Tumor vasculature as target for therapeutic intervention

1. Introduction

2. Tumor microenvironment

3. Angiogenic (growth) factors

and integrins

4. Inhibition and attack of

angiogenesis

5. Conclusion

6. Expert opinion

Review

Tumor vasculature as target fortherapeutic interventionMarcel HAM Fens, Gert Storm & Raymond M Schiffelers†

Utrecht University, Utrecht Institute for Pharmaceutical Sciences (UIPS), Department of

Pharmaceutical Sciences, Faculty of Science, The Netherlands

Importance of the field: Solid tumors rely on efficient oxygen and nutrients

transport for their growth, development and survival. Many tumors can stim-

ulate new blood vessel formation. Because this angiogenic vasculature is

aberrant from normal host vasculature, several strategies have been explored

that specifically target tumor blood vessels.

Areas covered in this review: Over the past decade, many molecules that act

on tumor vasculature have been identified. They can be divided into three

groups based on their mechanism of action: i) antiangiogenic molecules cause

tumor growth arrest; ii) vasoactive agents induce hyperabnormalization of

the tumor vasculature, improving conventional drug accumulation in the

tumor; iii) vascular disrupting agents that cause blood vessel congestion,

resulting in massive secondary tumor cell necrosis. Many investigational drugs

from these classes are currently being evaluated to assess their role in

tumor therapy.

What the reader will gain: The underlying principle of each of the strategies is

discussed, and the (pre)clinical results of the investigational drugs in this class

are highlighted.

Take home message: To fully exploit the therapeutic potential of these drugs,

it appears necessary to combine them with conventional anticancer agents,

improve their selectivity for tumor vasculature, and develop biomarkers that

predict the tumor sensitivity for these vascular strategies.

Keywords: angiogenesis, cancer, tumor vasculature, vascular active agents,

vascular disrupting agents, vascular normalization

Expert Opin. Investig. Drugs (2010) 19(11):1321-1338

1. Introduction

Tumorigenesis is the process in which normal cells transform into tumor cells. Nor-mally there is a tightly regulated balance between cell proliferation and programmedcell death. In cancer, this balance is disrupted and cell division occurs in an uncon-trolled and often rapid fashion. Multiple mutations are necessary for tumorigenesisto occur. In fact, a series of mutations within certain classes of genes are requiredbefore a normal cell transforms into a tumor cell [1]. Mutations in oncogenes,tumor-suppressor genes, and mRNA genes are at the root of tumorigenesis, sincethese genes play vital roles in cell division, apoptosis, and DNA repair [2]. The muta-tions usually are somatic events, although germ-line mutations can cause heritableor familial cancer. Tumors can be benign or (pre)malignant. Benign tumors donot spread to other parts of the body or invade other tissues, and they are rarely athreat to life. In contrast, malignant tumors can invade other organs, spread to dis-tant locations (called metastasis), and become life-threatening. Cancer is by defini-tion malignant, and most tumors form a solid cell mass. The swiftly growingmalignant tumor cells initially obtain oxygen and nutrients from host tissues untila tumor reaches the size of a few cubic millimeters. Beyond this point, the tumor

10.1517/13543784.2010.524204 © 2010 Informa UK, Ltd. ISSN 1354-3784 1321All rights reserved: reproduction in whole or in part not permitted

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needs to create its own blood vessel network to allow furthergrowth. Tumors organize this by inducing sprouting of newblood vessels from pre-existing vasculature in a process calledangiogenesis [3,4]. In addition to cancer, other diseases are alsoassociated with (pathological) angiogenesis. Moreover, angio-genesis is required in physiological processes like growth,development, the menstrual cycle and wound healing. Patho-logical angiogenesis differs from physiological angiogenesiswith respect to the expression of angiogenic regulators.In addition to angiogenesis, further mechanisms that pro-

vide tumors with oxygen and nutrients have been reported,such as the creation of microvascular channels in a processcalled vasculogenic mimicry and vessel co-option [5,6]. Theexpression profiles are not as well coordinated in tumors asin normal tissues. In addition, angiogenic tumor blood vesselslack mechanisms that silence the angiogenic process. Anexample is the full coverage by functional perivascular cellsof normal vessels during growth that induces vascular quies-cence [3,7]. Tumor blood vessels are composed of endothelialcells, pericytes and basement membrane, similar to normalblood vessels, but are structurally and functionally abnormal.They are unusually dynamic, and undergo sprouting, prolifer-ation, remodeling or regression. The vessels have irregulardiameters, branching patterns, are tortuous, and lack the nor-mal hierarchical arrangement of arterioles, capillaries andvenules [8-10]. The intratumoral blood flow is chaotic, variable,frequently sluggish, and at times might be stationary or evenexperience a reversal in the direction of flow [11,12]. Moreover,tumor vessels lack the tight endothelial monolayer essentialfor normal barrier function. Tumor endothelial cells andcapillaries may be disorganized, loosely connected, branched,overlapping, sprouting, and form a defective cellular lining ofthe vessel wall (Figure 1) [13,14]. This results in leakinesswhich, together with the limited number or absence offunctional lymphatic vessels, increases interstitial fluid pressure

(IFP) [13,15,16]. Furthermore, endothelial cells require growthfactors, like VEGF, for survival and express ubiquitous and uni-que cell markers [17]. These characteristics of angiogenic bloodvessels are used to develop cancer medicines that specificallytarget angiogenic factors and inhibit of angiogenesis [18].

