Ablation of Breast Cancer Stem
Cells with Radiation1
Steven P. Zielske*,†, Aaron C. Spalding*,2,
Max S. Wicha‡and Theodore S. Lawrence*
*Department of Radiation Oncology, University of
Michigan, Ann Arbor, MI, USA;†Department of Radiation
Oncology, Wayne State University, Detroit, MI, USA;
‡Department of Internal Medicine, University of Michigan,
Ann Arbor, MI, USA
Tumorradioresistanceleadstorecurrenceafterradiation therapy.Theradioresistant phenotypehasbeenhypothesized
to reside in the cancer stem cell (CSC) component of breast and other tumors and is considered to be an inherent
property of CSC. In this study, we assessed the radiation resistance of breast CSCs using early passaged, patient-
derived xenografts from two separate patients. We found a patient-derived tumor in which the CSC population
was rapidly depleted 2 weeks after treatment with radiation, based on CD44+CD24−lin−phenotype and aldehyde
dehydrogenase 1 immunofluorescence, suggesting sensitivity to radiotherapy. The reduction in CSCs according to
phenotypic markers was accompanied by a decrease in functional CSC activity measured by tumor sphere frequency
and the ability to form tumors in mice. In contrast, another patient tumor sample displayed enrichment of CSC after
irradiation, signifying radioresistance, in agreement with others. CSC response to radiation did not correlate with the
level of reactive oxygen species in CSC versus non-CSC. These findings demonstrate that not all breast tumor CSCs
are radioresistant and suggest a mechanism for the observed variability in breast cancer local recurrence.
Translational Oncology (2011) 4, 227–233
Radiation therapy is a mainstay of breast cancer treatment. Radiation
therapy given after surgery in early stage breast cancer patients has
been shown to significantly increase the probability of both local
control and survival . Postmastectomy irradiation in locally ad-
vanced breast cancer similarly improves local control and survival be-
yond both chemotherapy and antihormonal therapy [2–4]. However,
tumors of a subset of patient recur locally despite best efforts. The
reason why residual tumor cells escape eradication by radiation is un-
clear but may partially be due to intrinsic radioresistance of cancer
stem cells (CSCs).
The CSC hypothesis is based on the observation that a small sub-
set of cells obtained from a tumor (cancer stem cells) are preferentially
capable of generating tumors in mouse models [5,6]. As stem cells,
they are defined as being able to self-renew and are the origin of other
cancer cells that contribute to the mass of the tumor. CSCs were first
including breast, pancreas, colon, glioblastoma, and others [7–14].
They are responsible for maintaining the tumor and have been hypoth-
esized to lead the invasive front of the tumor and contribute to meta-
Resistance to radiation and chemotherapy has been reported to be
a defining characteristic of CSCs from various tumor types, including
glioma, breast, and colon cancers [15–20]. Diehn et al.  report
breast CSCs harbor lower levels of reactive oxygen species than the
non–stem cell component, and this contributes to radioresistance of
breast CSCs. A stem cell–like population of the MCF7 breast cancer
cell line has been shown to be more resistant to radiation than the rest
of thepopulation.Breasttumors areenrichedwithCD44+CD24−
CSCs in neoadjuvant chemotherapy–treated patients . However,
Address all correspondence to: Steven P. Zielske, PhD, Department of Radiation
Oncology, 540 E. Canfield, Wayne State University, Detroit, MI 48201.
1S. Zielske was supported by a LUNGevity Foundation – American Cancer Society
Postdoctoral Fellowship in Lung Cancer and the Elsa Pardee Foundation. This work
was supported by the Flow Cytometry and Histology Core facilities of the UM Com-
prehensive Cancer Center.
2Current address: The Norton Cancer Institute Radiation Center and The Brain Tu-
mor Center, Norton Healthcare, Louisville, KY.
Received 8 October 2010; Revised 2 May 2011; Accepted 4 May 2011
Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1944-7124/11/$25.00
Volume 4 Number 4August 2011pp. 227–233
others have shown that CD44+CD24−breast CSCs are reduced in
neoadjuvant chemotherapy–treated patients , and we have found
a similar decrease in cyclophosphamide-treated xenografts .
