Cell culture

Human ovarian cancer cell lines PA-1, OVCAR-3, TOV112D, and SKOV-3 were purchased from the American Type Culture Collection (Rockville, MD), and SNU840 was obtained from the Korean Cell Line Bank (Seoul, Korea). A2780 was kindly gifted by Prof. Benjamin K. Tsang (University of Ottawa, Canada). Induced ovarian surface epithelial cell line IOSE385 was generously gifted from Prof. Young Kee Shin (Seoul National University, Korea) and SNU3297 and SNU3236 were provided by Prof. Ja-Lok Ku (Seoul National University, Korea), respectively. PA-1 was cultured in MEM (WelGENE, Seoul, Korea), and other cancer cell lines were cultured in RPMI1640 (WelGENE). TOV112D and normal ovarian cell lines were grown in DMEM/F12 (Gibco-BRL, Gaithersburg, MD). All media were supplemented with 10% fetal bovine serum (WelGENE), 100 Units/ml penicillin, and 100 µg/ml streptomycin (Gibco-BRL). All cells were cultivated at 37 °C in humidified conditions with 5% CO2. For assays, all cell lines were treated with CAY10566 or SCD1 siRNA for 24–48 h in a 1% serum-containing medium.

Reagents and antibodies

CAY10566 was purchased from Cayman Chemical (Ann Arbor, MI). Oleic acid-BSA conjugate and fatty acid-free BSA were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies used for western blotting were as follows: P-PERK (Thr981), PARP, and cleaved caspase-3 from Santa Cruz Biotechnology (Santa Cruz, CA), IRE1a and ATF4 from Cell Signaling Technology (Danvers, MA), SCD1 from Abcam (Cambridge, UK), CHOP (GADD153) from Thermo Fisher Scientific (Waltham, MA), and GAPDH from Ab Frontier (Seoul, Korea).

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted using RNAiso Plus (TaKaRa, Tokyo, Japan), and the concentration of RNA was determined by Nano Drop2000 (Thermo Fisher Scientific). Complementary DNAs were synthesized from 1 µg of total RNA with oligo-dT primers and PrimeScript Reverse Transcriptase (TaKaRa). PCR was performed using QuantiSpeed SYBR No-ROX Kit (PhileKorea, Seoul, Korea) and the following specific primers: SCD1 sense 5’-CGA CGT GGC TTT TTC TTC TC-3’, antisense 5’-GGG GGC TAA TGT TCT TGT CA-3’ and GAPDH sense 5’-GAG TCA ACG GAT TTG GTC GT-3’, antisense 5’-TTG ATT TTG GAG GGA TCT CG-3’. The amplification conditions were as follows: an initial denaturation step at 95 °C for 3 min, followed by 45 cycles of denaturation at 94 °C for 5 s, annealing at 60 °C for 15 s, extension at 72 °C for 10 s, and a final extension step at 72 °C for 10 min. The relative gene expression levels were calculated using the comparative Ct method, and GAPDH was used as a reference gene.

Cell viability assay

Cell viability was examined by MTT assay. For CAY10566 treatment, cells were seeded into 96-well plates at a density of 4,000–10,000 cells per well. The cells were treated with various concentrations of CAY10566 or DMSO (solvent control) for 24–48 h. For siRNA transfection, cells were seeded into 6-well plates to be 60–80% confluent at transfection. After 24 h, the cells were transfected with SCD1 siRNA or scrambled siRNA (negative control), incubated overnight, and then cultured into 96-well plates for 24–48 h. At the end of the treatment, cells were incubated with 50 µl of MTT solution (2 mg/ml) for 3 h at 37 °C in humidified conditions with 5% CO2 and subsequently solubilized in 100 µl of DMSO for 30 min. The optical density was measured at 540 nm using a Multiskan Ascent plate reader (Thermo LabSystems, Helsinki, Finland).

Gas chromatography

The desaturase activity of SCD1 was measured by gas chromatography. CAY10566-treated cells were harvested by centrifugation and freeze-dried using a freeze dryer (LABCONCO, Kansas City, MO). The extraction and methylation of fatty acids were conducted as previously described [17]. For GC analysis, fatty acids were converted to their methyl esters (FAMEs). Gas chromatography was performed using an Agilent 7890 A GC system (Agilent Technologies, Wilmington, DE) equipped with a DB-23 capillary column (60 mm x 0.25 mm x 0.25 μm; Agilent Technologies). The GC conditions were as follows: the initial temperature was 50 °C for 1 min, then raised to 130 °C at 15 °C/min, to 170 °C at 8 °C/min, to 215 °C at 2 °C/min, and held for 10 min. The injector temperature was set at 250 °C, and the detector temperature was set at 280 °C. Pentadecanoic acid (C15:0) was used as an internal standard for quantification. The ratios of palmitoleic acid (C16:1) to palmitic acid (C16:0) and oleic acid (C18:1n9c) to stearic acid (C18:0) were determined for this study.

Small-interfering RNA (siRNA) transfection

The SCD1 siRNA target sequence was 5’-GAGAUAAGU UGG AGA CGA UUU-3’. Scrambled siRNA was used as a negative control (Genolution, Seoul, Korea). Cells were transfected with the siRNA oligonucleotides (100 nM) using Lipofectamine RNAiMAX (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s protocols. At 24 h post-transfection, the cells were used for cell proliferation assay, flow cytometry analysis, and western blot analysis.