In 1971, Judah Folkman proposed tumor angiogenesis tobe a promising therapeutic target to attack tumors [19]. Sincethen, the search for antiangiogenic therapies has emerged;but it was not until 1990 that the first compound to inducea specific angiostatic effect was discovered [20]. Since the early1990s, the research field has expanded drastically and to date,several antiangiogenic compounds have been successfullytested in clinical trials and are FDA-approved (see Table 1).At the same time, most therapeutics act on targets that seemcritical for both physiological and pathological angiogenesis.Consequently, clinical side effects, for example of anti-VEGF therapy, are seen [21]. Existing antiangiogenic thera-peutics can only be successfully used for certain types ofcancer, and often need to be administered in combinationwith relatively aspecific and toxic conventional chemothera-peutics. Thus, there is a need for novel approaches to specifi-cally attack tumors’ blood supply to increase tumor therapyefficacy. In this overview, the most important mediators ofangiogenesis are discussed, together with the three moststudied strategies to attack tumor vasculature.

2. Tumor microenvironment

The tumor microenvironment, or stroma, is essential fortumor proliferation, angiogenesis, invasion and metastasis.Progression of tumors is the product of an evolving crosstalkbetween different cell types within the tumor and its sur-rounding supporting tissue [22]. Tumors comprise a varietyof cell types other than tumor cells that are crucial fortumor progression. The microenvironment of tumors con-sists, besides tumor cells, of blood vessels, basement mem-brane, pericytes, fibroblasts, immune cells, and componentsof the extracellular matrix (ECM) [23]. As described in previ-ous section, tumor vasculature is aberrant and has developedimperfectly. But the other components of the microenviron-ment are also abnormal in tumors and play an importantrole in stimulating angiogenesis.

A fundamental structural and functional component ofblood vessels is the basement membrane (BM), also calledbasal lamina, which is an amorphous, dense, and sheet-like structure that shares similarities with the ECM [24].Vascular BMs have been demonstrated to play a crucial rolein the regulation of tumor angiogenesis. The BM providesthe cylinder-shaped framework of blood vessels, the inside ofwhich is lined with endothelial cells and the outside withpericytes (also known as mural cells or specialized smoothmuscle cells) for perivascular support. In tumors, pericytesform an incomplete covering around the abluminal surfaceof endothelial cells and show an altered expression of markerproteins [8,25].

Article highlights.

. Tumors can stimulate new blood vessel formation tosupply oxygen and nutrients needed to grow.

. This angiogenic vasculature is different from normal hostvasculature and can be a target for therapeuticintervention.

. Three classes of drugs attack tumor vasculature:� Anti-angiogenic molecules cause tumor growtharrest.

� Vasoactive agents induce hyper-abnormalization ofthe tumor vasculature.

� Vascular disrupting agents cause blood vesselcongestion.

. To fully exploit these drugs combination withconventional anticancer agents, improved selectivity anddevelopment of biomarkers that predict the tumorsensitivity seems essential.

This box summarizes key points contained in the article.

Tumor vasculature as target for therapeutic intervention

1322 Expert Opin. Investig. Drugs (2010) 19(11)

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Besides tumor cells, fibroblasts and immune cells arecrucial for the generation and regulation of angiogenesis-stimulating and -inhibiting growth factors and cytokines [22].However, the precise role of these activated fibroblasts andthe origin and initiation of tumor growth are still poorlyunderstood [26,27]. More is known about the role of theimmune cells in tumor development and angiogenesis. It isbelieved that chronic inflammation contributes to tumori-genesis [28], but also to progression of established tumors, aswell as to metastasis [29]. Macrophages appear one of themost prominent cell types in this respect. Within the tumormicroenvironment, they are known as tumor-associatedmacrophages (TAMs) and promote angiogenesis, ECMremodeling and tumor cell motility [30,31].

All mediators in the tumor microenvironment that con-tribute to tumor progression are interesting candidates fortherapeutic intervention. Nevertheless, many questions aboutthe precise role of these mediators remain unanswered andfurther research is required to optimally manipulate themfor tumor therapy.

3. Angiogenic (growth) factors and integrins

Angiogenesis strongly depends on specificmolecular interactionsbetween vascular cells and components of the ECM. In addition,a variety of growth factors, chemokines and cytokines drives theangiogenic process. These regulators of angiogenesis can bedivided into two main categories: pro- and antiangiogenic fac-tors. In normal tissues, a delicate balance exists between bothtypes of factors [32]. The balance tips in favor of the stimulatorsin both pathological and physiological angiogenesis. However,in pathological angiogenesis the imbalance persists and angio-genesis is consistently stimulated [32,33]. Continuous overproduc-tion of stimulating growth factors results in the ‘angiogenicswitch’, where new vasculature is formed in and around thetumor, allowing it to grow exponentially from a small dormant

and avascular tumor to a large, well-vascularized tumor [34].Pro-angiogenic factors include VEGF, basic fibroblast growthfactor (bFGF), hypoxia-induced factor-1a (HIF-1a), EGF,PDGF, TGF, angiopoietins (ANG), and interleukins (ILs).Among these, VEGF is considered one of the key regulators ofboth physiological and pathological angiogenesis [35-37]. VEGFis abundantly produced by hypoxic tumor cells, macrophagesand other cells of the immune system, and affects vasodilationand vascular permeability [38]. However, angiogenesis is notsolely dependent on VEGF and may be driven by additionalangiogenic factors like FGF, TGF or placenta growth factor(PlGF) [39,40].

