A YKL-40 Neutralizing Antibody Blocks Tumor Angiogenesis ... · Therapeutic Discovery A...

Therapeutic Discovery

A YKL-40–Neutralizing Antibody Blocks Tumor Angiogenesisand Progression: A Potential Therapeutic Agent in Cancers

Michael Faibish1, Ralph Francescone1, Brooke Bentley3, Wei Yan2, and Rong Shao1,2,3

AbstractAccumulating evidence has indicated that expression levels of YKL-40, a secreted glycoprotein, were

elevated in multiple advanced human cancers. Recently, we have identified an angiogenic role of YKL-40 in

cancer development.However, blockade of the function ofYKL-40,which implicates therapeutic value, has not

been explored yet. Our current study sought to establish a monoclonal anti–YKL-40 antibody as a neutralizing

antibody for the purpose of blocking tumor angiogenesis and metastasis. A mouse monoclonal anti–YKL-40

antibody (mAY) exhibited specific binding with recombinant YKL-40 and with YKL-40 secreted from

osteoblastoma cells MG-63 and brain tumor cells U87. In the functional analysis, we found that mAY inhibited

tube formation of microvascular endothelial cells in Matrigel induced by conditioned medium of MG-63 and

U87 cells, as well as recombinant YKL-40. mAY also abolished YKL-40–induced activation of the membrane

receptor VEGF receptor 2 (Flk-1/KDR) and intracellular signaling mitogen-activated protein (MAP) kinase

extracellular signal–regulated kinase (Erk) 1 and Erk 2. In addition, mAY enhanced cell death response of U87

line to g-irradiation through decreased expression of pAKT andAKT and accordingly, abrogated angiogenesis

induced by the conditioned medium of U87 cells in which YKL-40 levels were elevated by treatment with

g-irradiation. Furthermore, treatment of xenografted tumor mice with mAY restrained tumor growth,

angiogenesis, and progression. Taken together, this study has shown the therapeutic use for the mAY in

treatment of tumor angiogenesis and metastasis. Mol Cancer Ther; 10(5); 742–51. �2011 AACR.

Introduction

The human cartilage glycoprotein-39 (YKL-40) is asecreted glycoprotein originally identified from themedium of a human osteosarcoma cell line, MG-63(1). Structural analyses of this 40-kDa molecule haverevealed that YKL-40 is a highly phylogenetically con-served chitin-binding glycoprotein, classifying it in thefamily of chitinase-like proteins. However, YKL-40lacks chitinase/hydrolase activity due to mutation ofan essential glutamic acid to leucine in the chitinase-3–like catalytic domain (2, 3). Whereas the biophysiologicactivity of YKL-40 is poorly understood, it is believed tobe associated with proliferation of connective tissuecells (4, 5) and activation of vascular endothelial cells(6). Accumulating evidence has shown that serum levels

of YKL-40 were elevated in a variety of chronic inflam-matory diseases (7, 8), suggestive of its pathologicfunction being connected with the process of extracel-lular matrix remodeling (9, 10).

Over the last decade, particular attention has been paidto the pathologic role of YKL-40 in development of abroad type of human cancers. For instance, the databaseof gene microarray analyses and serial analysis of geneexpression (SAGE) shows significantly higher expressionlevels of YKL-40 in carcinoma tissues from ovary, brain,and breast than those expressed in adjacent normaltissues (11, 12). Furthermore, a multitude of clinicalstudies have found that high serum levels of YKL-40were associated with metastasis and short survival in anumber of human cancers, such as breast, colorectal,ovarian, leukemia, and brain carcinoma (13–18), indicat-ing that serum levels of YKL-40 may serve as a newcancer biomarker. Although there is mounting evidenceshowing elevated expression of YKL-40 in human can-cers, little is known regarding its mechanisms underlyingcancer progression and metastasis. Recently, we haveidentified YKL-40 as a tumor angiogenic factor capableof stimulating angiogenesis of microvascular endothelialcells in culture as well as in xenograft models (19).Furthermore, the expression levels of YKL-40 inhuman breast cancer were found to positively correlatewith blood vessel formation. These findings have mark-edly enhanced our understanding of the molecular

Authors' Affiliations: 1Molecular and Cellular Biology Program, MorrillScience Center, 2Department of Veterinary and Animal Sciences, Univer-sity of Massachusetts, Amherst; and 3Pioneer Valley Life Sciences Insti-tute, Springfield, Massachusetts

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: R. Shao, Pioneer Valley Life Sciences Institute,3601 Main Street Springfield, MA 01199. Phone: 413-794-9568; Fax: 413-794-0857. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-0868

