Cell Culture

Mouse forestomach carcinoma (MFC) (Item No. ICell-M035) cells were purchased from iCell Bioscience Inc. (Shanghai, China). Human gastric cancer cell lines (NCI-N87) (Item No. CL-0169) were purchased from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). Cells were cultured in RPMI1640 complete medium containing 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin, and 100 U/mL penicillin.

Isolation of Primary PBMC and T lymphocytes

Primary PBMC and T lymphocytes were isolated by density centrifugation using mouse peripheral blood mononuclear cell isolation solution KIT (No. LDS1090, Tbdscience, China) and mouse peripheral blood lymphocyte separation KIT (No. LTS1092, Tbdscience, China), respectively. In brief, 2 mL of mouse blood samples were slowly added to 2 mL of the solution and centrifuged for 30 min at 450 g. After centrifugation, the centrifuge tube was divided into four layers from top to bottom. The ring-shaped milky mononuclear cell layer, or lymphocyte layer, in the centrifuge tube was carefully aspirated and transferred to a new centrifuge tube, and 10 mL washing solution was added. The cells were centrifuged at 250 g for 10 min, the supernatant was discarded, repeated twice, and the resulting cells were resuspended.

Cell Co-culture and Cell Grouping

The MFC, NCI-N87, PBMC, and T cells at the logarithmic growth stage were inoculated into 96-well plates with a density of 4 × 104/mL (100 μL/well), and cultured in a 5% CO2 incubator at 37 °C and 95% humidity. A co-culture system between PBMC and GC cells was developed. The ratio of NCI-N87 or MFC to PBMC was 1:1, and the total number of cells per well was 5 × 104, and then co-cultured for 24 h under the above conditions. 1-MT (No. HY-16724, MedChemExpress, USA) and LY294002 (No. HY-10108, MedChemExpress, USA) were purchased from MedChemExpress.

Groups 1: (1) control group (MFC cells or NCI-N87 cells); (2) GC cells (MFC cells or NCI-N87 cells) + IDO inhibitors (adding 1-MT at the same time, 5 mmol/L, incubation for 24 h) group; (3) GC cells (MFC cells or NCI-N87 cells) + PBMC co-culture (MFC or NCI-N87 to PBMC ratio 1:1, incubation for 24 h) group; (4) GC cells (MFC cells or NCI-N87 cells) + PBMC co-culture + IDO inhibitor (1-MT, 5 mmol/L, incubation for 24 h) group, 4 replicates per group, and constant temperature culture at 37 °C and 5% CO2.

Groups 2: (1) T cell group; (2) T cell + oe-IDO group; and (3) T cell + oe-IDO + LY294002 (PI3K pathway inhibitor) group.

CCK-8 Assay Detection for Cell Proliferation

MFC, NCI-N87, PBMC, and T cells at the logarithmic growth stage were taken and washed with PBS and collected after trypsin digestion. After PBMC suspension, cell suspension was collected and centrifuged at 250 g for 5 min. The supernatant was removed, and an appropriate amount of medium was added to make a single-cell suspension. The cell density was adjusted to 2 × 105 cells/well, and 2 mL/well was inoculated into a 6-well plate and incubated at 37 °C with 5% CO2. After co-culture for 24 h, the supernatant (PBMC) was discarded, and CCK-8 solutions with a volume of 110 μL were added to each sample and incubated for 2 h at 37 °C and 5% CO2. At 450 nm wavelength, the optical density (OD) value of each hole was detected with an enzyme labeler.

Flow Cytometry Detection for Cell Apoptosis

Cell apoptosis was detected using flow cytometry by the Annexin V-APC double staining apoptosis detection kit (Item No. KGA1030, Jiangsu KeyGEN BioTECH Corp., Ltd., China). After co-culture, the supernatant (PBMC) was discarded. The cells were washed with 1× PBS and suspended in 100 μL binding buffer. Add 5 μL Annexin V-FITC and 5 μL PI, and incubate at room temperature in the dark for 10 to 15 min. Finally, 500 μL of binding buffer was added to the sample, and the apoptosis rate was detected by flow cytometry (Cytoflex, Beckman, USA) within 1 h.

Enzyme-linked Immunosorbent Assay (ELISA)

The contents of TNF-α, IL-1β, IL-6, IL-8, and INF-γ in the cell supernatant were detected by ELISA kits according to the instructions. In brief, except for blank wells, the sample wells were incubated with horseradish peroxidase (HRP)-labeled antibodies for 60 min at 37 °C, and then 50 μL of each substrate A and B were added and incubated at 37 °C in the dark for 15 min. Finally, the OD values of each well were determined at a wavelength of 450 nm. Mouse TNF-α ELISA KIT (Item No. ZC-390240), Mouse IL-1β ELISA KIT (Item No. ZC-37974), Mouse IL-6 ELISA KIT (Item No. ZC-37988), Mouse IL-8 ELISA KIT (Item No. ZC-37953), Mouse IFN-γ ELISA KIT (Item No. ZC-37905), and Mouse IDO ELISA KIT (Item No. ZC-39078) were purchased from Shanghai ZCIBIO Technology Co., Ltd. (China).

