Expression of CCL18 in BC (BLCA dataset) was checked using The Cancer Genome Atlas (TCGA) data. TCGA, launched by the National Institutes of Health (NIH, Bethesda, MD, USA), is a publicly funded project that comprises a comprehensive ‘atlas’ of cancer genomic profiles. The gene expression levels of CCL18 in 404 patients with BC compared with 28 normal samples from TCGA database and GTEx were analyzed using the GEPIA web server (14). The Pathology Atlas from the Human Protein Atlas (www.proteinatlas.org/pathology) was used to perform analyses based on the mRNA expression levels of CCL18 in BC tissue and the clinical outcomes (survival). The data in the Pathology Atlas are based on the integration of publicly available data from TCGA. In addition, gene expression profiling studies involving several clinical samples were performed to analyze the expression of CCL18 in a dataset available through the Gene Expression Omnibus (GSE31684; http://www.ncbi.nlm.nih.gov/geo/) (15).

The present study was approved by the Research Ethics Committee of the First Affiliated Hospital, Nanchang University (Nanchang, China). Informed consent was obtained from all patients. All specimens were handled and anonymized according to ethical and legal standards. Immunohistochemical analysis of CCL18 was conducted using 64 primary BC tissues and 30 adjacent noncancerous bladder tissues. Detailed information on the clinical features of all patients in this study is presented in Table I.

The 5637 human BC cell line was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China) and was maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator containing 5% CO₂. The small molecule CCR8 inhibitor R243 (cat. no. AOB2014) was purchased from AOBIOUS, Inc. (Gloucester, MA, USA). For chemokine treatment, 5637 cells pretreated with or without R243 (5 µM) for 6 h at 37°C/5% carbon dioxide (16), were exposed to 50 or 100 ng/ml CCL18 (PeproTech, Inc., Rockville, MD, USA) for 36 h.

For transfection, 5637 cells were plated in 6-well plates at 2×10⁵/well. The human CCR8 shRNA plasmid and non-target shRNA (NT shRNA) were obtained from Invitrogen; Thermo Fisher Scientific, Inc. The targeted sequences were: Human CCR8 (5′-CCGGGGATTATACACTTGACCTCAGTGTCGACACTGAGGTCAAGTGTATAATCCTTTTTTG-3′) and non-target (NT) shRNA (5′-CCGGCAACAAGATGAAGAGCACAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3′). Once the 5637 cells reached 50–60% confluence, 1.25 µg/ml CCR8 shRNA and NT shRNA were transfected using Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A total of 6 h post-transfection, the medium was replaced with fresh medium containing 10% FBS. NT shRNA was used as the negative control.

A total of 24, 48 and 72 h post-transfection, total RNA was extracted from 5637 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using a Takara PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan) according to manufacturer's protocols. qPCR was performed using SYBR Premix Ex Taq kit (Takara Bio, Inc., Otsu, Japan) in the ABI PRISM^® 7500 real-time PCR system (Thermo Fisher Scientific, Inc.). The thermocycling conditions were initial denaturation (1 cycle, 95°C, 30 sec), PCR reaction (40 cycles, 95°C, 5 sec, and 60°C, 30 sec for annealing and elongation). The data of RT-qPCR were normalized against an internal control β-actin and relative expression levels were evaluated using the 2^−∆∆Cq method and then expressed as fold changes (17). The oligonucleotide sequences of the RT-qPCR primers are listed in Table II.