Moreover, angiogenesis not only depends on growth factorsand their receptors but is also influenced by receptors forECM proteins. In general, cell adhesion to the ECM is medi-ated by integrins, heterodimeric transmembrane proteins thatconsist of a diverse family of multiple a and b subunits [41].Integrin-mediated adhesion leads to intracellular signalingevents that regulate migration, proliferation, invasion andcell survival [42]. Of the wide spectrum of integrin subunitcombinations expressed on the surface of cells, av-integrins(avb3 and avb5) have been identified as having a preferentialexpression pattern on vascular cells during angiogenesis andvascular remodeling. av-integrins are receptors for a wide vari-ety of ECM ligands with an exposed RGD (Arg--Gly--Asp)sequence, including vitronectin, fibronectin, and fibrinogen.Even though av-integrins are selectively expressed by specificcell types, they are known to be also expressed on a small per-centage of activated leukocytes, macrophages, and osteoclasts,where they contribute to immune function and bone resorp-tion. In addition, tumor cells of several invasive cancers,such as melanoma and glioblastoma, express avb3 integ-rins [43,44]. Endothelial cells that are exposed to growth factorsthat play a role during angiogenesis express high levels ofav-integrins [45]. In fact, av-integrins may serve as potentialtumor markers and are useful as diagnostic or prognostic

A. B.

Figure 1. Aberrant tumor vasculature. A. Tumor vessels with irregular diameters and branching patterns, tortuous appearance

and lack of the normal hierarchical arrangement of arterioles, capillaries and venules. B. A more close-up view of the vascular

abnormalities in tumors at the (sub)cellular level such as the lack of a tight endothelial monolayer (green), complete pericyte

coverage (yellow) and basement membrane (black) degradation. (Readers are referred to the full-colour version online at

10.1517/13543784.2010.524204.)

Fens, Storm & Schiffelers

Expert Opin. Investig. Drugs (2010) 19(11) 1323

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Table

1.Ove

rview

ofmolecu

lesinvo

lvedin

theinhibitionofangiogenesisandpossible

subsequenttumorbloodve

sselnorm

aliza

tion.

Group/origin

Name(tradename)

Description

Size

Target

Clinicaldevelopment

Refs.

Phase

FDA

approved

Matrix

derived

Endostatin

CollagenXVIIIfragment

20kDa

Angiogenesis*

I/II

no

[51-53]

Endostatin-likefragment(EFC

XV)

CollagenXVfragment

22kDa

Angiogenesis

N.A.

N.A.

[54]

Tumstatin

CollagenIV

fragment

28kDa

Angiogenesis

N.A.

N.A.

[55]

Thrombospondin

1Glycoprotein

450kDa

Angiogenesis

N.A.

N.A.

[56]

Thrombospondin-1-m

imetic(ABT-510)

9-aminoacidpeptide

994g/m

ol

Angiogenesis

I/II

no

[57,58]

Thrombospondin

2Glycoprotein

150--1

60kDAa

Angiogenesis

N.A.

N.A.

[56]

Arresten

CollagenIV

fragment

26kDa

Angiogenesis

N.A.

N.A.

[59]

Canstatin

CollagenIV

fragment

24kDa

Angiogenesis

N.A.

N.A.

[60]

Endorepellin

Perlecanfragment

85kDa

Angiogenesis

N.A.

N.A.

[61]

Non-m

atrix

derived:

Growth

factors

andcytokines

Interleukins

Cytokines

Various

Angiogenesis

N.A.

N.A.

[48]

Interferons

Cytokines

Various

Angiogenesis

N.A.

N.A.

[62]

Pigmentepithelial-derivedfactor(PEDF)

Serpin

superfamily

member

50kDa

Angiogenesis

N.A.

N.A.

[63]

Fragments

ofcoagulationfactors

Angiostatin

Fragmentofplasm

in38kDa

Angiogenesis

IIno

[64]

Antithrombin

IIIGlycoprotein

58kDa

Angiogenesis

N.A.

N.A.

[65]

Prothrombin

kringle

2Prothrombin

fragment

22kDa

Angiogenesis

N.A.

N.A.

[66]

Plateletfactor-4

Chemokine

7.8

kDa

Angiogenesis

N.A.

N.A.

[67]

Other

Chondromodulin

Glycoprotein

25kDa

Angiogenesis

N.A.

N.A.

[68]

Tissueinhibitors

ofmetalloproteinnases(tim

ps)

Proteins

Various

Angiogenesis

N.A.

N.A.

[69]

2-M

ethoxyestradiol(2Me2)

Estrogenfragment

302g/m

ol

Angiogenesis

I/II

no

[70,71]

Vasostatin

Calreticulin

fragment

22kDa

Angiogenesis

N.A.

N.A.

[72]

Pex

MMP-2

fragment

29-32kDa

Angiogenesis

N.A.

N.A.

[73]

Prolactin

fragments

Prolactin

fragments

16kDa

Angiogenesis

N.A.

N.A.

[74]

Troponin

1Cartilagederivedprotein

21--2

3kDa

Angiogenesis

N.A.

N.A.

[75]

*Angiogenesisincludes(m

ultiple)knowntargets.



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indicators [46,47]. Moreover, blocking of avb3 integrins intumors via specific antagonists was shown to promote tumorregression [48], indicating the crucial role of av-integrins intumor endothelial maintenance and development. Surpris-ingly, ablation of the gene for av-integrin subunits in knock-out mice, although causing lethality, allows considerabledevelopment including extensive vasculogenesis and angio-genesis [49]. Nevertheless, angiogenesis and tumor vasculatureseem very potent and accessible targets for tumor therapy.

