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Journal of the National Cancer Institute, Vol. 98, No. 24, December 20, 2006 ARTICLES 1777 Background: If cancer arises and is maintained by a small population of cancer-initiating cells within every tumor, understanding how these cells react to cancer treatment will facilitate improvement of cancer treatment in the future. Cancer-initiating cells can now be prospectively isolated from breast cancer cell lines and tumor samples and propagated as mammospheres in vitro under serum-free conditions. Methods: CD24 − /low /CD44 + cancer-initiating cells were isolated from MCF-7 and MDA-MB-231 breast cancer monolayer cultures and propagated as mammospheres. Their response to radia- tion was investigated by assaying clonogenic survival and by measuring reactive oxygen species (ROS) levels, phosphory- lation of the replacement histone H2AX, CD44 levels, CD24 levels, and Notch-1 activation using fl ow cytometry. All statis- tical tests were two-sided. Results: Cancer-initiating cells were more resistant to radiation than cells grown as mono- layer cultures (MCF-7: monolayer cultures, mean surviving fraction at 2 Gy [SF 2Gy ] = 0.2, versus mammospheres, mean SF 2Gy = 0.46, difference = 0.26, 95% confi dence interval [CI] = 0.05 to 0.47; P = .026; MDA-MB-231: monolayer cultures, mean SF 2Gy = 0.5, versus mammospheres, mean SF 2Gy = 0.69, difference = 0.19, 95% CI = − 0.07 to 0.45; P = .09). Levels of ROS increased in both mammospheres and monolayer cul- tures after irradiation with a single dose of 10 Gy but were lower in mammospheres than in monolayer cultures (MCF-7 monolayer cultures: 0 Gy, mean = 1.0, versus 10 Gy, mean = 3.32, difference = 2.32, 95% CI = 0.67 to 3.98; P = .026; mammo- spheres: 0 Gy, mean = 0.58, versus 10 Gy, mean = 1.46, difference = 0.88, 95% CI = 0.20 to 1.56; P = .031); phosphorylation of H2AX increased in irradiated monolayer cultures, but no change was observed in mammospheres. Fractionated doses of irradiation increased activation of Notch-1 (untreated, mean = 10.7, versus treated, mean = 15.1, difference = 4.4, 95% CI = 2.7 to 6.1, P = .002) and the percentage of the can- cer stem/initiating cells in the nonadherent cell population of MCF-7 monolayer cultures (untreated, mean = 3.52%, versus treated, mean = 7.5%, difference = 3.98%, 95% CI = 1.67% to 6.25%, P = .009). Conclusions: Breast cancer – initiating cells are a relatively radioresistant subpopulation of breast cancer cells and increase in numbers after short courses of fractionated irradiation. These fi ndings offer a possible mechanism for the accelerated repopulation of tumor cells observed during gaps in radiotherapy. [J Natl Cancer Inst 2006;98: 1777 – 85 ] One view of cancer is that it may arise from a single cell that has the ability to self-renew and thus to maintain the growth of a tumor, whereas the majority of its cellular progeny does not. There is increasing evidence that such a cell population exists and that these cells can be prospectively identifi ed in brain tu- mors ( 1 ) , breast cancer ( 2 ) , prostate cancer ( 3 ) , and melanoma Affi liation of authors: Department of Radiation Oncology, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA. Correspondence to: Frank Pajonk, MD, PhD, Department of Radiation Oncology, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave., Los Angeles, CA 90095-1714 (e-mail: fpajonk@mednet.ucla.edu ). See “ Notes ” following “ References. ” DOI: 10.1093/jnci/djj495 © The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org. The Response of CD24 − /low /CD44 + Breast Cancer – Initiating Cells to Radiation Tiffany M. Phillips , William H. McBride , Frank Pajonk ( 4 ) . A considerable effort is going into determining unique prop- erties of these cells with the assumption that this cell population, more