Biomarker Discovery by Interrogating Lung Cancer Lipid Metabolome

Kentucky has the highest lung cancer incidence and mortality in the nation, with the Appalachian population of special concern where mortality rates are even higher than in Kentucky as a whole. The 5-year survival rates for advanced stage lung cancer are very poor (50%). Unfortunately NSCLC is typically asymptomatic, and there are no routine, high specificity low cost screening methods available for early detection in individuals at risk; a reliable screening method applicable to remote populations at risk, such as in Appalachia is urgently needed. It has been shown that the number of lipid microparticles (MP such as microvesicles and exosomes) circulating in the blood increases in patients with lung cancer or other malignancies. These MP are derived from endosomal membranes of various cells from different tissues, and are important in interorgan communication such as immunomodulation and tumor progression. In preliminary work, we have shown that the lipid composition of plasma MP are distinct among healthy subjects, those with early stage non-small cell lung cancer (NSCLC), and individuals with breast cancer. We seek to extend this work to the Appalachian population to determine the mechanistic link between lung cancer tissues and plasma MP and to discern a robust set of lipid components that discriminate early stage NSCLC from healthy subjects and subjects with other diseases. The use of ultra-high resolution mass spectrometry and informatics package we have pioneered makes such a quest possible for the first time. These findings will be used to develop a simple blood-based screening tool that utilizes the complete lipid profile of circulating MP, for at-risk populations for lung cancer, including those in the Appalachia. The markers will comprise a panel of multiple lipid species, which has a much greater sensitivity and specificity than a single or a few lipid biomarker species. We will achieve our objectives by fulfilling the following specific aims. SA1 - To discern microvesicle/exosome lipid components released by paired non-cancerous & cancerous lung tissue slice cultures. Paired NC and CA lung tissue slices (obtained from surgical patients recruited by Lane’s P01 project) will be cultured in 13C6-glucose so that the lipid marker(s) that are derived from CA lung tissues and incorporated into MP that are released can be tracked based on the 13C labeled lipid patterns. Such detailed metabolic interrogation of the lung tissue slices is unique and will be instrumental in establishing a mechanistic link of lung cancer metabolism to plasma MP lipid markers acquired in SA3 as it directly measures tumor tissue derived lipid components under very well defined conditions. SA3 - To characterize plasma microvesicle/exosomal lipids of lung cancer patients & non-lung cancer subjects for discerning lung cancer markers. We will obtain plasma MP and analyze their lipid profiles by FT-MS from 100 NSCLC subjects. These profiles will be compared with those from healthy non-smokers and smokers (already acquired from our previous study) for distinct patterns. Further comparisons will be made using data obtained from subjects with chronic obstructive pulmonary disease (COPD) but not lung cancer and from breast cancer patients in a separate study to establish specificity. Furthermore we will compare the lipid profile of plasma MP from cancer subjects with those released from cultured CA lung tissue slices, in order to define the plasma MP lipids originated from CA lung tissues. This should further substantiate the specificity while enhancing the sensitivity of the plasma lipid profile as lung cancer markers. If successful, the data generated from this pilot proposal will serve as preliminary work for a multi-investigator R01 proposal and/or a lung spore proposal to NCI to validate the biomarker patterns uncovered, and to explore the biological function(s) of exosomal lipid markers in lung tumor development.