Soluble receptor-mediated selective inhibitionof VEGFR and PDGFR� signaling during physiologicand tumor angiogenesisFrank Kuhnert*, Betty Y. Y. Tam*, Barbara Sennino†, John T. Gray‡, Jenny Yuan*, Angeline Jocson*, Nihar R. Nayak§,Richard C. Mulligan‡, Donald M. McDonald†, and Calvin J. Kuo*¶

*Division of Hematology, Stanford University School of Medicine, 269 Campus Drive, CCSR 1155, Stanford, CA 94305; †Cardiovascular Research Institute,Comprehensive Cancer Center, and Department of Anatomy, University of California, San Francisco, CA 94143; ‡Department of Genetics, HarvardMedical School, 200 Longwood Avenue, Boston, MA 02115, and §Department of Gynecology and Obstetrics, Stanford University Schoolof Medicine, 300 Pasteur Drive, Stanford, CA 94305

Communicated by Gerald R. Crabtree, Stanford University School of Medicine, Stanford, CA, April 22, 2008 (received for review September 17, 2007)

The simultaneous targeting of both endothelial cells and pericytes viainhibition of VEGF receptor (VEGFR) and PDGF� receptor (PDGFR�)signaling, respectively, has been proposed to enhance the efficacy ofantiangiogenic tumor therapy. Clinical and preclinical modeling ofcombined VEGFR and PDGFR� signaling inhibition, however, has usedsmall molecule kinase inhibitors with inherently broad substratespecificities, precluding detailed examination of this hypothesis.Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (AdFlk1-Fc) in combination with a soluble ectodomain of PDGFR� (AdsPDGFR�) allowed highly selective inhibition of these pathways. Theactivity of Ad sPDGFR� was validated in vitro against PDGF-BB and invivo with near-complete blockade of pericyte recruitment in theangiogenic corpus luteum, resulting in prominent hemorrhage, thusdemonstrating an essential function for PDGF signaling during ovar-ian angiogenesis. Combination therapy with Ad PDGFR� and sub-maximal doses of Ad Flk1-Fc produced modest additive antitumoreffects; however, no additivity was observed with maximal VEGFinhibition in numerous s.c. models. Notably, VEGF inhibition via AdFlk1-Fc was sufficient to strongly suppress tumor endothelial andpericyte content as well as intratumoral PDGF-B mRNA, obscuringadditive Ad sPDGFR� effects on pericytes or tumor volume. Thesestudies using highly specific soluble receptors suggest that additivitybetween VEGFR and PDGFR� inhibition depends on the strength ofVEGF blockade and appears minimal under conditions of maximalVEGF antagonism.

combination therapy � pericytes

The central role of angiogenesis in promoting tumor progressionand metastasis is now well appreciated and is governed by a

balance between stimulators and inhibitors of angiogenesis, asproposed by Hanahan and Folkman (1). VEGF-A signaling con-stitutes the dominant regulatory pathway for developmental andtumor angiogenesis (2, 3). In randomized clinical trials, the human-ized monoclonal anti-VEGF-A antibody Avastin provided an over-all survival benefit for colon cancer patients when combined withconventional chemotherapy (4), thus validating angiogenesis inhi-bition as an effective anticancer strategy.

Targeting endothelial cells (ECs) or the principal angiogenicfactor VEGF-A alone may not be itself sufficient for maximalantiangiogenic effects (2). Progressive and refractory disease oftensupervenes in patients, perhaps mediated by VEGF resistance viathe production of alternative angiogenic mediators such as FGFsand reactivation of tumor angiogenesis (5–7). Additionally, non-ECtypes such as mural, stromal, and hematopoietic cells positivelycontribute to tumor angiogenesis, albeit indirectly (2, 8). Pericytes(PCs), in particular, have been recently appreciated as criticalregulators of vessel formation, stabilization, and function (9). Theamount of PC coverage of vessels for various tumors ranges fromextensive to none (10). Nevertheless, the protective and stabilizing

functions of PCs on tumor vasculature have been clearly demon-strated (11–13).

