Genotypic and phenotypic characterization of side population of gastric cancer cell lines

Abstract

The side population (SP) of tumor cell lines shares characteristics with tumor stem cells. The objective of this study was to phenotypically and genotypically characterize the SP of gastric cancer cell lines. SP cells were obtained from AGS and MKN45 gastric cancer cells using Hoechst 33342 staining and fluorescence-activated cell sorting. The cells were subsequently studied morphologically at cytology and immunocytochemistry, on the transcriptional level via gene array, and in cell culture using recultivation assays. Genes differentially expressed in SP cells were evaluated at immunohistochemistry in tissue samples from 486 patients with gastric cancer. The SP cells were smaller and rounder then non-SP cells. SP cells self-renewed in recultivation experiments and differentiated into SP and non-SP cells. Recultivated SP and non-SP cells exhibited distinct phenotypes in culture insofar as cell shape and colony formation. SP cells demonstrated increased levels of the stem cell markers CD133 and Musashi-1. Transcriptional analyses demonstrated that SP cells express genes that encode for stem cell properties including FZD7, HEY1, SMO, and ADAM17. It was observed that ADAM17 and FZD7 are differentially expressed in human gastric cancer, and FZD7-positive cancers are associated with significantly shorter patient survival. In conclusion, human gastric cancer cell lines enclose a phenotypically and genotypically distinct cell population with tumor stem cell features. Phenotypic characteristics of this distinct cell population are also present in gastric cancer tissue, and correlate with patient survival.

Figure 1

A side population can be differentiated at FACS analysis even after recultivation. AGS cells were stained with Hoechst 33342 and displayed in a Hoechst red (horizontal axis) versus Hoechst blue (vertical axis) diagram before (A and B) and after (C and D) recultivation for 6 days and a second FACS analysis. The SP region contains 0.5% and 5.3% of the cells, respectively, defined by gate P2 at the lower left (A and C), whereas this characteristically low staining pattern disappears in the presence of verapamil: 0.1% and 0.0% of cells, respectively (B and D).

Figure 2

Recultured SP and non-SP cells exhibit different growth patterns. After FACS analysis of AGS cells, SP and non-SP cells were recultivated and photographed every other day. SP cells formed circular colonies starting from single clusters of round SP cells. Non-SP cells demonstrated more fibroblastlike morphologic features and formed loose groups of slightly pleomorphic cells. Original magnification: ×200.

Figure 3

SP and non-SP cells demonstrate cytologically different phenotypes. Cells cytocentrifuged onto glass slides (AGS; A and B) and cells embedded in agarose (MKN45; C and D) were stained with H&E and demonstrated different phenotypes of SP cells (A and C) and non-SP cells (B and D). Original magnification: ×400 (A–D).

Figure 4

SP and non-SP cells demonstrate different immunophenotypes. Expression of various putative stem cell markers was studied at immunocytochemistry. For AGS (A) and MKN45 (B) cells, the number of CD133- and Musashi-1 immunoreactive cells was significantly different. Immunostaining was with anti-CD133 and anti–Musashi-1 antibodies. Hematoxylin counterstain. Original magnification: ×400 (A and B).

Figure 5

Expression of ADAM17 and FZD7 in nonneoplastic gastric tissue. A, B, C, and G: Expression of ADAM17. D, E, F, and H: Expression of FZD7. Foveolar epithelium did not express ADAM17 (B) or FZD7 (E). ADAM17 was observed in a few scattered cells of the gastric glands (C). FZD7 was expressed in inflammatory cells of the lamina propria (E), whereas the gland epithelium was immunonegative (F). ADAM17 (G) and FZD7 (H) were also expressed in bottom cells of the intestinal metaplasia. Hematoxylin counterstain. Original magnification: ×100 (A, D, G, and H); ×400 (B, C, E, and F).

Figure 6

Expression of ADAM17 and FZD7 in gastric cancer tissue. ADAM17 and FZD7 are expressed in intestinal-type gastric cancer cells. All images are from the same case. H&E (A), pan-cytokeratin (B), ADAM17 (C), and FZD7 (D). Hematoxylin counterstain. Original magnification: ×600 (A–D).

Figure 7

Overall survival in dependence of FZD7-expression. Kaplan-Meier curves depict overall survival in dependence of FZD7-expression in gastric cancer. The P value was calculated using the log-rank test.

Figure 8

mRNA expression of ADAM17 and FZD7 in gastric tissue. A: ADAM17 mRNA is highly significantly up-regulated in gastric carcinoma compared with nonneoplastic mucosa (P < 0.001). B: FZD7 mRNA was slightly but not significantly increased in gastric cancer tissue compared with nonneoplastic mucosa (P = 0.448).