2.1 Cell culture

The human oral tongue squamous carcinoma cell line Cal27, human epidermal keratinocyte cell line Hacat, and mouse acute monocytic leukemia cell line RAW264.7 were purchased from the China Center for Type Culture Collection. All of the cell lines were cultured in Dulbecco's modified Eagle's medium (VivaCell; Shanghai; China) with 10% fetal bovine serum (VivaCell; Shanghai; China), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified 5% CO2 atmosphere at 37 °C.

2.2 Reagents

Recombinant human CXCL1 and recombinant mouse EGF were purchased from R&D Systems Inc. (cat. no. 275-GR) and Abcam (cat. no. ab206643), respectively. AG1478 and SB225002 were purchased from Selleck Biotechnology Co., Ltd. (cat. no. S2728; cat. no. S7651). The antibodies used included rabbit anti-β-actin (cat. no. ab115777; Abcam), rabbit anti-E-cadherin (cat. no. ab40772 Abcam), rabbit anti-N-cadherin (cat. no. ab76011 Abcam), rabbit anti-CXCL1 (cat. no. ab206411 Abcam), rabbit anti-total EGFR (Zen Bio Science Co., Ltd), rabbit anti-phospho-EGFR (cat. no. R24173; Zen Bio Science Co., Ltd), rabbit anti-total P65 (cat. no. R25149; Zen Bio Science Co., Ltd), and rabbit anti-phospho-P65 (cat. no. 310013; Zen Bio Science Co., Ltd). DyLight 488 (A23220) and 800 (A23920) were purchased from Abbkine Scientific Co., Ltd. Anti-mouse PE CD86 (12-0862-81) and anti-mouse APC CD206 (17–2061-80) were purchased from Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA. The anti-CXCL1 (YT2074) antibody for immunohistochemical staining was obtained from Immunoway, USA. The secondary antibody kit (GK600705) for IHC was purchased from Gene Tech, Shanghai, China.

2.3 Tumor- and TAM-conditioned medium preparation

Cancer cell lines were cultured in complete medium, and the medium was replaced by serum-free medium when the cell density reached approximately 90%. After 48 h, the conditioned medium (CM) was harvested, centrifuged at 2000×g for 10 min, filtered through 0.22 mm filters, and stored at 80 °C. Regarding TAM-conditioned medium, Raw264.7 cells were stimulated with tumor-conditioned medium for 48 h, and then serum-free medium replaced the conditioned medium. After 24 h, the conditioned medium (CM) was harvested, centrifuged at 2000×g for 10 min, filtered through 0.22 mm filters, and stored at 80 °C. Tumor- or TAM-conditioned medium was defined as TCM or TAM-CM.

2.4 Immunohistochemistry (IHC)

Five healthy tissues and ten OSCC tissues were obtained from the Department of Oral Pathology of China Medical University. Human participants were not included in the study. Both healthy tissues and OSCC tissues were obtained using the paraffin-embedded method. Immunostaining was performed using the avidin–biotin-peroxidase method. Sections were stained using primary anti-CXCL1 (1:100) antibody. Then, a secondary antibody kit was used. Finally, sections were stained with Mayer’s hematoxylin, and sections were observed using a light microscope (Nikon Eclipse Ts2R, Japan).

2.5 Chemotactic migration and Matrigel invasion assays

For assessment of tumor cell migration, tumor cells (1 × 105) in 200 μl of serum-deprived medium were cultured in the upper chamber, and conditioned medium from macrophages stimulated with TCM or TCM and CXCL1 (100 ng/ml); TAM supernatant containing EGF (100 ng/ml) or EGF (100 ng/ml) and AG1478 (5 μM) with 10% FBS was added to the lower chamber. Moreover, cell invasion was assessed using a Transwell chamber (pore size 8 mm, Jet Biofil; Guangzhou; China) with Matrigel coating (BD Biosciences). For chemotactic migration and invasion, the non-migrated cells were removed from the surface of the upper chamber after 24 h, while after 48 h, the noninvasive cells were also scraped. The representative migrated or invaded cells were treated with 4% paraformaldehyde (product no. P0099; Beyotime Institute of Biotechnology) for 20 min and then with 0.5% crystal violet (product no. C0121; Beyotime Institute of Biotechnology) for 15 min. Cells were counted using a light microscope (Nikon Eclipse Ts2R, Japan) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.6 Scratch wound healing assay

