novel paracrine modulation of notch dll4 …...fibulin-3 promotes angiogenesis in high-grade gliomas...

Microenvironment and Immunology

Novel Paracrine Modulation of Notch–DLL4 Signaling byFibulin-3 Promotes Angiogenesis in High-Grade Gliomas

Mohan S. Nandhu1, Bin Hu2, Susan E. Cole3, Anat Erdreich-Epstein4,5, Diego J. Rodriguez-Gil6, andMariano S. Viapiano1

AbstractHigh-grade gliomas are characterized by exuberant vascularization, diffuse invasion, and significant

chemoresistance, resulting in a recurrent phenotype that makes them impossible to eradicate in the longterm. Targeting protumoral signals in the glioma microenvironment could have significant impact againsttumor cells and the supporting niche that facilitates their growth. Fibulin-3 is a protein secreted by gliomacells, but absent in normal brain, that promotes tumor invasion and survival. We show here that fibulin-3 is aparacrine activator of Notch signaling in endothelial cells and promotes glioma angiogenesis. Fibulin-3overexpression increased tumor VEGF levels, microvascular density, and vessel permeability, whereas fibulin-3 knockdown reduced vessel density in xenograft models of glioma. Fibulin-3 localization in humanglioblastomas showed dense fiber-like condensations around tumor blood vessels, which were absent innormal brain, suggesting a remarkable association of this protein with tumor endothelium. At the cellularlevel, fibulin-3 enhanced endothelial cell motility and association to glioma cells, reduced endothelial cellsprouting, and increased formation of endothelial tubules in a VEGF-independent and Notch-dependentmanner. Fibulin-3 increased ADAM10/17 activity in endothelial cells by inhibiting the metalloproteaseinhibitor TIMP3; this resulted in increased Notch cleavage and increased expression of DLL4 independentlyof VEGF signaling. Inhibition of ADAM10/17 or knockdown of DLL4 reduced the proangiogenic effects offibulin-3 in culture. Taken together, these results reveal a novel, proangiogenic role of fibulin-3 in gliomas,highlighting the relevance of this protein as an important molecular target in the tumor microenvironment.Cancer Res; 74(19); 5435–48. �2014 AACR.

IntroductionHigh-grade gliomas are the most common primary tumors

in the central nervous system (CNS) and one of the mostaggressive and difficult to treat forms of cancer (1). Glioblas-toma (GBM), the most common form of adult glioma, has aparticularly dismal prognosiswith amedian survival of approx-

imately 15 months (2). Multiple factors contribute to this pooroutcome, including tumor isolation within the CNS, cellularheterogeneity within the tumor, and the highly invasive natureof GBM cells (1).

GBMs are, in addition, among the most vascularized type oftumors (3). Aberrant GBM blood vessels exhibit glomeruli ofproliferating endothelial cells, reduced astrocyte and pericytecoverage, and thrombotic obstructions. The resulting capillar-ies are "leaky" and have abnormal flow, resulting in tumorhypoxia despite the increasedmicrovascular density (4, 5). Thelocal environment around these abnormal blood vessels formsthe anatomic and functional niche for glioma stem cells (GSC),which proliferate and disperse just beyond the reach of con-ventional therapeutics.

Antiangiogenic treatments have been an attractive strategyto disrupt the supportingmicroenvironment of GBM (4, 6). Theanti-VEGF antibody bevacizumab is widely used for adjuvanttherapy of recurrent GBM, enhancing quality of life andextending progression-free survival. However, results fromrecent clinical trials suggest lack of significant benefits forbevacizumab in primary GBM (7), highlighting the need forbetter antiangiogenic approaches. More importantly, mount-ing evidence suggests that antiangiogenic treatments maytrigger dispersion of residual tumor, making the treatment ofrecurrent GBMevenmore challenging (8). Novel approaches to

1Department of Neurosurgery, Brigham and Women's Hospital and Har-vard Medical School, Boston, Massachusetts. 2Department of Neurolog-ical Surgery, The Ohio State University, Columbus, Ohio.3Department ofMolecular Genetics, The Ohio State University, Columbus, Ohio. 4Depart-ment of Pediatrics, Children's Hospital Los Angeles, Keck School ofMedicine and theUniversity of Southern California, LosAngeles, California.5Department of Pathology, Children's Hospital Los Angeles, Keck Schoolof Medicine and the University of Southern California, Los Angeles,California. 6Department of Neurosurgery, Yale University School of Med-icine, New Haven, Conneticut.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M.S. Nandhu and B. Hu contributed equally to this article.

Corresponding Author: Mariano S. Viapiano, Department of Neurosur-gery, Brigham and Women's Hospital, 4 Blackfan Circle, Boston, MA02115. Phone: 617-525-5650; Fax: 617-713-3050; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-0685

�2014 American Association for Cancer Research.

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hinder simultaneously tumor vascularization and invasion,which are supported by multiple common molecular mechan-isms, would be a welcome strategic addition for combinationtherapy of GBM.

