An Exploration Of Small Extracellular Vesicle Nucleic Acid Cargo As A Liquid Biopsy Analyte For Breast Cancer

Small extracellular vesicles (sEVs) are lipid membrane vesicles, secreted by almost all cell types and in cancer may be involved in modifying the tumour microenvironment and promoting tumour progression. In addition, sEV release is elevated in cancer and as such, their cargo is under investigation as a liquid biopsy analyte in patients with cancer. The aim of this thesis was to compare methods for the isolation of sEVs from limited blood plasma volumes and to explore their DNA and RNA cargo in comparison with circulating tumour DNA (ctDNA) towards developing a multi analyte liquid biopsy (MALBA) test.

Two commercial kits ExoEasy (QIAGEN) and exoGAG (Nasasbiotech) were explored using sEVs from in vitro cell culture models; exoGAG gave a higher recovery. The cell models also illustrated the uptake and retention of cell line specific sEVs DNA and RNA. In 20 patients with breast cancer (BC) somatic copy number alterations were analysed by shallow whole genome sequencing (sWGS) in sEV DNA and matched cfDNA. ichorCNA analysis of sWGS data, showed a higher mean tumour fraction in DNase I treated sEV DNA (34.1%) than matched cfDNA (31.2%). In 2 HER2 negative patients ERBB2 amplification was detected in sEV DNA that could have guided treatment. Candidate RNAs identified from TCGA data, showed no difference between cancer and healthy derived sEVs. However, in 5 metastatic patients, sEV transcriptomics revealed 248 differently expressed genes compared to healthy controls, including ones implicated in the endosomal pathway and miRNA processing – ITSN1, AGAP1, PUS10. Pathway analysis showed a significant number of differentially expressed markers associated with the regulation of cellular processes (p = 1.368e-3). In conclusion, results presented in the thesis show that sEVs harbour DNA and RNA cargo and can provide additional information to plasma cfDNA warranting further investigation of sEV cargo as a liquid biopsy analyte in a larger series of patients.