Mini Grant: Exosomal Protein Biomarker Profiling of Extant Plasma Samples Acquired from a KLCRP Radon/Tobacco Smoke Exposure Project

Fan/Hahn SPECIFIC AIM Lung cancer is the single most deadly and preventable form of human cancer. Kentucky has the highest lung cancer incidence and mortality rates in the nation. Robust biomarkers are effective means for lung cancer prevention and improved survival. In a collaborative effort of Drs. Ellen Hahn, David Orren, and Teresa Fan, a translational pilot study was initiated to explore biomarkers of exposure to radon and/or TS/SHS, which is funded by Kentucky Lung Cancer Research Program (KLCRP). This research leverages on Drs. Hahn’s, Orren’s, and Fan’s expertise respectively on recruiting healthy homeowners, genotoxicity, and exosomal lipid biomarker discovery for lung cancer to explore synergistic effect of radon and TS on chromosomal aberrations as well as links in plasma exosomal lipid biomarkers for radon/TS exposure and lung cancer. Our preliminary data on 105 total subjects suggest a good power of plasma exosomal lipids in classifying subjects with low radon/low TS (30), high radon/low TS (38), low radon/high TS (29), and high radon/high TS (8). To further improve the power of plasma exosome-based classifiers and query their link to lung cancer biomarkers, we aim to explore plasma exosomal protein profiles in the 4 cohorts of radon/TS exposures in this rapid response grant proposal. Some of these proteins have been shown to be promising diagnostic and prognostic markers of NSCLC. Relevant protein markers can also be used to normalize the lipid data to correct for variations in exosome abundance and plasma contamination in the preparation. Our current multivariate classification analysis is based on normalization of lipid mass intensity by the number of nanoparticles or total protein in the plasma exosome preparations. These normalization methods can be problematic due to the inaccuracy of nanoparticle counting or contamination of non-exosomal plasma proteins. The necessary protein analysis will be carried out according to the following specific aim. Specific Aim 1: To profile a panel of 16 protein targets by Reverse Phase Protein Array We will employ a high throughput Reverse Phase Protein Array (RPPA) method 4,5 for profiling a panel of 16 protein targets. This method calls for delivering sub nanoliters of protein lysates using a protein microarray printer as 1-200 µm spots onto a nitrocellulose pad bonded on the surface of a glass slide. A 16-pad ONCYTE® Film slide (Grace Bio-Labs) can accommodate up to 300 samples per pad and 16 protein targets simultaneously using appropriate antibodies. This method of analyzing specific protein targets is superior to conventional Western blot (WB) technique in speed (days versus months), sample requirement (nL versus µL), and quantitative accuracy. For the small amounts of the exosomal protein lysates that we have and the time constraints, it is not feasible to profile these many protein targets using WB. The information gained from this study can lead to better classification of radon/TS exposure by combining lipids with protein markers and by more accurate normalization of marker abundance. As such, despite the small numbers of subjects surveyed, the data may be publishable and can certainly be used as strong preliminary evidence for an extramural proposal for a larger-scale biomarker study.