Targeting Olfactomedin-like 3 Inhibits Tumor Growth by Impairing … · Therapeutic Discovery Targeting Olfactomedin-like 3 Inhibits Tumor Growth by Impairing Angiogenesis and Pericyte

Therapeutic Discovery

Targeting Olfactomedin-like 3 Inhibits Tumor Growth byImpairing Angiogenesis and Pericyte Coverage

Marijana Miljkovic-Licina, Philippe Hammel, Sarah Garrido-Urbani, Boris P.-L. Lee, Mehdi Meguenani,Chiraz Chaabane, Marie-Luce Bochaton-Piallat, and Beat A. Imhof

AbstractAntiangiogenic drugs have been used as anticancer agents to target tumor endothelial cells or pericytes.

Because of limited efficacy of the current monotherapies, there is a strong demand for the dual targeting of

endothelial cells and pericytes. Here, we identify Olfactomedin-like 3 (Olfml3) as a novel proangiogenic cue

within the tumor microenvironment. Tumor-derived Olfml3 is produced by both tumor endothelial cells and

accompanying pericytes and deposited in the perivascular compartment. Blockade of Olfml3 by anti-Olfml3

antibodies is highly effective in reducing tumor vascularization, pericyte coverage, and tumor growth. In vitro,

Olfml3 targeting is sufficient to inhibit endothelioma cell migration and sprouting. Olfml3 alone or through

binding to BMP4 enhances the canonical SMAD1/5/8 signaling pathway required for BMP4-induced

angiogenesis. Therefore, Olfml3 blockade provides a novel strategy to control tumor growth by targeting

two distinct cell types within the tumor microenvironment using a single molecule. Mol Cancer Ther; 11(12);

2588–99. �2012 AACR.

IntroductionTumor growth is dependent on angiogenesis, the

growth of new blood vessels from the pre-existing vas-culature into the tumor mass (1, 2). The tumor microen-vironment contributes to this pathologic vascularizationprocess via a complex network of extracellular matrixmolecules and various cell types (3). Most cancer thera-pies are directed against tumor cells; however, a growingnumber of cancer therapeutics target cells of the tumormicroenvironment (4). Particularly, endothelial cells aretargeted via inhibition of the VEGF signaling pathway,but with limited long-term benefits and eventual devel-opment of resistance to the therapy (5). The resistance isdue to various mechanisms, including upregulation ofalternative proangiogenic signaling pathways, enhancedinvasive/metastatic activity of tumor cells, increasedrecruitment of proangiogenic bone marrow–derived cellsto tumor, and increased pericyte coverage, to rendertumor endothelial cells more resistant to antiangiogenictherapy (6). Therefore, new antiangiogenic drug targetsaimed at cells of the tumor microenvironment are neces-sary to complement existing therapies.

A growing body of evidence suggests that bone mor-phogenetic proteins (BMP) are critical growth factors inthe endothelial adaptation associatedwith tumorprogres-sion (7). BMPs are the largest subgroup of the TGF-b superfamily of signaling molecules. Although initially charac-terized by their ability to induce ectopic bone formation(8), these proteins were recently shown to be principalregulators of blood vessel formation (9). BMPs, notablyBMP2 and BMP4, are proangiogenic factors via inductionof VEGF expression (10) and recruitment of endothelialprogenitor cells (11). The proangiogenic effects of BMP4are mediated via activation of VEGF/VEGFR2 andangiopoietin/Tie2 signaling (12). However, endothelialmigration and sprouting can also be regulated throughcanonical BMP signaling independently of VEGF (13,14). In addition to autocrine effects on tumor cells, aparacrine effect of BMP4 on the vascular network hasalso been reported. Notably, melanoma cell–derivedBMP4 induces endothelial cell migration and tubeformation (15). During progression of hepatocellularcarcinoma, hypoxia-induced BMP4 mediates endothe-lial cell activation (16). Therefore, the BMP4-inducedangiogenic response involves several mechanisms.

TheOlfactomedin-like 3 (Olfml3) gene encodes a secretedextracellular protein, also known asONT1 inXenopus andchicken, mONT3 in mice, and HNOEL-iso or hOLF44 inhumans (17–20). Olfml3 belongs to a family of olfactome-din domain–containing proteins with distinct roles inembryonic patterning, cell-cycle regulation, tumorigene-sis, and ability to modulate critical signaling circuits suchas Notch or Wnt pathways (21, 22). Xenopus ONT1 servesas a scaffold protein that recruits the BMP1/Tolloid pro-teases to their substrate chordin, a BMP antagonist (19).

Authors' Affiliation: Department of Pathology and Immunology, CMU,University of Geneva, Switzerland

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

Corresponding Author: Beat A. Imhof, Department of Pathology andImmunology, CMU, rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.Phone: 41-22379-5747; Fax: 41-22379-5746; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-12-0245

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(12) December 20122588

on August 21, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst September 20, 2012; DOI: 10.1158/1535-7163.MCT-12-0245

http://mct.aacrjournals.org/

This function of Olfml3 may prove relevant to angiogen-esis. However, an interaction of Olfml3 with other BMPs,particularly those with prominent proangiogenic activityin tumors such as BMP4, has not been shown.Here, we provide the first report on Olfml3 protein

being a proangiogenic factor that interactswith BMP4 andpromotes angiogenesis. We also show that inhibition ofOlfml3 in tumor endothelial cells andpericytes effectivelyreduces tumor angiogenesis and growth in vivo.

Materials and MethodsCell lines and cultureThe t.End.1Vhigh cells were isolated in-house andmain-

tained as described previously (23). Authenticated Lewislung carcinoma cells (LLC1; European Collection of CellCultures) were cultured in Dulbeccos’ Modified Eagles’Media (Life Technologies), supplemented with 10% FBS.Smooth muscle cells (SMC) were isolated in-house fromporcine carotid artery using enzymatic digestion (S-SMC)or tissue explantation (R-SMC) as described previously(24). Human umbilical vein endothelial cells (HUVEC)were isolated in-house and cultured in EGM-2 Bullet kit(Lonza).

Tumor modelAll studies were conducted in accordance with the

ethical approval and recommendations of the VeterinaryOffice of Geneva state, according to the Swiss FederalLaw. To generate tumor growth inhibition model, a sus-pension of 0.5 � 106 LLC1 tumor cells in 200 mL PBS wasimplanted subcutaneously into the flank of femaleC56BL/6J mice (8- to 10-week-old). Mice were then trea-tedwith 25 to 50 mg of control and total rabbit IgG; 50 mg ofanti-Olfml3AþB affinity-purified against both Olfml3 pep-tides, and 25 mg of anti-Olfml3A or anti-Olfml3B affinity-purified against each peptide intraperitoneally everythird day starting from day 1. When tumors reached anaverage size of 1 cm3 (days 8–9), mice were sacrificed andtumors were harvested for evaluation of tumor growth.