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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mechanisms of YKL-40 in the regulation of tumor angio-genesis and progression.Tumor angiogenesis is an integral component of solid

tumor growth and metastasis. Elevated levels of angio-genic factors, such as VEGF and basic fibroblast growthfactor (bFGF), in cancer tissues directly correlate withtumor angiogenesis and tumor progression (20–22). Tosupport these angiogenic properties, multiple comple-mentary studies using neutralizing antibodies, includinga humanized anti-VEGF antibody (bevacizumab, Avas-tin), showed profound impacts in restriction of tumorgrowth (23–25). Unfortunately, to date, functional inhibi-tion of YKL-40 in human diseases and animal modelshas not been explored. Our current study sought toestablish a monoclonal anti–YKL-40 antibody as a neu-tralizing agent for the purpose of blocking tumor angio-genesis and metastasis. Our findings highlight thepotential therapeutic benefits of anti–YKL-40 treatmentin patients with a wide spectrum of cancers that over-express YKL-40.

Materials and Methods

Cell culturesU87 cells were purchased fromAmerican Type Culture

Collection in October 2008, andMG-63 cells were derivedfromDuke University Cell Culture Facility, Durham, NC,in September 2003. Both cell lines were kept in liquid N2

until recently grown in Dulbecco’s Modified Eagle’sMedium (Invitrogen) in the presence of 10% FBS. Nofurther authentication was done during this study.

Purification of recombinant YKL-40Full-length human YKL-40 cDNA with a 6xhistidine

tag was subcloned into a pFastBac1 vector (Invitrogen).Following transformation and amplification in DH10BacE. coli, bacmid DNA containing YKL-40 was transfectedinto Sf9 insect cells by using Cellfectin reagent (Invitro-gen) and subsequently baculoviral medium was pro-duced. An Ni-NTA column was used to purifyrecombinant YKL-40 according to manufacturer’sinstruction (Invitrogen) and YKL-40 pure protein wasfinally produced through a PD-10 desalting column(Millipore).

Anti–YKL-40 antibodiesPolyclonal anti–YKL-40 (rAY) was generated from the

immunization of rabbits with a short peptide of YKL-40encoding C-terminus of YKL-40. Crude serum was thenpurified through an Econo-Pac serum IgG purification kit(BioRad). mAY was created from a hybridoma technol-ogy in whichmice were initially immunizedwith YKL-40recombinant protein as an antigen. Five positive hybri-doma clones were screened twice with ELISA assay usingrecombinant YKL-40–precoated 96-well plates. Twoclones with highest binding activity were further evalu-ated by immunoblotting and functional analysis. Finally,1 clone with strong binding activity and neutralizing

activity was selected and grown for the study. Its culturemedium was consequently collected and concentratedbefore purification using an Affi-Gel protein A MAPS IIkit (Bio-Rad).

YKL-40 gene knockdownDNA oligos (19 bp) specifically targeting YKL-40

sequences 50 GACTCTCTTGTCTGTCGGA 30 [short inter-fering RNA (siRNA) 1] or 50 GGTGCAGTACCTGAAG-GAT 30 (siRNA 2) were selected and engineered intotemplates (64 oligonucleotides) containing these oligoswere subcloned into a retroviral pSUPER-puro-vector.293T retroviral packaging cells were transfected withpSUPER siRNA constructs in the presence of pCL10A1 vector using Fugene 6 (Roche). Forty-eight hoursafter transfection, the supernatant was harvested andfiltered through 0.45-mm pore size filter and then theviral medium was used to infect MG63 cells. Selectionwith 1 mg/mL of puromycin was started 48 hours afterinfection and the puromycin-resistant cell populationswere used for subsequent studies.

Migration assayHuman microvascular endothelial cells (HMVEC; 2 �

105; ref. 26) were preincubated with serum-free mediumovernight and then transferred onto Transwell (8-mm, 24-well plates) precoated with collagen IV (100 mg/mL). Thelower chamber of Transwell included conditioned med-ium of MG-63 cells expressing YKL-40 siRNA or controlvector. After 4 hours of incubation, HMVECs in theTranswell membrane were fixed and stained. Averagecell numbers were calculated from 5 different fields ineach sample.

Tube formation assaysHMVECs (0.1� 105cells) were transferred onto 96-well

Matrigel (BD Bioscience) in the presence of YKL-40 (100ng/mL) or conditioned medium of tumor cells. After 16hours of incubation, tube-forming structures were ana-lyzed. Averages of tubules were calculated from 3 fieldsin each sample.

