A total of 208C57BL/6 mice (age, 5–6 weeks; weight, 16±2 g), including the same number of male and female mice, were obtained from Beijing Weitong Lihua Laboratory Animal Technology Ltd. Co., Beijing, China. The mice were raised in the specific pathogen-free animal laboratory of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China), where the housing conditions were strictly controlled (temperature, 18–24°C; humidity, 40–60%; and light/dark cycle, 12/12 h). The carcinogen 4NQO (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was dissolved and diluted in sterile distilled water to 50 µg/ml. All experiments followed the guidelines of the welfare committee of the Fourth Hospital of Hebei Medical University.

After 1 week of adaptive feeding, the 208 mice were assigned to the control group (n=64) or to the 4NQO model group (n=144). The experiment lasted 28 weeks. The control mice were administered sterile distilled water during these 28 weeks, while the 4NQO model mice were fed with 4NQO (50 µg/ml) as drinking water from the 1st to the 15th week, and then with sterile distilled water from the 16th to the 28th week. The body weight of all mice was tested once every 2 weeks in order to assess their physical condition. In the period ranging from week 16 to 28, 9 mice from the control group and 20 mice from the 4NQO model group were sacrificed by removing the cervical vertebra every 2 weeks.

For every sacrifice, samples derived from 3 control mice and 7 4NQO model mice were subjected to H&E staining and immunohistochemistry tests. Following sacrifice, the thoracic cavity and abdominal cavity of the mice were exposed, and the esophageal tissues were separated and dissected longitudinally, rolled up from cardiac end to tongue end, and then fixed with pins. Subsequently, the esophagi were fixed in 4% formaldehyde, embedded in paraffin and sectioned into 4-µm-thick sections. Sections were stained with H&E. The H&E staining result was defined according to the highest pathological stage for each sample.

Immunohistochemistry tests were performed to detect the expression of B7-H4 as well as the infiltration of cluster of differentiation CD4⁺T, CD8⁺T and CD11b⁺ macrophage cells in esophageal tissues. The sections were dewaxed, rehydrated and exposed to 3% H₂O₂ solution for 20 min to block endogenous peroxidase. Then, antigens were hot-retrieved with high pressure (B7-H4, 4.5 min heating time and pH=6.0 citrate buffer; CD4 and CD8, 8 min heating time and pH=8.0 EDTA buffer; and CD11b, 10 min heating time, and pH=6.0 citrate buffer). Sections were incubated with primary antibodies against B7-H4 (1:500; GTX62090; GeneTex, Inc., Irvine, CA, USA), CD4 (1:50; orb4241; Biorbyt, Ltd., Cambridge, UK), CD8 (1:50; orb10325; Biorbyt, Ltd.) or CD11b (1:100; cb1211; Abcam, Shanghai, China) at 4°C overnight. Standard protocols for immunohistochemistry were followed as described previously (25). Scores of B7-H4 expression were derived from the multiplication of the area and intensity scores (26). The prevalence of CD4⁺T, CD8⁺T or CD11b⁺ macrophage cells was semi-quantitatively evaluated according to the number of each type of cells present per vision field (magnification, ×400) (27). Of note, the pathological grade of the observation field was in line with the H&E pathological result in each sample.

For every sacrifice, tissue samples derived from 3 control mice and 7 4NQO model mice were subjected to RT-qPCR analysis to evaluate the gene expression of B7-H4 and associated molecules. Total RNA was extracted from the tissue samples using TRIzol Reagent (Sigma-Aldrich; Merck Millipore) according to the manufacturer's protocol. Total RNA (2 µg for each sample) was reverse transcribed to cDNA with a Reverse Transcription System (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. qPCR was performed in a total volume of 20 µl, and the reaction mixture contained cDNA product (1 µg), specific primers (0.2 µmol/l), SYBR-Green qPCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and nuclease-free water. The sequences of the primers were as follows: B7-H4 sense, 5′-CACTCCTATCTCTCTCCTGTTGCCT-3′ and antisense, 5′-CACATCTTGTTCCTGTCTGCTCTCT-3′; interleukin (IL)-6 sense, 5′-CCATCCAGTTGCCTTCTTGG-3′ and antisense, 5′-CTCCTCTCCGGACTTGTGAA-3′; IL-10 sense, 5′-CTGAAGACCCTCAGGATGCG-3′ and antisense, 5′-TGGCCTTGTAGACACCTTGG-3′; transforming growth factor (TGF)-β sense, 5′-GCTGAACCAAGGAGACGGAA-3′ and antisense, 5′-AGAAGTTGGCATGGTAGCCC-3′; interferon (IFN)-γ sense, 5′-AGCCTCAGGAAGCGGAAAAG-3′ and antisense, 5′-TGAGTTCAGTCAGCCGCTTG-3′; STAT3 sense, 5′-TTGAGTCGCTCACGTTTGAC-3′ and antisense, 5′-GGCACTTGTCTAACAACCAACC-3′; and GAPDH sense, 5′-CTTTGGCATTGTGGAAGGGCTC-3′ and antisense, 5′-GCAGGGATGATGTTCTGGGCAG-3′. The number of specific transcripts was normalized to the housekeeping gene GAPDH. The qPCR cycling conditions were as follows: 95°C for 10 min, followed by 42 cycles of 95°C for 15 sec, 55°C for 15 sec and 60°C for 1 min. Differences in the expression levels of the genes were determined by calculating the fold-change in expression according to the 2^−ΔΔCq method (28).