In situ mRNA hybridizationThe digoxigenin- and fluorescein-labeled (Roche) RNA

probes were prepared after PCR amplification of mousePECAM-1 and Olfml3 genes as described in Supple-mentary Methods. In situ mRNA hybridization was con-ducted on frozen sections of LLC1 tumors as previouslydescribed (25).

Immunohistochemistry and TUNEL assayHUVECs were grown on glass slides and immunohis-

tochemistry was conducted as detailed in SupplementaryMethods. LLC1 tumors were processed for and stainedby immunohistochemistry as previously described (25).Samples were incubated with rabbit anti-Olfml3AþB

serum, rat monoclonal anti-PECAM-1 (26), mouse mono-clonal anti-a-SMA (27), and mouse monoclonal anti-NG2(clone 132.38; Millipore), mouse anti-Ki-67 (BD Pharmin-gen); and rabbit anti-HIF-1a (Santa Cruz). For apoptotic

cell labeling, the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay (Roche)was conducted according to the manufacturer’s instruc-tion. ImageJwasused for quantification of vessel diameterand colocalization analysis as described in Supplementa-ry Methods. Quantification of area density and pericytecoveragewas conducted usingMetamorph6.0 (MolecularDevices) as described in Supplementary Methods.

In vitro wound-healing assayTransient transfection of t.End.1Vhigh cells was con-

ducted using Amaxa-Nucleofector (Lonza) withStealth-Select siRNAs (Life Technologies) as describedin Supplementary Methods. The efficiency of Olfml3silencing was evidenced by real-time reverse tran-scription (RT)-qPCR as described in SupplementaryMethods. t.End.1Vhigh cells (1.5 � 104) and R-SMCs(8 � 104) were seeded onto rOlfml3-FLAG-coated (BDBiosciences) plates, and wound-healing assays wereconducted as described previously (25).

In vitro sprouting assayThe t.End.1Vhigh cells (1.2 � 104 cells/gel) were seeded

in suspension into fibrin gels (28), and sprouting assayswere conducted as described previously (25).

ELISAMaxisorb immunoplates (Nunc) were coated with

rBMP4 (2 mg/mL). Wells were blocked with 1% bovineserum albumin (BSA) and incubated with rOlfml3-FLAGat 0.5 mg/mL in PBS containing 0.05% Tween-20 and 0.5%BSA. Biotinylated-M2 antibody (2 mg/mL) was added.BoundM2was detected using streptavidin-HRP (JacksonImmunoResearch Laboratories) and substrate ReagentPack (R&D Systems). Optical densities at 450 nm wereread using a kinetic microplate reader and SoftMAXPro(Molecular Devices).

Pull-down assay of rBMP4 by rOlfml3-FLAGrBMP4 (R&D Systems) was incubated with anti-FLAG

M2-Agarose beads (Sigma-Aldrich) loaded with or with-out rOlfml3-FLAG (1 mg) in TBS, 0.1% NP-40, and 0.05%BSA. Beads were eluted with nonreducing SDS samplebuffer. Samples were subjected to SDS-PAGE and silverstaining was conducted using SilverQuest (Invitrogen).

Western blottingHUVECs were serum-starved in OptiMEM (Invitro-

gen), and 50 ng/mL rBMP4 (R&D Systems) and/or50 ng/mL of rOlfml3-FLAGwere added. Cells were lysedwith lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150mmol/L NaCl, 10 mmol/L MgCl2, and 0.5% Triton X-100] containing a cocktail of protease and phosphataseinhibitors (Sigma-Aldrich). Blots were incubated withanti-phospho-SMAD1/5/8 or anti-SMAD1 (Cell Signal-ing) and revealed using the horseradish peroxidase(HRP)-labeled anti-rabbit antibodies (Jackson Immuno-Research Laboratories), visualized using an enhanced

Novel BMP4 Agonist to Promote Tumor Angiogenesis

www.aacrjournals.org Mol Cancer Ther; 11(12) December 2012 2589




chemiluminescence system and a quantitative imagingsystem LAS4000Mini (Fujifilm).

Statistical analysisAll data are presented as means � SD unless indicated

otherwise. For comparisons of 2 means, the Student t test(2-sided, paired) was used. For multiple mean compar-isons, one-way or two-way ANOVA followed by theBonferroni test was used. All computations were doneusing GraphPad Prism. Results were considered statisti-cally significant at P < 0.05.

ResultsDual expression ofOlfml3 in tumor endothelium andaccompanying pericytes

To mimic molecular and functional properties of endo-thelial cells during tumor angiogenesis, we previouslyisolated 2 subpopulations of an endothelioma cell linewith molecular characteristics of angiogenic (t.End.1-Vhigh) and resting (t.End.1Vlow) cells (23). The t.End.1Vhigh

cells expressedhigh levels of the integrinaVb3 anddid notendocytose acetylated low-density lipoprotein (Ac-LDL),whereas t.End.1Vlow cells expressed low levels of aVb3integrin and efficiently endocytosed Ac-LDL. In addition,t.End.1Vhigh cells showed increased migration andsprouting in 3-dimensional (3D)-fibrin gels (23). Thesecells were exploited as a starting point for the transcrip-tomic profiling that yielded several new candidate geneswithout previously known proangiogenic activity (25).One of the most promising candidate genes in this cate-gory was Olfml3, as its expression was found to berestricted to t.End.1Vhigh cells (Supplementary Fig.S1A).Accordingly, in vivoOlfml3 expressionwasdetectedon vessels undergoing angiogenesis in Matrigel plugs(Supplementary Fig. S1B). To evaluate Olfml3 expressionin tumor vessels, LLC1 cells were s.c. implanted in wild-typemice (Fig. 1).While PECAM-1 transcripts were abun-dantly expressed along the tumor endothelium, Olfml3transcripts were expressed by a subset of endothelial cells(PECAM-1þ) and vessel-associated pericytes (PECAM-1�; Fig. 1A). Tumor cells themselves did not expressOlfml3mRNA (Supplementary Fig. S1C). Double stainingof tumors for Olfml3 and PECAM-1 revealed that Olfml3protein is enriched in the extracellular space of endothelialcells andpericytes of a subset of tumor vessels (Fig. 1B). Tovalidate vascular-specificOlfml3 expression, tumorsweretriple-stained for Olfml3, PECAM-1, and the pericytemarkers a-smooth muscle actin (a-SMA) or nerve/glialantigen-2 (NG2), respectively (Fig. 1C and D). Olfml3expression was detected in both a-SMAþ and NG-2þ

pericytes of PECAM-1þ vessels, whereas it was absentfrom PECAM-1þ vessels not covered by a-SMAþ cells(Fig. 1C). Two-color images of LLC1 tumor sections wereused to quantify colocalization of Olfml3 and PECAM-1,a-SMA, or NG2 staining respectively (Fig. 1E). Weobserved that approximately 25% of Olfml3 colocalizedwith either PECAM-1 or a-SMA whereas approximately55% colocalizedwithNG2 (Fig. 1E). These data illustrated

that endogenous Olfml3 is expressed by both tumorendothelial cells and pericytes. To determine whetherOlfml3 is produced by pericytes embracing establishedtumor vessels or de novo-forming vessels, we isolated 2distinct SMC populations having pericyte-like character-istics: resting, spindle-shaped (S-SMC), and activated,rhomboid (R-SMC) cells (24). R-SMCs displayedenhanced proliferative, migratory, and proteolytic activ-ities and were less differentiated than S-SMCs (24, 29).ActivatedR-SMCs expressed significantly higher levels ofOlfml3 than S-SMCs (Fig. 1F). Therefore, Olfml3 expres-sion levels correlate with the activation state of bothendothelial cells and pericytes, implying a potential func-tional importance of Olfml3 during activation and matu-ration phases of angiogenesis.

