tumor response to ionizing radiation combined with antiangiogenesis or … · review tumor response...

Review

Tumor Response to Ionizing Radiation Combined withAntiangiogenesis or Vascular Targeting Agents:Exploring Mechanisms of Interaction1

Phyllis Wachsberger,2 Randy Burd, andAdam P. DickerDivision of Experimental Radiation Oncology, Department ofRadiation Oncology, Kimmel Cancer Center, Jefferson MedicalCollege of Thomas Jefferson University, Philadelphia, Pennsylvania19107

AbstractRecent preclinical studies have suggested that radio-

therapy in combination with antiangiogenic/vasculature tar-geting agents enhances the therapeutic ratio of ionizingradiation alone. Because radiotherapy is one of the mostwidely used treatments for cancer, it is important to under-stand how best to use these two modalities to aid in thedesign of rational patient protocols. The mechanisms ofinteraction between antiangiogenic/vasculature targetingagents and ionizing radiation are complex and involve in-teractions between the tumor stroma and vasculature andthe tumor cells themselves. Vascular targeting agents areaimed specifically at the existing tumor vasculature. Anti-angiogenic agents target angiogenesis or the new growth oftumor vessels. These agents can decrease overall tumorresistance to radiation by affecting both tumor cells andtumor vasculature, thereby breaking the codependent cycleof tumor growth and angiogenesis. The hypoxic microenvi-ronment of the tumor also contributes to the mechanisms ofinteractions between antiangiogenic/vasculature targetingagents and ionizing radiation. Hypoxia stimulates up-regu-lation of angiogenic and tumor cell survival factors, givingrise to tumor proliferation, radioresistance, and angiogene-sis. Preclinical evidence suggests that antiangiogenic agentsreduce tumor hypoxia and provides a rationale for combin-

ing these agents with ionizing radiation. Optimal schedulingof combined treatment with these agents and ionizing radi-ation will ultimately depend on understanding how tumoroxygenation changes as tumors regress and regrow duringexposure to these agents. This review article explores thecomplex interactions between antiangiogenic/vasculaturetargeting agents and radiation and offers insight into themechanisms of interaction that may be responsible for im-proved tumor response to radiation.

IntroductionIonizing radiation is an effective modality for the treatment

of many tumors. It is a widely used treatment for cancer, withover half of all cancer patients receiving radiation therapyduring their course of treatment (1). Although widely used, aneed remains to improve the cure rate by radiation therapyalone. The most frequent treatment is to combine cytotoxicchemotherapeutic agents with radiation (2, 3). The cytotoxicityof chemotherapeutic agents, however, is not limited to tumorcells because treatment of tumors with these agents can result insignificant normal tissue toxicity.

There has been a recent rapid development of two newclasses of drugs termed antiangiogenesis and vascular targetingagents that target the relatively genetically stable tumor-associ-ated vasculature (endothelial cells) rather than the geneticallyunstable tumor cell (4). The importance of targeting tumorvasculature development and function first became apparent inthe 1970s through the seminal studies of Judah Folkman (5),who demonstrated that angiogenesis is important for the growthand survival of tumor cells. The relationship between angiogen-esis and tumor growth suggests that both tumor cells and theirsupporting endothelial cells are potential targets for cell killingand should be considered when planning cancer treatment (6).At least four theoretical advantages exist for considering tumorendothelial cells as targets for cancer therapy: (a) endothelialcells are more easily accessed by antiangiogenic/vascular tar-geting agents compared with drugs that act on tumor cellsdirectly and have to penetrate large bulky masses; (b) antian-giogenic/vascular targeting agents may avoid tumor drug resist-ance mechanisms because they are directly cytotoxic to endo-thelial cells; (c) angiogenesis occurs in very limitedcircumstances in adults (wound healing and ovulation), thusantiangiogenic therapies targeting specific receptors on prolif-erating tumor endothelium potentially are safe and should avoidnormal tissue toxicities; and (d) because each tumor capillarypotentially supplies hundreds of tumor cells, targeting of thetumor vasculature should lead to a potentiation of the antitu-morigenic effect (7).

Investigations of antiangiogenic/vascular targeting agentsthat have been conducted in preclinical and clinical trials indi-cate that tumor cures are limited when these agents are used as

Received 12/18/02; revised 2/5/03; accepted 2/11/03.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by National Cancer Institute Grant P30CA56036-03;a Bridge Grant (to A. P. D.) from the Office of the Dean, ThomasJefferson University; seed grants (to P. W. and A. P. D.) from theRadiation Therapy Oncology Group (RTOG); and a grant to the RTOG[RTOG from the Commonwealth of Pennsylvania, Tobacco SettlementAct (to R. B. and A. P. D.)]. A. P. D. has unrestricted grants fromMerck, Pharmacia, AstraZeneca Pharmaceuticals, and Entremed Phar-maceuticals. P. W. has an unrestricted grant from AstraZeneca Pharma-ceuticals. R. B. has unrestricted grants from Aventis and AstraZenecaPharmaceuticals.2 To whom requests for reprints should be addressed, at Thomas Jeffer-son University, Department of Radiation Oncology, 111 South 11thStreet, Philadelphia, PA 19107. Phone: (215) 955-5238; Fax: (215) 955-5825; E-mail: [email protected].

1957Vol. 9, 1957–1971, June 2003 Clinical Cancer Research

Research. on July 26, 2021. © 2003 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

the sole method of treatment (8). Some recent preclinical studiessuggest that the combination of radiotherapy and angiogenicblockade enhances the therapeutic ratio of ionizing radiation bytargeting both tumor cells and tumor vessels (9–12). At present,there is great interest in combining antiangiogenic/vascular tar-geting strategies with conventional cytotoxic therapies such asradiotherapy to improve therapeutic gain. The mechanisms bywhich tumor response to radiation is enhanced by these newagents, however, are not currently understood. In light of theknown fact that oxygen is a potent radiosensitizer, the combi-nation of ionizing radiation and antiangiogenic/vascular target-ing agents would appear to be a counterintuitive approach totumor cure because a reduction in tumor vasculature would beexpected to reduce tumor blood perfusion and reduce oxygenconcentration in the tumor. Some recent studies, however, in-dicate that oxygen levels may actually increase after treatmentwith antiangiogenic agents and ionizing radiation (13–16).

