Investigating the biomarker potential of extracellular vesicle nucleic acids in cancer, and the role of extracellular vesicle DNA in cell-to-cell communication

Extracellular vesicles (EVs) are nanosized vesicles released by almost all cells. Interest in EVs has increased dramatically in recent years. Although several aspects of these vesicles such as composition, origin, purpose and potential uses have already been well evaluated, many possible avenues of research remain open. One aspect yet to be fully understood is the nucleic acids contained within EVs. In this dissertation, we endeavoured to expand the current knowledge surrounding EV-DNA and EV-RNA by evaluating their potential diagnostic abilities in cancer models. As EVs can be isolated from body fluids and reflect their cell of origin, they are great candidates for liquid biopsies. Here, the ability to detect SMARCB1 mutations in EV-DNA of AT/RT cell lines and mouse plasma samples was investigated. Additionally, the ability to detect a FLI1 translocation in EV-RNA of a Ewing sarcoma cell line was demonstrated. Furthermore, EVs have important roles in cellular communication as well as tumour development and metastasis. Therefore, in the next step, the transfer of EV-DNA to recipient cells was investigated by establishing an EdU labelling method which allowed for the visualisation of EV-DNA with confocal microscopy. Here, the transfer and internalisation of EV-DNA by recipient cells was demonstrated. Additionally, the co-localisation of EV-DNA with donor cell components like Rab5+- and Rab7+ endosomes as well as transfer of donor EV-DNA to the recipient cell nucleus were observed. In parallel to the aforementioned projects, the evaluation of an EV isolation method that combined two previously published methods of PEG precipitation and SEC was carried out, with hope of optimising a method that would circumvent the need for ultracentrifugation of our starting materials. Since the emergence of disadvantages of UC, several EV isolation methods have been developed - all with their own advantages and disadvantages. Here, EVs isolated by the PEG/SEC method were compared to EVs isolated using UC alone, and analysed for characteristics such as EV yield and nucleic acid content. The work presented in this doctoral thesis has set the foundation for future research projects based in these areas and contributes to better understanding of EVs and their nucleic acid cargo.