Antiangiogenic agents increase breast cancer stemcells via the generation of tumor hypoxiaSarah J. Conley, Elizabeth Gheordunescu, Pramod Kakarala, Bryan Newman, Hasan Korkaya, Amber N. Heath,Shawn G. Clouthier, and Max S. Wicha1

Comprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109

Edited by Kornelia Polyak, Dana-Farber Cancer Institute, Boston, MA, and accepted by the Editorial Board December 23, 2011 (received for review January14, 2011)

Antiangiogenic therapy has been thought to hold significant poten-tial for the treatment of cancer. However, the efficacy of suchtreatments, especially in breast cancer patients, has been called intoquestion, as recent clinical trials reveal only limited effectiveness ofantiangiogenic agents in prolonging patient survival. New researchusing preclinical models further suggests that antiangiogenic agentsactually increase invasive and metastatic properties of breast cancercells. We demonstrate that by generating intratumoral hypoxia inhumanbreast cancer xenografts, the antiangiogenic agents sunitinibandbevacizumab increase thepopulationof cancer stemcells. In vitrostudies revealed that hypoxia-driven stem/progenitor cell enrich-ment is primarily mediated by hypoxia-inducible factor 1α. We fur-ther show that the Akt/β-catenin cancer stem cell regulatory path-way is activated in breast cancer cells under hypoxic conditionsin vitro and in sunitinib-treated mouse xenografts. These studiesdemonstrate that hypoxia-driven cancer stem cell stimulation limitsthe effectiveness of antiangiogenic agents, and suggest that to im-prove patient outcome, these agents might have to be combinedwith cancer stem cell-targeting drugs.

antiangiogenesis | HIF-1α

Angiogenesis has been a long-standing therapeutic target inmalignant tumors, an idea pioneered by Judah Folkman (1).

Preclinical studies have shown that inhibition of the vascular en-dothelial growth factor (VEGF) pathway impedes tumor growthand, clinically, the VEGF-neutralizing antibody Avastin (bev-acizumab) and VEGF receptor tyrosine kinase inhibitors (sor-afenib and sunitinib) have been used as anticancer treatments inseveral tumor types including breast cancer (2). However, clinicaland preclinical observations indicate that these therapies mayhave limited efficacy. Although these agents typically produceinhibition of primary tumor growth, lasting responses are rare,with only a moderate increase in progression-free survival andlittle benefit in overall survival (3). In addition, when anti-angiogenic agents are administered on an intermittent schedule,such as with sunitinib (4 wk on, 2 wk off), tumor regrowth issometimes seen during drug-free periods (4) or upon discontin-uation of the treatment (5). In light of these limited clinical ben-efits demonstrated, a U.S. Food and Drug Administration panelhas recently recommended that the approval of bevacizumab fortreatment of advanced breast cancer be revoked. Interestingly,recent reports describe increased tumor invasiveness and metas-tasis in response to VEGF inhibitors or VEGF gene inactivationin preclinical mouse models of cancer (6, 7).The administration of antiangiogenic agents has been shown to

generate intratumoral hypoxia, and hypoxia has been shown tomodulate each step in the metastatic process (8). Moreover, thetranscription factors hypoxia-inducible factors 1 and 2 alpha(HIF-1α and HIF-2α) have been linked to the stimulation ofcancer stem cells (CSCs) in glioblastoma (9–11). Because CSCshave tumor-initiating capabilities and a high metastatic potential(12), we hypothesized that hypoxia induced by administration ofantiangiogenic agents might accelerate tumor growth and me-tastasis by increasing the CSC population. We demonstrate thatadministration of antiangiogenic agents such as the VEGF re-ceptor tyrosine kinase inhibitor sunitinib and the anti-VEGF an-tibody bevacizumab increases the CSC population in breast

cancer xenografts as a consequence of the generation of tumorhypoxia. The increase in CSCs in response to hypoxia was medi-ated through HIF-1α through the activation of the Wnt pathwayvia Akt/β-catenin signaling.