The question of whether CSCs are radiation resistant or sensitive is
important given radiation’s effectiveness in reducing local recurrence
and improving survival. In our investigation, we find a patient-
derived tumor that displays radiosensitive CSCs, in contrast to the
expected radioresistance we and others define in other tumor sam-
ples. These data are based on the phenotypic and functional analysis
of the CSC fraction in irradiated tumor xenografts. Our data suggest
that breast CSCs are not uniform in their response to radiation, and
this may account for differential chances of recurrence after radia-
Tumors and Mice
MC1 and UM2 cells have been previously described [8,25]. MC1
cells were derived from a pleural effusion and are estrogen and proges-
terone receptor negative and HER-2−. UM2 cells were derived
from an ovarian metastasis and are estrogen and progesterone receptor
positive and HER-2−. Both lines were maintained exclusively as
xenografts in NOD.CB17-Prkdcscid/J (NOD/SCID) mouse (Jackson
Laboratory, Bar Harbor, ME) mammary fat pads. Samples used in
these experiments were less than 10 in vivo passages removed from
Tumors were produced in the mammary fat pad of NOD/SCID
mice by injecting 5 × 105cells, or numbers as indicated, in a 1:1 so-
lution of Matrigel (BD Biosciences, San Jose, CA) and serum-free
Dulbecco modified Eagle medium. Single-cell suspensions of tumors
were made by mincing the tumor and incubating in 300 U/ml colla-
genase, 100 U/ml hyaluronidase (Stem Cell Technologies, Vancouver,
Canada) in medium 199 for 15 minutes at 37°C, followed by trit-
urating through a 16-gauge needle/syringe. The digestion was stopped
by addition of fetal bovine serum (FBS) to 5% volume, cells were
filtered through a 100-μm cell strainer (BD Biosciences) and cen-
trifuged, and the pellet was resuspended with Hanks balanced salt
solution and 5% FBS and passed a second time through a 100-μm cell
strainer. Cells were pelleted and then resuspended in 15% dimethyl
sulfoxide in FBS for storage in liquid nitrogen unless analyzed imme-
diately. Tumor volume was calculated using the equation (π/6)ab2,
where a = the long dimension and b = the short dimension of
the tumor. All animal experiments were performed in accordance
with University Committee on Use and Care of Animals principles
Cells and tumors were irradiated with a Philips 250 orthovoltage
unit at approximately 2.5 Gy/min in the Irradiation Core of the Uni-
versity of Michigan Cancer Center. Dosimetry was carried out using
an ionization chamber connected to an electrometer system, which is
directly traceable to a National Institute of Standards and Technol-
ogy calibration. Mice were either placed in a Lucite restrainer or anes-
thetized with ketamine/xylazine and positioned such that the apex
of each tumor is at the center of a 2.4-cm aperture in the secondary
collimator and irradiated with the rest of the mouse being shielded
For analysis of the CSC phenotype based on CD44 and CD24,
106unfixed cells were washed and resuspended in 100 μl of PBS and
2% bovine serum albumin (BSA). We added fluorescently labeled anti-
CD44 and CD24 antibodies together with anti–H-2kdantibody and
a lineage cocktail composed of anti-CD3, CD10, CD16, CD18, and
CD140b antibodies (BD Biosciences); the cells were incubated at 4°C
for 15 minutes; and then the cells were washed with PBS, 2% BSA
before resuspending in PBS, 2% BSA, and 0.25 μg/ml propidium io-
dide (PI). PI+cells (dead cells) were gated out before analysis. Samples
for analysis were run on a BD Biosciences FACSCalibur instrument,
and data were analyzed using FCS Express software (De Novo Software,
Los Angeles, CA). Cell sorting was done on a BD Biosciences FACSAria
or FACSVantage SE instrument.
The ALDEFLUOR assay was performed according to the kit
manufacturer’s instructions and as previously described  (Stem
Eight-micrometer-thick sections were cut from formalin-fixed tu-
mors and deparaffinized, and the aldehyde dehydrogenase 1 (ALDH1)
epitope was unmasked by incubating in citrate buffer at 95°C for
20 minutes. Sections were blocked with Tris-buffered saline, 1% BSA,
10% normal goat serum for 1.5 hours before incubating in 1:100 anti-
ALDH1 antibody (BD Biosciences) overnight at 4°C. Washed slides
were then incubated in 1:1000 AlexaFluor-488–conjugated secondary
antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature.
