Gastric Cancer Stem Cells In Cell Line SGC-7901

Objectives: Gastric cancer is the most aggressive solid malignancy in China. It's possible that gastric cancer contains cancer stem cells responsible for its malignancy. The purposes of this study were⑴to verify the hierarchical structure within the SGC-7901 cell line.⑵to establish a method to propagate gastric cells with stem cell properties and identify the cells obtained in the assay.Methods:⑴Limit diluting assay was conducted to observe the multiplication ability of single cells.⑵SGC-7901 cells were cultured in the so-called serum free media( DMEM/F12 medium supplemented with EGF, b FGF,Insulin and B27 )at a density of 1000 cells/ml for 4 days to form spheres.⑶Colony formation assay and tumor formation assay were conducted to identify the propagation ability of both sphere cells and adherent cells in vitro and in vivo.⑷Microarray experiment was used to search for differentially expressed genes .Real-time flurogenetic quantitative PCR(RFQ-PCR) and Flow cytometry were used to verify the results of Microarray.Results: Limit diluting assay showed that only a subpopulation of SGC-7901 cells could survive, multiply and finally form stable cell lines in serum containing media. Also only a subpopulation of SGC-7901 cells could form floating spheres in serum free media. Colony formation assay and tumor formation assay showed that sphere cells were more tumorigenic than adherent cells. Microarray experiment revealled that compared with adherent SGC-7901 cells, sphere cells highly expressed stem cell associated proteins: ABCG2, HES1 and KRT153. RFQ-PCR was conducted to test the expression level of the three proteins mentioned above as well as protein CD44. The result was consistent with that of Microarray. Flow cytometry was also used to test the expression of both ABCG2 and CD44 in spheres and adherent cells. As expected, CD44 expression didn't differ much between the two while sphere cells expressed higher level of ABCG2.Conclusions: This study not only verified the hierarchical structure within the SGC-7901 cell line, but also established a way to propagate spheres with stem cell properties. The propagation capacity of SGC-7901 spheres was higher than that of cells cultured in serum containing medium both in vitro and in vivo. As revealed by micro array results and confirmed by RFQ-PCR and Flow cytometry results, sphere cells highly express stem cell associated proteins: ABCG2, HES1 and KRT153, which might provide a way to identify gastric cancer stem cells.