Isolation, Culture And Biological Characteristics Of Esophageal Cancer Stem Cells

Part I:The study of isolation and biological characteristics of cancer stem cells from esophageal cancer cell linesObjective:Te study on the cultivation method for cancer stem cells (CSCs) in esophageal cancer cell lines KYSE-150 and TE-1 with serum-free medium(SFM)and identify whether resulting spheroid cells display cancer stem cells characteristics.Methods:KYSE-150 and TE-1 cells was cultured in serum-free medium. The cells were observed to generate the spheres and differentiation in serum-supplemented medium. MTT assay and invasive assay were applied the examine cell invasive ability of both KYSE-150 and TE-1 spheres and parental cells. CD44+、CD271+、CD44+CD271+ expression of tumor spheroid cells was also analyzed by flow cytometry.Results:After SFM culture, KYSE-150 and TE-1 cells formed spheres, and could continuously passage in vitro. Cell spheres differentiation recurred when serum was supplemented in SFM. CD44+、CD271+、CD44+CD271+ in KYSE-150 cell were [(64.92±11.04)%、(28.27±6.79)%、(24.07±5.39)%],which were significantly higher than that those in parent cell [(37.04±6.30)%、(3.69±0.49)%、(1.64±0.11)%,P<0.05)]. CD44+、CD271+、CD44+CD271+ in TE-1 cell were (6.41±0.87)%, (2.58±0.17)%、 (0.27±0.53)%, which were significantly higher than that those in parent cell.[(1.87±0.18)%、(0.44±0.10)%、(0.093±0.025)%,P<0.05].Conclusions:Esophageal cancer cell spheres can be isolated from KYSE-150、 TE-1 by culture with SFM. Molecular markers CD44, CD271 may be the cell surface maker for these cancer stem cells.Part II:The study of isolation and biological characteristics of cancer stem cells from esophageal cancer cell linesObjective:Te study on the cancer stem cells population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether resulting stem-like spheroid cells display radiation resistance characteristics.Methods:Serum-free medium (SFM) suspension was used the culture esophageal cancer stem cell lines and enrich the esophageal stem-like spheres, RT-PCR assay was used to detect the stem cell gene expression in the spheroid cells. Radiosensitivity of stem-like spheres and parental cells were evaluated by clonogenic assays. Further, different cells after irradiation at different doses was tested the evaluate the change of spheres formation, CD44+CD271+ expression of tumor stem-like spheroid cells was also analyzed by flow cytometry after irradiation.Results:The cells were observed to generate the increased spheres in serum-free medium with the increase of cell passage. Radiation can increase the rate of two types of cells generating the stem-like spheres. KYSE-150 stem-like spheres’number: generation 1 was (25±2) pcs; generation 2 was (37±2) pcs; generation 3 was (47±3) pcs. TE-1 stem-like spheres’number:generation 1 was (15±3) pcs; generation 2 was (24±3) pcs; generation 3 was (36±4) pcs. The average survival fraction(SF2) of the suspension cultured KYSE-150、TE-1 stem-like spheres after 2 Gy of radiation were 0.81±0.03、0.87±0.01,while the average SF2 of KYSE-150、TE-1 parental cells were 0.69±0.04、0.80±0.03 (P<0.05). In the esophageal parental cells, irradiation dose dependently induced G2 arrest. Stem-like esophageal spheres were resistant to irradiation-induced G2 arrest without significant changes in percentage population of irradiated stem-like cells. Under irradiation 0、4、8Gy:the CD44+CD271+ cell percentage for KYSE150 parental cells were (1.08±0.03)%、(1.29±0.07)% and (1.11±0.09)%; the CD44+CD271+ cell percentage for TE1 parental cells were (1.16±0.11)%、(0.97±0.08)% and (1.45±0.35)%. The difference was not statistically significant (P>0.05). Under irradiation 0、4、8Gy:the CD44+CD271+ cell percentage for KYSE-150 stem-like spheres were (35.83±1.23)%、(44.9±1.67)% and (57.77±1.88)%; the CD44+CD271+ cell percentage for TE1 stem-like spheres were (16.07±0.91)%、(22.67±1.12)% and (33.27±1.07)%. The 4Gy and 8Gy irradiated KYSE-150 and TE-1 stem-like spheres group were compared with OGy irradiated group, the differences were statistically significant (P<0.05).Conclusions:The CSCs in KYSE150 and TE1 spheres is more than those in KYSE150 and TE1 parental cell lines. The KYSE150 and TE1 stem-like spheres are more radioresistant than their parental cells which may suggest that CSCs is related with the radioresistance.