Washed slides were mounted with ProLong Gold antifade reagent with
4′,6-diamidino-2-phenylindole dihydrochloride (Invitrogen) for stain-
Reactive Oxygen Species
Reactive oxygen species (ROS) were detected in cells using 5-
(and 6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate (H2DFFDA;
Molecular Probes, Eugene, OR). A single-cell suspension of MC1 or
UM2 cells was incubated in 10 nM H2DFFDA for 30 minutes at
37°C. Cells were then washed in 2% BSA and PBS and labeled with
antibodies for the detection of CD44+CD24−CSC as previously men-
were set based on the negative control (incubation without H2DFFDA).
A positive control was produced by incubating cells in 100 μM H2O2
Tumor Sphere Formation
Tumor spheres were grown on ultra-low-attachment six-well
plates at 2 × 104and 1 × 105cells per well in 1:1 Dulbecco modified
Eagle medium–F-12 (Hyclone, Logan, UT) containing 5% FBS, 2 mM
glutamine, 4 μg/ml heparin (Stem Cell Technologies), 20 ng/ml epider-
mal growth factor (R&D Systems, Minneapolis, MN), 20 ng/ml basic
fibroblast growth factor (R&D Systems), and B-27 (Invitrogen). Cells
Error bars and P values were generated using GraphPad Prism 5
software (GraphPad Software for Science, Inc, San Diego, CA). Error
bars represent SEM. Two-tailed Student’s t test was used for P value
calculations unless otherwise noted.
Radiation Sensitivity of Breast Cancer Stem CellsZielske et al. Translational Oncology Vol. 4, No. 4, 2011
We assessed the effect of radiation on the content of CSC and non-
CSCpopulations oftwopatient-derivedbreasttumors (MC1andUM2)
in an in vivo model with the hypothesis that radioresistant CSC would
be enriched by radiation, whereas radiosensitive CSC would be de-
pleted. Breast CSCs have been shown to be enriched in the CD44+
CD24−lin−population of a tumor, according to Al-Hajj et al. ,
and more recently, Ginestier et al.  have used the enzymatic-based
ALDEFLUOR assay to identify CSCs with ALDH enzyme activity.
Depletion of CSCs in Breast Xenografts by Radiation
MC1 and UM2 breast tumor xenografts were given 8 Gy as a single
dose to elicit a response that would result in decreased tumor volume
but was not expected to be a curative dose. Irradiated tumors were
removed 2 weeks after treatment for analysis. In UM2 tumors, we
found an increase in the proportion of CD44+CD24−lin−cells, from
2.6% ± 0.8% in untreated controls to 11% ± 3% in irradiated tumors
(P < .05; Figure 1A). These data suggest UM2 CSCs are relatively
resistant to radiation compared with the bulk population of cells, in
agreement with other data on breast CSC [21,22,26].
We next analyzed MC1 tumors for CSC content after radiation.
We found a rapid and progressive decrease in the proportion of CSCs
in irradiated MC1 tumors (Figure 1B). Control MC1 tumors had an
average of 2.5% ± 0.7% CD44+CD24−lin−. Two weeks after 8-Gy
treatment, the proportion of CD44+CD24−lin−cells dropped to
0.31% ± 0.14% (P < .05; Figure 1B). The loss of cells was progres-
sive, with 0.84% ± 0.14% (P < .05, analysis of variance) CD44+
CD24−lin−present in MC1 tumors 1 day after radiation (not
shown). There was also a decrease in ALDEFLUOR-positive MC1
cells (not shown). These data suggest MC1 tumor CSC are sensitive
to radiation compared with the non-CSC population.