Cell viability assayU87 cells were treated with 10 Gy g-irradiation in the

presence of 10 mg/mL mIgG or mAY. At 48-hour culture,the cells were imaged for cell detachment. Ninety-sixhours following culture, the cells were subjected to liveand dead analysis using a Live/Dead kit in which ethi-dium homodimer is used to detect dead cells and calceinis used to test live cells (Invitrogen).

Immunoprecipitation and Western blot analysisThe samples were prepared as described before (27).

The lysates were then incubated with an anti-pY20 anti-body at 4�C overnight followed by incubation withprotein A sepharose beads at 4�C for 4 hours. The immu-nocomplex was extensively washed and the sampleswere run on SDS-PAGE. Then proteins were transferred

YKL-40–Neutralizing Antibody and Tumor Angiogenesis

www.aacrjournals.org Mol Cancer Ther; 10(5) May 2011 743




to a PVDF (polyvinylidene difluoride) membrane (Invi-trogen) and incubated with one of several antibodies:anti-YKL-40 (1:200), Flk-1/KDR (1:200), phosphorylatedform of extracellular signal–regulated kinase (pErk) 1,Erk 2 (1:5,000; Santa Cruz), pAKT (ser473, 1:500), AKT(1:500, Cell Signaling Technology), and actin (1:1,000;Sigma). Specific bands were detected using an enhancedchemiluminescence kit (Pierce, VWR). Specific bandswere scanned and analyzed with NIH ImageJ software.Active forms of pFlk-1/KDR, pErk, and pAKT werenormalized with corresponding total nonphosphorylatedforms.

ImmunocytochemistryCells were fixed with 4% paraformaldehyde for 10

minutes and permeabilized with 0.5% Triton X-100 for10minutes at 4�C. Then, the samples were incubatedwithprimary antibodies in PBS-base blocking solution con-taining 0.1% bovine serum albumin, 0.2% Triton X-100,0.05% Tween-20, 7.7 mmol/L NaN3, and 10% goat serumfor one hour. Primary antibodies included purified rabbitrAY 1:100 or preimmune serum overnight at 4�C. Asecondary antibody including goat anti-rabbit Alex Fluor488 antibody (1:1,000; Invitrogen) was added for 1 hour atroom temperature. Fluorescence was determined under amicroscope.

Tumor xenografts in miceAll animal experiments were carried out under the

approval of InstitutionalAnimalCare andUseCommitteeof the University of Massachusetts. Severe combinedimmunodeficient mice (SCID)/Beige mice were subcuta-neously injectedwithU87 cells (7.5� 106) in 0.2mLof PBS.From week 3, when mice developed palpable tumors,mice received either a mAY (5 mg/kg body weight) ormIgG (5 mg/kg) twice a week for 3 weeks. Tumorgrowth from these injected cells was monitored weeklyfor 5 weeks before the animals were humanely sacrificed.Tumorsweremeasuredand tumorvolumewas calculatedas follows: volume ¼ length � width2 � 0.52.

ImmunohistochemistryFrozen tumor tissues from animals were cut to 6-mm

thickness and processed for the staining of CD31. In brief,the samples were incubated with 3%H2O2 for 30 minutesto block endogenous peroxidase activity followed byincubation with blocking buffer containing 10% goatserum for 1 hour. Then, a rat anti-CD31 monoclonal anti-body (1:500, BD Biosciences) was incubated at room tem-perature for 2 hours and a goat anti-rat secondaryantibody (1:100) conjugated with horseradish peroxidasewas added. Finally, DAB (3,30-diaminobenzidine) sub-strate (Dako)was introduced for severalminutes and afterwashing, methyl green was used for counterstaining.

StatisticsData are expressed as mean � standard error (SE) and

differences among groups were determined using 1-way

ANOVA analysis. The 0.05 level of probability was usedas the criterion of significance.

Results

YKL-40, a secreted glycoprotein, acts as an angiogenicfactor to promote tumor angiogenesis (19). To develop amonoclonal antibody as a potential neutralizing agentthat can block angiogenic activity of YKL-40, we isolatedand purified 6x histidine–tagged recombinant proteinYKL-40 as an antigen for immunization of mice(Fig. 1A). The purified recombinant YKL-40 retainedthe ability to activate endothelial cell angiogenesis in vitro(data not shown and see later). Hybridoma cells derivedfrom mice were grown to produce anti–YKL-40 antibody(mAY) and YKL-40–binding activity of mAY was testedusing immunoblotting (Fig. 1B). mAY can specificallyrecognize both recombinant YKL-40 and tumor-secretedYKL-40 of osteoblastoma cells MG-63 and brain tumorcells U87, both of which express YKL-40 (Fig. 1B). Thisbinding specificity was identical to a rAY which wasgenerated from rabbits immunized with a short peptideencoding C-terminus of YKL-40. As expected, mAY didnot interact with samples from HMVECs that do notexpress YKL-40, confirming the unique ability of mAYto react with YKL-40.