than any other, will determine the outcome of cancer treat- ment. Because such cells might be expected to share properties with adult stem cells in normal tissues, they are often termed can- cer stem cells ( 5 ) . However, in spite of old ( 6 ) and more recent ( 1 , 2 , 7 – 9 ) evidence that cancer stem cells exist, there is still a dearth of good phenotypic markers for such cells. In addition, many of the self-renewing cancer cell populations that are stud- ied may also contain early progenitor cells that are derived from cancer stem cells but are also able to initiate and maintain tumor growth. Therefore, we join others ( 2 ) in preferring to use the term cancer-initiating cells. In breast cancer, a population of CD24 − /low /CD44 + cells has been isolated that is highly enriched for cancer-initiating cells ( 2 ) . This population is 1000 times more tumorigenic than cell populations that are depleted of CD24 − /low /CD44 + cells, and in- jection of as few as 200 cells leads to tumor formation in SCID mice ( 2 ) . Breast cancer – initiating cells can be established from patients’ surgical specimens or breast cancer cell lines and can be propagated in vitro as nonadherent mammospheres ( 7 ) . Stem cell properties in normal tissues are tightly regulated by the Wnt, Shh, and Notch signaling pathways ( 10 , 11 ) . In addition, overexpression of Notch-1 was observed in breast cancer speci- mens, and the level of expression was associated with prognosis ( 12 ) . Activation of the Notch-1 pathway is initiated by the bind- ing of Notch-1 ligands, e.g., Jagged-1, to the extracellular do- main of Notch-1. This binding causes a conformational change in Notch-1 that allows the protease tumor necrosis factor alpha con- verting enzyme to cleave the extracellular domain of the mole- cule. Notch-1 is thereafter processed by γ -secretase – regulated intramembrane proteolysis, which allows the intracellular do- main of Notch-1 (Notch-1 ICD) to translocate into the nucleus where it binds to and activates the transcriptional repressor CBF1. Activation of Notch-1 signaling leads to increased transcription of ErbB2 ( 13 ) , cyclin D1 ( 14 ) , CDK2 ( 14 ) , and Notch-4 ( 15 ) . ErbB2 is related to radiation resistance ( 16 ) , whereas cyclin D1 and CDK2 promote the transition from G1 to S phase of the cell cycle and thus promote proliferation. Notch-1 signaling promotes the self-renewal of mammary stem cells ( 17 ) , and there is strong evidence that Notch-1 is involved in the carcinogenesis of breast cancer ( 17 ) . In addition, Notch-1 maintains the malignant pheno- type of Ras-transformed cells ( 15 ) , and overexpression of Notch induces mammary tumors in mice ( 18 ) . 1778 ARTICLES Journal of the National Cancer Institute, Vol. 98, No. 24, December 20, 2006 Radiation therapy (RT) is an integral part of the multimodal treatment concept for breast cancer. Its success depends on the complete elimination of all cancer stem cells. Radiation oncolo- gists have been advocating the existence of stem cells in normal tissues and cancers for decades ( 6 ) . Accelerated repopulation — the increase in the rate of growth as a result of time between treatments ( 19 , 20 ) — is a cancer stem cell – related phenomenon that occurs during fractionated RT. During accelerated repopula- tion, each day of a treatment gap decreases the effi cacy of RT by approximately 0.6 Gy, making it one of the major reasons for lo- cal failure of RT. Accelerated repopulation was fi rst described for head and neck epithelial tumors ( 21 ) , but it also occurs in breast cancer even though it may be diffi cult to detect ( 22 – 24 ) . In this study, we investigated the radiation response of CD24 − /low / CD44 + breast cancer – initiating cells, the population of cancer cells that are likely to be critical for success or failure of cancer