GBM samples were obtained from patients with GBM admitted to the Keelung Chang Gung Memorial Hospital, Linkou Chang Gung Memorial Hospital, and Taipei Medical University-Shuang-Ho Hospital. The characteristics of these patients are summarized in Supplementary Tables S1 and S2 and Supplementary Fig. 1 The pathologies of the human brain tumour samples were determined according to WHO classification.[21, 22] Isocitrate dehydrogenase 1 (IDH-1), glial fibrillary acidic protein (GFAP), and O6-methylguanine-DNA methyltransferase (MGMT) were used as the diagnostic and prognostic makers for GBM.[23, 24]
GBM cell lines U87MG, A172, and T98G were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Further, Pt#3 and Pt#5 were isolated from patients with GBM, and P1S was obtained from another patient, who exhibited therapeutic resistance right from the onset of treatment. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (GE Healthcare Life Sciences, South Logan, UT, USA), 100 µg/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific) at 37 ºC in a 5%-CO2 incubator. TMZ-resistant U87MG-R and Pt#3-R cells were established via long-term treatment with TMZ as described previously.[2, 4]
Establishment of Sp1 knockout cells
Sp1 knockout cells were prepared using clustered regularly interspaced short palindromic repeats (CRISPR) strategy under the assistance of Biotools Co., Ltd. (New Taipei City, Taiwan). Expression vectors for guide RNA (U6-gRNA) and Cas9 gene (CMV-p-Cas9), which are ampicillin-resistant, were obtained using the Escherichia coli strain DH5α as a host. Surrogate reporter vectors, which are kanamycin resistant, were also purified from the transformed E. coli. Cells were transfected with plasmids by using Lipofectamine® LTX & PLUS™ Reagent (15338-100, Thermo Fisher Scientific). Briefly, 10 µL of LTX Reagent were diluted in 100 µL of Opti-MEM Medium (Thermo Fisher Scientific), and 2.5 µg of plasmid DNA (Cas9: sgRNA: surrogate = 1:1:0.5) were diluted in 100 µL of Opti-MEM Medium. sgRNA was designed: 5’-AGGAGTTGGTGGCAATAATGGGG-3’. After hygromycin selection, Sp1-knockout cell colonies were identified and the depletion was confirmed by Western blotting.
Chemicals in Supplementary Table 3 were dissolved in the appropriate solvent according to the manufacturers’ instruction for stock solutions. Other materials were described in the respective paragraphs in this section. EP1, EP3, and EP4 antagonists were provided by Ono Pharmaceutical Co., Ltd (Osaka, Japan).
Chemicals and reagents for targeted metabolomics
All eicosanoids and deuterated IS were purchased from Cayman Chemical (Ann Arbor, MI, USA). HPLC-grade acetonitrile (ACN) and methanol (MeOH) were purchased from Merck (Darmstadt, Germany). MilliQ water (Millipore, Bradford, USA) was used in all experiments. Acetic acid was purchased from Sigma–Aldrich. CNW Poly-Sery MAX SPE cartridges were from ANPEL Co. (Shanghai, PRC). The stock solutions of standards were prepared at the concentration of 0.1 mg/mL in MeOH. All stock solutions were stored at -20°C. The stock solutions were diluted with MeOH to working solutions before analysis.
Sample preparation for targeted metabolomics
Tissue samples (about 50 mg) and 10 µL of BHT (butylated hydroxytoluene)/MeOH solution (W/V, 4.8 g/100 mL) were subjected to protein precipitation by adding 100 µL of MeOH containing deuterium-labeled IS, at a final concentration of 50 ng/mL each of PGE2-d4, 6-keto PGF1α-d4, 5-HETE-d8 and 100 ng/mL of 9-HODE-d4. Samples were extracted with 500 µL of MeOH using a tissuelyzer at 50 Hz for 30 sec (3 times) followed with 5 ultrasonication cycles (1 min treatment and 1min break). The supernatant was transferred to new tubes after centrifuged at 12000 rpm for 10 min at 4°C, and followed by diluting with pure water to 15% MeOH concentration, followed by solid phase extraction (SPE) pretreated with MeOH and equilibrated with H2O. The extract was dried and then re-dissolved in 100 µL of MeOH, followed by filtering the solution with a 0.22 µm membrane filter before UPLC-MS/MS analysis.
