Article

Ablation of Breast Cancer Stem Cells with Radiation

Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA.
Translational oncology (Impact Factor: 2.88). 08/2011; 4(4):227-33. DOI: 10.1593/tlo.10247
Source: PubMed
ABSTRACT
Tumor radioresistance leads to recurrence after radiation therapy. The radioresistant phenotype has been hypothesized 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.

Full-text

Available from: Steven Zielske, Mar 21, 2014
Ablation of Breast Cancer Stem
Cells with Radiation
1
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
Abstract
Tumor radioresistance leads to recurrence after radiation therapy. The r adioresistant phenotype has been hypothesized
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, 227233
Introduction
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 [1]. Postmastectomy irradiation in locally ad-
vanced breast cancer similarly improves local control and survival be-
yond both chemotherapy and antihormonal therapy [24]. 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
discovered in acute myeloid leukemia and subsequently in solid tumors,
including breast, pancreas, colon, glioblast oma, and other s [714].
They are responsible for maintaining the tumor and have been hypoth-
esized to lead the invasive front of the tumor and contribute to meta-
static seeding.
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 [1520]. Diehn et al. [21] report
breast CSCs harbor lower levels of reactive oxygen species than the
nonstem cell component, and this contributes to radioresistance of
breast CSCs. A stem celllike population of the MCF7 breast cancer
cell line has been shown to be more resistant to radiation than the rest
of the population [22]. Breast tumors are enriched with CD44
+
CD24
CSCs in neoadjuvant chemotherapytreated patients [20]. However,
Address all correspondence to: Steven P. Zielske, PhD, De partment of Radia tion
Oncology, 540 E. Canfield, Wayne State University, Detroit, MI 48201.
E-mail: szielske@med.wayne.edu
1
S. 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.
2
Current 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
DOI 10.1593/tlo.10247
www.transonc.com
Translational Oncology
Volume 4 Number 4 August 2011 pp. 227233 227
Page 1
others have shown that CD44
+
CD24
breast CSCs are reduced in
neoadjuvant chemotherapytreated patients [23], and we have found
a similar decrease in cyclophosphamide-treated xenografts [24].
The question of whether CSCs are radiation resistant or sensitive is
important given radiations 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 tumorxenografts.Ourdatasuggest
that breast CSCs are not uniform in their response to radiation, and
this may account for differential chances of recurrence after radia-
tion therapy.
Methods
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
[25]. UM2 cells were derived
from an ovarian metastasis and are estrogen and progesterone receptor
positive and HER-2
[25]. Both lines were maintained exclusively as
xenografts in NOD.CB17-Prkdc
scid
/J (NOD/SCID) mouse (Jackson
Labo ratory, Bar Harbor, ME ) mammary fat pads. Samples used in
these experiments were less than 10 in vivo passages removed from
original derivation.
Tumors were produced in the mammary fat pad of NOD/SCID
mice by injecting 5 × 10
5
cells, 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 s trainer (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-μmcell
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)ab
2
,
where a = the long dimension and b = the short dimension of
the tumor. All animal experiments were perfor med in accordance
with University Committee on Use and Care of Animals principles
and guidelines.
Irradiation
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 Inst itute 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
from radiation.
Flow Cytometry
For analysis of the CSC phenotype based on CD44 and CD24,
10
6
unfixed 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 antiH-2k
d
antibody 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,
LosAngeles,CA).Cellsortingwas done on a BD Biosciences FACSAria
or FACSVantage SE instrument.
The ALDEFLUOR assay was performed according to the kit
manufacturers instructions and as previously described [25 ] (Stem
Cell Technologies).
Immunofluorescence
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-488conjugated 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-
ing nuclei.
Reactive Oxygen Species
Reactive oxygen species (ROS) were detected in cells using 5-
(and 6)-carboxy-2,7-difluorodihydrofluorescein diacetate (H
2
DFFDA;
Molecular Probes, Eugene, OR). A single-cell suspension of MC1 or
UM2 cells was incubated in 10 nM H
2
DFFDA 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-
tioned, excluding PI staining. Flow cytometry was performed, and gates
were set based on the negative control (incubation without H
2
DFFDA).
A positive control was produced by incubating cells in 100 μMH
2
O
2
and H
2
DFFDA.
