2.1 Patients and Tissue Samples

All tissue samples were collected from ESCC patients who underwent esophagectomy followed by two- or three-field lymph node dissection at our hospital. A total of 93 patients received surgical resection between January 2009 and December 2010. The clinicopathological characteristics of these patients are presented in Table 1. Informed consent was obtained from each patient, and this study was approved by the Ethics Committee of our hospital.

2.2 Cell Lines

ESCC cell lines (TE-1 and Eca-109) were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All cells were cultured in DMEM high-glycemic medium supplemented with 10% fetal bovine serum at 37°C with 5% CO₂.

2.3 Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from tissues and cells using TRIzol Reagent (Sigma). Reverse transcription and qRT-PCR were performed with the Bestar one-step qRT-PCR kit (SYBR Green). β-actin was used as an internal control. The primers at exon–exon junctions were designed using Primer Blast. During the design process, we changed the Exon junction span option to Primer must span an exon–exon junction, which means that at least one primer crosses the junction between exons. In this way, even if genomic DNA contaminates cDNA, it will not be amplified, ensuring accurate quantification. The primer sequences used in this paper were shown in Table 2.

2.4 Immunohistochemistry (IHC) Staining

ESCC tissue slides were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. Subsequently, they were blocked with 5% BSA. The slides were then incubated overnight at 4°C with a primary antibody against FOXA3 (Sigma, rabbit, 1:1000) and subsequently with a secondary antibody for 30 min. The expression levels, along with the staining intensity, were assessed based on the German immunohistochemical score (GIS). The percentage of positive cells was classified as 0 (negative), 1 (< 10% positive cells), 2 (11%–50% positive cells), 3 (51%–80% positive cells), or 4 (> 80% positive cells). Staining intensity was classified as 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong). The final immunoreactive GIS was defined as the product of both grading results (percentage of positive cells × staining intensity). According to scores averaged, the expression of FOXA3 was divided into two groups: high expression group [2, 3] and low expression group (0, 1).

2.5 Lentivirus Infection

Recombinant lentiviral vectors containing green fluorescence protein (GFP) were obtained from HanBio Technology Co. Ltd. (Shanghai, China). The resultant plasmid was sequenced to confirm the authenticity of the insert. A total of 5 × 10⁵ Eca-109 and TE-1 cells were seeded on six-well plates and further incubated for 12 h to reach 30% confluence, and then infected with lentiviral vectors expressing either vector control (LV-Con) or LV-FOXA3 by replacing the infection medium containing recombinant vectors at a multiplicity of infection (MOI) of 20 plaque-forming units (pfu) per cell. Three days later, the GFP density contained by lentivirus was detected to assess the infection efficiency, and cells were harvested for qRT-PCR and Western blot.

2.6 Western-Blot Assay

The protein was extracted from cell lysis using RIPA lysis buffer, and equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membranes. Then, the membranes were blocked with 5% skimmed milk for 1 h and incubated with the primary antibody against FOXA3 (ab108454, 1:500, Santa Cruz Biotechnology) or GAPDH (1:5000, Santa Cruz Biotechnology) at 4°C overnight. After being washed three times, the membranes were incubated with the secondary antibody for 1 h. The blots were visualized with the enhanced chemiluminescence substrate.

2.7 Colony Forming Assay

Each group of cells was seeded onto petri dishes and maintained for 2 weeks, respectively. Then, cells were fixed in 4% paraformaldehyde and stained with crystal violet. The visible colonies were counted manually after taking photographs.

2.8 Cell Counting Kit 8 (CCK-8) Assay

Cells were seeded onto the 96-well plates at a density of 1000 cells per well and incubated for 24, 48, and 72 h at 37°C. Cells were then incubated with 10-μL CCK-8 solution (R&D Systems) for 2 h. Cell counts were measured in triplicate by measuring absorbance at 450 nm using the microplate readers (BioTek Instruments). And then the cell proliferation curve was plotted according to the OD value.

2.9 5-Ethynyl-2′-Deoxyuridine (EdU) Assay

Cell proliferation was measured using the EdU assay kit (RiboBio, China). First, cells were seeded onto the 96-well plates and incubated with EdU (50 μM) solution for 2 h at 37°C. Cells were then fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. After washed with PBS, cells were incubated with 1× Apollo staining reaction (100 μL) in the dark for 30 min. Subsequently, cells were stained with Hoechest33342 (100 μL) for 30 min to visualize cell nucleus. The images were obtained using the fluorescence microscope (OLYMPUS, Japan). Cell proliferation was analyzed based on the percentage of EdU-positive cells for each sample.

2.10 Wound Healing Assay

Cells were seeded on 6-well dish at a density of 3 × 10⁶/mL and grown until reaching 90% confluence. A 200-μL pipette tip was used to create a straight scratch in each well, and the images were captured at 0 and 48 h under a microscope to observe wound closure.

2.11 Transwell Assay

Tumor cell migration assay was performed using a Transwell chamber with 8-μm pores in 24-well plates. Cells (2 × 10⁵/mL) were suspended in serum-free medium and seeded into upper chambers, while the lower chambers were filled with medium containing 10% FBS. After 24 h of incubation, cells remaining in the upper chamber were removed. Cells that had migrated onto the membrane were fixed and stained with crystal violet. Quantification was based on counting the cells in five randomly selected fields under a microscope at 100× magnification.

