The Further Purification Of Breast Cancer Stem Cells By Suspension Sphere-culture Combined With Irradiation And Preliminary Studies On Mechanism Of Radiation Resistance

According to the cancer stem cell hypothesis, CSCs were not only the cause of cancer, but responsible for metastasis[8], recurrences [9] and resistance to conventional therapies[10,11]. Recent increasing publications provide much support for CSCs radioresistance. CSCs can be sorted by flow cytometry according to specific surface markers [6] or inherent properties such as Dye efflux of Hoechest33342 [7], however, injury of sorting process to cells and cytotoxicity of Hoechest 33342 dye on cells are not conducive to further study. The undifferentiated condition of stem cells could be kept by Serum-free suspension culture containing growth factors, at which stem cells can propagated and formed cell spheres which enriched for cancer stem cells. In breast cancer, the mammosphere culture system has now been used to identify, and enrich for, putative stem cells using breast cancer cells, however, mammosphere cells exists heterogeneity and contain limited proportion of breast cancer stem cells (BrCSCs). On the other hand, It is known that BrCSCs are resistant to radiation exposure. Hence, we speculate that BrCSCs with enhanced radiation resistance can be further purified by culturing in the suspension sphere-culture system combined with irradiation. Purpose:1) To explore the feasibility of the suspension sphere-culture combined with irradiation in high purification of BrCSCs.2) To explore the radioresistance characteristics of purified mammosphere cells.3) To explore the reliability of purified mammosphere cells used as Cell Model to study on radiation Resistance of BrCSCs.Materials and MethodsCell CultureHuman breast cancer MCF-7 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (HyClone) as monolayer culture. AS mammosphere culture, single-cell suspensions of MCF-7 cells were suspended at a density of 50,000 cells per milliliter in serum-free Dulbecco's modified Eagle's medium/F-12 containing 5μg/mL bovine insulin (Sigma),0.4% bovine serum albumin (BSA, Sigma),2% B27 (Invitrogen),20 ng/mL basic fibroblast growth factor (bFGF, Protech) and 10 ng/mL epidermal growth factor (EGF, Sigma), and culture in 37℃and 5%CO2 condition. To propagate spheres in vitro, spheres cultured for 7 days were collected by gentle centrifugation, dissociated to single cells as described by Dontu et al[12], and then cultured at a density of 10,000 cells/mL to generate mammospheres of the next generation.Analysis of Surface Marker ExpressionUsing an Epics Altra flow cytometer (Beckman Coulter), the expression of CD44 and CD24 was evaluated on cells cultured in mammosphere culture system for 1,2,3,4, 5 days,1 week, or in monolayer culture. Cells derived from monolayer cultures or mammospheres were trypsinized into single cell suspension, counted, washed with PBS, and stained with monoclonal antibody specific for human cell surface markers: CD44-FITC (ebioscience), CD24-PE (ebioscience), or ESA-PE (biolend). Isotype-matched labelled controls were also used in the analysis. Cells were labeled on ice for 30 minutes and washed twice before analysis in the cytometer. Three independent experiments were performed.IrradiationCells were irradiated with using 6-MV X-rays produced by a Varian 2100C linear accelerator at Southern Medical University. Does rate was 300cGy/min, and cells were irradiated from a vertical direction for the time required to generate a does curve of 0,2,4,6,8Gy. Corresponding controls were sham irradiated.Trypan Blue Exclusion TestSingle cell suspension was diluted into 106cells/ml, and 100μl 0.4% trypan solution was added into 900μl single cell suspension and mixed. Dye-excluded cells were observed under light microscopyTumorigenecity in Vivo of Mammosphere CellsIndicated numbers of monolayer culture or one-week-old mammosphere cells were resuspended in 1:1 (vol/vol) media and matrigel (BD Biosciences), and injected subcutaneously in the flank region of 5-week-old female NOD/SCID mice mice. 17β-Estradiol (sigma) was dissolved in ethanol and diluted in DMEM/F12, and 0.05 mg was inoculated i.m. every week beginning immediately after cell implantation.Assays of Cell Cycle Response and Apoptosis to RadiationTo assess the apoptosis levels of cells after, FITC-annexin V/propidium iodide staining for detecting apoptotic cell death was performed at 24 hours after radiation with a kit according to the manufacturer's instructions.Cells of monolayer culture in logarithmic phase of proliferation or mammospheres are seeded in 6-well plates (3×105 cells/well) in 3 mL. In order to reduce the error caused by different culture media, all cells in 6-well plates are cultured with fresh mammosphere culture medium for 2 hours before 4Gy irridiation. Cells are harvested at 24 h after irradiation and washed with PBS. Cells were fixed with 70% ethanol for 12 hours, Cells are then incubated with RNAase and PI (Sigma) and evaluated by flow cytometry. Cells treated with sham-irradiated was analyzed as corresponding controls.Clonogenic AssayFor clonogenic assays, cells derived from monolayer cultures or mammospheres were trypsinized (monolayer cultures) or mechanically dissociated with a Pasteur pipette (mammospheres) into single cell suspension, resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, seeded corresponding number of cells in 