GBM invasion results from concerted mechanisms of tu-mor cell adhesion, motility, and remodeling of the extracellu-lar matrix (ECM). Our laboratory was the first to describe theECM protein fibulin-3 (gene EFEMP1) in GBMs and to demon-strate the key protumoral role of this extracellular proteinin gliomas (9, 10). Fibulin-3 is a matrix protein detected inthe skin andelastic tissues but absent in normal brain, includingthe brain vasculature (11). Expression of fibulin-3 is associatedwith tumor progression toward a more malignant, metastaticphenotype: this protein is unchanged or downregulated in seve-ral primary solid tumors but upregulated in the late, metastaticstages of carcinomas (12, 13). In gliomas, fibulin-3 expressioncorrelates with tumor grade and is particularly increased inprimary and recurrent GBMs. Fibulin-3 promotes tumor inva-sion and supports survival of GBMcells challenged by apoptoticstimuli. Mechanistically, this protein promotes activation ofNotch signaling (9), being one of the first paracrine activators ofthis pathway described in cancer.

The Notch pathway is a highly conserved signaling mech-anism that involves activation of Notch receptors by ligandsfrom the Delta-Like and Jagged families located on adjacentcells. This pathway is highly active in gliomas (14), where ithas been correlated with tumor cell proliferation, survival toapoptosis, self-renewal of tumor stem cells, and invasion (15).In addition, activation of Notch in endothelial cells by itsligand DLL4 regulates the formation of tumor blood vessels(16) and is critical to stabilize a functional vascular network(17). Notch and DLL4 are upregulated in subsets of GBMspresenting an angiogenic phenotype (18), and Notch–DLL4signaling has been shown to mediate glioma resistance toanti-VEGF therapy (19). Whether fibulin-3 could regulateNotch–DLL4 signaling and be involved in glioma vasculariza-tion is unknown.

We demonstrate here thatfibulin-3 promotes vascularizationof high-grade gliomas in vivo and may play a role in the asso-ciation of glioma cells to blood vessels for tumor growth anddispersion. Moreover, we show that fibulin-3 activates endo-thelial Notch–DLL4 signaling independently of VEGF andthrough a novel TIMP3/ADAM-dependent mechanism, result-ing in increased proangiogenic behavior of endothelial cells.

Materials and MethodsCells and tissue specimens

The rat GBM cell line CNS1 and human GSCs GBM8 andGBM34 were cultured as previously described (9, 10). GSCswere validated for self-renewal, tumorigenicity in lownumbers,and multilineage differentiation, and were always used in earlypassages. Primary cultures of human brain microvascularendothelial cells (HBMEC) were prepared from normal braintissue obtained during expedited autopsy and cultured inRPMI1640 with 10% FBS, 2 mmol/L L-glutamine, 1 mmol/Lsodium pyruvate, 20 mmol/L HEPES, 50 U/mL penicillin, and50 mg/mL streptomycin. The cells used in this study were

originally described as HBMEC-3 (20) and validated for expres-sion of factor VIII-reactive antigen and uptake of acetylatedlow-density lipoprotein. We further validated HBMECs forexpression of endothelial markers and negligible expressionof fibulin-3 (Supplementary Fig. S1), and authenticated themby short-tandem repeat DNA profiling (Idexx Bioresearch).Human umbilical vein endothelial cells (HUVEC) wereobtained from ATCC and cultured in the same medium asHBMECs. Human GBM tissues were provided by the Cooper-ative Human Tissue Network funded by the NCI. Normal braintissues were provided by the Brain and Tissue Bank forDevelopmental Disorders, funded by the National Institute ofChild Health and Human Development.

DNA constructs, lentivectors, and siRNAsFull-length fibulin-3 cDNA (gene EFEMP1, Genbank

# BC098561.1) was cloned into pcDNA3.1(þ) (Life Techno-logies) and then subcloned into the lentiviral expressionvector pCDH-EF1-copGFP (System Biosciences) for consti-tutive expression in glioma cells (10). Full-length TIMP3 cDNA(Genbank # BC014277.2) was cloned into pcDNA3.1-V5/6xHisfor transient expression. Fibulin-3 short hairpin RNA (shRNA)have been previously described and validated; these shRNAswere cloned into the lentiviral vector pLVET-tTR-KRAB-eGFP for doxycycline-dependent coexpression of shRNAs andeGFP, as described (9). A Notch reporter construct carryingfirefly luciferase under control of 4xCBF1-binding elements(pGL2Pro-CBF1-Luc) and the truncated, constitutively activemouse Notch-1 intracellular domain (pSG5-FLAG-NICD) havealso been described (9). siRNA oligonucleotides against DLL4were purchased from Qiagen and validated at the mRNA andprotein levels (Supplementary Fig. S1).

Animal proceduresAll animal experiments were approved by the Institutional

Animal Care and Use Committees at The Ohio State Univer-sity (Columbus, OH) and Harvard Medical School/Brighamand Women's Hospital (Boston, MA). A total of 7.5 � 104

GFP-expressing CNS1 cells (2.5 � 104 cells/mL) were implant-ed intracranially in the striatum of Lewis rats as previouslydescribed (10) and allowed to form tumors for 15 days beforehistologic processing. To assess vessel leakiness, animalswere injected in the tail-vein with 200 mL of 1 mg/mL tetra-methylrhodamine-labeled dextran (average molecular weight155 kDa, Sigma-Aldrich), euthanized after 5 minutes, and thebrain immediately fixed and processed for cryosectioning.Human GSCs were first transduced with the lentiviral vectorcarrying inducible fibulin-3 shRNA/eGFP and selected byfluorescence as described (9). A total of 5 � 104 cells (2.5 �104 cells/mL) were implanted in the striatum of athymic miceand the shRNA induced with 1 mg/mL doxycycline in thedrinking water, starting 3 days after tumor implantation (9).Animals were euthanized and tumors harvested for cryosec-tioning and histologic analysis 21 days after implantation.Blood vessels were stained with endothelial markers (CD31 inmouse and RECA1 in rat) and analyzed within the tumorboundaries (identified by GFP fluorescence). Vessels wereidentified as elongated structures of arbitrary length >25 mm,