For every sacrifice, samples derived from 3 control mice and 6 4NQO mice were used to evaluate the expression of B7-H4 and associated proteins. The proteins were extracted from homogenates of esophageal tissues and quantified using the Bicinchoninic Acid kit (Sigma-Aldrich; Merck Millipore), as described previously (29). Western blot analysis was performed as described previously (30). All proteins were subjected to 10% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The transfer times were 40 min, 1, 1.5 and 1.5 h for β-actin, B7-H4, STAT3 and phosphorylated (p)-STAT3, respectively. The membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck Millipore) at 37°C for 1 h and incubated with primary antibodies, including rabbit anti-mouse β-actin (1:5,000; 600-401-886s; Rockland Immunochemicals, Inc., Philadelphia, PA, USA), anti-B7-H4 (1:2,000; GeneTex, Inc.), anti-STAT3 (1:2,000; 79D7; Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-p-STAT3 (1:500; 3771; Cell Signaling Technology, Inc.) monoclonal antibodies overnight at 4°C, followed by incubation with a fluorescent rabbit antibody (1:5,000; sc2020; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at 37°C. Finally, the gray value was detected with an Odyssey fluorescence scanner (LI-COR Biotechnology GmbH, Bad Homburg, Germany).

ELISA for IL-6 evaluation was detected according to the protocol of the Mouse IL-6 ELISA kit (Dakewe Biotech Co., Ltd., Beijing, China). A total of 50 µl protein solution was required for each sample. Protein samples and IL-6 standard solutions were added to enzyme-labeled wells. Subsequently, 100 µl biotinylated antibody was added to each well and the plates were incubated for 90 min at 37°C. After washing five times, 100 µl streptavidin-horseradish peroxidase was added to each well and incubated for 30 min at 37°C. Then, 100 µl 3,3′,5,5′-tetramethylbenzidine was added to each well and incubated for 20 min at 37°C. Finally, the reaction was stopped by 100 µl/well stop solution. The absorbance value was detected at 620 nm.

Results are presented as the mean ± standard deviation. All the experimental data were analyzed with GraphPad Prism 5.0 statistical software package (GraphPad Software, Inc., La Jolla, CA, USA). Spearman correlation analysis was performed to compare the correlation between different parameters. Student's t-test and Kruskal-Wallis analysis were performed to compare the difference between ≥2 groups. P<0.05 was considered to indicate a statistically significant difference.

Firstly, with the prolongation of the time of inducing cancer, the physical conditions of 4NQO model mice became poor, and their body weight increased more slowly compared with that of control mice (Fig. 1A). In addition, 1 control mouse and 4 4NQO model mice died without additional intervention and were excluded from the study.

Secondly, the esophageal tissues of all mice were dissected. Among these tissues, the esophagi of control mice exhibited smooth surface, normal thickness and flexibility, without any abnormal convex or concave appearance. However, the esophagi of 4NQO model mice exhibited exacerbation of the pathological conditions with the prolongation of the time (Fig. 1B). There were white patches scattered in the epidermis of esophagi from weeks 16 to 18. Subsequently, the esophageal tissues commenced to appear thicker and rough, with abnormal touchable protuberances from weeks 20 to 22. Furthermore, the thickness of the esophagi increased further and the surface became uneven, with an increased number of obvious masses of different size from weeks 24 to 28. In the present study, H&E staining revealed that, during the whole process of carcinogenesis, the pathological stage of the esophageal epithelium underwent normal, low-grade intraepithelial neoplasia (LGIN) and high-grade intraepithelial neoplasia (HGIN), as shown in Fig. 1C. Normal esophageal epithelial cells displayed regular distribution, natural nuclear size and polarity. In LGIN, however, the mucosal epithelium became thicker, and less than half of the cells lost polarity, their nuclei became enlarged and were hyperchromatic. Furthermore, in HGIN, the epithelial layer became even thicker, and >1/2 of cells lost polarity and presented a more enlarged nucleus. Additionally, carcinoma in situ, which occurs when the cells in the whole epithelium lose polarity and become immature (31), was also observed in HGIN. All the above pathological characteristics were similar to those observed in humans (31).