PDGF� receptor (PDGFR�) signaling is critical for the recruit-ment of PCs or perivascular progenitor cells during developmentaland tumor angiogenesis (12, 14, 15). EC-derived PDGF-BB, as theclassical PDGFR� ligand, is essential for embryonic PC recruit-ment (16), and a requisite role of local extracellular PDGF-BBconcentration gradients for proper integration of PDGFR�-positive PCs into the wall of tumor vessels has been demonstrated(17). As an alternative pathway, angiopoietin 1 (Ang1) and itsreceptor Tie2 have been implicated as mediators of blood vesselstability and mural cell recruitment during embryonic (18, 19) andtumor angiogenesis (20).

The tumor vessel-stabilizing functions of PCs have promptedintensive efforts to target both tumor vasculature PCs and ECsfor combinatorial antiangiogenesis therapy. Combination treat-ment with small molecule receptor tyrosine kinase inhibitors(RTKI) such as SU6668 and Gleevec targeting VEGF andPDGFR� signaling, respectively (21, 22), produced increasedefficacy as compared with either monotherapy at all stages ofpancreatic islet cancer and achieved regression of late-stagetumors in the RIP-Tag2 model (23, 24); similar additivity hasbeen reported for the VEGF inhibitor SU10944 and Gleevec(25). Most recently, in human clinical trials, broad-spectrumsmall molecule RTKIs such as sorafenib and sunitinib targetingboth VEGF receptor 1–3 (VEGFR1–3) and PDGFR�/� amongothers have demonstrated efficacy in renal cell carcinoma,gastrointestinal stromal tumor (GIST), and hepatocellular car-cinoma (26–28). However, the use of relatively promiscuousRTKIs does not allow inhibitory effects to be unequivocallyascribed to PDGFR� inhibition, and highly specific reagents areneeded to test the PC hypothesis. Further, the potential abilityof VEGF to stimulate endothelial PDGF-BB expression (29) andthe extensive reciprocal cross-talk between ECs and PCs (9)suggest that inhibition of these two pathways might not be fullyadditive or synergistic.

We previously used adenovirus to achieve reversible andsystemic expression of soluble VEGFRs in mice, producingstringent VEGF inhibition and potent inhibition of tumorgrowth and angiogenesis (30, 31). Here, we generated adenovi-

Author contributions: F.K., B.Y.Y.T., and C.J.K. designed research; F.K., B.Y.Y.T., A.J., andC.J.K. performed research; J.T.G., J.Y., and R.C.M. contributed new reagents/analytic tools;F.K., B.Y.Y.T., B.S., N.R.N., D.M.M., and C.J.K. analyzed data; and F.K., B.Y.Y.T., and C.J.K.wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

¶To whom correspondence should be addressed. E-mail: cjkuo@stanford.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0803194105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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ruses expressing soluble PDGFR� ectodomains (Ad sPDGFR�)to inhibit PC-recruitment pathways during physiological andpathological angiogenesis and elucidate the specific contribu-tions of inhibition of VEGFR and PDGFR� signaling duringcombination antiangiogenic cancer therapy.

ResultsCharacterization of Adenoviruses Expressing Soluble PDGFR� Ectodo-mains. We previously demonstrated extensive antiangiogenic andantitumor efficacy after adenoviral expression of soluble ectodo-mains of the VEGFRs Flt-1 (Ad Flt1) and Flk1 (Ad Flk1-Fc) (30,31). Accordingly, we constructed an analogous adenovirus express-ing a soluble PDGFR� ectodomain with C-terminal His6 tag (AdsPDGFR�). Single i.v. administration of purified Ad sPDGFR�(5 � 108 pfu) to adult C57BL/6 mice produced robust plasmaexpression of the respective ectodomains from hepatic transductionfor �30 days (Fig. 1A), consistent with the typical expressionkinetics of adenovirus in immunocompetent mice (31). The solublerecombinant PDGFR� ectodomain was purified from adenoviralsupernatants by using the C-terminal His6 tag and potently inhib-ited PDGF-BB-induced Akt phosphorylation in NIH 3T3 cells (Fig.1B) (32). As an additional control, we constructed Ad Tie2-Fcexpressing a soluble Tie2 ectodomain fused to IgG2� Fc. AdTie2-Fc also produced sustained plasma expression in C57BL/6mice (Fig. 1C), and the corresponding purified Tie2-Fc fromadenovirus supernatant neutralized Ang1-stimulated Akt phos-phorylation in human umbilical vein ECs (HUVECs) (Fig. 1D).