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using the image analysis software ImageJ, and quantified inat least 3 separate sections per tumor. In some cases, tumorsand contralateral brain tissue were recovered after 14 daysand dissociated with collagenase/hyaluronidase as described(21). Brain microvascular endothelial cells were isolated fromthese homogenates by flow cytometry, using fluorescentlylabeled anti-CD31 antibody.

Cell culture experimentsFor cell–cell adhesion experiments, CNS1 cells were seeded

at 5 � 104 cells per well in 48-well plates and allowed to formmonolayers overnight. HBMECs (1 � 104 cells/well) labeledwith the red-fluorescent cell tracker CM-DiI (Life Techno-logies) were added to the monolayers the following day andwashed off with PBS after 2 hours. The number of red-fluo-rescent adhered cells per well was counted using fluores-cence microscopy. For cell migration experiments, HBMECs(5 � 103 cells/well) were seeded in Transwell inserts (8 mmpore) precoated on their underside with fibronectin (10mg/mL) to induce cell motility. Cells were then allowed tomigrate toward CNS1 cells or purified fibulin-3 (300 ng/mL,Origene Technologies) for 8 hours. Cells were subsequentlyfixedwithmethanol, stained, and counted. For invasion experi-ments, the Transwell inserts were first covered with 50 mLMatrigel (7.5 mg/mL) and cells were allowed to migratethrough this barrier for 24 hours. For coculture experiments,fluorescent HBMECs (5 � 103 cells/well) were first seededin 24-well plates containing an aligned-nanofiber substrate(Nanofiber Solutions) that restricted cell movement and facil-itated cell tracking (22). Glioma cells (5 � 104 cells/well) wereseeded on Transwell inserts (0.4 mm pore) hanging on top ofthe nanofibers. Both cell types were cocultured in HBMECculture medium overnight. HBMECs were imaged by time-lapse fluorescence microscopy using an Olympus IX81 micro-scope adapted with an environmental chamber. Migrationof individual HBMECs was quantified using the softwareImagePro 6.1 (Media Cybernetics). For tubulogenesis assays,HBMECs were seeded at 1� 104 cells per well in 96-well platesprecoated with 50 mL Matrigel and allowed to aggregateovernight. The following day, cells were stained with Calcein-AM (Life Technologies) and imaged by fluorescence micro-scopy. Tubules were identified and analyzed using the softwareImageJ loaded with the Skeletonize and AnalyzeSkeleton plu-gins. For endothelial cell sprouting assays, we followed apreviously described method (23). Briefly, HBMECs (5 � 104

cells/well) were cultured in ultra-low attachment 96-wellplates (Corning Life Sciences) to form floating spheroids of250 to 300 mm diameter. Spheroids were manually seeded in96-well plates on a Matrigel substrate (50 mL/well) and incu-bated for 5 to 6 days to measure total cell sprouting fromeach spheroid. In all experiments, transfection with cDNAs orsiRNAs was performed 48 hours before cell culture assays.

Biochemical assaysCells were recovered from culture, lysed, and processed for

Western blotting using standard protocols (antibodies arelisted in Supplementary Table SI). For semiquantitative real-time (RT)-PCR, cells were processed using TRIzol reagent (Life

Technologies) and total RNA was purified by ethanol precip-itation (primers are listed in Supplementary Table SII). ForNotch reporter assays, HBMECs were transfected with theNotch reporter construct and Renilla luciferase as loadingcontrol (9). Reporter-transfected cells were treated with puri-fied fibulin-3 for 16 hours and processed to quantify luciferaseactivity. To measure alpha-secretase (ADAM10/17) activity,HBMECs were lysed in 50 mmol/L Tricine buffer (pH 7.5)containing 100 mmol/L NaCl, 10 mmol/L CaCl2, 1 mmol/LZnCl2, and 0.1%Triton X-100. Lysates (50mg total protein) wereincubated with a fluorogenic ADAM10/17 substrate peptide(TACE substrate III, 10 mmol/L; R&D Systems) and develop-ment of fluorescence was followed with a microplate reader asrecommended by the peptide manufacturer. Cultures weretreated for 1 to 16 hours with purified fibulin-3 (300 ng/mL),purified TIMP3 (1 mg/mL; Sigma-Aldrich), gamma-secretaseinhibitor DAPT (25 mmol/L; Tocris Bioscience), and alpha-secretase inhibitor TAPI-2 (1 mmol/L; Cayman Chemical)

ImmunohistochemistryFrozen human tissues were mounted in cryoprotectant and

sectioned at 20 mm. Sections were allowed to air-dry and thenfixed for 20minutes at room temperature using buffered acidicalcohol (8.5 mmol/L sodium acetate buffer, pH 5, in 90%ethanol). Fixed sections were washed with PBS, blocked withPBS containing 5% normal goat serum and 0.1% Triton X-100,and incubated with primary antibodies (16 hours) and sec-ondary antibodies (2 hours) following standard procedures.Stained sections were imaged using a confocal microscopeZeiss LSM710.

