Abstract P3-09-04: Exosome-Specific microRNA Signatures in Combination with Characteristic Surface Markers on the Circulating Exosomes Themselves Provide New Insights into the EMT

Abstract

Abstract Background: Recent discoveries have established that cancer tumors exhibit distinct microRNA (miR) expression profiles compared to normal tissues. Detection of miRs in the peripheral blood of cancer patients is possible due to their high stability. This stability is attributed to encapsulation of the miRs inside microvesicles (MVs) where they are protected from endogenous RNase activity in circulation. The tumor cell-secreted MVs of primary interest are circulating exosomes, a subpopulation of MVs distinguished by their relatively small size: 40 to 100 nm in diameter. The isolation of circulating tumor-derived exosomes from the other cell-secreted MVs in peripheral blood is the critical barrier to successful development of a robust assay for cancer-specific miR signatures. We describe the development of such an assay to: (1) capture/isolate circulating exosomes based on characteristic surface markers that correlate with different cell types, and (2) characterize exosome-specific miR signatures based on these surface markers. Materials and Methods: We apply sequential ultracentrifugation to isolate cell-secreted MVs, followed by asymmetric flow field-flow fractionation to selectively separate exosomes based on their intrinsically small size and characteristic surface markers. Isolated exosomes are then screened for surface markers by selectively capturing them on microarrays printed with antibodies against a library of known cell-surface markers for breast cancer. For the assessment of exosome-specific miR signatures, we have devised an antibody microarray assay that also enables the in situ characterization of miR profiles of the captured exosomes locally by qRT-PCR analysis confined to subarrays of printed spots in 50-µl microwells of a custom-designed, multi-well microarray. Assay development has been carried out using two human cancer cell lines (MCF 7 and MDA MB 231) and a non-malignant cell line (MCF 10a) representing pre-malignant cells and cancer of the breast at different epithelial and mesenchymal states. Results: We have used light scattering and cryo-transmission electron microscopy to characterize the size, size distribution, and morphology of exosomes as a sub-population of the secreted MVs from each of the three cell lines. We have also screened for surface markers by selective capturing the exosome sub-populations derived from these cell lines on antibody microarrays, and correlated these surface markers with the miR content of MVs using qRT-PCR analysis. Discussion: Our results demonstrate that correlating the miR signatures of circulating exosomes with characteristic surface markers on these exosomes leads to robust distinctions between the three cell lines that could not be achieved using either analysis or characterization alone. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-04.