Cell culture
The HeLa, SiHa (human cervical cancer cell line), TC-1(HPV-16 E6/E7 and c-Ha-Ras co-transformed mouse lung epithelial cell line) and U937 (human monocyte cell line) were bought from American Type Culture Collection. HeLa, SiHa and TC-1 cells were cultured with DMEM/F12 and U937 in RPMI 1640 (HyClone Laboratories, Logan, UT, USA), containing 10% fetal bovine serum (Gibco Cell Culture, Carlsbad, CA, USA) and 1% Antibiotic-Antimycotic (Gibco Cell Culture, Carlsbad, CA, USA). Cells were passaged depending on their densities. The temperature of the incubator was stabilized at 37°C and CO2 concentration was 5%.
Co-culture of cervical cancer cells with U937 cells
To identify whether IFN-γ, rapamycin or kynurenine treated cancer cells impact the phagocytic activity and polarization of human monocyte / macrophage cell line U937, HeLa and SiHa cells were pretreated with recombinant human IFN-γ protein (rhIFN-γ, 10ng/ml, PeproTech), rapamycin (2μmol/l, Sigma), or kynurenine (500μmol/l, Sigma) for 48 hours, supernatant was discarded and cells were washed with PBS. Then, fresh medium and U937 cells were added to the plate and cervical cancer cells were co-culture directly with U937 cells for 48 hours at the ratio of 1:1. After 48 hours, cells were harvested and analyzed by FCM.
Western blot
Cells were washed with PBS for three times, and lysed with lysis buffer (Beyotime Biotechnology, Shanghai, China), containing protease inhibitor cocktail (MedChemExpress, Shanghai, China) and phosphatase inhibitor cocktail (MedChemExpress). Protein concentrations were detected using a BCA protein assay kit (Beyotime Biotechnology). After that, protein was diluted with loading buffer (Beyotime Biotechnology) and heated to 95°C for 10 minutes; denatured protein was stored at -20°C. For western blot, equal amounts of protein calculated according to the concentration were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels (Epizyme Scientific, Shanghai, China), transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA), blocked by 5% non-fat milk for 2 h at room temperature, and incubated with corresponding primary LC3B, IDO1 and GAPDH antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. The membrane was washed three times with TBS-T for 15min and incubated with HRP-linked Anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. After washing for three times with TBS-T, protein bands were wetted with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Darmstadt, Germany) and detected by Luminescent Image Analyzer LAS 4000 (FUJIFILM, Japan).
Flow cytometry (FCM)
HeLa, SiHa or U937 cells collected from wells were centrifuged at 1500 rpm for 6 min, and incubated with APC-conjugated anti-human CD45, PE-conjugated anti-human CD86, and PE/CY7-conjugated anti-human CD163, FITC-conjugated anti-human CD80, and BV421-conjugated anti-human CD206 (eBioscience, San Diego, CA, USA). Specifically, HeLa and SiHa cells were fixed, permeabilized, and then stained with APC-conjugated anti-human IDO1 antibody. After that, the cells were washed twice with PBS, and resuspended for FCM analysis. In parallel, the isotopic IgG antibodies were used as controls.
In animal experiment, tumor tissue was mechanical cut, digested with collagenase, and filtrated by sieve to prepare monoplast suspension. Cells were centrifuged at 1500 rpm for 6 min, and incubated with Percp-conjugated anti-mouse CD45, APC-conjugated anti-mouse CDF4/80, BV605-conjugated anti-mouse CD11b, FITC-conjugated anti-mouse CD80, PE-conjugated anti-mouse CD86, Pecy7-conjugated anti-mouse CD206 (Biolegend, San Diego, CA, USA)
Data were collected in FACS Calibur flow cytometer (Beckman Coulter CyAn ADP or Beckman Coulter Cytoflex, North Carolina, USA) and analyzed with FlowJo 7.6. Each experiment was performed for three times independently. Statistical analysis was performed by using isotype matched controls as references. Typically, less than 1% positive cells were permitted beyond the statistical marker in the appropriate controls.
DAP Green autophagy detection
Cells were seeded in an appropriate dish overnight. Discard the supernatant and wash the cells with culture medium once. Add the diluted DAP Green solution (0.1μmol/l, Dojindo Laboratories, Japan), incubate at 37℃ for 30 minutes. Discard the supernatant and wash the cells with culture medium twice. Then add the medium in different group and treat cells for 4 hours. Discard the supernatant, dye the nucleus with DAPI (Sigma-Aldrich, USA) for 10 minutes and wash the cells with culture medium twice. Observe fluorescence and take pictures under a fluorescence microscope (Leica, Munich, Germany). Multiple fields of view were randomly selected through fluorescence microscope observation, and then the number of autophagosomes was calculated.
