Abstract
Abstract Exosomes are small nanovesicles (30-100 nm) secreted from the cell surface of most cell types. The vesicles are secreted at increased levels by cancer cells and can be detected in body fluids of cancer patients. Exosomes function in paracrine delivery of proteins, RNA, and DNA into recipient cells. By this, exosomes are speculated to be involved e.g. in pre-metastatic niche formation at distant sites. Here we present a characterization of exosome vesicles isolated from bladder cancer cells as examined by electron microscopy, dynamic light scattering and by western blotting of common exosome protein markers. The expression of 671 unique human miRNAs by qPCR was analyzed in both exosomes and donor cells from: 1) cultivated normal urothelial cells (NHU), 2) T24 bladder carcinoma cell line (non-metastatic) and its metastatic isogenic derivates FL3 and SLT4 and 3) UMUC3 bladder carcinoma cell line (moderate metastatic) and its highly metastatic isogenic derivative LUL2. Several miRNAs were detected in the vesicles yet absent in their parental donor cell indicating a possible preferential vesicular packaging. miRNAs miR92a, -106a, -20a, -21, -16, and let-7a/b/d/e were detected among the top100 ranked miRNAs in all vesicles. Among the top30 enriched exosomal miRNAs, miR-223 and-383 were detected in all cancer vesicles, and miR-921 in the highly metastatic vesicles (FL3, SLT4, LUL2). The release of miRNAs (miR-21, -16, and let7b) from FL3 cells into the culture media was time-dependent and a fraction of these miRNAs were stable to RNaseOne degradation indicating possible protection inside the vesicles. Examination of miRNAs in human plasma samples also suggested resistance towards endogenous RNAse degradation as well as towards repetitive freeze/thawing. The profiling of miRNA expression by Affymetrix microarray analysis of crude exosome-containing fractions of human plasma samples (10 healthy controls and 10 cases of metastatic bladder cancer) showed significant upregulation in cancer plasma of e.g. miR-92a, -106a, -23a, -20a, -21, some of which were the same as the miRNAs highly expressed in the tumor vesicles. The significant increase was confirmed by q-RT-PCR for miR-92a and -23a and validated on an independent set of plasma samples (8 healthy controls and 8 metastatic cancer cases). Our results suggest that specific miRNAs are encapsulated into exosomes in bladder cancer and that these may be detected in circulation in advanced stages of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3387. doi:1538-7445.AM2012-3387
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