Patient data and tissue specimens

Patients diagnosed with primary GC who underwent radical D2 resection and healthy individuals seen at the First Affiliated Hospital of Jiamusi University between 2017 and 2019 were enrolled in this study. Patients who were underage (< 18 years), on antitumour treatment before surgical treatment or had underlying complications such as endocrine, cardiovascular, haematological or infectious disease or other cancers were excluded from the study. Blood samples, tumour and adjacent normal tissue samples were obtained from GC patients who consented to this study. All patients were disease-free after surgery and had a minimum of one year of follow-up. Finally, eighty GC patients and twenty healthy individuals were enrolled in this study. Overall survival (OS) was calculated from the date of pathological diagnosis to the date of death, and disease-free survival (DFS) was calculated from the date of radical resection to the date of disease recurrence or death. Human neutrophils and platelet-free plasma (PFP) were isolated by density gradient centrifugation as previously described [18]. The protocol for this study was approved by the ethics committee of the First Affiliated Hospital of Jiamusi University.

Cell culture and transfection and preparation of hypoxic-conditioned medium (CM)

The human primary GC cell lines AGS and HGC-27 were purchased from Procell Life Science&Technology Co.,Ltd. (Wuhan, China). GC cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% foetal bovine serum (FBS; Gibco, USA) and 1% penicillin‒streptomycin solution (Beyotime, Beijing, China). Incubation was performed at 37 °C in 5% CO2 with 1% O2 or 20% O2 in a humidified environment. For siRNA transfection, the cells were transfected with specific siRNAs using Lipofectamine™ 3000 (Invitrogen, USA) according to the manufacturer’s instructions. The oligonucleotide sequences of the siRNAs were listed below:

HMGB1, F: 5'-GGCCCGUUAUGAAAGAGAATT-3', R: 5'-UUCUCUUUCAUAACGGGCCTT-3'; Control, F: 5'-UUCUCCGAACGUGUCACGUTT-3', R: 5'-ACGUGACACGUUCGGAGAATT-3'.

Hypoxic conditions medium (hypoxic-CM) of GC cells were prepared when cells cultured to 90% confluence at 37 °C under 20%O2 and 5% CO2 and recultured for 6 h, 12 h or 24 h in medium without FBS at 37 °C under 1% O2 and 5% CO2. The supernatant was collected and centrifuged at 500 × g for 10 min at 4 ℃ to remove cell debris for subsequent experiments.

Treatment of neutrophils

Neutrophils isolated from healthy individuals were suspended in RPMI 1640 medium supplemented with 1% FBS and incubated with normoxic-CM, hypoxic-CM, hypoxic-CM (HMGB1 siRNA), recombinant human HMGB1, recombinant human chemokine (C-X-C motif) ligand 1 (CXCL1) or recombinant human CXCL2 for 4 h. For experiments that used inhibitors, neutrophils were pretreated with inhibitors for 1 h prior to the addition of hypoxic-CM from GC cells. The inhibitors used in this study included an HMGB1 antagonist (HY-N0184, MCE, USA), a p38 MAPK pathway inhibitor (HY-12839, MCE, USA), a p65 NF-KB pathway inhibitor (HY-138537, MCE, USA), an ERK pathway inhibitor (HY-112287, MCE, USA), a TLR2 antagonist (HY-112146, MCE, USA), a TLR4 antagonist (HY-107575, MCE, USA), a TLR9 antagonist (HY-131952, MCE, USA), and a RAGE antagonist (HY-P2268).

ELISA

The amounts of MPO-DNA complexes in human and mouse plasma were quantified using capture ELISA as previously described [45]. The chemokine levels in the CM of normoxic or hypoxic GC cells at different times were measured using capture ELISA (CXCL1/KC, CXCL2/MIP2 and HMGB1 ELISA kits, Jingkbio, Shanghai, China) according to the manufacturer’s instructions.

