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
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. , 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 . 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
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
cells in HER2
tumors were enriched
during the course of therapy with docetaxel or doxorubicin plus cyclo-
phosphamide . However, CD44
cells in HER2
were decreased during treatment with lapatinib, an inhibitor of epi-
dermal growth factor receptor/HER2. We and others have found a
reduction in CD44
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.
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