Cell migration ability was assessed in vitro by wound healing. Cal27 cells (1 × 10^6/well) were seeded in 6-well plates treated with conditioned medium from macrophages stimulated with TCM or TCM and CXCL1 (100 ng/ml) or TAM-CM including EGF (100 ng/ml) or EGF and AG1478 (5 μM). After the tumor cells were grown to confluency, a scratch was created with a 200 µl pipette tip. Then, PBS was used to wash the plates several times. The plates were incubated for 24 h, and the tumor cells that migrated to heal the artificial wound were photographed with a light microscope (Nikon Eclipse Ts2R, Japan) and assessed with ImageJ software. The healing rate was calculated as (%) = (initial average scratch area − average scratch area at 24 h)/initial average scratch area × 100%.

2.7 Real-time PCR (RT‒PCR)

RT‒PCR was used to measure the gene expression levels of CD206, CD86, Arg-1, EGF, CXCL1, cell transfection- and EMT-related markers in Raw264.7 cells and Cal27 cells. Total RNA was isolated by TRIzol Reagent (Takara Bio, Inc., Otsu, Japan), and extracted RNA was reversed transcribed by PrimeScript RT (Takara Bio, Inc., RR036A, Otsu, Japan). RT‒qPCR was performed using the SYBR Premix® Ex TaqTM Kit (Takara Bio, Inc, RR820A, Otsu, Japan) with the 7500 Fast System (Thermo Fisher Scientific). RT‒qPCR thermocycling conditions were as follows: 40 cycles of 95 °C for 30 s, 95 °C for 5 s, 60 °C for 34 s, 95 °C for 15 s, 60 °C for 60 s and 95 °C for 15 s. The internal control of the experiment was β-actin. Primers for β‑actin, CXCL1, EGF, E-cadherin, N-cadherin, Vimentin, CD206, CD86, and Arg-1 were obtained from Shanghai Sheng gong Biological Technology Co., Ltd. All primer sequences are shown in Table 1.

Table 1 Primers used for RT‒PCR2.8 Western blot analysis

Cal27 cells were washed with PBS and then lysed using RIPA buffer (product no. P0013B; Beyotime Institute of Biotechnology) including 1 mM PMSF (Product no. ST507; Beyotime Institute of Biotechnology) to extract total proteins. Next, the protein concentration was determined by a BCA protein assay kit (product no. P0012s; Beyotime Institute of Biotechnology). Total proteins were separated using 10% or 12% SDS‒PAGE, and electrophoresis was run at 150 V. The separated proteins were transferred to a PVDF membrane at 220 mA. The PVDF membrane was incubated with 5% nonfat dry milk at room temperature for 1 h to block the immunoblots and probed at 4 °C overnight with specific primary antibodies, including rabbit anti-β-actin (1:2000), rabbit anti-CXCL1 (1:1000), rabbit anti-E-cadherin (1:1000), rabbit anti-N-cadherin (1:1000), rabbit anti-total EGFR (1:1000), rabbit anti-phospho-EGFR (1:1000), rabbit anti-total P65 (1:1000), and rabbit anti-phospho-P65 (1:500). Then, the membranes were incubated with the secondary anti-rabbit antibody DyLight 800 goat anti-rabbit IgG (1:2000) at room temperature for 1 h. The gray values of the bands were analyzed by Odyssey CLX (LI-COR, Lincoln, NE, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.9 Enzyme-linked immunosorbent assay (ELISA)

