4.1. Sample collection
This study included 20 patients diagnosed with MIBC at Kyushu University Hospital (Fukuoka, Japan) between 2019 and 2021. Of these 20 patients, nine and four patients received chemotherapy as neoadjuvant and primary/salvage treatments, respectively. Tumor tissues and blood samples were banked at −80°C until extraction of DNA or protein. Written informed consent was obtained from all patients, and the study was approved by the institutional review board (#2020-254). The study was conducted in accordance with the principles described in the Declaration of Helsinki and the Ethical Guidelines for Epidemiological Research enacted by the Japanese Government.
4.2. Targeted exome sequencing
Tumor DNA was extracted from tissues obtained by transurethral resection (TUR) or radical cystectomy with a DNeasy blood and tissue kit (Qiagen, Hilden, Germany). Germline DNA (gDNA) was extracted from blood collected before undergoing chemotherapy. Targeted exome sequencing was performed using the Ion Ampliseq Comprehensive Cancer Panel (CCP; #4477685; Thermo Fisher, Waltham, MA, USA), which consisted of four primer pools totaling nearly 16,000 primer pairs covering 1.23 Mb of 409 genes (Supplementary Table S3 (18). DNA (10 ng) from 20 tumors and matched gDNA was used for libraries. Libraries were generated by the Ion Torrent Ion Chef system (Thermo Fisher) with the Ion Ampliseq Library kit plus (Thermo Fisher) and sequenced by an Ion PGM Sequencer (Thermo Fisher). We analyzed sequences using Ion Reporter Software 220.127.116.11 (Thermo Fisher, https://ionreporter.thermofisher.com/ir/). The mean depth of read coverage for the target genes was 103 × (interquartile range, 48.5–139). Mutations reported as “pathogenic” or “likely pathogenic” in dbSNP (https://www.ncbi.nlm.nih.gov/snp/), and mutations with Polyphen2 score > 0.9 (http://genetics.bwh.harvard.edu/pph2/) were determined to be pathogenic (19). A mutation was defined as having a 5.0% allele frequency or greater, and tumor mutation burden (TMB) was calculated by dividing the number of mutations by the sequence length of the comprehensive cancer panel (1.23 Mb). Public dataset of the TCGA study (BLCA) were obtained from cBioPortal (www.cbioportal.org), and the frequency of mutation in TP53, ATM, ERCC, RB1, DPYD, FANCC genes was analyzed (20, 21).
4.3. DYPD mRNA expression analysis in BC tissues
Microarray data from a multi-institutional cohort of 223 MIBC patients treated with NAC followed by radical cystectomy were used (22). GC or GCarbo was administered in 169 patients, and MVAC in 54 patients. Molecular subtypes (basal, claudin-low, luminal, and luminal-infiltrated) were previously reported in this cohort (22). Public dataset of the TCGA study (BLCA) were obtained from cBioPortal (www.cbioportal.org), and the gene expression of DPYD was analyzed (20, 21).
TUR sections were deparaffinized and permeabilized with xylene, then activated with 0.01 M citric acid for 10 min. Blocking with 3% hydrogen peroxide for 15 min and Blocking-One (Nacalai Tesque, Kyoto, Japan) for 30 min was performed. Anti-dihydropyrimidine dehydrogenase (DPD) antibody (1:50, ab54797; Abcam, Cambridge, UK) was diluted with Blocking-One and used as the primary antibody with 90 min incubation. Histofine Simple Stain MAX-PO (MULTI) (Nichirei Bioscience Inc, Tokyo, Japan) was used as the secondary antibody with 45 min incubation. The signals were developed by DAB tablet (Fujifilm, Tokyo, Japan) for 20 min, followed by counterstaining with Histofine Mayer’s hematoxylin (415081; Nichirei Bioscience) for 2 sec. (23) After dehydration, sections were encapsulated and observed.