4. Inhibition and attack of angiogenesis

4.1 IntroductionAlthough endogenous growth factors that stimulate angiogen-esis (like VEGF and bFGF) have been well studied, the activ-ities associated with endogenous inhibitors are still poorlyunderstood [50].

4.1.1 InhibitorsThe endogenous inhibitors often are divided into two groups:matrix-derived inhibitors and non--matrix-derived inhibitors.Many endogenous inhibitors of angiogenesis are fragmentsof larger ECM molecules. These fragments become releasedupon proteolysis of the ECM and the vascular basementmembrane by enzymes such as MMPs, cathepsins and elasta-ses [51]. In addition to the matrix-derived molecules, a hetero-geneous group of other, non--matrix-derived, endogenousinhibitors exists that contains molecules originating from hor-mones, clotting factors and immunomodulatory proteins.Table 2 shows an overview of the two classes of endogenousangiogenesis inhibitors and the development stage of the smallnumber of compounds that entered clinical trials. The factthat numerous endogenous inhibitors of angiogenesis existpossibly indicates that these form a critical line of defenseagainst the conversion of dormant tumors into a malignantphenotype [52]. Yet a better understanding of the mechanismof action of these drug candidates is required. Endogenousinhibitors of angiogenesis are one group of prospective antitu-mor candidates. Many more, usually exogenous, antivascularapproaches have been (and still are) investigated for theirtherapeutic potential.

4.1.2 Vascular attackEven though the importance of angiogenesis and tumor vas-culature for tumor growth was acknowledged, up to the early1980s the majority of research focused on anticancer thera-peutics that still aimed at killing tumor cells directly. Then,Julia Denekamp proposed that attacking the tumor vascula-ture would be much more efficient as it seemed to require alower number of cells to be targeted [53,54]. By attacking ormodulating the easily accessible and genetically stable vas-culature, large numbers of tumor cells could be affected oreven killed. Consequently, tumor progression could (tempo-rarily) be arrested. Three major strategies in tumor vascularattack and modulation have been studied and described

extensively: tumor vessel inhibition and normalization; tumorvessel further abnormalization (manipulation); and tumorvessel disruption.

Inhibition of pro-angiogenic growth factors may induceangiogenic arrest and is the most widely studied strategy. Itaims at a long-term inhibition of tumor growth (but not neces-sarily regression), and is often explored in combination withconventional chemotherapeutics. Less than a decade ago, tumorvessel normalization was described as a possible consequence ofthe angiogenic inhibition therapy, providing new opportunitiesfor therapy. The second antivascular strategy aims at a hyperab-normalization of tumor vasculature. The main objective of thisapproach is to change the pathophysiology of solid tumors byhyperabnormalization of tumor vasculature (increased leaki-ness) via vasoactive agents. Consequently, increased local drugaccumulation could be acquired, leading to a more efficacioustumor response. The third strategy centers on pulsed disruptionof tumor vasculature leading to massive secondary tumor cellnecrosis. Blood vessel disruption is achieved by so called vascu-lar disrupting agents (VDAs) that specifically bind to tubulin.Subsequently, endothelial cells ‘round up’, leading to coagula-tion and finally to massive tumor cell death. These three thera-peutic antivascular strategies are discussed in the followingsections (2.2 -- 2.4).

4.2 Tumor blood vessel inhibition and normalizationThe majority of tumor vasculature-related research focused oninhibition of angiogenesis, often referred to as antiangiogenictherapy. The main target molecules for antiangiogenic ther-apy are stimulators of angiogenesis and players of the key sig-naling pathways [55,56]. Inhibitory approaches range fromtargeting one specific inhibitor, like monoclonal antibodiesfor a specific growth factor, to multitarget kinase inhibitorsthat affect a multitude of signaling cascades. In addition,other types of therapies were also shown to be potentiallyantiangiogenic, such as metronomic chemotherapy [57].

Apart from inhibition of angiogenesis leading to tumorgrowth arrest, antiangiogenic therapy may induce tumorblood vessel normalization. In the early 1970s, this wasdescribed for the first time by the group of Hellmann [58,59].They found that the antimetastatic drug razoxane (also calledICRF 159) induced tumor vasculature normalization, pre-venting tumor hemorrhages, blood-borne tumor-cell dissemi-nation and (pulmonary) metastasis [60]. Nevertheless, razoxanetargeting cancer cells rather than acting directly on endothelialcells [61]. In 2001, Rakesh Jain introduced tumor vasculaturenormalization induced by antiangiogenic therapeutics [62].The aim of vessel normalization is to restore the balancebetween stimulators and inhibitors of angiogenesis and conse-quently to regularize vascular architecture [32] (Figure 2). Bynormalizing the tumor vasculature, delivery of oxygen andtherapeutic agents to tumors could be improved [63]. Higherlocal drug levels probably increase therapeutic efficacy oftumor cell-specific drugs. Increased tumor oxygen levels intumors has proven to facilitate both radiation therapy and



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Table

2.Ove

rview

ofendogenousinhibitors

ofangiogenesis.

Name(tradename)

Description

Size

Target

Norm

aliza

tion

Clinicaldevelopment

Refs.