Because the mechanisms of interaction between ionizingradiation and antiangiogenic/vascular targeting agents are notfully understood, the ideal way to use this potentially powerfulcombination for tumor cure has yet to be determined. Thepurpose of this review is to examine possible mechanisms ofinteraction between antiangiogenic/vascular-induced cell deathand cell death resulting from ionizing radiation, in the expecta-tion of aiding in the design of rational protocols for treatmentwith antiangiogenic/vascular targeting agents and radiation.

Role of Angiogenesis in Primary Tumor Growthand Metastasis

A competent and expanding vascular supply is a necessarycomponent of the progressive growth of solid tumors becausecells in solid tumors, like normal tissue, must receive oxygenand other nutrients to survive and grow (17). The connectionbetween oxygen supply and tumor growth was first made in the1950s by radiobiologists who were aware of oxygen as a radio-sensitizer and the fact that hypoxic cells in tumors are resistantto radiation therapy (18). Histological analyses of human androdent tumors performed by Thomlinson and Gray (18) were thefirst studies to suggest that regions of viable cells exist close totumor blood vessels and that these walls or cords of viabletumor cells correspond in thickness to the distance that oxygencan diffuse (1–2 mm3). The “tumor cord” model implied thathypoxic cells exist in a state of oxygen and nutrient starvation atthe limits of the diffusion range of oxygen, and it was hypoth-esized that tumor cells could proliferate and grow only if theywere close to a supply of oxygen from tumor stroma. In the1970s, Folkman (5) corroborated the earlier findings of radio-biologists and proposed the importance of tumor vasculature asa viable target for anticancer therapy. He reported that a tumorwithout an adequate blood supply would grow only to a fewthousand cells in size or around 1–2 mm3, which is the distancethat nutrients can enter tumor cells by passive diffusion (5). Toincrease in size beyond this passive diffusion-limited state, thegrowing tumor mass must acquire new blood vessels. A switchto the angiogenic phenotype allows the tumor to expand rapidly.This so-called “angiogenic switch” (19) is regulated by envi-ronmental factors and by genetic alterations that act to either

up-regulate proangiogenic factors (i.e., VEGF3 and bFGF) andtransforming growth factors (TGF-� and TGF-�) and/or down-regulate inhibitors of angiogenesis [i.e., angiostatin, endostatin,thrombospondin, and IFN-� (20)]. Tumor angiogenesis is amultistep process of degradation of the extracellular matrix,migration and proliferation of endothelial cells from postcapil-lary venules, and, finally, tube formation (21). More than 20endogenous activators and inhibitors have been identified in thisprocess (7). The initial step in the process is the activation ofquiescent endothelial cells by binding of tumor-produced orstromal-produced growth factors to endothelial receptors. VEGFis the most potent and specific growth factor for endothelial cellactivation (22). Evidence for the importance of VEGF-inducedangiogenesis in tumor growth was demonstrated by use ofneutralizing antibodies or a dominant-negative soluble receptorto inhibit VEGF action and growth of primary and metastaticexperimental tumors (10, 23). VEGF also functions as a pow-erful antiapoptotic factor for endothelial cells in new bloodvessels (24). VEGF is secreted by almost all solid tumors (25).Leukemias are now also considered to be angiogenesis-depen-dent malignancies and have been shown to express VEGF(26–28). Factors that lead to up-regulation of VEGF expressionand secretion include external stresses such as ionizing radiationproduced in the application of radiation therapy and tumormicroenvironment factors such as hypoxia or a decrease in pH(29, 30). Hypoxia is the most potent stimulus for induction ofVEGF, which occurs by activation of Src kinase. Src kinaseactivation leads to an increase in HIF-1� and consequent up-regulation of VEGF expression (31, 32). Growth factors stim-ulating VEGF production include IGF-I and -II, epidermalgrowth factor, and PDGFs. Two specific signal pathways areknown to mediate the up-regulation of VEGF: (a) the phosphati-dylinositol 3�-kinase/Akt (protein kinase B) signal transductionpathway, which leads to stabilization of HIF-1� (33, 34); and(b) the MAPK pathway, in which activation of extracellularsignal-regulated kinase increases transcription of the VEGFgene (see Fig. 1 for summary of signaling mechanisms; Ref. 35).Mutant ras oncogenes and loss or mutation of the tumor sup-pressor gene, p53, can also result in increased VEGF expressionand angiogenesis (36, 37).

Although the growth of solid tumors depends on angiogen-esis for generation of a vascular network, the amount of newlyformed vessels in the tumoral stroma does not necessarily leadto increased blood flow (38). This inequality exists becausenewly formed microvessels in most solid tumors are abnormalwhen compared with the morphology of the host tissue vascu-lature (39). Endothelial cells lining tumor blood vessels differ inmany respects with regard to gene expression from those ofnormal vasculature (40). Because of lack of adequate vascular

3 The abbreviations used are: VEGF, vascular endothelial growth factor;VEGFR, VEGF receptor; bFGF, basic fibroblast growth factor, TGF,transforming growth factor; IGF, insulin-like growth factor; PDGF,platelet-derived growth factor; MAPK, mitogen-activated protein ki-nase; IFP, interstitial fluid pressure; HIF-1�, hypoxia-inducible factor1�; PG, prostaglandin; EBRT, external beam radiation therapy; EGFR,epidermal growth factor receptor; COX, cyclooxygenase; rhAngiostatin,recombinant human angiostatin.

