Comparative expressional analyses between a diffuse-type gastric cancer cell line HSC-60 and its highly metastatic subline 60As6 by microarray

Although peritoneal dissemination is a major obstacle in treating diffuse-type GC patients, the mechanism is still unclear. To address this phenomenon from the viewpoint of molecular biology, we established a highly peritoneal-metastatic cell line, 60As6, from a diffuse-type GC derived cell line, HSC-60, through a six-times iterative in vivo selection consisting of an orthotopic inoculation of the tumor cells and the isolation of highly-metastatic ones from the ascites of immunodeficient mice. To assess the biological differences between HSC-60 and 60As6 cells, we compared gene expression profiles for both cell lines by microarray analysis. Down-regulation of three gastric pit cell differentiation markers and some cytokeratins were found in 60As6. By contrast, several stem cell markers were up-regulated in this cell line. The expression profile suggests that 60As6 has a strong mensencymal phenotype, which is a characteristic of epithelial cell-derived CSCs. We conducted microarray analyses two times by using GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). The procedures were conducted according to the suppliers’ protocols. The expression value (average difference: AD) of each gene was calculated using GeneChip Analysis Suite version 5.0 software (Affymetrix). The mean of AD values in each experiment was 1000 to reliably compare variable multiple arrays. By Wilcoxon u-test (p < 0.05) and a 2-fold change, 684 genes were selected as specific genes for 60As6 and 60As6Luc cells, and 1150 genes were selected as specific genes for HSC-60 and HSC-60Luc cells.