Statistical analysisAll in vitro experiments were repeated at least in triplicate

with three independent replicates per group. Results areshown as means � SEM. Animal studies were performedwith five animals per experimental condition. Multivariateresults were analyzed by one- or two-factor ANOVA with aTukey multiple comparison test. A value of P < 0.05 was takento indicate statistically significant differences.

ResultsFibulin-3 expression correlates with tumorvascularization in rodent models of high-grade glioma

We have previously shown that fibulin-3 correlates withoverall size and invasion of intracranial gliomas: Fibulin-3–overexpressing gliomas are significantly larger and more inva-sive than controls (10), while tumors with downregulatedfibulin-3 are smaller and less dispersed (9). Interestingly, grossinspection of these tumors often showed increased hemor-rhage in fibulin-3–overexpressing tumors and reduced bleed-ing in fibulin-3–deficient tumors, independently of their size,which prompted the hypothesis that fibulin-3 could alsoregulate tumor vascularization.

To test this hypothesis, we first analyzed microvasculardensity of CNS1-derived tumors implanted in their syngeneichosts (Lewis rats). Fibulin-3–overexpressing tumors exhibitedsignificantly higher microvascular density and increased aver-age vessel length (Fig. 1A and B). Identical results were

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Figure 1. Fibulin-3 correlateswith increased tumor vascularization and vessel permeability. CNS1-derived gliomaswere processed for immunohistochemistryto identify blood vessels within the tumor parenchyma. In separate experiments, animals were injected with rhodamine-labeled dextran to quantifyvessel leakiness. A, representative image of control and fibulin-3–overexpressing (fibulin-3) tumor sections stained with RECA1 (bar, 100 mm). B,quantification of microvascular density, stratified by vessel length (�, P < 0.05; ��, P < 0.01 by repeated measures ANOVA). C, representative images fromtwo independent control and fibulin-3–overexpressing tumors, showing total tumor distribution (dashed line) and dextran accumulation (arrows) in thetumor parenchyma. D, quantification of integrated dextran fluorescence (IF) along the anteroposterior axis of the tumors; each line represents a separateanimal (RU, relative units). All sections were imaged under the same illumination conditions by automated imaging software. E, average signal offluorescent dextran in control and fibulin-3–overexpressing tumors (P ¼ 0.0175 by the Student t test).

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obtained with the same tumors implanted as xenografts innude mice (data not shown). Furthermore, intravenous injec-tion of fluorescent dextran showed remarkable accumulationof extravascular dextran in the parenchyma of fibulin-3–over-expressing tumors (Fig. 1C–E), indicating an increase ofvessel permeability typical of aberrant tumor neovasculature.Conversely, GSCs transduced for conditional knockdown

of fibulin-3 resulted in tumor xenografts that were visuallyless hemorrhagic (Fig. 2A) and had significantly reducedvascular density in the tumor parenchyma and borders (Fig.2B and C). Taken together, results from fibulin-3 overexpres-sion and knockdown strongly suggested that fibulin-3secreted by glioma cells is a positive regulator of tumorvascularization.

Fibulin-3 forms a fibrillar wrapping around gliomablood vesselsAlthough fibulin-3 can be detected throughout the tumor

parenchyma (9), the results above prompted us to investigatewhether this protein could have preferential localization neartumor vessels. Indeed, confocal imaging of human GBM sec-tions with well-preserved vasculature revealed that fibulin-3was intensely localized around tumor blood vessels (Fig. 3Aand B) but was absent in normal brain vessels (Fig. 3C), even inbrain tissue adjacent to tumor. Higher magnification imagesrevealed well-defined fibulin fibrils that tightly wrappedaround small tumor capillaries (Fig. 3D) and larger vessels(Fig. 3E and F and Supplementary Movie S1). These strikingfibrils were detected with different antibodies that recognizenonoverlapping domains of fibulin-3 and do not cross-reactwith other perivascular fibrillar proteins (e.g., elastin or fibril-lin), indicating that they were not staining artifacts.We were intrigued about the source of these fibrils because

fibulin-3 is absent in normal brain endothelium. Analysis offibulin-3 mRNA in HBMECs, which do not express a detectableamount of fibulin-3 (Supplementary Fig. S1), showed that theirfibulin-3 expression was unchanged during coculture withglioma cells (Supplementary Fig. S2). Moreover, microvascularendothelial cells recovered from GBM-bearing mice revealedthat mouse fibulin-3 mRNA expression was also extremely lowand undistinguishable between endothelial cells from thetumor, the contralateral brain side, or na€�vemouse brain tissue(Supplementary Fig. S2). Together, these results strongly sug-gested that fibulin-3 in human GBM is likely secreted by GBMcells and accumulates around tumor blood vessels, where itcould cause the proangiogenic effects described above.