ResultsTo determine whether antiangiogenic agents stimulate an increasein breast CSCs in vivo, we treated tumor-bearing mice with themultireceptor tyrosine kinase inhibitor sunitinib malate (Sutent;Pfizer). Previous studies have demonstrated strong growth in-hibition of established primary tumors in mice treated with thisagent (13). We compared the effect of sunitinib on tumors usingboth early and late treatment times. MDA-MB-231 and SUM159human breast cancer cells were implanted in the mammary fatpads of non-obese diabetic/severe combined immunodeficient(NOD/SCID) mice. Group A received vehicle control, and groupB received sunitinib treatment (60 mg/kg daily) starting whentumors reached 4 mm in diameter (late treatment). Mice in groupCwere given continuous sunitinib therapy (60mg/kg daily) startingthe day after tumor implantation (early treatment). A sustainedsunitinib therapy regimen of 60 mg/kg/d given continuously haspreviously been demonstrated to result in optimal tumor inhibitionwith minimal toxicity (14). As expected, significant inhibition oftumor growth was observed after sunitinib treatment of estab-lished tumors compared with controls (Fig. 1A). Sustained suni-tinib therapy beginning 1 d after tumor implantation resulted ina delay in the onset of tumor formation as well as a decrease intumor size (Fig. 1A). Staining for the endothelial marker CD31revealed significantly fewer blood vessels in tumors from sunitinib-treated mice compared with controls (Fig. 1B and Fig. S1), whichwere smaller and less vascularized than the control tumors (Fig.1C). We have previously demonstrated that a subpopulation ofcells that displays stem cell properties can be isolated from normalhuman breast tissue and breast carcinomas, by virtue of their in-creased expression of aldehyde dehydrogenase (ALDH) activity asassessed by the Aldefluor assay (15).Many breast cancer cell lines,includingMDA-MB-231, SUM159, andMCF-7 cells, also containanAldefluor+ population that displays stem cell properties in vitroand in NOD/SCID xenografts (12). We therefore determined theeffects of sunitinib treatment on the proportion of Aldefluor+ cellsin themouse xenografts. Treatmentwith sunitinib for 35 d initiatedafter MDA-MB-231 tumors reached 4 mm in diameter signifi-cantly increased (P < 0.01) the percentage of Aldefluor+ tumorcells, by 4.8-fold (Fig. 1D). The percentage of Aldefluor+ cellsfrom mice treated continuously beginning 1 d after implantationfor 75 d (group C) was also significantly increased compared withthe control, by 2.4-fold (P < 0.01). Sunitinib treatment also

Author contributions: S.J.C. and M.S.W. designed research; S.J.C., E.G., P.K., B.N., A.N.H.,and S.G.C. performed research; H.K. contributed new reagents/analytic tools; S.J.C. ana-lyzed data; and S.J.C. and M.S.W. wrote the paper.

Conflict of interest statement: M.S.W. is a consultant for Pfizer and OncoMed Pharma-ceuticals and holds equity in OncoMed Pharmaceuticals.

This article is a PNAS Direct Submission. K.P. is a guest editor invited by theEditorial Board.1To whom correspondence should be addressed. E-mail: mwicha@umich.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1018866109/-/DCSupplemental.

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resulted in growth inhibition of SUM159 xenografts (Fig. 1A).When cells from SUM159 tumors treated continuously for 55d were tested by the Aldefluor assay, there was a 4.6-fold increase(P < 0.05) in the proportion of Aldefluor+ cells.Although the increase in the ALDH+ cell population in suniti-

nib-treated tumors suggests that this drug increases breast CSCs,the ability of residual cancer cells to initiate tumors upon reim-plantation in secondary mice is a more definitive assay. We there-fore assayed the ability of serial dilutions of cells isolated from theprimary tumors to generate tumors when implanted in secondaryNOD/SCID mice (Fig. 1E and Fig. S2). Tumor cells isolated fromsunitinib-treated mice exhibited significantly increased tumor-ini-tiating capacity and growth in secondary mice compared with cellsisolated from control tumors. When 50,000 cells were injected,tumors grew equally well from control and sunitinib-treated pri-mary tumors. However, when smaller numbers of cells were injec-ted into secondary animals, those from sunitinib-treated miceshowed a 2.5-fold increase for 5,000 cells (P < 0.05) and a 6-foldincrease for 500 cells (P < 0.05) in tumor size compared with cellsfrom control animals. The results from these Aldefluor assays andtumor regrowth experiments indicate that sunitinib increases theAldefluor+, tumorigenic population of tumor cells.To further confirm that disruption of the VEGF pathway leads

