Antagonizing Integrin b3 Increases Immunosuppression in …1 510 PyMT-BO1-GFP-Luc cells in 50 mL PBS...

Microenvironment and Immunology

Antagonizing Integrin b3 IncreasesImmunosuppression in CancerXinming Su1, Alison K. Esser1, Sarah R. Amend1, Jingyu Xiang1, Yalin Xu1,Michael H. Ross1, Gregory C. Fox1, Takayuki Kobayashi1, Veronica Steri2,Kirsten Roomp3, Francesca Fontana1, Michelle A. Hurchla1, Brett L. Knolhoff1,4,Melissa A. Meyer1,4, Elizabeth A. Morgan5, Julia C. Tomasson1, Joshua S. Novack1,Wei Zou4,7, Roberta Faccio6, Deborah V. Novack7, Stephen D. Robinson2,Steven L. Teitelbaum4,7, David G. DeNardo1,4, Jochen G. Schneider3,8, andKatherine N.Weilbaecher1

Abstract

Integrin b3 is critical for tumor invasion, neoangiogenesis, andinflammation, making it a promising cancer target. However,preclinical and clinical data of integrin b3 antagonists havedemonstrated no benefit or worse outcomes. We hypothesizedthat integrin b3 could affect tumor immunity and evaluatedtumors inmicewithdeletionof integrinb3 inmacrophage lineagecells (b3KOM).b3KOMmice had increasedmelanomaandbreastcancer growth with increased tumor-promoting M2macrophagesand decreased CD8þ T cells. Integrin b3 antagonist, cilengitide,

also enhanced tumor growth and increased M2 function. Weuncovered a negative feedback loop in M2myeloid cells, whereinintegrin b3 signaling favored STAT1 activation, an M1-polarizingsignal, and suppressed M2-polarizing STAT6 activation. Finally,disruption of CD8þ T cells, macrophages, ormacrophage integrinb3 signaling blocked the tumor-promoting effects of integrin b3antagonism. These results suggest that effects of integrin b3therapies on immune cells should be considered to improveoutcomes. Cancer Res; 76(12); 3484–95. �2016 AACR.

IntroductionIntegrins are heterodimeric cell surface receptors that directly

bind components of the extracellular matrix (ECM) and partic-ipate in cell migration and other important cellular functions(1, 2). Integrin b3 can heterodimerize with integrinav oraIIb andis critical for mature osteoclastic bone resorption, platelet aggre-gation, and angiogenic endothelial cell function (3, 4). Tumorcells expressing high levels of integrin b3 exhibit enhanced pro-liferation andmetastasis (5–7). In vitro and in animal models, the

integrinavb3 antagonist cilengitide was shown to decrease tumorcell proliferation, migration, and neoangiogenesis (8). Micegenetically deleted for integrin b3 (Itgb3�/�) have decreasedmetastasis (9). Surprisingly, primary tumor growth is enhancedin Itgb3�/� mice (10). However, cilengitide can enhance tumorgrowth and angiogenesis if applied suboptimally in preclinicalmodels (11). In clinical trials, integrin b3 antagonists did notdemonstrate improved outcomes in many cancer types (12–14).These findings suggest a need to better understand the role ofintegrin b3 in the tumor microenvironment before translatingencouraging preclinical discoveries to patients.

Wepreviously reported that integrin b3deletion inplatelets hasno effect on primary tumor growth and angiogenesis, but specificdeletion of integrinb3 inmyeloid cells via lysozymeMpromoter–driven Cre recombinase (LysM-Cre) results in enhanced tumorgrowth (15), consistent with both the phenotype observed inItgb3�/�mice (10) and results frombonemarrow transplantationstudies (16). Interestingly, in endothelial cells, acute depletion ofintegrin b3 transiently inhibits tumor growth and angiogenesis,but long-term deletion has no effect on primary tumor growth(17). These results suggest that integrin b3 signaling in myeloidcells ismost likely to account for the enhanced tumor growth seenin genetic and pharmacologic studies.

Fibroblasts, stromal cells, myeloid cells/macrophages, andlymphocytes are very common tumor-infiltrating cells that playkey roles in tumor progression and metastasis (18). Of these,macrophages are among themost abundant recruited host cells intumors (19). For many solid tumor types, including breast, brain,and skin cancer, high densities of tumor-associated macrophages(TAM) are generally associated with poor clinical outcome

1Department of Medicine, Division of Oncology, Washington Univer-sity School of Medicine, St. Louis, Missouri. 2School of BiologicalSciences, University of East Anglia, Norwich Research Park, Norwich,United Kingdom. 3Luxembourg Centre for Systems Biomedicine(LCSB), University of Luxembourg, Luxembourg. 4Department ofPathology and Immunology, Washington University School of Medi-cine, St. Louis, Missouri. 5Department of Pathology, Brigham andWomen's Hospital, Harvard Medical School, Boston, Massachusetts.6Department ofOrthopedic Surgery,WashingtonUniversity School ofMedicine, St. Louis, Missouri. 7Deparment of Medicine, Division ofBoneandMineralDiseases,WashingtonUniversitySchoolofMedicine,St. Louis, Missouri. 8Department of Internal Medicine II, SaarlandUniversity Medical Center, Homburg/Saar, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.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: 10.1158/0008-5472.CAN-15-2663

�2016 American Association for Cancer Research.

