A total of 162 patients [62.75±9.76 years (mean age ± standard deviation); 87 male and 75 female] with lung adenocarcinoma, whose diagnosis (pathological stage I or II, pre-operative assessments) was confirmed by histological examination between February 2009 and July 2011 at the Hebei General Hospital (Shijiazhuang, China), were enrolled in the present study. All the patients underwent pulmonary resection by video-assisted thoracoscopic surgery (VATS). The patients were divided into a recurrence group (n=50) and a non-recurrence group (n=112) according to their recurrence and metastasis status within 2 years following surgery. Staging prior to and following surgery was based on histopathological analysis according to the International Union Against Cancer tumor-node-metastasis staging system (18). The study was approved by the Medical Ethics Committee on Human Research of Hebei General Hospital (no. 2005-123). Informed written consent was obtained from each patient. Patients were followed up for 2 years post-operatively.

General anesthesia with double-lumen endotracheal intubation and one-lung ventilation was used for all patients during surgery. For the VATS procedure, three working ports were established for the insertion of thoracoscopic instruments; these were located at the 7th or 8th intercostal space at the mid-axillary line, the 4th or 5th intercostal space at the anterior axillary line, and the 8th or 9th intercostal space at the scapular line. Specimen bags were used for removing the samples. No muscles (latissimus dorsi or serratus anterior muscles) or ribs were cut. A proportion of the lung tissues were fixed in 10% formalin, for 48–72 h at 4°C for immunohistochemistry, while others were stored at −80°C for subsequent use in reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses.

The 5-µm-thick tissue sections were deparaffinized in xylene for 20 min twice and hydrated in a graded series of alcohol baths (100% for 5 min twice, 95% for 5 min twice, 90% for 5 min once and 80% for 5 min once). The sections were then washed with PBS, immersed in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity at 37°C prior to being washed again with PBS and microwaved (medium power, 92–98°C) for 20 min for antigen retrieval. Subsequently, the sections were incubated with goat anti-human CCL20 monoclonal antibody (dilution, 1:100; cat. no. AF360; R&D Systems, Inc., Minneapolis, MN, USA) and mouse anti-human CCR6 monoclonal antibody (dilution, 1:200; cat. no. MAB195; R&D Systems, Inc.) overnight at 4°C. The sections were subsequently incubated with secondary anti-goat horseradish peroxidase-conjugated antibody (dilution, 1:500; cat. no. HAF019; R&D Systems, Inc.) or secondary anti-mouse horseradish peroxidase-conjugated antibody (dilution, 1:500; cat. no. abs20002A; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), respectively, for 30 min at room temperature. The sections were washed with PBS then incubated for 3–5 min with 3,3′-diaminobenzidine (dilution, 1:1,000; cat. no. K3468 Dako; Agilent Technologies, Inc.), followed by counterstaining with hematoxylin for 30 sec at room temperature and were dehydrated and mounted. Negative controls were performed in all cases by omitting the primary antibody.

The slides were independently evaluated and scored under a CX41 light microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan) by two pathologists without knowledge of clinical data who randomly captured and assessed 20 fields of view. The staining intensity was assigned a scored between 0 and 3, as follows: 0, negative; 1, weak; 2, moderate; or 3, strong. The percentages of positively stained tumor cells were counted and grouped into four levels (0–25, score 0; 26–50, score 1; 51–75, score 2; 76–100%, score 3). Samples with >50% positive cells were defined as having high expression. The total immunostaining score was calculated as the sum of each intensity score multiplied by the corresponding percentage, for example CCR6 index = (total staining intensity score of CCR6 × total positive tumor cells % score for CCR6)/100.

In total, 50 mg of each tissue sample was subjected to total RNA isolation using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. To ensure that total RNA fulfilled the requirements for the qPCR, the purity and quantity of RNA for each sample was analyzed by nanodrop (a minimum A260/A280 ratio of >1.8 was applied for all samples) and RNA integrity was assessed by formaldehyde modified gel electrophoresis. cDNA was synthesized using PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China). DNase (Takara Biotechnology Co., Ltd.) was used prior to the reverse transcription reaction. The following primer sequences were used: CCL20 forward, 5′-ATGTGCTGTACCAAGAGTTT-3′ and reverse, 5′-CAAGTCTGTTTTGGATTTGC-3′; CCR6 forward, 5′-CCATTCTGGGCAGTGAGTCA-3′ and reverse, 5′-AGCAGCATCCCGCAGTTAA-3′; GAPDH forward, 5′-ATCCCATCACCATCTTCCAG-3′ and reverse, 5′-GAGTCCTTCCACGATACCAA-3′. qPCR was performed using a thermocycler for 35 cycles according to the following program: 5 min at 95°C, 15 sec at 95°C, 30 sec at 58°C and 30 sec at 72°C. The PCR mixture was prepared using Eastep^® qPCR Master Mix (Shanghai Promega Biological Products Co., Ltd., Shanghai, China) according to the manufacturer's protocol. Expression levels of each mRNA were determined using the 2^−ΔΔCq method (19) using GAPDH (Takara Biotechnology Co., Ltd.) as an endogenous control. The experiment was repeated three times.

Protein samples were prepared using radioimmunoprecipitation assay buffer. The total protein concentration was measured by using the BCA method and 60 µg of each sample was subjected to 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. Subsequent to blocking at room temperature for 1 h with 5% fat-free milk powder with 1% Triton X-100 in TBS (20 mmol/l Tris-Cl, 150 mmol/l NaCl, pH 7.4), the films were incubated at 4°C overnight with antibodies against CCL20 (dilution, 1:200; cat. no. AF360; R&D Systems, Inc.), CCR6 (dilution, 1:200; cat. no. MAB195; R&D Systems, Inc.) and β-actin (dilution, 1:2,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Corresponding secondary anti-mouse horseradish peroxidase-conjugated antibody (dilution, 1:5,000; cat. no. abs20002A; DakoCytomation, Glostrup, Denmark) was then applied for 1 h at room temperature. The bands were detected on an X-ray film following the application of Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.). Relative optical density of all bands was analyzed by GelPro Analyzer (version 4.0; Media Cybernetics, Inc., Rockville, MD, USA).