Ad sPDGFR� Induces Hemorrhage During Corpus Luteum (CL) Angio-genesis. The effects of Ad sPDGFR� on physiologic angiogenesiswere validated in a murine ovarian CL angiogenesis model. Afterovulation, the basement membrane between the theca and granu-losa layers undergoes rapid dissolution, and the thecal vesselsinvade the avascular granulosum and form a dense vascular net-work surrounding the luteinized granulosa cells to sustain CLdevelopment and function, accompanied by robust endothelialproliferation and PC recruitment (33, 34). Using this model, weadministered Ad sPDGFR� i.v. 24 h before hormonal induction ofovulation [human choriogonadotropin (hCG) injection], and weexamined the ovaries 48 h after ovulation. As comparative controlsfor Tie2 and VEGF blockade, Ad Tie2-Fc and Ad Flk1-Fc wereadministered in parallel, and Ad Fc encoding an IgG2� Fc fragmentwas used as negative control.

Ovaries from Ad Fc (Fig. 2 A, E, and I) and Ad Tie2-Fc-treatedanimals (Fig. 2 C, G, and K) appeared grossly and histologicallynormal, whereas VEGF inhibition with Ad Flk1-Fc resulted in amarked reduction in ovary size (Fig. 2B). These Ad Flk1-Fc-treatedovaries contained mostly small antral follicles, only a very few fully

developed CL [Fig. 2F; supporting information (SI) Fig. S1] withoccasional central necrosis (Fig. 2 J), consistent with previousreports documenting an essential role for VEGF in CL develop-ment (35).

In contrast, ovaries from the Ad sPDGFR� treatment group,although comparable in size to the Ad Fc control, displayedprominent multifocal hemorrhage (Fig. 2D). In the Ad sPDGFR�-treated ovaries, the number of fully developed CL was markedlyreduced, and invariably, these CL displayed extensive luteal hem-orrhage (Fig. 2 H and L; Fig. S1). Furthermore, numerous blood-filled follicles that had failed to ovulate exhibited extensive hem-orrhage in peripheral luteinized granulosa cell layers (Fig. 2H).

Ad sPDGFR� Potently Suppresses CL PC Recruitment. Strikingly, AdsPDGFR� treatment resulted in a 93% reduction of CL NG2� PCcontent, without alterations of PC density in the preexisting thecalvasculature (Fig. 3 H, L, N, and O). The lack of PC investment inthe developing Ad sPDGFR� CL vasculature was confirmed byusing desmin as a second PC marker (Fig. 3O). CL PCs did notdisplay immunoreactivity for smooth muscle actin (SMA) undereither basal or inhibited conditions, whereas immunostaining forPDGFR� demonstrated complete tissue penetration of ovariantissue with the soluble PDGFR� ectodomain, allowing for theneutralization of local PDGF-BB gradients (36) (data not shown).Taken together, the quantitative Ad sPDGFR�-mediated suppres-sion of PC recruitment during physiologic CL angiogenesis dem-onstrated both the potency of this reagent and the requirement ofPDGFR�-mediated PC recruitment for development of the CLand its associated vasculature. In contrast, oral administration ofGleevec, an Abl-inhibitory RTKI with activity against PDGFR�(37), engendered smaller effects on CL PC coverage and CLmorphology, suggesting lesser PDGFR antagonism (Fig. S2).

The PC-selective Ad sPDGFR� phenotype contrasted withVEGF inhibition via Ad Flk1-Fc, which induced near-total sup-pression of luteal immunoreactivity for both the EC marker CD31and the PC marker NG2 (Fig. 3 B, F, J, and O). The potent AdFlk1-Fc inhibition of both ECs and PCs illustrates the strongdependence of PC recruitment on EC function. Ad sPDGFR�reduced CL CD31� microvessel density by �45% (Fig. 3 D and O),suggestive of significant reciprocal regulation of EC by PDGFR��

PCs. Inhibition of Tie2 signaling by Ad Tie2-Fc reduced CL CD31�

Fig. 1. Characterization of PDGFR� and Tie2-Fc adenoviruses. Tail vein i.v.administration of Ad sPDGFR� (A) or Ad Tie2-Fc (C) (109 pfu) to adult miceproduced plasma expression of their respective ectodomains for �30 days asdetermined by Western blotting. Affinity-purified PDGFR�-His (200 ng/ml)and Tie2-Fc (5 �g/ml) ectodomains from adenoviral supernatants inhibitedPDGF-BB- (10 ng/ml) and angiopoietin-1- (Ang1, 200 ng/ml) induced Aktphosphorylation in NIH 3T3 and HUVEC cells, respectively (B and D). IDE,insu-lin-degrading enzyme.

