HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells

Significance Uncontrolled cell proliferation and abnormal blood vessel formation result in regions of breast cancers that are hypoxic (deprived of oxygen). Hypoxia-inducible factors (HIFs) stimulate the expression of genes that enable cancer cells to invade and metastasize, leading to patient mortality. In this paper, we report that HIFs stimulate the production of CD47, a protein on the cell surface that enables cancer cells to avoid destruction by macrophages. CD47 is also important for maintaining cancer stem cells, which are a small population of cells that are required for the formation of primary tumors and metastases. Reduction of HIF activity or CD47 levels in breast cancer cells led to increased killing by macrophages and depletion of cancer stem cells. Increased expression of CD47 has been reported to enable cancer cells to evade phagocytosis by macrophages and to promote the cancer stem cell phenotype, but the molecular mechanisms regulating CD47 expression have not been determined. Here we report that hypoxia-inducible factor 1 (HIF-1) directly activates transcription of the CD47 gene in hypoxic breast cancer cells. Knockdown of HIF activity or CD47 expression increased the phagocytosis of breast cancer cells by bone marrow-derived macrophages. CD47 expression was increased in mammosphere cultures, which are enriched for cancer stem cells, and CD47 deficiency led to cancer stem cell depletion. Analysis of datasets derived from thousands of patients with breast cancer revealed that CD47 expression was correlated with HIF target gene expression and with patient mortality. Thus, CD47 expression contributes to the lethal breast cancer phenotype that is mediated by HIF-1.

Increased expression of CD47 has been reported to enable cancer cells to evade phagocytosis by macrophages and to promote the cancer stem cell phenotype, but the molecular mechanisms regulating CD47 expression have not been determined. Here we report that hypoxia-inducible factor 1 (HIF-1) directly activates transcription of the CD47 gene in hypoxic breast cancer cells. Knockdown of HIF activity or CD47 expression increased the phagocytosis of breast cancer cells by bone marrow-derived macrophages. CD47 expression was increased in mammosphere cultures, which are enriched for cancer stem cells, and CD47 deficiency led to cancer stem cell depletion. Analysis of datasets derived from thousands of patients with breast cancer revealed that CD47 expression was correlated with HIF target gene expression and with patient mortality. Thus, CD47 expression contributes to the lethal breast cancer phenotype that is mediated by HIF-1.
antitumor immunity | immune evasion | tumor-initiating cells | tumor microenvironment | "don't eat me" signal T he pathogenesis of breast cancer reflects not only the consequence of somatic mutations that dysregulate oncogenes and tumor suppressor genes, but also the effect of the changing tumor microenvironment, particularly the development of intratumoral hypoxia. The median pO 2 within primary breast cancers is 10 mmHg (1.4% O 2 ) compared with 65 mmHg (9.3% O 2 ) in normal breast tissue (1). Exposure of breast cancer cells to reduced O 2 availability induces the activity of hypoxia-inducible factors (HIFs), which are heterodimeric transcriptional activators, consisting of an O 2 -regulated HIF-1α, HIF-2α, or HIF-3α subunit and a constitutively expressed HIF-1β subunit, that control the expression of hundreds of target genes (2). Increased expression of the HIF-1α subunit, detected by immunohistochemistry in primary breast cancer biopsies, is associated with a significantly increased risk of metastasis and mortality (2).
To give rise to a primary tumor, a tumor relapse, or a metastatic tumor, a breast cancer cell must possess two important characteristics: first, the cell must avoid destruction by the immune system and, second, the cell must possess stem-cell-like properties. Hypoxia induces the breast cancer stem cell (CSC) phenotype (19,20) through functional and physical interactions of HIF-1 with the coactivator TAZ (20,21) and by HIF-dependent expression of pluripotency factors (22). Hypoxia also induces immune evasion by several HIF-dependent mechanisms (23,24). A major mechanism by which cancer cells evade the innate immune system is by expression of CD47, which is a cell-surface protein that interacts with signal regulatory protein α (SIRPα) on the surface of macrophages to block phagocytosis (25,26). Expression of calreticulin (CRT) on the surface of cancer cells is the primary trigger for phagocytosis by binding to low-density lipoprotein-related protein (LRP) on the surface of macrophages (27). The prophagocytic signal triggered by CRT-LRP ligation is counterbalanced by the antiphagocytic signal triggered by CD47-SIRPα ligation (28). Analysis of circulating tumor cells isolated from the blood of patients with breast cancer revealed that CD47 expression identified a subpopulation of cells with the capability to generate tumor xenografts in mice (29). Here, we report that CD47 expression is induced in a HIF-dependent manner when human breast cancer cells are exposed to hypoxia. Modest inhibition of CD47 expression was sufficient to increase the phagocytosis of breast cancer cells and decrease the number of breast CSCs. Human breast cancer database analysis revealed that high CD47 expression is correlated with increased HIF target gene expression and decreased patient survival.