Phase

FDA

approved

AZD2171/Cediranib

(Recentin)

Tyrosinekinase

inhibitor

450.5

g/m

ol

VEGFR-2

tyrosinekinases

yes

IIIno

[91,92]

ZD6474/Vandetanib

(Zactim

a)

Tyrosinekinase

inhibitor

475.4

g/m

ol

VEGFR-2/EGFR/RETR

tyrosinekinases

yes

IIyes(orphan)

[93]

AG-013736/Axitinib

Tyrosinekinase

inhibitor

386.5

g/m

ol

VEGFR1-3/PDGFRb/cKIT

tyrosinekinases

yes

II/III

yes(orphan)

[94]

Bay57-9352/Telatinib

Tyrosinekinase

inhibitor

585.7

g/m

ol

VEGFR

2&3/PDGFRb/c-kit

tyrosinekinases

?I

no

[95]

SU11248/Sunitinib

(Sutent)

Tyrosinekinase

inhibitor

398.5

g/m

ol

allVEGFRs&PDGFRs,

KIT,

RET,CSF-1R,flt3

?III

yes

[96]

PTK787/ZK222584(PTK/ZK)/Vatalanib

Tyrosinekinase

inhibitor

346.8

g/m

ol

allVEGFRstyrosinekinases

?I/II

no

[97]

Bay43-9006/Sorafenib

(Nexavar)

Multikinase

inhibitor

464.8

g/m

ol

multikinases(Raf/Mer/Erk

pathway)

?II/III

yes

[98]

SU5416/semaxamib/semaxinib

Tyrosinekinase

inhibitor

238.3

g/m

ol

VEGFG

1andVEGFR-2

yes

I/II

no

[99,100]

AEE788

Tyrosinekinase

inhibitor

440.6

g/m

ol

VEGFR-2/EGFR/HER-2

?I

no

[101]

Anginex

33-m

erpeptide

3.9

kDa

galectin-1

yes

-no

[102]

EMD121974(Cilengitide)

Cyclic

pentapeptide

588.7

g/m

ol

av-integrins

?I/II

no

[103,104]

ADH-1

(Exherin)

Cyclic

pentapeptide

684.7

g/m

ol

N-cadherin

?I/II

no

[105]

ATN-161

Pentapeptide

687.7

g/m

ol

b-integrins(a5b1)

?I/II

no

[106]

Bevacizumab(Avastin)

Monoclonalantibody

149kDa

VEGF-A

yes

II/III

yes

[107-109]

HuMV833

Monoclonalantibody(Igg4k)

145kDa

VEGF-A

?I

no

[110]

IMC-1C11

Monoclonalantibody

?VEGFR-2

?I

no

[111]

VEGFtrap/Aflibercept

IgG(1)fusionprotein

96.9

Da

VEGF-A,PlGF

?II/III

no

[112]

MEDI-522/LM609/Etaracizumab(Vitaxin)

Monoclonalantibody(Igg1)

116kDa

avb3integrin

?II

no

[113,114]

17E6

Monoclonalantibody

?av

-integrins

?-

no

[115]

M200/Volociximab

Monoclonalantibody

145.5

kDa

a5b1integrin

?I/II

no

[116]

YW152F

Humanizedphageantibody

?DLL4-Notch

?-

no

[117]



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many chemotherapeutic agents and limit metastasis [32].Moreover, many antiangiogenic drugs are temporarily able toinhibit angiogenesis, but little is known about their long-term capacity to normalize tumor blood vessels. This wouldbe apparent from measurements on a variety of tumor physiol-ogy parameters -- including local blood perfusion and volume,microvascular density (MVD), IFP, pericyte coverage of theblood vessels, permeability--surface area product, and energyconsumption, as well as systemic response measurements suchas circulating endothelial cells (CECs). Indeed, such measure-ments showed that molecules that block VEGF signaling caneffectuate tumor vasculature normalization [32,64]. An overviewof molecules inhibiting angiogenesis is given in Table 1.

Currently, FDA-approved antiangiogenic agents andagents in clinical trials mainly comprise anti-VEGF signalingpathway kinase inhibitors and monoclonal antibodies [65,66].In a monotherapy setting, these agents account for vessel nor-malization and temporary progression-free antitumor effects.However, to obtain improved therapeutic effects, these agentsare generally used in combination therapy. By using combina-tion therapies with conventional chemotherapeutics, radiationtherapy, or both (chemoradiation), tumors could be treatedmore successfully [67].

4.3 Hyperabnormalization of tumor vasculatureA different approach to treat solid tumors is by hyperabnorm-alization of the tumor vasculature. Contrary to vessel normal-ization, this strategy aims to stimulate the vasculature byvasoactive agents to make vessels hyperpermeable (Figure 3).As a result of the hyperpermeability, higher amounts of che-motherapeutics can enter the tumor site. Unfortunately, pop-ular vasoactive agents such as TNF and histamine that caninduce these permeability changes are toxic upon systemicadministration. Low metronomic systemic doses of vasoactiveagents can strongly reduce this toxicity, but most experi-mental and clinical strategies make use of isolated perfusiontechniques or local administration. With the isolated perfu-sion technique, the blood circulation in the limb or organthat contains the tumor is isolated [68]. After perfusion withthe vasoactive agent followed by the chemotherapeutic,

15 -- 25 times increased concentrations of chemothera-peutics were measured compared to systemically administereddrugs [69]. An additional advantage of the isolated perfusiontechnique is minimal systemic leakage, with limited toxicityas a result.