Fig. 2. Effect of Ad sPDGFR� on CL angiogenesis. Prepubescent female micewere administered the indicated Ads (5 � 108 pfu) 1 day before hCG-inducedovulation. As demonstrated in whole-mount images of ovaries, Ad sPDGFR�

induces prominent CL hemorrhage (D) compared with Ad Fc control-treatedanimals (A). Histological analysis revealed a large number of arrested folliclesdisplaying large central cavities entirely filled with blood and widespreadhemorrhaging throughout the luteal tissue in developed CL only in AdsPDGFR�-treated mice (H and L vs. Ad Fc, and E and I). Ad Flk1-Fc (B, F and J)but not Ad Tie2-Fc (C, G and K) suppresses CL development. (Magnification:A–H, �25; I–L, �200.)

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microvessel density by 32% without significantly decreasing PCcontent, further indicating the specificity of the Ad sPDGFR�phenotype (Fig. 3 C, G, K, and O).

sPDGFR� Ectodomains Inhibit PC Recruitment to Nascent Tumors.Recent studies suggested a requisite role of PDGFR� signaling formural cell recruitment to the tumor microvasculature (17, 38),although fully conditional strategies were not used. Pretreatment ofC57BL/6J mice with Ad sPDGFR� 24 h before s.c. Lewis lungcarcinoma (LLC) implantation dramatically decreased the PCcontent of tumor microvessels (�95%), as evidenced by SMA anddesmin staining, while producing a more modest �20% reductionin CD31� endothelial content (Fig. 4). In contrast, Ad Flk1-Fctumors displayed a �90% reduction in CD31� microvessel densityaccompanied by an �80% decrease in PC content (Fig. 4), paral-leling results in the CL (Fig. 3) and again indicating an obligate

suppression of PC after primary EC inhibition. Ad Tie2-Fc reducedtumor CD31� microvessel density by �20% without significanteffects on PC (Fig. 4).

Comparative and Combinatorial Antitumor Effects of Soluble PDGFR�and VEGFR Ectodomains. We further exploited the efficacy andspecificity of these adenoviral reagents to test the effects of com-bined blockade of VEGFRs and PDGFR� on tumor growth andangiogenesis in preestablished (200-mm3) s.c. tumor models. Twodifferent doses of Ad Flk1-Fc were used: a submaximal dose (7.5 �107 pfu for T241, 1 � 108 pfu for LLC) and a maximal dose (5 �108 pfu), associated with �25% and 60–70% tumor inhibition,respectively, as monotherapy. We observed additive effects be-tween submaximal doses of Ad Flk1-Fc and maximal doses of AdsPDGFR� in both the LLC and the T241 fibrosarcoma tumormodels (Fig. 5A and Fig. S3). However, when the stringency ofVEGF inhibition was increased via maximal doses of Ad Flk1-Fc(5 � 108 pfu), no additivity was observed with Ad sPDGFR� (5 �108 pfu) in (Fig. 5B and Fig. S4). These results indicated that thebeneficial effects of combined VEGF/PDGFR� inhibition areminimal under conditions of stringent VEGF blockade.

Likewise, combination Ad Flk1-Fc and Ad Tie2-Fc treatment oreven triple treatment combinations with Ad sPDGFR� did notelicit additive inhibitory effects on tumor growth as compared withAd Flk1-Fc monotherapy (Fig. S4). As monotherapy, AdsPDGFR� was less efficacious than Ad Flk1-Fc (37–45% versus62–75% inhibition). Ad Tie2-Fc was ineffective in all three models(Fig. S4), and adenoviruses expressing Tie1, ephrin-B2, or EphB4ectodomains or full-length Ang1 or Ang2 exhibited modest to noefficacy in LLC (B.Y.Y.T. and C.J.K., unpublished work).

