Identification of characteristic compounds of moderate volatility in breast cancer cell lines

In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.


Introduction
The national epidemiological profiles of cancer burden in the Global Burden of Disease study estimated that there will be 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018; lung cancer is the most commonly diagnosed cancer (11.6% of the total cases), closely followed by breast cancer (11.6%), prostate cancer (7.1%), and colorectal cancer (6.1%) for incidence. [1] The detection and diagnosis of cancer at earlier stages apparently determines further treatments, and periodic health screening using various techniques, such as X-rays, [2] PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0235442 June 29, 2020 1 / 14 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 blood tests, [3] mammography, [4] ultrasonography, [4] computed tomography, [5] positron emission tomography, [6] and magnetic resonance imaging, [4] is highly recommended. Regarding definitive diagnosis, an invasive tumour tissue biopsy followed by immunohistochemistry (IHC) of cancer-specific markers, such as oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 (a nuclear protein associated with cellular proliferation for breast cancer) is a common technique. [7] However, considering physical stress of patients and the cost for cancer diagnosis, simpler, easier, and faster diagnostic methodologies are still required. Among the reported cancer-diagnostic methodologies, non-invasive techniques using breath, [8] urine, [9] and hair [10] are approaches that must be of great benefit for initial cancer diagnosis owing to their convenience and rapidity. Our previous report showed that dog could distinguish colorectal cancer patients from non-cancer individuals by smelling their exhaled breath. [11] This strongly suggests the diagnostic benefit of breath as non-invasive technique and presence of cancer-related volatiles in breath, which could be useful for the breath-related research fields. Thus far, a number of volatiles have been identified in exhaled breath or headspace of cell culture media by gas chromatography-mass spectrometry (GC-MS). [12,13] However, it still remains unclear whether volatiles present in breath are specifically derived from cancer disorders, since distinguishing cancer patients' breath from that of the ones without cancer was achieved by pattern analysis (metabolomics) of volatile compounds, but not by analysis of individual compounds. [14,15] Moreover, it is challenging to reproducibly and precisely determine the volatile levels in exhaled breath due to their low levels (in some cases, below the limit of detection by GC) and environmental contaminations such as ingested food and smoking. [16] Most of the experiments on volatiles in breath from cancer patients so far have been conducted using the solid-phase micro-extraction (SPME) GC-MS method, [12,13] targeting the compounds with high volatility (boiling point: 50˚C-250˚C) [17] in breath or "headspace" of fluids. It is likely that fatty acids and lipids may be precursor candidates responsible for the generation of endogenous volatiles. [18] These findings led us to hypothesis that cancer-specific precursors with low volatility (boiling point: 250˚C-400˚C) may still occur in biological fluids. Thus, since breast cancer is one of the serious problems to be addressed, the aim of this study was to identify such compounds of moderate volatility by targeting breast cancer cells. A Porapak Q (ethylvinylbenzene-divinylbenzene copolymer) resin that is commonly used as GC absorbent was used for the solid-phase extraction of volatiles in the culture media, since the resin displayed an extensive absorption of diverse volatiles with middle to high boiling points. [19,20] GC or GC-MS analysis, in combination with liquid chromatography-time-of-flight (LC-TOF)/MS, were performed to identify compounds that were specifically present in culture media of several breast cancer cell lines (Fig 1A).