1958 Antivascular Therapy and Ionizing Radiation



maturation (41), vessels are often dilated and tortuous (42), havean incomplete or missing endothelial lining, and have inter-rupted basement membranes that result in an increased vascularpermeability with extravasation of RBCs and blood plasmaexpanding the interstitial fluid space (39). Moreover, blood flowis often erratic, with stasis or even reversal of blood flow withinindividual vessels (43). Extravasation of macromolecules anddevelopment of high IFP often results in vascular collapse (42,44, 45). Consequently, oxygen availability to the tumor cellsshows great variability. Many human tumors exhibit hypoxicregions that are heterogeneously distributed within the tumormass (46) Low perfusion rates and hypoxia may then coexistwith high nonfunctional vascular density, creating hypoxic re-gions (41). In these regions of hypoxia, endothelial cells mayup-regulate survival factors to maintain their integrity and pre-vent apoptosis (47). Thus, so-called “angiogenic hot spots” orlocalized regions of intense angiogenesis may be created andmay be associated with failure of radiotherapy (48). However, itis not clearly known whether hypoxia in the tumor producesangiogenic hot spots or whether these regions of intense angio-genesis are a property of the genomic instability of the tumorcells themselves, resulting in up-regulation of proangiogenicmolecules.

Ionizing Radiation and HypoxiaRadiation-induced cell death is usually attributed to DNA

damage to tumor cells, thus triggering cell death by apoptosisand/or necrosis. Oxygen is known to be a potent radiosensitizerand, through interaction with the radicals formed by radiation, isessential for the induction of radiation-induced DNA damage.Cells irradiated in the presence of air are about three times moresensitive than cells irradiated under conditions of severe hy-

poxia (49). Hypoxia, then, contributes to radiation resistance. Asalready discussed in an earlier section, the “tumor cord” modelof early radiobiologists implied that hypoxic cells exist in a stateof oxygen and nutrient starvation at the limits of the diffusionrange of oxygen, and it was hypothesized that tumor cells couldproliferate and grow only if they were close to a supply ofoxygen from tumor stroma [i.e., 1–2 mm3 (18, 50)]. Today,however, it is known that the width of viable regions of tumorcells can vary quite widely, depending on tumor microenviron-mental factors such as pO2 and respiratory metabolism of viablecells (51). Experimental and clinical studies have provided bothdirect and indirect evidence for the presence of hypoxic cells intumors (52, 53). Regions of viable cells exist in proximity totumor blood vessels, whereas regions of necrosis are observed atincreased distances from blood vessels. Viable hypoxic cells, atvery low oxygen tensions, are believed to exist at the edges ofnecrotic regions. Hypoxia resulting from diffusion-limited pro-cesses is known as chronic hypoxia. It is also known that inaddition to these radiobiologically hypoxic cells, there is adistribution of tumor cells at intermediate oxygen levels that caninfluence response to low-dose fractionated radiotherapy (54,55). Hypoxia can also be the result of intermittent blood flowarising from the abnormal tumor microvasculature (56–58).This acute hypoxia is distinct from that of chronic hypoxiabecause the affected cells would be expected to have intermit-tently perfused areas that could give rise to tumor regrowth (55).The effect of hypoxia on tumor control is that the existence ofviable cells in a microenvironment at low oxygen tensions cangive rise to populations of tumor cells that are not only radiore-sistant but also highly angiogenic and are potentially resistant toantiangiogenic therapy as well (59, 60).

Fig. 1 Signaling mechanisms involved in VEGF-induced tumor angiogenesis. Endothelial cell sur-vival and neovascular processes are induced byVEGF/VEGFR-2 signaling via the phosphatidyli-nositol 3�-kinase/Akt signaling axis. VEGF is up-regulated by external environmental stress (i.e.,ionizing radiation) and internal tumor microenvi-ronmental stress (i.e., hypoxia).

1959Clinical Cancer Research



Radiation and Angiogenesis: A Vicious CycleTumor vasculature is abnormal, and the endothelial cells

lining tumor blood vessels have different phenotypic propertiesfrom those of normal vasculature (41). Consequently, increasedtumor angiogenesis, as indicated by increased microvessel den-sity or by increased VEGF expression, does not necessarilycorrelate with increased blood flow and oxygen availability.This situation, together with the existence of heterogeneoushypoxic regions within tumors, makes it difficult to predict howtumor angiogenesis will affect response to radiation therapy in aparticular tumor. As already noted, hypoxia leads to radiationresistance because of lack of oxygen to facilitate DNA damageby radiation-induced free radicals. Hypoxic conditions also cre-ate a microenvironment in which tumor cells become less an-giogenesis dependent, more apoptosis resistant, more capable ofexisting under hypoxic conditions, and more malignant becauseof the development of genomic instability and mutant genotypesimpacting on apoptosis/survival signaling pathways (61). Hy-poxic tumor cells are particularly known to up-regulate HIF-1�,which increases the expression of VEGF (30, 62). All thesefactors suggest diminished sensitivity to antiangiogenic therapyas well as radiation therapy.

Strong evidence exists that cytotoxic therapy alone, such asradiation, can result in intensification of angiogenic processes(48). Direct up-regulation of VEGF after irradiation of variouscancer cell lines has been reported (10). This response is part ofthe overall cellular response to stress and is associated with theinduction of a variety of transcription factors that can activatetranscription of cytokine, growth factor, and cell cycle-relatedgenes. The products of these genes regulate intracellular signal-ing pathways through tyrosine kinases, MAPKs, stress-activatedprotein kinases, and ras-associated kinases. These multiple path-ways affect tumor cell survival or alter tumor cell proliferation.With regard to angiogenesis, radiation exposure can result inactivation of the EGFR, which, in turn, can activate the MAPKpathway (63). MAPK signaling is linked to increased expressionof growth factors, such as TGF-� and VEGF. It is possible thatradiation therapy itself contributes to radioresistance by up-regulating and intensifying angiogenic pathways. The increasedtumor cell proliferation that is often seen after radiation may bethe result of up-regulated angiogenic pathways (48) as well as

increased proliferation in the tumor stem cell compartment (64).Although many tumors reoxygenate after radiation, in tumorsthat are unresponsive to radiation therapy, up-regulated angio-genesis after radiation may lead to factors contributing to radi-ation resistance such as increased vascular permeability, in-creased IFP, decreased tumor perfusion, increased oxygenconsumption, increased hypoxia, and up-regulated survivalpathways (14, 45, 65). These factors all contribute to makingradiation therapy less effective in some tumors (62, 66, 67).

