We began by asking whether low ROS concentrations that appear to be critical to self renewal of hematopoietic stem cells (HSCs)3, 5
are also a property of mammary epithelial stem cells9, 10
by isolating CD24med
mammary cells, a population enriched for mammary repopulating units (MRUs), and CD24high
progenitor cells by flow cytometry (Supplementary Fig. S1
) and measuring intracellular concentrations of prooxidants using 2′-7′-dichlorofluorescein diacetate (DCF-DA) staining3
. Cells in the MRU-enriched population contained significantly lower concentrations of ROS than the progenitor-enriched cells in two different strains of mice (). Specifically, the MRU-enriched populations displayed low to intermediate ROS levels, while the progenitor-enriched populations contained more uniformly high levels of ROS. Similarly, analysis of the two populations with MitoSOX Red, a highly selective detection method for mitochondrial superoxide, revealed lower superoxide levels in the MRU-enriched population (). In order to assess if mammary repopulating activity was related to intracellular concentrations of ROS, we transplanted CD24med
cells based on their levels of DCF-DA staining. Mammary stem cells with both low and intermediate ROS levels gave rise to epithelial outgrowths when transplanted into cleared fat pads (Supplementary Table 1
). Similar heterogeneity of ROS concentrations was recently demonstrated in HSC-enriched populations5, 11
, where it may have functional significance in modulating the HSC-niche interaction12
Analysis of ROS levels in normal mammary and breast cancer stem cells and their progeny
Given the conservation of low ROS levels in several types of normal tissue stem cells, we hypothesized that CSCs in some tumors may also contain lower concentrations of ROS than their non-tumorigenic progeny. In order to investigate ROS biology in human CSCs, we began by examining the expression of genes involved in ROS metabolism in primary human breast CSCs and NTCs. Using microarray data from human breast CSC-enriched populations and NTCs13
and a curated list of genes involved in ROS metabolism5
(see methods), Gene Set Enrichment Analysis (GSEA)14
revealed that the expression of ROS genes was highly overrepresented in the CD44+
breast CSC-enriched population compared to NTCs (p
<0.001; Supplementary Fig. S2
). The ROS genes identified as the core enriched genes by GSEA included a number of important antioxidant genes (Supplementary Table 2
). Thus, gene expression profiles of human breast CSC-containing populations suggest that they contain higher levels of antioxidant defense systems than NTCs.
Next, we directly assessed ROS levels in human tumor subpopulations. To do this the CD44+
breast CSC-enriched population and the corresponding “Not CD44+
NTC population were purified from surgically resected breast tumors (Supplementary Fig. S3
). DCF-DA staining revealed that the CSC-enriched population in the human breast tumors we examined contained significantly lower levels of prooxidants than the NTC population. In some breast tumors, the vast majority of cells in the CSC-containing fraction displayed a low ROS phenotype compared to NTCs () while in others it was restricted to a significant subset of CSCs (Supplementary Fig. S4
). We found a similar enrichment of cells with low ROS concentrations in a head and neck tumor (Supplementary Fig. S4
). Thus, CSC-enriched populations from some human tumors contain lower average intracellular ROS levels than corresponding NTC populations.
Recently, we have demonstrated that Thy1+
cells in the majority of spontaneously developing breast tumors from MMTV-Wnt-1
mice are highly enriched for tumorigenic activity15
(Supplementary Fig. S5a
). We therefore asked whether the CSC-enriched population in this model system also displays a low ROS phenotype. ROS analysis using DCF-DA revealed that in tumors in which the Thy1+
population was enriched for CSCs, this population contained a significantly higher fraction of cells with low prooxidant levels than the “Not Thy1+
non-tumorigenic population (). Thy1+
cells contained two main sub-populations of cells based on ROS concentration, with the low ROS subpopulation being significantly overrepresented compared to NTCs (Supplementary Fig. S5b
). In order to confirm the presence of CSCs within the low ROS subpopulation, we transplanted CSCs based on their DCF-DA staining and found that both the low and high ROS subsets of Thy1+
cells gave rise to tumors in recipient animals (Supplementary Table 3
). Thus, a subset of CSCs from these MMTV-Wnt-1
tumors displayed low baseline levels of ROS compared to NTCs.
It is well established that cell killing after exposure to ionizing radiation (IR) and a subset of cytotoxic chemotherapeutics is partially mediated by free radicals16
. Given our observations of increased expression of ROS defense genes in CSCs, we were therefore interested in testing whether CSC-enriched populations develop less DNA damage after IR than NTCs. In order to examine DNA damage immediately after irradiation, we purified Thy1+
cells and NTCs from MMTV-Wnt-1
tumors by flow cytometry and irradiated them on ice. Cells were then either left on ice or incubated at 37°C, before being analyzed using the alkaline comet assay17
. While untreated cells did not display significantly different levels of DNA damage, there were fewer DNA-strand breaks in the Thy1+
cells than NTCs immediately after exposure to IR (). These findings are consistent with the hypothesis that enhanced expression of ROS defenses in CSCs contributes to reduced levels of DNA damage after irradiation.
Thy1+CD24+Lin- CSC-enriched cells develop less DNA damage after irradiation than non-tumorigenic cells
Since the alkaline comet assay mainly measures single strand breaks and since double strand breaks are important for IR-induced lethality18
, we also analyzed levels of double strand breaks as reflected by phosphorylated histone 2AX (H2AX) nuclear foci after in vitro
irradiation. As with the comet assay, we again observed significantly lower levels of DNA damage in Thy1+
CSC-enriched cells than in NTCs (Supplementary Fig. S6
). We also measured phosphorylated H2AX foci after in vivo
irradiation of MMTV-Wnt-1
tumors, and again found that Thy1+
cells contained fewer foci than NTCs (). Thus, consistent with their lower baseline levels of ROS, Thy1+
CSC-enriched cells isolated from these tumors developed less DNA strand breaks than NTCs after exposure to IR.