Tumor Sphere Formation
Tumor spheres were grown on ultra-low-attachment six-well
plates at 2 × 10
4
and 1 × 10
5
cells per well in 1:1 Dulbecco modified
Eagle mediumF-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
were incubated for 2 weeks at 37°C in 5% CO
2
before counting resulting
tumor spheres.
Statistics
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 Students t test was used for P value
calculations unless otherwise noted.
228 Radiation Sensitivity of Breast Cancer Stem Cells Zielske et al. Translational Oncology Vol. 4, No. 4, 2011
Page 2
Results
We assessed the effect of radiation on the content of CSC and non-
CSC populations of two patient-derived breast tumors (MC1 and UM2)
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. [8],
and more recently, Ginestier et al. [25] 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; F igure 1A). These data suggest UM2 CSCs are r elatively
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 pr oportion of CD44
+
CD24
lin
cells dropped to
0.31% ± 0.14% (P < .05; Figure 1 B). The loss of cells was progres-
sive, with 0.84% ± 0.14% (P < .05 , analysis of vari ance ) 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 [27]. 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
after radiation.
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 nece ssarily 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, 2011 Radiation Sensitivity of Breast Cancer Stem Cells Zielske et al. 229
Page 3
maintain similarities to CSC [28]. 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
4
in irradiated
tumors (P < .01; Figure 3). In MC1 tumors, tumor sphere frequency
was reduced 12-fold to 4.3 ± 0.3 × 10
5
after radiation compared
with 5.2 ± 1.0 × 10
4
in 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 MC 1 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
untreated controls.
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 × 10
4
cell dose) injected with
irradiated tumorderived 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)atall
cell doses except IR no. 1 at 3 × 10
4
, which showed the same 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 ra diation 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
non-CSCs.
Characterization of Recurrent Tumors
Recurrent MC1 t umors 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 showe d 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 th e proportion of CSCs initially infused of 0.2% in IR no. 1
and 0.02% in IR no. 2 (Figure 5; P < .05). Furthermore, the proportion
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.
230 Radiation Sensitivity of Breast Cancer Stem Cells Zielske et al. Translational Oncology Vol. 4, No. 4, 2011
Page 4
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 level of ROS in the cell [21]. Low levels of ROS are associated with
increased expression of free radical scavengers and radiation resistance.
We measured basal ROS in MC1 and UM2 cells using a flow cytometric
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 p roportionally
lower, at 34% the level of non-CSCs. There was no significant dif-
ference in ROS levels between MC1 and UM 2 CSCs or be tween
MC1 and UM2 non-CSCs (P > .05). These data are consistent with
those of Diehn et al. [21] 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.
Discussion
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 in vivo passage MC1 tumors contain CSCs with
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 det ection of
CD44
+
CD24
CSC. *P < .05 compared with control.
Figure 6. Reactive oxygen species in CSC. MC 1 and UM2 cells
were stained with H
2
DFFDA 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 H
2
O
2
.
Translational Oncology Vol. 4, No. 4, 2011 Radiation Sensitivity of Breast Cancer Stem Cells Zielske et al. 231
Page 5
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 wee ks after treatment.
In contrast, CSCs in UM2 xe nografts were pref erentially enriched
2 weeks after radiation treatment. Importantly, the loss of CSCs
in MC1 xenografts was accompanied by a functional defect in the
ability o f cells derived from treated tumors to produce tumor
spheres or recurrent tumors in seconda ry NOD/SCI D 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 re established baseline proportions of CSCs. R OS levels
were lower in CSC than in non-CSC, in agreement with Diehn
et al. [21], 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-
clusi on are limited. In a glioma xenograft model, radiation therapy
resulted in enrichment of CD133
+
glioma CSCs [17]. Radiation re-
sistance was attributed to increased activity of the DNA damage
checkpoint response. Of note is that gli omas 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 [22].
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 [19,2932].
In addition, the behavior of cells in culture may be different from that of
a tumor [33]. Similar findings were reported by Woodward et al. [26],
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 [20]. 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 sugge st a possible contribution to the radiation response, but
other mechanisms are l ikely 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.
Acknowledgments
The authors thank G. Dontu, C. Ginestier, and J. Dutcher for help-
ful discussion and technical expertise.
References
[1] Zhao L, Wang L, Ji W, Wang X, Zhu X , Hayman JA, Kalemkerian GP,
Yang W, Brenner D, Lawrence TS, et al. (2009). Elevation of plasma TGF-β1
during radiation therapy predicts radiation-induced lung toxicity in patients with
nonsmall-cell lung cancer: a combined analysis from Beijing and Michigan. Int J
Radiat Oncol Biol Phys 74,13851390.
