5.1. Animal model experimental design
Mice were eight-week-old male C57-BL/6J-219 mice (18–20 g) (Beijing Vital River Laboratory, Beijing, China) and IL-10+/− and IL-10F/+ mice (Stock code 004333, Jackson Laboratory, Bar Harbor, ME, USA). The experiments were performed after protocol approval by the ethics committee of China-US Hormel Cancer Institute, Henan, China and were conducted in accordance with the current guidelines for laboratory animal care. C57-BL/6J-219 (Access No: CUHCI2015006) and IL-10-mutant mice (Access No: CUHCI2015007) were randomly assigned to five groups of 10 animals each (n = 10). IL-10−/− and IL-10F/+ heterozygous mice were crossed to obtain IL-10+/+, IL-10−/+ and IL-10−/− mutant mice. After one week of acclimation, mice were administered ethanol (5 g/kg/day) in drinking water for two weeks prior to infection with H. pylori, and alcohol administration was continued throughout the experiment. To facilitate H. pylori colonization, pantoprazole (20 mg/kg) was administered by gavage 3 times to lower gastric acidity. Each mouse was administered a suspension of H. pylori SS1 strain containing 108 CFUs/mL by gavage 3 times per week. Mice (n = 5) were euthanized on consecutive weeks of 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40 weeks. A control group was maintained without any treatment. Doses of alcohol ranging from 0.5–5.0 g/kg/day were administered to mice (n = 5) to determine the optimal alcohol dose to induce gastric tumorigenesis.
5.2. Patients and gastroendoscopy
All gastric patients were subjected to gastroendoscopy and examination in the Second Affiliated Zhengzhou University Hospital and Henan Cancer Hospital (Zhengzhou, Henan, China). Blood samples and tissue biopsies were obtained from consenting patients from the antral and corpus portions of the stomach during gastrointestinal endoscopy and gastric surgery. The patients who donated the primary tumors were completely informed and provided written consent (Access No: CUHCI2015009).
5.3. Immunohistochemistry (IHC)
Paraffin-embedded gastric tissues were analyzed by immunohistochemistry (IHC). Serial sections (4–6 mm each) were deparaffinized in xylene and rehydrated in an alcohol concentration gradient and evaluated with antibodies to detect H. pylori (1:100), pepsinogen I and II (1:100 each), MPO (1:100), F4/80 (1:100), vimentin (1:100), PCK-26 (1:100), NKX6.3 (1:100), IL-10 (1:100) and SMA-α (1:100). Sections were subsequently incubated with their respective secondary antibodies for 30 minutes at room temperature. The signal was visualized with peroxidase-labeled streptavidin complexes by DAB, and the sections were briefly counterstained with hematoxylin. The immunohistochemical localization pattern was also recorded by digital imaging (Nikon Ti-DS, Japan). ImageScope (11.1.1.752) software program was used, and the labeling index was calculated as a percentage of positive cells relative to the total number of counted cells.
5.4. Quantitative enzyme and activity assays
Quantitative enzyme assays included measurement of Nitrite (Cayman, Michigan, USA, Cat number 780001), Aldehyde (Abcam, Cambridge, MA, USA, Cat number ab138882), urease (Cat number C013-2), malondialdehyde (MDA; Cat number A003-1), myeloperoxidase (MPO; Cat number A044), carbonyl (Cat number A087), lipoperoxidase (LPO), catalase (Cat number A007-1) and alcohol dehydrogenase (ADH; Cat number A083-1) were performed according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
5.5. Enzyme-linked immunosorbent assay
Serological assessment of CD-8 (Abcam, Cambridge, MA, USA, Cat number ab 238264), H. pylori (BioCheck, San Francisco, CA, USA, Cat number BC-1051), and H. pylori CagA (MyBiosource, San Diego, CA, USA, Cat number MBS263962) were measured by enzyme-linked immunosorbent assay (ELISA). Commercial ELISA kits were used to analyze serological values of CD8, H. pylori and H. pylori CagA according to manufacturer’s instructions (Cusabio, US).
