Controlling glycolysis to generate characteristic volatile organic compounds of lung cancer cells.

Characteristic volatile organic compounds (VOCs) are anticipated to be used for the identification of lung cancer cells. However, to date, consistent biomarkers of VOCs in lung cancer cells have not been obtained through direct comparison between cancer and healthy groups. In this study, we regulated the glycolysis, a common metabolic process in cancer cells, and employed solid phase microextraction gas chromatography mass spectrometry (SPME-GC-MS) combined with untargeted analysis to identify the characteristic VOCs shared by cancer cells. The VOCs released by three types of lung cancer cells (A549, PC-9, NCI-H460) and one normal lung epithelial cell (BEAS-2B) were detected using SPME-GC-MS, both in their resting state and after treatment with glycolysis inhibitors (2-Deoxy-D-glucose, 2-DG/3-Bromopyruvic acid, 3-BrPA). Untargeted analysis methods were employed to compare the VOC profiles between each type of cancer cell and normal cells before and after glycolysis regulation. Our findings revealed that compared to normal cells, the three types of lung cancer cells exhibited three common differential VOCs in their resting state: ethyl propionate, acetoin, and 3-decen-5-one. Furthermore, under glycolysis control, a single common differential VOC-acetoin was identified. Notably, acetoin levels increased by 2.60-3.29-fold in all three lung cancer cell lines upon the application of glycolysis inhibitors while remaining relatively stable in normal cells. To further elucidate the formation mechanism of acetoin, we investigated its production by blocking glutaminolysis. This interdisciplinary approach combining metabolic biochemistry with MS analysis through interventional synthetic VOCs holds great potential for revolutionizing the identification of lung cancer cells and paving the way for novel cytological examination techniques.

Distinguish Esophageal Cancer Cells through VOCs Induced by Methionine Regulation.

Detection of exhaled volatile organic compounds (VOCs) is promising for noninvasive screening of esophageal cancer (EC). Cellular VOC analysis can be used to investigate potential biomarkers. Considering the crucial role of methionine (Met) during cancer development, exploring associated abnormal metabolic phenotypes becomes imperative. In this work, we employed headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) to investigate the volatile metabolic profiles of EC cells (KYSE150) and normal esophageal epithelial cells (HEECs) under a Met regulation strategy. Using untargeted approaches, we analyzed the metabolic VOCs of the two cell types and explored the differential VOCs between them. Subsequently, we utilized targeted approaches to analyze the differential VOCs in both cell types under gradient Met culture conditions. The results revealed that there were five/six differential VOCs between cells under Met-containing/Met-free culture conditions. And the difference in levels of two characteristic VOCs (1-butanol and ethyl 2-methylbutyrate) between the two cell types intensified with the increase of the Met concentration. Notably, this is the first report on VOC analysis of EC cells and the first to consider the effect of Met on volatile metabolic profiles. The present work indicates that EC cells can be distinguished through VOCs induced by Met regulation, which holds promise for providing novel insights into diagnostic strategies.

HS-SPME-GC-MS Untargeted Analysis of Normal Rat Organs Ex Vivo: Differential VOC Discrimination and Fingerprint VOC Identification.

The investigation of volatile organic compounds (VOCs) in human metabolites has been a topic of interest as it holds the potential for the development of non-invasive technologies to screen for organ lesions in vivo. However, it remains unclear whether VOCs differ among healthy organs. Consequently, a study was conducted to analyze VOCs in ex vivo organ tissues obtained from 16 Wistar rats, comprising 12 different organs. The VOCs released from each organ tissue were detected by the headspace-solid phase microextraction-gas chromatography-mass spectrometry technique. In the untargeted analysis of 147 chromatographic peaks, the differential volatiles of rat organs were explored based on the Mann-Whitney U test and fold change (FC > 2.0) compared with other organs. It was found that there were differential VOCs in seven organs. A discussion on the possible metabolic pathways and related biomarkers of organ differential VOCs was conducted. Based on the orthogonal partial least squares discriminant analysis and receiver operating characteristic curve, we found that differential VOCs in the liver, cecum, spleen, and kidney can be used as the unique identification of the corresponding organ. In this study, differential VOCs of organs in rats were systematically reported for the first time. Profiles of VOCs produced by healthy organs can serve as a reference or baseline that may indicate the presence of disease or abnormalities in the organ's function. Differential VOCs can be used as the fingerprint of organs, and future integration with metabolic research may contribute to the development of healthcare.