[2] Jiang X, Sun Y, Chen S, Roy K, and Price BD (2006). The FATC domains of
PIKK proteins are functionally equivalent and participate in the Tip60-dependent
activation of DNA-PKcs and ATM. JBiolChem281, 1574115746.
[3] Hamilton JP, Sato F, Greenwald BD, Suntharalingam M, Krasna MJ, Edelman
MJ, Doyle A, Berki AT, Abraham JM, Mori Y, et al. (2006). Promoter methyl-
ation and response to chemotherapy and radiation in esophageal cancer. Clin
Gastroenterol Hepatol 4, 701708.
[4] Woodward WA, Chen MS, Behbod F, and Rosen JM (2005). On mammary
stem cells. J Cell Sci 118, 35853594.
[5] Wicha MS, Liu S, and Dontu G (2006). Cancer stem cells: an old ideaa par-
adigm shift. Cancer Res 66, 18831890; discussion 1895 1886.
[6] Soltysova A, Altanerova V, and Altaner C (2005). Cancer stem cells. Neoplasma
52, 435440.
[7] Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J,
Minden M, Paterson B, Caligiuri MA, and Dick JE (1994). A cell initiating
human acute myeloid leukaemia after transplantation into SCID mice. Nature
367, 645648.
[8] Al-Hajj M, Wicha M S, Benito-Hernandez A, Morrison SJ, and Clarke MF
(2003). Prospective identification of tumorigenic breast cancer cells. Proc Natl
Acad Sci USA 100, 39833988.
[9] Galli R, Binda E, Orfanelli U, Cipel letti B, Gritti A, De Vitis S, Fiocco R,
Foroni C, Dimeco F, and Vescovi A (2004). Isolation and characterization of
tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res
64, 70117021.
[10] Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke
MF, and Simeone DM (2007). Identification of pancreatic cancer stem cells.
Cancer Res 67, 10301037.
[11] OBrien CA, Pollett A, Gallinger S, and Dick JE (2007). A human colon cancer
cell capable of initiating tumour growth in immunodeficient mice. Nature 445,
106110.
[12] Ricci-Vitiani L, Lombardi DG, Piloz zi E , Biffoni M, Todaro M, Peschle C,
and De Maria R (2007). Identification and expansion of human colon-cancer
initiating cells. Nature 445, 111115.
[13] Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, and Dirks
PB (2003). Identification of a cancer stem cell in human brain tumors. Cancer
Res 63, 58215828.
[14] Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman
RM, Cusimano MD, and Dirks PB (2004). Identification of human brain tu-
mour initiating cells. Nature 432, 396401.
[15] Ishii H, Iwatsuki M, Ieta K, Ohta D, Haraguchi N, Mimori K, and Mori M (2008).
Cancer stem cells and chemoradiation resistance. Cancer Sci 99,18711877.
[16] Eyler CE and Rich JN (2008). Survival of the fittest: cancer stem cells in ther-
apeutic resistance and angiogenesis. J Clin Oncol 26, 28392845.
232 Radiation Sensitivity of Breast Cancer Stem Cells Zielske et al. Translational Oncology Vol. 4, No. 4, 2011
Page 6
[17] BaoS,WuQ,McLendonRE,HaoY,ShiQ,HjelmelandAB,Dewhirst
MW, Bigner DD, and Rich JN (2006). Glioma stem cells promote radio-
resistance by preferential activation of the DNA damage response. Nature
444,756760.
[18] Dylla SJ, Beviglia L, Park IK, Chartier C, Raval J, Ngan L, Pickell K, Aguilar J,
Lazetic S, Smith-Berdan S, et al. (2008). Colorectal cancer stem cells are en-
riched in xenogeneic tumors following chemotherapy. PLoS One 3, e2428.
[19] Fillmore CM and Kuperwasser C (2008). Human breast cancer cell lines con-
tain stem-like cells that self-renew, give rise to phenotypically diverse progeny
and survive chemotherapy. Breast Cancer Res 10, R25.
[20] Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, Hilsenbeck SG,
Pavlick A, Zhang X, Chamness GC, et al. (20 08). Intrinsic resistance of
tumorigenic breast cancer cells to chemothe rapy. J Natl Cancer Inst 100,
672679.