Autocrine effects of Olfml3 on endothelial cells andR-SMCs

To define the Olfml3-dependent vascular functions, wefirst tested whether Olfml3 mediates endothelial andpericyte cell migration. As t.End.1Vhigh cells migrate effi-ciently in wound-healing assays (23, 25), we investigatedthe consequences of Olfml3 gene silencing (Supplemen-tary Fig. S2A) on themigration of t.End.1Vhigh cells in thisassay. The Olfml3-silenced t.End.1Vhigh cells displayed asignificantly decreased migration rate into the denudedarea (Fig. 2A). Olfml3 silencing did not affect endothelialcell proliferation (not shown). This reduced migratoryability of Olfml3-silenced cells was partly compensatedwhen recombinant Olfml3-FLAG–tagged protein(rOlfml3-FLAG; Supplementary Fig. S2B) was coated onplates (Fig. 2B). Conversely, t.End1Vhigh cells treatedwithanti-Olfml3AþB antibody migrated less efficient than cellstreated with control rabbit immunoglobulin G (Supple-mentary Fig. S2C). Correspondingly, rOlfml3-FLAG pro-moted t.End.1Vhigh cell migration compared with controlFLAG peptide or DJAM-C-FLAG (Fig. 2C). The rOlfml3-FLAG also promoted migration of R-SMCs (Fig. 2D) andhad no effect on their proliferation (not shown). Thesedata identified Olfml3 as a novel autocrine regulator ofendothelial and pericyte-like cell migration.

The promigratory action of Olfml3 on t.End.1Vhigh cellssuggested that Olfml3 might also exert an effect on endo-thelial cell sprouting. As t.End.1Vhigh cells form a capil-lary-like network of ramified cords in 3D fibrin gels (23),we used this assay to study the effect of Olfml3 depletionon t.End.1Vhigh cell sprouting (Fig. 2E–G).Comparedwithmock- or control siRNA–treated t.End.1Vhigh cells (Fig.2E), the number of Olfml3-silenced cells that initializedsprout protrusions at early time points (24–32 hours) wassignificantly decreased (Fig. 2E and F). In addition, totallength of the vascular network in Olfml3-silenced cellswas reduced at later time points (72 hours; Fig. 2G andSupplementary Fig. S3A). Accordingly, the number ofsprouting cells (24 hours) and total length of the vascularnetwork (48 hours) of anti-Olfml3AþB–treated cells werereduced compared with control IgG-treated cells (Sup-plementary Fig. S3BandS3C).Anti-Olfml3 antibodieshad

Miljkovic-Licina et al.

Mol Cancer Ther; 11(12) December 2012 Molecular Cancer Therapeutics2590




no effect on t.End1Vhigh cells that have already sprouted(72 hours; Supplementary Fig. S3C). These findings sug-gest that abrogation of Olfml3 was sufficient to attenuateendothelial cell sprouting, further supporting its potentialrole in angiogenesis.

Anti-Olfml3 antibodies reduce LLC1 tumor growthand angiogenesisTo testwhetherOlfml3 promotes tumor angiogenesis in

vivo, we generated rabbit anti-Olfml3 antibodies by inject-ing simultaneously two 13-aa long peptides comprisingepitopes in the coiled-coil (peptide A) and the olfactome-din-like domains (peptide B; Supplementary Fig. S4A).Both peptides are identical in the mouse and human

Olfml3 protein sequences (Supplementary Fig. S4B). Theanti-Olfml3 antibodies recognized the peptides A and B,respectively (Supplementary Fig. S4C), and rOlfml3-FLAG (Supplementary Fig. S2B).

The Olfml3 antibodies were affinity-purified againstboth Olfml3 peptides (anti-Olfml3AþB) and evaluated forthe ability to block tumor growth and angiogenesis inthe LLC1 mouse model. Treatment with anti-Olfml3AþB

antibodies significantly decreased the tumor weightcompared with control rabbit IgG treatment (Fig. 3A).To determine which Olfml3 structural domain mightbe necessary for this effect, we affinity-purified theOlfml3 antibodies against either the Olfml3 peptide A(anti-Olfml3A) or peptide B (anti-Olfml3B) and used

Figure 1. Increased Olfml3expression in tumor endothelial cellsand pericytes. A, in situ mRNAhybridization of LLC1 tumors withOlfml3 (green) and PECAM-1 (red)RNA probes shows Olfml3expression on tumor vessels (arrows;inset, arrowheads) and vessel-associated pericytes (insets, stars).No staining with sense Olfml3 RNAprobe (sense). Olfml3-expressingendothelial cells (antisense) arePECAM-1þ (overlay). Pericytesexpress Olfml3 but not PECAM-1(insets, stars). B, Olfml3 (red) andPECAM-1 (green) immunostaining oftumors shows Olfml3 expression ontumor vessels (arrows) and pericytes(overlay/inset, stars). Pericytesexpress Olfml3 but not PECAM-1(insets, stars). C, Olfml3 (light blue),PECAM-1 (green), and a-SMA (red)immunostaining shows Olfml3expression on tumor vessels andaccompanying pericytes (arrows).No Olfml3 staining on PECAM-1þ

vessels not covered bya-SMAþ cells(stars). D, Olfml3 (light blue), PECAM-1 (green), and NG2 (red)immunostaining shows Olfml3expression on tumor vessels andaccompanying pericytes (arrows).DAPI, 40,6-diamidino-2-phenylindole; nuclear counterstain(blue; A–D, overlays). E, left, mergedtwo-color images of LLC1 tumorsshow the amount of colocalization ofOlfml3 and PECAM-1, a-SMA, orNG2 staining (white). Right,quantification of colocalization ofOlfml3 and PECAM-1, a-SMA, orNG2 staining. Error bars represent�SD. F, relative Olfml3 mRNA levelsin activated R-SMCs versus restingS-SMCs. Error bars represent�SD (2experiments, each group intriplicates). ��, P < 0.001. Barscorrespond to 30 mm (A and B),20 mm (C and D), 10 mm (B, inset; E),and 5 mm (A, insets).






them for tumor treatment. Both antibodies significantlyreduced tumor growth by 38% and 52%, respectively,with no significant difference observed between eithertreatment (Fig. 3B). The rate of tumor vascularization

measured by PECAM-1þ area density was decreased bytreatment with either anti-Olfml3A or anti-Olfml3B (Fig.3C and D). The antibodies showed different efficacy ofreducing tumor vascularization. Anti-Olfml3B reduced