Liquid chromatography and mass spectrometry
UPLC-MS/MS analyses were conducted on an Agilent UPLC-MS/MS system consisting of 1290 UPLC-system coupled with an Agilent 6470 triple-quadrupole mass spectrometer (Agilent Technologies). For analysis, 3 µL of the extract were injected. Chromatographic separation was achieved on an Agilent ZORBAX RRHD Eclipse XDB C18 column (2.1×100 mm, 1.8 µm particles) using a flow rate of 0.659 mL/min at 45°C during a 13 min gradient (0-12 min from 68 % A to 20 % A, 12-13 min 5 %A), while using the solvents A, water containing 0.005% formic acid, and B, acetonitrile containing 0.005% formic acid. Electrospray ionization was performed in the negative ion mode using N2 at a pressure of 30 psi for the nebulizer with a flow of 10 L/min and a temperature of 300°C, respectively. The sheath gas temperature was 350°C with a flow rate of 11 L/min. The capillary was set at 3500 V and the nozzle voltage was 500 V. Multiple reaction monitoring (MRM) has been used for quantification of screening fragment ions.
Data preprocessing: Peak determination and peak area integration were performed with MassHunter Workstation software (Version B.08.00, Agilent Technologies) while auto-integration was manually inspected and corrected if necessary. The obtained peak areas of targets were corrected by appropriate IS and calculated response ratios were used throughout the analysis.
The ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis was performed using an UHPLC system (1290, Agilent Technologies, Santa Clara, CA, USA) with a UPLC BEH Amide column (1.7 µm 2.1*100mm, Waters, Milford, MA, USA) coupled to TripleTOF 6600 (Q-TOF, AB Sciex, Framingham, MA, USA). The mobile phase consisted of 25 mM NH4OAc and 25 mM NH4OH in water(pH=9.75)(A) and acetonitrile (B) were carried with elution gradient as follows: 0 min, 95% B; 7 min, 65% B; 9 min, 40% B; 9.1 min, 95% B; 12 min, 95% B, which was delivered at 0.5 ml per min. The injection volume was 2 µL. The Triple TOF mass spectrometer was used for its ability to acquire MS/MS spectra on an information dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, 12 precursor ions whose intensity greater than 100 were chosen for fragmentation at collision energy (CE) of 30 V (15 MS/MS events with product ion accumulation time of 50 msec each). ESI source conditions were set as following: Ion source gas 1 as 60 Psi, Ion source gas 2 as 60 Psi, Curtain gas as 35 Psi, source temperature 650℃, Ion Spray Voltage Floating (ISVF) 5000 V or -4000 V in positive or negative modes, respectively.
Enzyme-linked immunosorbent assay (ELISA)
AA and PGE2 in culture media of wild type and TMZ-resistant U87MG cells were determined using Arachidonic Acid ELISA Kit (E4602, Biovision, Milpitas, CA, USA) and Prostaglandin E2 ELISA Kit (514010, Cayman, Ann Arbor, MI, USA), respectively, according to the manufacturers’ instruction.
Promoter reporter assay
Plasmids containing each promoter region were transfected into targeted cell lines. Cells were harvested with diluted Cell Culture lysis 5X Reagent (Promega, San Luis Obispo, CA, USA). 10 µL of sample were then mixed with 10 µL of luciferin (Promega). Luminometer (HIDEX, Tampa, FL, USA) was used to measure the promoter activity of the mixture.
3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cells (2 × 104 cells/well in a 24-well plate) were treated with different doses of drugs for 4 days. After the treatment, 300 µL of fresh medium containing 0.5 mg/mL MTT reagent (Sigma-Aldrich, St. Louis, MO, USA) were added to each well and incubated for 15 min at 37°C. Medium was removed and the crystals were dissolved in 300 µL of DMSO (Sigma-Aldrich). The absorbance was measured at 550 nm by using an iMark Microplate Absorbance Reader (Bio-Rad, Hercules, CA, USA).
Transmission electron microscopy (TEM)
To prepare the sample for TEM, cells were washed by PBS and fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M cacodylate (MERCK Millipore, Billerica, MA, USA) at room temperature. The samples were then sent to Taipei Medical University Core Facility (Taipei, Taiwan) for further preparation and imaging.
RNA-Seq and bioinformatics
After total RNA extraction, samples were subjected to genomic sequencing. The gene expression influenced by Sp1 for 1.5 folds was sorted, and the functional grouping was performed using the Ingenuity Pathway Analysis (IPA) system (https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis). Metabolism genes were sorted using Cancer Cell Metabolism Genes . Heatmaps were prepared based on the level of expression using ToppCluster (https://toppcluster.cchmc.org/).