Another method to increase tumor accumulation is deliveryvia drug carrier systems like liposomes and micelles. In con-trast to the isolated perfusion technique, no surgery is needed;and, in principle, all vascularized solid tumors can be reached.In the tumor vessel manipulation strategy, combination ther-apy is essential since the vasoactive agents are not killing(tumor) cells. Inflammatory modulators like TNF, histamine,and IL-2 are the most popular vasoactive agents. TNF is aninflammatory cytokine that is principally produced by acti-vated macrophages and monocytes [70]. Despite direct effectsof TNF on tumor cells, manipulation of the tumor vascula-ture by TNF accounts for the strongest antitumor effects.Changes in tumor vasculature after exposure to low-doseTNF comprise hyperpermeability, hemorrhagic necrosis,extravasation of erythrocytes, edema and vessel congestion [71],leading to an enhanced more homogeneous distribution ofsystemically administered (liposomal) drugs throughout solidtumors [72,73]. Studies have shown the enhanced efficacy ofsystemically and locally (isolated limb perfusion [ILP])administered TNF in combination with conventional chemo-therapeutics such as doxorubicin and melphalan or radiationtherapy [73-75].

The inflammatory modulator histamine (Hi) is mainlyformed and stored in the granules of mast cells and basophils.Histamine, a low-molecular-weight molecule, causes edema insmall vessels by locally increasing the lymph flow into theextracellular space and by promoting hyperpermeability ofthe endothelium [76]. This mechanism could be exploited toincrease intratumoral drug levels. Besides its direct tumorgrowth inhibitory effects, Hi also resulted in hole formationand destruction of the endothelial lining immediately uponILP. Combination with chemotherapeutic agents is stillrequired to obtain an antitumor response.

Lastly, IL-2, a cytokine that is produced by activatedT cells, is known to be able to cause vascular leakage and

Figure 2. Schematic overview of the tumor blood vessel normalization strategy. By inhibition of angiogenic growth factors,

tumor blood vessels could be normalized, resulting in regularized tumor vasculature.



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edema and may therefore also be a good candidate to increasedrug accumulation at the tumor site [77]. Table 3 depicts anoverview of these abnormalizing agents that have beenapplied in both preclinical and clinical settings. In a clinicalsetting, combination therapy of melphalan and TNF is awell-established approach to treat patients with unresectableadvanced sarcoma and advanced in-transit melanoma [78-80].

4.4 Tumor blood vessel disruptionOver the last decade, the specific disruption of existing tumorvasculature has received a lot of attention. Vascular disruptionaims to cause the rapid and selective shutdown of the estab-lished vasculature, resulting in secondary tumor cell death [81].The compounds known to initialize this kind of antitumoraction are called vascular disrupting agents (VDAs), previ-ously known as vascular damaging or targeting agents.Usually, VDAs are divided into two groups:

. Ligand-directed compounds utilizing specific antibodiesor peptides such as antibody-TF (tissue factor) andantibody-cytokine conjugates

. Small-molecule compounds such as ZD6126, combre-tastatin A4 phosphate, AVE8062, OXI4503, and5,6-dimethylxanthenone-4-acetic acid (DMXAA). Sev-eral of these are currently undergoing clinical trials(see Table 4 for overview of compounds and statusof development).

The majority of VDAs bind to microtubules -- dynamiccytoskeletal structures that switch between phases of relativestability to phases of alternating rapid shortening andgrowth [82]. Some tumor cells may be sensitive to cytoskeletaldisruption, but probably not after short exposure (in vivo half-life of VDAs free-injected regularly is very short). In vitroexperiments in which these effects are measured use exposuretimes that are generally much longer. In contrast to tumorand other cells, endothelial cell microtubules are markedlysensitive to VDAs, causing an increased dynamic instability.This instability, as a result of small Rho GTPase activation,

rapidly induces endothelial cells to become contractile (‘roundup’) [83,84]. Therefore, the immediate effect of VDAs resultingin morphological change overrules the mitotic arrest of tumorcells that requires prolonged exposure. These morphologicalchanges are accompanied by the disruption of cell--cell junc-tions, leading to an increased vascular permeability and expo-sure of basal lamina (Figure 4) [85,86]. Tumor selectivity of theVDAs is conferred by differences in the pathophysiology oftumor vessels versus capillaries in normal tissue [87,88].

As a result of this tumor endothelial rounding-up, localthrombi are formed and consequently blood vessels becomecongested or collapse. This leads to severe blood flow reduc-tion and catastrophic oxygen and nutrient deprivation forlarge numbers of tumor and stromal cells. Within hours afterthe onset of starvation, massive tumor cell necrosis occurs,reaching > 90% of total tumor mass. Although the centralregion of tumors can be completely killed, usually a typicalthin viable rim remains. A couple of explanations are offeredfor this peripheral layer of viable tumor cells: one is that adja-cent normal tissue still contains functional blood vessels thatare able to supply the nearby tumor cells with oxygen andnutrients via diffusion [89]. Another explanation is that inthe interior of tumors, the high interstitial pressure and lowpressure in VDA-damaged tumor vasculature causes vascularcollapse [81,87]. Together with a lower vessel density in thetumor core compared with the periphery, this could alsoform an explanation for the remaining viable rim.

It is certain that these residual viable tumor cells can rapidlyrepopulate the tumor, generally resulting in complete tumorregrowth in mere days after therapy, allowed by (accelerated)tumor progression [81]. By attacking this remaining proliferat-ing tumor rim, and accordingly hitting both tumor popula-tions at the periphery and in the central area of the tumor,the vascular disruption strategy could potentially benefitfrom combining VDAs with standard chemotherapeuticssuch as cisplatin [90,91], paclitaxel [92] and radionuclides [93]

or radiation therapy [94,95].Although preclinical animal studies have shown promising

results, ongoing clinical trials revealed adverse effects such as

Figure 3. Schematic overview of the tumor blood vessel manipulation strategy. By administration of vasoactive agents tumor,

blood vessels could be further abnormalized, resulting in increased hyperpermeability and consequently improved tumor

drug accumulation.