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(19, 20).Macrophage recruitment and polarization are dynamicprocesses in the tumor microenvironment because of the com-plex milieu of chemoattractant and polarization signals in vivo(20). TAMs that express high levels of MHCII usually have atumoricidal, antitumor, M1 phenotype, whereas TAMs expres-sing scavenger receptors, such as CD163, CD204, and CD206,are associated with a tumor-promoting and immunosuppres-sive M2 phenotype (19, 21). Lymphocyte-secreted factors, suchas IL4, IL10, IL13, IFNg , TNFa, and immunoglobulins, are verystrong regulators of macrophage polarization and function(20, 22). M2 TAMs can suppress immune responses by decreas-ing intratumor CD8þ T-cell numbers and have potent proan-giogenic activity through secretion of factors like VEGF (19, 23,24). STAT1 is a dominant transcriptional factor that regulatesM1 macrophage polarization. Lipopolysaccharide (LPS)-acti-vated TLR4 signaling and IFNg promote macrophage M1 polar-ization through activation of STAT1 (25, 26). STAT6 is requiredto drive M2 macrophage polarization in the presence of IL4 orIL13 (25, 26). STAT1 and STAT6 signaling events regulate andinhibit each other and control the polarization and function ofmacrophages (25, 26).

b3 integrins play key roles in myeloid lineage osteoclast dif-ferentiation and function and in preclinical models of pathologicinflammation (3, 6). Because loss of integrin b3 in myeloid cellsresults in enhanced tumor growth (15), we hypothesized thatintegrin b3 may be involved in macrophage polarization andfunction in the tumor microenvironment. Here, we present datauncovering an antitumoral role for integrin b3 signaling inthis context. Integrin b3 signaling controls the balance betweenantitumor and protumor immune cells through effects onSTAT6/STAT1 signaling, which in part explains the mixed resultsof integrin antagonists in the clinic. Taken together, our findingshighlight the important role of TAMs when designing clinicaltrials with integrin b3–targeted treatments in cancer.

Materials and MethodsMice

All animal studies were performed according to the guidelinesestablished by the Animal Studies Committee at the WashingtonUniversity in St. Louis (St. Louis, MO).Wild-type (WT), LysM-Creknockin mice and Stat6�/�mice are from The Jackson Laboratory(all C57BL/6J background). Itgb3flox/flox (15) and Itgb3�/� mice(27) on a pure C57BL/6J background were previously reported.All mice are housed under pathogen-free conditions according tothe guidelines of theDivisionofComparativeMedicine,Washing-ton University School of Medicine. Unless noted, all mice wereused at 6 to 10 weeks of age.

Cell linesIn 2013, the parental MMTV-PyMT cells (PyMT-B6; kindly

provided by D.G. DeNardo) were isolated from a fully invasivemammary tumor that spontaneously aroseatday120 inaC57BL/6background MMTV-PyMT mouse, a mouse model that representsan antiestrogen-sensitive, luminal B breast cancer. The tumor wascollagenase treated, grown in single-cell suspension on a collagen-coated plate, and cloned to establish the parent PyMT-B6 cell line.Parent PyMT-B6 cells were injected into the mammary fat pad(MFP) tissue of a female C57BL/6J mouse, and after reaching atumor size approaching 1 cm, tumor cells were collagenase treatedand cultured in a cell culture dish. The cultured tumor cells were

intracardially injected into a 6-week-old female C57BL/6Jmouse to establish bone metastases. Twelve days after intra-cardiac inoculation, the bone tumor was harvested and culturedin a cell culture dish with DMEM media plus 10% FBS toestablish the PyMT-BO1 subline, which when compared withthe parent PyMT-B6 cells, had a higher incidence of inducingbone metastases after either orthotopic MFP or intracardiacinjection. The PyMT-BO1 cells were infected with lentiviruscontaining the GFP-firefly luciferase genes as described previ-ously (28). GFP-expressing PyMT-BO1 cells were FACS sorted,cultured, and validated for luciferase expression; this cell linewas named PyMT-BO1-GFP-Luc. PyMT-B6, PyMT-BO1, andPyMT-BO1-GFP-Luc cells were evaluated by qPCR, and allexpress the PyMT, Esr1, Esr2, and Itgb3 genes. These cell lineswere tested as CD45 negative and pan-keratin positive by FACSin 2013 and 2015.

The B16-F10 C57BL/6 murine melanoma cell line (ATCC) wasmodified to express firefly luciferase (B16F10-Luc) as describedpreviously (29). In 2015, this cell linewas tested as CD45negativeand integrin b3 positive by FACS.

Mice tumor modelsFor MFP injection, 1 � 105 PyMT-BO1-GFP-Luc cells mixed

with BD Matrigel (BD Biosciences) were injected into MFP tissueof 8-week-old female mice. For subcutaneous injections, 1 � 106B16F10-Luc cells in 200 mL PBS were injected into the flank ofWTand b3KOMmice. Tumor growth wasmeasured at each indicatedtime point. Tumor size (mm3) was calculated by measuring thelongest (L) and shortest (S) distance of tumor tissue, with thisformula: tumor size ¼ 0.51 � L � S2.

For intracardiac injections, the left ventricular chamber of6-week-old mice was injected with 1 � 105 B16F10-Luc cells or1 � 105 PyMT-BO1-GFP-Luc cells in 50 mL PBS as describedpreviously (30). Bioluminescence imaging (BLI) was used toquantify tumor growth after injection. For the cilengitide treat-ment, mice were given cilengitide (5 mg/kg, Selleckchem) byintraperitoneal injection for the indicated time point. For anti-CSF1 antibody (clone 5A1) treatment, mice were given threedoses of antibody (1, 0.5, and 0.5 mg per mouse) by intraperi-toneal injection. For CD8þ T-cell depletion, mice were given anintraperitoneal injection of 100 mg anti-CD8a (53-6.7; BioLe-gend) or rat IgG2b k-chain isotype-matched control antibody(RTK4530; BioLegend) on the appropriate days.