Phagocytosis assays
HeLa and SiHa cells were planted in 24-well plates and treated with rhIFN-γ(10ng/ml, PeproTech), epacadostat(50nmol/L, MedChemExpress), kynurenine(500μmol/l, Sigma) or tryptophan free medium() as shown in results for 48 hours. Then cells were harvested and re-suspended in PBS supplemented with 5 μmol/l of 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE, eBioscience, San Diego, CA, USA) for 8 min at 37°C with 5% CO2. After washed with PBS, the CFSE-labelled cells were co-cultured with U937 cells for 2 hours at the ratio of 1:1, then cells were harvested and incubated with APC-conjugated CD45 antibody to label U937. The phagocytic ratio was tested by flow cytometry. CFSE+CD45+ cells were regarded as U937 cells which had swallowed CFSE+ cancer cells.
Lentivirus transfected in HeLa and SiHa cells
HeLa and SiHa cells were transfected with IDO1 overexpression lentivirus or negative control lentivirus respectively. Briefly, HeLa and SiHa cells were seeded at a density of 5 × 105 cells/well in 6-well plates and adhered overnight. At the density of 50%, cells were transfected in triplicate with lentivirus at the MOI of 1, 10, and 100. Choose appropriate MOI based on the fluorescence intensity. Puromycin was continuously used to filter the successfully transfected cells for 2 weeks until the purity was more than 90%. TC-1 cells were transfected with mouse IDO1 lentivirus in the same way.
High-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS) quantification of Tryptophan and kynurenine
Qualitative assessment of tryptophan metabolites by MS (metabolomics) was performed by the Institute of Biomedical Sciences, Fudan University. HeLa and SiHa cells treated with IFN-γ or transfected with IDO1 overexpression lentivirus were washed with PBS, trypsinized and collected in 1.5ml centrifuge tube. Cells were lysed by adding 300μl deionized water, freezed and thawed for three times. Add 900μl methanol to the lysis solution, well mixed and then centrifuged at 20000g for 10 minutes. Collected the supernatant and volatilized to get dry power. Samples and tryptophan/kynurenine standards were detected in TSQ-Vantage triple quadrupole mass spectrometer (Thermo), using a ShimazuLC (LC-20AB pump) system and a C18 column (250mm×2.1mm I.D., 3μm particle size, ULTIMATE). Selected reaction monitoring (SRM) scan mode was applied, and transitions evaluated were Trp, 205.09–188.0, Kyn, 209.063–94.2. The results were analyzed in Analyst Software.
Animal mode and treatment
Animals used in this study were approved by the Ethical Committee of the Obstetrics and Gynecology Hospital, Fudan University. Four-week-old female C57BL/6 mice were obtained from Animal Laboratory (Shanghai, China). After one week’s adaptation, 16 mice were randomly divided into two groups, injected subcutaneously on the back with 200μl TC-1 cells (5×10^6 cell per mouse, IDO1 overexpression or negative lentivirus transfected). When palpable, tumors were measured every other day and tumor volume was calculated as 1/2(length*width*width). Mice were euthanized when the tumor width was over 20mm or ulceration of tumor was observed. The tumor tissue was minced, digested and subjected for FCM.
In epacadostat experiment, 28 mice were randomly divided into four groups, injected subcutaneously on the back with 200μl TC-1 cells (5×10^6 cell per mouse, IFN-γ overexpression+PBS, IFN-γ overexpression+epacadostat, negative lentivirus+PBS, negative lentivirus+epacadostat). In IFN-γ and kynurenine group, 26 mice were randomly divided into three groups, injected subcutaneously on the back with 200μl TC-1 cells (5×10^6 cell per mouse). Mice in different groups were intraperitoneal injected with epacadostat (50mg/kg), IFN-γ (100ng/animal), or kynurenine (100mg/kg) every day after the tumor was palpable. Tumor volume was detected every three days.
Statistical analysis
All of the data are shown as mean ± SEM. Comparison between controls and treatments was analyzed by Student’s t-test. Comparison between groups more than two was analyzed by One-way ANOVA. All analyses were performed using Graphpad Prism software (Graphpad Software, San Diego, CA, USA) for Windows. Differences were considered to be significant at P<0.05.