cxcr4 protein epitope mimetic antagonist pol5551 ......death. the protein epitope mimetic (pem)...

Small Molecule Therapeutics

CXCR4 Protein Epitope Mimetic AntagonistPOL5551 Disrupts Metastasis and EnhancesChemotherapy Effect in Triple-Negative BreastCancerJingyu Xiang1, Michelle A. Hurchla1, Francesca Fontana1,2, Xinming Su1, Sarah R. Amend1,Alison K. Esser1, Garry J. Douglas3, Chidananda Mudalagiriyappa4, Kathryn E. Luker5,6,7,Timothy Pluard8, Foluso O. Ademuyiwa9, Barbara Romagnoli3, G�erald Tuffin3,Eric Chevalier3, Gary D. Luker5,6,7, Michael Bauer3, Johann Zimmermann3, Rebecca L. Aft4,Klaus Dembowsky3, and Katherine N.Weilbaecher1

Abstract

The SDF-1 receptor CXCR4 has been associated with earlymetastasis and poorer prognosis in breast cancers, especially themost aggressive triple-negative subtype. In line with previousreports, we found that tumoral CXCR4 expression in patients withlocally advanced breast cancer was associated with increasedmetastases and rapid tumor progression. Moreover, high CXCR4expression identified a group of bone marrow–disseminatedtumor cells (DTC)-negative patients at high risk for metastasis anddeath. The protein epitope mimetic (PEM) POL5551, a novelCXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had nodirect effects on tumor cell viability, but reduced migration ofbreast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on

primary tumor growth, but significantly reduceddistantmetastasis.When combined with eribulin, a chemotherapeutic microtubuleinhibitor, POL5551 additively reduced metastasis and prolongedsurvival inmiceafter resectionof theprimary tumor comparedwithsingle-agent eribulin. Hypothesizing that POL5551 may mobilizetumor cells from their microenvironment and sensitize them tochemotherapy,weused a "chemotherapy framing"dosing strategy.When administered shortly before and after eribulin treatment,three doses of POL5551 with eribulin reduced bone and livertumor burden more effectively than chemotherapy alone. Thesedata suggest that sequenced administration of CXCR4 antagonistswith cytotoxic chemotherapy synergize to reduce distant meta-stases. Mol Cancer Ther; 14(11); 2473–85. �2015 AACR.

IntroductionThe chemokine receptor CXCR4 plays an important role in the

trafficking and homing of hematopoietic stem cells (HSC) andleukocytes. Its ligand SDF-1 (also called CXCL12) is produced by

activated osteoblasts, bone marrow and lung stromal cells, andendothelial cells (1). During embryogenesis, the CXCR4-expres-sing HSCs migrate from fetal liver and home to the bone marrowvia chemotactic CXCR4–SDF-1 interactions (2). Mice with tar-geted genetic deletion of CXCR4 or SDF-1 are embryonic lethaldue to impaired cardiovascular development and failure of HSCto home to the bonemarrow (3–5). SDF-1 binding toCXCR4 alsomaintains the adult HSC niche; pharmacologic antagonism ofCXCR4 causes HSC to rapidly accumulate in the peripheralcirculation (6).

Cancer cells, especially subpopulations with stem-like or met-astatic properties, often use pathways typical of HSC and competefor their niche (7, 8). Accordingly, CXCR4 is overexpressed in anumber of cancer types, including breast, lung, colon, glioblas-toma, and multiple myeloma (9). During metastasis, CXCR4activation promotes the migration of tumor cells to SDF-1–richdistant organs, where they interact closely with resident stromaand extracellular matrix through both direct cell-to-cell contact(adhesion molecules) and soluble factors (chemokines, cyto-kines; refs. 10–12). The establishment of these tumor–stromalinteractions has been shown to promote tumor cell-cycle arrest,survival, and resistance to chemotherapy in hematologic malig-nancies, such as multiple myeloma and chronic myelogenousleukemia (particularly with multiple myeloma; ref. 13), and insolid tumors (14). Similar to CXCR4 inhibition in HSC, CXCR4

1DivisionofMolecularOncology,DepartmentofMedicine,WashingtonUniversity School of Medicine, St. Louis, Missouri. 2Division of Boneand Mineral Diseases, Musculoskeletal Research Center, WashingtonUniversity School of Medicine, St. Louis, Missouri. 3Polyphor Ltd.,Allschwil, Switzerland. 4Department of Surgery, Washington Univer-sity School ofMedicine, St. Louis, Missouri. 5Department of Radiology,Center for Molecular Imaging, University of Michigan Medical School,Ann Arbor, Michigan. 6Department of Microbiology and Immunology,University of Michigan Medical School, Ann Arbor, Michigan. 7Depart-ment of Biomedical Engineering, University of Michigan MedicalSchool, Ann Arbor, Michigan. 8Saint Luke's Cancer Institute, KansasCity, Missouri. 9Division of Oncology, Department of Medicine,Washington University School of Medicine, St. Louis, Missouri.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Katherine N. Weilbaecher, Washington UniversitySchool of Medicine, 660 South Euclid Avenue, Campus Box 8069, St. Louis,MO 63110. Phone: 314-454-8858; Fax: 314-454-8979; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0252

�2015 American Association for Cancer Research.

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antagonists can mobilize tumor cells out of these protectivestromal niches into the peripheral blood, which could enhancesusceptibility to anoikis, and to conventional chemotherapy. Inaddition, CXCR4 antagonists may disrupt metastasis of tumorsthat use CXCR4 to home to bone and other SDF-1–rich distantorgans. In contrast with their role in HSC and cancer cell mobi-lization, POL5551 and other CXCR4 inhibitors have also beenshown to blunt mobilization of some other cell types, such asmesenchymal stem cells to sites of injury or inflammation,reducing for example neointimal formation (15, 16).

Breast cancer is themost prevalent cancer inwomenworldwide.More than 90% of the mortality of breast cancer patients isassociated with metastasis and relapse. CXCR4 expression isdetectable in breast cancer of various subtypes and has beenassociated with early metastasis and poorer prognosis, especiallywith triple-negative breast cancer (TNBC), a particularly aggres-sive subtype (17). The presence of microscopic disseminatedtumor cells (DTC) in the bone marrow is also associated withearly metastasis and poorer survival (18). As CXCR4 can beexpressed by both primary tumors and circulating tumor cells,the interaction between tumor CXCR4 and the niche-attractantSDF-1 has been proposed to be an important player of earlymicrometastasis (19, 20).