Significance
Uncontrolled cell proliferation and abnormal blood vessel formation result in regions of breast cancers that are hypoxic (deprived of oxygen). Hypoxia-inducible factors (HIFs) stimulate the expression of genes that enable cancer cells to invade and metastasize, leading to patient mortality. In this paper, we report that HIFs stimulate the production of CD47, a protein on the cell surface that enables cancer cells to avoid destruction by macrophages. CD47 is also important for maintaining cancer stem cells, which are a small population of cells that are required for the formation of primary tumors and metastases. Reduction of HIF activity or CD47 levels in breast cancer cells led to increased killing by macrophages and depletion of cancer stem cells.

Results
Hypoxia Induces Increased CD47 Expression in a HIF-Dependent Manner.
Because cell-surface expression of CD47 is required for its antiphagocytic capacity, flow cytometry was performed. MCF7-DKD and HCC1954-DKD subclones showed decreased CD47 cell-surface expression compared with the respective NTC subclones under both hypoxic and nonhypoxic (20% O 2 ) conditions ( Fig. 1 F and G). In HCC1954-NTC and SUM159 cells, hypoxia increased CD47 cell-surface expression (Fig. 1H). It was not possible to perform flow cytometry on acriflavine-treated cells due to the inherent fluorescence of the drug. Taken together, the data in Fig. 1 demonstrate that CD47 mRNA and protein expression are induced by hypoxia in three breast cancer cell lines in a HIF-dependent manner, leading to increased cell-surface expression of CD47 in HCC1954 and SUM159 cells.
To determine whether DNA sequences encompassing either site alone functioned as a hypoxia response element (HRE), a 55-bp oligonucleotide spanning HIF binding site 1 or site 2 ( Fig.  2A, Bottom) was inserted into pGL2-promoter, which contains a basal SV40 promoter driving expression of firefly luciferase, to generate reporters pGL2-HRE1 and pGL2-HRE2, respectively. As a negative control, we used pSV-Renilla, which encodes Renilla luciferase driven by the SV40 promoter alone. Hypoxia significantly increased the ratio of firefly:Renilla luciferase in HCC1954 and SUM159 cells that were cotransfected with either pGL2-HRE1 or pGL2-HRE2 and pSV-Renilla (Fig. 2C), demonstrating that each of the 55-bp oligonucleotides functions as an HRE. Taken together, the data presented in Fig. 2 indicate that HIF-1 binds directly to the CD47 promoter to activate gene transcription. , and SUM159 (Right) cells were exposed to 20% or 1% O 2 for 16 h and chromatin immunoprecipitation (ChIP) assays were performed using IgG or antibodies against HIF-1α or HIF-1β. Primers flanking the entire sequence shown in A were used for qPCR and results were normalized to IgG at 20% O 2 (mean ± SEM; n = 3). *P < 0.05, **P < 0.01 vs. 20% O 2 (Student's t test). (C) Cells were cotransfected with pSV-Renilla and firefly luciferase reporter pGL2-HRE1 or pGL2-HRE2, containing an oligonucleotide encompassing HIF binding site 1 or site 2, respectively, and exposed to 20% or 1% O 2 for 24 h. Luciferase activity (firely:Renilla luciferase ratio) was determined and normalized to 20% O 2 (mean ± SEM; n = 3). **P < 0.01 vs. 20% O 2 (Student's t test).
HIF Deficiency Increases the Phagocytosis of Breast Cancer Cells. We observed decreased expression of CD47 on the surface of HIFdeficient human breast cancer cells (Fig. 1 F and Fig. 3A). Phagocytosis was significantly increased in MCF7-DKD and HCC1954-DKD cells compared with the respective NTC subclone, under both hypoxic and nonhypoxic conditions (Fig. 3 B and C). Hypoxia modestly increased phagocytosis of both NTC and DKD subclones, which suggests the existence of a prophagocytic signal that is induced by hypoxia in a HIF-independent manner.
