Biological Characteristics Of CD55^hig Side Population In Human Breast Cancer Line MCF-7

Objective:Cancer stem cells lie in cancer tissues, which possess the ability of self-renew and differentiation potential power,They are in the initial stage of forming cancer and keeping the growth of cancer. Therefore they are the origin of relapse and transference of cancer. We can have a good knowledge of the characteristics of cancer through sorting and appraising cancer stem cells,so as to seek new targeted drug to cure cancer, and improve cure rate. In this study we try to detect the mean fluorescent intensity of CD55 high expression (CD55hig)and CD55 low expression(CD55low) side population cells from human breast cancer line MCF-7 by flow cytometer,to separate CD55hig and CD55low cells and observe the cell appearance,adherence efficiency,clone formation and cell cycle disposition.So we can explore whether the CD55hig cells possess characteristics of cancer stem cells,and then find a new method for isolate breast cancer stem cells and provide theoretical basis.Method:Human breast cancer MCF-7 cells were cultured by DMEM as complete medium supplemented with 10% fetal calf serum,log phase growth cells were trypsinizated to single cell suspension, the first group was dyed by Hoechst33343, cancer stem cells surface had ABC transporter proteins which could efflux Hoechst33342, making it possible for stem cells light staining.The second group dyed by Hoechst33343 and blocked with verapamil was chosen as control group,Verapami could block the function of ABC transport protein on cancer stem cells surface.This study detcted the proportion of SP(side population) and MP(main population) population by flow cytometer. Then SP and MP cells were labeled with anti-CD55 monoclonal antibody, meanwhile anti-CD55 control antibody labled SP and MP cells was chosen as control group, detecting the mean fluorescent intensity and comparing the difference between them. We used anti-CD55 monoclonal antibody labling unsorted MCF-7 cells, detected the proportion of CD55hig and CD55low side population cells and sorted them to detect their biological characteristics:1. detected adherence efficiency:CD55hig and CD55low cells were planted in the 24 ports culture dishes with DMEM supplemented with 10% fetal calf serum,104 cells every port, to observe the cell apperance and adherence efficiency at 6h,12h,18h and 24h. Cell adherence efficiency (%)= adherence cells/planted cells X 100%; 2. detected cloning efficiency:two hunderd CD55hig cells and two hunderd CD55low cells were inoculated in 10cm culture capsule. After one week, we could see the cloning cells.Then the clonies number was counted:Cloning efficiency= clones cells/painted cells×100%; 3. cell cycle:CD55hig, CD55low and unsorted MCF-7 cells were labeled by PI, cell cycle was detected by flow cytometry.Results:1.4.34±0.59% SP cells were contained in MCF-7 after dyed by Hoechst33342. The percentage of SP significantly degrated when blocked by verapamil(0.1±0.07%). The percentage of MP cells was 33.66±7.89%; 2. The mean fluorescent intensity of SP and MP cells labeled with anti-CD55 monoclonal antibody was 100.85±4.57 and 50.51±4.75, respectively. MCF-7 cells were labeled with anti-CD55 monoclonal antibody and sorted CD55hig and CD55low cells. The proportion of CD55hig cells and CD55low cells was 2.12±0.39% and 20.10±5.29%, respectively; 3. The adherence efficiency of CD55low cells at 6h,12h,18h and 24h were 40.87±1.55%,55.40±2.46%, 85.10±3.36% and 96.3±0.96%, the adherence efficiency of CD55hig cells at 6h, 12h,18h and 24h were 29.53±1.20%,46.40±2.99%,77.2±1.07% and 95.5±1.1%. The adherence efficiency of CD55low cells were higher than CD55hig cells at 6h,12h and 18h. There was statistical difference between the two groups, (P<0.05). The adherence efficiency of CD55hig cells and CD55low cells at 24h didn't appear marked difference(P>0.05); the CD55hig cells were mostly spherica and CD55low cells were spindle after planted 12h; 4. The Cloning efficiency of CD55hig cells at one week was 20.04±1.07%, which was lower than CD55low cells. There was statistical difference between the two groups(P< 0.05); 5. The percentage of G0/G1 in CD55hig cells was 85.4±3.37%, which was higher than CD55low cells(58.6±2.55%) and unsorted MCF-7 cells (70.73±4.21%), P<0.05. The percentage of S in CD55hig cells was 13.93±3.45%, which was lower than CD55low cells(40.36±2.56%) and unsorted MCF-7 cells (24.93±2.86%), P<0.05.Conclusions:1. Hypochromatic sp cells are detect in human breast cancer line MCF-7 cells dyed by Hoechst33342. The percentage of SP cells obviously decreased when blocked by verapamil. SP cells contain cancer stem cells.2. The mean fluorescent intensity of SP is higher than MP cells when labeled with anti-CD55 monoclonal antibody. Unsorted MCF-7 cells are labeled with anti-CD55 monoclonal antibody by flow cytometer, we find a small amount of CD55hig cells.3. CD55hig cells are more nonadherent, quiensent and clonogenetic. So CD55hig cells have biological characteristics of cancer stem cells.