Isolation of peripheral blood mononuclear cells (PBMCs)

To determine the cytotoxic effect of CAY10566 on normal cells, buffy coats from healthy donors were collected under the approval of Seoul National University Hospital Institutional Review Board (C-1307-008-502). Human peripheral blood mononuclear cells were isolated from buffy coats by density-gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Marlborough, MA) according to the manufacturer’s instructions. In brief, buffy coats were diluted in PBS, carefully layered on Ficoll-Paque PLUS, and centrifuged at 400 x g for 30 min at 20 °C. The PBMC layer was transferred to a clean centrifuge tube, washed twice with PBS, centrifuged at 200 x g for 15 min at 20 °C, and suspended in RPMI1640 supplemented with 10% FBS, 100 Units/ml penicillin, and 100 µg/ml streptomycin.

Flow cytometry analysis

To analyze both floating and adherent cells, culture media containing floating cells were collected into a round-bottom tube (BD Falcon, San Jose, CA), and adherent cells were trypsinized, washed with cold PBS, and collected by centrifugation at 4 °C. All cells were stained with Annexin V-FITC and PI using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. The stained cells were analyzed using a BD FACS Canto II flow cytometer (BD Biosciences) with BD FACS Diva software (BD Biosciences).

Western blot analysis

Cells were lysed with the lysis buffer containing the premade 2X lysis buffer (150 mM NaCl, 10 mM Tris-HCI (pH 7.4), 1 mM EDTA, and 1 mM EGTA), 1% Triton X-100, 1 mM PMSF, 0.1% DCA, and 1X EDTA-free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The concentration of protein was determined using BCA Protein Assay Kit (Thermo Fisher Scientific). 10–20 µg of proteins were loaded onto 6–15% SDS-PAGE gels for separation and transferred to nitrocellulose membranes (GE Healthcare Life Sciences) for detection by immunoblotting. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20, incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies for 2 h at room temperature. Signals were visualized with the enhanced chemiluminescence detection kits, WESTSAVE up (AbFrontier) and ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences).

Organoid establishment

This study was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 2108-237-1251). Ovarian cancer tissue and ascites samples were collected with the consent of patients. Organoids were established with ovarian cancer acites as our previous study [24, 25]. The cells isolated from ascites were embedded in the in phenol red-free Matrigel Growth Factor Reduced Basement Membrane Matrix (BD Bioscience, CA, USA) and cultured for approximately 28 days. The culture conditions included consistent media changes every 2–3 days using Advanced DMEM/F12 (Gibco, MD, USA) supplemented with HEPES (10mM; Gibco, Gaithersburg, MD, USA), 1× GlutaMax (Gibco, Gaithersburg, MD, USA), 1× N2 (Invitrogen, CA, USA), 1× B27 (Invitrogen, CA, USA), β-Estradiol (1 mM; Sigma-Aldrich, St. Louis, USA), nicotinamide (1 mM; Sigma-Aldrich, St. Louis, USA), recombinant human Noggin (10 ng/mL; PeproTech, Rocky Hill, NJ, USA), recombinant R-Spondin1 (10 ng/mL; PeproTech, Rocky Hill, NJ, USA), EGF (10 ng/mL; Invitrogen, CA, USA), FGF2 (10 ng/mL; PeproTech, Rocky Hill, NJ, USA), FGF10 (10 ng/mL; PeproTech, Rocky Hill, NJ, USA), Y-27,632 dihydrochloride (10 µM; Sigma-Aldrich, St. Louis, USA), SB431542 (0.5 µM; Sigma-Aldrich, St. Louis, USA), and N-acetylcysteine (1mM; Sigma-Aldrich, St. Louis, USA). Bright field images of organoids were taken using EVOS M5000 (ThermoFisher Scientific).

Organoid viability assay

For organoid cell viability assay, organoids MTT assay was carried out as our previous study [24]. Briefly, fully grown organoids embedded in Matrigel were seeded in 24-well plates and each well was treated with 500 µl of MTT reagent initially. Following a 3-hour incubation, 200 µl of DMSO was added, and the Matrigel was dissociated by pipetting. The plates were placed on an orbital shaker and incubated for 30 min at room temperature. Subsequently, the optical density values were measured at a wavelength of 540 nm with a Multi-Scan Spectrum (Thermo Scientific, NH, USA).

Tissue microarray (TMA) construction and immunohistochemistry (IHC) staining

At Seoul National University Hospital, specimens of patients who have agreed to donate human materials are stored in the pathology department after surgery. We were provided with tissue microarray (TMA) blocks with personal information removed through hospital regulations. (IRB: 1807-037-956). All of the cases had available formalin-fixed paraffin-embedded (FFPE) specimens of primary and metastatic tumors and adjacent normal tissue which were obtained from biopsy. TMAs of ovarian cancer specimens were constructed using two cores (2 mm in diameter) from each specimen embedded in recipient paraffin blocks which include multiple sections of normal and cancer tissues of 72 ovarian cancer patients in Seoul National University Hospital using a trephine device (Superbiochips Laboratories, Seoul, Republic of Korea). TMAs were sectioned at a thickness of 4 μm and stained according to the manufacturer’s recommendations using the Benchmark XT Staining Systems (Ventana, Tucson, AZ). IHC staining was conducted using a rabbit polyclonal antibody against SCD1 (1:200, bs-3787R, Bioss, Woburn, MA). The intensity of positively stained cells was scored as follows: 0 (negative), 1 (weekly positive), 2 (moderately positive), 3 (strongly positive).

Statistical analysis

All data were expressed as mean ± SEM of three independent experiments. The statistical significance of differences was determined using student’s t-test for two groups and two-way ANOVA with Šídák’s multiple comparisons test for over three groups. All statistical analyses were performed using IBM SPSS Statistics 22 software (SPSS Inc., Chicago, IL) and GraphPad Prism 9 (GraphPad Software, La Jolla, CA). For all analyses, differences with p-values < 0.05 were considered statistically significant.