Fibulin-3 promotes endothelial cell migration andtubulogenesisWe next investigated whether fibulin-3 secreted by glioma

cells could induce proangiogenic behavior in brain microvas-cular endothelial cells.HBMECs coculturedwith CNS1 cells showed increased rates

of migration and invasion toward fibulin-3–overexpressingglioma cells compared with controls (Fig. 4A and B). Time-lapse videomicroscopy specifically demonstrated increasedvelocity of individual HBMECs cocultured with fibulin-3–over-expressing CNS1 cells (Fig. 4C). Moreover, HBMEC had

increased adhesion to fibulin-3–overexpressing glioma cells(Fig. 4D) and, reciprocally, these glioma cells showed increasedadhesion to preformed HBMEC tubules (Figs. 4E and Supple-mentary Fig. S3). Finally, conditioned medium from fibulin-3–overexpressing glioma cells increased number and length ofHBMEC tubules in vitro (Fig. 4F) but had negligible effects onendothelial cell proliferation (data not shown). Taken together,these results suggested that paracrine stimulation from fibu-lin-3–secreting glioma cells increased the overall ability ofHBMEC to migrate, form tubular structures, and associatewith glioma cells, all of which likely contributed to theincreased vascularization observed in vivo.

One possible explanation of these results would be increasedVEGF expression in glioma cells transfected with fibulin-3cDNA. Indeed, VEGF mRNA levels were higher in fibulin-3–overexpressing CNS1 cells (Fig. 4G) and their correspondingintracranial gliomas (Fig. 4G) compared with controls. There-fore, we reexamined the angiogenic behavior of HBMECs usinghighly purified fibulin-3. Remarkably, soluble fibulin-3 wassufficient to increase HBMEC migration (Fig. 4I) and tubuleformation (Fig. 4J) in the absence of VEGF from glioma cells.Moreover, fibulin-3–enhanced tubulogenesis was maintainedin the presence of concentrations of the VEGFR inhibitoraxitinib that abolished basal tubule formation (Fig. 4K).Together, these results suggested that while fibulin-3 increasesVEGF levels in gliomas, its proangiogenic effects are, at least inpart, independent of VEGF.

Fibulin-3 activates DLL4-Notch signaling to promoteangiogenic behavior of endothelial cells

Because Notch signaling is a critical mechanism thatregulates tumor vascularization and fibulin-3 is a paracrineactivator of Notch in glioma (9), we next explored whetherthis pathway would be the mediator of fibulin-3 in ourmodel.

Fibulin-3 caused remarkable activation of Notch signalingin HBMECs as shown by increased production of the Notchintracellular domain (NICD, Fig. 5A), activation of an NICD-dependent reporter construct (Fig. 5B) and increasedexpression of Notch-regulated genes (Fig. 5C). These effectsof fibulin-3 were inhibited by the gamma-secretase inhibitorDAPT, which blocks Notch cleavage and production ofNICD. DAPT also abolished fibulin-3–stimulated migrationof HBMECs (Fig. 5D) and tubulogenesis (Fig. 5E). Becausefibulin-3 is not expressed in HBMECs, fibulin-3 knockdownexperiments were instead performed in HUVECs, resultingin significant decrease of Hes5 and MASH mRNAs (Supple-mentary Fig. S4). Together, these results indicated thatactivation of endothelial Notch signaling is the likely mech-anism underlying the proangiogenic effects of fibulin-3.

To further understand this mechanism, we focused on themajor endothelial Notch ligand, DLL4. Treatment with fibulin-3 increased the expression of DLL4 in HBMECs and gliomacells (Fig. 6A and B). Fibulin-3 also increased themRNA level ofthe transcription factor Foxn4 that is known to upregulateDLL4 expression (24), but not the transcription factor Klf4 thatis a negative regulator of DLL4 (Fig. 6A; ref. 25). Furthermore,knockdown of DLL4 reduced HBMEC tubulogenesis and






Figure 2. Knockdown of fibulin-3correlates with reduced tumorvascularization. Human glioma stemcells (GBM8 and GBM34) weretransduced for doxycycline-dependentinduction of fibulin-3 shRNA andimplanted intracranially. Fibulin-3knockdown was initiated after surgeryand tumorswere collected after 21days.A, representative GBM34 tumorsshowing differences in grosshemorrhage between controls(control shRNA) and fibulin-3–knockdown tumors (fibulin-3 shRNA).Arrows indicate the approximateposition of the fibulin-3–deficienttumors. B, representative images ofcontrol and fibulin-3–knockdowntumors showing the tumor parenchyma(green) and vessels stained with anti-CD31 (red). C, quantification of bloodvessel density and stratification ofdensity by vessel length (�, P < 0.05;��, P < 0.01; ��, P < 0.001 by repeatedmeasures ANOVA).

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abolished the protubulogenic effect of fibulin-3 (Fig. 6C).These results suggested that the effects of fibulin-3 not onlyrequired activation of Notch receptors but also the specificpresence of the Notch ligand DLL4.

Loss of DLL4 also caused significant effects when weevaluated the effect of fibulin-3 on endothelial cell sprout-ing, although not as marked as in the tubulogenesis assays.DLL4 knockdown caused a drastic increase in HBMEC

Figure 3. Fibulin-3 associates with blood vessels in human GBMs. Fresh-frozen sections of GBM and normal human brain cortex were briefly fixed andprocessed for immunohistochemistry. A–C, sections of primary GBM (A), recurrent GBM (B), and age-matched normal brain (C) were probed with arabbit anti-fibulin-3 antibody (Ab3911) and tomato lectin to detect vessels (left), or with a mouse monoclonal anti-fibulin-3 antibody (mAb3-5) andanti-CD31 (right). Notice the intense staining of blood vessels in both cases (bars, 200 mm). Cell nuclei were counterstained with DAPI (blue). Punctate,non-specific green fluorescence was observed in normal brain tissue in the absence of primary antibodies, likely due to lipofuscin accumulation. D–F,high-magnification images revealed a striking wrapping of fibulin-3 fibrils around single capillaries (D) and larger blood vessels (E; bars, 50 mm). F, orthogonalprojections of the image in E show the fibrils associated with endothelial cells.






