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Title: Fibroblast uptake of prostate cancer vesicles
Author: Cocks, Alexander
ISNI:       0000 0004 7968 6817
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2019
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Prostate tumours are typically accompanied by aberrantly activated stroma, populated by myofibroblastic cells. These stromal cells support angiogenesis, and tumour growth in preclinical models. The tumour-derived factors responsible for the onset of stromal myofibroblasts remains poorly defined, but the secretion of transforming growth factor beta-1 (TGFβ1) is strongly implicated in the differentiation of various precursors, such as fibroblasts. Like other epithelial cancers, prostate cancer cells secrete small extracellular vesicles, these carry TGFβ1 on their outer surface, and can deliver to fibroblasts, driving their differentiation into myofibroblasts. Unlike stimulation with soluble TGFβ1, the vesicle generated myofibroblast is analogous to those naturally occurring at the cancer site. The vesicle form of TGFβ1 delivery therefore is TGFβ1 dependent yet the phenotype arising is distinct from that driven by soluble TGFβ1. This observation suggests that vesicles are likely to co-deliver other factors to the fibroblast that collectively generate the in vivo-like myofibroblast differentiation response. Because uptake of acquired vesicles is documented as important in many other biological systems, we hypothesised that vesicle entry into fibroblasts was an important aspect of vesicle-mediated communication, and relevant for the differentiation response. The aim of this project was to define the uptake process of prostate cancer vesicles by fibroblasts, to determine the intracellular fate of the vesicles once internalised, and the importance of cell entry in the complex differentiation process. To achieve this, we developed labelling techniques to fluorescently tag vesicles and used these to monitor vesicle uptake and intracellular trafficking in fibroblasts by fluorescence microscopy and flow cytometry. Prostate cancer vesicles can be flexibly fluorescently labelled with the novel maleimide linked Alexa dyes. Using Alexa labelled vesicles, we found that fibroblast uptake primarily occurs through Clathrin-mediated endocytosis, and we reveal a role for vesicle-cell surface interaction in the uptake process. The vesicles were observed in early endocytic compartments, then transit through maturing endosomes reaching lysosomes within 2 hours of cellular uptake. Vesicle labelling with intraluminal fluorescent dyes revealed the requirement of vesicle v internalisation by fibroblasts to occur prior to luminal cargo delivery to the cell cytosol, and this escape occurs before the vesicles reach lysosomes. Vesicle internalisation by fibroblasts is independent of the TGFβ1 mediated stimulation of the cell and occurs even if fibroblast differentiation is blocked. This study concludes that prostate cancer vesicles can deliver their intraluminal contents to fibroblasts after clathrin dependent vesicle endocytosis, but before reaching the lysosome. Any effect the intraluminal cargo has on the fibroblast phenotype would be independent of the TGFβ1 mediated effects.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: R Medicine (General)