Animal models

The animal protocol was designed to minimize pain or discomfort to the animals. Male BALB/c nude mice (6–7 weeks old, weighing 18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology, and housed in a specific pathogen-free mouse facility. All procedures were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Jiamusi University. Then, we established a well-used lipopolysaccharide (LPS)-induced NET model as previously described [25]. Briefly, LPS (10 μg/mouse, Beijing) was intraperitoneally injected to induce systemic inflammation in BALB/c nude mice. Deoxyribonuclease I (DNase I, 100 U/mouse, Roche) or a p38 MAPK signalling pathway inhibitor (1 mg/kg, MCE, USA) was injected intraperitoneally 24 h before injection of LPS as the NET inhibitor.

In the xenograft models, BALB/c nude mice were injected with AGS cells (2 × 106 cells/mouse) subcutaneously into the right axilla. DNase I (100 U/mouse, Roche) and the p38 MAPK signalling pathway inhibitor (1 mg/kg, MCE, USA) were administered intraperitoneally daily after AGS injection. Mice were sacrificed at day 27 after GC cells injection. Tumour volumes were calculated by measuring the length (L) and width (W) of the tumours: Tumour volume = π/6 × L × W2.

Neutrophil chemotaxis assay

Neutrophils (1 × 105 cells) isolated from healthy individuals were seeded in RPMI 1640 medium supplemented with 1% FBS in the upper chamber of the Transwell apparatus. The lower chamber contained normal CM or hypoxic-CM from GC cells collected at different times. After the cells migrated for 3 h, nonmigrated cells were removed by scraping with cotton-tipped applicators. The migrated cells on the underside of the chamber membranes were fixed, stained and counted under a microscope.

Cell proliferation and colony formation assay

For the cell proliferation assay, GC cell monolayers were incubated with cell-free NETs in the presence or absence of DNase I for 12 h at 37 °C in 5% CO2 and 20% O2 in a humidified chamber. Then, treated GC cells (2 × 104 cells/mL, 2 mL/well) were seeded in 12-well plates and cultured with 10% FBS medium and counted using a haemocytometer at the indicated endpoints.

For the colony formation assay, treated GC cells (1 × 103) were seeded in 6-well plates at 37 ℃ in a 5% CO2 chamber. The medium was refreshed every 3 days for approximately 10 days, and the colonies were fixed, stained with 1% crystal violet and finally counted.

Cell invasion and wound healing assays

The cell invasion assay was performed with the Boyden chamber invasion system as previously described [46]. Briefly, a Boyden chamber (Corning, USA) containing 8 μm pores was coated with Matrigel (Corning, USA). Human GC cells were cultured in the absence or presence of cell-free NETs (0.5 μg DNA/mL) for 12 h. Then, cells (4 × 104) were seeded into the upper chamber, and medium without FBS or with 2% or 10% FBS was seeded into the lower chamber. After 20 h of incubation at 37 ℃ in an atmosphere of 5% CO2 and 95% air, nonmigrated cells were removed by scraping with cotton-tipped applicators. The migrated cells on the underside of the chamber membranes were fixed, stained and counted under a microscope.

For the wound healing assay, GC cells were stimulated with cell-free NETs for 12 h at 37 ℃ and then digested and seeded in 6-well plates with medium containing 10% FBS. When the GC cells were approximately 90% confluent, a 200 μl sterile pipette tip was used to scratch the cell surface, followed by three washes with PBS. Thereafter, the cells were incubated in medium supplemented with 1% FBS. Images of the scratch were acquired at different time points under a microscope. The cell migration rate was calculated as follows: (width at 0 h–width at different time points)/width at 0 h.

Western blot assay

Western blotting was performed as previously described [47]. Briefly, proteins were extracted from tissue samples or neutrophils using RIPA lysis buffer containing proteinase inhibitors. Equal amounts of protein (20 μg) were separated by 10% or 12% SDS‒PAGE. Following electrophoresis, proteins were transferred to a PVDF membrane, blocked in 5% nonfat milk, and incubated with primary antibodies at 4 ℃ overnight. Antibodies against HIF-1α and citH3 were purchased from Abcam (UK), and antibodies against ERK1/2, p-ERK1/2, p65 NF-кB, p-p65 NF-кB, p38 MAPK, p-p38 MAPK, Akt, p-Akt, STAT3, p-STAT3 and GAPDH were purchased from Affinity Technology (USA). After washing with TBST three times, the membrane was incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Bioworld Technology) at RT for 1 h. The protein bands were visualized by enhanced chemiluminescence and analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). GAPDH served as the loading control.