Figure 2. Effects of Olfml3 targetingand rOlfml3-FLAG on t.End.1Vhigh

cell migration and sprouting. A, left,in vitro migration assays usingmock, control siRNA (ctrl siRNA,0.5 mmol/L) or Olfml3 siRNA–treated (Olfml3 siRNA, 0.5 mmol/L)t.End.1Vhigh cells. Confluent cellmonolayers were wounded(yellow). Cells migrated into thewounded area after 16 hours(violet). Right, quantification ofmigration distance (mm) of mock,control-, or Olfml3 siRNA–treatedt.End.1Vhigh cells. B, rescuedmigratory ability ofOlfml3-silencedt.End.1Vhigh cells on rOlfml3-FLAG–coated plates (1 mg/mL)when compared with noncoatedcontrol (0 mg/mL). C, coatedrOlfml3-FLAG promotest.End.1Vhigh cell migrationcompared with control FLAGpeptide (1 mg/mL) or DJAM-C-FLAG (1 mg/mL). D, coated rOlfml3-FLAG promotes migration ofR-SMCs compared with FLAGpeptide (1 mg/mL). E, in vitrot.End.1Vhigh sprouting assays in 3Dfibrin gels. Control siRNA–treatedcells start sprouting after 24 hours(arrows) to form a vascular-likenetwork (32–72 hours). TargetingOlfml3 delays sprouting(arrowheads) by 32 hours (arrows).Bars correspond to 10 mm.F, quantification of sprout-formingt.End.1Vhigh cells at early timepoints (24 and 32 hours). Olfml3targeting (Olfml3 siRNA) reducedthe total number of sprouting cellscompared with mock or controlsiRNA–treated cells.G, quantification of total length ofvascular-like network oft.End.1Vhigh cells treated withmock, control, or Olfml3 siRNAs,normalized to total number of cells/condition. At later time points (48and 72 hours), targeting Olfml3reduced the length of the vascular-like network compared withcontrols. Error bars represent �SD(5 experiments; each group intriplicates; A–D, F, G). �, P < 0.05;��, P < 0.01; ��, P < 0.001; ns,nonsignificant (A–D, F, G).






tumor vascularization by 55%, whereas anti-Olfml3A hadsmaller but still significant effect (25%; Fig. 3D), suggest-ing that both structural domains of the protein are nec-essary for its proangiogenic activity.However, when the 2

Olfml3 antibodies were co-injected, no synergistic inhibi-tion of tumor vascularization was observed (not shown).To further analyze the vascular effects ofOlfml3 targeting,we determined average vessel diameter in tumors of

Figure3. Inhibitory effects of anti-Olfml3 antibodieson tumorgrowth andvascularization. A, left, 9-day-old LLC1 tumors inmice treatedwith rabbit IgG (control)or anti-Olfml3AþB. Right, reduced tumor weight in mice treated with anti-Olfml3AþB compared with control. Error bars represent �SEM (3 experiments; 4–5mice/group; 2 tumors/mouse). �, P < 0.05. B, left, 9-day-old tumors in mice treated with control, anti-Olfml3A, or anti-Olfml3B. Right, reduced tumor weight inmice treated with either anti-Olfml3A or anti-Olfml3B compared with control. Error bars represent �SEM (2 experiments; 4–5 mice/group; 2 tumors/mouse).�, P < 0.05; ��, P < 0.01; ns, nonsignificant. C, representative images compare the dense vasculature (PECAM-1, green) of control tumors and prunedvasculature after treatments with anti-Olfml3A or anti-Olfml3B. DAPI, 40,6-diamidino-2-phenylindole; nuclear counterstain (blue). D, quantificationof vessel density in control, anti-Olfml3A-, or anti-Olfml3B-treated tumors measured as a ratio of the total pixel count of PECAM-1 to DAPI and normalized tocontrol. E, quantification of average vessel diameter in control, anti-Olfml3A-, or anti-Olfml3B–treated tumors. F, left, tumor cell proliferation (Ki-67, green)in control-, anti-Olfml3A-, or anti-Olfml3B–treated tumors. Right, quantification of Ki-67þ cells normalized to DAPI. G, left, level of apoptosis (TUNEL, green) incontrol-, anti-Olfml3A-, or anti-Olfml3B–treated tumors. Right, quantification of TUNELþ cells normalized to DAPI. H, level of hypoxia (HIF-1a, red) incontrol-, anti-Olfml3A-, or anti-Olfml3B–treated tumors. Five images at 3 planes analyzed in 8 to 10 tumors per group (D, E; right, F, G). Error bars represent�SEM (2 experiments; 4–5 mice/group; 2 tumors/mouse; D, E; right, F, G). �, P < 0.05; ��, P < 0.01; ��, P < 0.001; ns, nonsignificant (D, E; right, F, G). Barscorrespond to 1 cm (A and B) and 20 mm (C, F–H).






control- and anti-Olfml3–treated mice. This quantitativeassessment revealed that anti-Olfml3–mediated treat-ment led to smaller, collapsed vessels (Fig. 3E) and thus,correlated to thedecreaseof vessel density (Fig. 3D). Thesefindings confirmed our hypothesis that Olfml3 promotestumor angiogenesis, whereas blocking its function leadsto reduced angiogenesis and tumor growth.

As tumor growth is the net result of tumor cell prolif-eration and apoptosis, we next determined the prolifer-ative and apoptotic index of tumors using Ki-67 immu-nostaining and the TUNEL assay, respectively (Fig. 3Fand G). Neither anti-Olfml3A nor anti-Olfml3B treatmentsignificantly affected the number of proliferating Ki-67þ

cells (Fig. 3F). In contrast, anti-Olfml3B–mediated treat-ment significantly increased the number of TUNELþ

apoptotic cells compared with control or anti-Olfml3A–treated tumors (Fig. 3G). This suggests that anti-Olfml3B

treatment leads to increased cell death, thus explainingdecreased tumor growth. To determine the level of tumorhypoxia, we next assessed the activity of hypoxia-induc-ible factor-1a (HIF-1a) in control- and anti-Olfml3–trea-ted tumors (Fig. 3H). Tumors of control-treated miceshowed the translocation of HIF-1a into the nucleus andto perinuclear areas (Fig. 3H). Both anti-Olfml3A- andanti-Olfml3B–treated tumors did not have HIF-1a immu-noreactive cells (Fig. 3H). This suggests that anti-Olfml3treatment does not inducemassive hypoxia considered asa detrimental effect selecting for tumor cell invasion andmetastasis (3).