Several CXCR4 antagonists have been developed for therapeuticapplications, including HSC mobilization, and as antitumor ther-apies (21). In preclinical studies, small-molecule inhibitors ofCXCR4 induced prostate cancer cell mobilization in vivo (22) anddecreased pulmonary metastases in models of both osteosarcoma(23) andmelanoma (24). POL5551 (Polyphor Ltd.) is a novel fullysynthetic cyclic peptide antagonist of CXCR4 developed usingprotein epitope mimetic (PEM) technology (25). A representativestructure of a PEMmolecule incorporating a b-hairpin is shown inSupplementary Fig. S1A. Like the FDA-approved CXCR4 inhibitorplerixafor, POL5551 competes with SDF-1 for the extracellularloop–binding site of CXCR4 (pharmacology summarized in Sup-plementary Table S1). POL5551 has a higher affinity for CXCR4and an increased HSC mobilization activity compared with pler-ixafor (26). At high doses inmice, POL5551mobilized HSC levelssimilar to that produced by G-CSF, a far greater mobilization thanachievedwithplerixafor, or thathas been reported for otherCXCR4antagonists (26). In mouse models, POL5551 has been demon-strated to inhibit neointima hyperplasia in a model of atheroscle-rosis (27) and to prolong survival when added to anti-VEGFtherapy in a model of glioblastoma (28).

In this study, we found that, in stage II/III breast cancerpatients who did not have detectable bone marrow DTC,tumoral CXCR4 expression could identify patients at risk forearly mortality and metastasis. We hypothesized that antago-nism of the CXCR4 receptor with POL5551 would reducemetastases and improve survival in CXCR4-expressing breastcancer, and addressed this hypothesis in preclinical models. Wefound that POL5551 inhibited tumor cell migration anddecreased adhesion-independent survival in vitro, although ithad no direct effect on tumor cell viability. In agreement,single-agent POL5551 reduced tumor cell metastasis to thedistant sites outside the chest wall and lung, despite havingno effect on the primary tumor growth, which suggests adisruption of the metastatic process rather than a direct anti-tumor effect. When combined with low-dose eribulin chemo-therapy, POL5551 further decreased distant metastasis andprolonged survival in an orthotopic breast cancer model. We

hypothesized that part of POL5551's additive effects onchemotherapy could be explained by the displacement ofcancer cells from chemoprotective niches in the tumor micro-environment. When administered shortly before and aftereribulin, POL5551 treatment further reduced bone tumor bur-den. Our study suggests that combining CXCR4 antagonism tochemotherapy may be beneficial to treat TNBC patients, andprovides supporting evidence to a newly initiated clinical trialwith the CXCR4 antagonist POL6326 (ClinicalTrials.gov:NCT01837095).

Materials and MethodsPatient clinical trial information

A total of 120 patients with newly diagnosed stage II–III breastcancer were enrolled on an open label randomized single-blindphase II trial (NCT00242203) to assess the efficacy of zoledronicacid (ZA) in reducing DTCs and improving bone health. Eighty-one patients who had evaluable data for both bone marrow DTCand primary tumor gene-expression profiling were chosen for thisstudy (Supplementary Table S3). Exclusion criteria includedprevious history of malignancy, Easter Cooperative OncologyGroup (ECOG) score of 2 or higher, and evidence of distantmetastases by CT scan (of pelvis, abdomen, and chest) or 99TC-MDP bone scan. The complete enrollment criteria and results ofthe study have been previously described (29, 30). Patients weremonitored every 3 to 4 months for the first 2 years after enroll-ment, then every 6 months until year 5, and then annuallythereafter. In the context of this trial, bone marrow aspirates werecollected from each anterior iliac crest before treatment initiation,and DTC identified by cytokeratin staining as previouslydescribed (31). Patients were considered to be positive for DTC(DTCpos), if at least one CK-positive cell was isolated from eitherBM aspirate (31). Gene-expression profiling was performed onthe tumor biopsies of 81 of these patients (GEO accession #:GSE71258). A cutoff value for CXCR4 expression was selectedusing the dedicated Cutoff Finder application (http://molpath.charite.de/cutoff/; ref. 32) to analyze the entire set of 81 patients.On the basis of the distribution of primary tumor CXCR4 expres-sion data, a cutoff value (10250) was determined: CXCR4 expres-sion signal above 10,250was defined as CXCR4High (10 patients),whereas below 10,250 was defined as CXCR4Low (28 patients).The overall survival (OS) by Kaplan–Meier curve and log-rank testwas analyzed between CXCR4High and CXCR4Low patients. Toinvestigate the association betweenCXCR4 expression andmicro-metastases at diagnosis, we then compared frequencies of DTCpos

inCXCR4high versusCXCR4lowpatients, and tested the associationby Fisher exact test and OR. To analyze the effect of CXCR4 onsurvival, we considered the gene-expression profiling and DTCstatus of the 38 of 81 patients that were assigned to the placeboarm of the trial. Of these, 16 patients had bone marrow micro-metastases. DTCneg (20 patients) were divided into CXCR4high

(n ¼ 5) and CXCR4low (n ¼ 15) and analyzed for OS by Kaplan–Meier curve analysis and log-rank test.

Cell lines and reagentsThe 4T1-FL-GFP BALB/c murine breast cancer cell line was

generously provided by Dr. David Piwnica-Worms (The Univer-sity of Texas MD Anderson Cancer Center, Houston, TX) andmodified to coexpress firefly luciferase and GFP as previouslydescribed (33).

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The bone metastatic MDA-MB-231 variant (34) was a kind giftof Dr. T. Guise (Indiana University, Bloomington, IN). CXCL12-CGLuc, NGLuc-CXCR4, and NGLuc-CXCR7 MDA-MB-231 cellswere generated as previously described (35).

Cells were cultured at subconfluence in DMEM þ 10% FBS þ0.5% penicillin–streptomycin (pen/strep). Low-passage stockswere used and regularly tested for Mycoplasma and luciferaseactivity and maintenance of growth characteristics.