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sprouting (Fig. 6D), as expected and described else-where (23). This time, fibulin-3 was able to antagonize inpart the effect caused by loss of DLL4 while having no

effect on sprouting on its own, suggesting that fibulin-3was still able to activate Notch signaling or reduce sprout-ing by a different mechanism. We interpreted these results

Figure 5. Fibulin-3 activates endothelial Notch to promote tubulogenesis. A, Western blotting of HBMECs incubated overnight in the presence offibulin-3 (300 ng/mL) showed increased Notch-1 cleavage and expression of Notch-regulated genes (Hes1, Hes5); these effects were inhibited by theg-secretase inhibitor DAPT (25 mmol/L; NICD, Notch-1 intracellular domain). B, HBMECs were transiently transfected with a Notch reporter plasmid andtreated with fibulin-3 and DAPT as in A. Results show increased Notch activation by fibulin-3. A truncated, constitutively active fragment of Notch(FLAG-NICD) was cotransfected with the reporter plasmid as positive control (��, P < 0.001 by one-way ANOVA and the Bonferroni post hoc test). Theinset shows expression of endogenous NICD (�110 kDa) and FLAG-NICD (�68 kDa) in the cells. C, semiquantitative RT-PCR of fibulin-3–treatedHBMECs showed significant increase of Notch-regulated genes (��, P < 0.01; ��, P < 0.001 by the Student t test for each gene). D, increased chemotaxisof HBMECs toward fibulin-3 was abolished by DAPT. E, similarly, DAPT also prevented the enhancing effect of purified fibulin-3 on endothelial tubuleelongation. Results in D and E, ��, P < 0.01; ��, P < 0.001 by two-way ANOVA.

Figure 4. Fibulin-3 promotes angiogenic behavior in endothelial cells. A and B, HBMECs cultured in Transwell inserts showed increased migration (A) andinvasion through Matrigel (B) toward fibulin-3–expressing CNS1 glioma cells. C, HBMEC motility on nanofiber scaffolds was quantified using time-lapsefluorescencemicroscopy; average cell velocities were 10.97� 0.19 mm/hour for HBMEC exposed to control glioma cells (N¼ 121) and 16.52� 0.29 mm/hourfor HBMEC exposed to fibulin-3–expressing cells (N ¼ 109; P < 0.01, by a paired t test). D, fluorescently labeled HBMECs added to monolayers ofCNS1 cells for 2 hours adhered more to fibulin-3–expressing glioma cells than control cells. E. similarly, fibulin-3–expressing glioma cells adhered more thancontrol cells to pre-formed HBMEC tubules. F, HBMECs cultured on Matrigel formed longer tubules in the presence of conditioned medium from CNS1overexpressing fibulin-3 (CNS1-fibulin3); bars, 300 mm. G, VEGF-A mRNA expression increased in CNS1 cells transfected with fibulin-3 cDNA or treatedwith purified fibulin-3 (300 ng/mL) overnight. H, VEGF-A mRNA was also higher in tumors derived from fibulin-3–overexpressing CNS1 compared withcontrol CNS1 (N¼ 3/group); VEGF levels in contralateral brain tissue were taken as baseline (�,P < 0.05; ��,P < 0.01 by repeatedmeasures two-way ANOVA).I and J, HBMECs cultured in Transwell inserts showed increased chemotaxis toward purified fibulin-3 (I), while HBMEC cultured on Matrigel showedincreased tubule length when treated with fibulin-3 overnight (J). A–G, �, P < 0.05; ��, P < 0.01; ��, P < 0.001 by the Student t test for each assay. K, atubulogenesis experiment was performed in the presence of purified fibulin-3 and increasing concentrations of the pan-VEGFR inhibitor axitinib. Fibulin-3promoted tubule elongation even at concentrations of axitinib that abolished control tubulogenesis (>5 nmol/L). Significant differences for each treatmentagainst maximum inhibition at 10 nmol/L axitinib are shown (��, P < 0.01; ��, P < 0.001 by 2-way ANOVA and the Tukey multiple comparison test).






as indication that fibulin-3 mechanism involves primari-ly Notch activation in HBMECs, while its effects on vessellengthening require DLL4-Notch signaling in formedtubules.

Activation of Notch–DLL4 signaling by fibulin-3 isADAM10/17 dependent

We finally examined how fibulin-3 was activating Notchsignaling in endothelial cells. Treatment of HBMECs with

Figure 6. The proangiogenic effectsof fibulin-3 require endothelialDLL4. A and B, treatment ofHBMEC with fibulin-3 (300 ng/mL)increasedmRNA (A) and protein (B)expression of DLL4. Fibulin-3 alsoincreased mRNA expression of theDLL4-regulatory factor Foxn4but not the transcription factor Klf4(�, P < 0.05; ��, P < 0.001 bytwo-way ANOVA). C, transienttransfection of DLL4 siRNAs inHBMEC reduced tubulogenesisand prevented fibulin-3protubulogenic effect (�, P < 0.05;��, P < 0.01 by two-way ANOVA).Bars, 300 mm. D, transient DLL4knockdown also stimulatedHBMEC sprouting, which wassignificantly reduced, but notcompletely abolished, by fibulin-3(��, P < 0.01; ��, P < 0.001 bytwo-way ANOVA). Bars, 120 mm.

