Characterization of cell-secreted microvesicles: Modulators of cell-cell communication.

摘要

Recent advances in the field of intercellular communication have revealed the importance of cell-secreted microvesicles (MVs) as an acellular mode of cell-cell communication. These lipid membrane-bound spherical vesicles 30 nm up to 1 mum in diameter, are secreted by normal and diseased cells and contain a variety of biomolecules including proteins and nucleic acids (messenger RNAs, microRNAs, transfer RNAs and other small RNAs). MVs contain a distinct composition of proteins, lipids and nucleic acids (primarily microRNAs and mRNAs) compared to their donor cells and have been implicated as mediators of intercellular communication in cancer by promoting tumor metastasis and modulating immune response. More recently their role as carriers of microRNAs during cognate immune interactions has also revealed their potential as regulators of the immune system during immune cell stimulation.;MicroRNAs (miRNAs) are small non-coding RNAs, approximately ~22 nucleotides in length that regulate gene expression at the post-transcriptional level. They have been found to play a critical role in many homeostatic and pathological processes. Presence of differential expression of miRNAs between tumor and normal tissues have motivated efforts to use miRNA expression patterns as diagnostic biomarkers for various cancers and prognostic indicators of therapeutics.;Besides tumors, miRNAs are also found circulating in blood and the discovery that MVs encapsulate these miRNAs to protect them from degradation in circulation, suggest that vesicle mediated transfer of miRNAs can be a mechanism for epigenetic re-programming of cells. MV-mediated transfer of specific miRNAs implicated in cancer invasion and metastasis has been reported, however the individual contributions from different MV sub-populations, comprising of smaller exosomes and larger shedding vesicles or from different cell sources in circulation remain to be determined.;The main goal of the research discussed in this dissertation was to analyze the miRNA composition of the MVs released from cancer and immune cells and compare their miRNA profile with the cells of origin as a function of change in cell state. We focused on the oncogene pathway-dependent secretion of the miRNAs within the MVs from two thyroid cancer cell lines to identify differences in the miRNA composition of the cells and the MVs in response to inhibition of the critical signaling pathways involved in thyroid cancer initiation and progression. We also compared the miRNA profiles of non-specific and antigen-specific cytotoxic T lymphocytes (CTLs) and investigated the miRNA composition of CTLs and MVs to identify miRNAs that are secreted within MVs in response to antigen-specific stimulation in a temporal manner. For this study we used artificial antigen presenting cells (aAPCs) for CTL activation that allows studies of MV secretion and their miRNA cargo in a controlled environment without the interference from other stimulating cells such as dendritic cells.;This dissertation also focused on the characterization of the MVs on the basis of their morphology, size distribution and abundance from different cell types and cell states. We used cryo-TEM to study the morphology and size distribution of the MVs from thyroid cancer cells under different stimulating conditions and a multiple myeloma cell line. A flow-based technique, asymmetric flow-field flow fractionation coupled with multi angle light scattering (A4F-MALS) was used to measure the size and abundance of the MV sub-populations from these cells. This work supports use of A4F-MALS as a potential biophysical technique for fractionation and characterization of MVs based on size, as well as for estimation of the concentrations of the MV sub-populations.;Overall, this dissertation provides a comprehensive study of cell-secreted MVs towards understanding their role in cell-cell communication.