Hypoxic and normoxic cultured human monocyte-derived macrophage dataset GSE4630 was downloaded from the GEO (Gene Expression Omnibus) database (https://www.ncbi.nlm.nih.gov/geo/). They included two hypoxic-cultured samples and two normoxic-cultured samples. The GC RNA sequencing data were downloaded from the TCGA (The Cancer Genome Atlas, TCGA-STAD, 375 tumor samples vs. 31 normal samples, https://portal.gdc.cancer.gov/). CIBERSORT was used to determine the relative frequencies of immune cells in each sample. The GC and normal sample microarray data were also obtained from the GEO (GSE54129, 111 tumor samples vs. 21 normal samples). The differentially expressed genes were identified by the “limma” package (P < 0.05, and |FoldChange| ≥ 2). JASPAR (http://jaspar.genereg.net/) and UCSC (http://genome.ucsc.edu/) were performed to predict the transcription factor.
Cell culture and human PBMCs isolation
The GC (HGC-27, NUGC3) cells, human normal gastric epithelial cell line (GES-1) were acquired from China Medical University (Shenyang, China). Mouse GC cancer cell (GAN-KP cells) was acquired from International Research Center for Medical Sciences, Kumamoto University . Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors in Liaoning Cancer Hospital & Institute. CD14+ cells were enriched by depleting CD8+, CD19+, CD56+ and CD14+ cells by MACS (magnetic-activated cell sorting, EasySep, STEMCELL Technologies, 19,059) columns with negative selection. PBMCs were differentiated into immature macrophages (M0 macrophages) using 50 ng/mL human macrophage colony-stimulating factor (hM-CSF, Sigma, 81,627–83-0) for 7 days (Fig. 1C). All cells were cultured in RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation, 189–02145) supplemented with 10% fetal bovine serum (FBS, Gibco 26,140–079) under 1% O2 or 20% O2 conditions as described previously [8, 10, 26]. In the co-culture model, macrophages and GC cells were added in a 1:1 ratio into a 10 cm dish (1 × 106 cells for each) and analyzed after 7 days of co-culture.
GC tissues and ethical approval
Between January 2009 and December 2012, gastric cancer and adjacent non-cancerous tissue samples were obtained from 526 patients subjected to adequate complete surgical resection (R0) of locally advanced GC surgical resection in Liaoning Cancer Hospital & Institute. All recruited patients had not received preoperative chemotherapy or radiotherapy. They signed a written informed consent before surgery. The follow-up of patients was closed on December 31, 2020. The study was approved by the Ethics Committee of Liaoning Cancer Hospital and Research Institute (20181226).
The immunohistochemistry (IHC) was performed based on previously published protocols . Immunohistochemistry was scored based on the intensity of staining and the proportion of positive cells. The blinded review was performed by two pathologists. Ki67 (1:1000, Abcam, ab15580), Caspase-3 (1:1000, Abcam, ab184787) CXCL8 (5 μg/mL, R&D Systems, AF-208-NA).
The slices were overnight incubated with primary antibody at 4 °C. Then, they were incubated with species-appropriate rabbit/mouse secondary antibodies coupled with AlexaFluor dyes (488, 594, 1:200, Invitrogen, A32814, A32723, A32740), DAPI (1:1000, Dojindo, KT013) at room temperature for 1 h. CXCL8 (5 μg/mL, R&D Systems, AF-208-NA), CD68, CD163, CD86 (1:200, Abcam, ab213363, ab182422, ab270719). Olympus Fluoview FL1200 confocal microscope was used to capture 20x and 60x images.
ELISA assay was used to measure the CXCL8 in the supernatant of macrophages cultured in hypoxic or normoxic conditions and IL-10 in the medium of GC cells. The procedure followed the instructions of the Human CXCL8 kit (R&D Systems, D8000C) and Human IL-10 ELISA Kit (Abcam, ab185986).
Western blot analysis
The RIPA lysis buffer was used to extract total proteins from cells and tissues (Beyotime, Shanghai, China). Then, 10% SDS-PAGE gel was used to separate the proteins and transferred using the PVDF membrane. The membranes were overnight incubated with the primary antibodies (Supplementary Table 2) at 4 °C. Subsequently, they were co-cultured with the secondary antibody for 1 h. The ECL system (Amersham Imager 600) and ImageJ software were applied to observe and calculate grayscale values .
Chromatin immunoprecipitation (ChIP)
ChIP enzymatic chromatin IP kit protocol (Cell Signaling Technology, China) was used to perform the ChIP assay, where 1 × 107 logarithmic phase cells were subjected to ChIP. The chromatin was immunoprecipitated with an anti-STAT1 (Santa Cruz Biotechnology, USA, 1:50) and mouse IgG (Santa Cruz Biotechnology, USA, 1:100) on rotators at 4 °C for 16 h.
