Breast Cancer Stem Cells With Radiation Induced G2 Phase Arrest May Contributed To The Resistance To Radiotherapy

Background Breast cancers which are common for women, affect women's health physically and mentally. According to WHO, there are 1.2 million patients suffer from breast cancer, with 500 thousand patients die from those. What's more, the morbidity of new cases has been increasing by two percent to eight percent every year since twentieth-century. Regionally speaking, developed countries such as America and British have the highest ratio of new cases, in which breast cancer rank first or second of malignant tumor. Although it is less severity in Asia, there are more and more young suffers. In our country, thirty five to forty five out of one hundred thousand patients suffer from breast cancer, which is seven percent to ten percent of tumor of our body. Furthermore, it has been increasing three percent yearly, and get first place of female malignant tumor.Theory of CSCs (cancer stem cells) which considers that there are only a minor part of tumor cells to keep functional growth and variety of cancer were presented recently, to provide new promise for cancer treatment. Although most part of tumor cells are eliminated in traditional tumor therapy which ignorant CSCs, patients are also faced recurrence due to the CSCs which include three features:self-renew, unlimited proliferation and differentiation. Generally speaking, several methods were used to differ CSCs from non- CSCs, that is, specific surface markers, side population (SP), ALDH (aldehyde dehydrogennase) and suspension culture. Among these, the former three can sort CSCs, however, they can't maintain undifferentiated condition of CSCs, for which to testify tumorigenecity in vivo is the most important measures called "gold standards".Al-Hajj et al firstly discovered BrCSCs (breast cancer stem cells) from patients' pleural effusion in 2003, and CD44+CD24-/low cells were quantified by flow cytometry. Then, Ponti enriched for BrCSCs by suspension culture, whose properties confirmed by NOD/SCID mouse in vivo were still maintained. With further study of CSCs, Bao et al confirmed that CD133+ cells are radio-resistance due to their ability of repairing damaged DNA. What is more, Phillips et al discovered that CSCs among MCF-7 cell lines resist to radiotherapy, followed by Xie et al who informed that CSCs can be enriched by suspension culture combined with radiation. The most important thing is that early studies tell us what damaged DNA caused by radiation would prevent cell cycles into other, especially Gl/S and G2/M to repair DNA.In a word, suspension culture can be used to purify BrCSCs, and CD44+ CD24-/low phenotype which can be measured by flow cytometry are an important marker for them. At present, although some reports about brain tumor CSCs were published, however, the mechanisms of radio-resistance are not sure. With no doubt, it is possible for us to investigate the radio-resistance whether associated with cell cycle.Objectives1) To explore the feasibility of the suspension sphere-culture in high purification of BrCSCs in MCF-7 cell lines.2) To explore the radioresistance characteristics of purified mammosphere cells. 3) To investigate the radiation-induced cell cycles of human breast cancer stem cells enriched by suspension culture.MethodsCell Culture Human 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 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),20ng/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, and then cultured to generate mammospheres of the next generation.Analysis of Surface Marker Expression Using an Epics Altra flow cytometer (Beckman Coulter), the expression of CD44 and CD24 was evaluated on cells cultured in mammosphere culture system for 4th day, 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 labeled 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.Tumorigenecity in Vivo of Mammosphere Cells Indicated numbers of monolayer culture or 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, according to 5×105 cells/mouse,5×104 cells/mouse, 1×103 cells/mouse and 5×102 cells/mouse.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.Clonogenic Assay For 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 14days 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/ [seeded cells x (PE/100)]. Three independent experiments were performed, each in triplicate.Assays of Cell Cycle Response to Radiation Cells of monolayer culture in logarithmic phase of proliferation or mammospheres are seeded in 6-well plates (3×105cells/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, and then cells are incubated with RNAase and PI (Sigma) and evaluated by flow cytometry. Cells treated with sham-irradiated was analyzed as corresponding controls.Western blotting According to instruction of protein extraction kit of Col. Novagen, protein of mamosphere and monolayer after irradiation were extracted, and then concentration was measured by BCA. Protein mixed with loading buffer according to ratio of two to one was boiled for 5min. Finally, protein which was shifted to nitrocellulose membrane after it was sorted by SDS-PAGE was analysis by Gel2000.7) Statistical Analysis In all experiments, differences among groups were analyzed by variance (ANOVA) method using SPSS (13.0).A probability level of 0.05 was chosen for statistical significance.Results1) Suspension culture Mamospheres which was isolated form each other and formed in serum-free culture system are irregular. What is more, they were the same in new generations.2) Mammospheres enriching for CD44+/CD24-/low cells Some cells in MCF-7 grew as clonal non-adherent spherical clusters. CD44+/CD24-/low cells in MCF-7 increased after culture in serum-free medium. The proportion of CD44+/CD24-/low cells increased from (8.50±5.98) percent of monolayer to (84.26±4.56) percent, with significantly difference.3) Tumourigenicity assay Putified cells were more tumourigenic than parental MCF-7 cells after 12 weeks injection. After 12 weeks, the tumor incidence of monolayer cells was 3/3,4/4,6/6 and 7/8 when 5×105,5×104 and 1×103 and 5×102 cells were injected, respectively. In parallel, the tumor incidence with ammosphere cells was 3/3,3/4,1/6 and 0/8 in monolayer.4) Results of clonogenic assay To test whether enrichment of CD44+/ CD24-/low cell population also conferred resistance to radiation-induced cytotoxicity, clonogenic assays were performed. The mammospheres and monolayer showed differential sensitivities to radiation. Mammosphere cells were generally more resistant than the corresponding monolayer cells to a 2 Gy irradiation.5) Cell Cycle Response to Radiation Proportion of cells in G2 phase after radiation was significantly higher than that before irradiation in mammospheres. However, there is not significant difference in monolayer. The proportion of G2 phases increased from 0.210±0.080 to 0.474±0.074 in mamosphere.6) Results of Pcdc25C There is not significant difference in CDC25C between before and after radiation. In contrast, pCDC25C (ser216) presented difference before and after radiation in mamosphere.Conclusions1) The suspension sphere-culture can enrich for BrCSCs in MCF-7 cell lines, in which CD44+CD24-/low cells can account to 84%. Thus, it can provide a promise for further study.2) CSCs are radio-resistance compared with non-CSCs, which can be confomed by clonogenic a3) Breast cancer stem cells with radiation induced G2 phase arrest may contribute to the resistance to radiotherapy.