Radiation and Antiangiogenic InteractionsThe existence of tumor microenvironmental factors, such

as hypoxia, that can promote the up-regulation of angiogenicand survival pathways and hence resistance to radiation therapyhas prompted studies combining antiangiogenic agents withradiation in an effort to overcome this resistance. Teicher et al.(15) were the first to show an increased response to single-doseradiotherapy with antiangiogenic agents. A number of preclin-ical studies have since indicated that antiangiogenic agents canenhance the tumor response to radiation [see Tables 1 and 2(9–11, 13, 14, 68–75)]. However, it is difficult to compare andinterpret relative efficacies of different antiangiogenic agentswith radiation because of the variability in experimental condi-tions reported in the literature such as tumor models, tumor hoststrain, starting tumor size, final tumor volume measured, anddosing and scheduling (see Table 2). As expected, differenttumor models can vary a great deal in their responses to anti-angiogenesis treatment. The differences in tumor response toantiangiogenic agents may occur because of differences in an-giogenic growth patterns arising from differences in the relativelevels of angiogenic growth factors such as VEGF that stimulateangiogenesis. For example, the U87 human glioblastoma xe-nograft model that appears in many studies is known to be ahighly vascularized aggressive tumor and exhibits high levels ofVEGF, which would theoretically make it more resistant totherapy compared with tumors with lower VEGF expression,and thus it would require higher concentrations of antiangio-genic agents for efficacy (76). Differences in tumor response toangiogenic agents may also result from the presence of differentpatterns of multiple angiogenic factors among different tumorsinfluencing tumor growth and angiogenesis. This would explain

Table 1 Antiangiogenic and vascular targeting agents: target and activity

Agent Target Activity

TNP-40 Protein myristoylation and dependent signalingpathways in endothelial cells

Up-regulation of apoptotic pathways in endothelial cells

Angiostatin Endothelial cell membrane receptor (possiblymembrane proton pump)

Inhibition of pH regulation and up-regulation of apoptoticpathways in endothelial cells

VEGF/VEGFR inhibitors Neovascularization processes; endothelial cellsurvival pathways; tumor cell survivalpathways indirectly and possibly directlythrough autocrine signaling loops

Inhibition of endothelial cell migration, tube formation,etc.; up-regulation of apoptosis in endothelial cellsthrough down-regulation of key signaling molecules,i.e. survivin, BCL-2

COX-2 inhibitors Prostaglandin synthesis Inhibition of VEGF synthesis in tumor cells; up-regulation of apoptotic signaling pathways in tumorcells and endothelial cells

EGFR inhibitors VEGF synthesis, apoptotic signaling in tumorcells and endothelial cells

Inhibition of tumor angiogenesis; up-regulation ofapoptosis in both tumor cells and endothelial cells

Vascular targeting agents(i.e. ZD6126)

Microtubular cytoskeleton in endothelial cells Vasculature shutdown leading to tumor necrosis




Table 2 Antiangiogenic agents in combination with radiation therapy (RT)

Antiangiogenic agent Tumor model Tumor host

Tumordiameter at

start oftreatment

(mm)Total radiation

doseEffect of RT � drug

(end point: tumor growth delay)Reference

no.

Fumagillin analogueTNP-470 Mouse mammary

carcinomaC3H/He mice 4 10 Gy/5 fractions Additive effect with 2–4 doses of

100 mg/kg drug after RT75

TNP-40 Human glioblastomamultiforme (U87)

Athymic nudemice

7 10 Gy/singlefraction

Greater than additive effect withdaily dose of 6.7 mg/kgbefore RT

73

AngiostatinRecombinant Adk3

(expressingangiostatin)

Rat C6 glioma Athymic nudemice

6–7 7.5 Gy/3 fractions Greater than additive with vectorgiven after RT

69

Angiostatin Murine Lewis lungcarcinoma (LLC)

C57BL/6 mice 12 40 Gy/2 fractions Significant tumor regression with25 mg/kg drug given

12

Human malignantglioma (D54)

Athymic nudemice

9 30 Gy/5 fractions Significant tumor regression with25 mg/kg drug given

Human squamous cellcarcinoma (SQ-20B)

Athymic nudemice

11 50 Gy/10 fractions Significant tumor regression with25 mg/kg drug given

Human prostateadenocarcinoma(PC3)

Athymic nudemice

11–12 40 Gy/10 fractions Significant tumor regression with25 mg/kg drug given

VEGFR-2 blockadeDC 101 Human small cell lung

carcinoma (54A)Athymic nude

mice8 5–24 Gy/5

fractionsAdditive effect in both models

with 6 � 20–40 mg/kg drug72

Human glioblastomamultiforme (U87)

Athymic nudemice

8 5–24 Gy/5fractions

Given during RT

SU5416 Murine glioblastoma(GL261)

C57BL6 mice 10–11,11–12

24 Gy/8 fractions Greater than additive effect with0.75 mg/kg drug given duringRT

68

SU6668 Murine mammarycarcinoma (SCK)

A/J mice 6–7 15 Gy/singlefraction

Greater than additive effect with100 mg/kg injected daily for 2days before and after RT

13

PTK787/ZK222548 Human coloncarcinoma

Athymic nudemice

7 12 Gy/4 fractions Greater than additive with 4 �100 mg/kg drug injected on 4consecutive days during RT

70

Anti-VEGF antibodiesAnti-vegf 164, 165 Murine Lewis lung

carcinoma (LLC)C57BL/6 mice 9–10 40 Gy/2 fractions Greater than additive with 10 �g

of drug given before eachfraction for all models

10

Human esophagealadenocarcinoma(Seg-1)

Athymic nudemice

9–10 20 Gy/4 fractions Greater than additive with 10 �gof drug given before each

Human squamous cellcarcinoma (Sq 20B)