4.5. Cell culture
RT112 and UM-UC13 human bladder cancer cell lines were provided by the Pathology Core of Bladder Cancer SPORE at MD Anderson Cancer Center, and cultured in MEM/EBSS (Hyclone, GE Healthcare, Chicago, IL, USA) containing 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (24). To establish gemcitabine-resistant sublines (RT112 Gem-R and UT-UC13 Gem-R), RT112 and UM-UC13 cells were treated serially with increasing concentrations of gemcitabine up to 10 µM and 0.1 µM, respectively. HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% FBS and 1% penicillin/streptomycin.
4.6. Plasmids and siRNA
C-terminal FLAG-tagged DPYD plasmid in the backbone of pcDNA3.1 was obtained from GenScript (cloneID: OHu19551; GenScript Biotech, Piscataway, NJ, USA). To establish DPYD c.1031C>T plasmid, the PrimeSTAR Mutagenesis Basal Kit (Takara, Kusatsu, Japan) was used. The primers, annealing temperature (Tm), and cycle number of mutagenesis reactions were as follows: 5’-GACACTGTCTTTGACTGTGCAACATCT-3’ and 5’-GTCAAAGACAGTGTCTCCAGCTCCAAG-3’, 55°C, and 30 cycles. Correct introduction of mutations was confirmed by Sanger sequencing. Specific siRNAs against DPYD (#1: SASI_Hs01_0018-0530/DPYD and #2: SASI_01_0018-0539/DPYD) and control siRNA (MISSION siRNA Universal Negative Control #1, SCI001) were obtained from Sigma–Aldrich. Transfection of plasmids and siRNAs into cells was performed using Lipofectamine LTX (Invitrogen, Waltham, MA, USA) and Lipofectamine RNAiMAX (Invitrogen), respectively, in accordance with the manufacturer’s instructions (25). Stable overexpressing cells were established by transfecting expression plasmid into HEK293 cells as described above, and culturing with medium containing G418 (Nacalai tesque) at 400 µg/mL for at least 2 weeks for selection.
4.7. Cell proliferation assay
Cell proliferation was calculated by cell counting or CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Cell counting was performed using a TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA) after incubation in the indicated conditions. For CellTiter-Glo assay, cells were seeded in a 96-well plate with phenol-red free medium and cultured under the indicated conditions. CellTiter reagents were added to the medium and incubated for 10 min, followed by measurement using a 2030 ARVO X2 (PerkinElmer, Waltham, MA, USA) (26). Cell survival rate when exposed to gemcitabine was corrected with that in the absence of gemcitabine. The proliferation ratio was calculated using the formula below. A proliferation ratio of >1.0 indicated resistance and that of <1.0 indicated sensitivity.
All values represent the results of three independent experiments.
4.8. Western blot analysis
Cells and tumor tissues were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP40). For tissue lysis, 0.1% SDS was added with protease inhibitor cocktail set 1 (Fujifilm). Protein samples were separated by SDS-PAGE gel and transferred onto PVDF membranes. After 30 min blocking with Blocking-One (Nacalai Tesque), membranes were incubated with primary antibody overnight at 4°C. The following antibodies were used: anti-DPD antibody (1:10,000, ab54797; Abcam), anti-GAPDH antibody (1:10,000, 14C10; Cell Signaling Technology, Danvers, MA, USA), and anti-α-tubulin antibody (1:10 000, PM054; MBL, Tokyo, Japan). Then membranes were incubated with secondary antibody (anti-mouse IgG, 1:20 000 [NA9310V, GE Healthcare]; anti-rabbit IgG, 1:20 000 [Jackson ImmunoResearch, West Grove, PA, USA]) for 1 h at room temperature. Each antibody was diluted in 1× PBST. ECL substrates (Clarity Western ECL substrate; Bio-Rad) were used for signal detection with an electronically cooled charge-coupled device camera (LAS4000; GE Healthcare). Quantification of protein expression was performed with imageQuant TL (GE Healthcare) (27). As internal controls, GAPDH or α-tubulin was used for cells, and total protein was used for patients’ tissue samples. Total protein was detected by staining membranes with CBB-R (CBB Stain One Super, 11642-31; Nacalai Tesque) for 15 min.