Flow cytometric analysisTumor tissues were prepared in a single-cell suspension for

FACS analysis (31), with minor modifications (SupplementaryInformation).

Primary macrophage culturePrimary bone marrow macrophages (BMM) were generated

from the femurs and tibias of mice as described previously (30)(Supplementary Information).

Microarray analysisMicroarraywas performedwith theGenomeTechnologyAccess

Center at Washington University School of Medicine (Supple-mentary Information). The microarray data in this article areavailable on theGEOdatabasewith accessionnumberGSE75882.

Integrin b3 and Immunosuppression in Cancer

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qPCRqPCR was performed using SYBR Advantage Mix (Bio-Rad)

with mouse-specific primers as specified in Supplementary TableS4 (Supplementary Information).

BLILive BLI analysis was described previously (30), with minor

modifications (Supplementary Information).

Western blot analysisAntibodies and protocol were used from Cell Signaling Tech-

nology (Supplementary Information).

Statistical analysisData are shown as mean � SD unless noted otherwise. All

experiments were analyzed using two-tailed unpaired Studentt test for two groups or one-way ANOVA with a Bonferronipost hoc test for three or more groups by Prism (GraphPadSoftware). P < 0.05 was considered significant.

ResultsMice with myeloid-specific–targeted disruption of integrin b3have enhanced primary tumor growth and increased M2/M1macrophage ratios

Previous studies have shown that Itgb3�/� mice haveenhanced primary tumor growth (10), but the mechanism islargely unknown. Here, we evaluated tumor growth in b3KOMmice lacking integrin b3 specifically in myeloid cells (LysM-Creþ/�, Itgb3flox/�) and in WT (LysM-Creþ/�, Itgb3flox/þ) miceusing immunocompetent allograft orthotopic tumor models.We found that in both orthotopic melanoma subcutaneoustumor model and a breast cancer MFP model, tumor growthwas enhanced in b3KOM mice (Fig. 1A–D). There was nodifference in the number of blood vessels between WT andb3KOM mice as measured by endomucin staining in size-matched tumor tissue (Supplementary Fig. S1).

TAMs, osteoclasts, and myeloid-derived suppressor cells areLysMþ myeloid cells that have been demonstrated to supporttumor growth through immunosuppression, promotionof bone resorption, and stimulation of neoangiogenesis(19, 32, 33). We asked whether loss of integrin b3 in myeloidcells altered immune cell infiltration of primary tumor tissue.Tumor-infiltrating immune cells from WT and b3KOM micewere analyzed by flow cytometry. To avoid effects from varyingtumor size, we analyzed similar size tumors before statisticallysignificant changes in tumor growth were observed (day 11 forB16F10 and PyMT-BO1). Myeloid cell populations weredefined by FACS as described previously (31) and by the FACSgating strategy detailed in Supplementary Fig. S2. In bothtumor types, although the overall number of tumor-infiltratingCD45þ cells and TAMs (CD45þ CD11bþ Ly6C� Ly6G� F4/80þ)was not significantly different between WT and b3KOM mice(Fig. 1E and F), b3KOM tumors displayed a significant increasein the number of tumor-promoting M2 TAMs (CD206hi) and asignificant decrease in M1 TAMs (MHCIIhi; Fig. 1E and F). Geneexpression of FACS-sorted CD11bþ cells from tumors estab-lished in b3KOM mice and WT mice revealed upregulation ofthe M2 TAM markers CD163, Fizz1, Arg1, and Ym1 in b3KOMCD11bþ cells as compared with WT CD11bþ cells (Fig. 1G).Furthermore, there was a significant decrease in CD8þ T-cell

number in b3KOM tumors as compared with WT tumors(Supplementary Table S1). These results suggest that geneticdeletion of integrin b3 in myeloid cells increases the M2/M1TAM ratio in both orthotopic melanoma and breast cancermodels.

Integrin b3 knockout M2 macrophages have enhanced tumor-promoting function

To determine whether the increased number of M2 macro-phages alone can explain the increased tumor growth seen inb3KOM mice, or whether these b3KOM M2 TAMs also displayenhanced function, we adoptively transferred ex vivo–polarizedWT and Itgb3�/� M2 macrophages directly into B16F10tumors. We found that the Itgb3�/� M2 macrophages weremore potent in promoting tumor growth than WT M2 macro-phages (Fig. 2A). To account for the effects of integrin loss onM2 cell migration and homing, we adoptively transferredBMMs through intracardiac injection into mice bearingPyMT-BO1 MFP tumors. Again, we observed that breast cancerbearing mice that received Itgb3�/� BMMs had larger tumors(Fig. 2B). Together, these data suggest that integrin b3 is anegative regulator of tumor-promoting function in M2-polar-ized macrophages.

Despite comparable numbers of Foxp3þ Tregs, we also foundthat CD8þ T-cell numbers were significantly decreased in b3KOMtumors compared withWT tumors (Supplementary Table S1). Todetermine whether CD8þ T cells contributed to the enhancedtumor growth in b3KOM mice, we depleted CD8þ T cells in WTand b3KOM mice by anti-CD8a antibody injection and moni-tored tumor growth. As expected, depletion of CD8þ T cellsenhanced tumor growth in WT mice; however, there was noadditional enhancement of tumor growth in b3KOM mice(Fig. 2C and D). These data show that CD8þ T cells in the tumormicroenvironment contributed to the enhanced tumor growthphenotype observed in b3KOM mice.

