Human tissues and blood specimens were obtained from patients with ESCC who underwent surgery at the Division of Thoracic Surgery, Department of Surgery, Kaohsiung Medical University Hospital (Kaohsiung, Taiwan) between January 2009 and December 2014. Patients who received neoadjuvant chemotherapy or radiotherapy were included, while patients with a history of other malignancies and those who underwent esophageal surgery were excluded from the present study. The tumor grading system was classified according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (7th edition; http://cancerstaging.org/), and lymph node metastasis was determined by examining the presence or absence of invading tumors in the lymph nodes.

Esophageal cancer tissues and paired adjacent normal esophageal tissues were resected from patients with ESCC (n=73), while blood samples were collected from a different cohort of patients with ESCC (n=26, including 25 males and one female; mean age, 54 years; age range, 35–77 years) and healthy subjects (n=26, including 25 males and one female; mean age, 52 years; age range, 38–69 years). The present study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (approval no. KMUH-IRB-20130627; Kaohsiung, Taiwan) and written informed consent was provided by all participants prior to this study.

The human ESCC cell line, KYSE70 was gifted by Dr Yi-Ching Wang at the Department of Pharmacology, National Cheng Kung University (Tainan, Taiwan), while the TE8 cell line was gifted by Dr Mien-Chie Hung at the University of Texas MD Anderson Cancer Center (Houston, Texas). KYSE70 cells were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.), while TE8 cells were maintained in DMEM/F12 (Invitrogen; Thermo Fisher Scientific, Inc.). All cells were maintained in a humidified incubator with 5% CO₂ at 37°C, and the cell culture media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin (all Biological Industries) and 0.25 µg/ml amphotericin B (Biological Industries). Recombinant human resistin was purchased from PeproTech, Inc.

Serum resistin levels in patients with ESCC and healthy controls were measured using the Human Resistin ELISA kit (cat. no. EK-028-36, Phoenix Pharmaceuticals, Inc.), according to the manufacturer's instructions.

The migratory ability of cells was assessed via the Transwell assay (Corning, Inc.). KYSE70 cells were seeded into Transwell inserts at a density of 5×10⁴ cells/insert and placed in 24-well plates with serum-free cell culture medium (Invitrogen; Thermo Fisher Scientific, Inc.) to form the upper chamber. Normal cell culture medium supplemented with 10% FBS was plated in the lower chamber. Following treatment with resistin (0, 25, 50 or 100 ng/ml) at 37°C for 24 h, cells on the upper chamber were removed using cotton swabs, while the migratory cells were fixed in 4% formaldehyde at room temperature for 15 min, and stained with 0.2% crystal violet at room temperature for 10 min, and observed under a light microscope with a 10X objective lens (total magnification, ×100). Quantification of cell migration was performed using ImageJ software ver. 1.53j (National Institutes of Health; http://imagej.nih.gov/ij/).

Cell proliferation was assessed via the XTT assay (X4626, Sigma-Aldrich; Merck KGaA). KYSE70 or TE8 cells were seeded into 96-well plates at a density of 5×10³ cells/well and treated with resistin (0, 25, 50 and 100 ng/ml) at 37°C for 24–72 h. Following treatment, XTT reagent was added into each well along with phenazine methosulfate (Sigma-Aldrich; Merck KGaA) at 37°C for 2 h, according to the manufacturer's instructions. Absorbance was measured at a wavelength of 475 nm (A1) using a spectrophotometry, with a nonspecific reference wavelength at 660 nm (A2) to calculate the final reading (ΔA=A1-A2).

IHC analysis was performed to analyze resistin expression in esophageal cancer tissues and paired adjacent normal esophageal tissues, using the Leica Bond-Max automated IHC stainer (Leica Microsystems, Inc.), according to the manufacturer's instructions and our previous report (23). IHC staining with anti-resistin primary antibody (1:100; cat. no. sc-376336; Santa Cruz Biotechnology, Inc.) was determined independently and blindly by two pathologists using a modified H-score (24). Briefly, the percentage of positively stained cells was categorized as follows: 0, 0–4%; 1, 5–24%; 2, 25–49%; 3, 50–74% and 4, 75–100%. The intensity of staining was categorized as follows: 0, negative; 1, weak; 2, moderate and 3, strong. The total score for each sample was the product of positively stained cells (A) and intensity of staining (B), according to their categorized scores (ranging from 0–12 by A × B).