Flow cytometry results were confirmed by using immunofluores-
cence on histologic sections to detect ALDH1, one enzyme active in
the ALDEFLUOR assay . ALDH1 was detected in untreated con-
trol tumors as widely distributed, with no discernable histologic pattern
(Figure 2). On treatment with radiation, the number of ALDH1+cells
in UM2 tumors increased, in agreement with flow cytometry results
showing enrichment of the CSC population. In contrast, the number
of ALDH1+cells in MC1 tumors was substantially decreased 2 weeks
These data suggest that breast CSCs derived from MC1 are sensi-
tive to the effects of radiation compared with the non-CSC popula-
tion. Radiation caused preferential loss of CSCs according to surface
phenotype, ALDH activity, and ALDH immunofluorescent staining.
In contrast, CSCs in UM2 cells were enriched by treatment with ra-
diation and thus radiation resistant compared with non-CSCs.
Functional CSC Activity in Irradiated Tumors
Only a subset of marker-positive cells have the capability to pro-
duce tumors in mice; therefore, the discordance in phenotypic mark-
ers after irradiation does not necessarily mean that there was a
functional decrease in tumor-initiating activity. One measure of func-
tional CSC activity is by the ability of cells to form tumor spheres
in vitro. Mammary tumor spheres retain tumorigenic potential and
Figure 1. Effect of radiation on CSCs. MC1 and UM2 xenografts were treated with radiation and the proportion of CSCs analyzed after
2 weeks using flow cytometry. (A) Flow cytometric detection of CD44+CD24−cells (upper left quadrant) in untreated and treated UM2
xenografts. (B) Flow cytometric detection of CD44+CD24−cells in untreated and treated MC1 xenografts. *P < .05 compared with
untreated, n = 2-15.
Translational Oncology Vol. 4, No. 4, 2011Radiation Sensitivity of Breast Cancer Stem CellsZielske et al.
maintain similarities to CSC . To determine whether there was a
loss of CSC activity in irradiated MC1 and UM2 tumors, we measured
the tumor sphere frequency.
Control UM2 tumors had a tumor sphere frequency of 8.5 ± 4.3 ×
10−5, which was increased 7-fold to 6.2 ± 1.2 × 10−4in irradiated
tumors (P < .01; Figure 3). In MC1 tumors, tumor sphere frequency
was reduced 12-fold to 4.3 ± 0.3 × 10−5after radiation compared
with 5.2 ± 1.0 × 10−4in control tumors (P < .01). Thus, radiation
caused a decrease in tumor sphere frequency in MC1 tumors, but an
increase in UM2 cells, in accordance with the effect seen on CSC
phenotypic markers in Figures 1 and 2.
We then chose further analysis of MC1 functional CSC activity in
a robust in vivo tumor initiation model to verify that loss of CSCs
occurred. To assess the functional state of tumor-initiating activity in
irradiated MC1 tumors compared with controls, we injected serial
dilutions of unsorted tumor cells from treated and untreated tumors
into mice. If stem cell activity is reduced in a treated tumor com-
pared with an untreated tumor, then the time required for tumor
formation should be delayed. Conversely, if treatment enriched for
stem cell activity, then recurrent tumors should appear sooner than
In mice injected with cells from radiation-treated tumors, median
time to formation of recurrent tumors was delayed up to 33 days
compared with controls (Figure 4A). In untreated controls, 100%
of mice (18/18, all cell doses combined) developed tumors within
45 days of cell injection. Several mice (2/4 and 2/6 from tumor IR
no. 1 and IR no. 2, respectively, at the 3 × 104cell dose) injected with
irradiated tumor–derived cells failed to produce tumors up to 100 days
after injection. The difference between the frequency of tumor forma-
tion between control and treated groups was significant (P < .05) at all
cell doses except IRno. 1 at3 × 104, whichshowed thesame trend(P =
.09). Differences in the time to grow a tumor were not due to injection
of nonviable cells because PI staining and flow cytometry revealed that
all samples were of equivalent viability, 85% to 92% (not shown).
These data not only show that radiation treatment resulted in a
decrease in marker-positive cells in tumors but also that this was
reflected as a decrease in functional CSC activity, providing confir-
mation that stem cells were lost to radiation treatment. Thus, radi-
ation was preferentially detrimental to MC1 CSCs compared with
Characterization of Recurrent Tumors
Recurrent MC1 tumors arising from injection of cells from
treated, primary tumors, were examined for abnormal growth rates
and whether the proportion of CSCs remained reduced or returned to
an equilibrium state similar to the original untreated, control tumors.
