated microRNA secretion in breast cancer
Authors: Glynn Claire , Khan Sonja , Brougham Cathy , Clancy Cillian , Joyce Doireann , Dockery Peter , Kerin Michael J. , Dwyer Roisin M. 
Published: May 2015

Abstract
Intercellular communication between breast tumour cells and stromal cells plays a fundamental role in cancer initiation and progression. Mesenchymal Stem Cells(MSCs) are multipotent cells with the proven ability to home to the site of breast tumours and integrate into the tumour architecture. The mechanisms of MSC interaction with breast cancer cells are poorly defined. Exosomes are microvesicles secreted by cells that have the capacity to transport genetic material including microRNAs(miRNAs), which are important regulators of gene expression. However, the miRNAs selectively packaged into exosomes, and their true role in the tumour microenvironment, is poorly understood.
Aim: Investigate the presence of MSCs in primary breast tumours, and identify exosome-encapsulated miRNAs secreted by breast cancer cells and MSCs in vitro.
Methods: Stromal cells were isolated from primary breast tumours and characterized based on MSC-associated cell surface antigens. Tumour stromal cells were cultured in appropriate conditions to investigate potential for differentiation into osteoblasts or adipocytes, followed by Von Kossa staining of calcium deposition, and Oil Red O staining of fat droplets respectively. MSCs isolated from healthy volunteers and breast cancer cell lines were cultured in exosome-depleted media for 48 hrs, and secreted exosomes isolated using ultracentrifugation. Transmission Electron Microscopy(TEM) and Western Blot were performed to confirm the presence of exosomes in the purified fraction. Extracted miRNA was subjected to global miRNA expression analysis using the Exiqon miRCURY™ LNA Array.
Results: A subpopulation of tumour stromal cells expressed the full panel of surface markers associated with MSCs. This subpopulation also had the capacity to differentiate into osteoblasts and adipocytes, demonstrated by calcium deposition and formation of fat droplets. Exosomes were successfully isolated from conditioned media of breast cancer cell lines and MSCs. TEM revealed microvesicles with the appropriate size of 40-100nm. Western Blot confirmed the presence of the exosome-associated protein CD63. miRNA array analysis identified 413 miRNAs encapsulated in MSC-secreted exosomes from a panel of 2089 analysed, while each of the breast cancer cell lines secreted from 381 to 394 miRNAs in exosomes. Of these, 287 were common to all four breast cancer cell lines. Interestingly, a subset of these miRNAs are known to target genes associated with cell cycle regulation and apoptosis (e.g. miR-15a, miR-204, miR-221, miR-223). A panel of the miRNAs also appeared to have differential expression with reference to epithelial subtype.
Conclusion: MSCs are present in primary breast tumours, and elucidating their interaction with breast cancer cells will be fundamental to understanding their role in the tumour microenvironment. Bidirectional transfer of exosome-encapsulated miRNAs may play an important role in this intercellular communication.
Citation Format: Claire Glynn, Sonja Khan, Cathy Brougham, Cillian Clancy, Doireann Joyce, Peter Dockery, Michael J. Kerin, Roisin M. Dwyer. Investigation of exosome-encapsulated microRNA secretion in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3590. doi:10.1158/1538-7445.AM2014-3590