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Objective: STAT-3 transcription factor participates in some biological phenomenon including inflammation, anti-tumor immunosuppression, cellular proliferation and differentiation. Moreover, it helps to maintain pluripotency of embryonic stem cells and there are some recent evidences of its footprint in stemness properties of cancer initiating cells. In the present study, the expression of STAT-3 was evaluated in the spheroid bodies derived from MKN-45 cell line as cancer stem like cells and the results compared to parental cells. Materials and Methods: Spheroids obtained from non adhesive culture of cells in serum free media supplemented with EGF, bFGF (20 ng/ml) and B27 (2%). Gastro-spheres were characterized by expression of CD24, CD44, CD133, EpCAM, CD71, CD73, CD90 as putative CSCs markers; Nanog, Oct-4, Sox-2, Klf-4 and c-Myc as stemness related genes and drug resistancy to DTX. Finally, the expression of STAT-3 tested by immunofuorescence staining and real- time PCR. Parental cells were used as control group. Results: Gastro-spheres (passage 4) showed higher expression of CD44, CD24, CD71, and over expressed Nanog, Oct-4, Sox-2, Klf-4 and c-Myc genes than parental cells. Immunostaining of STAT-3 demonstrated that both spheroid cells and parental cells expressed STAT3 at protein level, however, its mRNA level in gastro-spheres was higher than parental cells. Conclusion: Spheroid formation provide an applicable method to isolate cancer stem like cells from MKN-45 cell line and the findings if support by further examinations may imply to roles of STAT-3 in GCSCs features, as recently seen in brain and glioblastoma CSCs. [ABSTRACT FROM AUTHOR]

Objective: Gastric cancer is one of the most principal causes of cancer-related death worldwide. To overcome therapeutic challenges, new investigations about cancer pathogenesis and tumor relaps have been tended to "cancer stem cells" (CSCs) as previously neglected targets. Identification and characterization of CSCs could help to develop novel therapeutic strategies in gastric cancer. Since the specific CSC makers have not been idefined in many of cancers, recently, functional approaches including spheroid body formation have been taken into consideration for enrichment of the CSCs. Materials and Methods: Tumour cells from adenocarcinoma cell line "AGS" were cultured in RPMI 1640. IN order to enrich the gastric cancer stem cells, cultured cells transfered to low attached plates and cultured in serumfree medium supplemented with growth factors: EGF, b- FGF and B27 to form spheres. Serial suspension cultures were done by passaging sphere derived cells into the new plates at the same condition each 8 days. After passage 4, cells were removed to be characterized by colonogenic assay, drug resistancy to cisplatin and docetaxel by MTS assay, marker expression evaluation for putative stemnessmarkers: CD44,CD133,CD24 and CD326 (EPCAM) at the protein level using flow cytometriv and sox2, oct4, klf- 4, nanog and c-myc at the level of mRNA. Results: Clonogenicity of subsphere-derived cells was significantly higher than parental cells (p<0.05). The screened cells showed more chemoresistance to cisplatin and docetaxel after 72 hours exposure Compared with non selected cells (p<0.05). In addition the stemness genes were up regulated in this population; indicating that their undifferentiated state was retained. Flowcytometric MFI index of Stemness-markers: CD44 and EPCAM, was also higher in sphere-derived group than control (parental tumour cells). Conclusion: Our results demonstrated that serial sphere formation assay could be a beneficial model for enrichment of cancer stem-like cells. However it needs to be confirmed by in vivo tumorigenicity models. [ABSTRACT FROM AUTHOR]