EVolving the Liquid Biopsy: Extracellular Vesicles for Assessing Liver Function and Disease

Author: Lauren Newman

Newman, Lauren, 2024 EVolving the Liquid Biopsy: Extracellular Vesicles for Assessing Liver Function and Disease, Flinders University, College of Medicine and Public Health

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Liver cells release extracellular vesicles (EVs) carrying diverse molecular information involved in homeostatic processes and the pathogenesis of metabolic associated fatty liver disease (MAFLD). Applying a liver-specific immunocapture method, this thesis explores the utility of circulating liver derived EV biomarkers to convey the impact of MAFLD on hepatic function and makes vital contributions to this approach for clinical sample analysis. Whereas solid tissue biopsy is preclusively invasive, EVs are an appealing tool to assess organ function and inform disease diagnosis, prognosis and predicted response to interventions via a minimally-invasive liquid biopsy; however, several challenges in EV isolation and analysis currently limits clinical translation.

EVs in biofluids, including blood, exist among a complex milieu of non-vesicular material that impacts the specific recovery and detection of target molecules. Enrichment of EV marker proteins and depletion of co-isolated matrix contaminants reflect the yield and purity of EV isolates to provide critical assessments of sample quality. Here, development and validation of a novel targeted proteomic assay is described that achieves sensitive, multiplexed, high-throughput and absolute quantification of EV markers and contaminants. Importantly for clinical studies, these improvements compared to standard protein detection methods, accommodates quality control on an individual sample level in limited sample volumes. The impact of donor characteristics and physiological variability, including diurnal, prandial and sex differences, on circulating EV abundance and cargo is investigated to establish basal ranges in global and liver-specific EVs. Increased abundance of circulating liver-specific EVs during the day highlights an important consideration for study protocols sampling liver-derived EV biomarkers.

EVs released by the liver constitute a minor fraction of the heterogenous population in the systemic circulation and methods for EV isolation poorly distinguish subpopulations containing markers of interest. Thus, advanced isolation methods targeted towards the tissue of origin are expected to improve the sensitivity and specificity of low abundance disease-associated biomarkers, including microRNA. Expression of liver-enriched miRNA with reported dysregulation in NAFLD (miR -122, -192, -128-3p) were compared in total plasma, global EVs and liver-specific EVs in mild and severe disease and healthy controls. Increased expression of each miRNA is associated with disease in liver-specific EVs but not the other less specific sources. Although liver-derived EVs carry a minor proportion of total plasma miRNA, this proportion increases significantly in disease. The results suggest changes are specifically occurring in the EV export of miRNA from hepatic cells and highlight the importance of removing background (non-EV and non-liver) signal for improved diagnostic performance.

Patients with MAFLD exhibit variability in exposure and response to medications resulting from differences in abundance of hepatic drug metabolising enzymes (DMEs) and drug target proteins. Key DMEs and MAFLD drug targets were readily detected and quantified in EVs isolated from liver tissue, supporting potential for in vivo monitoring of interactions of drugs with the liver. Moderate-strong correlation (r>0.6) of 13 from 14 DMEs between EVs and paired tissue supports utility for reporting on between-subject variability. The work described in this thesis advances the analytical framework for applications of liver-specific EV liquid biopsy in clinical samples and contributes to best practice in characterisation and reporting aligned with international standards in the field. These contributions lay the foundation for future development of EV-based precision medicine strategies for MAFLD and beyond.

Keywords: extracellular vesicles, liquid biopsy, biomarkers, chronic liver disease, proteomics

Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2024
School: College of Medicine and Public Health
Supervisor: Andrew Rowland