Cell culture
Human breast cancer cell lines MCF-7 (JCRB0134) and YMB-1 (JCRB0823), and fibroblast cell line KMST-6 (JCRB0433) were obtained from Japanese Collection of Research Bioresources (JCRB Cell Bank, Osaka, Japan). Another human breast cancer cell line SK-BR-3 (No.  was obtained from American Type Culture Collection (ATCC, Summit Pharmaceuticals International, Tokyo, Japan). Both JCRB and ATCC provide these cell lines with the confirmation of a negative result of micomycoplasma testing and STR-profiling to characterise the cell lines. Although International Cell Line Authentication Committee (ICLAC) Database of Cross-contaminated or Misidentified Cell Lines reports a risk that YMB-1 is a substrain of breast cancer cell line ZR-75-1, the aim of this study regarding characteristic compounds commonly present in breast cancer cell lines would overcome the issue. MEM media (Wako, Osaka, Japan) for MCF-7 and KMST-6, D-MEM media (Thermo Fisher Scientific, Waltham, MA, USA) for MCF-7, SK-BR-3, and KMST-6, and RPMI 1640 media (Thermo Fisher Scientific) for YMB-1 and KMST-6 were used with supplementation of 10% fetal bovine serum (Sigma). Cells were maintained at 37˚C in humidified atmosphere containing 5% CO 2 . Each cell line was individually seeded and grown in a culture flask (175 cm 2 , IWAKI, Shizuoka, Japan) up to a sub-confluence of 70%-80%. After this growing period, the cells were transferred to a new culture flask (Multi-Flasks 5-layer 875 cm 2 , Corning, NY, USA) at 5.0 × 10 6 cells/flask. The culture medium of 250 mL/bottle was refreshed every two days and collected into 5L-polypropylene bottle (Steritainer, Sekisui, Osaka, Japan) for 4 days as the medium collecting period (total volume: 500 mL) for solid-phase Porapak Q extraction. Collected culture medium was stored at -40˚C until extraction.

Extraction of volatile compounds in culture media with solid-phase Porapak Q resin
Volatile organic compounds in culture media of cell lines were extracted using a solid-phase Porapak Q resin according to the procedures described by Fukamachi et al. [21] Medium collected from the cell line culture (500 mL) was injected to a column packed with Porapak Q resin (50-80 mesh, Waters Co., Milford, MA, USA) with a bead volume of 20 mL (50 mm length × 20 mm I.D.). After washing the column with 100 mL of deionized water, diethyl ether (100 mL)-extraction was performed. Contaminated water in the ether eluate was removed by the addition of excess anhydrous sodium sulphate (Nacalai Tesque), followed by the evaporation of ether at 42˚C in a water bath. The obtained ether-concentrate (500 μL) was stored at -30˚C prior to GC or LC analysis.

GC-FID and GC-MS analyses
Ether concentrate was analysed using a Shimadzu GC-18A equipped with an FID detector (Shimadzu Co., Ltd., Kyoto, Japan) or a Shimadzu GC-MS QP2010 Plus on a DB-FFAP capillary column (30 m × 0.32 mm I.D. with 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA, USA). An aliquot (5 μL) of the concentrate was injected into GC system in splitless mode at a constant carrier gas (He) pressure of 70 kPa with a linear velocity of 40 cm/s. GC separation on the abovementioned capillary column was performed at temperatures programmed at 40˚C for 5 min to 240˚C at 5˚C/min. MS conditions were as follows: electron ionization (EI), positive at 70 eV; mass range, m/z 40-500; ion source temperature, 200˚C; interface temperature, 220˚C, and scan event time, 0.3 s. GC-MS identification of target was done by a matching (>90% of similarity) mass spectra with the NIST 14 library database, as well as matching of retention index (RI) [22] of target with that of the standard.

H 2 O 2 -oxidation of unsaturated fatty acids and identification of volatiles by SPME-GC-MS
H 2 O 2 oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid was performed as follows ( Fig 1B); each unsaturated fatty acid (10 ppm) was added to 10 mL of 0.2% H 2 O 2 solution in a teflon-capped 50 mL-vial and the vial stood for 6 days at 37˚C. A blank experiment in 0.2% H 2 O 2 solution without fatty acid was also performed at the above-mentioned incubation conditions. Extraction of volatile compounds in the headspace gas of the teflon-capped 50 mLvial was performed with SPME fibre coated with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, SUPELCO Co., Bellefonte, PA, USA). The fibre was conditioned at 270˚C for 1 h. After the vial was placed in a water bath at 37˚C for 20 min, the SPME fibre was exposed to the headspace of the vial for 45 min. The fibre was, then, transferred to an injection port of GC-MS at 250˚C for 10 min for desorption of SPME-absorbed compounds. GC-MS was conducted with a Shimadzu GC-MS QP2010 Plus on a DB-WAX capillary column (30 m × 0.32 mm I.D. with 0.5 μm film thickness, Agilent Technologies). Chromatographic and MS conditions were the same as aforementioned. GC-MS identification of targets was done by matching (>90% of similarity) of mass spectra with the NIST 14 library database, as well as matching of RI of targets with that of the standards. Statistical analyses. The statistical significance between two groups was analysed using unpaired two-tailed Student's t-test. All analyses were conducted with a GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).