To establish an adequate in vitro model that canrecapitulate YKL-40 angiogenesis in vivo, we used acoculture system consisting of endothelial cells andtumor cells that express YKL-40. Reduced expression

Figure 1. Anti–YKL-40 antibodies recognize both YKL-40 protein secretedfrom tumor cells and purified recombinant YKL-40. A, recombinant YKL-40was generated by a baculoviral system. His-tagged YKL-40 was purifiedthrough an Ni-NTA affinity-binding column followed by a desalting PD-10column. Collected samples were analyzed by immunoblotting with an anti-6xhistidine (anti-6xhis) antibody. B, YKL-40 levels in cell conditionedmedium collected from MG-63 cells, U87 cells, and HMVECs, as well asrecombinant YKL-40, were detected by immunoblotting using rAY andmAY.

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levels of YKL-40 fromMG-63 cells were achieved througha gene knockdown approach with a stable inhibition ofYKL-40. siRNA 2, in contrast to siRNA 1, dramaticallyinhibited the expression of YKL-40 (Fig. 2A and Supple-mentary Fig. S1) as 30% to 50% of YKL-40 was reduced insiRNA 2 cells compared with control or siRNA 1 cells. Inan attempt to characterize the angiogenic activity of YKL-40, we measured tube formation and cell migration ofHMVECs, the assays that commonly recapitulate angio-genic property in vitro. Conditionedmedium fromMG-63

control cells and siRNA 1 cells promoted the develop-ment of tubules 4.2-fold more than control endothelialmedium in the absence of the conditioned medium.Notably, YKL-40 gene knockdown of siRNA 2 reducedthe tubules to 50% comparedwith those seen in control orsiRNA 1 cells (Fig. 2B). Consistent with this result, theseconditioned media gave rise to the same effect on cellmigration as the tube formation (Fig. 2C), revealing aparacrine effect of YKL-40 in the induction of angiogen-esis in vivo. To determine the ability of mAY to suppressthe angiogenic activity of YKL-40, we used thetube formation assay and found that mAY fully inhibitedtubules induced by the conditioned medium of MG-63cells (Fig. 3A). Likewise, mAY abolished the tubulesdeveloped by conditioned medium of U87 cells in adose-dependent manner (Fig. 3B). In addition, mAYalso blocked angiogenesis induced by recombinantprotein YKL-40 (Fig. 3C and Supplementary Fig. S2),validating the neutralization of mAY on the angiogenicproperty of YKL-40 present in tumor cell conditionedmedium. All of these data show that mAY acts as aneutralizing antibody to effectively inhibit YKL-40–induced angiogenesis in vitro.

To explore mechanisms of mAY in the inhibition ofYKL-40–induced angiogenesis, we examined expressionof VEGF receptor 2 (Flk-1/KDR) in endothelial cells, oneof the most important membrane-associated tyrosinekinase receptors which mediate endothelial cell angio-genesis (27). Treatment of HMVECs with recombinantYKL-40 resulted in elevated expression of Flk-1/KDR in adose- and time-dependent manner (Fig. 4A). Likewise,this induction was also observed in the cells treated withU87 conditioned medium, but it was diminished in thepresence of mAY (Fig. 4A). Interestingly, YKL-40 alsonotably induced tyrosine phosphorylation of Flk-1/KDRand downstream effector mitogen-activated protein(MAP) kinase pErk 1 and pErk 2 (Fig. 4B and C). How-ever, treatment with mAY significantly reduced pFlk-1/KDR, pErk 1, and pErk 2 (Fig. 4B and C), as pFlk-1reduced to basal levels and pErk 1 and pErk 2 werereduced by 40% to 45% compared with control or mIgGtreatment, suggesting an interruption of the angiogenicsignaling cascades through Flk-1/KDR to MAPK Erk 1and Erk 2. To test whether or not Erk is the only onedownstream effector of Flk-1/KDR, we treated theHMVECs with Flk-1/KDR kinase inhibitor SU1498(12.5 mmol/L) and found that the blockade of Flk-1/KDR activity failed to suppress Erk phosphorylationinduced by recombinant protein YKL-40 (100 ng/mL;data not shown). The data suggest that other upstreammediators of Erk apart from Flk-1/KDR also regulate itsactivation.

