| The origin of cancer stem cells (CSCs) is usually believed to be a normalstem cell. Therefore, they possess similar biological characters and potentialreasons for chemoresistance, metastasis and tumor recurrence. Endoglin(CD105) and thymus cell antigen1(Thy1, CD90) are the identification ofstandards of the mesenchymal stem cells (MSCs) that announced by theInternational Association for Cell Therapy (ISCT). A growing number ofstudies have demonstrated MSCs tropism toward primary and metastatictumor locations. High expression of CD105in breast tumor is associated withpoor overall and disease-free survival, and can be an independent predictor.CD105is also an important therapeutic target in metastatic breast cancer. Ithave shown that CD90is a potential marker for liver CSCs in Yang’s study.Consistently with a possible role in tumor initiation, EMT inducers such asTWIST1and SNAIL1are frequently detected at the in situ ductal carcinoma(DCIS) stage, long before metastatic dissemination begins. Expression ofTWIST1and RAS in luminal-committed mammary epithelial cells issufficient to promote breast carcinogenesis. EMT induction has also beenfound to endow cells with stem-like properties. Since the combination ofCD105and CD90is accepted to identify MSCs, we considered the relevancebetween EMT and these two makers and to investigate the breast cancerrecurrence.Objective: To select breast cancer bone metastatic cell lineMDA-MB-231. The purpose was to find a group of cells with mesenchymalstem cell-like characteristics. To sort cells by CD105and CD90two cellsurface markers using flow cytometry. This study was performed to observethe proliferation and migration capacity of the cell subpopulations by thegrowth curve and transwell migration experiments, and detect the stem cell-related gene expression, to explore whether they have the stem cellscharacteristics, and to explore the relevance of breast cancer stem cells andEMT. Furthermore, the present study would support the CSCs theory andinvestigate the breast cancer metastasis and recurrence, in order to cure thedisease.Method: Usually stem cells have characteristics such as, slow-cyclical,undifferentiated, and self-renewal with fast proliferation rate. Human breastcancer bone metastatic MDA-MB-231cell line was selected.1. At the logarithmic growth phase, cells were stained by CD105-RPEand CD90-FITC antibodies, and then using a flow cytometry analysis to detectthe cell surface marker expression, subsequently CD105~+/CD90~+andCD105~-/CD90~-two groups of subpopulations were sorted out.2.The CD105~+/CD90~+subpopulation, CD105~-/CD90~-subpopulation andMDA-MB-231cell group(we called parental population) were culturedseparately. Each population of cells was seeded in96-well culture plate with asame concentration at the logarithmic growth phase. SRB assay was selectedto compare and observe the proliferative capacity among the three sub-groups.Counting the adherent cells at different time in a period of9days after seedingcells, and then to make the growth curve.3. To compare the migratory capacity of the three subsets, migrationexperiment was performed by using transwell plate.4. Since stem cells are slow cyclical and contain more quiescent cells.DNA content/cell cycle analysis of each population was performed and theproportion of G0/G1, S and G2/M phases were analyzed by flow cytometry.5. The expression of stemness genes oct3/4, nanog, sox2and klf4weredetected by using reverse transcription-polymerase chain reaction (RT-PCR)and quantitative real-time PCR technology in each cell subpopulation.6. Statistic data were analyzed by SPSS13.0software. Data wereprocessed as the mean±SD. The statistical significance of measurement wasdetermined by One-way ANOVA or Kruskal-Wallis H test, and comparing ofmean differences between groups by LSD, while counting material by using a Chi-square test (χ~2test). A p-value<0.05was considered statisticallysignificant.(P*<0.05, P**<0.01)Results:1. After labeling with CD105-RPE and CD90-FITC antibodies, therewere4.93%CD105positive cell subpopulation detected by flow cytometryanalysis,3.31%of CD90positive cell subpopulation and0.99%ofCD105~+/CD90~+double positive cell subpopulation, while CD105~-/CD90~-double negative cell subpopulation was90.77%.2. The cell growth curve of double positive, double negative and parentalpopulation was revealed: the proliferation rate of double positive populationwas significantly higher than the other two populations.3. The results of transwell migration experiment:The number of cells which passed through the polycarbonatemembranes after12hours: double positive population was3.4000±0.96609cell/membrane, parental population was1.9000±1.19722cell/membrane,double negative population was1.2000±0.91894cell/membrane. The numberof double positive population cells which passed through the membrane wasthe most(P＜0.05) compared to the other two populations, and there was nosignificant difference (P＞0.05) between double negative and parentalpopulations.The number of cells which passed through the polycarbonatemembranes after24hours: double positive population was7.7000±1.63639cell/membrane, parental population was4.3000±0.82327cell/membrane,double negative population was2.9000±0.91894cell/membrane. The numberof double positive population cells which passed through the membrane wasthe most(P＜0.05) compared to the other two populations, and the doublenegative population was the least (P＜0.05).When cells of the three populations attached to the lower chamber after72hours, the numbers of adherent cells were counted as:44.0000±3.60555cell/well of double positive population,10.0000±2.64575cell/well