The association between esophageal pathological stage and time of carcinogenesis induction was analyzed (Table I). In the present study, no mice exhibited abnormal pathological conditions in the control group. 4NQO model mice, on the contrary, exhibited all types of precancerous conditions (LGIN and HGIN). Furthermore, with increasing time, the pathological degree of their esophagi markedly increased (P<0.0001). There was no difference in the pathological stage between male and female mice, according to the results of Kruskal-Wallis analysis.

Overall, these findings indicated that 4NQO was a carcinogen able to induce SCC formation in mice, and the precancerous conditions exacerbated with increased time of induction. Therefore, the expression and significance of B7-H4 in the mouse model of SCC induced by 4NQO were evaluated.

B7-H4 was expressed in the cytoplasm and membrane of the epithelial cells in the mucosal layer (Fig. 2). According to the final scores (low or high expression), there were only 2 of 22 normal samples (9.1%) with B7-H4 high expression. As to the precancerous conditions, the B7-H4 high-expression rate was 40.7% (11/27) in LGIN specimens and 81.0% (17/21) in HGIN cases (Table II). Spearman correlation analysis demonstrated that the expression of B7-H4 was positively correlated with the pathological stage (P<0.0001), but not with the gender of the mice (P=0.2959).

CD4, CD8 and CD11b are membrane molecules mainly expressed in CD4⁺T, CD8⁺T and macrophage cells (32). As represented in Fig. 2, the infiltrations of these three types of lymphocytes were predominantly located in the submucosa of esophageal epithelia instead of the mucosa layer. It was speculated that the reason for this observation maybe that the extent of the lesion was not severe enough to be infiltrated by lymphocytes into the mucosa layer. In addition, it was observed that, during the precancerous condition, the effect of lymphocytes on the lesion was indirect, which may be due to the secretion of cytokines. The association between B7-H4 expression in the mucosa and lymphocytes infiltration in the pertinent submucosa was also analyzed. As indicated in Table II, the results of Spearman correlation analysis revealed that B7-H4 expression was positively correlated with CD11b⁺ macrophages infiltration (P=0.0002), but not with CD4⁺T (P=0.8507) or CD8⁺T cells infiltration (P=0.3166).

Collectively, these data indicated that B7-H4 protein expression increased significantly and was positively correlated with pathological variables and macrophages infiltration, but was not associated with gender or with CD4⁺T or CD8⁺T cell infiltration during the carcinogenesis process induced by 4NQO. Thus, the present data suggested that B7-H4 probably is important in promoting the progression of esophageal precancerous lesions, which may be attributed to its association with certain type of cytokines secreted by macrophages.

To evaluate the association between B7-H4 expression and esophageal precancerous lesions or cytokines secreted by macrophage cells, B7-H4, IL-6, IL-10, TGF-β, IFN-γ and STAT3 gene expression was assessed by RT-qPCR. Firstly, there was no obvious change in the expression of these genes in the control mice from the 16th to the 28th week (Fig. 3). Therefore, the control value of one gene comes from the data of all esophagi of the control mice during the whole carcinogenesis. Secondly, it was shown that the expression level of B7-H4, IL-6, IL-10, TGF-β and STAT3 genes in the esophagi of 4NQO model mice increased significantly compared with that of control mice, while IFN-γ gene expression did not change obviously (Fig. 4). Additionally, there were significantly positive correlations between B7-H4 gene expression and IL-6 (r=0.5952, P<0.0001), IL-10 (r=0.6561, P<0.0001), TGF-β (r=0.6586, P<0.0001) and STAT3 (r=0.5799, P<0.0001) gene expression. Therefore, the present data supported the hypothesis that B7-H4 participated in the formation of esophageal precancerous lesions via interaction with the IL-6, IL-10, TGF-β or STAT3 signal transduction pathways.

The present study focused on the association between B7-H4 expression and the IL-6/STAT3 signaling pathway. The protein levels of B7-H4, STAT3, p-STAT3 and IL-6 were determined by western blotting and ELISA. No obvious changes in the expression levels of these proteins were observed in the control mice from weeks 16 to 28 (Fig. 5). Thus, the control value of one protein came from the data of all esophagi of control mice during the whole carcinogenesis in the following statistical analysis. As presented in Fig. 6, in comparison with control mice, B7-H4 and IL-6 protein expression was upregulated in the model group during the esophageal carcinogenesis process, and increased significantly in weeks 26 and 28 (B7-H4: P=0.0159 and 0.0025, respectively; IL-6: P=0.0265 and 0.0057, respectively). Additionally, p-STAT3 levels also increased significantly in the 28th week (P=0.0127), despite the fact that the change in STAT3 expression was not significant (P=0.1033). During the whole process of carcinogenesis, B7-H4 expression was positively correlated with IL-6 and p-STAT3 expression (r=0.6145, P<0.0001; and r=0.6180, P<0.0001, respectively). Overall, these results implicated that the high expression of B7-H4 and its function enhancing esophageal precancerous lesions is associated, at least in part, with IL-6/STAT3 activation.