POL5551 was supplied by Polyphor. POL5551 has a template-bound b-hairpin peptide structure and belongs to a series ofpotent CXCR4 antagonists developed by Polyphor usingthe proprietary PEM technology (36). The peptide backbone ofPOL5551 was synthesized using a standard fluorenylmethoxy-carbonyl (Fmoc) solid-phase strategy on highly acid-labilechlorotrityl chloride resin (100–200 mesh, Novabiochem, 01-64-0114). A disulfide bond was installed on the resin, andmacrocyclization was performed in solution. The peptide waspurified by preparative reverse-phase HPLC. Conversion intoacetate salt was performed using an ion-exchange resin, and thefinal preparation of POL5551 was recovered by lyophilization. Inmice, POL5551 was well tolerated when exposed at intravenousbolus doses up to 120 mg/kg, with little effect on blood pressure,autonomic nervous, hepatic or cardiac tissues, and no effect onmortality during subsequent 3 days after dosing was observed(27). The plasma half-life of POL5551 inmice after subcutaneousadministration is about 30 minutes (26), the exposure profilefollowing subcutaneous administration of a single 30-mg/kg doseto mice is provided in Supplementary Fig. S1B. For in vitroexperiments, POL5551 was dissolved in PBS to desired concen-tration. For in vivo studies, POL5551 (20 mg/kg) was diluted insaline and administered by subcutaneous injection.

Eribulin (trade name:HALAVEN)was purchased fromEisai Co.Eribulinwas dissolved in PBS in vitro to desired concentration. Forin vivo studies, eribulin was diluted in saline and administered byi.v. injection once a week at 0.1 mg/kg for primary mammary fatpad therapy and 0.2 mg/kg for metastatic therapy.

Split luciferase assayFor the split-luciferase assay, CXCL12-CGLuc or unfused

CGLuc MDA-MB-231 cells (2 � 104 cells per well in the 96-wellplate) were coincubated overnight with NGLuc-CXCR4 orNGLuc-CXCR7 MDA-MB-231 cells in DMEM with 0.5% FBS/0.5% penicillin/streptomycin, followed by incubation with indi-cated concentrations of POL5551 for 6 hours. Bioluminescencefrom Gaussia luciferase complementation was measured 4 hourslater using the BioLux Gaussia Luciferase Assay Kit (New EnglandBiolabs) according to the manufacture's protocol.

MTT assayThe MTT assay was performed as described previously (37).

Scratch wound assayMDA-MB-231 cells (105 cells/well in 24-well plate) were seed-

ed to form a confluent monolayer. After overnight serum starva-tion (0.5%FBS), awoundgapwas createdby scratchwith apipettetip and POL5551 (0.1–5 mmol/L) was added. Images of cells weretaken with a Nikon Eclipse TE300 invertedmicroscope connectedto a Magnafire camera model S99802 (Optronics), as previouslydescribed (38). The extent of gap closure was measured after 24hours using ImageJ (NIH).

Survival assayTo test for survival, MDA-MB-231 cells were plated to 6-well

ultra-low attachment plates at a cell density of 5� 105 per well in0.5% FBS DMEM. After 48 hours of incubation with SDF-1 (12.5ng/mL and 50 ng/mL) and in the presence or absence of POL5551(8 mmol/L), 1:10,000 aliquot of the cells were plated to 6-wellplates and grown in10%FBSDMEMfor aweek.Cellswerefixed in10% buffered formalin and stained with 0.5% crystal violetdissolved in 1% SDS. Cell density was quantified by measuringthe absorbance at 570 and 630 nm by a plate reader (BioTek;ref. 39).

Animal studiesBALB/c and NOD-scid-IL2R gammanull (NSG) mice were

obtained from The Jackson Laboratory. Animals were housedunder pathogen-free conditions according to the guidelines of theDivision of Comparative Medicine, Washington University (St.Louis, MO). All animal experiments were approved by theWashington University Animal Studies Committee.

For xenograft experiments, 6- to 8-week-old female NSG micewere inoculated with 5� 105 MDA-MB-231 cells in Matrigel (BDBiosciences) in the #9 mammary fat pad to generate orthotopicbreast tumors. As an experimental model of bone metastasis, 1�105 4T1 or MDA-MB-231 cells were injected into the left cardiacventricle, as previously described (40).

In neoadjuvant–adjuvant regimens, POL5551 was adminis-tered at a dose of 20 mg/kg s.c. twice a day from day 7 as amonotherapy, or from day 10 in combination with eribulin.Eribulin was administered on days 10, 17, and 24 (0.1 mg/kg,i.v.). For the "framing dosing" experiment, on day 10, POL5551(20 mg/kg) was administered s.c. 4 hours before, 4 hours after,and again 18 hours after chemotherapy with erubulin (0.2mg/kg,i.v.). A simulated exposure profile following administration ofPOL5551 using this dose regimen is shown in Supplementary Fig.S1C. Vehicle-treated controls received saline solution (i.v. or s.c. asappropriate, see individual experiments for details).

Bioluminescence imagingBioluminescence imaging was performed using an IVIS 100

device (Caliper Life Sciences), as previously described (40).

Microcomputed tomographyTibiae and femurswere removedpost-mortem, scanned in a17-

mm holder using microcomputed tomography (mCT-40; ScancoMedical), and evaluated as described previously (38).

RadiographyOsteolytic lesion was imaged by an X-ray imaging system

(Faxitron). Quantification of the osteolytic lesion area was com-pleted using Image-Pro Plus (MediaCybernetics).

Complete blood countWhole blood was analyzed on a Hemavet Automated Coulter

Counter (CBC Tech).

ImmunohistochemistryImmunohistochemistry staining was completed by a routine

ABC method according to protocol (available online). Antigenretrieval was by the microwave heating method with 10 mmol/Lsodium citrate buffer (pH, 6.0). The anti-human keratin 18

Preclinical Evaluation of POL5551 in Breast Cancer Models

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antibody (Spring Bioscience) was used at 1:200. The anti-F4/80antibody and anti-CD31 antibody (Abcam) were used at 1:500and 1:400, respectively. Anti-rabbit (PerkinsElmer, Inc.) or rat(Jackson ImmunoResearch Laboratories, Inc.) secondary anti-body was used at 1:800 and SA-HRP antibody (Jackson Immu-noResearch Laboratories, Inc.) was used at 1:1,600. Images ofhistology slides were acquired using a Nanozoomer digital slidescanner (Hamamatsu Photonics), and image data analysis wasdone using Visiomorph software (Visiopharm).

