Human gastric cancer cell lines (MGC80-3 and HGC-27), human promyelocytic leukemia cell line (HL-60), and mouse gastric cancer cell line (MFC) were purchased from the Institute of Biochemistry and Cell Biology of the Shanghai Institutes for Biological Sciences at the Chinese Academy of Sciences (Shanghai, China). MGC80-3 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA). HGC-27, HL-60, and MFC cells were cultured in RPMI-1640 medium containing 10% FBS. HL-60 cells were induced to differentiate into neutrophil-like cells (HL-60N) by 1.25% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) for 5 days. All the cells were cultured in a humidified incubator with 5% CO₂ at 37°C. For the cell co-culture experiment, gastric cancer cells were seeded into plates in the presence or absence of HL-60N cells (1:3 ratio).

MGC80-3, HGC-27 and MFC cells were seeded onto 10-cm plates at a density of 8×10⁵cells/plate for 24 h in complete culture medium. The cells were then washed with phosphate-buffered saline (PBS) twice and cultured in serum-free medium for another 24 h. The supernatants were collected and the cell debris was removed by centrifugation at 3,000 × g for 10 min. The CM (designated as GC-CM) was filtered through a 0.22-mm filter and stored at −80°C until use. HL-60N cells were seeded in 25-cm² culture cell flasks at a density of 1×10⁵ cells and treated with GC-CM (1:1 ratio, v/v) for 24 h, then washed with PBS twice and cultured in serum-free medium for another 24 h. The CM (designated as HL-60 CM) was collected, filtered and stored at −80°C.

For the cell co-culture, 3×10³ GC cells were seeded into each well of 96 well-plates in the presence or absence of HL-60N cells (1:3 ratio). For the CM treatment, the same density of gastric cancer cells was seeded into each well of 96 well-plates and treated with or without HL-60N CM (1:1 ratio, v/v). At different time-points (1, 2 and 3 days) a methyl thiazolyl tetrazolium (MTT) assay was performed. Twenty microliters of MTT (5 mg/ml) was added into each well and the cells were further incubated for 4 h. Then, the media were discarded and 150 µl DMSO was added. The plate was shaken at room temperature for 15 min and the absorbance was read at 490 nm on an automatic microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Following the co-culture with HL-60N cells or treatment with HL-60N CM for 48 h, the cells were cultured in six-well plates at a density of 500 cells/well for 8 days. The medium was changed every two days. At the end of the experiment, the cell colonies were fixed with formaldehyde and stained with crystal violet for 30 min. The number of cell colonies was counted under a microscope.

After the co-culture with HL-60N cells or treatment with HL-60N CM for 48 h, the cells were collected and seeded into the upper chamber (8 µm) at a density of 1×10⁵ cells/well (Corning Inc., Corning, NY, USA). The lower chamber was filled with 500 µl culture medium supplemented with 10% FBS. Nine hours later, the cells on the upper surface of the membrane were removed with a cotton swab. Then, the lower cells were fixed with formaldehyde and stained with crystal violet for 30 min. The number of migrated cells was counted under a microscope.

The diluted basement Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was added into each chamber and let to polymerize at 37°C for 30 min. The cells were seeded into the upper chamber at a density of 2×10⁵ cells/well. The lower chamber was filled with 500 µl culture medium supplemented with 10% FBS. The cells were allowed to invade to the lower membrane for 30 h. Subsequently, the cells on the upper surface of the membrane were removed with a cotton swab. The lower cells were then fixed with formaldehyde and stained with crystal violet for 30 min. The number of migrated cells was counted under a microscope.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA using miScript reverse transcription kit (Bio-Rad, Hercules, CA, USA). The relative expression of target genes was detected on a Bio-Rad CFX96 quantitative real-time PCR system with the SYBR Green method (β-actin served as an internal control). The sequences of the primers are listed in Table I.

The cells were washed twice with PBS and lysed with RIPA buffer containing 1% protease inhibitors. Equal amounts of proteins were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes, followed by blocking with 5% non-fat milk for 1 h. The membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-T-ERK (4695S; Cell Signaling Technology, Beverly, MA, USA), anti-p-ERK (4370S; Cell Signaling Technology), anti-E-cadherin (H-108; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Slug (9585S; Cell Signaling Technology), anti-vimentin (5741S; Cell Signaling Technology) and anti-GAPDH (MB001; Bioworld Technology, St. Louis Park, MN, USA). After incubation with the secondary antibodies (Bioworld Technology) at 37°C for 1 h, the bands were visualized with an ECL chemiluminescent detection system.

The HL-60N cells were co-cultured with the HGC-27 cells or treated with the supernatants from the HGC-27 cells for 24 h. The CM of the HL-60N cells was collected. The protein levels of IL-1β, IL-6, IL-8 and TNFα were assessed using ELISA kits (Bangyi Biotechnology, Shanghai, China), according to the manufacturer's protocol. A volume of 100 µl of culture supernatants was added to each well and the absorbance was assessed at 450 nm. ELISA was performed in triplicate.

Mice (615; 6–8 weeks old) were purchased from the Tianjin Institute of Hematology and Blood Diseases Hospital (Tianjin, China). The bone marrow was collected and filtered through a 200-mesh metal screen and centrifugated at 800 × g for 5 min. After the lysis of the red cells, the single cell suspensions were washed once with PBS containing 2% FBS and incubated with anti-Ly6G-PE and anti-CD11b-PE Cy7 antibodies for 30 min at 4°C. The CD11b⁺Ly6G⁺ neutrophils were isolated on a flow cytometric cell sorter (BD FACS Aria II; BD Biosciences). The present study was approved by the Animal Care and Use Committee of Jiangsu University.