to an increase in CSCs, we used bevacizumab, a humanized anti-body to VEGF, to block angiogenesis in human breast cancerxenografts. MDA-MB-231 cells were implanted in the mammaryfat pads of NOD/SCID mice. When tumors reached 4 mm in di-ameter, either vehicle control or 5 mg/kg of bevacizumab wasadministered twice weekly. Bevacizumab treatment effectivelyabrogated tumor growth (Fig. S3 A and B) and resulted in lessvascularization (Fig. S1). There was approximately a twofold in-crease in the percentage of Aldefluor+ cells in tumors from bev-acizumab-treated mice compared with control tumors (Fig. S3C).To determine whether the increase represents an increase in theabsolute number of CSCs, cell viability was determined by trypan

blue exclusion. On average, control tumors yielded 17 ± 6 millioncells with 72% viability, whereas the sunitinib-treated tumorsyielded 3± 1million with 68% viability (Fig. S1B). Similarly, whenbevacizumab was tested, control tumors had 31 ± 8 million cellswith 93 ± 2% viability, whereas bevacizumab-treated tumorsyielded 6± 2 million cells with 92± 3% viability (Fig. S3D). Therewas no significant difference between the viability of cells fromcontrol and drug-treated tumors. This suggests that both the ab-solute number and proportion of CSCs in these tumors increase inresponse to the antiangiogenic therapies.To gain additional insight into the mechanism by which hypoxia

regulates breast CSC populations, we assessed the spatial re-lationship between ALDH1+ cells and areas of hypoxia withinmammary tumors. Whereas tumors from control animals exhibi-ted little or no hypoxia as determined by pimonidazole adduct(Hypoxyprobe) staining, tumors from sunitinib-treated mice dis-played multiple areas of intense hypoxia (Fig. 2A). Hypoxyprobepositivity was found specific to zones of low oxygen because themost intense staining coincided alongside necrotic zones in bothbevacizumab and sunitinib as well as very large control tumorswith necrosis (Fig. S4). Double labeling with anti-ALDH1 anti-body and pimonidazole adduct staining was carried out. WhereasALDH1+ cells were scattered throughout tumors from controlanimals, we observed high-density areas of ALDH1+ cells withinhypoxic regions of tumors from sunitinib-treated mice (Fig. 2B).Tumors from sunitinib-treated mice exhibited approximately aneightfold greater number of ALDH+ cells within hypoxic areas,but no significant difference within normoxic zones as determinedby staining (Fig. 2C). This localization strongly suggests that theincrease in Aldefluor+ tumor cells seen following sunitinibtreatment occurs as a direct effect of the hypoxia induced by theangiogenic inhibition. Because sunitinib is a potent multikinaseinhibitor, we further examined whether the effect of sunitinib onCSCs is mediated by the generation of tumor hypoxia or occurs viadirect mechanisms. The antiproliferative effect of sunitinib in

Fig. 1. Sunitinib malate induces hypoxia in breast tumors in vivo and results in an increase in the tumor stem/progenitor cell population. (A) MDA-MB-231 orSUM159 cells were injected into the inguinal fat pads of NOD/SCID mice. One day after injection, mice were given either vehicle (group A) or sunitinib (60 mg/kg) (group B). Group C received the vehicle control until tumors reached an average size of 4 mm in diameter, and then were administered sunitinib (60 mg/kg) daily. Data are shown as averages ± SD. n = 8–10 (MDA-MB-231); n = 4–6 (SUM159). Treated tumors were significantly smaller than control tumors at endpoint. *P < 0.01. (B) CD31 staining of blood vessels (green) and DAPI nuclear staining (blue) of tumors from a control mouse and a sunitinib-treated mouse.(Scale bars, 200 μm.) (C) Representative tumors displaying a lack of vasculature in sunitinib-treated mice compared with control tumors. (D) The percentage ofALDH+ cells in tumors was determined by Aldefluor assay. n = 8. *P < 0.05. (E) Serial dilutions of cells obtained from primary SUM159 tumors treated withcontrol or sunitinib were implanted in secondary NOD/SCID mice. Primary tumors treated with sunitinib grew secondary tumors more rapidly than the controltumors. Data are shown as averages ± SD. n = 4–5. *P < 0.05.