CD47 Deficiency Increases the Phagocytosis of Breast Cancer Cells. To determine whether CD47 mediates protection of breast cancer cells against phagocytosis, we transfected SUM159 cells with an expression vector encoding one of five different shRNAs targeting CD47. Remarkably, we were not able to establish stable subclones that expressed three of the shRNAs and expression of the other two shRNAs (shCD47-2 and shCD47-4) caused relatively modest (50-75%) inhibition of CD47 mRNA (Fig. 4A) and cell-surface protein (Fig. 4B) expression. However, phagocytosis was significantly increased in the CD47-knockdown subclones (Fig. 4C) and CD47 levels were inversely correlated with the extent of phagocytosis. Taken together, the data presented in Fig. 4 demonstrate that CD47 expression protects breast cancer cells against phagocytosis by bone marrow-derived macrophages.
CD47 Promotes the Breast CSC Phenotype. CD47 is preferentially expressed on bladder, liver, and pancreatic CSCs compared with the bulk cancer cells (non-CSCs) (33)(34)(35)(36). To investigate whether CD47 plays a role in breast CSCs, we first analyzed CD47 mRNA levels in SUM159 cells, which were cultured as standard adherent monolayers or as nonadherent spheroids (mammospheres), which are highly enriched for CSCs (37). CD47 mRNA levels were twofold higher in mammosphere cultures (Fig. 5A). Exposure of breast cancer cells to hypoxia for 3 d induces increased mammosphere formation in a HIF-1α-dependent manner (20). Knockdown of CD47 expression significantly decreased mammosphere formation, with the greatest reduction observed in the shCD47-4 subclone (Fig. 5B), which had the greatest inhibition of CD47 expression ( Fig. 4 A and B). Analysis of aldehyde dehydrogenase (ALDH) activity in breast cancer cells also identifies a subpopulation that is enriched for CSCs (38) and the percentage of ALDH + cells increases in response to hypoxia (19,20). Hypoxia markedly increased the percentage of ALDH + cells in the NTC subclone, whereas knockdown of CD47 expression significantly decreased the percentage of ALDH + CSCs at both 20% and 1% O 2 (Fig. 5C). Taken together, the data presented in Fig. 5 demonstrate that CD47 expression plays an important role in promoting the breast CSC phenotype. We and others have previously reported that knockdown of HIF-1α blocks hypoxiainduced enrichment of breast CSCs, as determined by ALDH expression or mammosphere formation assay (19,20). Thus, CD47 loss-of-function phenocopies HIF-1α loss of function with respect to CSC maintenance.

CD47 Expression Is Associated with HIF Target Gene Expression and
Patient Mortality. To test whether the results obtained from the analysis of breast cancer cell lines are relevant to patients with breast cancer, we analyzed gene expression data from 1,040 primary human breast cancer samples that are publicly available in The Cancer Genome Atlas (TCGA) database (39). To determine whether HIFs regulate CD47 gene expression in human breast cancers, we compared CD47 mRNA levels with the levels of P4HA1, P4HA2, ANGPTL4, SLC2A1, CXCR3, VEGFA, PLOD1, PLOD2, L1CAM, and MET mRNA, which are all products of genes that are HIF regulated in breast cancer cells (18,20). CD47 mRNA levels, like those of other HIF target genes, were most highly expressed in breast cancers of the basal-like molecular subtype (Fig. 6A). Statistical analysis revealed that CD47 mRNA levels were significantly correlated with the levels of 8 of 10 mRNAs encoded by HIF target genes (Fig. 6B), which is comparable to correlations between members of this group (10,15,20). Expression of CD44, which is a marker of breast CSCs (40) and a HIF target gene (41), was also significantly correlated with CD47 expression in the 1,040 breast cancers (Fig. 6B). HIF target gene expression in human breast cancers is significantly associated with patient mortality (18). To determine if CD47 mRNA expression was also a prognostic factor in human breast cancer, we used two published datasets that contain both gene expression and patient survival data (42,43). Patients were A B C
stratified into two groups based on whether CD47 mRNA levels in the primary tumor were greater or less than the median value. Increased CD47 mRNA expression levels were associated with a significantly decreased probability of overall survival in the two independent datasets, which together comprised 1,954 patients with breast cancer (Fig. 6C).

