Cell lines, mice and drugs
A mouse colon cancer CT-26 cell line was kept in the laboratory (Xinxiang Medical University, Xinxiang, China). IDO inhibitor D-MT was purchased from Merck KGaA. Oxaliplatin was purchased from Hengrui Pharmaceutical Co., Ltd. The Balb/c mice (6-8 weeks old) were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were fed in pathogen-free conditions, housed under a 12 h light/dark cycle at a temperature of 25±2˚C. The animal study was approved by the Ethics Committee of Xinxiang Medical University.
Animal experiments
The CT-26 cells were adjusted to 1x107 cells/ml, and a 0.1-ml cell suspension was injected subcutaneously into the upper part of the right leg of mice to establish the colon cancer-bearing mouse model. Next, the 24 mice were randomly divided into the PBS, D-MT, oxaliplatin and D-MT + oxaliplatin groups. On the 7th day of the establishment of the model, PBS, D-MT (1 mg/mouse), oxaliplatin (50 μg/mouse), D-MT (1 mg/mouse) and oxaliplatin (50 μ g/mouse) were injected once a day for 7 days. A further 7 days after the last treatment, the tumors were separated and weighted. Finally, each tumor was divided into two parts, one was used to extract the protein for western blotting, and another was fixed in 4% formalin for immunofluorescence (IF) detection. The spleens were also separated from the mice for flow cytometry. In addition, 7 days after establishing the mouse model of colon cancer, the 40 mice were randomly divided into the PBS, D-MT, oxaliplatin and D-MT + oxaliplatin groups and administered the appropriate treatment. Mouse survival was observed and recorded every day.
All the mice were giving euthanasia by cervical dislocation after sera collection.
Western blotting
The protein of cells or tumor tissues was extracted in lysis buffer (Beyotime Institute of Biotechnology), and then the concentrations were quantified according to the instructions of the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Based on the concentration, 30 μg protein was isolated by 10% SDS-polyacrylamide separation gel before being transferred onto PVDF membranes. The membranes were then incubated with the primary antibodies [Tubulin, IDO, p-signal transducer and activator of transcription 3 (p-STAT3), signal transducer and activator of transcription 3 (STAT3), matrix metallopeptidase 2 (MMP2), cleaved caspase-3 or lc3b; Cell Signaling Technology, Inc.)], diluted according to the manufacturer’s instructions. After 2 h, the membranes were washed with 1xTBST and incubated with the secondary antibodies for 1 h. Finally, the specificity of antigen-antibody complexes was detected using enhanced chemiluminescence reagent (Cell Signaling Technology, Inc.) and the images were visualized by Fusion FX Spectra imaging system.
IF detection
Indirect IF was performed as previously described [28]. Briefly, the tumor slides were incubated with the appropriate antibody (CD3, CD4 and CD8) at 4˚C overnight in a wet box. The tumor slides were restored to 25˚C at least 30 min after being incubated with the secondary antibody for 30 min. Finally, the slides were stained with DAPI and the images were captured using a fluorescence or a laser confocal microscope. The intensities of positive cells were analyzed using a scale of 0-3+: 0, no staining identified; 1+, <25% positive cells; 2+, 25-75% positive cells; 3+, >75% positive cells.
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)
The cell apoptosis in tumor tissues was detected by TUNEL assay kit (Beyotime Institute of Biotechnology), according the manufacturer’s instructions. Briefly, the TUNEL detection solution was dropwise added onto the surface of tumor section. Following incubation in the dark for 60 min at 37˚C, the sections were washed with PBS for 10 min, 3 times. Finally, the sections were dried and sealed with anti-fluorescence quenching solution, and the positive cells were observed using the fluorescence microscope. In addition, Hoechst 33342 solution (Beyotime Institute of Biotechnology) was used for staining living cells, according to the manufacturer’s instructions.
Flow cytometry
The concentration of spleen cells was adjusted to 1x107 cells/ml, and each tube was added into 100 μl cell suspension. The cells were incubated with antibodies for CD3, CD4, CD8 and CD49b (BioLegend, Inc.) at 4˚C for 30 min. The ratio of cells was detected using flow cytometry (CytoFLEX; Beckman Coulter, Inc.).
Enzyme-linked immunosorbent assay (ELISA).
One week after the last treatment, the mice in each group were sacrificed and the sera collected. The concentration of tumor necrosis factor alpha (TNF-α) or interferon gamma (IFN-γ) was analyzed by ELISA kits (RayBiotech), according to the manufacturer’s instructions.
Statistical analysis
Measurement data are expressed as the mean ± SD of three independent experiments. Data were analyzed by SPSS 19.0 (IBM Corp.). One-way ANOVA was performed to test the difference among the different groups, and the Kaplan-Meier method with a log-rank test was used to analyze survival. P<0.05 were considered to indicate a statistically significant difference.