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SUM159 and MDA-MB-231 was first evaluated in cells bymethylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Cellviability decreased as sunitinib concentration increased, with anIC50 of ∼30 μmol/L after 48 h (Fig. S5). The maximum plasmaconcentration achieved by 80mg/kg of sunitinib inmice (higher thanour 60-mg/kg dose) was reported to be 1.6 μM (16), below theconcentration needed to inhibit cell viability in vitro, suggesting thatthe in vivo treatment does not select for CSC survival over non-CSCs.We next analyzed sunitinib-treated cells by Aldefluor assay todetermine whether the drug directly affects the CSC population. Incontrast to the increase in Aldefluor+ cells seen in vivo, the addition

of 1 μmol/L did not have a significant effect on the Aldefluor+ cellscompared with controls (Fig. S6). Bevacizumab does not have directcytotoxic effects on tumor cells (17). Together, these findings sup-port the hypothesis that antiangiogenic drugs stimulate the CSCpopulation by generating hypoxia within tumors rather than throughdirect effects.To elucidate the molecular mechanisms mediating hypoxia-in-

duced CSC expansion, we determined whether we could simulatethese effects in vitro. To test the effect of low oxygen onbreast CSCsin vitro, three human mammary carcinoma cell lines were grownunder 21% O2 (normoxia) or 1% O2 (hypoxia). Cells from thesethree lines were cultured at subconfluency for 2, 4, or 6 d and, aftereach time point, cells were assayed for ALDH activity by Aldefluorassay. As shown in Fig. 3, the percentage of Aldefluor+ cells in eachof the cell lines increased approximately two- to threefold whencultured under low oxygen. This increase was seen in as little as2 d of hypoxia treatment, and was sustained for the 6-d treatmentperiod. Todemonstrate that the effect of hypoxia on theAldefluor+population reflects an increase in the CSC population rather thana direct effect on ALDH expression, we analyzed ALDH1A1mRNA expression in Aldefluor+ and Aldefluor− populations byRT-PCR. As shown in Fig. S7, hypoxia had no effect on ALDHA1expression in Aldefluor+ or Aldefluor− populations. This indicatesthat the increase in the Aldefluor+ population induced by hypoxiareflects an actual increase in Aldefluor+ cells rather than merely inALDH1A1 expression.Cellular responses to low oxygen are principally regulated by

the transcriptional activity of HIFs. We therefore investigatedwhich of the HIF α subunits might mediate the increase in CSCsunder hypoxia. Immunoblotting of Aldefluor+ and Aldefluor−cells grown under hypoxia shows a robust increase in HIF-1α (Fig.4A). The increase in HIF-1α was most significant in the Alde-fluor+ cells. HIF-2α was not detectable in either population.siRNA oligos directed against HIF-1α and/or HIF-2α weretransfected into SUM159 cells, and the cells were then grown for3 d under hypoxia. Complete knockdown of HIF-1α is shown inFig. 4B. The CSC population in each sample was assessed byAldefluor assay (Fig. 4C). Compared with untransfected cells orcells transfected with nontargeting siRNA oligos, knockdown ofHIF-1α completely abrogated the increase of the Aldefluor+population induced by hypoxia. As expected, knockdown of HIF-2α did not significantly block the effects of hypoxia, and knock-down of both HIFs did not decrease the CSC population anyfurther than with HIF-1α siRNA alone. Although there was noincrease in cell death following hypoxia or knockdown of HIF-1α(Fig. S8A), there were ∼30% fewer cells in untransfected orcontrol siRNA-transfected cells grown under hypoxia (Fig. S8B),suggesting that cells grow at a slower rate under hypoxic con-ditions. When HIF-1α was knocked down, there was no longera difference in the growth of the cells under hypoxia.The Akt/Wnt/β-catenin pathway has been reported to be a key