therapy. We characterized the radiation sensitivity of these cells and the size of this cell population after clinical fractions of radiation and ex- plored possible mechanisms for the failure of radiotherapy. M ETHODS Cell Culture MCF-7 and MDA-MB-231 breast cancer cells (American Type Culture Collection; Manassas, VA) were cultured in log- growth phase in modifi ed Eagle medium (MEM) (supplemented with 0.1 mM nonessential amino acids and 1 mM sodium pyru- vate; Cellgro, Kansas City, MO) and Dulbecco’s modifi ed Eagle medium (DMEM) (Cellgro), respectively, supplemented with 10% heat-inactivated fetal calf serum (FCS) and 0.01 mg/mL bovine insulin (Sigma, St Louis, MO) at 37 °C in a humidifi ed atmosphere (5% CO 2 ). To obtain cancer-initiating cells and to propagate them as mammospheres, cells fl oating in the superna- tant of 2-day-old cultures were collected by centrifugation for 5 minutes at 500 g , washed in Hanks’ buffered salt solution, and resuspended in phenol red – free DMEM – F12 (Cellgro) supple- mented with 0.4% bovine serum albumin (BSA, Sigma), 5 μ g/mL bovine insulin (Sigma), 20 ng/mL basic fi broblast growth factor 2 (bFGF, Sigma), and 10 ng/mL epidermal growth factor (EGF, Sigma) at a density of 1000 cells/mL. Growth fac- tors were added to the mammosphere cultures every 3 days. To mimic mammosphere culture conditions in cells grown as mono- layer cultures, cells were plated in MEM or DMEM media con- taining 10% FCS supplemented with 5 μ g/mL bovine insulin, 20 ng/mL bFGF, and 10 ng/mL EGF. Irradiation For clonogenic assays, cells derived from monolayer cultures or 5-day-old mammospheres were enzymatically dissociated with trypsin – EDTA (monolayer cultures) or mechanically disso- ciated with a Pasteur pipette (mammospheres), both passed through a 40- μ m sieve, and immediately irradiated (10 6 cells/ mL) at room temperature with a 137 Cs laboratory irradiator (Mark I, JL Shephard, San Fernando, CA) at a dose rate of 4.95 Gy/minute for the time required to generate a dose curve of 0, 2, 4, 6, and 8 Gy. Corresponding controls were sham irradiated. Colony- forming assays were performed immediately after irradiation by plating cells into triplicate 100-mm culture dishes. After 28 days, cells were fi xed with 75% ethanol and stained with 1% crystal violet, and colonies containing more than 50 cells were counted. To generate a radiation survival curve, the surviving fraction at each radiation dose was normalized to that of the sham-irradiated control, and curves were fi tted using a linear – quadratic model ( surviving fraction = e ( − α dose − β dose 2 ) , in which α is the number of logs of cells killed per gray from the linear portion of the survival curve and β is the number of logs of cells killed per [gray] 2 from the quadratic component) ( 25 ) . Three independent experiments were performed. To evaluate H2AX phosphorylation, single-cell suspensions were irradiated as above with 0, 2, or 10 Gy. Cells were harvested by centrifugation (500 g for 5 minutes at 4 °C) at 5 and 60 min- utes after irradiation. To measure reactive oxygen species (ROS) accumulation, 100 000 cells were treated with 0, 2, or 10 Gy. Cells were imme- diately analyzed as described below. To measure Notch-1 activation and Jagged-1 expression, cells were treated with single and fractionated doses of radiation. Cells (400 000 per dish) were plated onto 100-mm tissue culture dishes and allowed to grow for 24 hours. Cultures were then irradiated as monolayers at room temperature with 3 Gy daily for 5 consecutive days (days 2 – 6) or with a single dose of 10 Gy on day 6. Control cells were sham irradiated. Nonadherent and ad- herent cells were harvested 48 hours after the last irradiation (on day 8). For primary mammosphere formation assays, cells were irradiated with 3 Gy daily for 5 consecutive days (days 2 – 6) or with a single dose of 10 Gy on day 6. Control cells were sham irradiated. 