Identification of characteristic compounds present in culture media of breast cancer cells by GC and GC-MS
For GC and GC-MS analyses of volatiles in the culture media, three breast cancer cell lines, MCF-7, SK-BR-3, and YMB-1, were cultured in MEM, D-MEM, and RPMI 1640 media, respectively. The culture media were collected at every 2 days for 4 days (total volume of 500 mL) and were subjected to solid-phase extraction using a Porapak Q resin. A fibroblast cell line, KMST-6, as a control cell was cultured in the aforementioned three media respectively and each was subjected to solid-phase extraction. Concentrated ether-extract (500 μL) from solid-phase Porapak Q resin was applied to the gas chromatograph with a flame-ionization detector (GC-FID) to identify the peaks specific for breast cancer cell lines, since FID can widely detect organic compounds without any consideration of detection efficiency (as in the case of MS ionization) between compounds. On comparing GC chromatograms of KMST-6 media with those of breast cancer cell media, we observed enhanced GC peaks that were common in all three different cell lines. However, enhanced peaks that did not commonly appear in the three different media (marked with asterisk in Fig 2) were excluded from the possibility of being breast cancer-specific volatile candidates; e.g., peaks at 10.8 min, 14.1 min, 33.8 min, 37.2 min, 38.0 min, 40.1 min, 48.6 min, and 54.1 min for MCF-7 (Fig 2a), peaks at 21.2 min, 35.8 min, and 38.9 min for SK-BR-3 (Fig 2b), and peaks at 10.8 min, 14.2 min, 17.2 min, 35.7 min, and 48.6 min for YMB-1 (Fig 2c). According to the report by Hanai et al., [23] two peaks (denoted as P1 and P2 in Fig 2) showing significantly and commonly high intensity in the three different breast cancer cell media were selected (Fig 2a-2c); GC peak intensities of P1 and P2 in cell cultures of MCF-7 were > 6-times higher than those in KMST-6 (n = 3, P < 0.05) (S1 Fig). In addition, P1 and P2 observed in MEM medium for MCF-7 (Fig 2a) were also detected when MCF-7 was cultured in D-MEM (S2 Fig). This indicated that the two peaks were generated from cancer cell growth, but not medium composition. Identification of P1 and P2 was performed using GC-MS with the aid of a similarity search using NIST library 14 software. As shown in Fig 3, P1 and P2 were identified as 1-hexadecanol (NIST similarity score of 93) and 5-(Z)-dodecenoic acid (NIST similarity score of 95), respectively. The two compounds of moderate volatility were also confirmed by their standards [1-hexadecanol: RI on FFAP column, 2379; 5-(Z)-dodecenoic acid: RI on FFAP column, 2520] (S3 Fig). To the best of our knowledge, this is the first finding showing that the two compounds obtained through GC analysis of the culture media were possible candidates for breast cancer cell lines.

