Immunohistochemistry and tissue samples

A total of 306 tissue samples were obtained from patients with esophageal squamous cell carcinoma (ESCC) after ethical approval by the institutional review board of Samsung Medical Center (Seoul, Korea). All patients provided informed consent for tissue donation. Immunostaining for the Twist1 protein was performed using an anti-Twist1 monoclonal antibody (Abcam, Cambridge, UK). Tissue microarrays composed of 306 ESCC samples with clinical data including age, sex, tumor size, depth of invasion (T), nodal status (N), metastasis (M), overall survival (OS) and disease-free survival (DFS) were used to identify the clinical significance of the target genes’ expression in patients with ESCC. Staging based on TNM classification was applied according to guidelines from the 2010 American Joint Committee on Cancer staging manual. Immunohistochemical staining was estimated according to our previous studies⁵⁴. Sections on microslides were deparaffinized with xylene, hydrated using a diluted alcohol series, and immersed in 0.3% H₂O₂ in methanol to quench endogenous peroxidase activity. Sections were then treated with TE buffer (Tris 10 mM and EDTA 1 mM, pH 9.0) for antigen retrieval. To reduce nonspecific staining, each section was treated with 4% skim milk in PBS with 0.1% Tween 20 (PBST) for 30 min. Sections were then incubated with primary antibodies: anti-Twist1 (ab50887, 1:200), anti-Prrx1 (HPA051084, 1:200), and anti-Tenascin-C (ab108930, 1:500) in TBST containing 4% skim milk for 60 min at room temperature. After three successive rinses with a washing buffer, sections were then incubated with an anti-mouse/rabbit polymer kit (Envision Plus, Dako, Carpinteria, CA, USA) for 30 min at room temperature. The chromogen used was 3-amino-9-ethylcarbazole (AEC, SK-4205, Vector, Burlingame, CA, USA). Sections were counterstained with Meyer’s hematoxylin and the virtual slide images were generated using Aperio^® AT2 virtual slide scanner (Leica, Wetzler, Germany). As described in detail previously, immunohistochemical scores were measured semiquantitatively⁵⁴. In brief, we evaluated the staining intensity and the proportion of positive cells and then generated staining scores as follows: (Score 1), weak staining in <50% or moderate staining in <20% of stromal cells; (Score 2), weak staining in ≧50%, moderate staining in 20–50% or strong staining in <20%; (Score 3), moderate staining in ≧50% or strong staining in ≧20%.