Gas chromatographic-mass spectrometry analysis of volatile organic compounds from cancer cell cultures - The effect of hypoxia

Early diagnosis of lung cancer improves patient outcomes which has led to a search for non-invasive diagnostic tests suitable for population screening. Volatile organic compounds (VOCs) in exhaled breath have shown potential, however, confirmation of the metabolic origins and disease specificity of candidate markers is required. Cell culture metabolomics can identify disease biomarkers and their origins. To date VOC profiles from in vitro cultured cancer cells have little similarity to cancer breath VOC profiles. In vivo, cancer cells experience hypoxia whereas in vitro cells are cultured under normoxic conditions. Since hypoxia influences cell metabolism, we hypothesize that cancer cells cultured under hypoxic conditions will have altered cell metabolism and produce VOC profiles more typical of cancer breathe. This study investigates the effect of hypoxia on metabolic reprogramming in A549 lung cancer cells cultured under standard normoxic (atmospheric oxygen) or hypoxic (2% oxygen) conditions. Results from quantitative RT-PCR demonstrated a significant upregulation in hypoxia of the glucose transporter (GLUT1) and the key TCA regulatory gene PDHK1, demonstrating that hypoxia plays a pivotal role in regulating metabolism in A549 cells. A ratio-metric assessment of Lipid Peroxidation (LPO) and the production of reactive oxygen species (ROS) showed an increase in LPO and a slight decrease in the production of ROS in hypoxic cultures, the combined effect of which may serve to equip the cells to adapt to and proliferate under low oxygen. Finally, the comparison of endogenous VOCs produced by A549 cells under hypoxic and normoxic conditions identified twelve VOCs unique to cells grown under hypoxic conditions including n-pentane, a marker of LPO and cancer, and 3-methyl hexane, which has been reported as a biomarker of cancer. This data is consistent with the hypothesis that a hypoxic tumour microenvironment may influence cell metabolism leading to a unique and diagnostic cancer VOC profile.Doctor of Philosoph

Cell culture metabolomics in the diagnosis of lung cancer - The influence of cell culture conditions

Lung cancer is the leading cause of cancer deaths. Unfortunately, lung cancer is often diagnosed only when it becomes symptomatic or at an advanced stage when few treatment options are available. Hence, a diagnostic test suitable for screening widespread populations is required to enable earlier diagnosis. Analysis of exhaled breath provides a non-invasive method for early detection of lung cancer. Analysis of volatile organic compounds (VOCs) by various mass spectral techniques has identified potential biomarkers of disease. Nevertheless, the metabolic origins and the disease specificity of VOCs need further elucidation. Cell culture metabolomics can be used as a bottom-up approach to identify biomarkers of pathological conditions and can also be used to study the metabolic pathways that produce such compounds. This paper summarizes the current knowledge of lung cancer biomarkers in exhaled breath and emphasizes the critical role of cell culture conditions in determining the VOCs produced in vitro. Hypoxic culture conditions more closely mimic the conditions of cancer cell growth in vivo. We propose that since hypoxia influences cell metabolism and so potentially the VOCs that the cancer cells produce, the cell culture metabolomics projects should consider culturing cancer cells in hypoxic conditions