Pharmacologic blockade of integrin avb3–enhanced tumorgrowth and increased M2 TAM infiltration

Recent clinical trials with cilengitide have failed to improvesurvival in glioblastoma, non–small cell lung cancer (NSCLC),metastatic melanoma, prostate cancer, and advanced nonre-sectable pancreatic cancer (12–14, 34–36). As the b3KOMmiceexhibit enhanced tumor growth and increased M2 macrophagenumbers (Fig. 1), we evaluated the effect of pharmacologicblockade of integrin b3 on TAMs by establishing PyMT-BO1MFP tumors in WT immunocompetent mice. Treatment withcilengitide, initiated when the tumors were fully established(day 10, �200 mm3), significantly enhanced tumor growth(Fig. 3A–C). Analysis of the tumor-infiltrating myeloid cellpopulation by FACS revealed that cilengitide treatmentincreased M2 TAMs in the tumor tissue (Fig. 3D). Interestingly,cilengitide administration to early-stage tumors (less than100 mm3) did not increase tumor burden; however, it did notdecrease tumor burden either (Supplementary Fig. S3). Day 10tumors had almost double the number of myeloid cells andexhibited 50% decreases in T-cell numbers compared with day6 tumors (Supplementary Fig. S3), making them potentiallymore susceptible to the myeloid cell effects of cilengitide. Theseresults show that cilengitide treatment can adversely affectbreast cancer tumor growth in immunocompetent mice andresults in increased tumor-infiltrating M2 TAMs.

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Tumor cells and M2 macrophages from b3KOM mice havediminished immune response signatures

To better understand the impact of integrin b3 signaling inmyeloid cells on the tumor microenvironment, we compared thegene expression profiles of FACS-isolated GFPþ PyMT-BO1 MFPtumor cells andM2 TAMs (CD11bþGr1� F4/80þ CD206þ) fromWT and b3KOM tumor tissue. For themicroarray experiment data

analysis, an unadjusted P value cutoff of less than 0.01 wasselected to generate lists of differentially expressed genes (DEG;accession number GSE75882). For the CD206hi TAMs WT versusb3KOMcomparison, 122 unique annotated geneswere identified(Supplementary Table S2). Term enrichment (produced by thePANTHER classification system) for genes upregulated in M2TAMs from the b3KOM tumor identified many immune-related

Figure 1.Mice with specific deletion of integrin b3 in myeloid cells promoted tumor growth with increased M2 TAM infiltration. A, growth curve of subcutaneously(S.C.) injected B16F10-Luc cells (1� 106) in 8-week-oldmaleWT (LysM-Creþ/�, Itgb3f/þ) andb3KOM (LysM-Creþ/�, Itgb3f/�)mice (n¼ 8). B, tumorweight. C, growthcurve of MFP-injected PyMT-BO1-GFP-Luc cells (1 � 105) in 8-week-old female WT and b3KOM mice (n ¼ 6). D, tumor weight. E and F, FACS staining wasperformed on cells from day 11 tumor tissue. TAMs were gated on CD45þ, CD11bþ, Ly6G�, Ly6C�, F4/80þ population. In this population, M1 TAMs were gated asMHCIIhi/CD206low cells, and M2 TAMs were gated as MHCIIlow/CD206hi cells. G, M2 TAM markers CD163, Fizz1, Arg1, and Ym1 mRNA expression in CD11bþ

cells isolated from day 11 tumor tissue (n ¼ 3). Data, mean � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






biologic processes to be significantly overrepresented (Fig. 4A). Amore diverse list of biologic processes was identified from thedownregulated genes. Among them, IFNb response genes werefound to be significantly downregulated in b3KOM M2 TAMs(Fig. 4A).

The list of DEGs generated from the comparison of CD206hi

TAMs in WT vesrus b3KOMmice was used for pathway analyseswith IPA Ingenuity. An upstream analysis was performed, andfour genes (Tgfb1, Hgf, Tnfa, and Vegf) were predicted to beupregulated in b3KOM with an activation Z-score greater than2.0 (Fig. 4B). TGFb1, HGF, TNFa, and VEGF may generallypromote tumor growth and are related to TAM function. Allgenes connected directly to these four upstream genes wereplaced in a network, which also included mir-10 (also calledmir-99a). Mir-10 was the most downregulated gene/miRNA inb3KOM and has been shown to inhibit TNF (37). Cytotoxic Tlymphocyte antigen-2a and -2b (Ctla2a, Ctla2b) were also

upregulated in b3KOM CD206hi TAMs; of the two, Ctla2a wasshown to inhibit effector T-cell function (38). Together, thesedata suggest that loss of integrin b3 results in a tumor-pro-moting, immunosuppressive gene expression signature inTAMs.

