Proteomic study of small extracellular vesicle protein biomarker profiles for breast cancer liquid biopsy

Abstract

Breast cancer (BC) is the leading cause of cancer-related death among women worldwide. Currently, the conventional method for diagnosing BC such as mammogram, is not reliable for detecting small lesions or dense breast tissue. Surgical biopsies cannot provide accurate and real-time information due to the complexity of the tumour. Small extracellular vesicles (sEVs) are one of EV subpopulations and secreted by all cell types, containing various biological cargoes that reflect their cellular origin. sEVs are an important intercellular communicator, participating in all stages of cancer metastasis, immunity, and therapeutic resistance. Studying sEVs in liquid biopsy for BC diagnosis is a new developing research area. Therefore, disease specific proteins contained in sEVs are considered as a superior choice for non-invasive liquid biopsy biomarker source in BC. In this thesis, I specifically aim to 1) Establish and optimise a method for isolating sEVs from BC cell lines and human BC plasma samples; 2) Find the most effective approach for proteomic analysis; 3) Identify potential sEV protein biomarkers using BC cells and plasma samples by LC-MS/MS proteomics for BC diagnosis and prognosis. sEVs derived from three BC cell lines (MDA-MB-231, MCF-7, and SK-BR-3), one normal breast cell line (MCF-10A), three BC patients' plasma, and three non-cancer controls were isolated using ultracentrifugation (UC), Total Exosome Isolation kits (TEI), and a combined approach of UC and TEI (UCT). In BC cell lines, the UC isolates showed a higher sEV purity and sEV marker expression, as well as a significantly higher number of sEV proteins. UC isolation identified 10 potential sEV protein biomarkers in BC cell lines. In BC plasma samples, the UCT isolates showed the highest proportion of sEV- related proteins and the lowest percentage of lipoprotein-related proteins. UCT isolates demonstrated 9 potential sEV protein biomarkers in BC plasma. In summary, I have demonstrated that the assessment of both quantity and quality of sEV isolation methods is important in selecting the optimal approach for the specific sEV research purpose depending on the sample types and downstream analysis. In addition, future validation of distinct sEV proteins identified in my study holds potential, for developing new diagnostic approaches in BC liquid biopsy and promoting the application of personalised medicine.