Review

Anti-VEGF/VEGFR Therapy for Cancer: Reassessing theTarget

Basel Sitohy1,2, Janice A. Nagy1,2, and Harold F. Dvorak1,2

AbstractJudah Folkman recognized that new blood vessel formation is important for tumor growth and proposed

antiangiogenesis as a novel approach to cancer therapy. Discovery of vascular permeability factor VEGF-A as theprimary tumor angiogenesis factor prompted the development of a number of drugs that targeted it or itsreceptors. These agents have often been successful in halting tumor angiogenesis and in regressing rapidlygrowing mouse tumors. However, results in human cancer have been less impressive. A number of reasons havebeen offered for the lack of greater success, and, here, we call attention to the heterogeneity of the tumorvasculature as an important issue. Human and mouse tumors are supplied by at least 6 well-defined blood vesseltypes that arise by both angiogenesis and arterio-venogenesis. All 6 types can be generated inmouse tissues by anadenoviral vector expressing VEGF-A164. Once formed, 4 of the 6 types lose their VEGF-A dependency, and so theirresponsiveness to anti-VEGF/VEGF receptor therapy. If therapies directed against the vasculature are to have agreater impact on human cancer, targets other than VEGF and its receptors will need to be identified on theseresistant tumor vessels. Cancer Res; 72(8); 1909–14. �2012 AACR.

IntroductionIt is just 40 years since Judah Folkman published his classic

New England Journal of Medicine article entitled "Tumor angio-genesis: therapeutic implications" (1). In that article, he setforth 3 bold postulates: (i) angiogenesis is essential for tumorgrowth beyond minimal size; (ii) tumors secrete a "tumorangiogenesis factor" (TAF) that is responsible for inducingangiogenesis; and (iii) antiangiogenesis is a potential thera-peutic strategy for treating cancer. Now, 4 decades later, afterconsiderable debate and not a little controversy, all 3 of thesepostulates are widely accepted. Generally speaking, tumorsdo need to induce a new vascular supply if they are to grow,though some make do, at least for a time, by coopting theexisting host vasculature (2). Folkman's RNA-like TAF candi-date proved to be a blind alley, but the discovery of vascularpermeability factor/VEGF (VPF/VEGF or more simplyVEGF-A), and the preparation of antibodies against it, providedan opportunity for testing Folkman's hypotheses (3, 4). VEGF-Ais widely expressed by nearly all malignant tumors and is

generally agreed to be the most important TAF. It acts onvascular endothelial cells within minutes to induce vascularpermeability and, longer term, reprograms gene expression,leading to endothelial cell proliferation and migration in vitro,and generation of new blood vessels in vivo. Moreover, a varietyof drugs that target VEGF-A or its receptors effectively preventthe growth of manymouse tumors and tumor xenografts (2, 5–7). Among these drugs are antibodies against VEGF-A or itsreceptors, engineered proteins that mimic VEGFRs, and small-molecule receptor tyrosine kinase (RTK) inhibitors that pref-erentially target VEGFR-2 (VEGFR-2/flk-1/KDR) with highaffinity.

Unfortunately, however, the striking benefits of anti-VEGF/VEGF receptor (VEGFR) therapy observed in treatingmouse tumors have not been translated to the clinic. All ofthese drugs have had only modest effects on human cancersand have not lived up to the great expectations that Folkmanand many others anticipated. For example, bevacizumab(Avastin; Genentech), a humanized antibody against VEGF-A,prolongs the life of patients with advanced colon cancer by,on average, 4 to 5 months, and then only when combinedwith triple chemotherapy (8). In fact, a recent editorial hasquestioned whether bevacizumab was "boon or bust" becauseof its limited effectiveness, serious (though uncommon) sideeffects, and high cost (9). Other U.S. Food and Drug Admin-istration–approved drugs that bind VEGF-A, or that targetVEGF-A receptors, have fared no better (10). Thus, we are leftwith the question, why doesn't anti–VEGF-A/VEGFR therapywork better against human cancers? But, before tackling thatquestion, it could be helpful to address some others first:What are tumor blood vessels? How do they form? What doesanti–VEGF-A/VEGFR therapy do to tumor blood vessels?

Authors' Affiliations: 1The Center for Vascular Biology Research and2Department of Pathology, Beth Israel Deaconess Medical Center andHarvard Medical School, Boston, Massachusetts

Current address for B. Sitohy: Division of Surgery and PerioperativeScience, Department of Orthopedics, Umea

�University, Umea

�, Sweden.