4.9. Quantitative real-time PCR (qRT-PCR)
RNA from cells was extracted with the RNeasy Mini kit (Qiagen). After treatment with DNase I (Qiagen), reverse transcription with 500 ng RNA using a PrimeScript RT Reagent Kit (Takara) was performed, and cDNA was stored at −80°C. mRNA expression was calculated using SYBR Premix ExTaq II (Takara) with a thermal cycler (StepOne plus; Applied Biosystems, Waltham, MA, USA) (28). Primers, Tm, and cycle numbers were as follows: DPYD; 5’-CAACGTAGAGCAGATGTTGCAC-3’ and 5’-GAGCTGTCATGCAGAAATGGTTT-3’, 18s ribosomal RNA (internal control); 5’-AAACGGCTACCACATCCAAG-3’ and 5’-CCTCCAATGGATCCTCGTTA-3’, 60°C, 40 cycles. All values represent the results of three independent experiments.
4.10. Plasma metabolite preparation
Metabolite extraction from cultured cells was described previously (29). To prepare nucleic acid-related metabolites, approximately 3×106 HEK293 cell pellets were added to 500 µL ice-cold 80% methanol, vortexed, sonicated five times (30-sec sonication and 30-sec cooling) with a BIORUPTOR (Cosmo Bio, Tokyo, Japan), and centrifuged at 21,500 ×g for 5 min at 4°C. The supernatants were collected, and 250 µL supernatant was evaporated to dryness, then dissolved in a mobile phase determined by each analytical condition and then subjected to LCMS measurement of nucleotides or nucleosides.
4.11. Liquid chromatography-mass spectrometry
Cellular metabolites were analyzed by liquid chromatography-mass spectrometry using an LCMS-8060 instrument (Shimadzu, Kyoto, Japan). For nucleotides, the prepared sample was separated on a SeQuant® Zic®-pHILIC column (150×2.1 mm, 5 µm particle size; Merck, Darmstadt, Germany) with mobile phases consisting of solvent A (10 mM ammonium bicarbonate, 0.1% ammonia, 100 µM medronate) (30) and solvent B (acetonitrile). The column oven temperature was maintained at 40°C. The gradient elution program was as follows: flow rate of 0.25 mL/min; 0–2 min, 70% B; 0–1 min, 70–47.5% B; 1–1.5 min, 47.5–30% B; 1.5–3.5 min, 30% B; 3.5–6.5 min, 70% B; and 6.6–12.5 min. The parameters for the heated electrospray ionization source (ESI) in negative ion mode under multiple reaction monitoring (MRM) were as follows: drying gas flow rate, 10 L/min; nebulizer gas flow rate, 3 L/min; heating gas flow rate, 10 L/min; interface temperature, 300°C; DL temperature, 250°C; heat block temperature, 400°C; CID gas, 270 kPa. However, for nucleosides, the sample was separated on a KINETEX C18 column (150 mm×2.1 mm, 1.7 µm; Phenomenex) with mobile phases consisting of solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile). The column oven temperature was 35°C. The gradient elution program was as follows: flow rate of 0.3 mL/min; 0–2min, 3% B; 2–5.5min, 3–6% B; 5.8–8.5 min, 100% B; and 8.6–15 min, 3% B. The parameters for the heated ESI in positive ion mode under multiple reaction monitoring (MRM) were the same as above. Data processing was performed using the LabSolutions LC-MS software program (Shimadzu).
4.12. Statistical analysis
All statistical analyses were performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R 2.13.0 (31). Statistical analyses were performed using Fisher test, Student’s t-test, one-way ANOVA, and Mann–Whitney U test. All P-values are two-sided. Levels of statistical significance were set at P < 0.05.
4.13. Ethical compliance
The study was approved by Kyushu University Institutional Review Board for Clinical Research (#2020-254).