Measurement of tumor volume showed that the rate of growth of
recurrent tumors derived from treated primary tumors was equivalent
to those derived from untreated tumors (Figure 4B; P < .05). Thus,
once tumors were established, there was no defect in growth.
We then examined the CSC content of recurrent tumors from
treated primary tumors to determine whether they returned to a state
equivalent to untreated controls (Figure 5). The proportion of CD44+
CD24−lin−cells in IR no. 1 and IR no. 2 recurrent tumors was 4.9% ±
1.7% and 1.2% ± 0.3%, respectively. This is significantly increased
from the proportion of CSCs initially infused of 0.2% in IR no. 1
of CSCs in IR no. 1 and IR no. 2 was not different from the average
Figure 2. Immunofluorescent detection of ALDH1. Control and irradiated UM2 and MC1 tumor sections were stained for ALDH1 2 weeks
after treatment. ALDH1 staining is in green on the upper panels, and DAPI staining of nuclei is in blue on the lower panels. Irradiated
tumors displayed fewer ALDH1-stained cells than untreated tumors.
Figure 3. Tumor spheres in control and irradiated tumors. The fre-
quency of tumor sphere-forming cells in control or irradiated UM2
and MC1 xenografts was determined. *P < .01, n = 3-4.
Radiation Sensitivity of Breast Cancer Stem Cells Zielske et al.Translational Oncology Vol. 4, No. 4, 2011
number found in control tumors of 2.5% ± 0.7% (P > .05). Although
the proportion of CSCs in IR no. 2 trended lower, it was not signifi-
cant. Neither was there a significant difference in the proportion of
CSCs between any recurrent tumor group (P > .05). Control tumors
displayed 5.2% ± 2.2% CSCs, similar to the proportion injected of
4.2%. Taken together, these data show that recurrent tumors do not
have a growth defect and reestablish the baseline proportion of CSCs
found in untreated tumors.
Reactive Oxygen Species
One potential mechanism contributing to radiation resistance of CSC
is the levelof ROS in the cell. Low levels of ROS are associatedwith
increased expression of free radical scavengers and radiation resistance.
method to determine whether it was consistent with the radiation sensi-
tivity and resistance observed in MC1 and UM2 cells, respectively.
CSCs contained lower levels of ROS than non-CSCs in MC1 and
UM2 cells (P < .05; Figure 6). In MC1 cells, the ROS levels of the
CSC population were 59% the level of non-CSCs, whereas in UM2
cells, the ROS levels of the CSC population were proportionally
lower, at 34% the level of non-CSCs. There was no significant dif-
ference in ROS levels between MC1 and UM2 CSCs or between
MC1 and UM2 non-CSCs (P > .05). These data are consistent with
those of Diehn et al.  in that the CSC populations had lower ROS;
however, in our samples, there was no correlation between ROS levels
in vitro and relative radiation resistance determined in vivo.
In this study, we have found that breast cancers can contain either
sensitive or resistant CSCs relative to the bulk tumor population.
More specifically,early invivo passage MC1tumorscontainCSCswith
relative sensitivity to radiation, whereas UM2 xenografts displayed
Figure 4. Functional assay for CSC activity. (A) MC1 cells from control and two different irradiated tumors (2 weeks after irradiation) were
injected into the mammary fat pad of mice at the indicated cell quantities. The number of days required for tumor formation was re-
corded and plotted. (B) Measurement of MC1 tumor growth. There was no statistical difference between the growth rate of each group
(P < .05).
Figure 5. Analysis of CSC in recurrent tumors. Recurrent tumor
xenografts derived from control or two irradiated primary xenografts
(IR#1 and IR#2) were subjected to flow cytometric detection of
CD44+CD24−CSC. *P < .05 compared with control.
Figure 6. Reactive oxygen species in CSC. MC1 and UM2 cells
were stained with H2DFFDA to detect ROS levels by flow cytometry.
CSC had statistically lower ROS levels than non-CSC (P < .05, N =
2). (+)ctrl signifies positive controls treated with H2O2.
