Fig 2: Immunohistochemically staining of WNT3A in metaplasia, HGIN and EAC. Immunohistochemical staining of WNT3A was performed and the staining index of WNT3A-positive cells (a) of squamous epithelial (SQ), metaplastic (MC), high-grade intraepithelial neoplasia (HGIN), and esophageal adenocarcinoma (EAC) cells in human specimens (b,c) were analyzed. The staining index used, describes staining as 1 < 30%, 2 < 60%, 3 < 100%. WNT3A protein localization, showed a higher staining index for SQ than MC, HGIN and EAC. Values are shown as mean ± S.E.M. (Kruskal-Wallis with Dunn’s correction, * - p < 0.05, *** - p < 0.001, nSQ = 30, nMC = 15, nHGIN = 7, nEAC = 15, bar-20 µm)

Fig 3: Response to Wnt3a in cells of Barrett’s sequence. Expression of the downstream target Axin2 was analyzed in EPC-1 and EPC-2, CP-A, CP-B, OE33 and OE19 cells by quantitative Real time RT-PCR (a). The carcinoma cell lines OE33 and OE19 were treated with 200 ng/mL rhWnt3a for 1 h. Cell specific FCS free medium served as control. Expression of the downstream targets Axin2 and CyclinD1 were analyzed by quantitative Real time RT-PCR (b, c). Along Barrett’s sequence, a strong increase of Axin2 expression was found with significant higher levels in OE19 as compared to EPC-1 (a). EPC-1, CP-A and OE33 cells responded to Wnt3a with an increased Axin2 expression. In contrast, EPC-2 and OE19 showed no altered Axin2 expression as compared to controls (b). Decreased levels of CyclinD1 after Wnt3a were detected in EPC-1, EPC-2, CP-A, and CP-B. Wnt3a treatment induced significant higher CyclinD1 levels in OE33 cells as compared to controls (c). Protein level expression of pAkt/Akt, pß-catenin/ß-catenin and pGSK3ß/GSK3ß after Wnt3a treatment was analyzed in EPC-1 and EPC-2, CP-A, CP-B, OE33 and OE19 cells by westernblot analyses. Higher levels of pß-catenin after Wnt3a treatment were found in OE33 cells (d). CP-B responded with an elevated pGSK3ß expression (e). A loss of pAkt was detected in CP-A and CP-B (f). Normalization was done with ß-Actin. Values are shown as mean ± S.E.M. (One-way-ANOVA with Bonferroni correction, **- p < 0.01 compared to EPC-1 (a), Two-way-ANOVA with Bonferroni correction, * - p < 0.05, ** - p < 0.01 compared to controls (b-f))

Fig 4: Expression of the extracellular ligands Wnt3a and Wnt5a along Barrett’s sequence. Expression of Wnt3a (a) and Wnt5a (b) was analyzed in EPC-1 and EPC-2 (squamous esophageal epithelium), CP-A (metaplastic epithelium), CP-B (epithelium with high-grade dysplasia), OE33 and OE19 (esophageal adenocarcinoma) cells by quantitative Real time RT-PCR. Highest Wnt3a expression was detected in EPC-1 and EPC-2 (a). Wnt5a was also expressed in EPC-1 and EPC-2, but highest levels of Wnt5a were found in CP-B (b). OE33 and OE19 expressed only marginal levels of Wnt3a and Wnt5a (a, b). Normalization was done with ß-Actin. Values are shown as mean ± S.E.M. (One-way-ANOVA with Bonferroni correction, * - p < 0.05, ** - p < 0.01, *** - p < 0.001 compared to EPC-1)