Athymic nudemice


Human glioblastomamultiforme (U87)

Athymic nudemice


VEGF mAb A.4.6.1 Human glioblastomamultiforme (U87)

Athymic nudemice

6 20 Gy, 30 Gy/single fraction

Greater than additive with 100 �gof drug given 6� on alternatedays prior to RT

14

Colon adenocarcinoma Athymic nudemice

6 20 Gy/30Gy/singlefraction

Additive with 100 �g of druggiven 6� on alternate daysprior to RT

EGFR inhibitorsZD1839 (Iressa) Human squamous cell

carcinomasSCC-1 Athymic nude

mice�5 21 Gy/7 fractions Synergistic effect with 0.5 mg

(orally) in 12 doses during RT71

SCC-6 Athymic nudemice

�5 21 Gy/7 fractions Additive effect with 0.5 mg drug(orally) in 12 doses during RT

C225 antibody Human head and neckcarcinoma (A431)

Athymic nudemice

8 18 Gy/singlefraction

Greater than additive effect with1 mg of drug in 3 dosesbefore and after RT

84

COX-2 inhibitorsSC�-236 Mouse sarcoma

(NFSA)C3Hf/Kam mice 6 25–80 Gy/single

fractionGreater than additive effect with 6

mg/kg drug before and duringand after radiation for 10consecutive days

74




the lack of efficacy of the single agent in antiangiogenesis trials.Differences in tumor response to antiangiogenic agents may alsobe influenced by the organ microenvironment in which thetumor is growing during the study. Studies of ectopic versusorthotopic tumor implantations have revealed differences inresponse to antiangiogenic treatments (73, 77).

The size of transplantable tumors at the start of an exper-iment is also an important factor to consider when examiningtumor response to radiation in murine systems. Tumor growthdelay studies reported in the literature vary greatly with regardto tumor size at start of treatment as well as tumor volume thatis permitted to be reached during treatment until a statisticallymeaningful effect is seen. Tumor size can affect oxygen tension,nutrient supply, and pH, which are all factors in determiningradiation response. Radioresistance is directly proportional totumor size (Fig. 1). As tumor size increases, oxygen tension andpH decrease because of a greater demand for oxygen andnutrients. Many murine tumors, even tumors as small as 6 � 6mm, have low oxygen tension (1–10 mm Hg) and are consideredhypoxic. As tumors become increasingly large, oxygen is un-available, and glycolysis dominates, leading to acidosis. Even-tually, the lack of oxygen and nutrients and chronic exposure tolow pH lead to tumor necrosis. When a tumor is allowed toreach a size beyond 2000 mm3 in volume, a volume largeenough to create hypoxia, necrosis, and consequent radioresis-tance, the comparison of antiangiogenic treatment plus radiationwith radiation alone under these conditions may result in anunderestimation of the tumor growth delay. In some tumormodels, for example, radiation resistance emerges at �250mm3. In tumors larger than 500 mm3, tumor oxygen tension isless than 2 mm Hg, and radiation resistance is significant(Fig. 2).

Another factor that may influence tumor growth delay isthe tumor bed effect, which can prolong tumor growth delayindirectly through the late effects of radiation on tumor stroma[vasculature/connective tissue (78)]. Other factors such as thetype of anesthetic or restraint used to irradiate the animal, aswell as animal and tumor temperature, should also be consideredbecause they all could affect perfusion. Special attention shouldbe paid to generalizations about relative efficacies of treatmentsbased on individual in vivo tumor growth delay studies. Withthese caveats in mind, some of the antiangiogenic strategies thathave been used in combination with radiation are reviewedbelow.

The list of antiangiogenic agents used in combination withradiation include angiostatin (9, 12), anti-VEGF or VEGFRantibodies and tyrosine kinase inhibitors (10, 13, 14, 68, 72),TNP-470 (15, 73, 79), COX-2 inhibitors (80–83), and anti-EGFR inhibitors (71, 84). The mechanisms of enhancement arebelieved to involve both direct and indirect targeting of tumorcells and/or endothelial cells (see Table 1). The antiangiogenicand antitumor effects have been reported to be additive as wellas synergistic. Possible mechanisms of enhanced tumor re-sponse include (a) indirect inhibition of vessel formation byinhibition of vascular growth factors and endothelial receptors(14), (b) direct radiosensitization of endothelial cells [endothe-lial cell apoptosis (72, 85, 86], (c) direct radiosensitization oftumor cells (i.e., tumor cell apoptosis) (87), and (d) decrease innumber of hypoxic cells [improved oxygenation (14, 15, 88)].

Radiation and VEGF/VEGFR Signaling PathwayInhibitors

Some studies targeting the VEGF/VEGFR signaling path-way in conjunction with radiation have been reported. Gorski etal. (10) found that antibodies to VEGF, when combined withionizing radiation in vitro, resulted in increased endothelial celldeath without affecting tumor cells. Greater than additive anti-tumor effects were noted in a variety of tumor model systems invivo. It was then proposed that enhancement of radiation re-sponse occurs through inhibition of VEGF-induced protectionagainst radiation damage to endothelial cells. This suggestionwas supported by a recent study showing that SU5416, aninhibitor of VEGFR kinase, increased the radiation-inducedapoptosis in endothelial cells (68). It has also been reported thatSU668, which inhibits VEGF, bFGF, and PDGF receptor ki-nases, which are angiogenic growth factors, caused apoptosis inendothelial cells in vivo (89). It was then proposed that VEGF,fibroblast growth factor, and PDGF may be required not only forneoangiogenesis but also for survival of existing endothelialcells and for maintenance of existing tumor vasculature.