It was established that serum levels of YKL-40 wereelevated in cancer patients treated with radiation therapy.Furthermore, these elevated concentrations positivelycorrelated with cancer recurrence and poor survival,suggesting that serum levels of YKL-40 serve as a prog-nostic biomarker (13–18). To test the hypothesis that

Figure 2. YKL-40 siRNA inhibits endothelial cell angiogenesis induced byYKL-40. A, Western blot. Conditioned media from MG-63 expressingsiRNA 1, siRNA 2, and vector control were measured for YKL-40, and celllysates were used for actin using immunoblotting followed by quantitativeanalysis. Intensity of YKL-40 and actin was analyzed with NIH ImageJsoftware and YKL-40 levels were normalized with actin expression.Subsequently, these levels were compared with the basal control arrangedas one unit. B, tube formation. DMEM serum-free medium of MG-63 cellsexpressing YKL-40 siRNA were collected after 24 hours and transferred toMatrigel. After a 16-hour incubation, tubules were imaged and tubedensity was analyzed quantitatively. n ¼ 5. Control represents DMEMserum-free medium and MG-63 control indicates DMEM serum-freeconditioned medium of MG-63 control cells. C, cell migration. Sameconditioned medium of MG-63 cells as described above was transferredto the bottom chamber of a Transwell to test its effects on migration ofHMVECs that were loaded onto the top chamber of the well. After 4 hoursof incubation, migrated cells were fixed and stained. The quantificationwas shown in the bottom. n ¼ 4. *, P < 0.05 compared with DMEM serum-free medium as controls; þ, P < 0.05 compared with MG-63 control orsiRNA 1. Bars, 100 mm.






g-irradiation–induced YKL-40 protects tumor cell death,we monitored the levels of YKL-40 in U87 cells exposedto g-irradiation and measured cell survival in thepresence of mAY. As anticipated, exposure of the cellsto g-irradiation resulted in increased concentrations ofYKL-40 (Fig. 5A). Once these cells were simultaneously

treated with mAY for 48 hours, they displayed a pheno-type indicative of failure to survive, as more than 80%of these cells were detached from culture plates com-pared with the counterparts that showed 30% of detachedcells in the absence of mAY (Fig. 5B). To assess whetherthese cells undergo apoptosis, culture was extended to96 hours and mAY treatment led to cell death around1.5-fold greater than control or mIgG-treated cells(Fig. 5B). To further determine whether the phosphoinosi-tide 3-kinase (PI3K)/AKT signaling cascade mediates theYKL-40–induced cell survival activity, we collected celllysates and measured the expression of these proteinsusing immunoblotting. Intriguingly, mAY treatment ledto decreases in activated AKT and nonactivated AKT by65% to 82%, but did not affect PI3K expression (Fig. 5C) orkinase activity (data not shown), suggesting that YKL-40–mediated cell survival is AKT dependent but PI3K inde-pendent. We next questioned whether the upregulation ofYKL-40 by g-irradiation is capable of prompting endothe-lial cell angiogenesis through a paracrine fashion, whichmaysupport tumor cell survival in vivo. Toaddress this,wecollected the conditionedmediumofU87 cells treatedwithg-irradiation and transferred to HMVECs for the tubeformation assay. The medium from g-irradiation treatedcells increased tubules 2-fold more than did the controlmedium (Fig. 5D). However, mAY completely abrogatedthe angiogenic effects of YKL-40 in HMVECs, stronglysuggesting that mAY be used as an efficacious agent forthe treatment of radiotherapy-resistant cancers.

Finally, we sought to explore the inhibitory effects ofmAY on tumor growth and angiogenesis induced byYKL-40 in vivo. SCID/Beige mice were subcutaneouslyinjected with U87 cells and once these mice developedpalpable tumors by week 3, they were treated with eithermAY or mIgG as a control (5 mg/kg body weight, sub-cutaneously) twice a week for 2 weeks. As shown inFig. 6A, treatment of mice with mAY significantly inhib-ited tumor growth by approximately 40% relative tomIgG treatment. To determine whether antiangiogenicactivity of mAY contributes to the tumor suppression, weexamined expression of CD31, a vascular endothelial cellmarker in tumor samples (Fig. 6B). Histologic analysisexhibited a remarkable reduction in blood vessel forma-tion in tumors from mice treated with mAY, as vesseldensity of mAY-treated tumors was only 28% that of thecontrol group. In parallel with this inhibited tumor angio-genesis, decreased incidence of ectopic tumors was alsoobserved (Fig. 6C). Two of 5 (40%)mice treatedwithmAYdisplayed the normal liver free from tumor cells, onemouse (20%) developed sparse liver metastases and theremainder (40%) exhibited extensive liver metastases. Incontrast, all mIgG-treated control mice (100%) developedmassive liver metastasis (Fig. 6C). No lung metastasiswas identified in either group (data not shown). Takentogether, these in vivo results support the notion thatmAY may serve as a powerful agent for the suppressionof tumor angiogenesis and metastasis in a variety ofadvanced cancers that overexpress YKL-40.