Patients were divided into low resistin expression (n=55) and high resistin expression (n=18) groups, and receiver-operating characteristic curve analysis was performed to determine optimal sensitivity and specificity for the cut-off point used in the present study (Fig. S1). For negative control in IHC staining, anti-resistin primary antibody was omitted and all other steps remained the same.

Resistin mRNA expression in ESCC tissues and adjacent normal tissues was analyzed using the Oncomine database (https://www.oncomine.org), which is a web-based data-mining platform with a collection of cancer microarray datasets for comparing differential expression of genes between cancer tissues and normal tissues (25). All data analyzed by Oncomine were log-transformed and are illustrated as median-centered boxplots (25).

Statistical analysis was performed using SPSS v17 software (SPSS, Inc.). For in vitro studies, data are presented as the mean ± SD from three independent experiments. Fisher's exact test was used to assess the association between resistin expression and the clinicopathological characteristics of patients with ESCC. Comparisons for the in situ or in vitro studies were calculated using either paired or unpaired Student's t-test where applicable, or one-way ANOVA followed by Dunnett's test, respectively. P<0.05 was considered to indicate a statistically significant difference.

IHC analysis revealed that resistin expression was significantly downreglated in esophageal tumor tissues compared with paired adjacent normal esophageal tissues from patients with ESCC (n=73) (P<0.001; Fig. 1). The present study assessed the association between resistin expression in ESCC tissues and the clinicopathological characteristics of the patients, who were stratified into high and low resistin expression groups determined by receiver-operating characteristic curve analysis (Fig. S1). As presented in Table I, resistin expression was significantly associated with body mass index (BMI) (P=0.042). However, no significant associations were observed between resistin expression and lifestyle factors (alcohol consumption, betal quid chewing or smoking), tumor characteristics (histopathological grade, tumor size or lymph node metastasis), treatment modalities (chemotherapy or radiotherapy) or patient outcomes (recurrence or death) (Table I).

The present study also analyzed resistin mRNA expression in ESCC tissues and normal esophageal tissues using the Oncomine database. The results demonstrated that resistin expression was higher in normal esophageal tissues compared with ESCC tissues (Fig. 2). These results suggest that the reduced resistin expression in ESCC tissues may serve as a diagnotic marker for clinical use.

Given that circulating resistin is released by adipose tissues or the associated tumor tissues (19), the present study investigated serum resistin levels in patients with ESCC compared with healthy individuals via ELISA. Despite the heterogeneous expression of serum resistin between individuals, the results demonstrated that the average serum resistin level was significantly lower in patients with ESCC (n=26) compared with the healthy individuals (n=26) (P=0.025; Fig. 3 and Table SI). Notably, the serum resistin levels observed in the present study were within a similar range as previously reported (26). These results support the IHC findings of differential resistin expression between ESCC tissues and adjacent normal esophageal tissues. In addition, the detection of serum resistin level may have the potential to be developed into a non-invasive liquid biopsy test for ESCC.

To investigate the biological effects of resistin on ESCC cell migration and proliferation, in vitro Transwell and XTT assays were performed, respectively. The results demonstrated that treatment with different concentrations of resistin significantly inhibited the migratory ability of KYSE70 cells in a dose-dependent manner (25 ng/ml vs. control, P=0.023; 50 ng/ml vs. control, P<0.001; 100 ng/ml vs. control, P<0.001; Fig. 4), while treatment with resistin had no significant effect on KYSE70 cell proliferation (Fig. 5), suggesting that resistin may have a unique role in cell migration other than affecting proliferation of ESCC.

Similar results of the effect of resistin on the migration (Fig. S2A and B) and proliferation (Fig. S2C) of TE8 cells were observed. However, whether the phenomena of resistin-reduced in vitro cell migration can be translated into the cancer progression of ESCC in clinical settings is yet to be investigated. These results also suggest that the effect of resistin on ESCC cell migration may result from a signaling pathway that is distinctive from the signaling for ESCC proliferation.