Serial Sphere Formation, A Method For Enrichment Of Gastric Cancer Stem Cells; An Experience On AGS Cell Line

Gastric cancer is one of the most principal causes of cancer-related death worldwide. To overcometherapeutic challenges, new investigations about cancer pathogenesis have been tended to cancerstem cells (CSCs) as previously neglected targets. Identification and characterization of CSCs,could help to develop novel therapeutic strategies for gastric cancer. Since the specific CSC makershave not been identified in many malignancies including gastric cancer, functional approaches,including spheroid forming assay, have been taken into consideration. This method relies onfunctional properties of CSCs, including: self-renewal, proliferation, growing under anchorageindependent and poor environmental conditions. Tumor cells from AGS cell line, transferred intothe low-attached plates and cultured in low cellular density serum-free medium supplemented bygrowth factors: EGF, b-FGF and B27 to form spheres. Serial suspension cultures were performed forenrichment of the gastric cancer stem cells by passaging sphere derived cells into the new plates atthe same condition each 8 days. After passage 3, cells were removed for characterization bycolonogenic assay, drug resistance to cisplatin and docetaxel by MTS assay. Putative stemnessmarkers: CD44, CD24 and CD326 were measured using flow cytometry; and SOX2, OCT4, KLF-4,NANOG and C-MYC expression evaluated by qRT-PCR. sphere cells in comparison with parentalcells were more clonogenic (p<0.01), more chemo-resistant to cisplatin and docetaxel (p<0.05). Thestemness genes were upregulated (p<0.05) and MFI(mean fluorescent intensity index) of CD44 andEPCAM, was also higher in sphere group. Our results demonstrated that serial sphere formationassay could be a beneficial model for enrichment of the CSCs. This finding supports the concept of internal hierarchial heterogeneity of cancers even in some cancer cell lines as well as AGS;however it needs to be confirmed by further techniques such as in vivo tumorigenicity.