Diagnostic, prognostic and therapeutic applications of extracellular vesicles in chronic obstructive pulmonary disease and lung cancer

Abstract

Chronic obstructive pulmonary disease (COPD) and lung cancer comprise the leading causes of lung disease-related mortality worldwide, with a common and preventable aetiological risk factor, smoking. There is a need for novel biomarkers to identify high risk populations and improve therapeutic strategies for these lung diseases.Extracellular vesicles (EVs) are nano-sized lipid bilayer membrane vesicles, secreted by normal and diseased cells, that play a vital role in intercellular communication through the exchange of their bioactive cargo (e.g. nucleic acids and small molecules) with recipient cells. EVs are highly stable in circulating bodily fluids, carrying disease-specific bioactive cargo reflective of their parental cell, making them attractive biomarkers for lung disease, as well as vehicles for novel therapeutics.The objectives of this PhD were to investigate novel diagnostic, prognostic and therapeutic applications of EVs and their packaged bioactive cargo (miRNAs, microbial DNA and small molecules) in cigarette smoking-related lung diseases (COPD and lung cancer).Methods5 main studies were undertaken in this PhD:1. Assessment of plasma EV miRNAs (Chapter 2) using qPCR and their dysregulation in 80 participants including healthy controls, healthy smokers, patients with stable COPD and patients with lung cancer.2. a) Assessment of plasma EV miRNAs (Chapter 3 – Part 1) using qPCR and their dysregulation in 20 COPD patients during an exacerbation event and 20 COPD patients during stable state.b) Assessment of sputum EV bacterial derived cargo (Chapter 3 – Part 2) for abundance and biodiversity using 16S rRNA sequencing in 5 stable COPD and 5 exacerbating COPD patients.3. Assessment of EVs containing bacteria-derived molecular cargo (Chapter 4) using 16S rRNA sequencing in lung tissue from lung cancer patients, with or without COPD. Bacterial abundance and biodiversity were analysed by sample type.4. Assessment of e-cigarette toxicity (Chapter 5 – Part 1) in bronchial epithelial cells (BECs) cultured and differentiated at an air-liquid interface (3D model), from patients with COPD who underwent lung cancer resection surgery. Bioassays measured cell cytotoxicity, DNA damage and inflammatory responses. EVs post-direct aerosol exposures were assessed (Chapter 5 – Part 2) for ability to induce cell cytotoxicity endpoints in unexposed BECs.5. Assessment of cytotoxicity induced by probiotic bacteria and their EVs (Chapter 6) compared to pathogenic bacteria and their EVs, as well as LPS in BECs cultured 2D.ResultsThe 5 studies identified the respective key findings:1. Significantly dysregulated plasma EV miRNAs were able to discriminate between healthy smokers, lung cancer and stable COPD patients.2. a) Significantly dysregulated plasma EV miRNAs were able to discriminate between COPD states (exacerbation and stable state).b) Unique bacterial species DNA in sputum EVs was also able to distinguish between exacerbation and stable COPD states.3. Distinct microbiota in lung EVs may be contributing to lung dysbiosis, disease pathogenesis, and progression.4. a) Demonstration that in BECs from patients with COPD, the cytotoxicity, DNA damage and inflammatory responses to e-cigarette aerosols were comparable to those elicited by cigarette smoke.b) EVs appear to act as vehicles for the transfer of aerosol exposure toxicity into recipient un-exposed BECs.5. Although probiotic bacterial EVs have the potential to deliver ‘protective’ cargo, these EVs elicited cytotoxicity in BECs, therefore further purification or modification may be needed for therapeutic application.ConclusionsEVs hold significant biomarker potential and novel therapeutic application in lung disease, for their ability to transport disease-specific bioactive cargo, reflective of parental cells, which remains highly stable in bodily fluids.Firstly, studies from this PhD support that EV bioactive cargo (miRNA) within plasma holds disease-specific information that can distinguish between lung disease states, with strong diagnostic and prognostic biomarker potential, which warrants further investigation for their translational application.Secondly, EVs containing bioactive cargo (microbial) unique to disease states highlights their biomarker potential (exacerbation vs. stable COPD), as well as novel therapeutic approaches (probiotic bacteria EVs) in mitigating lung dysbiosis. Further, EVs containing tumour specific microbial cargo have potential clinical utility as novel screening biomarkers to distinguish patients at high risk for cancer development and progression. Thirdly, e-cigarettes may not be a ‘safer’ smoking cessation alternative to cigarette smoking for smokers with pre-existing lung disease. Furthermore, EVs appeared to act as vehicles that transferred aerosol toxicity.Overall, this thesis has clearly demonstrated innovative applications of EVs in lung disease, for use in the identification of potential clinically useful diagnostic and prognostic biomarkers, and therapeutic applications.