Discussion
Growing evidence indicates that in various human cancers, CD47 expression is required to avoid innate immune surveillance and elimination by phagocytosis. Blocking the interaction of CD47 with its receptor, SIRPα, on macrophages enables phagocytosis and inhibits tumor growth (35,36,(44)(45)(46). However, the molecular mechanisms regulating the expression of CD47 by cancer cells have not been delineated. In this study, we demonstrate that hypoxia, which is a critical microenvironmental stimulus in advanced breast cancers, induced HIF-dependent expression of CD47, leading to decreased phagocytosis of cancer cells by macrophages and induction of the breast CSC phenotype, which promote cancer progression and patient mortality (Fig. 6D).
Despite the recent interest in targeting CD47 for cancer therapy (46), remarkably little is known about the transcriptional regulation of the CD47 gene. The results presented here represent to our knowledge the first identification of a transcriptional regulator of CD47 expression in breast cancer cells and further studies are required to determine whether HIF-1 cooperates with other transcription factors that are induced by the tumor microenvironment, such as CREB, NF-κB, SMAD2, or STAT3. Increased NF-κB activity was observed in hepatocellular carcinoma cells that developed sorafenib resistance (36), which is of interest because the antiangiogenic effects of sorafenib induce intratumoral hypoxia that causes HIF-1-dependent induction of breast CSCs (19). Immunosuppressive functional interactions of HIF-1 and CREB have been proposed in T cells (47), but may also occur in cancer cells.
Intratumoral hypoxia is a common finding in breast cancer and HIFs activate the transcription of a large battery of genes encoding proteins that promote multiple steps in tumor progression, including tumor growth and vascularization, stromal cell recruitment, extracellular matrix remodeling, premetastatic niche formation, cell motility, margination and extravasation of circulating tumor cells, and CSC specification/maintenance (2). Here, we find that HIFs also play an important role in immune evasion by activating the expression of CD47, an antiphagocytic signal. In three different breast cancer cell lines, which represent luminal/ER + (MCF7), HER2-enriched (HCC1954), and basal-like/triple-negative (SUM159) subtypes of breast cancer, the expression of CD47 mRNA and protein was induced by hypoxia in a HIF-dependent manner. A recent study reported that CD47 protein was detected in 5% of hormone receptor positive, HER2 − breast cancers (48). Our bioinformatic analysis suggests that CD47 expression is likely to be higher among triple-negative breast cancers, most of which fall into the basal-like molecular subtype that is characterized by increased expression of the HIF transcriptome (18,39 increased expression of LOX and LOXL4 in MDA-MB-231, whereas LOXL2 expression was induced in MDA-MB-435 cells (12). We also observed heterogeneity with respect to requirements for HIF-α subunits, as CD47 induction required only HIF-1α in MCF7 cells, but both HIF-1α and HIF-2α in HCC1954 cells. Similarly, we previously reported that hypoxia-induced LOXL2 expression required only HIF-1α in MDA-MB-435 cells, whereas expression of LOX and LOXL4 in MDA-MB-231 cells required HIF-1α and HIF-2α (12). Heterogeneity at the level of gene expression is recognized as a major obstacle to successful cancer therapy. However, in the cases described above, treatment with a HIF inhibitor, such as acriflavine, successfully blocked all hypoxia-induced gene expression (Fig. 1D). Cells express varying levels of prophagocytic and antiphagocytic signaling proteins, and it is the integration of both signals that determines whether the target cell will be phagocytosed. The relatively modest changes in CD47 cell surface levels that resulted from expression of shRNA targeting HIF-1α and HIF-2α or CD47 led to significant changes in phagocytosis, indicating a fine balance between pro-and antiphagocytic signaling, such that a hypoxic tumor microenvironment may significantly tip the balance toward immune evasion. Several prophagocytic signals have been identified, including cell-surface expression of phosphatidylserine and CRT (49,50). CRT is required for the phagocytosis of cancer cells when CD47-SIRPα interaction is blocked (28). CRT expression was induced by exposure of cardiomyocytes to hypoxia and reoxygenation (51), suggesting that the increased phagocytosis of NTC and DKD subclones after hypoxic exposure may be due to HIF-independent CRT expression, but further studies are required to test this hypothesis. A recent report demonstrated that CD47 blockade also promotes T-cell-mediated elimination of immunogenic tumors (52) and HIF-1 is known to inhibit effector T cells through the production of adenosine (23,47), suggesting that HIF inhibitors may improve the response to CD47 blockade both by inhibiting CD47 expression and by disinhibiting T-cell-mediated antitumor immunity.