Combination PDGFR� and VEGFR2 Ectodomain Effects on TumorVasculature. To explore the basis for the lack of additivity betweenhigh-dose VEGFR/PDGFR� inhibition, effects on the tumor vas-culature were examined in these preestablished tumor models. AdFlk1-Fc treatment of LLC reduced CD31� EC by �80% (Fig. 5C).Ad sPDGFR� treatment reduced CD31� content by �45% (Fig.5C), consistent with the notion that the targeting of PC recruitmentpathways alone can suppress EC via cross-talk. Paralleling tumorgrowth, combined Ad Flk1-Fc and Ad sPDGFR� treatment did notresult in additional reduction of CD31� EC content versus AdFlk1-Fc alone. Although Ad sPDGFR� reduced SMA� PC cov-erage by 70%, Ad Flk1-Fc was itself sufficient to decrease PCcontent by �60%, with lack of statistically significant additiveeffects on PC via the combination (P � 0.59 vs. Ad sPDGFR�, P �0.1 vs. Ad Flk1-Fc) (Fig. 5C), providing a mechanistic explanationfor the observed lack of additivity on tumor growth.

The Ad sPDGFR�-induced decreases in ECs coupled with theAd Flk1-Fc-induced decreases in PCs are consistent with the notionof substantial and mutually reinforcing cross-talk betweenVEGFR� ECs and PDGFR�� PCs. These observations alsosuggest a potential mechanism for the observed lack of additivity onthe combination of PDGFR� and VEGF inhibition because thelatter already strongly inhibits PC recruitment. Indeed, in T241tumors, for which ECs but not tumor cells themselves constitute thepredominant source of PDGF-BB (39), Ad Flk1-Fc pretreatmentsignificantly (�90%) reduced PDGF-B RNA (Fig. 5D). Thus,VEGF inhibition can indirectly inhibit PC recruitment via primarysuppression of ECs and accompanying endothelial PDGF-BBproduction in the absence of PDGFR�-targeted inhibition. Weadditionally observed lack of additivity on EC or PC contentbetween triple combinations of Ad Tie2-Fc, Ad sPDGFR�, and AdFlk1-Fc vs. Ad Flk1-Fc monotherapy (Fig. S5).

DiscussionThe participation of PDGF-BB and its cognate receptor PDGFR�during embryonic and adult PC recruitment has rendered thispathway an extremely attractive target for antiangiogenic cancer

Fig. 3. Suppression of CL PC recruitment by Ad sPDGFR�. (A–N) Immunoflu-orescence staining of CL sections for ECs (CD31, green) and PCs (NG2, red) wasperformed on ovaries obtained from female mice (n � 4) pretreated with theindicated adenoviruses before ovulation. Ad sPDGFR� potently inhibits therecruitment of NG2-positive PCs to the nascent CL vasculature as comparedwith Ad Fc control virus, without affecting the preestablished thecal andstroma vasculature (H and N vs. E and M). Ad Tie2-Fc demonstrates minimaleffect on PC content (G), whereas Ad Flk1-Fc reduced ECs (B and J), withresultant secondary inhibition of PC recruitment (F). (Magnification: A–L,�100; M and N, �400.) (O) Differential effects on ECs and PCs in adenovirus-treated CL. EC (CD31) and PC content (NG2, desmin) were determined fromimmunostained images by automated pixel quantitation (n � 4 mice pervirus). Ad Flk1-Fc potently suppressed endothelial CD31 immunoreactivitywith consequent lack of NG2 signal, whereas Ad sPDGFR� selectively inhibitedPC recruitment (, P � 0.02 vs. Ad Fc) with a trend toward suppression of CD31signal (P � 0.05 vs. Ad Fc). Sections are from the experiment in Fig. 2.

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therapy, particularly in concert with VEGF antagonism (23, 24).However, examination of the roles of PDGFR� signaling in bothtumor angiogenesis and normal physiology has been hampered bythe lack of facile and robust methods for specific, conditionalinactivation of this receptor system in adult organisms. In thecurrent work, we addressed this question by using adenoviralexpression of ligand-binding ectodomains of PDGFR�, first vali-dating bioactivity in a CL angiogenesis assay before combinatorialevaluation with VEGF inhibition in tumor models.