6-well plates, and then incubated for 3 hours before irradiation. Immediately following irradiation, the cells were incubated for lOdays at 37℃in a 5% CO2 environment to allow the colony formation. After 10 days, the colonies were fixed with pure ethanol and stained with 1% crystal violet. Colonies containing>50 cells were counted as clonogenic survivors. Plating efficiency (PE)=colonies observed/ number of cells plated, Surviving fraction (SF)= colonies counted/[cells seeded x (PE/100).Three independent experiments were performed, each in triplicate.Using GraphPad Prism 5 software, the average data were fitted into single-hit multitarget formula:S=1-(1-e-D/D0)N, where S is the fraction of cells surviving a dose, D0 called the "mean lethal dose", is the dose on the straight-line portion of the survival curve to decrease the survival to 37%. The "quasi-threshold dose" or Dq, which is the intercept of the extrapolated high dose, was also calculated. N is referred to the extrapolation number which is a parameter to measure the width of shoulder of the survival curve.Detection of Intracellular ROSThe levels of intracellular reactive oxygen species (ROS) were analyzed on the basis of the oxidative conversion of cell permeable 2',7'-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) upon reaction with hydroxyl radical, hydrogen peroxide, or peroxynitrite[13]. Monolayer cells or mammosphere cells were collected, suspended in diluted DCFH-DA solution (10μM in 1L RPMI 1640) at a density of 1×106 cells/ml, and then incubated at 37℃in a 5% CO2 condiction for 20 min, shaken to mix suspension every 5 minutes. The cells were washed thrice with RPMI 1640 and analysed at 1 hour after washing by flow cytometry. An equal volume of RMPI1640 media without DCFH-DA served as an negative control and the addition of 50μg Rosup served as an internal positive control.Real Time RT-PCR of ATM GeneCells from MCF-7 monolayer culture or mammospheres were harvested into Trizol (Invitrogen), and RNA was extracted following the manufacturer's protocol. Four micrograms of total RNA per 20μl of reaction volume was reverse transcribed into cDNA using the SuperScript First-Strand Synthesis System (Invitrogen). Real-time polymerase chain reactions (PCRs) were performed and monitored using the SYBR Green PCR Mastermix and the ABI Prism 7500 Sequence Detection System (Perkin Elmer/Applied Biosystems, Rotkreuz, Switzerland, http://www.perkinelmer.com). cDNA samples (5μl for total volume of 20μl per reaction) were analyzed for ATM gene and for the reference gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). The relative mRNA expression was then presented in relation to MCF-7 group.Statistical AnalysisIn all experiments, differences among groups were analyzed by variance (ANOVA) method or Student's t test using SPSS (13.0). Bonferroni method was used for multiple comparisons.A probability level of 0.05 was chosen for statistical significance. Results1) Mammospheres enriching for CD44+/CD24"/low cells:Some cells in MCF-7 grew as clonal nonadherent spherical clusters. CD44+CD24-/low cells in MCF-7 increased after culture in serum-free medium. At fifth day, the proportion of CD44+CD24-/low cells accounted for (77.53±5.98)%, however, the proportion did not significantly increase with following passaging, it peaked at (79.40±3.40)% when passaged for 5 generations.2) The role of radiation in enrichment for CD44+CD24-/low cells:Trypan blue exclusion test showed that a few dye-excluded cells could be found in mammosphere cells treated with 8Gy or 2Gy×4 times irradiation. the proportion of CD44+CD24-/low cells increased significantly when mammosphere cells were treated with 8Gy or 2Gy×4 times, (93.57±2.81)% and (98.50±1.08)%, respectively3) Tumourigenicity assay:Putified cells were more tumourigenic than parental MCF-7 cells4) Responding of purified mammosphere cells to radiation:Purified mammosphere cells exhibited lower frequencies of early apoptotic events after 2Gy exposure than monolayer cells:(12.0±1.75)% in monolayer cells and (6.40±0.44)% in purified mammosphere cells, the difference is statistically significant (t=-5.37, P=0.006) The proportion of cells in G2 phase of mammospheres was significantly higher than that of monolayer cells (t=-7.62, P=0.002). Purified mammosphere cells showed higher mean survival fraction at 2 Gy (SF2Gy), which represents a typical daily radiation dose in clinical radiotherapy, than monolayer cells (t=-3.6, P=0.024). In addition, purified mammosphere cells had higher N and Dq values than monolayer cells.5) Detection of intracellular ROS levels:To detect intracellular ROS levels of mammosphere cells and their parental MCF-7 cells, the oxidant-sensitive dye DCFH-DA was used. Purified mammosphere cells contained significantly lower concentration of ROS than their parental MCF-7 cells (t=6.305, P=0.003) additionally, Purified mammosphere cells contained significantly higher proportion of cells with low ROS level (t=-32.125,P=0.000).6) ATM gene expression levels:ATM gene expression levels were evaluated in mammosphere cells and parental MCF-7 using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Mammospheres expressed higher level of ATM gene than its parental MCF-7 cells (t=-4.97, p=0.008).ConclusionsCD44+/CD24-/low breast cancer stem cells with enhanced radiation resistance can be further purified by culturing in the suspension sphere-culture system combined with irradiation.