Recent data indicate a hierarchical organization of many solid cancers, including breast cancer, with a small number of cancer initiating cells (CICs) that have the ability to self-renew and exhibit multi-lineage potency. We, and others, have demonstrated that CICs in breast cancer and glioma are relatively resistant to ionizing radiation if compared to their non-tumorigenic counterparts. However, the extent of the remaining self-renewing capacity of CICs after fractions of radiation is currently unknown. We hypothesized that CICs, in contrast to their non-tumorigenic counterparts, not only survive fractions of ionizing radiation but also retain the CIC phenotype as defined by operational means.
We used two marker systems to identify breast CICs (CD24-/low/CD44high, or lack of proteasome activity) and performed sphere-forming assays after multiple clinical fractions of radiation. Lineage tracking was performed by membrane staining. Cell cycle distribution and RNA content were assessed by flow cytometry and senescence was assessed via β-galactosidase staining.
We demonstrated that irradiated CICs survived and retained their self-renewal capacity for at least four generations. We show that fractionated radiation not only spared CICs but also mobilized them from a quiescent/G0 phase of the cell cycle into actively cycling cells, while the surviving non-tumorigenic cells were driven into senescence.
The breast CIC population retains increased self-renewal capacity over several generations and therefore, we conclude that increases in the number of CICs after sublethal doses of radiation have potential clinical importance. Prevention of this process may lead to improved clinical outcome.
Recent experimental data provide evidence for a hierarchical structure of many solid cancers [1-7] including breast cancer . Using surface markers, a small population of highly tumorigenic breast cancer initiating cells (CICs) has been prospectively identified, which exhibits a cancer stem cell phenotype, thus being able to self-renew and to give rise to progeny of multiple different lineages of differentiated cells . We previously demonstrated that this population of cells was relatively resistant against ionizing radiation if compared to their non-tumorigenic counterparts . Two other groups have confirmed our data [10,11]. Additionally, Han and Crowe recently identified a side population in MCF7 and T47D breast cancer cells , which initiated tumors in vivo and was more resistant to ionizing radiation than the non-side population. Furthermore, we, and others, have reported that the population of CICs increases during the course of fractionated radiation [9,13]. However, the extent of the remaining self-renewing capacity of this CIC population after fractions of radiation is currently unknown. This information is crucial for evaluating the significance of radiation resistance of CICs.
Breast CICs were first prospectively identified in patient derived samples by Al-Hajj et al. using antibodies against ESA, CD24, and CD44 , and later validated for established breast cancer cell lines . The labeling of CICs using antibodies or enzyme substrates  has been an invaluable tool to study the features of CICs. Nevertheless, both techniques have disadvantages, which make it difficult to study living CICs in-situ or in-vivo. To overcome this problem, we recently developed an imaging system for breast and glioma CICs that allows for identification and tracking of CICs without the need for antibodies or exogenous enzyme substrates . It is based on the observation that CICs lack 26S proteasome function. In cell lines engineered to express a fusion protein between the fluorescent protein ZsGreen and the C-terminal degron of murine ornithine decarboxylase (cODC), CICs can be identified via fluorescent imaging due to the accumulation of ZsGreen-cODC protein while non-tumorigenic cells degrade this protein immediately after translation.
In the present study, we hypothesized that CICs, in contrast with their non-tumorigenic counterparts, not only survive fractions of ionizing radiation but also retain the CIC phenotype as defined by operational means and may even become more aggressive than populations of non-irradiated control CICs. Using two distinct marker systems to identify breast CICs (CD24-/low/CD44high, or lack of proteasome activity) and sphere forming assays, developed by Dontu et al., to enrich for normal mammary stem cells/progenitors , we demonstrated that cycling cell populations were enriched for CICs after irradiation with fractions of 2 or 3 Gray (Gy). Irradiated CICs not only survived but also retained their self-renewal capacity for at least four generations. CICs were resistant to radiation-induced apoptosis and were arrested in the G2 phase of the cell cycle, while non-tumorigenic cells were prone to radiation-induced apoptosis. Finally, in lineage-tracking experiments we observed that fractionated radiation not only spared CICs but also mobilized CICs from a quiescent/G0 phase of the cell cycle into actively cycling cells, while the surviving non-tumorigenic cells were driven into senescence.