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Table

3.Ove

rview

ofmolecu

lesinvo

lvedin

bloodve

sselhyp

erabnorm

aliza

tion.

Group(origin)

Administration

Combinationtherapy

Target/cancertype

Clinicaldevelopment

Refs.

Phase

FDA

approved

TNF-alpha

ILP

Melphalan

Soft

tissuesarcoma

IIIno*

[130,131]

ILP

Melphalan

Melanoma

IIIno*

[129,132]

ILP

Actinomycin

DSoft

tissuesarcoma(rat)

-no

[133]

ILP

Doxorubicin

(anddoxilz )

Osteosarcomaandsoft

tissuesarcoma(rat)

-no

[134]

IHP

Melphalan

Soft

tissuesarcoma(rat)

-no

[135]

Systemic

No

fibrosarcoma(m

urine)

-no

[136]

Systemic

Doxorubicin

(anddoxil)

Soft

tissuesarcoma(rat)

-no

[122]

Systemic

Doxorubicin

(anddoxil)

Melanoma(m

urine)

-no

[123,137]

Systemic

Doxorubicin

(anddoxil)

Osteosarcoma(rat)

-no

[138]

Systemic

Cyclophosphamide,doxorubicin,5-fluorouracil

Sarcoma(m

urine)

-no

[139]

Systemic

Adriamycin

(+liposomal)

fibrosarcoma(m

urine)

-no

[140]

Systemic

Cisplatin

Osteosacromaandsoft

tissuesarcoma(rat)

-no

[141]

Systemic

(liposomal)

Doxorubicin

(anddoxil)

Soft

tissuesarcoma(rat)

-no

[142]

Systemic

(liposomal)

Radiation

Humancolonxenograft

-no

[124]

Systemic

(MLV

)Interleukin

1a

Melanoma(m

urine)

-no

[143]

TNF-NGR

Systemic

(i.p.)

No

Lymphomaandmelanoma(m

urine)

-no

[144]

Systemic

(i.p.andi.v.)

Doxorubicin

andmelphalan


urine)

-no

[145]

Systemic

(i.p.)

EMAP-IIandcisplatin


urine)

-no

[146]

TNF-RGD

Systemic

(i.p.)

Melphalan

Lymphoma(m

urine)

-no

[147]

Histamine

ILP

Melphalan

Soft

tissuesarcoma(rat)

-no

[126]

ILP

Doxorubicin

Soft

tissuesarcoma(rat)

-no

[148]

IHP

Melphalan

Soft

tissuesarcoma(rat)

-no

[149]

Interleukin-2

ILP

Melphalan

Soft

tissuesarcoma(rat)

-no

[127]

*TNFisnotFD

Aapprovedbutsince

1998approvedforILPTNF+melphalanin

bulkymelanomasandirresectable

soft

tissuesarcomasin

Europe.

z Doxil/CaelyxisaFD

Aapprovedliposomaldoxorubicin

form

ulation.



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Table

4.Ove

rview

ofva

sculardisruptingagents

testedin

(ongoing)clinicaltrials.

Group(origin)

Name(tradename)

Description

Size

Clinicaldevelopment

Refs.

Phase

FDA

approved

Colchicinederivatives

N-acetylcolchinol-O-phosphate/ZD6126

Colchicineanalogue,prodrug

437g/m

ol

I/II

no

[165-167]

Combretastatins

CombretastatinA4phosphate

(CA4P)/(Zybrestat)

CombretastatinA4prodrug

440.3

g/m

ol

II/III

no

[168,169]

AC-7700/AVE8062

Combretastatinanalogue

?II/III

no

[170]

combretastatinA1phosphate

(CA1P)/(OXI4503)

SerinecombretastatinA4prodrug

?I

no

[171]

Flavanoids

5,6

dim

ethylxanthenone-4-aceticacid

(DMXAA)/(ASA404)

AnalogueofFA

A282.3

g/m

ol

II/III

no

[172,173]

Other

MN-029

Tubulin

inhibitor

?I

no

[174]

E7010/ABT-751

Oralsulphonamide,b-tubulin

inhibitor

371g/m

ol

I/II

no

[175,176]

TZT-1027/Soblidotin

Dolastatin10derivative,tubulin

bindingagent

702g/m

ol

I/II

no

[177,178]

NPI-2358

Diketopiperazine,tubulin

bindingagent

?I

no

[179]

MPC-6827(Azixa)

Tubulin

bindingagent

?I/II

no

[180]

EPC2407

4-Arylchromene,tubulin

bindingagent

?I

no

[174]

BNC105

Tubulin

bindingagent

?I

no

CYT997

Tubulin

bindingagent

?I

no



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myocardial necrosis (as evidenced by cardiac enzyme release),in addition to declines in left ventricular ejection fraction andmyocardial infarction. These side effects were encounteredduring dose escalation of ZD6126. In addition, markedly dif-ferent individual therapeutic responses were noted. Althoughangiogenesis inhibitors also produce side effects on the cardio-vascular system, these usually feature internal bleeding andblood pressure increase.

5. Conclusion

Over the past decade, three strategies to address tumor vascu-lature have evolved: inhibition and normalization, hyperab-normalization, and disruption. Few molecules have made itto approval as drugs, and this seems related to a variety of hur-dles such as tumor-induced resistance and systemic toxicity,which introduce severe limitations for most molecules. Ingeneral, the efficacy of monotherapies that target tumor vas-culature is poor, adverse effects are commonly seen, and sen-sitivity of individual tumors towards antivascular strategiesis unclear.

