Extracellular vesicles in chronic lymphocytic leukaemia: exploration of their miRNA cargo as biomarkers

The lymph node microenvironment provides essential signals for the proliferation and survival of chronic lymphocytic leukaemia (CLL) cells and contributes to resistance to chemotherapy. There is no readily available access to lymph node tissue and currently no markers of leukaemic cell activity specifically due to stimulation within lymph nodes. Extracellular vesicles (EVs) are produced by CLL cells and their cargo, which includes miRNA, mRNA and proteins, is important for intercellular signalling. Following CD40L/IL-4 stimulation EVs are enriched in miR-363-3p and miR-374b. These miRNA are only detectable at lower levels in plasma from normal subjects and in this thesis the idea that microRNAs might be predictive biomarkers reflecting leukaemic activity in the tumour microenvironment was investigated. To pursue the hypothesis that plasma levels of miR-363 might correlate with disease activity I established real-time PCR assays and showed that patients had higher miR-363 levels than normal subjects and confirmed this result in a repository study using samples from patients in the ARCTIC and CLEAR clinical trials. There were no associations between miR-363 levels and prognostic markers. Numbers of EVs, measured by dynamic light scattering are higher in CLL patients than normal subjects. To examine the source of circulating miRNA size exclusion chromatography was carried out followed by real-time PCR and showed that circulating miR-363 was derived from both plasma and particle bound fractions in healthy subjects but in patients a greater proportion was found in the particle fractions. Finally, I investigated the function of miR-363 in CLL. In contrast to T-cells miR-363 did not appear to have effects on the expression or function of CD69 in CLL B-cells. Overall, numbers of EVs and miR-363 levels associate with CLL but not with survival. An observation, which may have implications for identifying disease associated miRNA and can be followed up is that there appears to be a disease specific distribution of circulating miR-363 between plasma protein and particle bound fractions.