Materials and methods
Human MCF-7 and T47D breast cancer cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). MCF-7-ZsGreen-cODC and T47D-ZsGreen-cODC were obtained as described in Vlashi et al. . All cells were cultured in log-growth phase in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) (supplemented with 10% fetal bovine serum (Sigma Aldrich, St Louis, MO, USA) and penicillin and streptomycin cocktail (Sigma Aldrich)) and were grown in a humidified incubator at 37°C with 5% CO2.
Cells were irradiated at room temperature with a 137Cs laboratory irradiator (Mark I, JL Shephard, San Fernando, CA, USA) at a dose rate of 4.95 Gy/minute for the time required to apply a prescribed dose. For fractionated radiation of 2 or 3 Gy, cells were irradiated on eight or five consecutive days, respectively. The single dose was applied at the same time as the last dose of fractionated radiation. Corresponding controls were sham irradiated. Cell proliferation, number of stem cells, and sphere forming assays were performed 48 h after the last fraction of radiation.
After fractionated radiation, CD24 and CD44 expression was analyzed in cells derived from monolayer cultures following incubation with trypsin-EDTA and passage through a 40 μm sieve. At least 105 cells were pelleted by centrifugation at 500 × g for five minutes at 4°C, resuspended in 10 μL of monoclonal mouse anti-human CD24-fluorescein isothiocyanate (FITC) antibody (BD Pharmingen, San Jose, CA, USA) and a monoclonal mouse anti-human CD44-phytoerythrin (PE) antibody (BD Pharmingen), and incubated for 20 minutes at 4°C. Corresponding isotype control APC-conjugated antibodies (BD Pharmingen) and isotype control PE-conjugated antibodies (BD Pharmingen) were used as controls (Additional file 1). Three independent experiments were performed.
Additional file 1. Figure S1. Control gate of CD24 and CD44 analysis (Figure 1). Fluorescence-activated cell-sorting (FACS) analysis was performed to measure non-specific binding of anti-mouse isotype control PE-conjugated and anti-mouse isotype control APC-conjugated antibodies, and effects of radiation treatment on cell auto-fluorecence.
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We had previously shown  that breast cancer stem cells could be identified via their low proteasome activity, which can be assessed by analyzing ZsGreen-cODC protein accumulation. For 48 h after the last fraction of radiation, cells were trypsinized and ZsGreen-cODC expression was assessed by flow cytometry. Cells were defined as ZsGreen-cODC positive if the fluorescence in the FL-1H channel exceeded the fluorescence level of 99.9% of the empty vector-transfected control cells (Additional file 2).
Additional file 2. Figure S2. Control gate of ZsGreen-cODC analysis (Figure 4). Cells stably transfected with an empty control vector were irradiated with 5 × 3 Gy (right panel) or sham irradiated (left panel), and cells were analyzed for green auto-fluorescence. Cells were defined as ZsGreen-cODC positive if the fluorescence in the FL-1H channel exceeded the fluorescence level of 99.9% of the empty vector control cells.
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Six day-old MCF-7-ZsGreen-cODC mammospheres were transferred on glass slides by centrifugation in a cytocentrifuge (Cytospin, Shandon Elliot, London, UK) and fixed with 4% formaldehyde. The cells were incubated in blocking buffer (2% Bovine Serum Albumin (BSA), in phosphate buffer saline (PBS)) and antibodies (anti-CD44 APC-conjugated antibody (BD Pharmingen) and anti-CD24 PE-conjugated antibody (BD Pharmingen)) were added for one hour at room temperature. Hoechst 33342 (5 μg/mL, Invitrogen) solution was added for nuclear staining. The slides were visualized with an Olympus IX71 inverted fluorescent microscope. Grayscale images were merged using the ImageJ software  and displayed in false colors.
Sphere forming capacity
After irradiation, cells were trypsinized and plated in selection media (pheno-red free DMEM-F12, 0.4% BSA (Sigma), 10 ml of B27 supplement (Invitrogen)/500 ml of media, 5 μg/ml bovine insulin (Sigma), 4 μg/ml heparin (Sigma), 20 ng/ml fibroblast growth factor 2 (bFGF, Sigma) and 20 ng/ml epidermal growth factor (EGF, Sigma)) into 96-well plates, ranging from 1 to 256 cells/well. Growth factors, EGF, bFGF and heparin, were added every three days, and the cells were allowed to form spheres for 20 days. The number of spheres formed per well was then counted and expressed as a percentage of the initial number of cells plated. Cells were also plated in selection media into 100 mm suspension dishes at 10,000 cells/ml, and allowed to form spheres for 15 days, these cells were used for secondary sphere forming experiments. The same protocol was used after the secondary sphere forming assays, in order to plate cells for analyzing the formation of tertiary and quaternary spheres. Three independent experiments were performed.
Determining the number of cell divisions
We used the red fluorescent lipophilic dye PKH26 for proliferation tracking. Cells were trypsinized to obtain a single cell suspension and stained with the membrane-labeling dye PKH26 according to manufacturer's protocol (Sigma Aldrich). Briefly, cells were washed twice with PBS to remove exogenous proteins. Immediately before staining, cells were dispersed in 1 ml of Diluent C (an iso-osmotic, salt-free staining vehicle) at a concentration twice the desired final staining concentration (20 × 106 cells/mL). In parallel, a 2× solution of the PKH26 dye was prepared by diluting 4 μl of the ethanolic dye stock solution (4 μM) into 1 ml of Diluent C in a separate tube. Rapidly dispensing and admixing an equal volume of 2× cell solution into the 2× dye solution initiated the staining. After two to three minutes at room temperature, the staining was stopped by adding an equal volume of fetal bovine serum (FBS). Stained cells were then washed twice in medium, counted, and plated at 200,000 cells per 100 mm culture dish. Homogeneous staining between CICs and non-tumorigenic cells was immediately verified by flow cytometry (FL-2 Channel) (Additional file 3), and the mean FL-2 fluorescence at this time was considered as time 0 (Mean FL-2(t0)). Forty-eight hours after the last fraction of radiation, cells were trypsinized, and the remaining PKH26 staining was analyzed by flow cytometry [Mean FL-2(tsample)]. The number of cell divisions was calculated using the following formula:
Additional file 3. Figure S3. Control gate of cell membrane PHK26 staining efficiency (Figure 5). After cell membrane staining with PKH26 fluorescence was analyzed in the total population (left panel), non-tumorigenic cells (middle panel), and CICs (right panel). No difference was found for PKH26 efficiency staining between non-tumorigenic cells and CICs.
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Cell cycle and G0 phase analysis
After fractionated radiation, cells were stained according to Darzynkiewicz et al. . Briefly, cells were trypsinized and rinsed with HBSS/1 mM HEPES/10% FBS. DNA was stained with 1 μg/ml Hoechst 33342 in HBSS/1 mM HEPES/10% FBS solution/50 μM Verapamil, 45 minutes at 37°C, followed by RNA staining with 3.3 μM pyronin Y (PY) for 45 minutes at 37°C. Finally, cells were rinsed with HBSS/1 mM HEPES/10% FBS solution, and resuspended in PBS. At least 100,000 cells were analyzed by flow cytometry. ZsGreen-cODC expression was analyzed in FL-1H, PY in FL-2H and Hoechst 33342 in FL-5A.
β-galactosidase staining for cellular senescence
β-galactosidase-positive cells were detected by the method of Dimri et al. . Briefly, cells were washed two times with PBS and then fixed with PBS/2% formaldehyde/0.2% glutaraldehyde solution for five minutes. Cells were then washed again two times with PBS. After the last wash, staining solution was added (1 mg/ml 5-bromo-4-chloro-3-inolyl-β-D-galactoside (X-gal) (20 mg/ml stock, in dimethyformamide), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2) and cells were incubated in a 37°C for 18 h. After incubation, cells were washed two times with PBS and visualized with an Olympus IX71 inverted fluorescent microscope. The percentage of β-galactosidase-positive cells was counted in 10 random fields at 20× magnification.
All data are represented as means +/- standard error means (SEMs). A P-value of ≤ 0.05 in a paired two-sided Student's t-test was considered to indicate statistically significant differences.