Figure 3.mAY blocks tube formation induced by cell conditioned mediumcontaining YKL-40. A, mAY inhibits tube formation induced by bothconditioned medium of MG-63 and U87 cells. MG-63 and U87 cells werepretreated with mAY or mIgG (10 mg/mL) for 24 hours and the conditionedmedium was transferred to HMVECs for the tube formation. The data werequantified. n ¼ 3. B, mAY-inhibited tube formation is dose dependent.Tube formation of HMVEC was determined in the presence of conditionedmedium of U87 cells pretreated with mAY at 5, 10, and 20 mg/mL or mIgGat 20 mg/mL for 24 hours. The data were quantified. *, P < 0.05 comparedmIgG. n ¼ 4. C, mAY blocks tube formation induced by recombinantYKL-40.mAY ormIgG (10 mg/mL) was introduced to serum-freemediumofHMVECs in the presence of recombinant YKL-40 from 50 to 250 ng/mL inMatrigel and quantification of tube formation was displayed. *, P andþ, P < 0.05 compared with non–YKL-40-treated controls andcorresponding mIgG treatments, respectively. n ¼ 4. Bars, 100 mm.

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Discussion

Our current study has established a monoclonal anti-body specific for YKL-40 and showed that it can neutra-lize YKL-40 function both in vitro and in vivo. This findingoffers immense value for the future development of ananti–YKL-40 humanized antibody not only in the treat-ment of cancer patients but also in the therapy of otherdiseases. Serum levels of YKL-40 have been long viewedas a biomarker in various types of human diseasesincluding cancers (15, 18, 28), inflammatory diseases(9), hepatic fibrosis (8), and atherosclerosis (29). Further-more, these increased concentrations of YKL-40 posi-tively correlate with the progression of these diseases,

indicative of a pathologic role played by YKL-40. Wehave recently identified that YKL-40 acts as a novelangiogenic factor to stimulate vascular endothelial cellmigration and vasculature generation in cancer, indepen-dent of VEGF activity (19). In concert with these pub-lished data, herewe found that YKL-40 secreted fromU87and MG-63 cells and recombinant YKL-40 possess thesame ability to promote endothelial cell angiogenesis invitro. However, mAY fully blocked this angiogenic activ-ity, strongly suggesting that the YKL-40–neutralizingantibody serves as a therapeutic agent for the treatmentof cancers and other diseases that overproduce YKL-40.

Although mAY showed a neutralizing activity onangiogenesis in vitro and in vivo, it failed to completely

Figure 4.mAY interrupts Flk-1/KDR andMAPK/Erk signaling pathways induced by YKL-40. A, mAY blocks YKL-40–induced Flk-1/KDR expression. HMVECspretreated with serum-free medium overnight were stimulated with 100 ng/mL recombinant protein YKL-40 from 0 to 40 hours or with 0 to 200 ng/mL YKL-40for 24 hours. Conditioned medium (CM) from U87 cells in the presence or absence of 10 mg/mL mIgG or mAY was transferred to HMVECs for 24 hours asdescribed in Fig. 3A. Subsequently, all of the cell lysates were tested for Flk-1/KDR expression by immunoblotting. Flk-1/KDR data were normalized with actinlevels and then compared with the basal serum-free level arranged as one unit. n¼ 4, mean� SE. B, mAY blocks YKL-40–induced tyrosine phosphorylation ofFlk-1/KDR. HMVECs were pretreated with serum-free medium overnight in the presence of mAY or mIgG (10 mg/mL) and then directly stimulated withrecombinant YKL-40 (100 ng/mL) for 10 minutes. n ¼ 2, mean � SE. For treatment with cell conditioned medium, HMVECs were pretreated with serum-freemedium overnight and then treated for 10 minutes with U87 cell conditioned medium that was exposed to mAY or mIgG (10 mg/mL) for 24 hours. Halfof the cell lysates were immunoprecipitated with an antibody against phosphorylated tyrosine protein (pY20) followed by immunoblotting using anantibody against Flk-1/KDR. The remaining lysates were used directly for immunoblotting against total Flk-1/KDR. IgG of the anti-pY20 antibody wasalso tested as loading control. pFlk-1/KDR was normalized with total Flk-1/KDR. n ¼ 3, mean � SE. C, mAY blocks YKL-40–induced MAPK Erk 1 andErk 2 activation. The same conditions of HMVECs as described in B were collected and immunoblotted with antibodies against pErk 1, Erk 2, and total Erkfollowed by quantification. pErk 1 and pErk 2 levels were normalized with total Erk 1 and Erk 2. SF, serum-free basal DMEM; Con, DMEM serum-freeconditioned medium alone. n ¼ 2, mean � SE.