Statistical analysisExperiments were analyzed using a two-tailed Student t test (2

groups), one-way ANOVA (>2 groups or repeated measures), ortwo-way ANOVA (two variables, P value refers to the interaction)using Prism5 (GraphPad Software, Inc.). Results were consideredto reach significance at P� 0.05 and are indicatedwith asterisks (�,P� 0.05; ��, P� 0.01; ��, P� 0.001). Data are presented asmeanvalues; error bars represent SEM. Mouse weight changes arepresented as mean and range.

ResultsHighCXCR4 expression associated with high rate ofmetastaseseven in patients without detectable bone marrow DTCs

The presence of microscopic DTC in bone marrow is anindependent poor prognostic factor for patients with localizedbreast cancer (18). As CXCR4 also correlates with metastaticpotential (41, 42), we investigated the prognostic implicationsof CXCR4 expression in the primary tumor and the presence ofbone marrow DTC, using gene-expression profiling of primarybreast tumors from patients enrolled in our previous phase II trialevaluating the effect of ZA and chemotherapy on DTC in patientswith locally advanced breast cancer (29, 31). In this study, 81patients had evaluable data for both bone marrow DTC andprimary tumor gene-expression profiling. From this dataset, wedetermined the cutoff value for high and low populations basedon the distribution of CXCR4 expression signal from all patients.We found thatDTCswere present in 5 of 10 (50%)CXCR4high and13of 28 (46%)CXCR4low patients, with no significant associationbetween the presence of DTCs and CXCR4 expression (Fisherexact test P ¼ 0.72; OR, 0.6; 95% confidence interval, 0.1–2.6;Supplementary Table S2). Both CXCR4high and CXCR4low popu-lations had comparable frequencies of DTCs relative to the totaltrial dataset (Supplementary Table S3; ref. 29), and DTC frequen-cies obtained in other published studies (43).

To analyze the effects of DTC and CXCR4 status on prognosis,we then considered the standard chemotherapy alone arm of thestudy (38 evaluable patients). The ZA treatment armwas not used

because it has been previously reported that treatment of ZA canincrease disease-free survival in patients with breast cancer (44–46). Eighteen of 38 (47.3%)of these patients had detectableDTCsin the bone marrow at diagnosis. We first evaluated the impact ofCXCR4 expression in the primary tumor (Fig. 1A and Supple-mentary Fig. S2) on survival, and found that high CXCR4 expres-sion was associated with poor survival, consistent with previousreports (47), and with a higher incidence of metastases (Table 1)to bone, lung, and liver (60%), than CXCR4low patients (25%).Importantly, metastases took over five times longer to develop inthe CXCR4low expression patients (median of 55 months fromdiagnosis) comparedwith those in the CXCR4high patients (medi-an of 10 months). We next evaluated CXCR4 expression in theDTC-negative population. We found that DTCnegCXCR4high

patients had a significantly lower survival (��, P ¼ 0.005; Fig.1B) than DTCnegCXCR4low. These data suggest that that lowCXCR4 expression and the absence of bone marrow DTC definea highly favorable prognosis group in patients with locallyadvanced, stage II and III breast cancer. Because we found thathigh CXCR4 expression was associated with metastasis and poorprognosis even in patients with no detectable bone marrow DTC,we hypothesized that blocking CXCR4 activity may reduce bothvisceral and bone metastases.

CXCR4-selective antagonist POL5551 decreases breast cancercell migration and promotes anoikis

We evaluated a novel fully synthetic cyclic peptide CXCR4antagonist, POL5551, for effects on breast cancer cell viabilityandmigration. Breast cancer cells can express bothof theCXCL12/SDF-1 receptors, CXCR4 and the alternative inhibitory receptorCXCR7 (48). To confirm POL5551's specificity for CXCR4 overCXCR7, we used a complementation-imaging model (35), inwhich the N-terminal portion of the Gaussia luciferase (GLuc) isfused to CXCR4 or CXCR7 whereas the C-terminal portion ofGLuc is fused to the ligand SDF-1. In the presence of coelenter-azine, reconstitution of GLuc and subsequent light productionserves as a quantitative measure of receptor-ligand binding andPOL5551 bioactivity (Supplementary Fig. S3A). POL5551 inhib-ited luciferase activity at low nanomolar concentration in humanMDA-MB-231 breast cancer cells expressing GLuc-engineeredCXCR4, but not GLuc-CXCR7, demonstrating specific bioactivityagainst CXCR4 and not CXCR7 (Fig. 2A).

We investigated the bioactivity of POL5551 on breast cancercell viability and migration, because the activation of the CXCR4receptor with ligand SDF-1 mediates many cellular functions,including chemotaxis and survival (1). Under standard cultureconditions, neither the chemokine SDF-1 nor the CXCR4

Figure 1.High CXCR4 expression associatedwith high rate of metastases even inpatients without detectable bonemarrow DTCs. A, OS of patients withprimary breast tumors that areCXCR4High (n ¼ 10) and CXCR4Low

(n ¼ 28). B, OS of patients thatare DTCnegative characterized byCXCR4High (n¼ 5) orCXCR4Low (n¼ 15)expression of the primary tumor.

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antagonist POL5551 had significant effects on MDA-MB-231viability (Fig. 2B). However, when cultured in anoikis-inducingsuspension conditions (0.5% FBS and ultra-low attachmentplates), SDF-1 promoted the survival of MDA-MB-231 cells in aconcentration-dependent manner, and POL5551 significantlydecreased the survival of tumor cells in suspension (Fig. 2E andF). Others and our own data have shown that CXCR4 antago-nists, such as AMD3100, can block SDF-1–mediated migrationof breast cancer cells (Supplementary Fig. S3B; ref. 49). In ascratch migration assay, POL5551 decreased chemotacticmigration of SDF-1–expressing MDA-MB-231 cells in a con-centration-dependent manner (Fig. 2C and D). These datashow that POL5551 had little direct bioactivity on breast cancercell viability, but inhibited SDF-1–dependent migration andresistance to anoikis.