In addition to growth factors and their receptors, otherfactors associated with endothelial cell survival exist such asmaintenance of association with perivascular cells (pericytesand vascular smooth muscle cells), integrin interaction with theextracellular matrix, and antiapoptotic proteins such as survivinand BCL2 (7, 47, 60). Endothelial cells, like other cells, alsodepend on intracellular pH-regulatory mechanisms to survive. If

Fig. 2 Model of tumor oxygen tension, pH, and radioresistance as afunction of size. As a tumor grows larger, the demand for oxygenincreases. However, the vascular network is unable to supply the tumorwith enough oxygen to maintain an oxygenated state, and the tumorbecomes hypoxic. To produce sufficient energy for cell survival, the rateof glycolysis and nutrient consumption is increased, and the pH be-comes acidic through lactic acidosis. The lack of oxygen and nutrientsand state of chronic low pH eventually lead to necrosis, and pH mayincrease. The increase in tumor burden and hypoxic low pH environ-ment result in tumor radioresistance. The theoretical curves are based onpO2 and pH data obtained in our laboratory using U87 glioma and DB-1melanoma xenografts.




they fail, metabolism ceases, and apoptosis ensues (90). It hasbeen proposed that angiostatin may compromise endothelialintracellular pH and hence survival by inhibiting a major protonpump in the cell membrane of endothelial cells (91).

Because most endothelial survival factors interact withVEGF function in complex pathways, VEGF seems to be thepredominant stabilizing factor (47). More recently, Guptaet al. (86) demonstrated that genetically engineered VEGF-producing xenografts were more resistant to the cytotoxiceffects of ionizing radiation than xenografts that do notproduce VEGF (86). Because the VEGF-null and the VEGF-producing xenograft cell lines exhibited identical radiosensi-tivities in vitro, their observations support the hypothesis thatradiation targets the tumor endothelial cells as well as thetumor cells under conditions in which protective survivalfactors are diminished. These observations further confirmearlier studies that suggest that VEGF mediates tumor ra-dioresistance by directly affecting the radiosensitivity of theendothelial cells.

Effects of COX-2 Inhibitors on Angiogenesis andRadiation Response of Tumors

Although the exact mechanisms for improved responsein tumors are not known, the rationale for using COX-2inhibitors with ionizing radiation is based on the angiogenictumor environment and the biochemical responses of tumorsto radiation (92). The COX-2 enzyme is induced in cells afterinjury or stress, such as radiation, and catalyzes the synthesisof PGs from arachidonic acid. PGs serve to protect cells andtissue from radiation damage (92) and promote angiogenesisby the up-regulation of factors such as VEGF (93). Therefore,pretreatment with COX-2 inhibitors before exposure to stressmay inhibit the inflammation response induced by PGs. Fur-thermore, COX-2 inhibitors at clinically relevant doses, suchas those achieved during the treatment of arthritis, may actdirectly on endothelial cells to inhibit proliferation and toincrease their intrinsic sensitivity to radiation (80). The abil-ity of COX-2 inhibitors to enhance radiation therapy has beendemonstrated in murine and human tumor models (83, 74,94 –96). For example, it was demonstrated that the radiationresponse of a murine sarcoma was increased if tumor-bearingmice were pretreated with a COX-2 inhibitor before radiationtherapy (74). Tumor growth delay by drug plus radiation wassignificantly increased over drug or radiation alone, and theTCD 50 (dose of radiation required for 50% tumor cure) wasreduced by almost half in the drug plus radiation group. Toelucidate the mechanism of sensitization, an intradermal in-jection of tumor cells was used to visualize and count newlyforming vessels. Neovascularization was shown to precedetumor growth, and pretreatment with a COX-2 inhibitor ef-fectively reduced neovascularization, resulting in reducedtumor growth.

There is also direct evidence that down-regulation of PGsby COX-2 inhibitors reduces angiogenesis and increases radio-sensitivity. In another study with a murine fibrosarcoma ex-pressing high levels of PGE2, radiosensitization was again ob-served by pretreatment of mice with a COX-2 inhibitor (95).COX-2 expression was not affected by treatment, and no in-

crease in the amount of tumor cell apoptosis was observed, butPGE2 was significantly decreased. PGE2 is a vasoactive com-pound that can affect tumor perfusion as well as stimulate tumorgrowth and induce angiogenesis. The effect of the COX-2inhibitor on PGE2-mediated angiogenesis was determined to bea key mechanism in the increased radiation response.

The concept that COX-2 inhibitors can reduce the radiationdose required to achieve tumor control caused great excitementamong radiation oncologists and stimulated the writing of manyclinical protocols. However, preclinical studies on human tumorxenografts showed that, although COX-2 inhibitors increasedthe effectiveness of radiation, inhibition of angiogenesis was nota factor in the improved response. Authors concluded that inhuman tumors, the intrinsic sensitivity to radiation was in-creased (96), possibly via a cell cycle mechanism (97). Anincrease in the incidence of apoptosis was also a factor (83).Experimental evidence in murine tumor systems indicates thatinhibition of angiogenesis by COX-2 inhibitors is a factor in theincreased radiation response. However, in human tumor models,other factors may play a predominant role in the increasedradiation sensitivity.

The existing approval of these agents for noncancerousconditions has allowed clinicians to use COX-2 inhibitors in alarge number of clinical trials in combination with radiation andchemoradiation. To date, these trials are still too immature toyield meaningful outcome data.

Effect of Radiation and Antiangiogenic Agents onTumor Oxygenation

Enhancement of tumor response to radiation plus antian-giogenic agents has also been explained by an increase in tumoroxygenation after treatment. Tumor oxygenation is a function ofperfusion and consumption of oxygen, including consumptionof oxygen by both tumor and endothelial cells. Antiangiogenicagents can theoretically improve tumor oxygenation by reducingthe number of oxygen-consuming tumor cells and endothelialcells, which, in turn, will reduce the overall demand for a givenamount of oxygen. Antiangiogenic agents can also theoreticallyincrease perfusion by reducing the number of immature, ineffi-cient vessels (41, 98–100) and by decreasing IFP (38, 101).However, IFP is related to many factors, including vascularleaking and vascular resistance. High vascular resistance mayreduce tumor IFP and, at the same time, reduce perfusion. Thus,a reduction of tumor IFP may not necessarily improve perfusionor oxygenation (102).

