Release of volatile organic compounds (VOCs) from the lung cancer cell line CALU-1 in vitro

As lung cancer cell line we tested the human, epithelial cell line CALU-1 which has been derived from a lung squamous cell carcinoma. This cell line builds numerous microvilli, a prominent rough endoplasmatic reticulum, lysosomes, and lipid inclusions. Furthermore, CALU-1 cells express a mutant K-ras gene. They are grown in DMEM high glucose (4.5 g/L) culture medium containing pyruvate (PAA) and supplemented with 10% FCS, penicillin (100 000 units/L), streptomycin (100 mg/L) and L-glutamine (293 mg/L). Cells were cultivated under standard conditions in a conventional incubator at 37°C in a humidified atmosphere with 92.5% air/7.5% CO₂. For VOC measurements 50 millions trypsinized cells were inoculated in 100 ml phenol red free medium (DMEM high glucose) in a cell culture fermenter, flushed with clean, synthetic air taken from a gas cylinder (50 L defined gas mixture, Linde, Stadl-Paura, Austria) containing 5% CO₂ for at least 10 min at a flow rate of 100 ml/min and sealed for 4 to 18 h. At the end of the incubation time 200 ml of air from the headspace was used for GC-MS analyses. Cell viability counts (trypan blue exclusion method) were performed at the end of the incubation period after.

Glass tubes (Gerstel, Mülheim an der Ruhr, Germany) filled with the following sorbents were used as traps for sample collection with simultaneous preconcentration: 25 mg Tenax TA (60/80 mesh), 35 mg Carboxen 569 (20/45 mesh), 250 mg Carboxen 1000 (80/100 mesh) (each from Supelco, Bellefonte, PA, USA). Sorbents were separated by glass wool. The sampled air was diluted 1:6 with dry and additionally purified synthetic air to avoid excess humidity for thermodesorption. The volume of collected sample originating from the fermenter was 200 ml with a total flow through sorption trap of 30 ml/min.

The sampled analytes were released from sorbents by thermal desorption in the TDS3 unit equipped with a TDSA2 auto sampler (both from Gerstel, Mülheim an der Ruhr, Germany). The flow rate of carrier gas through the sorption trap during desorption was 90 ml/min. The initial temperature was 30°C and was increased to 300°C by a heating rate of 100°C/min (held for 10 min). Liquid nitrogen was used for cryofocusing the desorbed analytes (-90°C). For subsequent sample injection into the capillary column the CIS-4 injector which contained the glass liner filled with Carbotrap B (Gerstel, Mülheim an der Ruhr, Germany) was heated with the rate 12°C/sec up to 320°C (hold 2 min in splitless mode).

The TD-GC-MS analyses were performed on a 6890N gas chromatograph equipped with a mass selective detector 5973N (both from Agilent Technologies, Waldbronn, Germany) with sample injection by means of thermal desorption (described in previous sections). The MS analyses were performed in a full scan mode, with a scan range of 20–200 amu. Ionization of the separated compounds was done by electron impact ionization at 70eV. The acquisition of the chromatographic data was performed by means of the Agilent Chemstation Software (GC-MS Data Analysis from Agilent, Waldbronn, Germany) and the mass spectrum library NIST 2005 (Gatesburg, USA) was applied for identification. The PoraBond Q capillary column 25 m × 0,32 mm × 5 μm (Varian, Palo Alto, CA, USA) was used. The oven temperature program was as follows: initial 50°C held for 5 min, then ramped 5°C/min up to 140°C held 5 min, again ramped 5°C/min to 280°C and held for 4 min. The constant flow rate of helium as a carrier gas was 2 ml/min.

2-Butanone, acrolein, methacrolein, 2-ethylacrolein, 2-methylpropanal, 3-methylbutanal, 2-methylbutanal, 2-methyl-2-butenal, hexanal, n-butyl acetate, methyl tert-butyl ether, ethyl tert-butyl ether, 2,4-dimethylpentane, 2,3,4-trimethylpentane, decane and tetrahydrofuran were each purchased from Sigma-Aldrich (Sigma-Aldrich, Steinheim, Germany). 2,3,3-Trimethylpentane, 2,3,5-trimethylhexane, 2,4-dimethylheptane and 4-methyloctane were purchased from ChemSampCo (ChemSampCo, LLC, Trenton, New Jersey, USA), acetaldehyde was purchased from Acros Organics (Acros Organics, Geel, Belgium) and acetonitrile was purchased from J.T. Baker (Mallinckrodt Baker B.V., Deventer, The Netherlands).

