Cell culture

Human nasopharyngeal epithelial cells NP69SV40T (NECs) (Catalog No. CL-0804; Pricella, Wuhan, China) were cultured in a cell-specific medium (Catalog No. CM-0804; Pricella). Human NPC cell lines HNE2 (Catalog No. IM-H434; Immocell, Xiamen, China) and HK-1 (Catalog No. CC0708; Cellcook, Guangzhou, China) were cultured in Dulbecco’s modified eagle medium (Thermo, Waltham, MA, USA) or Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Thermo) and 1% penicillin-streptomycin (P/S, Pricella), respectively. Human THP-1 monocytes (Catalog No. CL-0233; Pricella) were cultured in RPMI-1640 supplemented with 10% FBS, 0.05mM β-mercaptoethanol (Sigma), and 1% P/S. All cells were incubated in a 37 °C incubator (Thermo) with 5% CO2 environment.

Mφ polarization

To induce M0-type Mφs, about 1 × 106 THP-1 monocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 185 ng/mL, Sigma) for 6 hours. To induce M2-type polarization, THP-1 monocyte-derived M0-type Mφs were subjected to interleukin (IL)-4 (20 ng/mL, Sigma) and IL-13 (20 ng/mL, Sigma) stimulation for 48 h.

Cell transfection

THP-1 monocytes were transfected with different small interfering RNAs (siRNAs) (si-NC, si-IRG1#1, si-IRG1#2, si-IRG1#3, si-TET2#1, si-TET2#2, or si-TET2#3) (GenePharma, Shanghai, China) using the HiPerFect Transfection Reagent (Qiagen, Hilden, Germany) in an Opti-MEM serum-free medium. Transfected cells were cultured in the appropriate medium for 24 h.

Cell co-culture

To analyze the influence of HK-1 cells on the polarization of Mφs, M0 type Mφs obtained by stimulating THP1 cells with PMA were used for co-culture. The experiment was divided into the following 4 groups: control (mono-cultured M0-type Mφs), M2 stimulation (mono-cultured M0-type Mφs stimulated with IL-4 and IL-13), NEC co-culture (M0-type Mφs co-cultured with NECs), and NPC co-culture (M0-type Mφs co-cultured with HK-1 cells). A non-contact co-culture transwell® system (Catalog No. 3450, Corning, USA) was employed to construct a co-culture system of NPC cells or NECs and Mφ.

To investigate whether IRG1-induced ITA production in TAMs mediates NPC cell malignant behaviors, the experiment was divided into the following 5 groups: control, ITA, TAM-si-NC, TAM-si-IRG1, and TAM-si-IRG1 + ITA groups. The ITA solution (5 mM) (Abmole, Shanghai, China) was prepared by dissolving 5 mg of ITA in 7.6864 mL of double-distilled water. For the control group, NPC cells (about 1 × 104/compartment) were seeded into the upper compartments and cultured with a normal medium added with phosphate-buffered saline (PBS). For the ITA group, NPC cells (about 1 × 104/compartment) were seeded into the upper compartments and stimulated with ITA (125 µM). For the TAM-si-NC and TAM-si-IRG1 groups, the upper compartments were cultured the same as the control group, but the lower compartments were cultured with TAMs transfected with si-NC or si-IRG1. For the TAM-si-IRG1 + ITA group, the upper compartments were cultured as in the ITA group, but the lower compartments were cultured as in the TAM-si-IRG1 group. The amount of PBS was the same as ITA.

For analysis of the function of TET2, M0-type Mφs were divided into the following groups: control, ITA, si-TET2, and si-TET2 + ITA groups. For the control group, PBS-treated NPC cells were co-cultured with M0-type Mφs. For the ITA group, ITA-stimulated NPC cells were co-cultured with M0-type Mφs. For the si-TET2 group, PBS-treated NPC cells were co-cultured with si-TET2-transfected M0-type Mφs. For the si-TET2 + ITA group, ITA-stimulated NPC cells were co-cultured with si-TET2-transfected M0-type Mφs. Flow cytometry assay.

Harvested Mφs were made into single-cell suspensions by trypsin digestion. After washing, Mφs were stained with CD206 APC (Catalog No. 141714, Biolegend, San Diego, California, USA) on ice away from light according to the manufacturer’s instructions. Flow cytometry analysis was performed on a BD FACSCanto II system (BD Biosciences, Frank, New Jersey, USA) and data were analyzed with FlowJo v10 software (FlowJo, Ashland, OR, USA).