Phone: 46907850000; Fax: 4690137455; E-mail:basel.sitohy@orthop.umu.se

Corresponding Author: Harold F. Dvorak, Department of Pathology,Beth Israel Deaconess Medical Center, 330 Brookline Avenue, RN227C,Boston, MA 02215. Phone: 617-667-8529; Fax: 617-667-3591; E-mail:hdvorak@bidmc.harvard.edu

doi: 10.1158/0008-5472.CAN-11-3406

�2012 American Association for Cancer Research.

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WhatAreTumorBloodVessels, andHowDoTheyForm?

Despite the great sums of money that companies, theNIH, and charitable organizations have invested in anti-VEGF/VEGFR approaches to cancer therapy, relatively littleattention has been paid to the types of new blood vessels thatthese drugs are expected to target. Tumor angiogenesis hasoften been regarded as if it were a single entity in which alltumor blood vessels were identical. However, it has long beenknown that tumor blood vessels are heterogeneous and differconsiderably among themselves with respect to organization,structure, and function (11). Studying several importanthuman carcinomas, we identified 6 distinctly different typesof blood vessels (12, 13). Further, to avoid the complexity ofthe tumor environment, we developed a means of generatingsurrogate forms of tumor blood vessels in the absence oftumor cells, making use of a nonreplicating adenovirus (engi-neered by Richard Mulligan) to express the most commonVEGF-A isoform, mouse VEGF-A164 (human VEGF-A165;ref. 14).

When injected into the tissues of immunodeficient mice,Ad-VEGF-A164 generates each of the 6 vessel types we hadfound in human cancers and does so by 2 distinct but inter-related processes, angiogenesis and arterio-venogenesis. Onthe angiogenesis side, preexisting venules transform over thecourse of a few days into "mother" vessels, which are greatlyenlarged, pericyte-poor vessels that result from proteolytic

degradation of venule basementmembranes and frompericytedetachment (Fig. 1; refs. 12–15). Freed of these restraints,normally cuboidal venules undergo dramatic enlargement thatis made possible by extensive endothelial cell thinning; in thisprocess, endothelial cells discharge the cytoplasmic vesiclesand vacuoles that compose their vesiculo-vacuolar organellesto the cell surface, providing the greatly expanded plasmamembrane that is needed to line a 4- to 5-fold expanded lumen(14, 16). Mother vessels are highly permeable to plasma pro-teins, largely by transcellular routes that involve residualvesicles and newly formed fenestrae. Over the course of daystoweeks,mother vessels evolve into daughter vessels of 3 types:(i) glomeruloid microvascular proliferations (GMP), poorlyorganized structures that, as their name implies, resemblerenal glomeruli macroscopically. GMP result from intralum-inal proliferation of endothelial cells and pericytes and theaccumulation of macrophages; (ii) vascular malformations,thought to represent mother vessels that have reacquired asmooth muscle cell coat; and (iii) typical capillaries that formas mother vessels sprout inwards instead of outwards, project-ing cytoplasmic processes into their lumens that form inter-connecting intraluminal bridges that subsequently separatemother vessels into smaller vessels by intussusception. Vas-cular malformations and capillaries, unlike mother vessels andGMP, are stable vascular structures that persist indefinitely.Arterio-venogenesis proceeds in parallel with angiogenesis asarterioles, arteries, and veins undergo extensive remodeling

Figure 1. Diagram of the angiogenic and arterio-venogenic blood vessels that are induced by Ad-VEGF-A164 in mouse tissues, identifying those that are(enclosed within the dash-lined box) or are not, susceptible to anti-VEGF therapy with rapamycin and aflibercept (VEGF Trap).

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and enlargement with proliferation of endothelial cells andsmooth muscle cells (Fig. 1; refs. 13, 14).

