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.