CXCR4 antagonist POL5551 decreases metastasis but has littleeffect on primary tumor growth

Because POL5551 decreased CXCR4-mediated migration andtumor cell survival specifically in suspension, we evaluated theeffect of POL5551 on local recurrence and distant metastasis.Orthotopic mammary fat pad (MFP) MDA-MB-231 tumors wereestablished in immunocompromised NSG mice. POL5551monotherapy was initiated when tumors were palpable (day 7)and continued until study termination. PrimaryMFP tumorsweresurgically resected by mastectomy on day 21 and mice werefollowed for the development of local recurrence and distantmetastasis by bioluminescent imaging (BLI; Fig. 3A). We con-firmed that the POL5551 dosing used in this study was sufficientto mobilize hematopoietic cells into the blood (22) and increaseWBC counts (Supplementary Fig. S4). AlthoughPOL5551 did notreduce primary tumor growth (Fig. 3B and C and SupplementaryFig. S4), distant metastases to chest and leg bones, as assessed byBLI, were significantly decreased in POL5551-treated mice (Fig.3D, E, andH).Metastasis to the liver was examined by both ex vivoBLI and histology; however, no significant differences were foundbetween the treatment groups (Fig. 3I; Supplementary Fig. S4Band S4C). Micrometastatic tumors in the lung, as quantified

Table 1. Metastasis profiling of CXCR4High and CXCR4Low patient groups

Percentage of patientswith distant metastasis

First metastasis detected(median months; range)

CXCR4High 60%; 6/10 a10; (4–35)CXCR4Low 25%; 7/28 55; (8–104)aP < 0.005; metastases detected in bone, liver, lung, and other organs.

Figure 2.CXCR4-selective antagonist POL5551decreases breast cancer cell migrationand promotes anoikis in vitro. A,complementation imaging. Binding ofSDF-1-CGLuc to NGLuc-CXCR4 orNGLucCXCR7 reconstitutes GLuc,producing light as a quantitativemeasure of ligand:receptor binding.SDF-1-CGLuc MDA-MB-231 cells werecoincubated overnight with NGLuc-CXCR4 or NGLuc-CXCR7 MDA-231 cells,followed by incubation with variousconcentrations of POL5551 for 6 hours(�� , P < 0.001). B, MDA-231 cells wereincubated with various concentration ofPOL5551 for 48 hours and cell viabilitywas assessed by an MTT assay. C and D,a wound gap was created by scratch inconfluent monolayers of SDF-1–secreting MDA-231 cells. Gap closurewas measured after 24 hours oftreatment with various concentrationsof POL5551 (� , P < 0.05; �� , P < 0.001). Eand F, cell survival after forcedsuspension culture with SDF-1 (12.5 and50 ng/mL) and POL5551 (8 mmol/L). Cellcolonieswere stainedwith crystal violet.The absorbance of crystal violet dyedissolved with 1% SDS was measured byplate reader at 570 to 630 nm(� , P < 0.05; �� , P < 0.01; �� , P < 0.001).






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histologically by measurement of human cytokeratin-18 (CK-18)–positive tumor cells, were significantly decreased inPOL5551-treated mice compared with vehicle controls (P ¼0.001; Fig. 3F and G).

To confirmPOL5551 inhibition ofmetastasis in the context of afully competent immune system and in a metastasis model, 4T1murine breast cancer cells were inoculated into the left cardiacventricle of BALB/c mice to establish disseminated metastaticdisease. Mice were treated with either POL5551 or saline fromday 3 to the end of the study (Fig. 4A). Histomorphometricanalyses showed that POL5551 treatment significantly decreasedtumor metastases into the kidney (P ¼ 0.03; Fig. 4D and E), andmicro-CT showed a significant reduction in tumor-associatedbone loss (P¼ 0.03; Fig. 4F andG). Thesefindingswere associated

with a nonsignificant trend toward reduction of bone and whole-body tumor burden when measured by BLI (Fig. 4B and C).Together, these data show that POL5551 as a single agent hadlittle direct effect on primary tumor burden, but decreased metas-tases and metastatic tumor burden in two breast cancer models.

Combination of eribulinwith POL5551 in the neoadjuvant andmetastatic settings improves survival compared with eribulinalone

In addition to investigating the effect of POL5551 administeredas a monotherapy, we also evaluated the effect of POL5551 incombination with cytotoxic chemotherapy. Eribulin is a micro-tubule-inhibiting chemotherapeutic agent, with strong activity inpatients with metastatic breast cancer that have failed both

Figure 3.CXCR4 antagonist POL5551 decreases metastasis, but has little effect on primary MFP tumor growth in a xenograft NSG model. A, MDA-MB-231 mammaryfat pad orthotopic model schema: MDA-MB-231 tumor cells were injected to the mammary fat pad of mice. Treatment with POL5551 (20 mg/kg, s.c., twicedaily, n ¼ 3) or saline (n ¼ 5) commenced on day 8 when the tumor became palpable and continued until the end of study. B and C, primary MFP tumor growth asmeasured by bioluminescence imaging, representative image showing the primary tumors on day 21. D and E, distant metastasis to the chest as measured bybioluminescence imaging, representative image showing the distant metastases on day 41 (� , P < 0.05). F, human CK-18 staining of MDA-MB-231 tumor cellsin lung tissue. G, quantification of CK18-positive cells in lung tissue; �� , P < 0.001; H, ex vivo BLI of bone metastases (� , P < 0.05). I, ex vivo BLI of liver metastases(ns, P > 0.05).