Impaired pericyte coverage of tumor vessels afteranti-Olfml3 treatment

Endothelial cell survival correlates with the extent ofpericyte coverage in tumor vessels (30). AsOlfml3was co-expressed in tumor endothelial cells and pericytes (Fig. 1),we investigatedwhether anti-Olfml3–mediated treatmentaffects pericyte density and coverage using PECAM-1,a-SMA, andNG2 as the readout. Numerous a-SMAþ andNG2þ pericytes were observed under control conditions(Fig. 4A and C). Following anti-Olfml3–mediated treat-ment, a-SMAþ/pericyte and NG2þ/pericyte densitydecreased by approximately 40% and 25%, respectively(Fig. 4B and D). Correspondingly, anti-Olfml3A- or anti-Olfml3B–mediated treatment reduced NG2þ/pericytecoverage by approximately 40% (Fig. 4E). These observa-tions indicate that targeting Olfml3 decreases the pericytecoverage in tumor vessels, implying Olfml3 involvementin the maturation of de novo-forming vasculature.

Olfml3 is a BMP4-binding proteinPrevious studies have shown thatXenopusOlfml3 inter-

acts with BMP1 and chordin through the coiled-coil andolfactomedin-like domains, respectively (19). We there-fore investigated a possible interaction of Olfml3 withBMPs known as either pro- or antiangiogenic cues withinthe tumor microenvironment (31). We used rOlfml3-FLAG for interaction studies with 3 different BMPs inELISA. rOlfml3-FLAG specifically bound recombinant

BMP4 (rBMP4) but not rBMP1 or rBMP9 (Fig. 5A), andrOlfml3-FLAG co-immunoprecipitated with rBMP4 (Fig.5B). To map the BMP4-binding regions on the Olfml3protein, anti-Olfml3A, anti-Olfml3B, and a commercialantibody raised against a distinct Olfml3 peptide (Olfml3peptide C) were used for binding studies (Fig. 5C). Bothanti-Olfml3A and anti-Olfml3B antibodies partiallyblocked the interaction of rOlfml3-FLAG with rBMP4(Fig. 5D). The third high-affinity antibody, targeting anonoverlapping epitope in the coiled-coiled domain, didnot blockOlfml3–BMP4 interaction (Fig. 5E). These resultssuggest that the coiled-coil (peptide A) and the olfacto-medin-like domain (peptide B) are equally required forthe interaction with BMP4, confirming our previoushypothesis of a single ligand for the 2 Olfml3 domains.Our results define a novel interaction between mouseOlfml3 and BMP4, a potent proangiogenic growth factor.

Olfml3 activates canonical SMAD1/5/8 signalingpathway in HUVECs

As BMP4 directly binds to Olfml3 (Fig. 5), we sought toinvestigate the possible effect of this interaction in BMP4downstream signaling. HUVECs were treated withrOlfml3-FLAG and/or BMP4, and subsequently bothnuclear translocation of SMAD1 and phosphorylation ofSMAD1/5/8 as readouts of the BMP4 pathway activitywere analyzed (Fig. 6). rOlfml3-FLAG alone inducednuclear translocation of SMAD1 after 15 minutes (Fig.6A). Likewise, nuclear translocation of SMAD1 wasobserved in BMP4-treated HUVECs (Fig. 6A). Upon chal-lenge ofHUVECswith rOlfml3-FLAGor BMP4, SMAD1/5/8 proteins were phosphorylated rapidly (Fig. 6B–D),whereas SMAD1/5/8 phosphorylation was not observedin untreated control cells (data not shown) or cells treatedwith the FLAG peptide (Fig. 6B and C). In the presence ofanti-Olfml3AþB antibodies, the ability of Olfml3 to induceSMAD1/5/8 phosphorylation is lost (Fig. 6B and C). Ofinterest, Olfml3 and BMP4 showed additive effects onpSMAD1/5/8 phosphorylation when combined (Fig. 6B–D). While SMAD1/5/8 phosphorylation reached a max-imum after 15 minutes of rOlfml3-FLAG exposure inHUVECs (Fig. 6D), rOlfml3-FLAG and BMP4 exposuregave rise to an increased and prolonged effect onSMAD1/5/8phosphorylation in time course experiments(Fig. 6C and D). These findings show that Olfml3 alone orin a complex with BMP4 acts as an enhancer of theSMAD1/5/8 signaling pathway in HUVECs.

DiscussionOlfml3 has been recently described as an extracellular

modulator of BMP signaling during embryogenesis (19).BMP4 signaling is shown to be critically involved in thedevelopment of blood vessels during embryogenesisand adult life (7, 9). To date, there have been no studiesshowing Olfml3 ability to regulate adult vascular remo-deling under normal or pathologic conditions. Herein,we show that Olfml3 expression is restricted to vesselsundergoing angiogenesis as shown in Matrigel plugs






and tumors. Olfml3 is expressed by angiogenic endo-thelial cells and pericytes and deposited in the perivas-cular compartment. Our findings provide a paradigmfor the contribution of a novel vascular cue withinthe tumor microenvironment that reinforces tumorgrowth.Intense a-SMA and NG2 staining in pericytes charac-

terize the phenotype of mature blood vessels (32). Here,we show that Olfml3 expression is concomitant witha-SMA and NG2 staining in tumor vessel–associatedpericytes. In addition, we found that both a-SMA- andNG2-expressing pericytes were depleted in tumor-bear-ingmice that are treatedwith anti-Olfml3 antibodies. Partof this depletion may reflect reduced pericyte prolifera-tion, but it is also possible that pericyte migration alongthe new tumor vessels is impaired after anti-Olfml3 treat-

ments. In support of this, we observed that Olfml3 pro-motes migration of activated pericyte-like cells (24).Therefore, Olfml3 may contribute to a proangiogenicmicroenvironment that supports remodeling and matu-ration of tumor vessels. Accordingly, anti-Olfml3 tumortreatments substantially affect both angiogenic endothe-lium and accompanying pericytes. In comparison, anti-VEGF treatments generally eliminate tumor vessels with-out removing pericytes (33). Upon cessation of anti-VEGFtherapy, empty sleeves of basement membrane andaccompanying pericytes provide a scaffold for rapidrevascularization of tumors (34). Of interest, increasedand tight pericyte coverage of newly formed vessels con-tributes to the resistance of tumors to anti-VEGF therapy(6). Thus, targeting Olfml3 has a potential advantageover anti-VEGF therapy by reducing tumor growth

Figure 4. Anti-Olfml3 antibodytumor treatment inhibits pericyteassociation with vessels. A, top, theabundance of pericytes (a-SMA, red)in control-, anti-Olfml3A-, or anti-Olfml3B–treated tumors. Bottom,insets of top panels at highermagnification. B, quantification ofa-SMAþ area density in control-,anti-Olfml3A-, or anti-Olfml3B–treated tumorsmeasured as a ratio ofthe total pixel count of a-SMA toPECAM-1 and normalized to control.C, top, the abundance of pericytes(NG2, red) in control-, anti-Olfml3A-,or anti-Olfml3B–treated tumors.Bottom, insets of top panels at highermagnification. DAPI (blue), 40,6-diamidino-2-phenylindole; nuclearcounterstain (A, C). D, NG2þ areadensity in control-, anti-Olfml3A-, oranti-Olfml3B–treated tumorsmeasured as a ratio of the total pixelcount of NG2 (red) to PECAM-1(green) and normalized to control. E,quantification of pericyte coverage incontrol-, anti-Olfml3A-, or anti-Olfml3B–treated tumorsmeasuredasa ratio of the total pixel count ofoverlapping NG2/PECAM-1 toPECAM-1. Ten individual images at 3planes analyzed in 8 to 10 tumors pergroup (B, D, E). Error bars represent�SEM (2 experiments; 4–5 mice/group; 2 tumors/mouse; B, D, E).�, P < 0.05; ��, P < 0.01; ns,nonsignificant (B, D, E). Barscorrespond to 20 mm (top; A, C) and10 mm (bottom; A, C).






