# Application of Thin-Film Microextraction to Analyze Volatile Metabolites in A549 Cancer Cells
## 2 Results and Discussion
### VOCs Released from A549 Cancer Cells  
Four Petri dishes were seeded with 250,000 A549 cells and cultivated over 4 days. After this time, viable cell count was checked with the Trypan blue dye exclusion method using an automatic cell counter (in triplicate for each biological replicate). The calculated cell loads were within the range of 1.83 \(\times\) 10\({}^{6}\)-2.16 \(\times\) 10\({}^{6}\) cells/mL, while relative standard deviations ranged from 3.6% to 5.4%. For the untargeted analysis of volatile metabolites produced by A549 cancer cells, the previously optimized protocol was used with extraction on HLB meshes in \"internal-headspace\" sampling mode at elevated temperature of 37 \({}^{\circ}\)C for over 90 min. Out of the 40 VOCs unambiguously identified by confirmation of retention time of standards, in addition to the NIST Mass Spectra Library Search, six metabolites were observed at statistically different levels in the headspace of the cell culture medium compared to the cell-free medium controls at levels above their respective Quantitation Limit. Carbon disulfide, 2,3-butanedione, pyrrole, pyridine and 2-methylpyrazine were found at higher levels, whereas benzaldehyde was the only VOC observed at a decreased level in cell culture medium (Figure 5). The concentrations of the mentioned VOCs measured in both sample types, along with calibration parameters, are given in the Table 1. Our findings are consistent with the literature (Table 2), as the diminishing profile of benzaldehyde was previously reported in studies with 2D and 3D cultures of the A549 cell line [17]. Moreover, this compound exhibited decreasing profile also in other cell lines, such as fHB, HUVEC, HDF, NCM460, HepGG2, RGP, VGP, Mm, SW1116, HeLa, SW480, compared to the respective control medium samples thoroughly reviewed in [20]. Uptake of aldehydes by cancer cells may be related to the increased activity of aldehyde dehydrogenase, which, amongst other biological functions, oxidizes the cytotoxic aldehydes into carboxylic acids. Intriguingly, [PERSON] et al. [21] indicated that only a certain number out of possible 19 isoforms of aldehyde dehydrogenase are active in particular tumor types. In turn, alcohols and ketones may originate from oxidation of alkanes by Cytochrome P450 oxidase enzymes, the activation of which in transformed cell lines was discussed by [PERSON] [22]. While the origin of straight chained alkanes is related to the free radical peroxidation of polyunsaturated fatty acids (PUFA) [23], there are justified doubts that this pathway can lead to the production of methylated hydrocarbons [24]. Two out of six mentioned compounds were found to be significant in other in vitro studies focused on VOCs analysis in A549 cells [10; 15; 16; 17; 18], and four compounds were significant in ex vivo studies with cancerous lung tissues [8; 19]. On the other hand, carbon disulfide and methylpyrazine were not reported for A549 cells nor for tumor tissues. Some compounds have consistent profiles amongst studies, such as decreasing levels of benzaldehyde, while other compounds do not exhibit such consistency. The reason for the observed divergence is currently unknown, but it may be related to different growth conditions, _inter alia_, adhesive cultures on the Petri dish in the RPMI medium vs. bottled 3D suspension in the DMEM medium, or different availability of oxygen, which influences the activity of enzymes and free radical oxidation. Relatively small number of analytes with statistical significance may be related to some extent to the data acquisition mode, where a single-quadrupole mass spectrometer operating in full scan mode was used to cover the widest range of \(m/z\) for appropriate untargeted analysis and identification of unknown analytes, instead of the selected ion monitoring (SIM) mode which drastically improves the sensitivity of MS analyses but focuses only on chosen \(m/z\) values.