Western blot

Proteins were isolated using RIPA buffer (Catalog No. P0013C, Beyotime, Shanghai, China), and the samples were quantified using the BCA kit (Catalog No. 23227). Equal amounts of total protein were separated and transferred to PVDF membranes. The membrane was blocked with 5% nonfat milk, followed by incubation overnight at 4 °C with antibodies against CD206 (Catalog No. FNab09812, Fine Biotech Co., Ltd, Wuhan, China), arginase-1 (Arg-1) (Catalog No. PA1783, Boster, Wuhan, China), IRG1 (Catalog No. 17805 S, Cell Signaling Technology, USA), β-actin (Catalog No. FNab00869, Fine Biotech Co., Ltd), and TET2 (Catalog No. orb537560, Biorbyt, Wuhan, China). After incubation with secondary antibodies, an ECL kit (Tanon, Shanghai, China) was utilized for the detection of protein bands.

Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-6, IL-10, and vascular endothelial growth factor (VEGF) in different culture media were estimated by the corresponding reagent kit as described by the manufacturer. Information on the kits used for this experiment is shown in Table 1.

Table 1 Information on the kits used for ELISAGas chromatography-mass spectrometry (GC-MS)

The levels of ITA in cell culture supernatant samples were measured according to a previous report [14]. Extracellular metabolites were extracted using the ice-cold methanol/water plus internal standard mixture (5:1, v/v). For quantification, GC-MS measurements were performed in selected ion monitoring mode on an Agilent 7890 A GC coupled to an Agilent 5975 C inert XL Mass Selective Detector (Agilent Technologies, Santa Clara, California, USA) using the following masses: m/z 301.2; 343.2; 358.2 (dwell times: 60 ms).

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted using Trizol (Catalog No. 15596-026, Ambion, Austin, Texas, USA). Total RNA (2 µg) was reverse transcribed using the Primescript™ RT reagent kit (Catalog No. RR047A, Takala, Dalian, China). The reaction of qPCR was executed with the TB Green® Premix Ex Taq™ II (Catalog No. RR820A, Takara). Relative mRNA levels were computed by the 2−ΔΔCt method [15]. Sequences for all primers are provided in Table 2.

Table 2 The sequences of all primers utilized for RT-qPCRColony formation assay

NPC cells were co-cultured with TAMs for 14 days. Fixation in 4% paraformaldehyde (Solarbio, Beijing, China) and then staining with 0.1% crystal violet (Solarbio) was performed on colonies. Images were photographed and counted per four fields under an Olympus BX51 microscope (Olympus, Tokyo, Japan).

Wound healing assay

NPC cells were plated in the upper compartments. After 24 h, a scraped cell-free area was made using a micropipette tip (200 µL) and serum-free medium was added, and the lower compartments were seeded with TAMs. Wound closure was observed and photographed at 0 and 24 h, followed by analyzing using the ImageJ software.

Transwell invasion assay

The upper compartment was added with 100 µL of Matrigel (BD Biosciences) and incubated for 4 hours at 37 °C. NPC cells (1 × 104 cells) resuspended in 200 µL of serum-free medium were seeded in the upper compartments, and the upper compartments were seeded with TAMs. After 24 h, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The cells were pictured by a microscope and counted using ImageJ.

T-cell functional assay

CD8+ T cells were generated from the spleen of OT-I mice using the EasySep™ mouse CD8+ T-cell isolation kit (Catalog No. 19853, STEMCELL, USA), followed by stimulating with anti-CD28 (5 mg/mL, Catalog No. BE0015-1, Bioxcell, Beijing, China) and anti-CD3 (0.5 mg/mL, Catalog No. BE0001-1, Bioxcell) pre-coated plates for 2 days and maintained in RPMI-1640 medium with 2-mercaptoethanol (50 mM, Catalog No. M6250, Sigma) and 1% ITS (50 mM, Catalog No. I3146, Sigma). T cells were expanded with IL-2 (0.4 ng/mL, Catalog No. 402-ML-020, R&D Syetems, Minnesota, USA). SIINFEKL peptide (Catalog No. HY-P1489, MCE, USA) was overexpressed in NPC cells in order to work as target cells.

For the T cell killing assay, NPC cells expressing SIINFEKL were co-cultured with CD8+ T treated with different conditioned medium (CM) from control, ITA, TAM-si-NC, TAM-si-IRG1, and TAM-si-IRG1 + ITA groups and allowed to growth for 2 days. For apoptosis analysis, NPC cells were double-stained with annexin V-FITC and propidium iodide (Catalog No. C1062L, Beyotime) according to the manufacturer’s instructions, and staining was detected using a BD FACSCanto II system flow cytometer (BD Biosciences).

For the production of luciferase reporter cells, 293T cells were co-transfected with pHIV-luc-zsgreen (Catalog No. 39196, Addgene, Massachusetts, USA), psPAX2 (Catalog No. 12260, Addgene), and pMD2.G (Catalog No. 12259, Addgene) plasmids. Seventy-two hours later, lentivirus was collected and filtered (0.45 mM filter). Lentivirus with pHIV-luc-zsgreen was utilized for the infection of SIINFEKL-expressing NPC cells. For analyzing the killing of T cells, the medium was removed after co-culturing target cells with T cells. Target cells were incubated individually in the blank group. D-luciferin (150 mg/mL, Sigma) was then added and incubated for 10 min. Measurement of luciferase activity was performed by a FLUOstar OPTIMA microplate reader (BMG LABTECH, Germany). Calculation of T cell killing capacity was as follows: cell lysis (%) = (value blank-value detection)/value blank.

