Isolation methodology is essential to the evaluation of the extracellular vesicle component of the senescence-associated secretory phenotype.

PhD ThesisSenescence is a state of proliferative arrest that is triggered through a range of harmful stimuli. Senescent cells are associated with the acquisition of an enhanced secretory profile known as the senescence-associated secretory phenotype (SASP), which has been demonstrated to confer a secondary senescence response upon neighbouring proliferating cells, in a process known as paracrine senescence. This work aims to characterise the contribution of a group of small lipid bilayer-bound particles, known as extracellular vesicles (EVs), to the induction of paracrine senescence, with a focus on emphasising the limitations of widely applied EV isolation methodologies in a senescence context. First, models of oncogene-induced (OIS) and replicative (RS) senescence were established in IMR90 and adult human mammary fibroblasts respectively, with phenotypic characterisation performed via a high content analysis (HCA) approach. EVs were then isolated from these models via differential ultracentrifugation (dUC), with increased EV release identified following senescence induction in both settings. Proteomic profiling via mass spectrometry (MS) indicated that soluble components of the SASP co-isolate during dUC based procedures, hindering the utility of this widely used technique within senescence research. Size-exclusion chromatography (SEC) was established to overcome this limitation, facilitating a further, more comprehensive assessment of OIS derived EVs via MS. Furthermore, SEC allowed demonstration that EVs isolated from OIS fibroblasts mediate the induction of paracrine senescence in both proliferating fibroblasts and breast cancer cells. Finally, in order to probe the mechanisms underlying these responses, potential mechanistic leads (NOTCH1 and ADAM10/17) were identified from EV proteomic profiles and validated to be constituents of 5 OIS derived EVs. The functional importance of these targets in paracrine senescence was also investigated. Overall, this work emphasises that EVs are distinct and functionally important mediators within the SASP, but that their investigation in a senescence setting requires careful attention to isolation rigor and stringency