DLL4 expression was identified in the cytoplasm and cellular membrane of cancer cells (Figure 2), and in the stromal cells (Figure 3). Ten representative tissue sections were observed by light microscropy and the percentage of DLL4 positive cancer cells was scored, averaged, and scored semiquantitatively. All immunostained slides were evaluated by two independent observers (SI and AT), who were unaware of the clinical data and disease outcome. If more than 10% of dominant staining see more intensity in tumor cells or stromal cells was identified, the patients were regarded as DLL4 positive. After evaluation, patients were divided into two groups according

to DLL4 expression positivity. Clinicopathological factors of gastric cancer were assessed according to the General Rules of Gastric Cancer in Japan [18]. Figure 2 DLL4 expression in gastric cancer cells. Right: DLL4 expression was identified in the cellular membrane of gastric cancer cells. DLL positivity was found in the cytoplasm

and cellular membrane of gastric cancer (yellow arrow). Left: DLL4 expression was not found in gastric cancer (negative control). Figure 3 DLL4 expression in brain and stromal cells of gastric cancer. DLL4 positive infiltrative cells were identified in cancer stroma (yellow arrow). Statistical analysis Statistical analysis of clinical features was performed using the χ2-test. Survival curves were constructed using the Kaplan-Meier method, and survival differences were analyzed by the generalized Wilcoxon Selleck GF120918 test. Multivariate

analysis was performed to determine prognostic factors. A p-value of less than 0.05 was considered to be statistically significant. Results DLL4 expression in gastric cancer tissues DLL4 positivity was identified in brain tissue as a positive control of DLL4 (Figure 1). DLL4 expression was primarily identified in the membranes and cytoplasm of cancer cells, regardless of tumor histology (Figure 2), as well as infiltrative cells in cancer stroma (Figure 3). 88 (49%) patients were classified as DLL4 positive (10% of DLL4 positive) group in cell lines; Casein kinase 1 41 (23%) were positive in the stroma. DLL4 expression in gastric carcinoma cell lines Immunohistochemical staining showed DLL4 expression in cytoplasm of the four gastric cancer cell lines (Figure 4). Cell lysates extracted separately from the nucleus and cytoplasm in the gastric cancer cell lines were loaded and probed with anti-DLL4 antibody. DLL4 protein was identified in cytoplasm of the all gastric cancer cell lines, but not in the nucleus (Figure 5). Figure 4 DLL4 expression in gastric cancer cell lines. DLL4 expression was identified in the cellular membrane and cytoplasm of gastric cancer cells. Figure 5 DLL4 protein detection in gastric cancer cell lines by Western blot analysis.