while simultaneously affecting tumor endothelium andpericytes. In addition, targeting perivascular Olfml3 mayaffect an intricate crosstalk between endothelial cells andpericytes. It is now widely accepted that pericytes limitthe effectiveness of antiangiogenic therapy by providingsurvival signals for endothelial cells (35). However,inhibition of platelet-derived growth factor receptor(PDGFR)b signaling, which is critical for pericyte recruit-ment, reduces pericyte coverage but has limited efficacyon endothelial cell regression (36, 37). Inhibition of PDGFbsignaling can indirectly reduce tumor vascularization butdoes not necessarily retard tumor growth (38). Severalstudies have shown that the dual targeting of endothelialcells and pericytes is more efficient than targeting eithercell type alone, even in established or drug-resistanttumors (39–41). Therefore, blockingOlfml3 holds promisefor more effective strategies to control tumor growth bytargeting a singlemolecule that affects 2 distinct cell typeswithin the tumor microenvironment.

Two different Olfml3 antibodies, recognizing either anepitope in the coiled-coil or in the olfactomedin-likedomain, substantially inhibited tumor growth and angio-genesis. We did not observe a synergistic effect of the 2antibodies, implying that both Olfml3 epitopes are func-tional and equally necessary for angiogenic outcome. This

is possible only if the 2 Olfml3 domains interact simulta-neously with a putative ligand. Accordingly, our bindingstudies indicated that Olfml3 interacts with BMP4, andboth Olfml3 antibodies were able to specifically interferewith the Olfml3–BMP4 complex. Previous studies byothers have shown that Xenopus ONT1 binds BMP1 andchordin through the coiled-coil and olfactomedin-likedomain, respectively, whereas it does not interact withBMP4 (19).We showhere thatOlfml3 does not bindBMP1or BMP9, both known for their antiangiogenic activities(42, 43). Together, these findings suggest that Olfml3creates specificity for BMP4 by 2 binding sites interactingsimultaneously. Inhibition of Olfml3–BMP4 interactionmight be a major mechanism by which anti-Olfml3 anti-bodies exert their antitumor and antiangiogenic effects.Nonetheless, we cannot dismiss the possibility that thereduction in tumor growth by anti-Olfml3B antibody wasin part a consequence of affecting tumor cell functionindirectly.

BMP4 itself signals to endothelial cells in an autocrinemanner via the canonical SMAD1/5/8 pathway (44, 45).Our data show thatOlfml3 itself promotes endothelial cellmigration and sprouting and activates SMAD1/5/8 sig-naling, implying a role for Olfml3 in the activation ofendothelial cells. In addition, Olfml3 binds BMP4 and this

Figure 5. Recombinant Olfml3binds rBMP4.A, bindingof rOlfml3-FLAG to rBMP4 detected by ELISAusing FLAG (M2) antibody. TherOlfml3-FLAG specificallyrecognizes BMP4 but not BMP1 orBMP9 in a dose-dependentmanner (0.1–1 mg/mL). HumanJAM-C-FLAG negative control (0.1mg/mL). B, immobilized rOlfml3-FLAG on M2-beads binds rBMP4.Silver-stainedSDSgel; left, input ofrBMP4 loaded for comparison(rBMP4; 21 kDa); middle, pull-down of rBMP4 by M2-beads;right, pull-down of rOlfml3-FLAGand rBMP4 by M2-beads (arrow).C, Olfml3 domains relative to anti-Olfml3A, anti-Olfml3B, andcommercial anti-Olfml3C epitoperegions. D, blocking of rOlfml3-FLAG binding to rBMP4 by anti-Olfml3AþB (A þ B), anti-Olfml3A (A)or anti-Olfml3B (B), but not by rabbitIgG (control). E, blocking ofrOlfml3-FLAG binding to rBMP4 byanti-Olfml3AþB, but not by rabbitIgG (control) or anti-Olfml3C. Errorbars represent �SD (5experiments; each group intriplicates; D, E). �, P < 0.05;��, P < 0.001; ns, nonsignificant(D and E).






potentiates SMAD1/5/8 signaling. Therefore, there are atleast 2 possible scenarios that can be proposed for Olfml3-dependent activation of endothelial cells through stimu-lation of the SMAD1/5/8 pathway. First, Olfml3 could

facilitate cell surface retention of BMP4 to promote itsinteraction with receptors, thereby enhancing BMP4-induced recruitment and activation of SMAD1/5/8 at theplasma membrane. Second, Olfml3 may promote BMP4

Figure 6. Olfml3 activates the canonical SMAD1/5/8 pathway. A, Olfml3 induces nuclear translocation of SMAD1/5/8. SMAD1 (red) immunostaining comparesSMAD1 cytoplasmic localization in control HUVECs with SMAD1 nuclear translocation in HUVECs treated with rOlfml3-FLAG (Olfml3; 100 ng/mL), rBMP4(BMP4; 50 ng/mL), or combination (Olfml3þBMP4) for 15 minutes. B, Olfml3 induces SMAD1/5/8 phosphorylation in HUVECs. Phospho-SMAD1/5/8immunostaining (red) compares levels of phoshoSMAD1/5/8 in control HUVECs (control, FLAG peptide) and levels of phospho-SMAD1/5/8 in HUVECstreated with rOlfml3-FLAG (Olfml3; 100 ng/mL), rBMP4 (BMP4; 50 ng/mL), or combination (Olfml3þBMP4) for 15 minutes. Olfml3 does not induce phospho-SMAD1/5/8 in the presence of anti-Olfml3AþB (Olfml3 þ anti-Olfml3) compared with control (Olfm3þ IgG). FITC-phalloidin (green) allows visualization of thecell scaffolds (A and B). DAPI (blue), 40,6-diamidino-2-phenylindole; nuclear counterstain (A and B). Scale bars represent 10 mm (A and B). C, quantification ofthe intensity of nuclear phospho-SMAD1/5/8 signals. rOlfml3-FLAG and rBMP4 combination (Olfml3þBMP4) shows an additive effect on SMAD1/5/8phosphorylation.Mean nuclear intensitywasmeasured from5 to 10 fields per group in 2 experiments. ��,P < 0.001; ns, nonsignificant. D, prolonged effect onSMAD1/5/8 phosphorylation using both recombinant proteins (Olfml3þBMP4), compared with the effect of rOlfml3-FLAG alone. HUVECs were treated withcontrol (0 minute); rOlfml3-FLAG (100 ng/mL) or rOlfml3-FLAG and rBMP4 (Olfml3 þ BMP4; 100 and 50 ng/mL, respectively) for 15, 30, and 45 minutes.






activity by dislodging BMP4 from a putative antagonist inthe extracellular space (46), as shown for pro-BMPactivityof Xenopus ONT1 or Twisted gastrulation (19, 47). Addi-tional studies are needed to elucidate the definitive modeof action bywhichOlfml3 stabilizes BMP4andpotentiatesendothelial cell signaling.