Gene expression analysis of isolated tumor cells established inWT versus b3KOM mice identified 130 unique annotated genes(Supplementary Table S2). Changes in tumor cell gene expressionreflected myeloid integrin b3–dependent changes to the tumormicroenvironment. An interaction network consisting of 53nodes was subsequently generated from the DEGs in tumor cellsfrom WT versus b3KOM mice (Fig. 4C). In this network, Stat1(downregulated in b3KOM) and IL1b (upregulated in b3KOM)are two highly connected nodes. Among the downregulated genesin tumor cells isolated from b3KOM mice, Stat1 and Cxcl10belong to the IFNg pathway. Taken together, these data indicatethat IFN-related pathways are downregulated in the b3KOM

Figure 2.Integrin b3 knockout M2 macrophages have enhanced tumor-promoting function, and b3KOM macrophages promote tumor growth in part through CD8þ T cells.A, in WT mice, at day 6 after subcutaneous B16F10 tumor establishment, 3 � 106 IL4-induced WT or Itgb3�/� M2 macrophages were injected into the tumortissue. Tumor weight was measured at day 14. B, at day 6 and day 9 after PyMT-BO1-GFP-Luc tumor cell injection, 5� 106 WT or Itgb3�/�ex vivo M2 macrophageswere intracardially (I.C.) injected into tumor-bearing mice. MFP tumor size and weight were measured. (n ¼ 6). 5M, 5 million. C, eight-week-old male WT andb3KOM mice were subcutaneously (S.C.) injected with 1 � 106 B16F10 tumor cells. Anti-CD8a antibody was given by intraperitoneal (i.p.) injection one daybefore tumor cell injection and 7 days after first antibody injection. Tumor growth was monitored and measured for 2 weeks. D, tumor weight measured on day14 (control, n ¼ 8; anti-CD8, n ¼ 5). Data, mean � SEM. �, P < 0.05; �� , P < 0.01; �� , P < 0.001. ns, nonsignificant.

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tumor microenvironment. STAT1 is an important regulator inIFN-related pathways (39), suggesting that integrin b3 may beinvolved in the regulation of STAT1 pathway genes in the tumormicroenvironment.

Integrin b3 favors STAT1 activation and suppresses STAT6signaling in macrophages

We found that macrophages lacking integrin b3 had down-regulation of IFN (STAT1) pathway genes in vivo (Fig. 4). Weevaluated the role of integrin b3 signaling when macrophageswere polarized in M1 (LPS-STAT1) or M2 (IL4-STAT6) condi-tions. After LPS treatment, Itgb3�/� BMMs had decreased phos-phorylation of STAT1 compared with WT (Fig. 5A and B). ThemRNA expression of the STAT1 downstream gene Ccl5 was alsodecreased in Itgb3�/� BMMs after LPS treatment (Supplemen-tary Fig. S4A). Spleen tyrosine kinase (SYK) phosphorylation isusually associated with integrin activation (40). Genetic orpharmacologic (cilengitide) disruption of integrin b3 signifi-cantly decreased SYK phosphorylation in macrophages (Fig. 5Aand C). We evaluated the effect of LPS on integrin b3 expressionand activation in BMMs and found that in a short time course,LPS had little effect on integrin b3 expression (SupplementaryFig. S5A). However, LPS had a potent effect on integrin b3activation as measured by binding of IntegriSense-680, a ligandfor activated integrin avb3 (Supplementary Fig. S5C). To deter-mine whether decreased STAT1 signaling was caused by the lossof integrin b3, we performed a rescue experiment. Integrin b3was transduced into Itgb3�/� BMMs and rescued the defect in

STAT1 phosphorylation after LPS treatment (Fig. 5D). To deter-mine whether the expression of other integrins was altered bygenetic disruption of Itgb3, we compared the mRNA expressionof other integrin genes between polarized and nonpolarizedWT and Itgb3�/� BMMs (Supplementary Fig. S6). Interestingly,the mRNA expression of some integrins was indeed affected,suggesting that integrin b3 disruption can modulate expressionof other integrins. That said, ITGAV and ITGB5, which togetherbind the same RGD motif as integrin avb3, were not signifi-cantly changed after disruption of ITGB3 and were thus unlike-ly to have compensatory effects in Itgb3�/� BMMs. Together,these results demonstrate that disruption of integrin b3 causeddecreased phosphorylation of both the integrin b3 signalingmolecule SYK and the M1-polarizing molecule STAT1 inmacrophages.

b3KOM mice had increased M2 macrophages with enhancedtumor-promoting function (Fig. 1), so we evaluated the M2-polarizing STAT6 signaling pathway in Itgb3�/� BMMs. Incontrast to WT, Itgb3�/� BMMs treated with the M2 polarizationfactor IL4 showed enhanced STAT6 signaling and increasedexpression of STAT6 downstream gene Ym1 (Fig. 5E and F andSupplementary Fig. S4B). Enhanced STAT6 signaling was alsoobserved in WT BMMs pretreated with cilengitide (Supplemen-tary Fig. S7). IL4 has been shown to increase integrin b3 mRNAexpression in BMMs (41). We found that while IL-4 inducedintegrin b3 gene and protein expression in WT BMMs, this wasabrogated in Stat6�/� BMMs (Fig. 5G and H). Taken together,we find that integrin b3 signaling regulates the balance of

Figure 3.Cilengitide treatment enhanced tumor growth and increased M2 TAM population. A, experimental schema. PyMT-BO1-GFP-Luc cells (1 � 105) were injected intoMFP of 8-week-old female WT mice (n ¼ 5). Starting at day 10 after tumor cell injection, mice were treated daily with cilengitide for 5 days. At day 14, allmice were sacrificed. i.p., intraperitoneal. B, tumor growth was measured at the indicated time points by BLI. C, tumor weight at day 14. D, TAM populations intumor tissue were analyzed by FACS. Data, mean � SEM. � , P < 0.05; �� , P < 0.01. ns, nonsignificant.