Figure 4.POL5551 monotherapy decreaseddistant metastasis in a disseminatedtumor model. A, an intracardiac tumorinjection model, 4T1-Luc murine tumorcells were injected into the left ventricleof the BALB/c mice. Treatment withPOL5551 (20 mg/kg, s.c., twice daily,n ¼ 7) or vehicle (saline, n ¼ 6) wasfrom days 3 to 11. Mice were sacrificedon day 11. B and C, BLI of 4T1-Luc tumorburden in leg bones (ns, P ¼ 0.23) andwhole body (ns, P ¼ 0.08). D, H&Estaining of kidney tissue, 4T1 tumorfoci/nodules are outlined in white toincrease visibility. E, quantification of4T1 tumor foci in kidney per section(n¼ 6/group; �� , P <0.01). F andG, mCTanalysis for calculation of trabecularbone to tissue volume ratio (BV/TV; n¼6/group; � , P < 0.05).






anthracycline and taxane chemotherapies (50–52). Eribulindecreased MDA-MB-231 viability in vivo (Supplementary Fig.S5A–S5C) and prolonged mice survival in a dose-dependentmanner (Supplementary Fig. S5D).However, the higher andmosteffective doses were associated with greater chemotherapy-induced weight loss (Supplementary Fig. S5E). Aiming at iden-tifying a combination regimen that would reduce toxicity andreveal synergistic effects with CXCR4 inhibition, we selected asubmaximal effective dose of eribulin (0.1 mg/kg) that yielded50% primary breast tumor reduction when used as single agent(Supplementary Fig. S5F). After orthotopic MFP tumors wereestablished, neoadjuvant treatment was initiated on day 10, andmastectomy was performed when all tumors reached 1 cm3, sothat metastasis-free survival after surgery could be fairly evaluatedfor all treatment groups. Because of the antitumor effect of

eribulin on primary growth, chemotherapy-treated mice requiredadditional time (roughly 5 extra days) to reach primary tumormass equivalent to the vehicle group. All doses of eribulin wereadministered before mastectomy, whereas POL5551 administra-tion continued after mastectomy. As expected, eribulin decreasedtheprimary tumor growth rate,whereas POL5551hadnodirect oradditive effects on primary MDA-MB-231 MFP tumors (Fig. 5A–C). In contrast, combination POL5551 plus eribulin treatmentsignificantly decreased distantmetastases to the chest and bone asmeasured by BLI (Fig. 5D and E). Finally, POL5551, in combi-nationwith eribulin, prolonged survival (58days) comparedwitheribulin alone (51 days) or vehicle controls (45 days; Fig. 5F).

Breast cancer cell interactions with the stromal environmentcan contribute to resistance to cytotoxic chemotherapy (11). Wehypothesized that antagonizing CXCR4 with POL5551 for short

Figure 5.Combination of eribulin with POL5551in the neoadjuvant setting improvessurvival compared with eribulin alone.A, experimental design: MDA-MB-231cells were mixed with Matrigel andimplanted s.c. into the right flank ofthe NSG mice. Ten days after tumorimplantation,mice (n¼ 10/group)weretreated as follows: A, vehicle saline (i.v.)on days 10, 17, and 24) plus (s.c., twicedaily) from day 10 to the day of surgery;B, eribulin alone (0.1mg/kg, i.v) on days10, 17, and 24; C, eribulin treatment(days 10, 17, and 24) plus POL5551(20 mg/kg, s.c., twice daily)administration daily from day 10 untiltermination. B, primary tumor sizemeasured by caliper. C, primary tumorweight at surgery (ns, P > 0.05). D,bioluminescence imaging of distantmetastasis to the chest (� , P < 0.05).E, bioluminescence imaging of distantmetastasis to the bone in eribulin aloneand combo group (�� , P < 0.001). F,Kaplan–Meier survival curve.

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periods of time around each dose of chemotherapy—a "framingdosing" strategy—could disrupt CXCR4/SDF-1–mediatedtumor–stromal cell interactions and potentially enhance chemo-therapy efficacy. To test this hypothesis, metastases were estab-lished by intracardiac inoculation of MDA-MB-231 cells, andPOL5551 was administered 4 hours before eribulin and 4 and18 hours after eribulin chemotherapy (Fig. 6A). After intracardiacinjection, MDA-MB-231 cells preferentially metastasize to theliver and leg bones (Fig. 6F). The rate of distant metastasis ofmice with successful left ventricle injectionwas 100%. In the hindleg bones, themetastasizing tumor cells produce severe osteolyticlesions, usually detectable by X-ray 2 weeks after inoculation.Compared with vehicle control and eribulin alone, POL5551 incombinationwith eribulinwas superior in decreasing bone tumorburden as measured by BLI (Fig. 6C). In the liver, althougherubulin was very effective in reducing tumor burden (BLI) by75%, this result was further reduced (86%) in the POL5551þ

eribulin group (Fig. 6B; P > 0.05 between eribulin-alone andcombo groups). In the bone, POL5551 in combination witheribulin produced a significant reduction in BLI, and fewer

tumor-associated osteolytic areas were detectable on X-ray, com-pared with the vehicle and eribulin-alone groups (Fig. 6D and E).Progressive metastatic tumor burden results in weight loss in thismodel, and the combination of POL5551 with eribulin wassuperior to eribulin alone in preventing tumor-associated weightloss (Fig. 6G; P < 0.05). Together, these data suggest that short,sequenced dosing of POL5551 with eribulin chemotherapy canprevent tumor-associated weight loss and decrease tumor burden.

DiscussionOur work shows that high CXCR4 expression in primary breast

cancer tissue defined a subgroup of patients that, despite the lackof bone marrow DTCs and other favorable characteristics, hadhigh and early incidence of metastasis and poor survival. It hasbeen proposed that CXCR4 antagonism could directly affecttumor cell survival at two stages of metastasis. Early in progres-sion, CXCR4 inhibitors may inhibit tumor cell homing from theprimary tumor to metastatic sites expressing high SDF-1. Aftertumor cells have disseminated to secondary sites and are protected

Figure 6.Reduction of bone tumor burden:POL5551 in combination with eribulin issuperior to eribulin alone. A, schema ofbone metastasis model utilizing"framing dose" strategy: MDA-MB-231bone metastases were established byintracardiac inoculation in NSG mice,and on day 10 mice were treated asfollowing: A, vehicle (n ¼ 4): saline (s.c.and i.v.); B, eribulin alone (n ¼ 5):eribulin (0.2 mg/kg, i.v.) alone andsaline (s.c); C, combination treatment (n¼ 5): POL5551 (20 mg/kg, s.c.) wasadministered 4 hours before eribulinand 4 and 18 hours after eribulin (0.2mg/kg, i.v.) chemotherapy. B and C, BLIof tumor burden in liver and bone(� , P < 0.05; �� , P < 0.001). D,representative X-Ray images of tumor-associated osteolytic lesion in leg bone.E, quantification of osteolytic lesionarea. F, representative image showingextent of metastatic burden in liver andleg bone on day 17 (� , P < 0.05;�� , P < 0.01). G, mouse weight lostbetween days 9 and 17. Statisticalanalyses are as compared with vehicletreatment (ns, P > 0.05; ��, P < 0.01).