6. Expert opinion

Over the past decade, angiogenesis has become one of themajor therapeutic targets in cancer, as this process appearsto be essential for solid tumor progression. The abnormaltumor blood vessel architecture and deviated molecular, bio-chemical and physiological characteristics have proven to bevaluable targets in the quest for successful cancer therapeutics.However, despite the discovery of numerous compounds withtumor vascular activity, only a limited number made it intoclinical testing and even fewer have been approved by theFDA to date. The large number of newly developed antian-giogenic compounds that did not make it to FDA-approvedproducts are an indication of the difficulties that this class ofcompounds faces to succeed.

Antiangiogenic therapies generally focus solely oninhibition of neovascularization, resulting in tumor growth

arrest and not necessarily in tumor regression. Therefore,most of the investigated candidate antiangiogenic drugs arecombined with other (mostly conventional) chemotherapeu-tics to improve efficacy. Often this combination is considerednecessary to obtain a positive therapeutic outcome [32].

In a similar manner, manipulation of the tumor vasculatureby vasoactive agents alone does not induce antitumor efficacy.These agents also require (conventional) anticancer drugs thatattack the tumor mass. Actually, their mechanism of action isprimarily to potentiate the activity of conventional agents.Clinical data on efficacy of VDAs is scarce. Animal studiessuggest that for this type of antiangiogenic compound toachieve striking and sustained antitumor effects, combinationtherapy strategies are also required.

Another major concern of these newly developed antiangio-genic medicines is the incidence of adverse effects in patients.The best-studied class of inhibitors of angiogenesis is that ofthe agents that target the VEGF pathway. For this class ofagents, it is increasingly recognized that diverse adverse effectsoccur that are related to disturbance of VEGF-dependentphysiological functions and homeostasis in the cardiovascularand renal systems, as well as wound healing and tissue repair.Although most adverse effects of VEGF inhibitors aremodest and manageable, some are associated with seriousand life-threatening consequences [96].

All vasoactive agents tested to this point appeared very toxicwhen administered systemically at effective doses. This makesthis strategy only practically achievable via isolated perfusion.This is a major drawback of this strategy, because it limits thenumber of applications. For VDAs, a variety of adverse effectshave been described. One of the biggest concerns is cardiotox-icity, seriously narrowing the therapeutic window [97].Another additional hazard could be that the VDA-inducedhypoxia results in induction of HIF-1a and other pro-angiogenic factors that ultimately stimulate blood vesselregrowth [85].

Taken together, it appears that the compounds that havebeen identified to specifically interfere with tumor vasculaturestill lack target specificity. The pronounced systemic toxicities

Figure 4. Schematic overview of the tumor blood vessel disruption strategy. By administration of vascular disrupting agents

(VDAs) tumor endothelial cells round up and consequently local coagulation occurs (pink) resulting in blood vessel congestion

and secondary tumor cell necrosis (dark blue). (Readers are referred to the full-colour version online at 10.1517/

13543784.2010.524204.)



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indicate that insulation of the molecule’s activity in the circu-lation and site-specific delivery to tumor vasculature couldsubstantially improve their therapeutic window.Apart from shortcomings on the side of antiangiogenic

compounds, factors on the side of the tumor could also playa prominent role, such as variability of tumor susceptibilityto antiangiogenic therapy [98]. Tumor vasculature does notnecessarily depend solely on endothelial cell proliferationand new blood-vessel sprouting. Additional mechanismshave been described that are able to provide tumors with oxy-gen and nutrients, [99] such as vasculogenic mimicry [6] bypass-ing tumor vasculature strategies. To address this, the role oftumor stromal cells and their activity in tumor biology is akey area of future research; this should result in biomarkersthat predict successful vascular therapeutic intervention.In addition, future improvements for tumor vasculature-

targeted therapy should lead to molecules that show a signifi-cant reduction in systemic toxicity, avoid development ofdrug resistance and at the same time are very effective andtumor (vasculature)-specific. Antiangiogenic therapy has,thus far, been unable to meet these requirements. VDAshave not yet been shown to be effective in humans

because of their toxicity as a result of an extremely small ther-apeutic window. For this type of drug, widening the gapbetween therapeutically active and toxic doses seems veryimportant. Hyperabnormalizing drugs on the other hand,have shown very promising clinical results, albeit only incombination with chemotherapeutics. However, clinicalsuccess was limited to situations where specific perfusion ofthe tumor tissue is possible, which significantly hamperswidespread applicability.

For all three classes of drugs, improved therapeutic out-come could be reached when tumor (vasculature) specificityis increased -- for example, by using efficient drug delivery sys-tems. Such drug delivery systems have already shown to beable to reduce systemic toxicity and thereby allowing higherlocal doses, but further improvements can be expected whenthese systems also target tumor vasculature.

Declaration of interest

All of the authors are employees of Utrecht University, andthe manuscript has been prepared in this function. Theauthors report no other conflicts of interest.

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AffiliationMarcel HAM Fens, Gert Storm &

Raymond M Schiffelers†

†Author for correspondence

Utrecht University,

Utrecht Institute for Pharmaceutical

Sciences (UIPS),

Department of Pharmaceutical Sciences,

Faculty of Science,

PO Box 80.082,

3508 TB Utrecht, The Netherlands

Tel: +31 30 2539392; Fax: +31 30 2517839;

E-mail: [email protected]



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Documents

Tumor vasculature as target for therapeutic intervention