The belief that there is an improved tumor response toangiogenesis inhibitors and radiation is derived from animaltumor models. Fig. 3 provides a hypothetical model of howoxygen tension could be increased by treatment with antiangio-genic drugs and radiation.

Tumor oxygen tension is a function of oxygen consump-tion (by both tumor cells and endothelial cells) and tumorperfusion. In nonmalignant tissue, the blood supply is orga-nized and efficient. Perfusion and oxygen consumption arebalanced so that the tissue is in an oxygenated steady state. Incontrast, malignant tissue contains dividing tumor cells thatoutgrow their blood supply (Fig. 3a). The overgrowth causes




Fig. 3 Hypothetical model for the rationale of combining antiangiogenic agents with radiotherapy. Nonmalignant tissue has a mature organized bloodsupply. Perfusion and oxygen consumption are balanced so that the tissue is in an oxygenated steady state. In normal tissue, the IFP is low. a, inmalignant tissue, an increase in tumor cell number increases oxygen consumption and demand. As oxygen tension falls, angiogenesis is induced bycytokines to compensate for decreased oxygen tension. However, the newly formed vessels are torturous and inefficient, which may furthercompromise perfusion. Production of VEGF increases tumor IFP. b, after irradiation, oxygenated cells are destroyed, leaving the radioresistanthypoxic cells. The hypoxic cells can reoxygenate during radiation therapy and improve radiation response and tumor control. c, in cases where tumorcontrol is not achieved, it has been proposed that in response to irradiation, protective cytokines are expressed by the tumor that reinitiate angiogenesis.Angiogenesis and vascular leakage induced by VEGF will compromise perfusion. d, in theory, pretreatment with antiangiogenic agents reduces thenumber of inefficient vessels and increases perfusion. More oxygenated tumor cells are killed by irradiation, which reduces oxygen consumption, andinduction of angiogenesis. No regrowth is observed, and the oxygenated steady state is maintained. The tumor response to fractionated radiotherapyis then greatly improved.




oxygen tension to fall, and angiogenesis is induced by cyto-kines (103). The newly formed vessels are torturous andinefficient, further compromising perfusion and resulting inhypoxia. Expression of VEGF increases endothelial cell per-meability, which can also result in high tumor IFP (21, 104),which has been correlated with tumor hypoxia (101).

Although many biological factors can contribute to tumorradiation response, tumor oxygen tension is one of the mostimportant parameters, and it will be considered here. As shownin Fig. 3b, oxygenated cells are more sensitive to radiation andare destroyed by irradiation, leaving the radioresistant hypoxiccells. During a course of radiotherapy, the hypoxic cells in thetumor may reoxygenate (105), and continued fractions of radi-ation can lead to tumor control. Studies in patient tumors andtumor models indicate that reoxygenation occurs because oftumor shrinkage (106, 107), decreased oxygen consumption(108), and increased perfusion (105, 108, 109).

In cases where radiation therapy is ineffective, it has beenproposed that angiogenesis induced by radiation may compro-mise tumor response (Fig. 3c). After a radiation insult, thesurviving hypoxic cells can produce protective cytokines suchas VEGF (10). Angiogenesis can reinitiate, and the tumor cellscan reoxygenate and proliferate (110). This scenario could the-oretically be avoided if treatment with antiangiogenic agentswas used before radiotherapy (Fig. 3d). A reduction in thenumber of inefficient vessels and in the number of oxygen-consuming tumor cells and endothelial cells by pretreatmentwith antiangiogenic agents would increase perfusion and reduceconsumption, giving rise to more oxygenated tumor cells thatare sensitive to radiation. This would prevent tumor regrowthand maintain the oxygenated state. This scenario would be idealfor fractionated radiotherapy because it prevents a reversionback to the hypoxic state during the course of therapy.

In a number of studies, hypoxia was reduced by angiogen-esis inhibitors (13–16). In particular, Lee et al. (14) noted thatvessel regression induced by an anti-VEGF antibody was asso-ciated with a decrease in hypoxia in a U87 xenograft. Theysuggested that the observed increase in oxygenation, despite adecrease in vascular density, was related to vascular reorgani-zation after inhibition of VEGF. One possible mechanism bywhich VEGF affects vascular organization is its effect on vas-cular permeability and IFP within a tumor. Its inhibition de-creases vascular permeability (111) and IFP, thereby reducingcompression of vessels and allowing for normalization of vas-cular architecture and perfusion and consequent normalizationof pO2. Improvement in tumor oxygenation may also be ex-plained by decreased oxygen consumption resulting from in-creased killing of tumor and endothelial cells (14). Lee et al.(14) concluded that the oxygenation status of a tumor at time ofradiation is probably not the major cause of the increasedradioresponse to anti-VEGF therapy because they observed sim-ilar enhanced responses under both normoxic and hypoxic con-ditions. They concluded that the critical factor in producingenhanced tumor response to radiation is more likely related tothe reduced vessel density induced by anti-VEGF treatmentrather than the state of oxygenation of the tumor at the time ofradiation.

Effect of Radiation and Antiangiogenic Agents onTumor Cell Apoptosis

Enhancement of tumor response to radiation plus antian-giogenesis agents has also been explained by increases in tumorcell apoptosis relative to tumor cell proliferation (87, 112–114).It has been observed that, on the tumor cell level, the prolifer-ation index is unaffected, but the apoptotic index is increasedafter treatment with antiangiogenic agents such as angiostatinand TNP-470 (87, 114). Tumor cell apoptosis may occur di-rectly by radiation-induced DNA damage to tumor cells orindirectly by antiangiogenic and radiation-induced damage toendothelial cells, upon which tumor cells ultimately depend.The molecular mechanism by which damage to endothelial cellsresults in increased rates of tumor cell apoptosis is not clear. Ithas been speculated that angiogenesis inhibition of tumor cell-expressed autocrine growth factors and receptors (115) or lossof endothelial-derived paracrine factors needed for tumorgrowth contributes to tumor cell cytotoxicity via apoptoticmechanisms (87, 113).