Prior methodology for inhibiting PDGF-BB/PDGFR�-mediatedrecruitment of PCs in the adult has included EC-specific deletionof PDGF-B, or fine genetic deletion of the PDGF-B C-terminalheparin-binding retention motif has circumvented embryonic le-thality of PDGF-B-null mutants (17, 36, 40, 41). However, thesedeficits originate in embryogenesis and may not generate fully nullphenotypes given the potential for incomplete deletion in theformer and neomorphic or hypomorphic effects in the latter.Pharmacologic approaches have included either small moleculeinhibitors of PDGFR� tyrosine kinase activity or anti-PDGFR�antibodies (12, 23, 42), although these have potential disadvantagesof promiscuous action against multiple RTKs in the former andpharmacokinetic limitations with the need for frequent and/orrepetitive administration in both cases. Nonetheless, the aforemen-tioned strategies have provided compelling evidence for therapeu-tic inhibition of PDGFR� signaling during tumor angiogenesis andfor PC developmental hierarchies.

Adenoviral administration of PDGFR� ectodomains representsan alternative and convenient strategy for adult inactivation, withpersistent and high-level circulating transgene levels from single i.v.injection. Inherently, the use of soluble PDGFR� as decoy, al-though affording specific blockade of PDGFR� signaling, does notdistinguish between effects mediated by sequestration of eitherPDGFR� ligand, PDGF-BB or PDGF-DD (43). Nonetheless, theadenoviral approach affords specific blockade of PDGF-B signal-ing, and the in vivo efficacy of Ad sPDGFR� is strongly supportedby near-quantitative PC suppression in the physiologic setting of CLangiogenesis. Single i.v. dosing of Ad sPDGFR� reduced CL PCcontent by 93%, in contrast to partial inhibition by the multitar-geted RTK inhibitor Gleevec. The blood vessel-destabilizing effectsof impaired PC recruitment have been previously demonstratedduring development and tumor angiogenesis (16, 17), and loss ofPCs likewise leads to profuse hemorrhaging during CL formation,although contribution of secondary EC effects cannot be ruled out.PDGF-BB and PDGF-DD are abundantly expressed in the ovary(43, 44), and the prominent hemorrhage in Ad sPDGFR�-treatedCL strongly suggests an essential requirement for PDGFR�-mediated signaling in providing vessel stability during CL angio-genesis and ovarian physiology. Moreover, our findings establishthe CL angiogenesis model as a robust assay system to evaluatePDGF antagonists.

Combinatorial therapy targeting both PDGFR� and VEGF isthought to produce improved antiangiogenic effects vs. eitheralone. Indeed, combination treatment with small molecule RTKinhibitors targeting VEGF (SU5416, SU10944) and PDGFR�(SU6668, Gleevec) exhibited enhanced efficacy in the RIP-Tagpancreatic cancer model (23) and various xenograft models (25),although these relatively promiscuous RTKIs did not allow inhib-itory effects to be unequivocally ascribed to VEGF or PDGFR�inhibition.

Here, the potent and specific inhibition engendered by AdsPDGFR� afforded an opportunity to test the effects of combinedPDGFR�/VEGFR inhibition on tumor angiogenesis. Consistentwith prior results with small molecule RTKIs (23, 25), we observedadditive effects when combining Ad sPDGFR� with submaximaldoses of Ad Flk1-Fc. However, combined high-grade treatmentwith Ad sPDGFR� and Ad Flk1-Fc did not produce appreciablesynergistic antitumor effects when tumor burden was used as anendpoint. Stringent VEGF inhibition by Ad Flk1-Fc alone de-creased PC number to a similar extent as Ad sPDGFR�, suggestinga basis for the lack of additional effects from Ad sPDGFR�. Wefurther present evidence in the T241 fibrosarcoma model for amechanism in which VEGF inhibition with primary suppression ofECs can suppress EC production of PDGF-BB with secondaryconsequences of impaired PC recruitment.

The current data do not exclude the potential for additivity inhuman patient populations. Ad sPDGFR� enhanced the efficacy ofsubmaximal doses of Ad Flk1-Fc (Fig. 5), a scenario that mayparallel the clinical situation where near-complete inactivation ofVEGF signaling may be difficult to achieve. We also cannot excludeclass-specific additive effects in the RIP-Tag vs. subcutaneousmodels used herein, given the different mechanisms of actionbetween small molecule inhibitors and soluble receptors. Interest-ingly, although the tumors used in the current studies expressVEGF in the tumor parenchyma (Fig. S6), Ad sPDGFR� sup-presses CD31� microvessel density more strongly than Ad Flk1-Fcin LLC variants in which VEGF is produced solely in tumor PCs andnot the tumor parenchyma (B.S. and D.M.M., unpublished work).Ad sPDGFR� exhibits strong activity in the B16Bl6 andCT26.CL25 metastasis models, suggesting the potential utility ofPDGFR� inhibitors such as orally bioavaliable small molecules forsuppression of hematogenous metastases (F.K. and C.J.K., unpub-lished results).