Translational Oncology Vol. 4, No. 4, 2011Radiation Sensitivity of Breast Cancer Stem CellsZielske et al.
radioresistant CSCs compared with the rest of the tumor. When MC1
xenografts were exposed to radiation, the proportion of CSCs based
on two phenotypic definitions (CD44+CD24−lin−flow cytometry
and ALDH1 immunofluorescence) preferentially decreased as early as
1 day after treatment and to a greater degree 2 weeks after treatment.
In contrast, CSCs in UM2 xenografts were preferentially enriched
2 weeks after radiation treatment. Importantly, the loss of CSCs
in MC1 xenografts was accompanied by a functional defect in the
ability of cells derived from treated tumors to produce tumor
spheres or recurrent tumors in secondary NOD/SCID mice. Thus,
the effect observed on phenotypic markers correlated with func-
tional activity. Recurrent MC1 tumors grew at a similar rate to con-
trols and reestablished baseline proportions of CSCs. ROS levels
were lower in CSC than in non-CSC, in agreement with Diehn
et al. , but the magnitude of difference was greater in the radio-
resistant sample (UM2).
There is a general perception that CSCs are inherently resistant to
radiation, extending the hypothesis that this is a general property of
cancer stem cells [17,22,26]. However, the data supporting this con-
clusion are limited. In a glioma xenograft model, radiation therapy
resulted in enrichment of CD133+glioma CSCs . Radiation re-
sistance was attributed to increased activity of the DNA damage
checkpoint response. Of note is that gliomas are clinically far more
resistant to radiation than breast cancer, so a difference between these
tumor types may not be surprising. In breast cancer, in vitro work
with the MCF-7 cell line has shown that radiation enriches for the
CD44+CD24−fraction of floating cells but not adherent cells .
Furthermore, MCF-7 mammospheres displayed greater survival and
less expression of γH2AX than adherent cultures exposed to radiation.
This important early study was limited to the breast cancer cell line,
MCF-7, and, to a lesser extent, MDA-MB-231, without explicit val-
idation that the cell phenotypes analyzed possessed cancer stem cell
activity, a question of continuing controversy in cell lines [19,29–32].
In addition, the behavior of cells in culture may be different from that of
a tumor . Similar findings were reported by Woodward et al. ,
using side population (SP cells) as a phenotypic definition of CSCs in
MCF-7 cells. Our analysis of UM2 xenografts extends theses studies by
showing enrichment of CSCs after in vivo irradiation using an early-
passage xenograft that has not been culture-adapted. MC1 xenografts,
however, supports the hypothesis that breast CSCs are not universally
radioresistant. Therefore, we feel our data do not contradict other find-
ings but may have produced different results because of a different cell
type and a more stringent model system.
The sensitivity of CSC to chemotherapy has also shown variability.
In one study, CD44+CD24−cells in HER2−tumors were enriched
during the course of therapy with docetaxel or doxorubicin plus cyclo-
phosphamide . However, CD44+CD24−cells in HER2+tumors
were decreased during treatment with lapatinib, an inhibitor of epi-
dermal growth factor receptor/HER2. We and others have found a
reduction in CD44+CD24−breast CSC after chemotherapy in labora-
tory and clinical analyses [23,24]. In a subset of glioblastoma tumors,
temozolomide treatment results in depletion of CSC, but in colon
cancer, chemotherapy enriches CSC [18,34]. Taken together, these
studies suggest that the relative resistance or sensitivity of CSCs to anti-
cancer therapy is a more complex question than originally thought.
The analysis of ROS levels indicates that CSCs contain lower
levels than non-CSCs do, suggesting increased an expression of free
radical scavengers that limit the impact of radiation damage. These
data suggest a possible contribution to the radiation response, but
other mechanisms are likely to have equal or greater impact. For
example, we also detected a difference in PCNA expression after
irradiation in UM2 versus MC1 cells (not shown) and cannot exclude
cell cycle as playing a role in the radiation response.
Elucidation of additional mechanisms for MC1 radiation sensitiv-
ity, as well as the frequency and extent of this phenomenon in the
patient population, is an important avenue of continued study that
could impact individualized therapies and new approaches aimed at
radiosensitization. The overall conclusion is that breast CSCs are not
universally radiation resistant but can respond uniquely to therapy,
and this should be a consideration in future work.
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