5.6. Real-time RT-PCR
Total RNA was extracted using the commercial RNA extraction kit (Ambion by Life Technologies, Van Allen Way Carsbad, USA, 92008) and cDNA synthesized by amfiRivert cDNA synthesis platinum master mix (GenDEPOT, Katy, TX, USA, Cat number R5600-200). Real-time PCR (qRT-PCR) was conducted using a 7500 FastDX (Applied Biosystems, MA, USA) and the power SYBR green PCR master mix (Applied biosystem, Warrington WA1 4SR, UK, Cat number 4367659). Primer IDs and sequences are shown in Supplementary Tables 2, 3 and 4.
5.7. Cytokine and chemokine protein measurement using a multiplex bead array.
Cytokine and chemokine protein levels in mouse (Cat number 740446) and human (Cat number 740118) serum were measured using a multiplex magnetic bead array kit (customized by BioLegend LEGENDplex, San Diego, CA, USA). The multiplex bead arrays were performed according to the manufacturer’s instructions with a minimum detectable concentration varying from 0.96–11.27 pg/mL. Legendplex (version: 7.0) software program was used to analyze the FACS data and the cytokine concentrations were calculated in pg/mL against standard values.
5.8. H. pylori CFU quantification within Mouse Stomachs and Histology
After 1, 3, or 6 weeks, mice were euthanized. Stomachs were halved longitudinally along the greater and lesser curvatures and rinsed in sterile phosphate-buffered saline. Each half was manually disrupted on ice in 750 μL of Iso-Sensitest (Oxoid, Basingstoke, UK) broth/15% glycerol. Cells were serially diluted and plated on Columbia blood agar base plates (Oxoid, Basingstoke, UK, Cat number CM0331), supplemented with 10% defibrinated horse blood (Solarbio, Beijing, CRP, Cat number S9050), and Dent supplement (Oxoid, Basingstoke, UK, Cat number SR0147E).
5.9. Urine collection and measurement (metabolic cage experiments)
Alcohol metabolism and urinary flow rate were determined by placing mice individually in metabolic cages. Mice were allowed a 3-day habituation period to adapt to the environment. Later, food and water intake, urinary flow rate, and body weight were recorded every day. Subsequently, a 12 h collection (9 p.m. to 9 a.m.) of urine was performed to obtain the urinary parameters, including volume, pH, and color. Data were analyzed for alcohol intake, metabolism, and excretion. Alcohol content in the serum and urine samples from mice and humans was measured according to the Enzychrom ethanol commercial assay kit’s instructions (BioAssay System, ECET-048, Hayward, CA, USA, Cat number ECET-048).
5.10. Mouse-derived xenograft model
A mouse-derived xenograft model was developed from gastric tumor tissue of mice treated with alcohol (Access No: CUHCI2016011). Then additional mice were subcutaneously implanted with tissues weighing 0.10-0.12 g and measuring ~3 mm. Animals were monitored periodically for their weight and tumor growth. A second passage was performed and the same protocol was followed as described above.
5.11. RNA-seq analysis
Total RNA of stomach tissues was prepared by using an RNeasy kit (QIAGEN) with an RNase free DNase set (QIAGEN). Sample preparation and sequencing were performed in the BGI (Beijing Genomics Institute, Shenzhen Beishan Industrial Zone, Shenzhen 518083, China) using an Illimina HiSeq sequencing system at 50 bp read length.
5.12. Cell culture
Jurkat T-lymphocytes, RAW264.7, NCIN87, AGS and HEK-293T cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM, RPMI-1640 media supplemented with 10% FBS (Invitrogen, Camarillo, CA, US), 100 U/mL penicillin, and 100 µg/mL streptomycin. All the cell lines were authentified for mycoplasma; with the passage number of 3-5. The cell cultures were maintained at 37 ºC under 5% CO2. Cells were seeded in 100 x 20 mm dish of DMEM/F12. Upon 70% confluence, cells were treated with acetaldehyde (500 µM), IL-1β and IL-10 (100ng/mL; Peprotech, Rocky Hill, USA, Cat number 200-01B), or infected with H. pylori strain (ATCC-43504) (1x103 bacteria/mL).