3-( 4 , 5 -Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assays to Measure Cell Proliferation For 3-( 4 , 5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, MCF-7 cells in monolayer culture were irradiated; incubated for indicated times in MEM media supple- mented with 10% FCS, 20 ng/mL bFGF, and 10 ng/mL EGF; washed twice with PBS; incubated with trypsin – EDTA; resus- pended in MEM (containing 10% FCS); counted; and plated in 100 μ L MEM (10% FCS) at 2000, 10 000, 15 000, and 20 000 cells per well into 96-well plates. After 7 days, 20 μ L of MTT solution (5 mg/mL in PBS) was added to each well, and cells were incubated for 4 hours at 37 °C. Then 50 μ L sodium dodecyl sulfate solution (20% sodium dodecyl sulfate, 0.01% HCl) was added to each well, and plates were incubated at 37 °C overnight. Absorbance was measured at 560 nm in a fl uorescence plate reader (Spectrafl uor, Tecan, San Jose, CA). Flow Cytometry to Measure CD24, CD44, and Jagged-1 Expression; Notch-1 Activation; and H2AX Phosphorylation CD24 and CD44 expression was analyzed in cells derived from monolayer cultures or in 5-day-old primary mammo- spheres following incubation in trypsin – EDTA or dissociation with a Pasteur pipette and passage through a 40- μ m sieve. At least 10 5 cells were pelleted by centrifugation at 500 g for 5 min- utes at 4 °C, resuspended in 10 μ L of monoclonal mouse anti- human CD24 – fl uorescein isothiocyanate (FITC) antibody (BD Pharmingen, San Jose, CA) and a monoclonal mouse anti- human CD44 – phytoerythrin (PE) antibody (BD Pharmingen), Journal of the National Cancer Institute, Vol. 98, No. 24, December 20, 2006 ARTICLES 1779 and incubated for 20 minutes at 4 °C. Ten independent experi- ments were performed. To measure Jagged-1 expression and Notch-1 activation, cells were permeabilized with 4% formaldehyde and pelleted by centrifugation as above. Cells were then incubated with 0.25 μ g of PE/Cy5-conjugated monoclonal mouse anti-human CD44 antibody, 10 μ L of monoclonal mouse anti-human CD24 – FITC antibody, and 200 μ L of either polyclonal rabbit anti-human Jagged-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or polyclonal rabbit anti-human Notch-1-ICD antibody (Cell Sig- naling, Danvers, MA) that had been diluted 1 : 200 in PBS con- taining 2% BSA for 20 minutes at 4 °C. Cells were then washed with PBS/4% BSA and incubated with a secondary, PE-con- jugated polyclonal goat anti-rabbit antibody (BD Pharmingen). For analysis of H2AX phosphorylation, cells were centrifuged for 5 minutes at 500 g and resuspended in 0.3 mL of PBS. To fi x the cells, 0.7 mL of ethanol (99%) was added to the tube while vortexing, and samples were stored for 30 minutes at − 20 °C. Cold Tris-buffered saline (TBS, pH 7.4, 1 mL) was added, and cells were pelleted by centrifugation at 500 g and resuspended in 1 mL cold TST (TBS containing 4% FBS and 0.1% Triton X-100) for 10 minutes to permeabilize and rehydrate the cells. Cells were pelleted again and resuspended in 200 μ L of monoclo- nal mouse anti- γ H2AX – FITC antibody (Upstate, Charlottesville, VA) diluted 1 : 500 in TST, incubated on a shaker platform for 2 hours at room temperature, and washed twice in TST. Three independent experiments were performed. Flow cytometry and cell sorting were performed on a FACScalibur fl ow cytometer (Becton Dickinson, Franklin Lakes, NJ). The CellQuest (Becton Dickinson) software package was used. Reactive Oxygen Species Formation Assay Cells derived from monolayer cultures or 5-day-old mammo- spheres were incubated with trypsin – EDTA or dissociated me- chanically using a Pasteur pipette, respectively, resuspended in modifi ed HBSS (10 mM HEPES, 1 mM MgCl 2 , 2 mM CaCl 2 , 2.7 mM glucose), passed through a 40- μ m sieve, counted, and diluted to a fi nal concentration of 10 6 cells/mL in 15-mL Falcon tubes (Becton Dickinson). Aminophenyl fl uorescein (Cell Tech- nology, Mountain View, CA) was added to a fi nal concentration of 10 μ M, and cells were incubated for 30 minutes in the dark and irradiated as indicated above. A total of 100 000 cells per well were plated into black 96-well plates, and fl uorescence was measured in a fl uorescence plate reader (Spectrafl uor, Tecan; ex- citation: 480 nm, emission: 520 nm). Fluorescence was normal- ized to the fl uorescence readings of untreated monolayer culture cells. Three independent experiments were performed, each in triplicate. Primary Mammosphere Formation Assay The ability of cells in the nonadherent population of mono- layer cultures to initiate mammosphere formation after irradia- tion was assessed by harvesting, washing, and resuspending nonadherent cells in phenol red – free DMEM – F12 medium (supplemented with 0.4% BSA, 20 ng/mL bFGF, and 10 ng/mL EGF). Cells were then passed through a 40- μ m sieve, counted, diluted, and plated into 96-well plates at clonal densities. Mam- mospheres were counted on day 5. Statistical Methods All data are represented as means and differences of the means with 95% confi dence intervals (CIs). P values of .05 or less, cal- culated using a paired two-sided Student’s t test, were considered to indicate statistically signifi cant differences. R ESULTS Response of CD24 − /low /CD44 + Breast Cancer – Initiating Cells to a Single Dose of Radiation We established nonadherent mammosphere cultures from both MCF-7 and MDA-MB-231 breast cancer cells and analyzed the percentage of CD24 − /low /CD44 + cells on day 5 by fl ow cytome- try. In general, by day 5, MCF-7 ( Fig. 1, A ) and MDA-MB-231 (data not shown) mammospheres showed dramatically elevated percentages of CD24 − /low /CD44 + cells. The responses of cells from monolayers and CD24 − /low / CD44 + -enriched mammospheres (day 5) to radiation were com- pared by clonogenic assay. The plating effi ciencies of MCF-7 cells derived from monolayer cultures and mammospheres with- out irradiation were similar (mean = 7.2%, 95% CI = 0.73 to 13.7, and mean = 11%, 95% CI = 8.8 to 13.2, respectively). How- ever, cells derived from MCF-7 mammospheres were more ra- dioresistant than cells derived from monolayer cultures (monolayer-derived cells: α = 0.79, β = 0.011, mean surviving fraction at 2 Gy [SF 2Gy ] = 0.2, versus mammospheres: α = 0.30, β = 0.044, mean SF 2Gy = 0.46, difference = 0.26, 95% CI = 0.05 to 0.47; P = .026, n = 9; Fig. 1, B ). Comparable results were found for mammospheres that were derived from MDA-MB-231 cells (monolayer-derived cells: α = 0.65, β = 0.0014, mean SF 2Gy = 0.5, versus mammospheres: α = 0.31, β = 0.035, mean SF 2Gy = 0.69, difference = 0.19, 95% CI = − 0.07 to 0.45; P = .09, n = 6). One hallmark of the recognition and repair of double-strand DNA breaks is phosphorylation of the replacement histone H2AX ( 26 ) . Single-cell suspensions from MCF-7 mammo- sphere and monolayer cultures were irradiated with 0, 2, or 10 Gy, and H2AX phosphorylation ( γ H2AX) was measured by fl ow cytometry at 5 and 60 minutes after irradiation (n = 2). MCF-7 cells derived from monolayer cultures showed a time- dependent increase of γ H2AX after irradiation, whereas cells derived from primary mammospheres showed little change in γ H2AX ( Fig. 1, C ). At 60 minutes, the increase in γ H2AX for cells derived from monolayer cultures was dose dependent, with 10 Gy being more effective than 2 Gy (monolayer cultures: rela- tive to control, 2 Gy, mean = 1.63-fold, not statistically signifi - cant, 10 Gy, mean = 3.2-fold, difference = 2.2, 95% CI = 1.46 to 2.92, P = .006, n = 3; mammospheres: relative to 0 Gy, 2 Gy, mean = 1.06-fold, not statistically signifi cant, 10 Gy