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 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+
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 functional activity. Recurrent MC1 tumors grew at a similar rate to controls and reestablished baseline proportions of CSCs. ROS 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 radioresistant 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 conclusion are limited. In a glioma xenograft model, radiation therapy resulted in enrichment of CD133+
glioma CSCs [17
]. Radiation resistance 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+
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 validation 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 [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 findings 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 cyclophosphamide [20
]. However, CD44+
cells in HER2+
tumors were decreased during treatment with lapatinib, an inhibitor of epidermal growth factor receptor/HER2. We and others have found a reduction in CD44+
breast CSC after chemotherapy in laboratory 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 sensitivity, 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.