5.13. DNA constructs, transfection, and electroporation
A lentiviral expression system was used to knockdown IL-10 target protein in RAW 264.7 cells. HEK-293T cells were cultured in DMEM containing penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS, Gemini Bio-Products, Calabasas, CA). Cell cultures were maintained at 37 °C under 5% CO2 in a humidified atmosphere. Cytogenetically tested and authenticated frozen cells were thawed and maintained for approximately two months. HEK 293T cells were cultured in DMEM with 10% FBS.
For knockdown of IL-10 expression in RAW264.7 cells or HEK 293T cells, transfection was performed with shRNA IL-10, or pLKO.1-mock DNA plasmids together with packaging vectors, pMD2.0G and psPAX (Addgene Inc., Cambridge, MA). Imfectin Ploy DNA transfection reagent (GenDepot, Barker, Tx, USA, Cat number 17200-101,) was used as directed by the manufacturer’s protocols. Transfection medium was changed at 4 h post-transfection, and the cells were cultured for an additional 36 h in fresh medium. Viral particles were harvested by filtration using a 0.45 mm syringe filter. Viral infection into RAW264.7 cells occurred with polybrene (1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA, Cat number TR-1003-G) for 24 h. Cell culture medium was replaced with fresh media, and the cells were cultured for an additional 24 h after selection with Puromycin (2 μg/mL; Gemini Bio Product, Cat number = 400128P). Furthermore, Bio-Rad electroporation system was used for knocking down the expression of IL-10 in Jurkat immune cells. Electroporation pulser cuvette (BioRad Gene Pulser, Cat number XCell, 617BR105102) was used with a protocol of voltage (V) = 10, Capacitance (µF) = 25, Resistance (Ω) = infinite, cuvette = 2mm (BioRad Gene Pulser Cuvette, Cat number 165-2086). Furthermore, NCIN87 gastric cancer cells lines were cultured at 37 °C under 5% CO2 in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum. Complete NKX6.3-cDNA (gene symbol= NKX6.3, Harvard Medical School, Clone ID = HsCD00461824) was cloned into the expression vector pCMV6-Myc-DDK (Origene). NCIN87 cells were transiently transfected with expression plasmids (15 μg total DNA) in 150 x 25 mm dishes using Imfectin Ploy DNA transfection reagent (GenDepot, Barker, Tx, USA, Cat number 17200-101), according to the manufacturer’s recommendations. Stable expression of NKX6.3 was confirmed in NCIN87NKX6.3 cells by Western Blot analysis. ShRNA and overexpression plasmid sequences are shown in Supplementary Tables 3.
5.14. Immunoblotting
Cells were washed with PBS (pH 7.4) and incubated with lysis buffer. Afterwards, samples were evaluated for anti-NKX6.3 (1:250), anti-Apoptosis Western-blot Cocktail (1:1000), anti-OXPHOS (1:1000);, anti-Pgm2 (1:250), anti-pkm2 (1:1000) and anti-GAPDH (1:250) as described [38, 39].
5.15. Tissue harvesting for cell sorting and FACS analysis
Mice stomach tissue was minced into 3-4 pieces and pressed firmly to force the fragments apart; this allowed cells to pass through a wire mesh (70 µM Nylon; Corning, Durham, USA, Cat number 431751,). The cell suspension was centrifuged at 250 x g for five minutes at room temperature. Each cell pellet was treated with ammonium chloride (0.17 M per 1 mL cell pellet) and incubated at room temperature for five minutes, followed by centrifugation at 250 x g for five minutes. The pellet was washed with PBS, and the cells were gently resuspended into Corning DMEM media. After washing with PBS containing 2% BSA, cells were incubated with a lineage cocktail containing biotinylated antibodies against CD4 (Biolegend, London, UK, Cat number 557307), CD8 (Biolegend; Cat number 553030), CD19 (Biolegend; Cat number 557398), CD11b (Biolegend; Cat number 561688), CD11c (Biolegend; Cat number 557400) and CD49b (Biolegend; Cat number 561067) at 4 °C for 30 minutes in the dark. After washing twice with PBS containing 1% FBS and 0.1% sodium azide, cell sorting was performed using a BD FACSAria II, San Jose, CA, SN: P69500099 cell sorter with BD FACSDiva Software version 8.0.2. Data were analyzed with BD FlowJo version 10 software.