Figure 4.Gene expression profiling of tumor cells and M2 macrophages from b3KOM mice reveals diminished immune response signatures. Pathway-based analysis ofmicroarray data. A, biologic process, term enrichment results produced by PANTHER classification system (GO) for both up- and downregulated genesin b3KOM tumors. B, upstream analysis of WT CD206hi macrophages versus b3KOM CD206hi macrophages, in which genes upregulated in b3KOM are shownin red, and genes downregulated in b3KOM are shown in green (the darker the more extreme the increase/decrease). For the edges, orange indicatespredicated activation, blue indicates predicted inhibition, yellow indicates findings inconsistent with the state of the downstream molecule, and gray indicatesthat the effect is unknown. Solid edges, direct interactions; dashed edges, indirect interactions. C, interaction network from the DEGs of WT tumor cellsversus b3KOM tumor cells. Solid gray edges, direct interactions; dashed gray edges, indirect interactions.

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p-STAT1 and p-STAT6. These results show an integrin b3–negative feedback loop within M2 macrophages, in whichintegrin b3 expression is induced by STAT6 signaling, but thatintegrin b3 signaling favors STAT1 activation and suppressionof STAT6 signaling (Fig. 6).

Myeloid cells are required for increased tumor growth aftercilengitide treatment

Because there were increased numbers of M2 tumor–promot-ing macrophages in cilengitide-treated tumors, we asked whetherthe direct effects of cilengitide on myeloid cells were required for

Figure 5.Integrin b3 signaling favors STAT1 activation and suppression of STAT6 signaling in macrophages. A, Western blots of p-SYK and SYK in LPS-stimulatedWT and Itgb3�/� BMMs. B, Western blots of p-STAT1 and STAT1 in LPS-stimulated WT and Itgb3�/� BMMs. C, Western blots of p-STAT1 and STAT1 in LPS-stimulated WT BMMs with or without cilengitide pretreatment. D, Western blots of p-STAT1 and total STAT1 in WT BMMs overexpressing integrin b3.Mþ pMX,macrophage treatedwith empty pMX vector; Mþ hb3, macrophage treatedwith human integrin b3–constructed pMX vector. E,Western blots of p-STAT6and total STAT6 in IL4 stimulated WT and Itgb3�/� BMMs. F, Ym1 mRNA expression after IL4 treatment of WT and Itgb3�/� BMMs. G, Itgb3 mRNA expressionlevel after IL4 treatment in Stat6�/� macrophages. H, Western blot analysis of integrin b3 expression in WT, Itgb3�/�, and Stat6�/� BMMs after 5 ng/mL IL4treatment for 48 hours.






enhanced tumor growth. We administered cilengitide to tumor-bearing b3KOMmice, where only LysMþ myeloid cells would beunresponsive to cilengitide. In contrast to the larger MFP tumorsobserved when cilengitide was administered to WT mice, wefound that cilengitide treatment significantly reduced tumorburden in b3KOMmice (Fig. 7A). These results show that integrinb3 expression on TAMs is required for some of the cilengitide-induced tumor-promoting effects.

Next, we used anti-CSF1 antibody to decrease the number ofmacrophages and myeloid cells in tumor-bearing mice and eval-uated the effect of cilengitide treatment. Coadministration ofcilengitide with anti-CSF1 resulted in no enhancement of tumorgrowth compared with cilengitide alone (Fig. 7B and C). Analysisof tumor-infiltrating cells demonstrated that anti-CSF1 antibodytreatment significantly decreased myeloid cell numbers in thetumor,with an expected concomitant increase inCD4þ andCD8þ

T-cell numbers in the tumors in WTmice (Fig. 7D–G). Anti-CSF1antibody also decreased tumor-infiltrating myeloid cells andincreased CD4þ and CD8þ T cells in tumor-bearing b3KOMmice(Supplementary Fig. S8). Taken together, these data show thatmacrophage/myeloid lineage cells are required for the tumor-enhancing effects of cilengitide and that coadministration ofcilengitide and antimacrophage therapy reverses the tumor-enhancing effects of integrin b3 blockade.

DiscussionIn this study,weprovidenewevidence for the role of integrinb3

in TAM polarization and function. We found that modulating

integrin b3 levels either genetically or pharmacologically resultedin enhanced tumor growth and increased M2 macrophage num-bers and tumor-promoting function in the tumor microenviron-ment. We found that integrin b3 signaling favored M1-polarizingSTAT1 signaling and suppressed M2-polarizing STAT6 signaling.However, integrin b3 gene expression was suppressed by STAT1and induced by STAT6 signaling, creating a negative feedbackloop for M2 polarization. Thus, loss of integrin b3 signalingpromoted an immunosuppressive tumor environment throughincreased M2 TAM polarization and function and decreasedCD8þ T-cell numbers. Finally, disruption of macrophagesblocked the tumor-promoting effects of cilengitide, demonstrat-ing an immediate way to improve the efficacy of integrin b3therapies in cancer.

M1 TAMs are essential participants in Th1 responses and havepotent immunostimulatory capacity (25). On the other hand,M2TAMs help maintain an immunosuppressive environment andpromote tumor growth by facilitating angiogenesis, tumor cellinvasion, metastasis, and chemotherapeutic resistance (19, 25,33, 42). We found that integrin b3 knockout TAMs had elevatedexpression of M2 macrophage markers and enhanced tumor-promoting function in vivo when compared with WT TAMs (Figs.1 and 2). We also noticed a significant decrease in CD8þ T-cellnumber in the tumor tissue when integrin b3 was disrupted inmyeloid cells (Supplementary Table S1). In the tumor microen-vironment, CD8þ T cells play a central role in antitumor cellularimmune responses. High CD8þ T cells, low CD4þ T cells, and lowCD68þ TAMs in the tumor microenvironment correlated withbetter survival for breast cancer patients (42). In vivo, b3KOM

Figure 6.Model of integrin b3 signaling pathway in TAMs. A, a negative feedback loop, in which integrin b3 expression is induced in M2 macrophages by STAT6activation, but integrin b3 signaling suppresses STAT6 andpromotes STAT1 activation. B, in the tumormicroenvironment, integrinb3 signaling is a negative regulatorof M2 TAM function. Blocking integrin b3 signaling enhances tumor-promoting function in M2 TAMs.

