Tissue samples and cell lines
Primary tumors were collected from 99 RCC patients at Hiroshima University Hospital (Hiroshima, Japan). The RCC patients with preoperative chemotherapy were excluded from this study. Informed consent was obtain from all patients. This study was approved by the Ethical Committee for Human Genome Research of Hiroshima University, Hiroshima, Japan (No. IRINHI66). The 7th edition of the TNM classification system was used to determine tumor staging [17].
For quantitative reverse transcription polymerase chain reaction (qRT-PCR), 12 pairs of tumor tissue and normal tissue specimens, which were immediately frozen in liquid nitrogen and stored at − 80°C after surgical removal, were used.
For immunohistochemistry, formalin-fixed, paraffin-embedded tissues from 87 RCC patients were used. Two tumor blocks in each patient, including the tumor and the tumor with non-neoplastic epithelial tissue, were evaluated by immunohistochemical staining.
Three RCC cell lines, including 786-O, ACHN, Caki-1 were used for the in vitro experiments. Cells were maintenance cultured in RPMI-1640 (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) plus 10% fetal bovine serum (FBS) (BioWhittaker, Walkersville, MD, USA) in a humidified incubator at 37°C with 5% CO2. All cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).
TCGA databases analysis
The RNA-Seq expression of target genes and clinicopathologic data of three cohorts, clear cell RCC (KIRC), papillary RCC (KIRP), and chromophobe RCC (KIRH), were downloaded from http://firebrowse.org/. The correlation between TDO2 and PDL1 gene expression and the mutation status of the top ten genes in clear cell RCC was explored with TIMER2.0, http://timer.comp-genomics.org/. The top ten mutated genes in clear cell RCC were retrieved from the NCI genome database (https://portal.gdc.cancer.gov/).
qRT-PCR analysis
Total RNA was isolated using ISOGEN (Nippon Gene, Toyama, Japan), and 1 µg RNA was used to synthesize cDNA with a PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). PCR was performed with a CFX Connect real-time PCR detection system (Bio-Rad) using the SYBR Green PCR Core Reagents Kit (Applied Biosystems; Thermo Fisher Scientific, USA). The 2−ΔCT method was used to calculate the relative expression levels as previously described [12]. ACTB served as an internal control.
Immunohistochemical analysis
Sections of 3 µm thickness were used for immunohistochemical analysis. The immunohistochemical staining procedures were performed as previously described [12]. The expression of TDO2 was scored as described in the previous study, which combined the intensity (1+, 2+, 3+) and the percentage (from 0 to 100%) of tumor cells expressing TDO2 [12]. Immunohistochemical staining of ALDH1, CD44, CD133, EGFR, HER2, p53, and PDL1 was performed as previously described [18]. Primary antibodies and dilution ratios are described in detail in Table S1.
Western blot analysis
Cell pellets were lysed in RIPA buffer (50 mM Tris, pH 7.4, 125 mM NaCl, 0.1% NP40, 5 mM EDTA and protease inhibitor cocktail [cOmplete™, Roche]). Western blot procedures were carried out as previously described [19]. The primary antibodies are listed in Table S1. Immunocomplexes were detected with an ECL Western Blot Detection System (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). GAPDH was presented as an internal control.
RNA interference
Short interfering RNA (siRNA) oligonucleotides targeting TDO2 and a negative control were purchased from Invitrogen (Carlsbad, CA, USA). We used two different TDO2 siRNA oligonucleotide sequences: siRNA1: 5′-AUACCUUGUACCUAUCACUCACAGU-3′ and siRNA2: 5′-CCCGACACUGGAUACCGAAGAUGAA-3′. Transfection was performed using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s protocol.
CRISPR-Cas9
To generate PTEN knockout ACHN and Caki-1 cells, we used All-in-One lentivector pLenti-U6-sgRNA-SFFVCas9-2A-Puro CRISPR/Cas9 (Cat. 380721110703, ABM Inc., Richmond, BC, Canada) as previously described [20]. The PTEN-sgRNA sequence was TGGGAATAGTTACTCCC. PTEN knockout ACHN and Caki-1 cells were selected and maintenance cultured in RPMI plus 10% FBS containing 2 µg/mL puromycin.
Proliferation, colony formation, and invasion assays
To examine the cell growth, 3000 cells were plated per well in 96-well plates. Cell growth was checked after 1, 2, and 4 days by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.
To generate colonies, 500 cells per well were seeded in 6-well plates with 2 mL RPMI 1640 plus 10% FBS. The plates were incubated at 37°C in 5% CO2. The media was changed every 2–3 days. The numbers of colonies were counted after 14 days.
In vitro invasion assays were performed as previously described [21]. Cells were seeded at 10,000 cells in RPMI 1640 without FBS in the upper chamber of a culture insert (8-µm pore size; Corning, NY, USA) covered with 50 µL Matrigel (1 µg/mL). The bottom of the culture insert was embedded in RPMI 1640 with 10% FBS. After 24 hours, the invaded cells on the lower surface of the insert were stained with CyQuant GR dye to determine the number of cells. Three independent experiments were carried out. The mean and SE were calculated for each of the experiments.
Statistical methods
Associations between clinicopathologic parameters and TDO2 expression in TCGA datasets were examined by Wilcox/Kruskal-Wallis test. Receiver operating characteristic (ROC) curve analysis was used to determine the cut-off value for the TDO2 expression score that correlated with clinicopathologic features. Correlations between TDO2 staining and clinicopathologic features and/or and various molecules were checked by Chi-square test. The Kaplan-Meier method was performed to examine the survival of patients with high and low TDO2 expression. The differences in the intergroup comparisons were tested by Student t-test. A p value of < 0.05 was considered to indicate statistical significance.
Statement
All methods and experiments were carried out in accordance with relevant guidelines and regulations.