Total proteins were extracted from cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins were quantified using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.), followed by western blot analysis. 40 µg proteins from cell lysates were subjected to 12% SDS-PAGE. Once proteins were transferred to nitrocellulose membranes, they were incubated with antibodies targeting β-actin (cat. no. 58169, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CCR8 (cat. no. ab32131, 1:500; Abcam, Cambridge, UK), E-cadherin (cat. no. 3195, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), matrix metalloproteinase (MMP)-2 (cat. no. 40994, 1:1,000; Cell Signaling Technology, Inc.) and vascular endothelial growth factor (VEGF)-C (cat. no. 2445, 1:1,000; Cell Signaling Technology, Inc.), followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721, 1:3,000; Abcam) for 1.5 h at room temperature. Protein bands were visualized by Millipore enhanced chemiluminescence (cat. no. WBKLS0500; EMD Millipore, Billerica, MA, USA). ImageJ 1.45 software (NIH) was used to perform densitometric analysis of each band.

Tissue sections from paraffin- embedded bladder cancer were dewaxed in xylene and rehydrated in a graded alcohol series. Following microwave-induced antigen retrieval (Tris-EDTA pH 9.0), the slides were washed with PBS and incubated with primary antibody against CCL18 (cat. no. ab104867, 1:200; Abcam) overnight at 4°C. The sections were incubated with secondary antibody at room temperature. for 30 min after washing with PBST. Color detection was performed by liquid DAB⁺ substrate chromogen system (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's protocols. Slides were counterstained with hematoxylin. Hemalum was used to stain nuclei of cells blue at room temperature for 20 sec. Immunohistochemical images of CCL18 expression were analyzed according to a previous study (18). Representative areas with 5 or 20 CCL18 cells per ×400 high-power field were chosen and images were captured randomly as references for grading. The densities of CCL18⁺ cells were graded as follows: Grade 1, <5 positive cells/microscopic field; grade 2, 5–20 cells/field; and grade 3, >20 cells/field. Grade 1 was considered to be low-level CCL18⁺, and grades 2 and 3 were considered to be high-level CCL18⁺.

Cell migration was determined using a wound-healing assay. Briefly, 5637 cells were seeded in six-well culture dishes and cultured at 37°C to form a confluent monolayer. After transfection with or without CCR8 shRNA for 24 h, and a wound was made by scratching the monolayer with a 10 µl pipette tip. The wounded monolayer was then washed three times with PBS to remove cell debris and treated with CCL18 for 24 h at 37°C. After scratching, the area of the cell-free scratch was imaged under an Olympus CKX41 inverted microscope (Olympus Corporation, Tokyo, Japan) at 0 and 24 h. Cell invasive capacities were measured using a Transwell assay (Corning Incorporated, Corning, NY, USA). Briefly, 5637 cells were treated with CCL18 and/or transfected with CCR8 shRNA for 24 h, and were then seeded in the upper chamber of the Transwell system. The upper chamber of the insert was precoated with 0.1 ml (300 µg/ml) Matrigel matrix (Corning Incorporated) for the invasion assay. The invaded cells on the underside of the membrane were counted. In this assay, prepared cells were seeded in the upper chamber with serum-free medium and the medium of the lower chamber was supplemented with 10% FBS as a chemoattractant. Following incubation for 24 h, the cells were fixed with 4% formaldehyde at 37°C for 30 min. Cells that did not invade through the pores were removed with a cotton swab. Cells that had invaded to the lower surface of the membrane were stained with crystal violet at 37°C for 15 min. Finally, images of five representative fields at ×100 magnification were randomly captured using a light microscope (Carl Zeiss AG, Oberkochen, Germany) and the number of cells in each well was semi-quantified.