The human alveolar epithelial A549 cell line was purchased from Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China) and grown in RPMI-1640 medium, supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. RPMI-1640 medium and 10% fetal bovine serum were purchased from Thermo Fisher, Scientific, Inc. Cultures were maintained on tissue culture-treated petri dishes (BD Falcon; BD Biosciences, Franklin Lakes, NJ, USA) in a 5% CO₂ incubator at 37°C.

Prior to western blot analysis, A549 cells were treated with CCL20 (500 ng/ml; R&D Systems, Inc.) for 0, 5, 15, 30 and 60 min. A549 cells with various treatments were harvested and lysed on ice in RIPA buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0. 2 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride and 0.5 % NP-40; cat. no. 89900; Thermo Fisher Scientific, Inc.) containing a protease inhibitor. The total protein concentration was measured using the BCA method. Proteins (60 µg per lane) were subjected to SDS-PAGE using a 10% gel, and blotted onto nitrocellulose membranes (Hybond ECL; GE Healthcare, Chicago, IL, USA). Membranes were blocked by incubation in TBS containing 5% nonfat dry milk and 0.1% Tween-20 for 2 h at room temperature and then incubated overnight at 4°C with rabbit anti-human p-ERK antibodies (dilution, 1:1,000; cat. no. sc-101760; Santa Cruz Biotechnology, Inc.), ERK antibodies (dilution, 1:1,000; cat. no. sc-292838; Santa Cruz Biotechnology, Inc.) and β-actin (dilution, 1:1,000; cat. no. sc-130656; Santa Cruz Biotechnology, Inc.). Blots were then washed with TBS/Tween-20 three times and incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-rabbit antibodies (cat. no. ab97051; Abcam, Cambridge, UK). The bands were detected on an X-ray film following application of Pierce ECL Western Blotting Substrate Thermo Fisher Scientific, Inc.). Relative optical density of all bands was analyzed using GelPro Analyzer 4.0.

The A549 cells were treated with various concentrations of CCL20 (10, 50 and 250 ng/ml) and PD98059 (20 µl/ml) (Cell Signaling Technology Biological Reagents Co., Ltd., Shanghai, China) at 37°C for 14 days. After macroscopically visible clones appeared, the culture was terminated; the cells were rinsed with PBS twice, fixed with methanol at 37°C for 15 min and subjected to Giemsa staining (Giemsa stain, modified; cat. no. GS1L-1L; Sigma-Aldrich; Merck KGaA, Darmstadt. Germany), performed according to manufacturer's protocol. The number of colonies was counted in 10 different fields under a CX41 light microscope (magnification, ×100; Olympus Corporation).

Analyses were performed using SPSS 17.0 for Windows (SPSS, Inc., Chicago, IL, USA). Data are expressed as percentages or as the mean ± standard deviation. The mean differences of continuous variables between groups were analyzed using an unpaired Student's t-test. The differences of categorical variables between groups were analyzed using the χ² test. The mean differences of continuous variables among groups were analyzed using one-way analysis of variance, and the Newman-Keuls post hoc test was used in the event of a significant F-ratio. All significant tests were two-sided and P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, CCL20 was expressed in the cell membrane and cytoplasm, whereas CCR6 was predominantly expressed in the cytoplasm of lung adenocarcinoma tissues. In cancer-adjacent tissues, there was scarcely any expression of CCL20 or CCR6. The percentages of high-expression samples (with >50% of cells stained) in the recurrence group were 76 and 66%, respectively, which were significantly higher compared with those in non-recurrence group (8 and 6%; P<0.001). The sum of the staining indexes (total immunostaining score) of CCL20 and CCR6 in the recurrence group were 149.3 and 134.4, respectively, significantly higher than those in non-recurrence group, which were 57.2 and 58.0, respectively (P<0.001; Tables I–III).

Compared with the non-recurrence group, the mRNA expression levels of CCL20 and CCR6 were increased by 82% (P<0.001) and 56% (P<0.001), respectively, in the recurrence group (Fig. 2). Furthermore, compared with the non-recurrence group, the relative protein expression levels of CCL20 and CCR6, measured by western blotting, were increased by 282 (P<0.001) and 188% (P<0.001), respectively, in the recurrence group (Fig. 3).

Phosphorylation of ERK in tumor cells was detected at 5 and 15 min after stimulation of the cells with 500 ng/ml CCL20, while the level of overall ERK remained unaffected. The level of phosphorylation was significantly increased at 5 and 15 min compared with that at 0 min (P<0.01), and markedly increased at 15 min compared with that at 5 min (P<0.01). The p-ERK protein level decreased at 30 and 60 min (Fig. 4).

The colony formation was significantly increased following stimulation with 50 or 250 ng/ml CCL20 compared with 0 and 10 ng/ml, in a dose-dependent manner (P<0.01; Table IV).

In the absence of CCL20 stimulation, there was no change in colony formation when cells were treated with specific ERK inhibitor compared with untreated cells (P=0.147), indicating no effect of PD98059 on the basal capacity of colony formation of A549 cells. However, treatment with PD98059 significantly attenuated the CCL20-induced increase in colony formation ability (0 vs. 20 µl/ml PD98059, P<0.001 for all concentrations of CCL20; Table IV).