Promoter-luciferase reporter assay
Promoter-Luciferase reporter assay confirmed the combination of STAT1 and IL-10 promoter sequence, NFKB1, and CXCL8 promoter sequence, respectively. The recombinant pGL-3 basic-plasmid contained truncated human IL-10 (or CXCL8) promoter (wild type, wt, − 2000 ~ + 99) or mutant IL-10 (or CXCL8) promoter (mutant type, mut). The plasmids were transfected with the POLO3000 transfection reagent (Research and science, China). After 48 h, the luciferase activity of each group was evaluated following the manufacturer’s instructions.
Colony formation assay
A total of 1 × 105 GC cells/each group were seeded into 6-well plates for 2 weeks. The colonies were washed three times using PBS, fixed with 4% paraformaldehyde, and stained with Diff-Quik III Kit (Muto Chemical, ZS0003). The stained colonies were observed under a light microscope (IX81 Olympus).
Cell counting assay
Cell proliferation was evaluated using the Cell counting kit (Cell counting kit 8, CCK-8). The cells were incubated at 37 °C for 24, 48, 72, and 96 h. Subsequently, the medium was discarded then the chromogenic solution at 10:1 was prepared. For incubation, a color-developing solution (10 μl) was added to the 96 well plates at 37 °C for 2 h. The optical density (OD) was detected using the UV spectrophotometer (BioTek Synergy H1) at 450 nm.
Gastric cancer cells were seeded in Transwell upper chambers coated with gelatin. The lower chambers with 600 μL RPMI-1640 with 20% FBS. After 24 h, the cells were fixed and stained using methanol, hematoxylin, and eosin (Sigma-Aldrich, St. Louis, MO, USA). The upper chamber was removed and the lower layer migrating cells counted under the microscope (IX81 Olympus).
Real-time imaging of cell migration
A total of 200 μl Matrigel (BD Biosciences, 356,235) pre-coated the bottom of the six-well plates, the cells were then inoculated for 24 h. Thereafter, a 6-well plate was cultured and imaged using KEYENCE BZ-X700 (KEYENCE, Japan), equipped with CO2 and temperature control chamber as well as a time-lapse tracking system. BZ-X Viewer software (KEYENCE) was used to capture phase-contrast images at intervals of every 10 min for 48 h, while the BZ-X Analyzer software (KEYENCE) was used to convert the continuous images into movie files. Furthermore, KEYENCE video editing and analysis software was used to analyze cell migration in the movies. Microsoft Excel 2010 was used to process the trace data to create XY coordinate graphs and distance measurements.
The cell concentration was adjusted to 1 × 106 cells/ml in PBS containing 2% FBS. The cell suspensions were incubated with antibodies (BioLegend, Cat# 137005, Cat# 333805, Cat# 506804, Cat# 505007; abcam, ab289967, ab52460) for 30 min on ice, washed with PBS containing 2% FBS, centrifuged twice, and suspended in PBS. Flow cytometry was performed with a FACSVerse instrument (BD Biosciences). The flow cytometry data were analyzed using FlowJo 3.3 software (Tree Star).
Xenograft mouse model
For subcutaneous tumor xenografts, an estimated 1 × 106 mouse GC cells/0.2 ml PBS (with or without CXCL8 which injected every 3 days) were subcutaneously injected into the right axillary region of a 5-week-old female BALB/ C mouse. After 28 days of injection, tumors were collected, measured volumes weighed, and photographed. The volume was calculated using the following formula (tumor volume = L*W*W/2).
For the intraperitoneally model, approximately 5 × 106 GC cells/0.1 ml PBS (with or without CXCL8) were injected into the mouse as mentioned above. Similarly, the mice were euthanized 28 days after injection, then the volume of ascites and the number of visible (> 0.1 cm) metastatic nodules in the peritoneal cavity were measured.
Other reagent or resource
Recombinant Human IL-8 (CXCL8, PeproTech, 200–08), Recombinant Human IL-10 (PeproTech, 200–10), Recombinant Human M-CSF (PeproTech, 300–25), Repertaxin (Sigma-Aldrich, 266,359), Fludarabine (Tocris, 3495/10), Sarsasapogenin (Selleck, S3607), Bay11–7082 (Selleck, S2913), Anemoside B4 (Selleck, S9081).
GraphPad Prism 9.0 (GraphPad Software Inc) and SPSS 24.0 statistical software (IBM) were used for statistical analyses of data. The student’s t-test was utilized to perform statistical analysis. P < 0.05 was considered statistically significant.
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