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Data: 27-gen-2012
Autori: Brunetto, Lidia
Titolo: Investigation of Cancer Stem Cells (CSCs)-derived exosomes and their influence on the tumor microenvironment
Abstract: Exosomes are membrane nanovesicles of endocytic origin that can be released to the extracellular environment by many different cells, included the tumor cells. Exosomes have been suggested to have a number of different functions and are believed to take part in the communication between cells. The aim of this thesis was to assess the composition and functions of Lung cancer stem cells (LCSCs)-derived exosomes, with focus on the content of typical proteins, microRNAs and cell-to-cell communication. We were able to set up an isolation protocol to obtain the exosomes from LCSCs supernatant. The exosomes have a distinct composition of surface proteins. By western blot analysis (WB) we characterized LCSCs-derived exosomes for the presence of typical exosomes markers. We found that, in addition to well known markers, such as Tsg101 and CD81, LCSC-derived exosomes are enriched in CD133 protein. Immunoprecipitation (IP) experiments revealed that by immunoprecipitating the culture SN of CD133 expressing cells with an antibody against CD133 protein, typical exosomal markers, such as Tsg101 and Rab5b, are contained in the precipitate. This suggests that CD133 is contained in bona fide exosomal particles and that these proteins might have a physiological role in the biological function of exosomes. The structure of CD133 protein shows homologies with proteins involved in plasma membrane fusion, suggesting, that CD133 might play an active role in the transport of biological information from one to another cell. Immunofluorescence (IF) experiments and FACS analysis revealed that exosomal vesicles, contained in the supernatant, are able to transfer CD133 protein from expressing to non-expressing cells. Therefore we established an experimental system in order to study a transport of proteins or genetic from cell to cell. Results of co-culture experiments, using a tetracycline-dependent regulatory system, showed an interchange of information between stably transduced cell lines. We wanted also investigate if CD133 enriched SN is able to alter the phosphorylation status of recipient cells. We found that the recipient cells, after incubation with CD133 enriched SN, show a marked increase in MAP kinase pathways. In addition, transfer experiments suggest, that miR146a contained in LCSC136-derived exosomes is shuttled to HEK293T recipient cells. Furthermore the transfer of miR146a is increased by CD133 overexpression in LCSC136 cells. In order to verify if CD133 containing exosomes, indeed transport microRNAs, we performed immunoprecipitation experiments of CD133 enriched SN, using CD133 antibody. We found, that the CD133 positive membrane fraction isolated by immunoprecipitation also contained miR146a. This result revealed that we didn t isolate just protein aggregates, but CD133 positive exosomes, which are able to transport microRNAs. In summary, CSC-derived exosomes are bioactive molecules mediating the interchange of informations and are able to stimulate target cells in order to develop tumor capacities. This function can be enhanced by the presence of CD133, which positively regulates the transport of bioactive molecules such as microRNAs between cells. These new findings suggest a new, pro-tumorigenic role to this well known cancer stem cell marker.
InArea 05 - Scienze biologiche

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