abolish tumor growth in our xenografted model. Thisinconsistent result may be attributed to a number ofpossibilities that occur in animal models. First, we startedthe treatment of animals with mAY from week 3 whentumors had established significant tumor mass thatpoorly responded to mAY. Second, the dose of mAYwe used (5 mg/kg body weight) in this study may beinsufficient to fully eliminate YKL-40 activity, as 10 timesof anti-VEGF receptor antibody (DC101) or doubleamount of anti-VEGF–neutralizing antibody (bevacizu-mab) were tested by other groups in xenograft tumormodels previously (30, 31). In addition, it is likely that avariety of angiogenic factors other than YKL-40, such asbFGF, which are produced by tumor cells, also mediate

vasculature formation in vivo (in our unpublished data).Finally, host tissue may play a role in facilitating angio-genesis as well because YKL-40 and other growth factorscould be derived from a number of infiltrating cellsincluding macrophages and neutrophils when tumorsdevelop and invade adjacent normal tissue (32, 33). Thus,mAY is incapable of blocking other proangiogenicand/or tumor-promoting factors that contribute to tumordevelopment. Nonetheless, our study has shown theeffectiveness of mAY in inhibition of tumor angiogenesisand growth.

Theevidence that tumor cell conditionedmediumexhib-itedmore pronounced impacts on tube formation than didrecombinant YKL-40 (Fig. 3) strongly suggests that other

Figure 5.mAY enhances tumor cell death responses to g-irradiation and decreases endothelial cell angiogenesis. A, g-irradiation induced YKL-40 expression.U87 cells were treated with 0, 3, 5, or 10 Gy of g-irradiation and 48 hours later, cell conditioned medium was subjected to testing YKL-40 levels and celllysates were examined for actin levels. YKL-40 levels were quantified by normalization with actin. The basal level of YKL-40 without g-irradiation wasset as one unit. n ¼ 2, mean � SE. B, mAY decreases cell viability in response to g-irradiation. Top, U87 cells treated with 10 Gy g-irradiation as describedabove in the presence of mAY or mIgG (10 mg/mL) were determined for cell viability in cultured condition. Attached and detached cells were quantified under amicroscope. Bottom, 96 hours following treatment with 10 Gy g-irradiation above, U87 cells were assayed for cell death as described in Materials andMethods. Red fluorescence, dead cells; green fluorescence, live cells. *,P < 0.05 compared with the control or mIgG-treated group. n¼ 5. Bars, 50 mm.C, mAYdecreases pAKT and AKT levels. U87 cells treated with g-irradiation for 48 hours as described in B were tested for pAKT (ser473), AKT, PI3K, and actin. pAKTand AKT levels were normalized with actin, and subsequently, these levels were compared with their basal expressions without g-irradiation arranged as oneunit. n¼ 2, mean� SE. D, mAY blocks g-irradiation–induced angiogenesis. Conditionedmedium of U87 cells as described in Cwas transferred to HMVECs fortesting tubules in Matrigel. n ¼ 4. *, P < 0.05 compared with cells without g-irradiation; þ, P < 0.05 compared with control or mIgG. IR, irradiation.

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potential angiogenic factors derived from tumor cellsmay also participate in tumor angiogenesis. However,this angiogenesis induced by the conditioned mediumcan be sufficiently blocked by mAY, implicating a centralrole that YKL-40 plays in regulating other angiogenicfactors. Indeed, we found in a separate study thatYKL-40 upregulates VEGF expression in U87 cells (34).Interestingly, mAY is able to fully eliminate tube forma-tion in the presence of tumor cell conditioned medium, incontrast to its inhibition in the presence of recombinantYKL-40, suggesting that blockade of YKL-40 in the condi-tioned medium also induces the release of other factorsfrom tumor cells which may profoundly impair tubedevelopment.