[21] Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, Qian D, Lam
JS, Ailles LE, Wong M, et al. (2009). Association of reactive oxygen species
levels and radioresistance in cancer stem cells. Nature 458, 780783.
[22] Phillips TM, McBride WH, and Pajonk F (2006). The response of CD24(/
low)/CD44
+
breast cancerinitiating cells to radiation. J Natl Cancer Inst 98,
17771785.
[23] Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H,
Panula SP, Chiao E, et al. (2009). Downregulation of miRNA-200c links breast
cancer stem cells with normal stem cells. Cell 138,592603.
[24] Zielske SP, Spalding AC, and Law rence TS (2010). Loss of tumor-initiating
cell activity in cyclophosphamide-treated breast xenografts. Transl Oncol 3,
149152.
[25] Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M,
Jacquemier J, Viens P, Kleer CG, Liu S, et al. (2007). ALDH1 is a marker of
normal and malignant human mammary stem cells and a predictor of poor clin-
ical outcome. Cell Stem Cell 1, 555567.
[26] Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, and Rosen
JM (2007). WNT/β-catenin mediates radiation resistance of mouse mammary
progenitor cells. Proc Natl Acad Sci USA 104, 618623.
[27] Armstrong L, Stojkovic M, Dimmick I, Ahmad S, Stojkovic P, Hole N, and
Lako M (2004). Phenotypic characterization of murine primitive hematopoietic
progenitor cells isolated on basis of aldehyde dehydrogenase activity. Stem Cells
22, 11421151.
[28] Liu JC, Deng T, Lehal RS, Kim J, and Zacksenhaus E (2007). Identification of
tumorsphere- and tumor-initiating cells in HER2/Neu-induced mammary tu-
mors. Cancer Res 67, 86718681.
[29] Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, Hur
MH, Diebel ME, Monville F, Dutcher J, et al. (2009). Breast cancer cell lines
contain functional cancer stem cells with metastatic capacity and a distinct mo-
lecular signature. Cancer Res 69, 13021313.
[30] Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, and Tang
DG (2005). Side population is enriched in tumorigenic, stem-like cancer cells,
whereas ABCG2
+
and ABCG2 cancer cells are similarly tumorigenic. Cancer
Res 65, 62076219.
[31] Pries R, Witrkopf N, Trenkle T, Nitsch SM, and Wollenberg B (2008). Poten-
tial stem cell marker CD44 is constitutively expressed in permanent cell lines of
head and neck cancer. In Vivo 22,8992.
[32] Kondo T, Setoguchi T, and Taga T (2004). Persistence of a small subpopula-
tion of cancer stemlike cells in the C6 glioma cell line. Proc Natl Acad Sci USA
101, 781786.
[33] Vargo-Gogola T and Rosen JM (2007). Modelling breast cancer: one size does
not fit all. Nat Rev Cancer 7, 659672.
[34] Beier D, Rohrl S, Pillai DR, Schwarz S, Kunz-Schughart LA, Leukel P ,
Proescholdt M, Brawanski A, Bogdahn U, Trampe-Kieslich A, et al. (2008).
Temozolomide preferentially depletes cancer stem cells in glioblastoma. Cancer
Res 68, 57065715.
Translational Oncology Vol. 4, No. 4, 2011 Radiation Sensitivity of Breast Cancer Stem Cells Zielske et al. 233
Page 7
  • Source
    • "CSCs from one patients were rapidly depleted 2 weeks after treatment with radiation, resulting in a significant decrease in tumor sphere frequency and tumorigenic capacity. In contrast, CSCs from the other patient showed enrichment after radiation and resistance to therapy, suggesting that CSC variance may exist in individual patients [56] . Therefore, therapeutics that target different CSC subtypes is likely required. "
    [Show abstract] [Hide abstract] ABSTRACT: In recent years, it has become increasingly apparent that noncoding RNAs (ncRNA) are of crucial importance for human cancer. The functional relevance of ncRNAs is particularly evident for microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). miRNAs are endogenously expressed small RNA sequences that act as post-transcriptional regulators of gene expression and have been extensively studied for their roles in cancers, whereas lncRNAs are emerging as important players in the cancer paradigm in recent years. These noncoding genes are often aberrantly expressed in a variety of human cancers. However, the biological functions of most ncRNAs remain largely unknown. Recently, evidence has begun to accumulate describing how ncRNAs are dysregulated in cancer and cancer stem cells, a subset of cancer cells harboring self-renewal and differentiation capacities. These studies provide insight into the functional roles that ncRNAs play in tumor initiation, progression, and resistance to therapies, and they suggest ncRNAs as attractive therapeutic targets and potentially useful diagnostic tools.