For the detection of interferon-gamma (IFN-γ) levels in supernatants, the ELISA MAX standard set mouse IFN-γ kit (Catalog No. MBS262163, MyBiosource Inc., California, USA) was used in accordance with the operating procedures.

Mφ phagocytosis

For the phagocytosis analysis, Mφs in control, TAM-si-NC, TAM-si-IRG1, and TAM-si-IRG1 + ITA groups were cultured for 24 h, followed by incubation with NPC cells labeled with MitoTracker Deep Red for 10 min. The control group was set up with the mono-culture of M0-type Mφs, and the other groups were consistent with the co-cultures described above. After washing, the fluorescent fractions within the Mφs were quantified by flow cytometry.

Animal experiments

All animal experiments were performed on 4-6-week-old mice (Gem Pharmatech (Nanjing, China), including huHSC-NCG (n = 24) and BALB/c (n = 12) mice. Mice were maintained under specific pathogen-free conditions a 12-hour light/dark cycle, room temperature of 23 ± 2 °C, and humidity of 30–70%. All procedures were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Xiangya Hospital, Central South University.

For subcutaneous xenograft experiments in huHSC-NCG mice, HK-1 cells (1 × 106 cells) were implanted in the right flank of huHSC-NCG mice. Fifteen days after the injection, huHSC-NCG mice were treated according to the following groupings: control (vehicle + isotype), ITA (ITA + isotype), anti-programmed cell death ligand-1 (PD-L1) (vehicle + anti-PD-L1), and ITA + anti-PD-L1 (n = 6 mice/group). Mice received intraperitoneal injections of vector/ITA and isotype/anti-PD-L1 treatments alternating daily. Mice were treated with 50 mg/kg ITA, and PBS was used as vehicle control. The isotype control (Catalog No. BE0089, Bioxcell) and anti-PD-L1 (Catalog No. BE0146 l, Bioxcell) were conducted (100 µg/mouse). For subcutaneous-tumor xenograft models in BALB/c mice, HK-1 cells (1 × 106 cells) were implanted in their right flank (n = 6 mice/group). Fifteen days after the injection, mice were treated based on the following groupings: control (vehicle) and ITA. Tumor sizes were measured every 4 days from day 12. Volume estimation was made according to the formula: 1/2 × (length×width2). After 50 days of cells inoculation, all mice were sacrificed and tumor tissues were collected for further analysis.

Immunofluorescence (IF) assay

Tissue samples were blocked with 5% bovine serum albumin for 30 minutes at room temperature, followed by overnight incubation at 4°C with the primary antibody. The fluorescence-conjugated secondary antibody was then incubated for 1 hour at room temperature in the dark. The primary antibodies included anti-F4/80 (Catalog No. FNab02922, Fine Biotech Co., Ltd) and anti-CD163 (Catalog No. A72933, EpiGentek, USA) antibodies. Visualization of cell nuclei was performed using 4’-6-diamidino-2-phenylindole(Beyotime). Confocal microscope (LSM710, Zeiss, Jena, Germany) was utilized for the acquisition of fluorescence images.

Immunohistochemistry (IHC)

Paraffin-embedded slides were deparaffinized, rehydrated and then subjected to antigen retrieval. Endogenous peroxidase was then executed using 3% H2O2 solution. Following blocking with 10% goat serum, the tissues were incubated with a primary antibody against CD8 (Catalog No. LS-C414175, LSBio, USA) at 4°C overnight. After incubation with a biotin-conjugated secondary antibody, the immunoreactive protein was detected by 3,3’-diaminobenzidine staining. Images of representative areas were taken using a microscope.

Dot-blot assay

M0-type Mφs were treated with low, medium, and high concentrations of ITA, respectively. The collected cells were subjected to genomic DNA extraction by a genomic DNA purification kit (Catalog No. K0512, Thermo). The extracted DNA (1 µg) was mixed with 6 × SSC (200 µL), followed by denaturation at 100 °C for 10 min. Spots were transferred to nitrocellulose membranes and dried. 5hmC (Catalog No. 39769, Active Motif, Carlsbad, CA) levels in cells were analyzed using standard protein blot analysis.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. Generally, a two-tailed t-test was utilized for the comparison of between 2 groups, and a one-way analysis of variance with Tukey’s post hoc test was used for multiple comparisons. The data from at least triplicate independent experiments were are expressed as standard error of the mean (SEM). P < 0.05 was considered statistically significant.