Our previous study has found that YKL-40 inducedcoordination of membrane-bound receptor syndecan 1and integrin avb3 and activated intracellular signalingcascade including FAK (focal adhesion kinase), Erk 1, andErk 2 (19). Here, we identified that YKL-40 not onlyincreased expression of Flk-1/KDR, a VEGF receptor 2that mediates VEGF angiogenesis (27) but also activatedthe tyrosine phosphorylated form of Flk-1/KDR, possiblyleading to a synergistic effect on the angiogenic signalingactivation. Erk 1 and Erk 2 were found to be the down-stream intracellular effectors. Notably, mAY abrogatedall these signaling cascades induced by YKL-40. Thesedata show the molecular mechanisms underlying YKL-40–induced angiogenic responses in endothelial cells andunderscore the neutralizing activity of mAY in the inhi-bition of angiogenesis. It remains to be determined thatwhether YKL-40–induced tyrosine phosphorylation ofFlk-1/KDR is dependent or independent of activationof other adjacent membrane receptor syndecan1 andintegrin avb3.

We found that g-irradiation induced YKL-40 expressionwhich not only protected cell death, but also elicited endo-thelial cell angiogenesis through a paracrine loop. Wefound that mAY sensitized the death responses of tumorcells to g-irradiation through a decrease of PI3K-indepen-dent AKT phosphorylation, a common survival pathwaywhich has been established previously (35–37). Consistentwith our results, MAPK and AKT were reported to medi-ate YKL-40–induced mitogenic signaling in connectivetissue cells (4). In addition, we interestingly found thatmAY nullified endothelial cell angiogenesis induced byU87conditionedmediumtreatedwith g-irradiation.There-fore, our studies offered mechanistic insight into radio-therapy resistance of cancers that express increased levelsof YKL-40 and show poor prognosis (18, 38). Furthermore,our findings shed light on the pronounced impacts ofYKL-40–neutralizing antibody on cancer progression inconjunction with radiation therapy.

In current cancer therapy, particular attention has beenfocused on a number of antiangiogenic drugs approvedby FDA (Food and Drug Administration) such as anti-VEGF antibody bevacizumab and VEGF receptor tyro-sine kinase inhibitors (sorafenib and sunitinib). However,the benefits of these antiangiogenic agents appear to betransitory in the treatment of several types of advancedcancers, as drug resistance, tumor regrowth, and exten-sive vascular recovery rapidly develop once the therapyis terminated (39–41). Furthermore, there are severalrecent studies showing the opposite effects of these anti-angiogenic drugs on tumor growth, angiogenesis, andmetastasis in xenografted tumor models. For example,treatment of xenotransplant models with an anti-VEGFreceptor 2 antibody unexpectedly resulted in extensivetumor revascularization, increased invasiveness, andrapidly ectopic dissemination (30, 42). In line withthis evidence, a short-term therapy with sunitnib andSU11248 (VEGF and platelet-derived growth factorreceptor kinase inhibitor) accelerated local tumor

Figure 6. mAY inhibits tumor growth, angiogenesis, and progression. A,U87 cells were injected subcutaneously into SCID/Beigemice, andmAY ormIgG (5 mg/kg body weight) was injected subcutaneously twice a weekfrom week 3. Tumor volume was evaluated, n ¼ 5. *, P < 0.05 comparedwith corresponding mIgG. B, tumor samples were collected and subjectedto immunohistochemistry of CD31 staining. Brown color, positive stainingfor blood vessels. Bar, 100 mm. Vessel density was quantified usingImageJ software. *, P < 0.05 compared with corresponding mIgG. C, liverwas removed from mice and processed for hematoxylin and eosinstaining. Dark blue cells are tumor cells. Bar, 100 mm.






invasion and multiple distant metastases after intrave-nous injection of tumor cells or removal of primarytumors (43). This immediately acquired adaptation tothe antiangiogenic therapies is believed to be associatedwith the angiogenic switch by which tumors undergorobust revascularization and malignant transformation(42). It would be quite interesting to know whether YKL-40 plays an active role in these evasion responses. Never-theless, the combined antiangiogenic approaches thatinclude antiangiogenic monoclonal antibodies and mem-brane receptor kinase inhibitors together with other che-motherapies pave the way toward a more efficacioustherapy for cancer patients.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by National Cancer Institute R01 CA120659and DOD W81XWH-06-1-0563 (to R. Shao)

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 September 14, 2010; revised February 16, 2011; acceptedFebruary 23, 2011; published OnlineFirst February 23, 2011.

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2011;10:742-751. Published OnlineFirst February 25, 2011.Mol Cancer Ther Michael Faibish, Ralph Francescone, Brooke Bentley, et al. and Progression: A Potential Therapeutic Agent in Cancers

Neutralizing Antibody Blocks Tumor Angiogenesis−A YKL-40

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