What Does Anti-VEGF/VEGFR Therapy Do toTumor (and Normal) Blood Vessels?Folkman conceived of antiangiogenesis primarily as a

means of preventing angiogenesis and, thereby, limitingfurther tumor growth. However, it was soon found thatanti-VEGF/VEGFR therapies not only prevented angiogen-esis, but additionally caused considerable pruning of pre-existing vessels (5, 17). All of the drugs used, whethertargeting VEGF-A or its receptors, had similar effects. Endo-thelial fenestrations, when present, disappeared, vascularsprouting and permeability were suppressed, and blood flowand vessel patency were extensively reduced. Overall vascu-lar density decreased (depending on the tumor type by asmuch as 70%). Changes were also noted in the normalvasculature, particularly a loss of endothelial cells fenestraein renal glomeruli and several endocrine organs (5, 17).These findings suggested an explanation for at least someof the side effects that have been found in patients receivinganti–VEGF-A/VEGFR drugs (9); they also provided strongevidence that VEGF-A was necessary for the maintenance ofthe normal adult vasculature. Some tumor cells, as well astumor-infiltrating macrophages, express VEGFRs, particu-larly VEGFR-1, and so anti-VEGF/VEGFR agents may act onthese cells as well (18). This action is more likely to be thecase for small-molecule RTKs, because large molecules suchas antibodies are unlikely to penetrate the vascular barrierefficiently.

ExplanationsCommonlyOffered for theFailureofAnti-VEGF/VEGFR Therapy to Work Better inCancer PatientsA number of explanations have been offered to explain the

modest effectiveness of anti–VEGF-A/VEGFR therapy incancer patients vis-�a-vis their more potent effects intumor-bearing mice (19). All of these explanations are likelyto have some validity. One obvious possibility is that cancerpatients are often elderly and very ill, in contrast with theyoung, relatively healthy tumor-bearing mice treated in thelaboratory. Further, higher dosing is possible in mice, whocannot complain about side effects. A second likely reasonfor the limited effectiveness of anti-VEGF/VEGR therapy isthat it does not result in the killing of all tumor cells; residualtumor cells rendered hypoxic by a compromised bloodsupply are stimulated to make increased amounts ofVEGF-A that may overwhelm anti-VEGF/VEGFR therapy,especially when accompanied by increased expression ofmatrix components that bind and sequester VEGF-A, pro-tecting it from anti-VEGF drugs (20). Hypoxic tumor cellsalso begin to make a plethora of other growth factors andcytokines, which have the capacity to replace VEGF andstimulate new blood vessel growth. Included among theseare FGF, PDGF-B, PDGF-C, HGF, EGF, IL-8, IL-6, Ang-2,SDF1a, PDGF-C, CXCL6, and others, as well as their recep-tors. Other possibilities are recruitment from bone marrow

of vascular progenitor cells and proangiogenic myelocytesthat can serve as a rich source of growth factors (19, 21).

Another explanation is that of "vascular normalization," alimited period of time during which tumors treated with anti-VEGF/VEGFR therapy exhibit a reduction in tumor vesseltortuosity, hyperpermeability, interstitial tissue pressure, andedema (6). The mechanisms responsible for vascular normal-ization are not yet fully understood and could reflect a returnto normality of abnormal tumor blood vessels and/or pruningof the most abnormal vessels, leaving behind those that areless abnormal. Whatever the mechanism, vascular normaliza-tion persists for only a short time, in both mouse and humancancers, and could have Janus-like effects on tumor growth.On the one hand, more normal blood vessels provide improvedblood flow and so might be expected to provide a window ofopportunity for improved drug delivery; however, a betterblood supply could also favor tumor growth (for a review ofthis issue, see refs. 6, 10).

Another Possible Explanation for the Limitationsof Anti-VEGF/VEGFR Therapy: DifferentialSensitivity of Tumor Blood Vessel Subtypes toAnti-VEGF/VEGFR Therapy

Many of the early, highly successful studies targeting VEGFor its receptors in mice followed protocols in which therapywas initiated concurrently with or soon after the transplant oftumor xenografts. Obviously, however, this treatment patterndoes not mimic the clinical situation in which tumors havebeen present for months or years prior to discovery and onsetof therapy. Bergers and Hanahan (19, 22) recognized thisdistinction, raising an important red flag that called attentionto the limitations of anti-VEGF/VEGFR therapy in treatingestablished tumors. They found that VEGFR inhibitors werehighly effective in preventing the development of the sponta-neous Rip-Tag tumor and in inhibiting its early growth, butthat they were of much less benefit in regressing tumorswith an established vasculature (19, 22). Thus, in mice as inpatients, anti-VEGF/VEGFR therapy was found to be lesseffective in advanced disease. Bergers andHanahan (19) attrib-uted the failure of late therapy to the maturing of the vascu-lature with increased pericyte coverage and found thataddition of an RTK inhibitor that targeted PDGFR-b (highlyexpressed on pericytes) improved anti-VEGFR therapy,though in other systems this has not been the case. Manyother reports indicate that "immature" vessels, variouslydefined, are preferentially susceptible to anti-VEGF/VEGFRtherapy (19, 22–24). However, there are exceptions to thispattern of differential sensitivity. For example, in some tu-mors nearly all blood vessels are pericyte or smooth musclecell coated and yet undergo extensive pruning in responseto anti-VEGF/VEGFR therapy (5).