All data are presented as the means ± standard deviation in at least three replicates per group. Statistical analysis was performed to determine the significance of the differences between groups using one-way analysis of variance (with post hoc Turkey's honest significant difference test) or Student's t-test. χ² analysis and Fisher's exact test were used to examine the association between CCL18 expression and the clinicopathological features of patients with BC. The effects of CCL18 expression levels on survival were estimated using the Kaplan-Meier method and were compared by the log-rank test. Wilcoxon-signed rank test was used to compare CCL18 expression between 30 paired cancerous and noncancerous tissues of patients with BC. All statistical analyses were performed using GraphPad Prism 7.00 software for Windows (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To assess whether CCL18 may be involved in the development of BC, this study first investigated the mRNA expression levels of CCL18 using TCGA dataset. As illustrated in Fig. 1A, the mRNA expression levels of CCL18 were significantly higher in BC tissues compared with in nontumor tissues. In addition, CCL18 mRNA expression was higher in BC tissues at stages III and IV compared with at stage II (Fig. 1B and C). Kaplan-Meier analysis indicated that higher CCL18 expression was associated with worse patient survival (log-rank test, P=0.0487; Fig. 1D). Subsequently, the expression of CCL18 was investigated in BC tissues using immunohistochemistry. As reported in a previous study (10), there was no epithelial staining for CCL18; however, inflammatory cells in the stroma were CCL18-positive. It was revealed that CCL18 expression was higher in the majority of cancerous samples compared with in noncancerous tissues (Fig. 1E). Furthermore, the protein expression levels of CCL18 were detected in tissues obtained from 30 patients with BC by immunohistochemical staining. The results demonstrated that CCL18 expression was significantly stronger in the majority of BC tissues compared with in paired normal tissues (Fig. 1F). As shown in Fig. 1G, CCL18 was weakly detected in stage I and II BC tissues, but was strongly detected in stage III and IV BC tissues in tissue samples employed in the present study. Subsequently, the association between CCL18 expression in BC cases and various clinical outcomes was analyzed. As shown in Table I, higher CCL18 expression was associated with higher pathological stages (P<0.05). These data suggested that CCL18 was elevated in BC tissues and may be associated with the progression of BC.

EMT is thought to serve a key role in the invasion and metastasis of numerous types of tumor (19). EMT is an evolutionarily conserved developmental program, which is believed to have a critical role in carcinogenesis and tumor metastasis by enhancing mobility and invasion. Our previous study revealed that VEGF-C, MMP-2 and MMP-9 are immunosuppressive factors, which serve important roles in BC invasion and metastasis (20). To determine whether CCL18 enhanced BC invasion and metastasis through EMT, and by altering MMP-2 and VEGF-C expression, 5637 BC cells were treated with rCCL18. Following treatment of 5637 BC cells with 50 or 100 ng/ml CCL18 for 36 h, the expression levels of E-cadherin were significantly lower, and the expression levels of MMP-2 and VEGF-C were higher compared with in the control group (Fig. 2).

CCL18 has numerous receptors, including PITPNM family member 3 (PITPNM3) and G protein-coupled estrogen receptor 1 (GPR30), which mediate CCL18-induced migration of cancer cells, and promote tumor invasion and metastasis (8,21). Recently, Islam et al reported that CCR8 is a functional receptor for CCL18 (13). To identify whether CCR8 was associated with CCL18-induced tumor invasion and EMT, this study examined the expression levels of E-cadherin, MMP-2 and VEGF-C in 5637 BC cells treated with a small molecule CCR8 inhibitor, R243, or transfected with CCR8 shRNA, followed by CCL18 treatment (Figs. 2 and 3). As shown in Fig. 2, R243 reversed the decreased levels of E-cadherin and increased levels of MMP-2 and VEGF-C caused by CCL18, without affecting CCR8 levels. The results of RT-qPCR confirmed that the mRNA expression levels of CCR8 were downregulated by CCR8 shRNA (Fig. 3A); CCR8 protein expression was also reduced in response to CCR8 shRNA (Fig. 3D). RT-qPCR analysis revealed that the mRNA expression levels of E-cadherin were increased, whereas those of MMP-2 and VEGF-C were decreased compared with in cells transfected with NT shRNA (Fig. 3B). Similar results were revealed by western blot analysis for the protein expression levels of E-cadherin, MMP-2 and VEGF-C (Fig. 3C and D). Subsequently, the migratory and invasive abilities of BC cells were reduced in CCR8-knockdown cells compared with in control cells (Fig. 4A-D).