| Objective:To isolate and cultivate human colon cancer cell line CW-2 stem cells .To investigate an effective method to enrich tumor stem cells by comparing the efficiency of 3 cell isolation methods.To observe the biological characteristics of human colon cancer cell line CW-2 stem cells.Methods:Isolation and cultivation human colon cancer cell line CW-2 stem cells by using simple serum-free medium, serum-free medium combination with oxaliplatin, and the second one further combined with flow cytometry (multi-combination).The enrichment efficiency of the 3 methods was analyzed and compared with flow cytometry, tumorigenicity assay in NOD-SCID mice and Transwell invasion assay in vitro.Then study their biological characteristics by cell cycle analysis,cell differentiation experiment, invasion assay in vitro and tumorigenicity assay in vivo.Results:Human colon cancer cell line CW-2 which were cultured in SFM rarely could survive,proliferate and form the suspended tumor stem cell spheres .The frequency of CD44~+EpCAM~+ cells were 57.39%, 73.85 %, and 89.57% separately in cells enriched by simple serum-free medium, serum-free medium combination with oxaliplatin and multi-combination.The tumor xenograft of cells enriched by multi-combination were formed earliest and grew fastest than the tumor xenograft of cells enriched by pure serum-free medium and serum-free medium combination with oxaliplatin. The volume of transplantation tumor from cells enriched by multi-combination were (209.25±31.54)mm~3, (500.00±72.90)mm~3,(852.75±157.51)mm~3,(2280.00±440.91)mm~3,(4897.00±1154.15) mm~3, (11070.00±3559.86) mm~3 separately on the 4th, 7th, 9th, 14th, 21st, and 28th day. Tumor-forming capability of the 3 group cells had statistically significant differences (P<0.05). The number of cells past through man-made BM to down-room were (98±7) cells per field view, (135±7) cells per field of view, (188±6) cells per field of view separately in cells enriched by simple serum-free medium, serum-free medium combination with oxaliplatin and multi-conbination. Invasion capability of the 3 group cells had statistically significant differences (P<0.05). So the cells enriched by multi-combination had most tumor stem cells than the cells enriched by other 2 methods.Tumor stem cells could differentiate into Non-tumor stem cells in serum-supplied medium.The G0/G1 phase cells of CD44~+EpCAM~+cells population accounted for 86.19%, the G2 + S phase cells accounted for 13.81%, the G0/G1 phase cells of unsorted cells(the traditional serum-supplied medium cultured cells ) population accounted for 69.34%, the G2 + S phase cells accounted for 30.66 %, the G0/G1 phase cells of CD44-EpCAM-cells population accounted for 56.04%, the G2 + S cells accounted for 43.96%.Proliferation capability of the 3 group cells had statistically significant differences (P<0.05).CD44 + EpCAM +cells population in the low proliferation level, the traditional serum-supplied medium cultured cells populations in the middle proliferation level,CD44-EpCAM-cells populations in high proliferation level. It is easy for CD44~+EpCAM~+cells to differentiate into adherent growth of non-tumor stem cells in Serum-supplied medium.The number of cells past through man-made BM to down-room were (0±0) cells per field view, (56±5) cells per field of view, (188±6) cells per field of view separately in CD44 - EpCAM - cells,unsorted cells,CD44 + EpCAM +cells.Invasion capability of the 3 group cells had statistically significant differences (P<0.05). CD44~+EpCAM~+cells had highest Invasion capability than the other 2 cells populations.The tumor xenograft from CD44~+EpCAM~+cells were formed early and grew fast.The tumor xenograft from unsorted cells were formed latively later, and growth slower than the tumor xenograft from CD44~+EpCAM~+ Cells.The volume of transplanted tumor from unsorted cells were (0.00±0.00)mm~3,(0.00±0.00)mm~3,(180.45±86.25)mm~3,(514.36±24.63)mm~3,(1161.75±49.88)mm~3 separately on the 4th, 7th, 14th, 21st, and 28th day.There are no tumor forming after CD44 - EpCAM - cells were injected all the time.Tumor-forming capability of the 3 group cells had statistically significant differences (P<0.05).CD44~+EpCAM~+ cells had highest tumor formation capacity among the 3 groups.CD44 and EpCAM expressed in cytomembrane of tumor xenograft.Histopathology of tumor xenograft similared with human colon cancer.Conclusion:Tumor stem cells can form suspended tumor stem cell spheres in serum-free medium. Simple serum-free medium, serum-free medium combination with oxaliplatin and Multi-combination can get TSC in different purity.Multi-combination is highest than pure serum-free medium and serum-free medium combination with oxaliplatin in enrichment capability of tumor stem cells and is a better method to enrich tumor stem cells. CD44~+EpCAM~+ tumor stem cells have biological characteristics of low generation, easy differentiation,high invasiveness , high tumorigenesis . |
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