Analysis of volatile organic compounds in exhaled breath after radiotherapy.

Radiotherapy uses high-energy X-rays or other particles to destroy cancer cells and medical practitioners have used this approach extensively for cancer treatment (Hachadorian et al., 2020). However, it is accompanied by risks because it seriously harms normal cells while killing cancer cells. The side effects can lower cancer patients' quality of life and are very unpredictable due to individual differences (Bentzen, 2006). Therefore, it is essential to assess a patient's body damage after radiotherapy to formulate an individualized recovery treatment plan. Exhaled volatile organic compounds (VOCs) can be changed by radiotherapy and thus used for medical diagnosis (Vaks et al., 2012). During treatment, high-energy X-rays can induce apoptosis; meanwhile, cell membranes are damaged due to lipid peroxidation, converting unsaturated fatty acids into volatile metabolites (Losada-Barreiro and Bravo-Díaz, 2017). At the same time, radiotherapy oxidizes water, resulting in reactive oxygen species (ROS) that can increase the epithelial permeability of pulmonary alveoli, enabling the respiratory system to exhale volatile metabolites (Davidovich et al., 2013; Popa et al., 2020). These exhaled VOCs can be used to monitor body damage caused by radiotherapy.

Distinguish oral-source VOCs and control their potential impact on breath biomarkers.

By means of glass bottle sampling followed by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) technique, the change characteristics of volatile organic compounds (VOCs) in breaths, between before gargling and after gargling, were investigated, respectively, in 41 healthy subjects and 50 esophageal cancer patients. Using an untargeted strategy, 143 VOC chromatographic peaks were enrolled in the statistical analysis. Based on the orthogonal partial least squares discriminant analysis (OPLS-DA), the VOC variations after gargling for each breath test group were obtained according to the combined criteria of variable importance in projection (VIP > 1.5), Wilcoxon signed-rank test (P < 0.05), and fold change (FC > 2.0). When gargled, the levels of indole, phenol, 1-propanol, and p-cresol in the breath of healthy people decreased; meanwhile, for esophageal cancer patients, the declined VOCs in breath were indole, phenol, dimethyl disulfide, and p-cresol. Particularly, these substances were previously reported as breath biomarkers in some diseases such as esophageal, gastric, thyroid, breast, oral, and lung cancers, as well as certain non-cancer disorders. The present work indicates that expiratory VOCs involve the prominent oral cavity source, and in the breath biomarkers study, the potential impact that originates from oral volatiles should be considered. In view of the present results, it is also proposed that gargle pretreatment could eliminate possible interference from the oral cavity VOCs that might benefit breath biomarker investigation. Gargle pretreatment helps to distinguish oral-source VOCs and control their potential impact on breath biomarkers.

Variable VOCs in plastic culture flasks and their potential impact on cell volatile biomarkers.

In order to find out cancer markers in human breath, in vitro cell culture is often used to study the characteristic volatile organic compounds (VOCs). In the cell culture process, disposable vessels are frequently adopted. However, these vessels are normally made of plastic, and they have the possibility to release some VOCs, which may interfere with the cell-specific volatiles and even can result in an incorrect conclusion. In this study, by using glass cell culture flasks as control, the headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analyses of the VOCs in plastic cell culture flasks were systematically carried out for the first time. A total of 35 VOCs were detected in five brands of flasks. In each flask, there were between 13 and 25 volatile compounds. Furthermore, the components and packaging bag of each flask were also sampled and analyzed by HS-SPME-GC-MS. The results show that the flask cap, septum, flask body, and packaging bag exhibit respectively different volatile behaviors. The former two parts release the most volatiles which have obvious contributions to the headspace gases in the flasks, while the flask body mainly liberates styrene. For different flasks packed within the same bag, the headspace analyses show that their residual VOCs are inconsistent with each other. Moreover, the residual VOCs in the same flask are variable in three consecutive days. These results indicate that the multiple flasks in parallel cell culture experiments, or the same flask with different cell culture durations, will produce an indelible disturbance to the cell-specific VOCs. In addition, among the 35 VOCs detectable in five brands of empty plastic flasks, 15 VOCs were previously reported as characteristic VOCs from lung cancer, melanoma, cervical cancer cells, or normal cells. This is an alert that, when using plastic flasks, it must be careful to treat the possible interference from the background VOCs in the flasks. This study demonstrates that the cell culture tool needs to be standardized, and the clean glass or metal vessels are strongly recommended for usage when studying cell volatile biomarkers. Graphical abstract.