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purified fibulin-3 for up to 1 hour did not have effect on NICDproduction, DLL4 expression, or activation of VEGFR2 andAkt (not shown). Longer incubations (�6 h) with purifiedfibulin-3 did not activate VEGFR2 or Akt but neverthelessresulted in increased production of NICD and increasedexpression of DLL4 and Hes1 (Fig. 7A). These effects wereinhibited with DAPT but not with axitinib, suggesting thatthey were dependent on Notch signaling but not VEGF sig-naling. Notch signaling depends not only on Notch ligands butalso on the enzymes that process the Notch receptors; becausefibulin-3 increases metalloprotease activity in GBMs (10),we hypothesized that this protein could also regulate secretaseactivity to increase Notch activation.We specifically focused on the alpha-secretase activity that

must precede gamma-secretase cleavage during activation ofNotch receptors. Alpha-secretase activity is mediated by themembrane-bound enzymes ADAM10 and ADAM17, whichcan be specifically inhibited with TAPI-2. Strikingly, preincu-bation of HBMEC with TAPI-2 prevented all enhancingeffects of fibulin-3 on Notch–DLL4 signaling (Fig. 7B and C).In the presence of TAPI-2, fibulin-3 did not upregulate Foxn4and DLL4 mRNAs and did not increase NICD levels or Hes1expression. These results confirmed that the effects of fibulin-3were mediated by activation of Notch receptors and couldbe blocked by inhibitors of the different Notch-processingenzymes.One of the few confirmed ligands of fibulin-3 is the

extracellular metalloprotease inhibitor TIMP3 (26), whichcan also bind ADAM17 to regulate angiogenesis (27). Usinga coimmunoprecipitation assay, we first confirmed thatpurified fibulin-3 could bind secreted TIMP3 in our model(Supplementary Fig. S5). Next, using a fluorogenic assay tomeasure ADAM10/17 activity in HBMEC lysates, we observedthat exogenous fibulin-3 caused a dramatic increase in thisenzymatic activity, which was completely abolished byTIMP3 (Fig. 7D). Moreover, purified TIMP3 was sufficient toprevent the enhancing effect of fibulin-3 on DLL4 expressionand Notch activation (Fig. 7E). Finally, TIMP3 also preventedthe protubulogenic effect of fibulin-3 in HBMECs (Fig. 7F).Together, these results strongly suggested that fibulin-3 likelyactivates Notch–DLL4 signaling by binding TIMP3 and inhi-biting its ability to inactivate ADAM10/17, leading toincreased cleavage of Notch and expression of DLL4 thatresult in the observed proangiogenic effects.

DiscussionThe contribution of fibulin family members to cancer devel-

opment, and in particular to tumor angiogenesis, is complexand depends on the tumor type and model analyzed (28). Bothtumor-promoting and tumor-suppressive effects have beendescribed for fibulin-1 (29, 30), fibulin-2 (31, 32), fibulin-3(10, 12, 33), and fibulin-5 (30, 34). This multiplicity of effectshas been attributed to the presence ofmultiple isoformswithinthe family, with different functions and differential regulationby extracellular signals (35).Fibulin-3 in particular has been described as an enhancer

of tumor progression and metastasis in pancreatic, cervical,

and ovarian carcinomas (12, 36–38), and a potential tumorsuppressor in colorectal, lung, and hepatic cancers (39, 40).We provided the first comprehensive description offibulin-3 in human gliomas (10), its paracrine Notch-acti-vating mechanism, and its ability to promote tumor growthand invasion (9, 10).

Our present results demonstrate that the effects of fibulin-3 on the glioma microenvironment are even more extensive:fibulin-3 secreted by glioma cells can activate endothelialNotch–DLL4 signaling in a paracrine manner and promoteangiogenic behavior. This is remarkable because fibulin-3was originally reported as an antiangiogenic protein (41) dueto the in vitro use of a short recombinant form lacking thecritical Notch-activating, N-terminal domain (9). This shortisoform, which was hypothesized but never observed in vivo(42), could also explain the lack of protumor effects of fibulin-3 in another glioma study (43), highlighting how critical theNotch pathway is to mediate fibulin-3 functions in thesetumors. Several studies have since demonstrated that fibulin-3 upregulation is in fact associated with angiogenesis incarcinomas (36–38). Moreover, fibulin-3 accumulation haslong been known to correlate with vascular proliferation inage-related macular degeneration in the retina (44), suggest-ing that this protein may have a proangiogenic role beyondthe context of cancer.