ECs and PCs exhibit extensive bidirectional paracrine cross-talk,wherein endothelium-derived PDGF-BB acts on PCs, whereasreciprocal PC secretion of VEGF-A and Ang-1 stimulates ECs (16,19, 45). In the current studies, we provide supporting in vivo datawhereby VEGF inhibition elicited primary effects on ECs that wereobligately accompanied by substantial decrements in PC content.Conversely, although selective PDGF inhibition with Ad sP-DGFR� primarily affected PCs, lesser although detectable second-

Fig. 4. Effect of Ad sPDGFR� on pathological angiogenesis in a pretreatment LLC model. Adenoviruses (5 � 108 pfu) were administered 1 day before the s.c.implantation of LLCs in C57BL/6J mice. Tumors were excised 12 days after implantation, and immunostaining for CD31 and PC markers �-SMA and desmin wasperformed (A). Ad sPDGFR� ectodomains potently suppress the recruitment of PCs to the growing tumor vasculature (n � 4; *, P � 0.03; #, P � 0.01 vs. Ad Fc)(B). (Magnification in A: �100.)

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ary decreases in the endothelium were noted. Selective manipula-tion of the endothelium and PCs by VEGF and PDGFR�inhibition, respectively, as manifested by the highly specific solublereceptor approach described herein, appears intrinsically self-limited by reciprocal cross-talk and indirect inhibition of the othercell type, limiting the efficacy of combination therapy particularlywhen maximal blockade is achieved.

Currently, significant interest exists in dual targeting of VEGFand PDGFR� in cancer patients, given the encouraging clinicalactivity of multitargeted small molecule RTK inhibitors such assorafenib and sunitinib that inhibit VEGFR2 and PDGFR�,among other kinases (26–28). The promiscuous nature of theseagents likely contributes to their unquestioned clinical efficacy butat the same time complicates the definition of relative contributionsfrom inhibition of diverse vascular and nonvascular RTK targets.Within the vascular RTKs, the present studies emphasize thesubstantial cross-talk between VEGF and PDGFR� signalingduring their action on tumor ECs and PCs. Accordingly, our studiessuggest that as increasingly efficacious VEGF inhibitors are devel-oped, additive benefits from superimposed PDGFR� antagonismmay diminish, given that primary EC suppression via VEGFblockade can be itself sufficient to inhibit PDGF-BB production

and PC recruitment in parallel. We further anticipate that the AdsPDGFR� reagent will greatly facilitate the continued investigationof adult physiologic and pathophysiologic functions of the PDGFfamily.

MethodsConstruction of Adenoviruses. PDGFR� ectodomain cDNA (corresponding toamino acids 1–527) was amplified from embryonic day 12.5 mouse embryo cDNAwith C-terminal His6 epitope tag, sequenced, and cloned into the E1 region ofE1�E3� Ad strain 5 by homologous recombination followed by Ad production in293 cells and CsCl gradient purification of virus as described (31, 46). The con-struction of Ad Flk1-Fc encoding the entire murine Flk1 ectodomain fused toIgG2� Fc and Ad Fc encoding IgG2� Fc has been described previously (31). AdTie2-Fc expresses the entire cognate murine ectodomains followed by fusion toC-terminal murine IgG2� Fc.

Adenovirus Administration and Detection of Plasma Transgene Expression.Adenoviral administration and analysis of transgene expression were performedas described in ref. 31. Antibodies can be found in SI Methods.

In Vitro Analysis of Soluble Ectodomain Function. Inhibition of Akt phosphory-lation by purified soluble Tie2 and PDGFR� ectodomains was demonstrated byusing HUVECs and NIH 3T3 cells, respectively. NIH 3T3 cells were incubated in the