Effects of Rapamycin and Aflibercept (VEGFTrap) onTumorSurrogateBloodVessels Inducedby Ad-VEGF-A164

In 2 recent articles, we investigated the sensitivity of tumorsurrogate blood vessels to anti–VEGF-A drugs (23, 25). When

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injected into the skin of immunodeficient mice, Ad-VEGF-A164

replicates at successive intervals large numbers of each ofthe 6 types of blood vessels that we had identified in humancancers. Therefore, by initiating treatment at different inter-vals before or after Ad-VEGF-A164 injection, we were able toevaluate the effects of drugs that targeted VEGF-A indirectly(rapamycin; ref. 25) or directly [aflibercept (VEGF Trap);ref. 23]. Rapamycin, acting on mTOR downstream of theVEGF-A–VEGFR-2 signaling pathway, prevented both angio-genesis and arterio-venogenesis and regressed early bloodvessels (mother vessels, GMP). VEGF Trap, a human solubledecoy receptor protein with high affinity for all of the VEGF-Aisoforms, as well as for VEGF-B and placental growth factor(26), had similar effects (Fig. 1). Up to about 1 month, mothervessels, GMPs, early vascular malformations, and developingfeeder arteries and draining veins regressed, at least to someextent, and vascular volume and vascular leakage were strik-ingly reduced. However, like rapamycin, VEGF Trap becameprogressively less effective when administered at later timesafter Ad-VEGF-A164 injection, when the great majority ofblood vessels induced by angiogenesis (vascular malforma-tions) and arterio-venogenesis (feeder arteries, draining veins)had developed fully. Thus, although all 6 types of tumorsurrogate blood vessels were induced by VEGF-A164, not allremained sensitive to anti–VEGF-A therapy. Results consistentwith ours were recently reported in a model of angiogenesisinduced by HIF-1a–overexpressing keratinocytes; antibodiesagainst VEGFR-2 were initially successful in preventing newblood vessel formation but were largely ineffective whenadministered 2 weeks later (27).

In an effort to explain our results mechanistically, weimmunostained Ad-VEGF-A164–induced blood vessels forVEGFR-2. Whereas early susceptible vessels (mother vessels,GMP) expressed high levels of VEGFR-2, late, rapamycin-, andVEGF Trap–resistant vessels (vascular malformations, feederarteries, draining veins) expressed extremely low and some-times undetectable levels of this receptor as determined byimmunohistochemistry (23), and by reverse transcriptase–PCR (H.F. Dvorak, unpublished data). Thus, a possible expla-nation for the insensitivity of vascular malformations, feederarteries, and draining veins to VEGF Trap is that their liningendothelial cells have lost dependence on exogenous VEGF-A,and that any remaining requirement for VEGF-A as a survivalfactor had been met by the pericytes or smooth muscle cellsthat closely enveloped them (28).

Our findings with rapamycin and VEGF-Trap on Ad-VEGF-A164–induced tumor surrogate blood vessels are likely to berelevant to anti-VEGF/VEGFR therapy for mouse tumors andhuman cancer. Personal review of many different tumors andof the published photomicrographs of others indicate thatvessels with the structure and functional properties of mothervessels predominate during early stages of the growth of mosttransplantable mouse tumors and tumor xenografts and alsoin such spontaneously arising tumors as the Rip-Tag2 model.Additional evidence for this conclusion comes from the findingthat anti-VEGF/VEGFR drugs potently reverse tumor vascularhyperpermeability (7, 23, 29, 30). In the pathologic angiogenesisinduced by tumors or Ad-VEGF-A164, mother vessels (and to a

lesser extent GMP) are the only hyperpermeable blood vesselsthat have thus far been identified in tumors (13, 14). The samereasoning suggests that the "vascular normalization" followinganti–VEGF-A/VEGFR treatment results, at least in part, fromthe pruning of hyperpermeable mother vessels. Left behind arerelatively unaffected pericyte- and smooth muscle–coatedvascular malformations, feeder arteries, and draining veins,that is, vessels that by MRI and histology seem relativelynormal in that they are not hyperpermeable and are smoothmuscle cell coated.

