Ulia Lopatin

The ability to acquire biomarkers from patients with lung diseases, such as lung cancer and chronic obstructive pulmonary disease, currently relies on lung biopsies. Lung biopsies are invasive and pose an increased risk to the patient. Hence, there is a critical need to develop a minimally-invasive method for sampling lung biomarkers. Biofluids such as plasma, urine, and saliva are easy to collect and the collection methods are minimally invasive. If lung biomarkers could be sampled from a patient with increased frequency, it would enable one to monitor the progression of lung disease and the response to treatment over time. This study aims to develop a method for isolating lung-derived extracellular vesicles (EVs) from biofluids to identify biomarkers. EVs contain cell-type specific macromolecules, giving insight into tissue specific cargo. The lung protein target for detecting the lung EVs, RAGE, was determined from tissue-specific RNA expression data. A dot blot was performed with a variety of cell lysates to assess the tissue specificity of the antibody used for lung enrichment. The presence of RAGE protein in the extracellular binding domain of EVs was confirmed through a NanoView assay. A western blot was performed to verify enrichment; this showed that the captured product was positive for flotillin, an EV marker. RNA was isolated from the RAGE positive EVs and sequenced to observe differential gene expression. Although our blots did not show RAGE in our enriched fraction, it was found that the RAGE capture enriched for several alveolar type 1 cell enriched genes and two lung enriched genes across replicates. While the developed method was able to capture EVs and demonstrate enrichment for five lung genes, the capture method should be further optimized and the experiment should be repeated to determine whether the captured EVs are suitably lung-enriched.