from chemotherapy, CXCR4 antagonists may mobilize tumorcells into the peripheral blood, as occurs with HSCs. Once incirculation, tumor cells are no longer protected by interactionswith stromal cells in the HSC niche and could become moresusceptible to therapeutic agents, including chemotherapy, result-ing in reduced metastases and tumor burden. We hypothesizedthat inhibition of CXCR4 could decrease metastasis. We foundthat the CXCR4 antagonist POL5551 blocked breast cancer cellmigration and resistance to anoikis in vitro, and reduced breastcancer metastasis despite having little effect on primary tumorgrowth in vivo. When administered in combination with eribulinchemotherapy, POL5551 decreased metastases and improvedsurvival compared with chemotherapy alone. We hypothesizedthat POL5551 could "mobilize" tumor cells from their stromaland vascular niches, and heighten sensitivity to chemotherapy.We found that only 3 doses of POL5551 given just before and justafter eribulin further decreased metastatic tumor growth com-pared with chemotherapy alone. Taken together, our findingssuggest that POL5551 is capable of disrupting interactionsbetween cancer cells and their environment that contribute totheir metastatic potential and resistance to chemotherapy.

It has been previously reported that POL5551 is a potentmobilization agent that can mobilize hematopoietic stem andprogenitor cells from bone marrow niche with greater efficiencythan AMD3100 (plerixafor; ref. 26). In confirmation of this, thePOL5551 dose we used in vivo rapidly mobilized leukocytes intothe circulation (Supplementary Fig. S6). Duringmetastatic dissem-ination, CXCR4-expressing cancer cells are thought to use theHSC-specific pathways to preferentially home to SDF-1–rich sites, suchas bone, lymph nodes, and lungs. In a mouse metastasis model,Shiozawa and colleagues (8) demonstrated that prostate cancercells can directly competewithHSC to occupy theHSCniche in thebone marrow through the CXCR4–SDF-1 pathway, and thatadministration of the CXCR4 antagonist AMD3100 mobilizedDTCs into the peripheral blood. Stromal environments in the boneor lung have been shown to be protective and contribute tochemoresistance to cytotoxic therapies (11);mobilizationof tumorcells from the protective microenvironment may, therefore,increase their susceptibility to cytotoxic chemotherapy.

Twomain chemokine receptors capable of SDF-1 binding havebeen found toplay some role inbreast cancer: CXCR4andCXCR7.Although CXCR7 may play a role in transendothelial migration(53), Hernandez and colleagues (54) demonstrated that CXCR7overexpression enhanced tumor growth and vascularization, butreduced invasiveness and metastases. Specifically designed toselectively target CXCR4, POL5551 showed no effect on SDF-1binding to overexpressed CXCR7 by a Gaussia luciferase comple-mentation assay in breast cancer cells. Moreover, in agreementwith previously published datasets (55), we did not detect CXCR7expression inMDA-MB-231 cells. We therefore consider it unlike-ly that CXCR7 might play a major role in the current set ofexperiments.

Targeting a physiologic characteristic of the cell of origin,together with a protumor gain-of-function, a number of clinicaltrials have used CXCR4 antagonists in hematologic malignancies,such as multiple myeloma, acute myelogenous leukemia, andlymphoma (14). In solid tumors, CXCR4overexpressionhas beenassociated with poorer prognosis, and functional studies haveshown that CXCR4 plays a critical role in the metastatic dissem-ination of solid tumor cells (56, 57). In accordance with thishypothesis, we found that, even in patients with no bonemarrow

micrometastases (DTC negative), high CXCR4 expression in theprimary tumor was associated with earlier metastases and poorerprognosis.

In the host, the CXCR4 gene plays an important role in anumber of cell types and physiologic processes, such as HSCsand leukocytes trafficking. In our previous work, we have shownthat targeted gene disruption of host hematopoietic CXCR4resulted in enhanced osteoclastogenesis and consequent tumorgrowth in bone, although no differences were found in lung andsubcutaneous tumor burden (58). Bypharmacologically targetingthe CXCR4 receptor with an antagonist, especially with an inter-mittent regimen, CXCR4 expression by normal tissues would notbe constantly disrupted, thus potentially having less sustainedeffects on leukocytosis. Indeed, mice did not show overt hemato-poietic toxicity as measured by serial blood counts following aframing dose of POL5551 in combination with eribulin (Sup-plementary Fig. S7). It should be noted that not all HSCs and/orDTCs are mobilized after a single dose of a CXCR4 antagonist, sorepeated dosing may have higher efficacy. In this study, we foundthat POL5551 did not induce bone loss and in fact resulted indecreased tumor-associated bone loss. This is likely due todecreased tumor burden in the bones, which is consistent withthe reduction of metastatic tumor burden in extra-skeletal sites(lungs and kidneys), and with the observed effects on the migra-tion and survival of isolated tumor cells. We also anticipate thatthe pharmacologic dosing of POL5551 would not affect osteo-clasts to the same extent as complete genetic disruption.

Amongbreast cancers, the TNBC subtypehas themost aggressivephenotype with a poorer prognosis (59–61). Unlike ER- or HER2-positive breast cancers, which benefit from targeted therapies, thetreatment of TNBC patients largely relies on surgery and systemicchemotherapy. The microtubule inhibitor eribulin has shownsignificant survival benefit in patients withmetastatic breast cancerrefractory to anthracyclines and taxanes (51, 62). As with mostantiproliferative drugs, in addition to the killing effect of chemo-therapy on rapidly dividing tumor cells, chemotoxicity oftenoccurs. In a dose-ranging experiment, in which we treated micebearingMDA-MB-231 tumorswith eribulin, a highdoseof eribulin(>0.5mg/kg) significantly improved survival, but to the expense ofgreater treatment-associated weight loss due to chemotoxicity. Inaddition, optimal eribulin dosing reduced tumor burden to a pointthatwasbelow the level inwhichour in vivobioluminescence assayswere able to consistently and confidently measure tumor burden,precluding our ability to detect any synergistic effects of POL5551.For these reasons, suboptimal and less toxic dosing of eribulin(0.1–0.2 mg/kg) was used for this preclinical study. In agreementwith previous research that has shown CXCR4 inhibition to sen-sitize tumor cells to chemotherapy (13, 63), we found that com-bining POL5551 with low-dose eribulin (0.1–0.2 mg/kg)decreased metastatic tumor burden compared with mice admin-istered eribulin alone. Together with other studies in publishedliterature, our preclinical data suggest that combining CXCR4antagonism and cytotoxic chemotherapy could decreasemetastasisbymobilizing tumor cells or otherwisedisrupting their interactionswith chemoprotective stromal environments. In the present study,POL5551 administered just before and after eribulin was sufficientto decrease tumor burden.