Our study conclusively identifies fibulin-3 as a proangio-genic signal secreted by glioma cells, encouraging future workto further elucidate itsmolecularmechanisms.We have shownhere that fibulin-3 activates ADAM10/17, likely by inhibitingTIMP3, and that this mechanism then promotes Notch acti-vation and DLL4 expression, both of which are needed tomediate the proangiogenic effects of fibulin-3. Blockade ofDLL4 upregulation by TAPI-2 (Fig. 7B) and DAPT (Fig. 7A)suggest that increased expression of this Notch ligand (andits regulatory factor Foxn4) would depend at least in part onNotch activation and could form a feed-forward loop aspreviously proposed (45). Whether fibulin-3 activity is, inaddition, sufficient to promote ligand-independent activationof Notch receptors as proposed in certain models (46) is stilluncertain, but is nevertheless an attractive hypothesis in thecontext of aberrant receptor activation in glioma. While themolecular mechanisms of fibulin-3 are not yet fully defined,our results show robust modulation of Notch signaling andoverlapping effects (i.e., VEGF upregulation, Notch activation,and DLL4 upregulation) that make fibulin-3 a strong positivemodulator of tumor angiogenesis. Fibulin-3 could thereforebe a major target to help disrupt Notch–DLL4 signaling, withsignificant impact in strategies to overcome anti-VEGF resis-tance (47). Moreover, because fibulin-3 has an establishedproinvasive role in gliomas (9, 10), targeting this protein coulddisrupt not only angiogenic compensation mechanisms butalso dispersion mechanisms in gliomas treated with anti-VEGF. As an additional benefit, targeting the proangiogenicrole of fibulin-3 could help devise strategies for eye vasculardiseases where this protein is clearly involved.

Finally, we consider important to remark on the distinc-tive fibulin-3 fibrils wrapping the blood vessels of humanGBMs. Expression of fibulin-3 in adult organs is diffuse and






restricted to the basal lamina of connective tissues (11). Apattern of vascular fibrils has been observed in embryoniclung tissue (48) but never in the CNS, underscoring the

unique association of fibulin-3 with the wall of glioma bloodvessels. Fibulins 4 and 5, which share considerable homologywith fibulin-3, form perivascular fibrils by association with

Figure 7. Fibulin-3 activates ADAM10/17 to trigger DLL4-Notch signaling. A, HBMECs treated with fibulin-3 (300 ng/mL, 6 hours) did not show activation ofVEGFR2 or Akt but exhibited increased production of NICD, upregulation of DLL4 andHes1, and activation of Erk1/2. These effects were blocked by preincubationwith DAPT (25 mmol/L) but not by axitinib (5 nmol/L). B, incubation of HBMECs with the ADAM10/17 inhibitor TAPI-2 prevented the enhancing effects of fibulin-3on expression of DLL4 and Foxn4. C, TAPI-2 also blocked fibulin-3–dependent increase of Notch cleavage, Hes1 expression, and Erk1/2 activation. D,incubation of HBMEC lysates with purified fibulin-3 dramatically increased ADAM10/17 activity on a fluorogenic substrate (RFU, relative fluorescent units);this effect was abolished by purified TIMP3 (1 mg/mL). E, purified TIMP3 prevented the enhancing effect of fibulin-3 on NICD production and expression of DLL4and Hes1. F, similarly, TIMP3 abolished the protubulogenic effect of fibulin-3. Results in B and F, ��, P < 0.01; ��, P < 0.001, by one-way ANOVA.

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elastin and fibrillin-1, but fibulin-3 does not associate withthese proteins (48, 49). This suggests that unique dockingmolecules, possibly restricted to embryonic and tumor-associated endothelial cells, may account for this unusualpattern of fibulin-3 that could help associate glioma cells tothe tumor vasculature. Interestingly, purified fibulin-3 canself-polymerize in vitro (Viapiano's Laboratory, unpublishedobservation), which could contribute in part to the forma-tion of the fibrils.In sum, the localization of fibulin-3 around glioma blood

vessels, together with its promotion of angiogenesis, asso-ciation of glioma and endothelial cells, and glioma celldispersion, suggest that this matrix protein may play mul-tiple key roles in tumor progression, enhancing both for-mation of new vessels and co-option of existing vessels bydispersing tumor cells. We propose fibulin-3 as a relevanttarget restricted to the tumor parenchyma that can beexploited to disrupt Notch-dependent proangiogenic andproinvasive mechanisms in glioblastoma.

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

Authors' ContributionsConception and design: M.S. Nandhu, B. Hu, S.E. Cole, M.S. ViapianoDevelopment of methodology: M.S. Nandhu, B. Hu, A. Erdreich-Epstein,D.J. Rodriguez-GilAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.S. Nandhu, B. HuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.S. Nandhu, B. Hu,D.J. Rodriguez-Gil,M.S. ViapianoWriting, review, and/or revision of the manuscript: M.S. Nandhu, B. Hu,S.E. Cole, A. Erdreich-Epstein, D.J. Rodriguez-Gil, M.S. ViapianoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.S. NandhuStudy supervision: M.S. Viapiano

AcknowledgmentsThe authors thank Jessica De Jesus and Brynn Hollingsworth (The Ohio

State University Wexner Medical Center) for valuable technical support.

Grant SupportThis work was supported by grants from the NIH (1R01CA152065-01) and the

National Brain Tumor Society (M.S. Viapiano), and the Discovery ResearchGrantfrom the American Brain Tumor Association (B. Hu).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 6, 2014; revised June 26, 2014; accepted July 25, 2014;published OnlineFirst August 19, 2014.

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2014;74:5435-5448. Published OnlineFirst August 19, 2014.Cancer Res Mohan S. Nandhu, Bin Hu, Susan E. Cole, et al. Promotes Angiogenesis in High-Grade Gliomas

DLL4 Signaling by Fibulin-3−Novel Paracrine Modulation of Notch

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