For the invasion assay, similar steps were followed using Transwell inserts pre-coated with Matrigel (8 μm, BD Biosciences) to simulate the extracellular matrix. All other procedures were the same as the migration assay.

2.12 Tumorigenesis by Subcutaneous Injection

All animal procedures were approved by the Medical Experimental Animal Care Commission in Navel Military Medical University. Six-week-old nude mice were randomly divided into two groups: Eca-109 (LV-Con) and Eca-109 (LV-FOXA3). Each group received a subcutaneous injection of cells (2 × 10⁶/mL) into the back. Six weeks after the injection, the mice were sacrificed, and the tumors were excised and weighed.

2.13 Statistical Analysis

Data were expressed as the mean ± standard error of the mean (SEM). Categorical variables were analyzed using the Chi-squared test, while continuous variables were assessed with the Mann–Whitney U test. Overall survival (OS) analysis was conducted using univariate and multivariate Kaplan–Meier survival analysis and Cox regression analysis. OS was described as the time frame between date of surgery to the date of death or the last follow up date: July 2015. Statistical significance level was p-values < 0.05. Statistical analyses were achieved using the SPSS version 22.0.

3.1 FOXA3 Is Down-Regulated in Human ESCC Tissues

We initially collected 11 human ESCC tissue samples and paired adjacent normal tissues to detect the expression of FOXA3 by RT-PCR. As shown in Figure 1A, FOXA3 expression was significantly lower in human ESCC tissues compared to the paired adjacent normal tissues (p = 0.022). The results from IHC staining also revealed the staining intensity of FOXA3 in 68/93 of normal tissues was noticeably stronger than that in tumors, as shown in Figure 1B. All these results suggested that FOXA3 might play an important role in the progression of ESCC.

3.2 Association of FOXA3 Expression With Clinicopathological Characteristics

To further explore the role of FOXA3 in the progression of ESCC, we analyzed the correlation between FOXA3 expression levels and the clinicopathological characteristics of ESCC patients (age, gender, tumor size, histology grade, TNM stage, T grade, and lymph node metastasis status). All of ESCC patients were divided into two groups according to FOXA3 expression level. As shown in Table 1, FOXA3 expression was inversely correlated with lymph nodes metastasis (p = 0.002) but did not show a significant association with age, gender, tumor size, or differentiation. In addition, FOXA3 was correlated with TNM stage (p < 0.05). However, due to the small number of cases in stages I and IV (5 cases each) and the fact that the primary difference between stages II and III was lymph node metastasis (N0 vs. N1–3), the correlation between FOXA3 expression and pTNM stage requires further investigation with more early- and advanced-stage cases.

Kaplan–Meier survival analysis indicated that higher FOXA3 expression was associated with better overall survival in ESCC patients (p = 0.001, Figure 2). Univariate Cox regression analysis revealed that both lymph node metastasis and low FOXA3 expression were significantly associated with poorer survival (p = 0.001 and p = 0.002, respectively, Table 3). Multivariate Cox regression analysis confirmed that lymph node metastasis and FOXA3 expression were independent prognostic factors for overall survival (p = 0.044 and p = 0.032 respectively, Table 4).

3.3 FOXA3 Inhibits the Proliferation of ESCC Cells

To investigate the functional role of FOXA3 in ESCC, we generated stable FOXA3-overexpressing cell lines and confirmed the upregulation of FOXA3 expression at both the mRNA and protein levels (Figure 3A,B). The influence of FOXA3 on ESCC cell viability was investigated using the CCK-8 assay. As shown in Figure 4A, FOXA3 overexpression significantly decreased the viability of ESCC cells. To further validate these findings, we performed colony formation and EdU assays, and the results demonstrated that FOXA3 overexpression markedly reduced ESCC cell proliferation (Figure 4B,C). These results collectively indicated that FOXA3 inhibited the proliferation of ESCC cells.

3.4 FOXA3 Inhibits the Tumorigenicity In Vivo

To investigate the effects of FOXA3 on tumorigenicity in vivo, Eca-109 cells transfected with FOXA3-overexpressing vectors were injected into the backs of nude mice. As shown in Figure 5, the average tumor weight in the FOXA3 overexpression group (0.6586 ± 0.1764 g) was significantly lighter than that in the control group (2.021 ± 0.1162 g, p < 0.05). This result suggested that FOXA3 could suppress tumor growth in vivo.

3.5 FOXA3 Inhibits Migration and Invasion of ESCC Cells

Given the correlation between lower FOXA3 expression and lymph node metastasis, we hypothesized that FOXA3 might also play a role in tumor metastasis. Tumor metastasis itself is the greatest cause of death in cancer patients and is an extremely complex, multi-step process that involves the migration and invasion of cancer cells, which is the key step in cancer metastasis. Thus, we evaluated the effect of FOXA3 overexpression on ESCC cell migration and invasion. As shown in Figure 6, FOXA3 upregulation significantly reduced cell migration. The results of the Transwell migration assay (Figure 7A) further confirmed that fewer cells migrated in the LV-FOXA3 group compared to the control group. Similarly, the invasion assays (Figure 7B) demonstrated a significant reduction in the invasive potential of both Eca-109 and TE-1 cells after FOXA3 overexpression. These results were in accordance with our clinical data, indicating the suppressive role of FOXA3 in the metastasis of ESSC.