Characterization and miRNA analysis of cancer cell-secreted microvesicles.

摘要

The development of minimally invasive clinical biomarkers for the detection and monitoring of human cancers would greatly reduce the worldwide health burden of this disease. To date, none of the biomarkers recommended by the American Society of Clinical Oncology can accurately predict the risk of cancer initiation, development, or a response to advanced treatment. Consequently, factors such as disease-free interval, previous therapy, site of disease and number of metastatic sites are used to monitor the impact of treatment on patients.;MicroRNAs (miRNAs) post-transcriptionally regulate gene expression and have been found to play a critical role in many homeostatic and pathological processes. Recent studies have systematically analyzed miRNA expression in cancer tumors and showed that these tumors exhibit distinct miRNA profiles compared to healthy tissues. Current technologies to isolate and analyze miRNAs are limited to tissue biopsies and include a myriad of assays which are time consuming, labor intensive, semi-quantitative and prohibitively expensive for routine clinical application. However, the recent breakthrough observation that epithelial tumor-derived miRNAs are present in blood has bolstered the study of circulating miRNAs as potential biomarkers for the initiation and progression of solid tumors.;Besides epithelial tumor derived miRNAs, blood contains a mixture of miRNAs originating from all the myriad of blood-cell types that are in circulation. Since cancer-related inflammation is likely to alter the miRNA expression of the cellular constituents of blood, systemic changes in the profiles of secreted miRNAs in the blood stream are confounded with tumor specific changes until the effects of cancer on each of the constituents of blood is accurately quantified. A way to bypass this complexity is to analyze only the blood miRNAs that are originating specifically from the tumor.;Extracellular miRNAs travel the body protected from degradation within cell secreted microvesicles (MVs) that range in size between 30-1,000 nanometers. Both healthy and tumorigenic cells secrete MVs into their surroundings and are found in all biofluids. Consequently, if MV miRNAs are to be proposed as a potential source of novel biomarkers for cancer, the accurate selection and characterization of tumor derived MVs from the rest of the biofluid's MV background is required to achieve consistent and robust measurements. Additionally, since MVs have been shown to contain distinct miRNA compositions to that of the donor cells, the analysis of the miRNAs contained within them is required for tracking changes during tumorigenesis.;The main goal of the research discussed in this dissertation was to characterize cancer cell secreted MVs and to compare their miRNA content with the cells of origin. This dissertation focuses on three distinct types of cancer secreted MVs: breast, thyroid, and multiple myeloma. In this dissertation, the abundance, on a per cell basis, was obtained for the mature miRNA content in cultured cells and in their secreted MVs. The results obtained are relevant for both the clinical applications of circulating MV biomarkers, as well as, for advancement in the understanding of the mechanistic processes involved in miRNA MV secretion.