regulator of breast CSC self-renewal (18), and activation ofβ-catenin by hypoxia has been shown to enhance metastatic po-tential of cancer cells (19). In addition, HIF-1α was reported toenhance β-catenin signaling in hypoxic embryonic stem cells (20).For these reasons, we investigated whether hypoxia induces Akt/Wnt/β-catenin signaling in human breast cancer cells. SUM159and MCF-7 cells were grown under normoxic or hypoxic con-ditions for 2 d, and total and activated (phosphorylated) Akt andβ-catenin levels were determined by Western blot. Although totalamounts of Akt were unchanged, hypoxia treatment resulted in anincrease in activated phospho-Akt in both cell lines (Fig. 5A).When β-catenin levels were assessed in SUM159, total amounts ofthe protein were unchanged following hypoxia, but an increase inthe activated phospho-S552 form of β-catenin was detected. De-spite an increase in phospho-Akt, no change was detected inβ-catenin levels inMCF-7 cells, suggesting that Akt may not signalvia the Wnt/β-catenin pathway in this cell line.To provide further evidence for cross-talk between Akt sig-

naling and the Wnt pathway, we used an LEF-1/TCF reportersystem tomonitor β-catenin transcriptional activity. SUM159 cellswere infected with an LEF-1/TCF lentiviral reporter driving GFPand luciferase. Cells were first sorted into GFP+ and GFP−

Fig. 2. Sunitinib malate induces hypoxia in breast tumors in vivo, andALDH1+ cells are concentrated in hypoxic regions. (A) Hypoxia in SUM159tumors was detected by immunofluorescence staining of pimonidazoleadducts in sections from control or sunitinib-treated animals. Staining showspimonidazole immunodetection (green). (Scale bars, 800 μm.) (B) Stainingshows pimonidazole (green) and ALDH1 (red) merged with DAPI-stainednuclei (blue). (Scale bars, 400 μm.) (Inset) Magnification: 5×. (C) Quantitationof ALDH1-positive cells in control tumors versus hypoxic and normoxic areaswithin sunitinib-treated tumors. Data are shown as averages ± SD. n = 5.*P < 0.05, **P < 0.01.

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populations by flow cytometry to isolate cells in which β-cateninsignaling was active or inactive under normoxia. Equal numbers ofcells were grown for 20 h under normoxia or hypoxia, and thetranscriptional activity of β-catenin was determined by luciferaseassay. As expected, luciferase reporter activity was ∼40-foldhigher in the GFP+ cells compared with GFP− cells under nor-moxic conditions (Fig. 5B). Whereas no significant difference wasdetected in GFP+ cells following hypoxia treatment, luciferaseactivity was increased by approximately twofold in GFP− cellsfollowing hypoxia treatment. To determine whether the activationof β-catenin following hypoxia treatment required the presence ofHIF-1α, we probed cell extracts following HIF-1α siRNA knock-down for phospho-S552-β-catenin. Indeed, no activation wasfound after hypoxia when HIF-1α was absent (Fig. 5C), demon-strating that HIF-1α is upstream of the β-catenin response.To extend these observations to themousemodels, we examined

the expression and localization of β-catenin in SUM159 tumorsfrom control and sunitinib-treated mice by immunohistochemistry.All tumors showed the presence of β-catenin staining. As shown inFig. 5D, β-catenin was primarily detected in the cytoplasm of tumorcells from control mice. In contrast, cells within sunitinib-treatedtumors displayed distinct nuclear localization of β-catenin, espe-cially prominent in hypoxic areas surrounding necrotic regions.Together, these experiments suggest that activation of the Akt/β-catenin signaling cascade may modulate the hypoxic response inCSCs induced by antiangiogenic agents.

DiscussionHypoxia Increases the Population of Breast CSCs. Using the Alde-fluor assay to identify populations enriched for CSCs, we deter-mined that growing human breast cancer cell lines under mildhypoxic conditions resulted in an increase in the CSC population.Furthermore, we demonstrate that this effect is mediated by HIF-1α. This is consistent with previous reports that knockdown ofHIF-1α reduces migration potential and formation of tumorspheres in glioma cells (21), expansion of CD133+ CSCs in glio-blastoma (9), and tumorigenicity of renal cell carcinoma (22).We further demonstrate that the increase in CSCs following