Su et al.





TAMs have elevated TGFb signaling downstream gene expression,suggesting increased TGFb in the b3KOM tumor microenviron-ment (Fig. 4B). In vitro, Itgb3�/� BMMs have higher levels of IL10mRNA expression after LPS treatment when compared with WTBMMs (data not shown). Finally, although CD8þ T-cell depletionin WT mice enhanced tumor growth, the same treatment inb3KOM mice resulted in no significant difference in tumor size(Fig. 2C and D), indicating that the enhanced tumor-promotingfunction of integrin b3–null TAMs is mediated by suppressiveeffects on the CD8þ T-cell response. Therefore, our data describe afunctional role for integrin b3 in the macrophage-dependentregulation of immunosuppression within tumors.

Several integrin b3 antagonists have been developed for clinicaluse in cancer, including peptide antagonists, such as cilengitide,and neutralizing antibodies, such as Vitaxin and c7E3 (43).Treating integrin avb3–expressing tumor cells with cilengitide invitro reduces tumor cell proliferation and invasion (8, 43). On thebasis of these data, integrin b3 inhibition initially appeared to bean exciting cancer therapeutic target, particularly after cilengitidetreatment displayed positive results in some animal tumor mod-els (8, 43). However, clinical trials with cilengitide on glioblas-toma, NSCLC, metastatic melanoma, prostate cancer, andadvanced pancreatic cancer showed no improvement in out-comes, even while cilengitide was well tolerated in patients(12–14, 34–36). We noticed that in WT mice with early-stage

mammary tumors (day 6, less than 100 mm3), cilengitide treat-ment did not increase or decrease tumor growth (SupplementaryFig. S3). However, in established mammary tumors (day 10,�200 mm3), cilengitide treatment significantly enhanced tumorgrowth (Fig. 3). We compared the number of tumor-infiltrating Tcells and myeloid cells in both early and established mammarytumors; established tumors had almost double the number ofinfiltrating myeloid cells and a 50% decrease in infiltrating T-cellnumbers compared with early-stage tumors (SupplementaryFig. S3). This result suggests that late-stage tumors with anincreased number of TAMs may be more susceptible to theprotumor effect of integrin b3 blockade. Importantly, cilengitidetreatment decreased tumor burden in mice genetically lackingintegrin b3onmyeloid cells (Fig. 7A). Cilengitide has been shownto inhibit tumor cell proliferation (8). We subsequently con-firmed that cilengitide treatment inhibits PyMT-BO1 cell prolif-eration in vitro (Supplementary Fig. S9A), a direct inhibitory rolethat may explain the decreased tumor burden we observed incilengitide-treated b3KOMmice. Taken together, tumor-promot-ing M2 macrophages played an important role in integrin b3disruption–induced enhancement of tumor growth (cilengitidetreatment and b3KOM). Moreover, when mice were pretreatedwith anti-CSF1 antibody to decrease myeloid cell numbers, thiscilengitide-induced tumor progression was significantly abrogat-ed (Fig. 7C). Thus, anti-integrin b3 (cilengitide) treatment has

Figure 7.Myeloid cells are required for increased tumor growth after cilengitide treatment. A, PyMT-BO1-GFP-Luc MFP tumor established on b3KOMmice (n¼ 5). From day10 after tumor cell injection, mice were treated daily with cilengitide for 5 days. Tumor growth was measured at the indicated time points. B, experimentalschema. PyMT-BO1-GFP-Luc cells (1 � 105) were injected into MFP of 8-week-old female WT mice at day 0. Anti-CSF1 antibody treatment started on day 6 byintraperitoneal injection of 1 mg of antibody per mouse, followed by 0.5 mg of antibody on day 9 and day 12. Cilengitide treatment started on day 9 for 5 dayswith a dosage of 5 mg/kg per mouse by intraperitoneal injection (n ¼ 7 or 8 per group). C, tumor burden was measured by BLI at day 15. D–G, tumor-infiltrating myeloid cells and T cells were measured by FACS at day 15. Data, mean � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. ns, nonsignificant.






several roles in the tumor microenvironment, directly inhibitingtumor proliferation and neoangiogenesis while indirectly pro-moting tumor progression through stimulation of macrophageM2 polarization and tumor-promoting function.

We found that LPS-induced phosphorylation of SYK and STAT1was strongly reduced in Itgb3�/� BMMs. SYK is an importantsignaling molecule to transduce integrin b3 outside-in signaling(40). In addition, it has been shown that p-SYK can activate STAT1(44). A similar decrease in SYK and STAT1 activationwas observedif WT BMMs were pretreated with integrin avb3 inhibitor cilengi-tide (Fig. 5A–C). Furthermore, Itgb3�/� BMMs and WT BMMspretreated with cilengitide displayed an increase in STAT6 activa-tion upon IL4 treatment (Fig. 5E and Supplementary Fig. S7).These data demonstrate that integrin b3 downstream signalingenhances STAT1 activation, the key M1-polarizing signal, andnegatively regulates STAT6 activation, the key M2-polarizing sig-nal. Thus, integrin b3 activation and ligand binding induce SYKphosphorylation and then STAT1 activation in macrophages. LPS(TLR4 signaling) administration to macrophages induced integrinb3activationandenhancedSTAT1signaling; disruptionof integrinb3 (such as b3KOM) diminished this enhancement of STAT1signaling. Defining the precise signaling pathway through whichintegrin b3, LPS, and STAT1 interact in macrophages is underway.

