Isolation And Characterization Of Cancer Stem Cells In Esophageal Sequamous Cell Carcinoma By Tumorsphere Formation And Using Cripto-1as A Surface Marker

Objective To isolate/enrich and characterize the cancer stem cells (CSCs) in esophagealsequamous carcinoma cells (ESCCs) cell lines using methods of tumor sphere formation andFluorescence-activated cell sorting with Cripto-1(CR-1) as a cell surface marker.Methods1. EC109cells were cultivated in poly-Hema coated dishes or commercial lowadhesion plates with serum-free stem cell medium (DMEM/F121:1) supplemented with1×B27and the cancer stem cells reforming into floating spheres were isolated. The expression ofstemness relative transcriptional factors of the spheres was measured by qRT-PCR. Consecutivepassage and plate clone formation was employed to estimate the self-renewal capacity.Immunofluorescence cytochemistry was used to detect the changes of stemness relativetranscriptional factors and epithelial markers expressed in spheres before and after differentiation.Xenograft assay in nude mice was performed to estimate tumorigenesis in vivo.2. To determinethe possibility of CR-1as a marker of ESCC CSCs, the relationship between the CR-1expressionand clinical pathological parameters was ascertained in clinical samples of ESCCs byImmunohistochemistry (IHC), and the survival curve was drawn. Using flow cytometry,CR-1HIGHand CR-1LOWsubpopulations were sorted out in EC-109and TE-1cells. The expressionof stemness genes and abilities of clony formation and invasion were compared between the twosubpopulations. The tumorigenesis in nude mice was observed with or without silencing theexpression of CR-1gene.Results1. Tumor spheres were usually generated at day5under the selective conditions.Compared with monolayer adherent cells, isolated tumor sphere cells highly expressed stemnessrelative transcriptional factors, and possessed self–renewal and differentiation potential. Thehigher tumorigenicity of tumor sphere cells in immuno-deficient mice was observed. The size ofsphere cell-derived xenograft tumors was larger than monolayer adheret cell-derived ones.2.Cripto-1was expressed in ESCC and positively related to the invasive depath and lymph node metastasis（p=0.023and0.032respectively） and negatively related to survival span（p﹤0.001）.The expression of Cripto-1was observed both in EC-109and TE-1ESCC cell line cells. Byfluorescence-activated cell sorting, CR-1HIGHcells were sorted out with percentage of2.27and1.96in EC-109and TE-1, respectively. Compared with CR-1LOW, CR-1HIGHsubpoplutionexpressed higher level of stemness genes, and exhibited higher abilities of clony formation andinvasion in vitro. The ability of initiating tumors in nude mice was significantly decreased aftersilencing the expression of CR-1gene by shRNA.Conclusion1. Tumor spheres generated from EC109cells under serum-free stem cell mediumand floating culture enrich cancer stem cells.2. CR-1is a potential prodector for malignancy andpoor prognosis in ESCC. Existing experiments have indicted that CR-1can be used as a cancerstem cell sueface marker of ESCC, but the intensive evaluation should be accomplished in future.