Measurement of EGF and CXCL1 levels in the supernatant of the TAMs and Cal27 cells was conducted using mouse EGF ELISA kits (BOSTER Biological Technology Co. Ltd., Wuhan, China; EK0326) and human CXCL1 ELISA kits (R&D Systems Inc., Minneapolis, MN, USA; cat. no. #DY275), respectively. For the standard solution or sample, 100 μl/well was added to the 96-well containing specific antibody incubated at room temperature for 2 h. After washing three times with 300 μl diluted washing buffer, each well was incubated with the diluted HRP conjugate at room temperature for 1 h. Then, the diluted washing buffer was used to wash each well three times. Each well was incubated with 100 µl chromogenic substrate in the dark for 30 min. Finally, 90 μl stop solution was added to every well, and the absorbance of each well was measured by a microplate reader at 450 nm. By constructing a standard curve, the results of the sample solution were obtained.

2.10 Immunofluorescence

The Cal27 cells were treated with 0.5% Triton solution for 10 min. PBS was used to wash the Cal27 cells three times. Then, the Cal27 cells were incubated with primary antibodies against one of the EMT markers (E-cadherin) at 4 °C overnight. The cells were washed three times with PBS again. The cells were incubated with the corresponding secondary anti-rabbit antibodies with DyLight 488 (1:200) at room temperature for 1 h. Finally, the cells were counterstained with DAPI at room temperature for 10 min. Moreover, tissue sections were incubated using primary anti-CXCL1 (1:100) antibody. Then, a secondary antibody kit was used. The staining was observed and photographed with a fluorescence microscope (Nikon Eclipse Ts2R, Japan).

2.11 Cell proliferation assay

The rate of cell proliferation was determined using a Cell Counting Kit-8 assay (product no. C0038; Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Cal27 cells were plated in 96-well plates at a ratio of 3 × 10^3/well. Then, the cells were incubated in TAM-CM with EGF (100 ng/ml) or EGF (100 ng/ml) and AG1478 (5 μM) for 24 and 48 h. Ten microliters of CCK-8 solution was added to every well, and the cells were cultured at 37 °C for 1 h. The absorbance of each well was measured at 450 nm (Tecan Mechelen, Belgium).

2.12 Colony formation assay

Cal27 cells were cultured in 6-well plates at a ratio of 1 × 10^3/well and stimulated with TAM-CM containing EGF (100 ng/ml) or EGF (100 ng/ml) and AG1478 (5 μM) for 2 weeks. The incubation medium was replaced every two days. Subsequently, the cells were treated with 4% paraformaldehyde and then with 0.5% crystal violet for 30 min, and colonies were counted with a light microscope.

2.13 Transfection of Cal27 cells

Cal27 cells (1 × 10^5/well) were seeded in 6-well plates and transfected with double-stranded small interfering RNA (siRNA) (Shanghai Gene Pharma Co., Ltd., Shanghai, China). Sense and antisense strands for siRNAs were as follows: si-CXCL1-1 (5′-ACUCAAGAAUGGGCGGAAATT-3′) (5′-UUUCCGCCCAUUCUUGAGUTT-3′); si-CXCL1-2 (5′-CCAAGAACAUCAAAGUGUTT-3′) (5′-ACACUUUGGAUGUUCUUGGTT-3′); and si-CXCL1-3 (5′-GAUGCUGAACAGUGACAAATT-3′) (5′-UUUGUCACUGUUCAGCAUCTT-3′). Cal27 cells transfected for 24 h were used for subsequent experiments.

2.14 Flow cytometry

After RAW264.7 cells were cultured with TCM or TCM and CXCL1 for 48 h, they were stained with anti-mouse PE CD86 (Thermo Fisher Scientific, Waltham, MA, USA) and anti-mouse APC CD206 (Thermo Fisher Scientific, Waltham, MA, USA) on ice for 20 min according to the dilution ratio recommended by the manufacturers. Data analysis was performed using FlowJo software.

2.15 Statistical analysis

Prism 8.0 for Windows (GraphPad Software, Inc., La Jolla, CA, USA) was used for data analysis. Measurement data are presented as the mean ± standard deviation (SD), and statistical significance between groups was determined by Student’s t test or analysis of variance (ANOVA). Western blotting, wound healing assay and Transwell assay results were assessed by ImageJ software. Differences were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.