It is important to understand effects on M2 TAMs and othermyeloid cells when administrating integrin b3–targeted therapy orantitumor immune therapy, particularly in patients whose tumorsexpress high numbers of infiltrating macrophages. InfiltratingTAMs in cancer are more likely to polarize towards an M2 pheno-type with tumor progression (21, 24). TAM burden correlates withpoor survival inmany cancer types (19, 20). TAM-targeted therapyis now being considered as an important facet of successful cancertreatment. We found that anti-CSF1 therapy prevented the increasein tumor growth associated with cilengitide treatment in breastcancer. Single-agent anti-CSF1 antibody was effective to decreaseTAMs by 30% to 50% and increase T cells in established tumors.However, it did not significantly decrease tumor burden. Mostbreast cancer cells, including PyMT-BO1 cells, secrete macrophagecolony-stimulating factor (M-CSF) 1 (CSF1). It is possible that anti-CSF1 antibody as dosed in our experiments did not completelyblock the M-CSF receptor (M-CSFR or CSF1R) signaling on tumor-infiltrating macrophages. Also, anti-CSF1 treatment targets bothtumor-promotingM2TAMsand tumor-suppressiveM1TAMs, thusdiminishing the antitumor properties of the latter. Accordingly,longer treatment times or blockade of MCSFR signaling pathwaysmayhave stronger effects on the tumormicroenvironment in termsof rebalancing the T-cell population and reducing tumor burden.

Taken together,wepropose thatM2TAMs are a keyplayer in thedecreased efficacy of integrin avb3–targeted therapies and thatconcurrent treatment with macrophage antagonists represents apossible strategy to improve their clinical effectiveness. Our

results suggest that when giving integrin b3–targeted therapy,myeloid cell function should be considered.

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

Authors' ContributionsConception and design: X. Su, S.R. Amend, S.L. Teitelbaum, D.G. DeNardo,J.G. Schneider, K.N. WeilbaecherDevelopment of methodology: X. Su, Y. Xu, M.A. Meyer, W. Zou,K.N. WeilbaecherAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Su, S.R. Amend, Y. Xu, M.H. Ross, F. Fontana,M.A. Hurchla, B.L. Knolhoff, M.A. Meyer, E.A. Morgan, S.D. Robinson,K.N. WeilbaecherAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Su, A.K. Esser, S.R. Amend, J. Xiang, Y. Xu, V. Steri,K. Roomp, F. Fontana, J.C. Tomasson, J.S. Novack, R. Faccio, S.D. Robinson,D.G. DeNardo, J.G. Schneider, K.N. WeilbaecherWriting, review, and/or revision of the manuscript: X. Su, A.K. Esser, Y. Xu,M.H. Ross, G.C. Fox, T. Kobayashi, K. Roomp, M.A. Hurchla, D.V. Novack,S.D. Robinson, J.G. Schneider, K.N. WeilbaecherAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Su, J. Xiang, Y. Xu, J.G. Schneider,K.N. WeilbaecherStudy supervision: X. Su

AcknowledgmentsThe authors thank Drs. William Frazier, Patrick Ross, Sheila Stewart, Michael

Tomasson, and Duanwen Shen for their valuable expert suggestions andcriticism and Crystal Idleburg, Lynne Collins, and Julie Prior for their experttechnical assistance. The authors also thank the Musculoskeletal ResearchCenter for histology and microCT (NIH P30-AR057235), the Molecular Imag-ing Center at Washington University (NIH/NCI P50-CA09056), DDRCC Mor-phology core (grant # P30 DK52574), The Barnes-Jewish Foundation, The St.Louis Men's Group Against Cancer, and Hope Center Alafi Neuroimaging Lab(NIH Shared Instrumentation grant # S10 RR027552).

Grant SupportX.Su,A.K.Esser, S.R.Amend, J. Xiang,Y.Xu, J. Tomasson, andK.N.Weilabecher

were supported by NIH RO1-CA097250. J. Xiang, Y. Xu, M. Hurchla, andK.N.Weilbaecher were supported by NIH PO1-CA100730. A.K. Esser, M.H. Ross,F. Francesca, and K.N. Weilabecher were supported by NIH RO1-CA154737.M.H. Ross was supported by the training grant NIH 5T32GM007067-39 andNIHT32AR060719. A.K. Esser was supported by the training grant NIH5T32CA113275-07. W. Zou and S.L. Teitelbaum received grant support fromNIH R01-AR057037, R37-AR046523, and Shriners Hospitals for Children grant85400-STL. J.G. Schneider received grant support from the German ResearchSociety (DFGSCH682/3-1), the FondsNationale de LaRecherchedeLuxembourg(CORE Itgb3VascIn), and the EU (CIG303682).

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 September 23, 2015; revised February 12, 2016; accepted March 7,2016; published OnlineFirst May 23, 2016.

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2016;76:3484-3495. Published OnlineFirst May 23, 2016.Cancer Res Xinming Su, Alison K. Esser, Sarah R. Amend, et al.

3 Increases Immunosuppression in CancerβAntagonizing Integrin

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