Given these findings, CSCs would be expected to preferentially survive exposure to IR in intact tumors. Mice bearing MMTV-Wnt-1
tumors were therefore treated with short, fractionated courses of IR and the percentage of the Thy1+
CSC-enriched population before and after irradiation was analyzed using flow cytometry. On average, we found an approximately 2 fold increase in the percentage of the Thy1+
CSC-enriched population compared with “Not Thy1+
NTCs in the irradiated tumors, suggesting that CSCs are relatively radioresistant compared with NTCs (). We found a similar increase in the fraction of CD44+
CSC-enriched population when we irradiated human head and neck cancer xenografts grown in immunodeficient mice (). Other investigators have documented similar radioresistance of CSCs in brain tumors19
and a breast cancer cell line20
. Thus, CSCs in some murine and human tumors are relatively radioresistant compared to their NTC counterparts.
Enrichment of CSCs after in vivo irradiation
Since a significant fraction of murine breast CSCs contained relatively low levels of ROS, we hypothesized that these cells may express enhanced levels of ROS defenses compared to their NTC counterparts. We were particularly interested in glutathione (GSH), a critical cellular reducing agent and antioxidant which has been implicated in chemotherapy and radiotherapy resistance of cancer cells21
. Since our prior analyses revealed heterogeneity within CSC-enriched populations and since single cell gene expression studies have revealed significant variation in gene expression in other stem cell populations22
, we investigated expression of critical GSH biosynthesis genes in MMTV-Wnt-1
CSCs and NTCs using single cell qRT-PCR. This analysis revealed significant overexpression of Gclm
<0.001) and Gss
<0.005) in a large fraction of cells within the CSC-enriched population, the former of which encodes the regulatory subunit of the enzyme (glutamate-cysteine ligase) that catalyzes the rate limiting step of GSH synthesis ()23
. Furthermore, Foxo1
, a transcription factor implicated in the regulation of an anti-ROS gene expression program in HSCs5
, was also overexpressed in CSCs compared to NTCs (p
<0.001; ). Other genes, including Hif1a
, and Foxo4
were not differentially expressed. Thus, genes controlling GSH biosynthesis were overexpressed by many cells within the CSC-enriched population isolated from this tumor.
Thy1+CD24+Lin- cells overexpress genes involved in ROS scavenging and pharmacologic modulation of ROS levels affects the radiosensitivity of Thy1+CD24+Lin- and “Not Thy1+CD24+” Lin-cells
In order to pharmacologically manipulate ROS levels separately in CSCs and NTCs, we employed in vitro
culture conditions that allowed both cell populations to produce colonies upon co-culture with irradiated feeder cells. We found that Thy1+
CSC-enriched cells were relatively radioresistant compared with NTCs ( and Supplementary Fig. S7
). When exposed to 2 Gy, a dose commonly administered clinically during daily treatments of breast cancer patients, 2.0-fold +/- 0.2 more CSC-enriched colonies survived than NTC colonies. Next, we attempted to radioprotect NTCs by exposing them to the nitroxide antioxidant tempol24
. Pretreatment with tempol radioprotected NTCs, and resulted in survival levels similar to those seen in the CSC-enriched population ().
Given the overexpression of genes involved in GSH synthesis by CSCs, we wished to assess the sensitivity of these cells to ROS elevation via pharmacologic depletion of GSH. Exposure of Thy1+
CSC-enriched cells to Buthionine Sulfoximine (BSO), which inhibits glutamate-cysteine ligase25
, decreased their colony forming ability by approximately three fold (). Finally, we asked if GSH depletion would radiosensitize CSCs. As shown in , BSO pretreatment of Thy1+
CSC-enriched cells led to significant radiosensitization. These data demonstrate the importance of low ROS levels and antioxidant defenses to CSC survival and radiosensitivity in these tumors.
Our data indicate that normal breast stem cells and a subset of CSCs in some tumors arising in both mice and humans contain lower levels of ROS than their cellular descendants. Taken together with previous reports of low ROS concentrations in other normal tissue stem cells, these findings suggest that stem cells in diverse systems have conserved this attribute, which likely helps to protect their genomes from endogenous and exogenous ROS-mediated damage. The mechanism leading to low ROS levels in some CSCs appears to be at least partially due to the increased production of free radical scavengers. Notably, there appears to be significant heterogeneity of ROS levels in both normal stem cell and CSC-enriched populations, which could reflect that the enriched populations contain both stem and non-stem cells and/or that ROS levels within stem cells can differ based on environmental factors that alter the balance of endogenous production and the expression of scavenging pathways. The low ROS subset found in the various stem cell populations may also represent a quiescent subpopulation. Heterogeneity of ROS levels may influence the extent to which CSC-enriched populations are resistant to therapies such as ionizing radiation.
In light of recent findings that CSCs in glioblastoma multiforme display enhanced DNA repair capabilities19
, it appears that CSCs may resist standard cytotoxic therapies through a combination of mechanisms, and that these may be unique to a given tumor. In the case of human CSCs, the frequency with which these cells display low ROS levels or enhanced DNA repair remains to be determined, particularly since the normal transformation precursor may be either stem or progenitor cells26, 27
and since ROS concentration ( and 5, 28
) and increased DNA repair capabilities appear to partially reflect differentiation state. Clinical therapies could likely be optimized by patient- and tumor-specific identification of CSC-resistance mechanisms and overcoming low ROS levels within CSCs may be a useful method for improving local and systemic oncologic therapies.