5.16. Measurement of mitochondrial activity and ROS production
Jurkat T-lymphocytes cells seeded in non-tissue culture plates were stimulated as described above. MitoTracker Green (0.3uM; Molecular probes, Isnvitrogen Oregon USA, Leiden Netherland, Cat number CM-H2Xros) (for total mitochondrial mass), Total ROS (DCFDA) (0.1uM; Sigma Aldrich, St. Louis, MO, USA, Cat number D6883-250MG), and MitoSox staining (5uM; MitoSox Red mitochondrial superoxide indicator, Invitrogen by Thermo-Fisher Scientific, Cat number M36008), were performed according to manufacturer’s instructions. For flow cytometry analysis, data were acquired with a BD FACSAria II, San Jose, CA, SN: P69500099 cell sorter with BD FACSDiva Software version 8.0.2. Data were analyzed with BD FlowJo version 10 software.
5.17. Cell proliferation and viability assay
Cells (1 × 103 cells/well) were plated in 96-well plates. After 24 hours, cells were treated with the acetaldehyde (500 µM), H. pylori (1 × 103 cells), or IL-1β (100 ng/mL) and incubated at 37 ºC for 24, 48, 72, or 96 hours. IncuCyteS3 live-cell imaging system (Essen BioScience, Tokyo, Japan, IC5047) was used to monitor cell proliferation. Later, IncuCyteS3:2019 software was used to quantify cell proliferation. This cell proliferation assay was performed in six replicates.
5.18. Apoptosis and cell cycle analysis via flow cytometry
For apoptosis assessment, an Annexin V-FITC (BD Pharmingen (BD Biosciences), Franklin Lake, NJ, USA, Cat number 556419) binding assay was performed in NCIN87NKX6.3 cells stimulated with acetaldehyde (500uM; Sigma Aldrich, St. Louis, MO, USA, Cat number 402788-100ML), IL-1β (100ng/mL; Peprotech, Rocky Hill, USA, Cat number 200-01B) and H. pylori (1x103). For cell cycle analysis, the cells from each experimental group were harvested, fixed overnight in 70% ethanol, and stained with propidium iodide (50 ug/mL; BD Pharmingen (BD Biosciences), Franklin Lake, NJ, USA, Cat number 556463) and RNase (Sigma-Aldrich, St. Louis, MO, USA, Cat number R5125-250MG) for 30 minutes before analysis on a BD LSRFortessa X-20, San Jose, CA, SN: R656385195 with BD FACSDiva Software version 8.0.2. Data were analyzed with BD FlowJo version 10 software.
5.19. Statistical analysis
The experiments were randomized, and investigators were blinded to histological examination during all experiments. All statistical analyses were performed using Graphpad Prism 5.0 software (San Diego, CA, USA), with differences between groups considered significant with a p value <0.001. Data are presented as mean values ± S.E.M. Histopathological scores and all other experimental data were compared using a t-test (two-sided) or one-way analysis of variance (ANOVA) followed by (post hoc) Newman-Keuls multiple and Turkey multiple comparison tests. The discriminatory ability of markers for gastric cancer was evaluated by Receiver Operating Characteristics Curve (ROC) providing the Area Under the Curve (AUC). All tests were two-sided and P-values≤ 0.01 were considered statistically significant. Statistical software IBM SPSS 20.0 (SPSS Inc., Chicago, IL) and R program package (Wirtschafts Universität, Wien, Austria) were used to perform these analyses.