Fig. 5. Effect of combination antiangiogenic therapy on preestablished tumor growth. (A) Under conditions of suboptimal inhibition of VEGF signaling (AdFlk1-Fc, 1 � 108 pfu for LLC, 7.5 � 107 pfu for T241), the addition of Ad sPDGFR� produces additive antitumor effects on LLC (n � 16) and T241 (n � 7) tumorgrowth vs. Ad sPDGFR� or Ad Flk1-Fc monotherapies. Final tumor volumes are presented (*, P � 0.02 vs. Ad Fc). (B) Adult mice bearing LLC (n � 7 per group);T241 fibrosarcoma (n � 6/group), or B16Bl6 (n � 7 per group) tumors were treated with a single adenovirus Ad Fc (109 pfu) or with adenovirus combinations,Ad Flk1-Fc/Ad Fc, Ad sPDGFR�/Ad Fc, or Ad Flk1-Fc/Ad sPDGFR� (5 � 108 pfu each) simultaneously. In all three tumor models, the combination of stringent VEGFblockade via Ad Flk1-Fc with PC-targeting Ad sPDGFR� did not augment the inhibition of tumor growth afforded by Ad Flk1-Fc alone. Only the AdFlk1-Fc-containing therapies significantly reduced tumor volume relative to Ad Fc (P � 0.008 for all three tumor models). *, P � 0.008 vs. Ad Fc. (C) Effect ofcombination antiangiogenic therapy on tumor vasculature. Endothelial (CD31) and PC (SMA) content was quantitated in LLC tumors treated with the indicatedAds. Ad Flk1-Fc/Ad sPDGFR� combination therapy does not further increase the reduction of LLC microvessel density achieved by Ad Flk1-Fc treatment (n � 4;

*, P � 0.05). (D) Quantitative real-time PCR analysis of PDGF-B mRNA in nascent T241 tumors. Stringent inhibition of VEGF signaling by Ad Flk1-Fc leads to asignificant reduction of total tumor PDGF-B mRNA expression, accompanying reduced PC content in Ad Flk1-Fc-treated tumors (n � 4; #, P � 0.03).

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presence or absence of 200 ng/ml soluble PDGFR� ectodomain for 10 min beforestimulation with PDGF-BB (10 ng/ml, R&D Systems). For HUVECs, cells wereincubated with 150 ng/ml Ang1 in the presence or absence of excess soluble Tie2protein (5 �g/ml) for 15 min. Inhibition of Akt phosphorylation was determinedby Western blotting of whole-cell extracts with primary antibody specific tophosphorylated Ser-473 Akt antibody (Cell Signaling Technology). Equal loadingwas determined by Western blotting with primary antibodies specific to GAPDHor insulin-degrading enzyme.

CL Angiogenesis Model. Three- to 4-week-old female C57BL/6 mice were firstinjected i.p. with 5 international units of pregnant mare serum gonadotropin(Sigma) followed by the i.v. administration of 5 � 108 pfu of Ad Fc, Ad Flk1-Fc, AdTie2-Fc, or Ad sPDGFR� 24 h later. One day after adenovirus infection, ovulationwas induced by treatment with 5 international units of hCG (Sigma). Forty-eighthours after hCG injection, ovaries were harvested and processed for hematoxylinand eosin histological analysis according to standard protocols. Immunofluores-cence analysis was performed as described below. Gleevec (100 mg/kg, Novartis)was administered twice daily by oral gavage from the day after pregnant mareserum gonadotropin treatment until the harvesting of the ovaries.

Tumor Modeling. Tumor modeling was performed as described in ref. 31. For adetailed description, see SI Methods.

Immunofluorescence Staining and Vessel Quantification. Immunoflurescencestaining and vessel quantification were performed according to standard proto-cols. For details and antibody information, see SI Methods.

Analysis of PDGF-B mRNA Expression. PDGF-B mRNA was quantified by SYBRGreen real-time PCR. For details, see SI Methods.

ACKNOWLEDGMENTS. We are indebted to members of the C.J.K. laboratoryand to Cecile Chartier for helpful discussion. F.K. is a fellow of the American HeartAssociation, and B.Y.Y.T. is a Fonds de la Recherche en Sante du Quebec Fellow.This work was supported by grants from the National Institutes of Health (1 R01CA95654-01, NS052830, and HL074267) and the Department of Defense (toC.J.K.), National Institutes of Health Grants HL-24136 and HL 59157 from theNational,Heart, Lung,andBlood Institute (toD.M.M.), andGrantCA082923fromthe National Cancer Institute (to D.M.M.). These studies were also supported byBurroughs Wellcome Foundation Scholar in the Pharmacological Sciences andKimmel Foundation Scholar awards (to C.J.K.).

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