hypoxic stress is at least partly regulated by the Akt/β-catenin sig-naling pathway. We previously demonstrated that Akt activationincreases breast CSC self-renewal through stimulation of the Wntpathway (18). Hypoxia increases levels of both phospho-Akt andphospho-S552-β-catenin in SUM159 cells. Moreover, nuclear trans-location of β-catenin was detected in tumor cells from sunitinib-treated mice, particularly in hypoxic regions near necrotic areas. Inaccordance with our findings, inhibition of the Akt pathway wasdemonstrated to reduce hypoxia-driven CD133+ glioma cell ex-pansion (9). HIF proteins have been reported to interact with theβ-catenin pathway in multiple ways depending on the cell type.HIF-1α modulates Wnt/β-catenin signaling in hypoxic embryonicstem cells and neural stem cells by enhancing β-catenin activationand increasing expression of the downstream effectors LEF-1 andTCF-1 (20). With respect to cancer cells, HIF-1α and HIF-2α are

Fig. 3. Hypoxia increases the Aldefluor+ stem cell population in breastcancer cell lines in vitro. MDA-MB-231, SUM159, and MCF-7 cells were grownunder 21% O2 (normoxia) or 1% O2 (hypoxia) for 2–6 d. The percentage ofALDH+ cells was assessed using the Aldefluor assay (STEMCELL Technolo-gies). Data are shown as averages ± SD. n = 3–5. *P < 0.05.

Fig. 4. HIF-1α mediates increase of CSCs by hypoxia. Aldefluor+ and Alde-fluor− cells were sorted by flow cytometry and plated. After 24 h, cells wereplaced under hypoxic conditions for 3 d, after which the levels of HIF proteinswere assessed. (A) Immunoblotting shows only HIF-1α is expressed following72 h of hypoxia. Cell lysate (0.5 μg) from HIF-2α–positive control was loadednext to 40 μg of SUM159 cell lysate to demonstrate the effectiveness of HIF-2αantibody. N, 21%O2; H, 1% O2. (B) SUM159 cells were transfected with siRNAoligos against HIF-1α or HIF-2α or nontargeting oligos for 24 h, and thenplaced under 1% or 21% O2 for 72 h. Immunoblotting demonstrates com-plete knockdown of HIF-1α after hypoxia treatment when HIF-1α siRNA wastransfected. (C) The percentage of Aldefluor+ cells increased under hypoxia inuntransfected cells and cells transfected with either control siRNA or HIF-2αsiRNA oligos. Knockdown of HIF-1α blocked the stimulation of Aldefluor+

cells following hypoxia. Data are shown as averages ± SD. n = 3. *P < 0.05.

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reported to have potentially antagonistic effects on β-catenin sig-naling. A recent study showed that HIF-2α binds β-catenin andenhances the transcriptional activity of β-catenin/TCF by recruitingp300 in HEK293 cells (23). However, HIF-1α has been shown tobind to and inhibit β-catenin–T-cell factor 4 (TCF-4) complexformation and transcriptional activity in colorectal cancer cells (24).Although at first glance these findings seem contradictory, theymaysimply reflect the differences between activities of HIF-1α in stemcells versus nonstem cells, because reported cancer studies onlyexamined the bulk population of cells rather than activity in therarer CSC population. The lack of Wnt activation in MCF-7 cells,which are a luminal breast cancer cell type, is consistent with pre-vious reports that CSCs in luminal breast cancers may be regulatedby alternate pathways such as Notch (25).Induction of HIF proteins may also be involved in epithelial-to-

mesenchymal transition (EMT). This could be another potential

mechanism for hypoxia-induced increase in the CSC population.Recent reports have linked EMT to stem cell characteristics inboth normal and tumor cells (26–28), and hypoxia has beendemonstrated to induce an EMT-like phenotype in cancer cells(29). The possibility of tumor cell plasticity and an EMT-inducedstem cell-like phenotype in response to tumor hypoxia thus war-rants further examination.