In conclusion, we provide the first evidence for Olfml3being anovel proangiogenic factor,which joins an intrigu-ing group of BMP modulators and affects angiogenesisduring normal and pathologic conditions. In particular,our study shows the functional importance of Olfml3during tumor angiogenesis, and it establishes the poten-tial value of this extracellular molecule as a target forantiangiogenic therapy specific for both endothelial cellsand pericytes. Given the potential side effects and resis-tance linked to current antiangiogenic therapies, a betterunderstanding of novel angiogenic signaling circuits canaccelerate the development of alternative andmore selec-tive strategies, which in turn may be used in combinationwith VEGF inhibitors to increase the efficacy of antiangio-genic tumor treatments.

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

Authors' ContributionsConception and design: M. Miljkovic-Licina, P. Hammel, B.A. ImhofDevelopment of methodology: M. Miljkovic-Licina, P. HammelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Miljkovic-Licina, P. Hammel, S. Garrido-Urbani, B. P.-L. Lee, M. MeguenaniAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M. Miljkovic-Licina, P. Hammel, B. P.-L.Lee, M.-L. Bochaton-PiallatWriting, review, and/or revision of the manuscript:M. Miljkovic-Licina,B. P.-L. Lee, M.-L. Bochaton-Piallat, B.A. ImhofAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Miljkovic-Licina, P. HammelStudy supervision: B.A. ImhofIsolation of SMCs and SMC migration assay: C. Chaabane

AcknowledgmentsThe authors thank Roland Stocker, Christiane Ody, and Yalin Emre for

critical reading of the manuscript and Stephane Jemelin and PhilippeHenchoz for technical help.

Grant SupportThe studywas supported by Institut Clayton de la Recherche grant and

SwissNational Science Foundation 31003AB-135701/1 grant to B.A. Imhofand 310030_130700 to M-L. Bochaton-Piallat.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 5, 2012; revised July 9, 2012; accepted September 12,2012; published OnlineFirst September 20, 2012.

References1. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications

of angiogenesis. Nature 2011;473:298–307.2. Folkman J. Is angiogenesis an organizing principle in biology and

medicine? J Pediatr Surg 2007;42:1–11.3. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.

Cell 2011;144:646–74.4. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of

angiogenesis. Cell 2011;146:873–87.5. Crawford Y, Ferrara N. Tumor and stromal pathwaysmediating refrac-

toriness/resistance to anti-angiogenic therapies. Trends PharmacolSci 2009;30:624–30.

6. Bergers G, Hanahan D. Modes of resistance to anti-angiogenic ther-apy. Nat Rev Cancer 2008;8:592–603.

7. Moreno-Miralles I, Schisler JC, Patterson C. New insights into bonemorphogenetic protein signaling: focus on angiogenesis. Curr OpinHematol 2009;16:195–201.

8. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW,et al. Novel regulators of bone formation: molecular clones andactivities. Science 1988;242:1528–34.

9. Moser M, Patterson C. Bone morphogenetic proteins and vasculardifferentiation: BMPingupvasculogenesis. ThrombHaemost 2005;94:713–8.

10. Vogt RR, Unda R, Yeh LC, Vidro EK, Lee JC, Tsin AT. Bone morpho-genetic protein-4 enhances vascular endothelial growth factor secre-tion by human retinal pigment epithelial cells. J Cell Biochem 2006;98:1196–202.

11. Smadja DM, Bieche I, Silvestre JS, Germain S, Cornet A, Laurendeau I,et al. Bone morphogenetic proteins 2 and 4 are selectively expressedby late outgrowth endothelial progenitor cells and promote neoangio-genesis. Arterioscler Thromb Vasc Biol 2008;28:2137–43.

12. Suzuki Y, Montagne K, Nishihara A, Watabe T, Miyazono K. BMPspromote proliferation and migration of endothelial cells via stimulationof VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling. J Biochem2008;143:199–206.

13. Valdimarsdottir G, Goumans MJ, Rosendahl A, Brugman M, Itoh S,Lebrin F, et al. Stimulation of Id1 expression by bone morphogenetic

protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation 2002;106:2263–70.

14. Wiley DM, Kim JD, Hao J, Hong CC, Bautch VL, Jin SW. Distinctsignalling pathways regulate sprouting angiogenesis from the dorsalaorta and the axial vein. Nat Cell Biol 2011;13:686–92.

15. Rothhammer T, Bataille F, Spruss T, Eissner G, Bosserhoff AK. Func-tional implication of BMP4 expression on angiogenesis in malignantmelanoma. Oncogene 2007;26:4158–70.

16. MaegdefrauU, Amann T,Winklmeier A, Braig S, Schubert T,Weiss TS,et al. Bone morphogenetic protein 4 is induced in hepatocellularcarcinoma by hypoxia and promotes tumour progression. J Pathol2009;218:520–9.

17. Zeng LC, Liu F, Zhang X, Zhu ZD, Wang ZQ, Han ZG, et al. hOLF44, asecreted glycoprotein with distinct expression pattern, belongs to anuncharacterized olfactomedin-like subfamily newly identified by phy-logenetic analysis. FEBS Lett 2004;571:74–80.

18. Sakuragi M, Sasai N, Ikeya M, Kawada M, Onai T, Katahira T, et al.Functional analysis of chick ONT1 reveals distinguishable activitiesamong olfactomedin-related signaling factors. Mech Dev 2006;123:114–23.

19. Inomata H, Haraguchi T, Sasai Y. Robust stability of the embryonicaxial pattern requires a secreted scaffold for chordin degradation. Cell2008;134:854–65.

20. Ikeya M, Kawada M, Nakazawa Y, Sakuragi M, Sasai N, Ueno M, et al.Gene disruption/knock-in analysis of mONT3: vector construction byemploying both in vivo and in vitro recombinations. Int J Dev Biol2005;49:807–23.

21. Tomarev SI, Nakaya N. Olfactomedin domain-containing proteins:possible mechanisms of action and functions in normal developmentand pathology. Mol Neurobiol 2009;40:122–38.

22. Grover PK, Hardingham JE, Cummins AG. Stem cell marker olfacto-medin 4: critical appraisal of its characteristics and role in tumorigen-esis. Cancer Metastasis Rev 2010;29:761–75.