Few clinical trials have so far targeted CXCR4 in solid tumors,despite encouraging preclinical studies and associations betweenexpression and prognosis. A phase I study using a peptide CXCR4antagonist, LY2510924, in patients with advanced cancer was

Xiang et al.





recently completed in the United States (64). However, no ded-icated breast cancer trial has so far been designed to evaluateCXCR4 antagonism. This study has provided important preclin-ical rationale and proof-of-principle data to examine the combi-nation of CXCR4 antagonism and eribulin chemotherapy inmetastatic breast cancer patients. To translate these findings frombench to bedside, a multicenter phase I clinical trial ("Doseescalation of POL6326 in combination with eribulin in patientswith metastatic breast cancer," ClinicalTrials.gov identifier:NCT01837095, sponsored by Polyphor), has been initiated. Thisstudy will evaluate POL6326, a close analogue of POL5551,which differs at only a single amino acid, has nearly identicalbioactivity profiles, but has slightly more favorable pharmacoki-netic properties than POL5551. Here, POL6326 will be evaluatedin combination with eribulin in a framing dose sequence similarto what we used in the preclinical studies presented in Fig. 6. Thisis the first clinical trial in breast cancer patients with a CXCR4antagonist in combination with chemotherapy. In addition,POL6326 is currently in clinical studies for HSC mobilization.

Although the overall magnitude of survival extension in thispreclinical study was modest (as compared with vehicle, mediansurvival was increased 12% with eribulin alone and 22% witherubulin þ POL555), it is important to note that a suboptimaldose of eribulin was used for this study. This was necessary topermit a level of tumor growth sufficient for detection by in vivobioluminescent assays. Thus, when optimal doses of eribulin areused, as in the ongoing phase I clinical trial above, additionalbenefit may be seen. Our preclinical data suggest that CXCR4–SDF-1 interactions play a critical role during breast cancer metas-tasis, and that CXCR4 antagonism administered in sequence withchemotherapy can disrupt tumor cell spread. Future studies toevaluate the impact ofCXCR4 inhibition on tumor cell dormancy,tumor microenvironment, and immune modulation will beimportant to better define which patients to treat and how tocombine CXCR4 antagonist with ongoing cancer therapies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Xiang, M.A. Hurchla, X. Su, G.J. Douglas, T. Pluard,E. Chevalier, K. Dembowsky, K.N. Weilbaecher

Development of methodology: J. Xiang, M.A. Hurchla, X. Su, K.E. Luker,G.D. Luker, K.N. WeilbaecherAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Xiang, M.A. Hurchla, F. Fontana, S.R. Amend,A.K. Esser, K.E. Luker, R.L. Aft, K.N. WeilbaecherAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Xiang, M.A. Hurchla, F. Fontana, S.R. Amend,G.J. Douglas, C. Mudalagiriyappa, F.O. Ademuyiwa, B. Romagnoli, J. Zimmer-mann, R.L. Aft, K.N. WeilbaecherWriting, review, and/or revision of the manuscript: J. Xiang, M.A. Hurchla,F. Fontana, S.R. Amend, A.K. Esser, G.J. Douglas, T. Pluard, F.O. Ademuyiwa,B. Romagnoli, G. Tuffin, E. Chevalier, G.D. Luker, M. Bauer, J. Zimmermann,K. Dembowsky, K.N. WeilbaecherAdministrative, technical, or material support (i.e., reporting or organ-izing data, constructing databases): J. Xiang, X. Su, J. Zimmermann,K.N. WeilbaecherStudy supervision: J. Xiang, K.N. WeilbaecherOther (review and discussion on concept, conduct, and data analysis):M. Bauer

AcknowledgmentsThe authors thank Michael Tomasson, John DiPersio, David DeNardo,

Melissa Meyer, Yu Zhu, and Michael Ross for their valuable expert suggestionsand criticism. The authors thank Yalin Xu, LynneCollins, and Julie Prior for theirtechnical assistance. The authors thank Dr. Mark Watson for the supervision ofthe gene array data generation.

Grant SupportThis research was supported by grants from NIH CA097250 (to K.N.

Weilbaecher, M.A. Hurchla, J. Xiang, X. Su, and F. Fontana), NIH CA100730(to K.N. Weilbaecher, J. Xiang, and A.K. Esser), NIH CA154737 (to K.N.Weilbaecher, A.K. Esser), NIH research fellowship 1F31CA174096-01A1 (toS.R Amend), NIH 1K12CA167540 (to F.O. Ademuyiwa), NIH R01CA172623(to R.L. Aft and C. Mudalagiriyappa), NIH CA170198, CA142750, andCA182333 (to G.D. Luker and K.E Luker). Targeted funding to purchase NSGmice for a subset of experiments was provided by Polyphor Ltd. This study wasalso supported by the Musculoskeletal Research Center for microCT (NIH P30AR057235), the P50 small animal imaging core for bioluminescence studies(CA94056), the DDRCC Morphology core (grant P30 DK52574), The Barnes-Jewish Foundation, The St. LouisMen's Group against Cancer, andHope CenterAlafi Neuroimaging Laboratory (NIH Shared Instrumentation grant S10RR027552).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 30, 2015; revised July 27, 2015; accepted July 28, 2015;published OnlineFirst August 12, 2015.

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2015;14:2473-2485. Published OnlineFirst August 12, 2015.Mol Cancer Ther Jingyu Xiang, Michelle A. Hurchla, Francesca Fontana, et al. Breast CancerMetastasis and Enhances Chemotherapy Effect in Triple-Negative CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts

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cxcr4 protein epitope mimetic antagonist pol5551 ......death. the protein epitope mimetic (pem)...

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