Defining a Hypoxic Niche for Breast Cancer Stem Cells. We in-vestigated the in vivo effects of hypoxia on CSCs induced by theantiangiogenic agents sunitinib and bevacizumab in mice bearinghuman breast cancer xenografts. As expected, pimonidazole stain-ing revealed extensive areas of hypoxia in tumors fromdrug-treated,but not control mice. Concomitant with the increase in hypoxia, thepercentage of CSCs within these tumors also increased, as de-termined by Aldefluor assay and reimplantation studies. Tumorstreated continuously from day 1 had an initial delay in tumor for-mation. However, tumors stopped growing once they reached anaverage size of ∼3 mm, suggesting that these tumors cannot con-tinue growing beyond that size without the formation of new vas-culature. Because the antiangiogenic effects on CSCs are related tothe hypoxia generated in the tumormicroenvironment, this effect isincreased in larger tumors that develop areas of hypoxia. Addi-tionally, immunohistochemical staining revealed that concentratedpopulations of ALDH1+ stem cells were predominantly localized inhypoxic regions within tumors from sunitinib-treated mice. Theincrease in CSCs following treatment with bevacizumab providesadditional evidence that blockade of the VEGF pathway, whethervia a VEGF RTKI or an anti-VEGF antibody, increases CSCsthrough the generation of tumor hypoxia. Our results are consistentwith a previous report that in vivo treatment with the mouseVEGFR2-targeting antibody DC101 increases the secondarysphere-forming capacity of cells from glioma xenografts (30). Theexistence of a hypoxic niche for breast CSCs is in agreement withreports that several types of normal tissue stem cells reside in hyp-oxic niches. In particular, a hypoxic microenvironment regulateshematopoietic stem cells within bone marrow, where the least dif-ferentiated cells reside in the least oxygenated areas (31–33). Ourresults are also in agreement with findings of a hypoxic CSC nichewithin glioblastoma tumors (10). Although an enrichment of CSCsin hypoxic microenvironments seems to contradict findings thatCSCs are often detected near blood vessels (10, 34, 35), this juxta-position may be explained by the intimate interactions betweenCSCs and vascular endothelial cells. In fact, emerging evidence hasimplicated a number of vascular-derived factors that can regulateCSCs (36, 37). In addition, tumor neovasculature often developsrapidly, resulting in structural and functional abnormalities ulti-mately leading to reduced oxygen transport. Thus, itmay be possiblefor CSCs to be concurrently in close proximity to tumor vasculatureand still be exposed to low oxygen levels.

Antiangiogenic Therapy and Cancer Stem Cells. The findings pre-sented here not only broaden our understanding of the role ofhypoxia in CSC biology, but also have significant clinical implica-tions. Angiogenesis has been a long-standing therapeutic target incancer. Currently, there are three major antiangiogenic therapiestargeting the VEGF signaling pathway approved for clinical use. Inmost cancer types, use of these therapeutics results in growth in-hibition of primary tumors and amoderate increase in progression-free survival. Unfortunately, responses are often transitory, andpatients relapse with more invasive metastatic disease resulting inlittle to no benefit in overall survival (3, 38). In addition, recentreports have described enhancement of tumor invasiveness andmetastasis in response to different classes of VEGF inhibitors orVEGF gene inactivation in various preclinical mouse models ofcancer (6, 7). Of importance, breast CSCs have been linked to tu-mor invasion and metastasis (12). Interestingly, pretreatment ofmice with sunitinib before tumor inoculation was shown to increasemetastasis of breast cancer cells, suggesting that this observed effectmay result from a “host” response to the drug (6). Indeed, ma-nipulation of the tumormicroenvironment likely induces a range ofresponses, in both the tumor as well as the host.We propose that an

Fig. 5. The β-catenin pathway is stimulated in response to hypoxia. (A)SUM159 and MCF-7 cells were grown under 1% (H) or 21% (N) O2 for 24 h.Immunoblotting was carried out for total and phospho-Akt and total andphospho-β-catenin. (B) SUM159 cells infected with the pGreenFire LEF/TCFlentivirus reporter were sorted by flow cytometry into GFP+ and GFP− pop-ulations, grown under 1% or 21% O2 for 20 h, and assayed for luciferase ac-tivity. GFP− cells displayed significantly higher luciferase activity in response tohypoxia. Data are shown as averages± SD. n= 6. *P< 0.01. (C) Immunoblot forphospho-β-catenin in SUM159 cell extracts following transfectionwith controlsiRNA or HIF-1α siRNA and 3-d hypoxia treatment. (D) Representative immu-nofluorescent staining for β-catenin in SUM159 xenografts from control orsunitinib-treated mice. Note the primarily cytoplasmic staining in cells fromcontrol tumors and intense nuclear staining in cells near necrotic regions (N)of sunitinib-treated tumors. (Scale bars, 100 μm.)