Two findings in our study suggested that hypoxia-induced expression of CD47 plays an important role that is independent of its antiphagocytic function. First, although CD47 mRNA and protein expression were induced by hypoxia in all three breast cancer cell lines analyzed, increased CD47 cell-surface expression and decreased phagocytosis were only induced by hypoxia in SUM159 cells. These data suggest that hypoxia-induced CD47 may fulfill another function that does not require cell-surface expression of the protein. Second, attempts at generating CD47deficient cells were only successful in establishing subclones with modest knockdown of CD47, suggesting that expression of the protein was required for clonal expansion. Using two established assays for breast CSCs, we demonstrated that CD47 expression was increased in breast CSCs (relative to bulk cancer cells) and was required for breast CSC maintenance, as decreased CD47 expression led to significantly reduced numbers of CSCs in a A C B Monolayer Mammosphere Fig. 5. CD47 expression promotes the breast cancer stem cell phenotype. (A) CD47 mRNA expression relative to 18S rRNA was analyzed by RT-qPCR using total RNA isolated from SUM159 cells that were grown in conventional adherent monolayer culture or as nonadherent mammospheres (described below). The results were normalized to monolayer cells (mean ± SEM; n = 3). ***P < 0.001 (Student's t test). (B) SUM159 subclones were exposed to 20% or 1% O 2 for 72 h in monolayer culture, harvested, seeded at a density of 5,000 cells per well on six-well ultra-low attachment culture plates, and cultured for 5 d in the presence of 20% O 2 (Upper). Mammospheres that were greater than 50 μm in diameter were counted by automated image analysis (Lower; mean ± SEM; n = 3). **P < 0.01 vs. NTC at 20% O 2 ; ### P < 0.001 vs. NTC at 1% O 2 (two-way ANOVA with Bonferroni posttest). (C) SUM159 subclones were exposed to 20% or 1% O 2 for 72 h in monolayer culture and the percentage of cells expressing aldehyde dehydrogenase (ALDH + ) was determined by flow cytometry (mean ± SEM; n = 3). *P < 0.05, **P < 0.01 vs. NTC at 20% O 2 ; ## P < 0.01, ### P < 0.001 vs. NTC at 1% O 2 (two-way ANOVA with Bonferroni posttest).
dose-dependent manner. Further studies are required to determine whether CD47 must be expressed at the cell surface to contribute to the maintenance of breast CSCs.
To generate a primary, recurrent, or metastatic tumor, a breast cancer cell must avoid immune destruction and give rise to both CSCs and differentiated breast cancer cells. A recent study found that among circulating tumor cells in the blood of women with metastatic breast cancer, the cells that were capable of initiating tumors when injected into mice expressed the cell surface proteins CD47, CD44, and MET (29). Remarkably, as in the case of CD47, the expression of CD44 and MET is induced by hypoxia in a HIFdependent manner (41,53). Furthermore, our analysis of gene expression data from over 1,000 human breast cancers revealed that CD47 expression was significantly correlated with the expression of CD44, MET, and other HIF target genes (Fig. 6B). We also found that CD47 mRNA expression in primary human breast cancers was significantly associated with patient mortality in two large and independent datasets (Fig. 6C). Similar results were recently reported regarding the immunohistochemical detection of CD47 protein expression in breast cancer biopsies (48).