All the results were expressed as the mean ± SD. Statistical analyses were performed using Student's t-test with GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

After DMSO induction for 5 days, the expression of CD11b was notably increased in the HL-60N cells, revealing that the HL-60 cells were successfully induced into HL-60N cells (Fig. 1A). To examine the potential role of the HL-60N cells in gastric cancer progression, we co-cultured the gastric cancer cells with HL-60N cells for 48 h and then examined their proliferating, migratory and invasive abilities. The results of the Transwell migration assay and the Matrigel invasion assay revealed that the co-culture with HL-60N cells markedly increased the migratory and invasive abilities of both the MGC80-3 and the HGC-27 cells (Fig. 1B). However, the results of the MTT and colony formation assays revealed that the gastric cancer cells co-cultured with the HL-60N cells had no significant change in their proliferating ability (Fig. 1C and D). Collectively, these results revealed that the co-culture with HL-60N cells promoted the migration and invasion of GC cells while it did not affect their proliferation.

To examine whether the HL-60N cells promoted gastric cancer progression through a cell contact-dependent manner, we collected CM from the HL-60N cells after being co-cultured with HGC-27 cells for 24 h. We then treated the HGC-27 cells with the CM for another 24 h. The metastatic potential of the HGC-27 cells was determined using Transwell invasion assay and Matrigel invasion assay. As shown in Fig. 2A, the CM from the HL-60N cells co-cultured with the HGC-27 cells enhanced the migration and invasion of HGC-27 cells to a greater extent than that from the HL-60N cells alone. The results of the MTT and colony formation assay revealed that there was no significant change in the proliferation of the HGC-27 cells after treatment with the CM from the activated HL-60N cells (Fig. 2C and D). Similar results were also obtained for the MGC80-3 cells (Fig. 2B and D). These results revealed that the HL-60N cells, in response to the stimuli from gastric cancer cells, promoted the migration and invasion of gastric cancer cells through a paracrine mechanism.

We next determined the expression of pro-inflammatory factors including the IL-6, IL-8, IL-1β and TNFα genes in HL-60N cells using qRT-PCR. We found that the co-culture with gastric cancer cells significantly upregulated the expression of IL-6, IL-8, IL-1β and TNFα in the HL-60N cells (Fig. 3A). In addition, treatment with the CM of gastric cancer cells also increased the expression of the IL-6, IL-8, IL-1β and TNFα genes in HL-60N cells (Fig. 3B). Moreover, the results of ELISA revealed that either the co-culture with gastric cancer cells or the treatment with CM of gastric cancer cells improved the expression levels of IL-6, IL-8, IL-1β and TNFα in HL-60N cells (Fig. 3C). Collectively, these results revealed that gastric cancer cells induced HL-60N cells to acquire a pro-inflammatory phenotype.

We then determined the expression EMT-related genes in gastric cancer cells. The results of the qRT-PCR analyses revealed that the co-culture with HL-60N cells or treatment with the CM of activated HL-60N cells significantly increased the expression of vimentin, ZEB-1, Slug, Sox2, Nanog and Oct4 in HGC-27 cells (Fig. 4A and B), indicating that the interaction with neutrophils promoted gastric cancer cell migration and invasion through the induction of EMT.

We further determined the activation of the ERK pathway in HGC-27 cells using western blot analysis. As shown in Fig. 5A, the activity of p-ERK started to increase at 15 min after treatment with the CM of activated HL-60N cells and was maintained at a high level 2 h after treatment, while the expression of total ERK exhibited no significant change. To further investigate whether the effects of the activated HL-60N cells on gastric cancer migration and invasion were ERK pathway-dependent, we pretreated the gastric cancer cells with the ERK inhibitor U0126 for 1 h and then incubated the cells with the CM of activated HL-60N cells. As shown in Fig. 5B, the inhibition of ERK activity with U0126 could reverse the promoting roles of the activated HL-60N cells in gastric cancer cell migration and invasion. In addition, the results of qRT-PCR revealed that the inhibition of ERK activity with U0126 upregulated the expression of E-cadherin while it downregulated that of vimentin, ZEB-1, Slug, Sox2, Nanog and Oct4 in gastric cancer cells (Fig. 5C). Moreover, the results of the western blot analysis revealed that the inhibition of ERK activity with U0126 upregulated the expression of E-cadherin while it downregulated that of vimentin and Slug (Fig. 5D). These results indicated that the interaction with neutrophils promoted gastric cancer cell migration and invasion through the activation of the ERK pathway.

To confirm the results of the in vitro experiments, we isolated neutrophils from the bone marrow of 615 mice by cell sorting. Consistent with the observations for the HL-60N cells, the mouse bone marrow neutrophils also exhibited an increased expression of IL-6, IL-1β and TNFα when treated with the CM of MFC cells (Fig. 6A). In addition, the CM of activated mouse bone marrow neutrophils could induce the activation of the ERK pathway in mouse gastric cancer MFC cells (Fig. 6B). Furthermore, the promoting roles of the activated mouse bone marrow neutrophils on MFC cell migration and invasion were decreased when the ERK pathway was inhibited (Fig. 6C). Collectively, these results revealed that the activated mouse neutrophils also promoted mouse gastric cancer cell migration and invasion.