Chromatin immunoprecipitation coupled with sequencing (ChIP-Seq)
U87MG cells were fixed with 1% formaldehyde for preserving the protein-DNA interactions, and DNA-protein complexes were harvested using the Simple ChIP enzymatic chromatin IP kit (#9003, Cell Signaling Technology, Danvers, MA, USA) followed by NextSeq 500 high throughput sequencing system (Illumina, San Diego, CA, USA) as described previously [7, 26]. XFe24 Seahorse Mitochondrial Respiration Mito Stress Test
Cells were treated with different conditions of TMZ, PGE2 or EP1-EP4 antagonists for 4 days. After the treatment, cells were trypsinized and 2x104 cells/well were seeded into the XFe24 Cell Culture Microplates (Agilent Technologies) and incubated for a day. Meanwhile, a sensor cartridge (detecting probes, Agilent Technologies) in Seahorse XF Calibrant at 37°C was hydrated in a non-CO2 incubator overnight for the following experiments. In the assay day, the cell-cultured medium was replaced with assay medium (DMEM without sodium bicarbonate, supplemented with 2% FBS and Penicillin/Streptomycin, pH: 7.4) and incubated for 1 h. Cells were then incubated at 37°C in a non-CO2 incubator that ready for experiments. Oligomycin (10 µM), FCCP (2 µM), and rotenone/antimycin A (5 µM) were prepared and placed into the sensor cartridge for the injection in the running procedure. The procedure of the assay was performed according to the guidelines from the XFe24 Seahorse Mitochondrial Respiration Mito Stress Test (Agilent technologies) . For the evaluations of FAO percentage, etomoxir (40 µM) were added into the medium, 90 min before running the assay. FAO-dependent oxygen consumption was calculated as [OCR from groups without etomoxir – OCR from groups treated with etomoxir].
Plasmids and transfection
GFP-Sp1 and PTGES2 plasmids (HG19428-ACG, Sino Biological, Wayne, PA, USA) were transfected into cells with Poly-JetTM Reagent (SignaGen Laboratories, Rockvillie, MD, USA) for overexpression. PTGS2 siRNA (Dharmacon, Lafayette, CO, USA) was transfected into cells with Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) for knockdown. PLA2G5, ABHD8, and PTGS2 promoter were designed as a 1000 bp sequence before the coding region. The information of the constructs was listed in Supplementary Table 4.
Protein samples were separated on SDS-PAGE and transferred onto the PVDF membrane (Bio-Rad). The PVDF membrane was blocked in 5% nonfat milk in TBST buffer at room temperature for an hour, and then incubated with specific primary antibodies (Supplementary Table 5) at 4°C overnight. After washing with TBST buffer, the membranes were incubated with the appropriate secondary antibodies for another one hour. Finally, the membranes were washed, and then developed by using T-Pro LumiLong Plus Chemiluminescent detection kit (T-Pro Biotechnology, New Taipei City, Taiwan).
The RNA sample was extracted by TRIzol (Thermo Fisher Scientific), and 1 µg of total RNA was subjected to real-time PCR reagent using Prime ScriptTM RT Reagent kit (Takara Bio. Inc, Shiga, Japan). The expression of each mRNA was determined using 2 × SYBR real time master mix (AB Sciex) and the specific primers (Supplementary Table 6). GAPDH was used as the internal control. SYBR green fluorescence was then monitored using an ABI 7000 Sequence Detection System (AB Sciex).
MitoPlates analysis for estimating the consumption of NADH/FADH-producing substrate
MitoPlates S-1 (Biolog, Hayward, CA, USA) were used according to the manufacturer’s instruction. To dissolve the substrates coated on MitoPlates, assay mixture containing saponin (30 µg/mL) was added into each well and the plate was incubated at 37°C for 1 h. After the treatment with PGE2, cells were trypsinized and 1.5x106 cells were mixed with 1X Mitochondrial Assay Solution (MAS) and added equally into each well. The mixtures were incubated at 37°C for 2 h and the absorbance was detected at 590 nm.
Xenograft animal model
TMZ-resistant U87MG-R (1 × 106 cells in 50 µL of DMEM) was injected into the back of 8-week-old CAnN.Cg-Foxn1nu/CrlBltw (BALB/c nude) male mice. Pt#3-R cells (1 × 106 cells in 50 µL of DMEM) were injected into the back of 8-week-old NOD.CB17-Prkdcscid/NcrCrl (NOD/SCID) male mice. A month after transplantation, mice were administrated with TMZ (10 mg/kg), ONO-8713 (15 mg/kg), and celecoxib (10 mg/kg) twice a week by intraperitoneal injection for another 8 weeks. After the treatment, the mice were then sacrificed and the tumors were excised by surgery to measure the weight and size. Tumor size was calculated according to the formula: 1/2 * long side * (short side)2.
The data obtained were represented as means ± S.E.M. Two-tailed unpaired Student’s t-test or two-way ANOVA (animal experiments) were used to analyse the differences between the control and experimental groups. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant in all comparisons.