Looking Back and Moving ForwardThe work reviewed here invites some conclusions but also

raises new questions. On the plus side, Folkman's postulateshave been affirmed. Targeting VEGF and its receptors offersbenefit to patients with at least some types of cancer andprovides proof-of-principle that attacking the vasculature is avalid approach to cancer therapy. At present, however, thesebenefits are limited and may have untoward consequences;just as inadequately treated bacterial infections can result inmutations that lead to antibiotic resistance, so residual tumorcells, rendered hypoxic but not killed by anti-VEGF/VEGFRtherapy, can reprogram their genomes to express increasedamounts of VEGF-A as well as other growth factors that canoverwhelm or bypass the VEGF/VEGFR axis and stimulatenew blood vessel growth. To mix metaphors, wounding a bearcan make him very angry!

A further conclusion is that tumor blood vessels areheterogeneous and that some are susceptible, whereasothers are highly resistant, to anti-VEGF/VEGFR therapy.Resistant surrogate vessels such as vascular malformations,feeder arteries, and draining veins develop gradually overtime after Ad-VEGF-A164 administration; they are relativelylarge vessels, coated by 1 or more layers of smooth musclecells, and are lined by endothelial cells that express very lowlevels of VEGFR-2. Late-appearing vessels of this type arelikely to predominate in human cancers, which are oftenpresent for months to years before they are discovered.

The mechanisms by which tumor blood vessels are actual-ly destroyed by anti-VEGF/VEGFR therapy require furtherinvestigation (18). An obvious possibility is that vessels linedby endothelial cells expressing high levels of VEGFR-2 andcoated with few pericytes require substantial amounts ofVEGF-A for survival and, therefore, undergo apoptosis whenthe VEGF-A/VEGFR axis is disrupted. However, VEGFR-2expression levels and pericytes may not provide the entireexplanation for resistance to anti-VEGF/VEGFR therapy.Pericyte-coated capillaries are not always spared by anti-VEGF/VEGFR therapy (5), and other mechanisms can beenvisioned for their destruction (18). In some cases, bloodvessel pruning could result from tumor cell killing; for example,dying tumor cells could release cytokines or toxic products thatdamage blood vessels. A possibility that has received littleattention is that anti–VEGF-A/VEGFR therapy could not onlyact on angiogenic capillaries but also act upstream on thelarger feeder arteries that supply the tumor microcirculation.The VEGF-A–VEGFR-2–eNOS–NO pathway has an important

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role in maintaining normal arterial tone, and, when inter-rupted, could cause constriction of the feeder arteries supply-ing tumors, leading to reduced downstream blood flow andnecrosis of both tumor blood vessels and the tumor cells theysupply. Consistent with this possibility is the finding thatpatients receiving anti–VEGF-A/VEGFR therapy commonlydevelop systemic hypertension (18). It would be ironic iftreating this hypertension, as is commonly done, actuallyinterfered with therapeutic benefit.Finally, going forward, it is important to think beyond VEGF

and its receptors as the sole vascular targets. We need toidentify new molecular targets on the endothelial cells liningthe large tumor blood vessels that supply and drain the tumormicrovasculature (vascular malformations, feeder arteries,and draining veins) and that have proved resistant to anti-VEGF/VEGFR therapy. Attacking these large vessels would cutoff the blood supply to the entire tumor and, therefore, have alarger effect than pruning the innumerable VEGF-A–sensitivemicrovessels. As any plumber knows, shutting off the watersupply at its source makes more sense than trying to turn off

every faucet in the house. Evidence for the potential of thisconcept comes from studies in which photodynamic occlusionof feeder arteries and draining veins led to eradication ofmouse ear tumors (31) and from findings that uterine arteryembolization ormyolysis can ablate uterinefibroids in patients(32). Progress is being made in this regard, and a number oftumor vessel markers worthy of further investigation havebeen identified (33, 34). We anticipate bright days ahead fortargeting the vasculature as a mode of cancer therapy.

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

Grant SupportThis work was supported by U.S. Public Health Service grants P01 CA92644,

CA-142262, by a contract from the National Foundation for Cancer Research(H.F. Dvorak), and by The Swedish Research Council and The Swedish Societyof Medicine (B. Sitohy).

Received October 12, 2011; revised December 5, 2011; accepted December 15,2011; published OnlineFirst April 11, 2012.

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