23. Aurrand-Lions M, Johnson-Leger C, Pepper MS, Imhof BA. Haeman-giomas are formed by cells expressing high levels of alphavbeta3integrin and lacking acetylated LDL uptake. J Pathol 2004;203:700–9.






24. Hao H, Ropraz P, Verin V, Camenzind E, Geinoz A, Pepper MS, et al.Heterogeneity of smooth muscle cell populations cultured from pigcoronary artery. Arterioscler Thromb Vasc Biol 2002;22:1093–9.

25. Miljkovic-Licina M, Hammel P, Garrido-Urbani S, Bradfield PF, Sze-petowskiP, ImhofBA. Sushi repeat protein X-linked2, anovelmediatorof angiogenesis. FASEB J 2009;23:4105–16.

26. Piali L, Albelda SM, Baldwin HS, Hammel P, Gisler RH, Imhof BA.Murine platelet endothelial cell adhesion molecule (PECAM-1)/CD31modulates beta 2 integrins on lymphokine-activated killer cells. EurJ Immunol 1993;23:2464–71.

27. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, GabbianiG. A monoclonal antibody against alpha-smooth muscle actin: anew probe for smooth muscle differentiation. J Cell Biol 1986;103:2787–96.

28. Pepper MS, Montesano R, Mandriota SJ, Orci L, Vassalli JD. Angio-genesis: a paradigm for balanced extracellular proteolysis during cellmigration and morphogenesis. Enzyme Protein 1996;49:138–62.

29. Brisset AC, HaoH, Camenzind E, BacchettaM,Geinoz A, Sanchez JC,et al. Intimal smoothmuscle cells of porcine andhumancoronary arteryexpressS100A4, amarker of the rhomboid phenotype in vitro. Circ Res2007;100:1055–62.

30. Franco M, Roswall P, Cortez E, Hanahan D, Pietras K. Pericytespromote endothelial cell survival through induction of autocrineVEGF-A signaling and Bcl-w expression. Blood 2011;118:2906–17.

31. David L, Feige JJ, Bailly S. Emerging role of bone morphogeneticproteins in angiogenesis. Cytokine Growth Factor Rev 2009;20:203–12.

32. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angio-genesis. Cell Tissue Res 2003;314:15–23.

33. Baluk P, Hashizume H, McDonald DM. Cellular abnormalities of bloodvessels as targets in cancer. Curr Opin Genet Dev 2005;15:102–11.

34. MancusoMR,DavisR,NorbergSM,O'BrienS,SenninoB,Nakahara T,et al. Rapid vascular regrowth in tumors after reversal of VEGFinhibition. J Clin Invest 2006;116:2610–21.

35. Erber R, Thurnher A, Katsen AD, Groth G, Kerger H, HammesHP, et al.Combined inhibition of VEGF and PDGF signaling enforces tumorvessel regression by interfering with pericyte-mediated endothelialcell survival mechanisms. FASEB J 2004;18:338–40.

36. Abramsson A, Lindblom P, Betsholtz C. Endothelial and nonendothe-lial sources of PDGF-B regulate pericyte recruitment and influencevascular pattern formation in tumors. J Clin Invest 2003;112:1142–51.

37. Song S, Ewald AJ, Stallcup W, Werb Z, Bergers G. PDGFRbetaþperivascular progenitor cells in tumours regulate pericyte differentia-tion and vascular survival. Nat Cell Biol 2005;7:870–9.

38. Sennino B, Falcon BL, McCauley D, Le T, McCauley T, Kurz JC, et al.Sequential loss of tumor vessel pericytes and endothelial cells afterinhibition of platelet-derived growth factor B by selective aptamerAX102. Cancer Res 2007;67:7358–67.

39. BergersG, SongS,Meyer-MorseN, Bergsland E, HanahanD. Benefitsof targeting both pericytes and endothelial cells in the tumor vascu-lature with kinase inhibitors. J Clin Invest 2003;111:1287–95.

40. Murphy EA, Shields DJ, Stoletov K, Dneprovskaia E, McElroy M,Greenberg JI, et al. Disruption of angiogenesis and tumor growth withan orally active drug that stabilizes the inactive state of PDGFRb/B-RAF. Proc Natl Acad Sci U S A 2010;107:4299–304.

41. Sennino B, Kuhnert F, Tabruyn SP, Mancuso MR, Hu-Lowe DD, KuoCJ, et al. Cellular source and amount of vascular endothelial growthfactor and platelet-derived growth factor in tumors determineresponse to angiogenesis inhibitors. Cancer Res 2009;69:4527–36.

42. GeG, FernandezCA,MosesMA,GreenspanDS.Bonemorphogeneticprotein 1 processes prolactin to a 17-kDa antiangiogenic factor. ProcNatl Acad Sci U S A 2007;104:10010–5.

43. David L, Mallet C, Keramidas M, Lamande N, Gasc JM, Dupuis-GirodS, et al. Bone morphogenetic protein-9 is a circulating vascularquiescence factor. Circ Res 2008;102:914–22.

44. Heinke J, Wehofsits L, Zhou Q, Zoeller C, Baar KM, Helbing T, et al.BMPER is an endothelial cell regulator and controls bone morphoge-netic protein-4-dependent angiogenesis. Circ Res 2008;103:804–12.

45. Zhou Q, Heinke J, Vargas A, Winnik S, Krauss T, Bode C, et al. ERKsignaling is a central regulator for BMP-4 dependent capillary sprout-ing. Cardiovasc Res 2007;76:390–9.

46. Umulis D, O'Connor MB, Blair SS. The extracellular regulation of bonemorphogenetic protein signaling. Development 2009;136:3715–28.

47. Oelgeschlager M, Larrain J, Geissert D, De Robertis EM. The evolu-tionarily conserved BMP-binding protein Twisted gastrulation pro-motes BMP signalling. Nature 2000;405:757–63.






2012;11:2588-2599. Published OnlineFirst September 20, 2012.Mol Cancer Ther Marijana Miljkovic-Licina, Philippe Hammel, Sarah Garrido-Urbani, et al. Angiogenesis and Pericyte CoverageTargeting Olfactomedin-like 3 Inhibits Tumor Growth by Impairing

Updated version

10.1158/1535-7163.MCT-12-0245doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2012/10/15/1535-7163.MCT-12-0245.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/11/12/2588.full#ref-list-1

This article cites 47 articles, 14 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/11/12/2588.full#related-urls

This article has been cited by 14 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/11/12/2588To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-12-0245

http://mct.aacrjournals.org/content/suppl/2012/10/15/1535-7163.MCT-12-0245.DC1

http://mct.aacrjournals.org/content/11/12/2588.full#ref-list-1

http://mct.aacrjournals.org/content/11/12/2588.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/11/12/2588


Documents

Targeting Olfactomedin-like 3 Inhibits Tumor Growth by Impairing … · Therapeutic Discovery Targeting Olfactomedin-like 3 Inhibits Tumor Growth by Impairing Angiogenesis and Pericyte