of parentalpopulation,7.3333±1.52753cell/well of double negative population. It showed that, the cells of double positive population passed through themembranes at72hours were the most(P＜0.05) compared to the other twopopulations, and there was no significant difference (P＞0.05) between doublenegative and parental populations.4. Cell cycle analysis of the three populations: the double positivepopulation showed that the proportion of cells at G0/G1phase was50.7%±1.58%, S phase was27.3%±2.54%, and G2/M phase was22%±1.76%.The double negative population showed that the proportion of cells at G0/G1phase was43.6%±1.62%, S phase was42.2%±2.21%, and G2/M phase was14.1%±1.37%. The parental population showed that the proportion of cells atG0/G1phase was48.2%±2.14%, S phase was38.2%±2.81%, and G2/M phasewas13.6%±1.43%. Comparison of the G0/G1phase of the three populations:double positive population to double negative population: χ~2=110.990; P＜0.01; double positive population to parental population: χ~2=38.591; P＜0.01;parental population to double negative population: χ~2=4.358; P＜0.01. Sincestem cells possess slow-cyclical characteristic and contain much quiescentcells, the result of cell cycle analysis showed that there is more cells in theG0/G1phase of double positive population, it supports that theCD105~+/CD90~+cells should contain much more stem-like cells.5. Stemness gene expression of the three populations were detected byRT-PCR and statistical data were analyzed by calculating gradation value.oct3/4expression of double positive population was0.300839±0.0003259,double negative population was0.104273±0.0004666and parental populationwas0.167838±0.0001645. The mean difference of three populations wascompared by One-way ANOVA, and it showed that F=257988.7, P<0.01. Themean differences between groups was compared by LSD and at the sameimaging condition, the gradation value of oct3/4expression was the highest indouble positive subpopulation, lower in parental population and lowest indouble negative population.nanog expression of double positive population was0.741022±0.0004544,double negative population was0.279969±0.0008109and parental population was0.571277±0.0004220. The mean difference of three populations wascompared by One-way ANOVA, and it showed that F=469563.0, P<0.01. Themean differences between groups was compared by LSD and at the sameimaging condition, the gradation value of nanog expression was the highest indouble positive subpopulation, lower in parental population and lowest indouble negative population.sox-2expression of double positive population was0.574776±0.0002915,double negative population was0.478683±0.0002005and parental populationwas0.553225±0.0002452. The mean difference of three populations wascompared by One-way ANOVA, and it showed that F=123480.3, P<0.01. Themean differences between groups was compared by LSD and at the sameimaging condition, the gradation value of sox-2expression was the highest indouble positive subpopulation, lower in parental population and lowest indouble negative population.klf4expression of double positive population was0.603706±0.0003567,double negative population was0.093214±0.0001857and parental populationwas0.371799±0.0001921. The mean difference of three populations wascompared by One-way ANOVA, and it showed that F=2959712.0, P<0.01.The mean differences between groups was compared by LSD and at the sameimaging condition, the gradation value of klf4expression was the highest indouble positive subpopulation, lower in parental population and lowest indouble negative population.6. Quantitative real-time PCR to stemness gene expression：Quantitative real-time PCR was performed using the ABI7500RealTime PCR System(PE Applied Bio Systems) to analyze the quantitativeamount of stemness gene mRNA of the three populations, following themanufacturer’s instructions. Acquired data were analyzed by software7500version2.0.6(PE Applied Bio Systems).The oct3/4CTmean remained with a value of27.327±0.05when thefluorescence was detected in double positive population, so as it with a valueof26.004±0.11in parental population, and23.617±0.09in double negative population. The expression level of oct3/4in double positive population was2.448folds higher than parental population, and2.517folds higher thandouble negative population(P＜0.05).The nanog CTmean remained with a value of29.805±0.04when thefluorescence was detected in double positive population, so as it with a valueof25.698±0.11in parental population, and27.923±0.10in double negativepopulation. The expression level of nanog in double positive population was1.657folds higher than parental population, and1.909folds higher thandouble negative population(P＜0.05).The sox2CTmean remained with a value of27.159±0.04when thefluorescence was detected in double positive population, so as it with a valueof25.104±0.12in parental population, and22.686±0.13in double negativepopulation. The expression level of sox2in double positive population was1.460folds higher than parental population, and1.481folds higher thandouble negative population(P＜0.05).The klf4CTmean remained with a value of27.977±0.03when thefluorescence was detected in double positive population, so as it with a valueof25.983±0.12in parental population, and26.236±0.15in double negativepopulation. The expression level of klf4in double positive population was1.817folds higher than parental population, and8.494folds higher thandouble negative population(P＜0.05).Conclusions:1. CD105and CD90correlate with cancer stem cells in breast cancer cellline MDA-MB-231.2. CD105~+/CD90~+subpopulaion possess mesenchymal stem cell-likecharacteristics. |