We have recently demonstrated that exposure of breast cancer cells to hypoxia or chemotherapy induces the breast CSC phenotype through multiple HIF-dependent molecular pathways (18,20,54). Induction of CD47 expression provides another mechanism by which hypoxia induces the CSC phenotype. In addition to interacting with SIRPα, CD47 engages in several other functional interactions (55) and further studies are required to determine the molecular mechanism by which the expression of CD47, localized to the cell surface or perhaps an intracellular compartment, promotes the specification and/or The relative expression levels of 10 mRNAs encoded by HIF target genes, as well as CD44 and CD47 mRNAs, are shown for 1,040 primary breast cancer tissue samples from The Cancer Genome Atlas (TCGA), which were grouped according to the expression levels of 50 mRNAs (PAM50) that define the luminal A, luminal B, HER2 + , basal-like, and normal-like breast cancer molecular subtypes (57). Color code for mRNA expression levels are as follows: red, greater than median; green, less than median. (B) CD47 mRNA expression in each primary breast cancer specimen was compared with the expression of CD44 mRNA and HIF-regulated mRNAs using Pearson's correlation test. *P < 0.05, ***P < 0.001; n.s., not significant. (C ) Kaplan-Meier curves were constructed to analyze the association of CD47 mRNA expression in the primary tumor with overall survival (OS) of patients with breast cancer using two independent datasets (42,43). Statistical analysis was performed using log-rank tests. (D) Intratumoral hypoxia induces HIF-1-dependent expression of CD47 in breast cancer cells, leading to decreased phagocytosis of cancer cells by macrophages and induction of the cancer stem cell (CSC) phenotype, which promote cancer progression and patient mortality. maintenance of CSCs. Genetic or pharmacologic inhibition of HIF activity reduced the percentage of CSCs (18)(19)(20)56), which may be due in part to the inhibition of CD47 expression. Increased expression of HIF-1α mRNA or protein, or HIF target gene mRNAs, in primary breast cancer biopsies is associated with increased patient mortality (2,18). Our data suggest that addition of HIF inhibitors to current treatment regimens may improve outcome in such high-risk patients in part by attacking CSCs and disinhibiting innate immunity.
Immunoblot Assay. Whole-cell lysates were prepared in modified RIPA buffer; proteins were separated by SDS/PAGE, blotted onto nitrocellulose membranes, and probed with primary antibodies against HIF-1α, HIF-2α, and CD47 (Novus Biologicals). HRP-conjugated secondary antibodies (GE Healthcare) were used. Chemiluminescent signals were detected using ECL Plus (GE Healthcare). Blots were stripped and reprobed with antiactin antibody (Santa Cruz).
ChIP Assay. Cells were exposed to 20% or 1% O 2 for 16 h, then cross-linked in the presence of 3.7% (vol/vol) formaldehyde for 10 min, quenched in 0.125 M glycine for 5 min, and lysed with SDS lysis buffer. Chromatin was sheared by sonication, and lysates were precleared with salmon sperm DNA/ protein A-agarose slurry (Millipore) and incubated with IgG or antibodies against HIF-1α (Santa Cruz) or HIF-1β (Novus Biologicals) in the presence of protein A-conjugated agarose beads overnight. After sequential washes of the beads, DNA was eluted in 1% SDS with 0.1 M NaHCO 3 , and cross-links were reversed by addition of 0.2 M NaCl. DNA was purified by phenolchloroform extraction and ethanol precipitation. Candidate binding sites were analyzed by qPCR using flanking primers with the following sequences: 5′-AGG AAC GGG TGC AAT GAG-3′ and 5-CTT CCA GGT CAC GTC CTG TC-3′.
Cell Surface Expression of CD47. Cells were harvested after 24-h exposure to 20% or 1% O 2 and a single cell suspension was prepared. Cells were incubated with Fc Block (BD Pharmingen) for 10 min, then stained with phycoerythrin (PE)-conjugated CD47 antibody or isotype control (R&D Systems), and subjected to flow cytometry (FACScalibur, BD Biosciences). Dead cells were gated out by side-scatter and forward-scatter analysis.
Mammosphere Assay. Cells were exposed to 20% or 1% O 2 in monolayer adherent culture for 72 h. Single cells were plated in six-well ultra-low attachment culture plates (Corning) at a density of 5,000 cells per well in complete mammocult medium (Stem Cell Technologies). Mammospheres (diameter ≥50 μm) were counted after 5 d. Mammosphere cultures were imaged using an Olympus phase-contrast microscope and mammosphere diameters were determined using ImageJ software (National Institutes of Health).
ALDH Assay. Cells were exposed to 20% or 1% O 2 for 72 h and harvested for Aldefluor assay (Stem Cell Technologies). Cells were suspended in assay buffer containing 1 μmol/L BODIPY-aminoacetaldehyde and incubated for 45 min at 37°C. As a negative control, for each sample an aliquot of cells was treated with the ALDH inhibitor diethylaminobenzaldehyde (50 mM). Samples were subjected to flow cytometry analysis (FACScalibur, BD Biosciences).
Statistical Analysis. Data are expressed as mean ± SEM and were analyzed using Student's t test for two groups or ANOVA with Bonferroni posttest for multiple groups. Kaplan-Meier curves were generated using two independent datasets containing gene expression and overall survival data on patients with breast cancer (42,43). The log rank test was performed to determine whether observed differences between groups were statistically significant. P values <0.05 were considered significant.