In vitro analysis of volatile organic compounds in search of potential biomarkers of lung cancer
Lung cancer is a leading cause of death from cancer worldwide. An early diagnosis andappropriate treatment are crucial in reducing mortality among people suffering from thedisease. Therefore, one of the main focuses of lung cancer studies is on advances in itsearly detection. One of the most promising is the analysis of volatile organic compounds(VOCs). VOCs are a diverse group of carbon-based chemicals that are present in exhaledbreath and biofluids, and may be collected from the headspace of these matrices. Differentpatterns of VOCs have been correlated with various diseases, cancer among them. Studieshave shown that various cancer cells in vitro produce or consume specific VOCs that can serve as potential biomarkers to differentiate them from non-cancer cells. The present study aimed at the detection, identification and semi-quantification of VOCsreleased or consumed by the adenocarcinoma human alveolar A549 cell line. For thispurpose, gas chromatography with mass spectrometric detection was combined with twopre-concentration techniques: monolithic material sorptive extraction (MMSE) orextraction of thermal desorption (TD) sorbent tubes with an Easy-VOCTM pump as asample loading tool. MMSE is a new technique for the extraction of VOCs from varioussamples and it is used for the first time for the analysis of VOCs from cell culturemedium. TD-GC-MS is a popular technique for VOCs analysis and it has been used forthe first time here with Bio-VOCTM pump in the studies of VOCs in vitro. The project alsoaimed at the comparison of the A549 VOC level trends to the trends of the normal humanlung fibroblasts NHLF (MMSE experiment) and normal human bronchial BEAS-2B cells(TD experiment). In addition, the VOC patterns between the growing and confluent cellsof the same cell line were compared for the first time. In the MMSE experiment, seven VOCs were produced and 14 VOCs metabolisedexclusively by the cancer cells. Among the released compounds were methylatedhydrocarbons (2,4-dimethyl-1-heptene; 4-methylundecane; 2,3,6,7-tetramethyloctane2,3,5-trimethylhexane and 2,3,5-trimethyldecane) and alcohols (cyclohexanol and 3-heptanol). The metabolised analytes were alcohols (4-decanol; 6-dodecanol; 2-ethylhexanol; 1-octanol), aldehydes (dodecanal; tetradecanal), ketones (acetophenone;cyclohexanone; 2-tetradecanone), phenols (phenol and 2-nitrophenol); an ether (2-methoxydiphenylmethane), an ester (pentanoic acid, 2,4-dimethyl-3-oxo-, methyl ester)and a hydrocarbon (tetradecane). 2,4-Dimethylheptane; 2,6-di-tert-butyl-1,4-benzoquinone; 1-phenylethanol and 2-pentadecanone were released by both A549 andNHLF cell lines. The cancer cells were observed, however, to emit the VOCs at a higherlevel than the fibroblasts. Benzaldehyde; 2-ethylhexanol; hexanal and 1-nonanol werefound to be consumed by both the cancer and NHLF cells, however, at a greater rate bythe former. In the TD experiment, 2,3,5-trimethylhexane and tert-butanol were producedexclusively by the A549 cells, while ethyl acetate solely by the BEAS-2B cells. Acetophenone, benzaldehyde and 2-methylbutanal were metabolised and acetone wasproduced at higher levels by the cancer cells than by the BEAS-2B cells. The possible use of the analysed VOCs as potential biomarkers of lung cancer isdiscussed, along with the suggestion and discussion of the possible metabolic pathwaysleading to the uptake and release of these VOCs by the analysed cells. Also the discussionof poor correlation between different in vitro studies, as well as between in vivo and in vitro studies of VOCs as potential biomarkers of cancer, is undertaken.
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