For quantification of compounds detected in headspace of cells and medium solutions the external standard calibration was performed. Preparation of gaseous standards was performed by evaporation of liquid substances in glass bulbs. Each bulb (Supelco, Bellefonte, PA, USA) was cleaned with methanol (Sigma-Aldrich, Steinheim, Germany), dried at 85°C for at least 20 hours, purged with clean nitrogen for at least 20 min and subsequently evacuated using a vacuum pump (Vacuubrand, Wertheim, Germany) for 30 minutes. Liquid standards (1–3 μL according to desired concentration) were injected through a septum, using a GC syringe. After the evaporation of standards the glass bulb was filled with nitrogen of purity 6.0 (i.e. 99,9999%, Linde, Vienna, Austria) in order to equalize the pressure (to the ambient pressure). Then the appropriate volume [μL] of vapour mixture was transferred using a gas tight syringe (Hamilton, Bonaduz, Switzerland) into Tedlar^® bags (SKC 232 Series, Eighty Four, PA, USA, SKC 232 Series) previously filled with 1,5 L of nitrogen (99,9999%) additionally purified by means of carbon molecular sieves (Carboxen 1000).

Viability after incubation of 50 millions of cells for 4 hours was 98.6 ± 1.1% and after incubation for 18 h 98.7 ± 0.7%. Thus, almost no cell death was caused under the given conditions which ensures that the release of potential VOCs is mostly due to living and not dying cells.

Among all detected compounds we observed altogether 60 substances that could be identified not only by spectral library match using NIST 2005 library but also by determination of retention time based on calibration mixtures of the respective pure compound (Table 1A). An exemplary chromatogram of the headspace of CALU-1 cancer cells is presented in Figure 1. The substances, for which identification was done only by means of spectral library match without confirmation of their retention times, are listed in Table 1B. The peaks for which the proper identification was not possible (too low library match and no confirmation by retention time) are not discussed at all. Generally, the applied TD-GC-MS method is characterized by good linearity (even for the lowest detected concentrations) with correlation coefficients being mostly higher than 0,99. The LODs for almost all compounds were as low as ppt_v level. The lowest LOD was observed for 2,3,3-trimethylpentane (127 ppt_v = 5,725*10^-4 μg/l) and 2,3,5-trimethylhexane (137 ppt_v = 7,583*10^-4 μg/l) but also polar compounds exhibited very low detection limits, e.g. 2-methylpropanal (143 ppt_v = 3,912*10^-4 μg/l) and acetonitrile (147 ppt_v = 1,166*10^-7 μg/l). Only two compounds of interest with LOD at the level of single ppb_v (the highest observed values) were acetaldehyde (1,517 ppb_v = 1,312*10^-3 μg/l), and 4-methyloctane (1,168 ppb_v = 5,578*10^-3 μg/l). More detailed informations are listed in Tables 2 and 3A-B). Such low limits of detection with simultaneous low errors (expressed by correlation coefficients) testify very good precision and sensitivity of the applied TD-GC-MS method.

In three independent experiments using TD-GC-MS after preconcentration of sampled air by means of adsorption on solid sorbents, the concentrations of 4 compounds could be shown to be increased (Table 2) and other 12 compounds to be decreased (Table 3A and 3B) in the headspace of CALU-1 cells as compared to the headspace of medium only. These compounds are those which were detected in all measured samples of cancer cells as well as medium. All other 44 (= 60-16) identified compounds are not discussed in detail since they appeared "irregularly", i.e., not consistently increased or decreased with respect to medium only. The only exception is 2-methyl-2-butenal which was always found > LOD in cell culture medium samples whereas its concentration was below LOD in every CALU1 cell sample. Because of the big variations and small sample population, statistical tests should not be used. Instead, the direct comparison of VOCs concentration detected in cell and medium samples was done. We discuss only those compounds which are present in every CALU1 cell sample at (consistently) higher or (consistently) lower level than in every medium sample. Ratios of mean concentrations from both kinds of samples are given in Tables 2 and 3A–B.

After 4 hours of incubation in the fermenter an increase of isopropyl alcohol could be found. However, concentration of 2-propanol increased in only 3 from 4 measurements with 4 h incubation, whereas for the samples incubated 18 h there is no significant difference between the measured amount of 2-propanol in headspace of cancer cells and in headspace of medium. After 18 h of incubation 2,3,3-trimethylpentane (mean ppb_v in cells/mean ppb_v in medium = 1,89), 2,3,5-trimethylhexane (1,71), 2,4-dimethylheptane (3,04) and 4-methyloctane (1,71) were increased significantly in the headspace of CALU-1 cells (Figure 2). Decreased concentrations from cancer cells after 4 h were found for the compounds methacrolein (0,10), 2-methylpropanal (0,08), 2-methoxy-2-methylpropane (0,39), 3-methylbutanal (0,01), 2-methyl-2-butenal (0) and n-butyl acetate (0,14) (Figure 3 and Figure 4A). After 18 h of incubation acetaldehyde (mean ppb_v in cells/mean ppb_v in medium = 0,22), acetonitrile (0,48), acrolein (0,57), methacrolein (0,12), 2-methylpropanal (0,07), 2-butanone (0,47), 2-methoxy-2-methylpropane (0,41), 3-methylbutanal (0,01), 2-methyl-2-butenal (0), 2-ethoxy-2-methylpropane (0,40), hexanal (0,37) and n-butyl acetate (0,17) were decreased significantly (Figure 3 and Figure 4B).