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increase in the CSC population would contribute to the overallaggressiveness of a tumor. Our findings that treatment with theantiangiogenic agents sunitinib or bevacizumab leads to an increasein CSCs provides a potential explanation for the limited clinicaleffectiveness of antiangiogenic agents. If this is the case, then im-proving the clinical efficacy of antiangiogenic treatments will re-quire the concurrent use of CSC-targeting agents.

Materials and MethodsCell Culture and Reagents. Culture media and conditions are described in SIMaterials and Methods. Sunitinib malate (Sutent) was a kind gift from Pfizer.The drugwas suspended in vehicle containing carboxymethylcellulose sodium[United States Pharmacopeia (USP); 0.5% wt/vol], NaCl (USP; 1.8% wt/vol),Tween 80 [National Formulary (NF); 0.4%wt/vol], benzyl alcohol (NF; 0.9%wt/vol), and deionized water adjusted to pH 6.0. Sunitinib was prepared weeklyand kept at 4 °C. Bevacizumab (Genentech) was diluted in saline solution.

Animal Studies. Allmouse experimentationwas conducted in accordancewithstandard operating procedures approved by the University Committee on theUse and Care of Animals at the University of Michigan. NOD/SCID mice werepurchased from The Jackson Laboratory. Cancer cells were injected into themammary fat pads of mice (2 × 106 MDA-MB-231, 1 × 105 SUM159). Drugtreatment and tumor digestion methodology is described in SI Materialsand Methods.

Aldefluor Assay and Flow Cytometry. The Aldefluor assay was carried outaccording to themanufacturer’s (STEMCELL Technologies) guidelines. Briefly,cells were suspended in Aldefluor assay buffer containing an ALDH substrate,bodipy-aminoacetaldehyde, at 1.5 μM, and incubated for 45 min at 37 °C. Todistinguish between ALDH+ and ALDH− cells, a fraction of cells was incubatedwith a 10-fold excess of an ALDH inhibitor, diethylamino-benzaldehyde. Thisresults in a significant decrease in fluorescence intensity of ALDH+ cells andwas used to compensate the flow cytometer.

Immunohistochemistry. Formalin-fixed, paraffin-embedded tissue was sec-tioned, dewaxed, and rehydrated through graded alcohol. Sections wereheated to 98 °C for 40 min in 0.01 M sodium citrate buffer (pH 6.0) for antigenretrieval. For specific methodology, see SI Materials and Methods.

Immunoblotting. Cells were lysed in RIPA buffer, boiled, subjected to SDS/PAGE,andtransferred toPVDF (Pierce). Blotswereblockedwith5%milkor2%BSA (forphospho-specific antibodies) and incubated with primary antibodies overnightat 4 °C. Blots were washed and incubated with secondary antibodies (GEHealthcare) and detected using SuperSignal West Pico Chemiluminescent Sub-strate (Pierce). SeeSIMaterialsandMethods formore information.Densitometricanalysis was carried out using ImageJ software (National Institutes of Health).

siRNA. ON-TARGETplus siRNA pools against HIF-1α or HIF-2α and a non-targeting pool were from Thermo Scientific. SUM159 cells were transfectedusing Dharmafect Reagent 1 (Thermo Scientific) overnight; media were thenchanged and cells were incubated under 1% O2 for 72 h before beingsubjected to Aldefluor assay.

Reporter Assay. pGreenFire TCF/LEF lentivirus reporter was obtained fromSystem Biosciences. mCMV pGreenFire reporter lacking a β-catenin/TCFbinding site was used as a control to select GFP+ cells. Cells were sorted byflow cytometry into 96-well plates and incubated for 20 h under 1% or 21%O2. Luciferase activity was measured using the ONE-Glo Luciferase Assay Kitfrom Promega.

ACKNOWLEDGMENTS. We thank Dr. J. Christensen (Pfizer) for providing suni-tinib; the University of Michigan Flow Cytometry and Vector Cores; and TahraLuther, Hsiu-Fang Lee, and Denise Poirier for their assistance. This work wassupported by Breast Cancer Research Foundation Grants N012653 andW011541, National Institutes of Health Grants CA-R01 129765 and CA-101860,and The Taubman Research Institute.

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