LC-TOF/MS analysis of 5-(Z)-dodecenoic acid and the related compounds specific for MCF-7 breast cancer cell culture medium
Considering that 5-(Z)-dodecenoic acid could be a possible biomarker for breast cancer, further experiments were performed to investigate whether 5-(Z)-dodecenoic acid-related unsaturated fatty acids were also abundant in MCF-7 cell culture media compared to those in KMST-6. LC-TOF/MS (but not GC-MS) analysis of MCF-7 cultured MEM medium was performed owing to aqueous properties of the targeted fatty acids in medium. As shown in Fig  4a-4g, seven peaks on extracted-ion chromatograms (EIC) corresponding to m/z values of unsaturated fatty acids (C10:1, C10:2, C12:1, C14:1, C14:2, C16:2, and C18:2) were detected, and five peaks (C10:1, C10:2, C12:1, C14:1, and C14:2) showed much higher intensity in MCF-7 compared to those in KMST-6 (Fig 4a-4e). With the aid of commercially available standards, C12:1 and C14:1 detected in MCF-7 were successfully identified as 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid, respectively, whereas the position of the unsaturated bond for other unsaturated fatty acids (except for C18:2 as linoleic acid) could not be defined due to unavailability of standards. acid-derived volatiles by 0.2% H 2 O 2 -accelerating oxidation of each acid in water (10 ppm) in a 50 mL-sealed vial for 6 days at 37˚C. As shown in Fig 5, GC-FID analysis of the 6-day-incubates of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid revealed that four peaks from 5-(Z)-dodecenoic acid and three peaks from 5-(Z)-tetradecenoic acid were newly and markedly obtained compared to GC chromatograms of the blank (without both acids) sample. GC-MS analysis in combination with RI matching [22] on DB-WAX column showed that all the identified compounds except T2 were volatile aldehydes (Tables 1 and 2). In addition, these identified aldehydes (heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal) were volatiles identified in the breath or blood from patients of various cancers as reported previously, [8,[25][26][27][28][29] e.g., heptanal in breath as a lung cancer-specific volatile. [8,25,26] This finding allowed us to speculate that the unsaturated fatty acids identified in this study may be one of the possible precursors of the reported cancer-specific volatiles.

Discussion
So far, research interests have been focused on the elucidation of "volatiles" in the exhaled breath of cancer patients, since the evidential report that dog could distinguish colorectal cancer patients by smelling their exhaled breath. [11] Thus, research on volatiles in breath from cancer patients [8,12] have been conducted using SPME extraction method, which is suitable for the recovery of highly volatile compounds from gas phase. In contrast, in the present study, we used a solid-phase Porapak Q resin for the recovery of a variety of volatiles, since compounds of moderate volatility such as fatty acids or lipids were targeted as precursors of these cancer specific volatiles. For the first time, we identified two compounds, 1-hexadecanol and 5-(Z)-dodecenoic acid, by GC-FID analysis in the culture media of MCF-7, a breast cancer cell line (Fig 2a). Considering the boiling points of 344˚C and 311˚C for 1-hexadecanol and 5-(Z)-dodecenoic acid, respectively, they could evaporate into gas phase, since compounds with a high boiling point of > 300˚C, e.g., β-naphthyl methylketone with boiling point of 301˚C, can behave as a synthetic strawberry-like flavour compound. [24] Thus, a direct analysis of the headspace of culture media of breast cancer cells is needed for further experiments, which is now in progress to confirm their vaporization into gas phase. Abaffy et al. reported that 1-hexadecanol was identified as one of the malignant melanoma-specific volatiles in melanoma biopsy. [31] Shigeyama et al. also revealed that 1-hexadecanol was newly produced in the saliva of oral squamous cell carcinoma patients, together with other alcohols and ketones as volatile metabolites.
In conclusion, in this study, we firstly demonstrated that 1-hexadecanol and 5-(Z)-dodecenoic acid (and its related unsaturated fatty acids including 5-(Z)-tetradecenoic acid) were important candidates in breast cancer cells but not fibroblast cells by GC-MS and/or LC-MS analyses of cell culture media. We also found that unsaturated fatty acids could generate the reported volatiles specific for breath from various cancer patients such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal by H 2 O 2 oxidation. Thus, the diagnostic targets, medium chain unsaturated fatty acids of moderate volatility, would overcome the issues on poor reproducibility and precision of volatile detection in exhaled breath. [16] In summary, the present findings clearly indicate that unsaturated fatty acids of moderate volatility may be over-produced in breast cancer cells, serving as potential precursors for the production of cancer-specific volatiles reported so far.