Real-time quantitative PCR

Total RNA was extracted from GC cells and neutrophils using TRIzol reagent (Roche, Switzerland), and 1 μg of RNA was reverse transcribed to cDNA by using reverse transcriptase (Thermo Fisher Scientific, USA). Real-time quantitative PCR was performed by using a SYBR Green I real-time detection kit (Thermo Fisher Scientific, USA) on a Bio-Rad CFX96 Detection System. Relative mRNA expression was normalized to β-actin expression. The primers used for amplification of the target genes are listed in supplementary Table 1.

Preparation of NETs

Cell-free NETs were isolated from neutrophils of GC patients as previously described with slight modifications [48]. Briefly, neutrophils (1 × 107 cells/ml) were cultured for 4 h at 37 °C in 5% CO2 in medium supplemented with 200 nM PMA (HY-18739, MCE, USA). The supernatant was centrifuged at 500 × g for 10 min at 4 °C to remove cell debris. Thereafter, the supernatant (NET-rich medium) was centrifuged at 12,000 × g for 15 min at 4 ℃. The resultant pellets (a mixture of chromatin and protein) were suspended in ice-cold 1 × PBS, and the DNA concentration in the medium obtained was measured using spectrophotometry (Biospec-nano, Japan).

Immunohistochemical assay

Briefly, 4 μm thick paraffin-embedded tissue sections were deparaffinized, rehydrated, treated with 0.3% hydrogen peroxide, and subjected to antigen retrieval with heat induction for approximately 10 min. The following primary were used for IHC staining: rabbit anti-CD66b (1:200, Ab197678, UK) and rabbit anti-CD31 (1:200, Ab76533, UK). The tissues were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ZSGB-bio, Beijing, China). The slides were finally stained with 3,3´-diaminobenzidine (DAB substrate kit, ZSGB-Bio, Beijing, China) and counterstained with haematoxylin. The specimens were analysed under a light microscope (Nikon, Tokyo, Japan) by pathologists.

Immunofluorescence staining

For tissue samples, 6 μm Optimal Cutting Temperature (OCT)-embedded tissue sections were fixed with ice-cold acetone for 15 min. The following primary were used for IF staining: rabbit anti-citH3 (1:500, Ab5103, UK) and mouse anti-MPO (1:500, Ab90810, UK). Tissues were then incubated with Alexa Fluor 594-conjugated goat anti-rabbit (1:500, Ab150080, UK) and Alexa Fluor 488-conjugated goat anti-mouse (1:500, Ab150113, UK) secondary antibodies.

For NET formation, neutrophils (5 × 105 cells) isolated from healthy individuals were seeded and incubated in glass-bottom poly-L-lysine-coated 24-well plates for 1 h at 37 ℃ in a 5% CO2 chamber and treated as described above. To detect and quantify NETs, the samples were incubated first with rabbit anti-citH3 and mouse anti-MPO primary antibodies and then with fluorescent secondary antibodies. All images were acquired using a confocal microscope (Zeiss, LSM 800, Germany) and analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Clinical data were available for all GC patients and healthy individuals. The survival of these patients was analysed using the Kaplan‒Meier method and compared using the log-rank test. Cox proportional hazards regression analysis was used to determine the effect of plasma MPO-DNA on OS and DFS. For in vitro and in vivo experiments, data are expressed as the means ± standard deviations (SDs). Data were analysed to assess distribution normality. For normally distributed data, statistical significance was analysed using Student’s t test and one-way analysis of variance (ANOVA). For nonnormally distributed data, statistical significance was analysed using the Mann‒Whitney test and the Kruskal‒Wallis test. All analyses were performed using GraphPad Prism v. 8.0 and SPSS 22.0 statistical software. P < 0.05 was considered statistically significant.