Western Blot

The proteins associated with the PI3K/Akt/mTOR signaling pathway in bladder cancer were detected by Western blot. The cell culture supernatant was collected, and the PBMC cells were obtained by centrifugation, quantified by bicinchoninic acid assay (BCA) protein concentration detection kit, and separated by polyacrylamide gel electrophoresis. After electrophoresis, the membrane was transferred to polyvinylidene fluoride (PVDF) membrane. After sealing 5% skim milk powder, add the primary antibodies, refrigerate at 4 °C overnight, wash the film with TBST after rewarming, add the secondary antibody and incubate for 2 h. The images were collected by gel imager, and the results were analyzed by Quantity One software. Antibody information: Goat Anti-Rabbit IgG (H + L) HRP, No. S0001, 1:5000, Affinity, China; β-actin, No. AC026, 1:50000, Abclonal, China; AKT, No. A17909, 1:2000, Abclonal, China; mTOR, No. A2445, 1:1000, Abclonal, China; p-AKT, No. AP0098, 1:1000, Abclonal, China; PI3K, No. AF6241, 1:2000, Affinity, China; p-mTOR, No. AP0094, 1:1000, Abclonal, China; p-PI3K, No. AF3241, 1:2000, Affinity, China.

Flow Cytometry Detection for T cell Proportion

The cell culture supernatant was collected, and the PBMC cells were obtained by centrifugation. Setting groups 1: GC cells +PBMC co-culture group (MFC to PBMC ratio 1:1, incubation for 24 h), GC cells +PBMC co-culture +IDO inhibitor group (1-MT, 5 mmol/L, incubation for 24 h). Groups 2: T cell group, T cell+oe-IDO group, and T cell+oe-IDO + LY294002 (PI3K pathway inhibitor) group. The cell density was adjusted to 3 × 105/well, 2 mL/well was inoculated in a 6-well plate, and incubated at 37 °C and 5% CO2. 24 h later, according to the group, the suspension was centrifuged 250 g for 5 min to obtain cell precipitation. 100 μL PBS suspension cells were added to each tube labeled with CD4, CD8, and CD25 antibodies (FITC anti-mouse CD4, No. 116003, Biolegend, USA; APC anti-mouse CD8a, No. 162305, Biolegend, USA; PE anti-mouse/rat/human FOXP3, No. 320007, Biolegend, USA; PerCP anti-mouse CD25, No. 102027, Biolegend, USA), incubated at 4 °C for 30 min in dark light, centrifuged at 300 g for 5 min, and the supernatant was discarded. 1 × True Nuclear Fixation Concentrate 500 μL was added to each tube and incubated at room temperature for 50 min. Centrifuge at 350 g for 5 min, then discard the supernatant. Each tube was washed twice with 250 mL 1 × True Nuclear Perm, centrifuged at 350 g for 5 min, 100 μL 1× True Nuclear Perm resuspension cells were added to each tube with the FOXP3 antibody label, incubated at 4 °C for 30 min, centrifuged at 350 g for 5 min, and the supernatant was discarded. Each tube was washed with 250 μL 1× True Nuclear Perm, centrifuged at 350 g for 5 min, the supernatant was discarded, and 300 μL 1× True Nuclear Perm resuspension cells were detected and analyzed by computer.

Establishment of GC Mouse Xenograft Model with MFC Cells

Twelve C57BL/6 mice were used in the tumor formation experiment of C57BL/6 mice to detect the tumor growth effect of GC in vivo. The sample size was calculated according to G*prower 3.1.9.7. At 80% efficacy, the total sample size required 12 mice to obtain a significant difference of p = 0.05. MFC cells were washed on a super-clean workbench with PBS and digested by 0.25% trypsin, then blown and mixed with a pipette gun and placed in a centrifuge with the centrifuge speed set at 1000 r/min and centrifuge time at 5 min. After centrifugation, the cell density was adjusted to 5 × 105/mL. Twelve C57BL/6 mice were disinfected with 75% ethanol in the right armpit and inoculated with 0.2 mL of MFC cell suspension. 5–6 days after the establishment of the mouse model, nodular nodules in the right axillary inoculation site of the mice could be seen by the naked eye, and the nodules in the mice reached 5 mm × 5 mm on the 7th day after the inoculation of MFC cells. In the MFC + IDO inhibitor (1-MT) group, IDO inhibitor was intraperitoneally injected at 100 mg/kg once every 7 days for 14 days. 21 days later, the C57BL/6 mice were sacrificed by the cervical detachment method. The tumor was taken out, the weight of the tumor was measured, photographs were taken, and tumor samples were collected for detection. This study was approved by the Experimental Ethics Committee of West China Hospital of Sichuan University and met the provisions of national experimental animal welfare ethics (Ethics number: 20231030001).

Immunohistochemistry

The tumor samples were sectioned at a thickness of 2 mm and mounted on a coated slide. After incubation at 60 °C for 30 min, heat-induced antigen repair was performed. The tissues were dewaxed and rehydrated by soaking in xylene and a series of ethanol in different concentrations. The endogenous peroxidase activity was blocked by H2O2, and goat serum sealer was added to the mixture at room temperature for 20 min. Ki-67, CD4+, CD8+, and CD25+ antibodies were added at 4 °C overnight. Wash with PBS for 3 times, 5 min each time; drop the second antibody, 37 °C for 30 min; wash with PBS for 3 times, 5 min each time. The antigen complex was shown by binding goat serum with the Envision Flex Kit. Image-Pro Plus software was used to digitally quantify the percentage of stained area in total patch area and staining intensity.

Statistical Analysis

SPSS 26.0 software was used for statistical analysis, and the data were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to evaluate the differences between multiple groups. A difference of p < 0.05 was considered statistically significant.