    Full-text · Article · Nov 2013 · Ai zheng = Aizheng = Chinese journal of cancer
  • Source
    • "Consistent with this finding, knockdown of CD44 expression has been found to induce breast CSCs to differentiate into regular tumor cells without the capacity to self-renew (defined as non-CSCs) [13]. Nevertheless, in certain breast cancer patients, CD44+/CD24- cells are still sensitive to radiotherapy, suggesting that not all breast CSCs are radioresistant [14] and that not all CD44+/CD24- cells are CSCs. Indeed, clinical data have indicated that there is no significant correlation between CD44+/CD24-/low tumor cell prevalence and tumor progression. "
    [Show abstract] [Hide abstract] ABSTRACT: Identification of cancer stem cells (CSCs) and their behaviors will provide insightful information for the future control of human cancers. This study investigated CD44 and CD24 cell surface markers as breast cancer CSC markers in vitro and in vivo. Flow cytometry with CD44 and CD24 markers was used to sort breast cancer MCF7 cells for scanning electron microscopy (SEM), tumor cell invasion assay, and nude mouse xenograft assay. Flow cytometry assay using CD44 and CD24 markers sorted MCF7 cells into four subsets, i.e., CD44+/CD24-/low, CD44-/CD24+, CD44+/CD24+, and CD44-/CD24-. The SEM data showed that there were many protrusions on the surface of CD44+/CD24-/low cells. CD44+/CD24-/low cells had many microvilli and pseudopodia. The CD44+/CD24-/low cells had a higher migration and invasion abilities than that of the other three subsets of the cells. The in vivo tumor formation assay revealed that CD44+/CD24- cells had the highest tumorigenic capacity compared to the other three subsets. CD44 and CD24 could be useful markers for identification of breast CSCs because CD44+/CD24-/low cells had unique surface ultrastructures and the highest tumorigenicity and invasive abilities.
    Full-text · Article · Jun 2013 · Cancer Cell International
  • Source
    • "Furthermore, pancreatic cancer stem cells are sensitive to gemcitabine because we found that secondary tumor initiation is delayed in response to gemcitabine. Thus, although many studies have found that cancer stem cells are relatively resistant to therapy, the current study and our previous findings regarding the sensitivity of some breast cancer stem cells to radiation [42] suggest that it is premature to conclude that cancer stem cell resistance is universal. Although the combination of gemcitabine and AZD7762 reduced both the percentage of marker-positive cells and their tumor-initiating capacity, after tumor initiation, tumor growth rates were similar across treatment groups (Figure 3A). "
    [Show abstract] [Hide abstract] ABSTRACT: Checkpoint kinase 1 (Chk1) inhibition sensitizes pancreatic cancer cells and tumors to gemcitabine. We hypothesized that Chk1 inhibition would sensitize pancreatic cancer stem cells to gemcitabine. We tested this hypothesis by using two patient-derived xenograft models (designated J and F) and the pancreatic cancer stem cell markers CD24, CD44, and ESA. We determined the percentage of marker-positive cells and their tumor-initiating capacity (by limiting dilution assays) after treatment with gemcitabine and the Chk1 inhibitor, AZD7762. We found that marker-positive cells were significantly reduced by the combination of gemcitabine and AZD7762. In addition, secondary tumor initiation was significantly delayed in response to primary tumor treatment with gemcitabine + AZD7762 compared with control, gemcitabine, or AZD7762 alone. Furthermore, for the same number of stem cells implanted from gemcitabine- versus gemcitabine + AZD7762-treated primary tumors, secondary tumor initiation at 10 weeks was 83% versus 43%, respectively. We also found that pS345 Chk1, which is a measure of DNA damage, was induced in marker-positive cells but not in the marker-negative cells. These data demonstrate that Chk1 inhibition in combination with gemcitabine reduces both the percentage and the tumor-initiating capacity of pancreatic cancer stem cells. Furthermore, the finding that the Chk1-mediated DNA damage response was greater in stem cells than in non-stem cells suggests that Chk1 inhibition may selectively sensitize pancreatic cancer stem cells to gemcitabine, thus making Chk1 a potential therapeutic target for improving pancreatic cancer therapy.
    Full-text · Article · Jun 2012 · Neoplasia (New York, N.Y.)
Show more