Preliminary Study On Cancer Stem-like Cells In Human Breast Cancer Cell Iines

BackgroundWith the development of cancer biology and animal cell culture technology , people realize that there exist a few cells in the cancer cells with the characteristic of self-renewing,multipotentiality ,steadily controlling,and so on.Those cells are the source of cancer recrudesce and metastasis. Thus"cancer stem cell"hypothesis is proposed.The domestic and international scholars make a lot of attempts to separate and appraise the cancer stem cells.Breast cancer is a cancer of obvious heterogeneity and one of the most common malignant tumors of women. The following experiment makes preliminary study about cancer stem-like cell quality and its enriching methods in different breast cancer cell lines by separating and appraising.ObjectiveUnder culturing different breast cancer cell lines and the related content of cancer stem-like cells,understanding CD44+CD24-/low cells proportion in different cell lines and exploring the methods for enriching cancer stem-like cells with chemotherapy drugs intervention and identification by flow cytometry .Methods1.Observed growth curve and compared the growth situation by culturing human breast cancer cell 1ines"MCF-7,MDA-MB-231"with different biological characteristics which were combined with anticancer regimens(TA scheme) or without.2.Detected the expression proportion of CD44+CD24-/low cells in the two breast cancer cell lines by flow cytometry.3.Under suspension culturing the above cell lines in serum-free medium,combining with or without anticancer regimens of TA scheme,observed clonal nonadherent spherical clusters. 4. The method of two independent samples t test was used to make sensitivity test of two above cells lines to chemotherapy drugs. The difference with"P<0.05"had statistical significant.Statistical software"SPSS13.0"was used to analysis the above datas.Results1.Compared growth curve of the cell lines"MCF-7,MDA-MB-231":The logarithm anagen phase was NO.5 day in MCF-7 cells and NO.3 day in MDA-MB-231 cells.The platean phase was NO.7 day in the former and NO.8 day in the latter.It suggests that the multiplial time of MDA-MB-231 cells was shorter,which grew quicker and existed bader biological behavior than MCF-7 cells.2.Compared the two cell lines growth curve under combining with anticancer regimens of TA scheme: Most of the two cells lines died after chemotherapy intervention in the first day. They grew slowly,while MCF-7 cells growth rate gradually recovered after the second day, which indirectly reflected these cells might exist few proportion of cells that had chemotherapy resistance,self-renewing ability and adapt to new micro-environment. The method of two independent samples T test was used to compare the sensitivity of anticancer regimens.The results in MCF-7 cells suggested it was not sensitive ( P>0.05) .The results in MDA-MB-231 cells showed obvious positive effect ( P<0.05) .3. Observed clonal spheres with serum-free suspension culturing of two above cell lines: Most of MDA-MB-231 cells in serum-free culture showed gradually dead and did not form clonal spheres,while there were clonal spheres in MCF-7 cells lines; The clonal spheres in MCF-7 cells were found in about NO. 6 day at the first time. When combined with anticancer regimens of TA scheme , majority of the two cells lines gradually died, and there no existed living cells.4.Detected the content of CD44+CD24-/low cytometry cells by flow cytometry: The results suggested that two above cell lines contained mainly CD44+CD24+ subsets and CD44-CD24+ subsets,the CD44+CD24-/low proportion of MCF-7 cells and MDA-MB-231 cells was respectively 2.07%and 0.20%. Conclusion1.The MDA-MB-231 cells line grew faster and had bader malignancy, it showed more sensitive than MCF-7 cells line to TA chemotherapy drugs.2.After TA chemotherapy drugs intervention, MCF-7 cells showed rapider recoverying after the first inhibition of growth rate, which indirectly reflected these cells might exist a small amount of cancer stem-like cells that resisted chemotherapy drugs.They could renew by themselves and adapt to new micro-environment.3.A small amount of cells in MCF-7 cell lines grew as monadherent spherical clusters in serum-free culture medium, indicating that they could form cell clones and had the characteristics of cancer stem-like cells.4. Although CD44 + CD24-/low phenotype cell proportion of two cell lines by flow cytometry was small, it was consisted with the proportion of cancer stem-like cells presented on the relevant literature: in the tumor organization there existed extremely few cancer cells acting as stem cell role .5.The CD44+CD24-/low phenotype might not be the only specific surface markers of breast cancer stem cell. There were still some experiments of tumor formation in animals to further verify them.