microRNA and non-targeted proteome analysis of liquid biopsies from the distal lung collected by Particles in Exhaled Air (PExA) reveals presence from extracellular vesicles

Abstract

Background. Early detection and longitudinal follow-up are essential for timely diagnosis and treatment for lung diseases. Currently, invasive methods are often required to examine the distal parts of the lungs. The growing need to explore the bio-molecular mechanisms in various lung diseases highlights the importance of non-invasive methods. The use of particles in exhaled air (PExA), a non-invasive technique for sampling of epithelial lining fluid from distal airways, is gaining attention. MicroRNAs (miRNAs) are crucial in modulating protein expression both intracellularly and intercellularly, often transported via extracellular vesicles. Dysregulated miRNAs have been linked to many pulmonary diseases, and their relative stability, especially when encapsulated in EVs, makes them promising biomarkers. Here we report for the first time multi-modal analysis of miRNAs and proteins in PExA, offering an opportunity to study the role of miRNAs in the pathophysiology of respiratory diseases in a non-invasive manner. Methods. Exhaled particles were collected from healthy subjects using the PExA 2.0 instrument utilizing the optimized PExA breathing maneuver. PExA samples collected on different types of impaction membranes were analyzed using a non-targeted mass spectrometry-based proteomics workflow optimized for single-cell detection, and a miRNAseq workflow optimized for low input starting material. Technical validation of a subset of the detected miRNAs was performed using custom-designed miRCURY LNA miRNA PCR assays. Pathway enrichment analyses for the detected proteins were performed using STRING. Results. Proteomic analysis consistently identified over 50 proteins across multiple types of impaction membranes, sample dilution series, and individuals down to a single PEx sPOT (24ng starting material). We observed a significant enrichment of proteins associated with extracellular vesicles, including blood microparticles, and secretory granules. miRNA-seq revealed 39 mature miRNAs, the majority of which have been previously reported to be detected in the airways. Some were also reported to be secreted by primary human airway epithelial cells via extracellular vesicles. miRNA-125b and the members of the let-7 family were among the most abundant miRNAs detected. Fluorometric assays showed significant RNase activity in both PExA and other lung-related samples, such as bronchoalveolar lavage fluid, suggesting that this activity originates from the airways and is independent of the sampling techniques used. The workflow for extraction and processing of the PExA collection membrane, tested with abundant synthetic miRNAs and analyzed using the miRCURY LNA miRNA PCR assay, yielded results comparable to control samples, indicating that the membrane material does not interfere with the assay. Conclusions. Using PExA, we identified several miRNAs reported to be dysregulated in pulmonary disorders. The enrichment of extracellular secretory components in the core protein list, along with the elevated RNAse activity in the respiratory tract, suggest that the detected miRNAs may be encapsulated within extracellular vesicles. These miRNAs are of particular interest due to their potential role in intercellular communication. Our findings suggest that PExA holds a potential as a non-invasive tool for studying extracellular vesicle-mediated miRNA cargo in the small airways.