Radiation and Vascular Targeting AgentsVascular targeting agents differ from antiangiogenic agents

in their mechanism of action. Vascular targeting agents takeadvantage of the unique properties of tumor endothelium toselectively target the tumor vasculature (116). They are directedat the immature, rapidly proliferating tumor endothelial cells inexisting tumor vasculature rather than the multistep processesinvolved in neovascularization. Vascular targeting strategiesinclude biological agents, such as targeted gene therapy andantibodies to neovascular antigens, and drug-based agents (117).The drug-based strategies have progressed the furthest in devel-opment (117). These agents include drugs related to flavoneacetic acid (118–121) and tubulin-binding compounds (122–127). Flavone acetic acid and its derivatives are believed towork by inducing tumor necrosis factor � (128–130), a cytokineknown to induce hemorrhagic necrosis in experimental tumormodels (131). The tubulin-binding compounds are believed towork by affecting the immature endothelial microtubular cy-toskeleton found in tumors, leading to abnormalities in endo-thelial cell shape, thrombus formation, rapid vasculature shut-down, reduction in tumor blood flow, and secondary inductionof tumor necrosis (132). However, after treatment with vasculartargeting agents, viable tumor cells have been found at the tumorperiphery (122, 123, 125, 132). It has been suggested thatincreased blood flow in the adjacent normal tissue, together withprobable rapid up-regulation of angiogenic factors such asVEGF, directly facilitates rapid growth and expansion of theremaining rim of viable tissue because of the extensive ischemicinsult (125, 133). Because the cells surviving the vascular tar-geting treatment are believed to be well oxygenated and conse-quently present an excellent target for radiation therapy, vascu-lar targeting has been studied in combination with ionizingradiation. A number of studies have reported an increased an-titumor effect with this combined complementary approach.Combretastatin A-4-disodium phosphate and ZD6126, potentand selective tubulin-binding agents against tumor vasculature,enhanced the radiation response of murine mammary carcinomaand sarcoma (123–125). Scheduling was found to be an impor-




tant factor in the efficacy of ZD6126 when combined withradiation (125). ZD6126 increased tumor cell killing in KHTmurine sarcoma when administered 24 h before radiation or �1h after radiation, but it was found not to be as effective ifadministered 1 h before radiation. It was suggested that bloodflow needs to be reestablished in the remaining viable tissue toobtain maximum radiosensitization of the tumor.

Overall Tissue Response to RadiotherapyThe respective roles of endothelial cells and tumor cells in

the overall tissue response to radiotherapy still remain contro-versial. In mouse models, Paris et al. (134) showed that a singlelarge dose of radiation administered to mouse gastrointestinalmucosa preferentially damaged endothelial cells of the gut mi-crovasculature that lie in close apposition to the epithelial cellslining the gut. Previous work showed that irradiation of micro-vascular endothelial cells resulted in the generation of ceramide,a compound that facilitates endothelial cell apoptosis (135). Inmore recent experiments, Paris et al. (134) showed that systemicadministration of bFGF, an endothelial cell survival factor, ordeletion of the acid sphingomyelinase gene (the gene uponwhich ceramide generation depends) can override the apoptoticsignal from ceramide, thus protecting gut endothelial cells andepithelial stem cells from the effects of whole body irradiation.They concluded that the endothelial cells may represent theprincipal targets for radiation and that the death of epithelialstem cells may be a secondary event dependent on endothelialcell death. In an analogous manner, it has been speculated thattumor cell death in response to radiotherapy may represent asecondary event after death of endothelial cells, on which tumorcells ultimately depend (85, 135). However, it should be pointedout that the concept of what is the radiation target in normaltissue may well depend on the radiation dose per fraction. In thisregard, it is well established that the effects of a single largedose, i.e., 15 Gy, are not equivalent in terms of normal tissueside effects to a more clinically relevant low-dose fractionatedradiotherapy regimen of 2 Gy times 8 (136).

Clinical Trials with Antiangiogenic Agents andRadiation Therapy

To date, only one clinical trial has been completed evalu-ating an antiangiogenic drug (angiostatin) with radiation ther-apy. rhAngiostatin is a protein consisting of the first 3 kringles(amino acids 97–357) of human proplasminogen with a singleamino acid substitution (Asn308 to Glu308) to prevent N-glyco-sylation. Previous studies using angiostatin in combination withionizing radiation have indicated that the antitumor activity ofhuman angiostatin is potentiated in a number of preclinicaltumor models, resulting in a significant reduction of tumorvolume without increase in toxicity.

A single-center, open-label, dose-escalation, Phase I clin-ical study at Thomas Jefferson University evaluated the safetyand pharmacodynamics of three dose levels (15, 60, and 240mg/m2/day) of rhAngiostatin i.v. protein in combination withEBRT for the treatment of cancer patients with solid tumors(137). Patients received rhAngiostatin i.v. 5 times/week, 30 minbefore EBRT (head and neck, thoracic, or pelvic regions), for aminimum of 25 EBRT fractions. This study had a unique design

in that it was carried out concurrently with a Phase I drug doseclinical trial at Thomas Jefferson University (137). As safedoses were achieved in the drug alone study, these were used forthe radiation study. Twenty-three patients were enrolled andevaluated for safety. Three patients were not evaluable, andthree who did not complete the minimum EBRT were excludedfrom response analysis. The 17 remaining patients with evalu-able tumors had advanced head and neck, thoracic, or pelviccancers. No added toxicity was observed in normal tissue con-tained within the radiation portal. Mild rash was noted in threepatients. No clinical thrombotic or bleeding events occurred inany patient. Tumor responses were demonstrated in 90% ofpatients who entered the trial with measurable disease in theradiation field. The conclusions from this Phase I trial are thatconcomitant administration of daily 10-min infusions ofrhAngiostatin and EBRT is safe and does not increase radiation-induced toxicity. Local durable tumor responses (National Can-cer Institute Response Evaluation Criteria in Solid Tumors)have been observed in this Phase I study, although this would beexpected with the use of radiation alone (137).

Summary and ConclusionsDespite the expectation that agents causing tumor vessel

regression should increase hypoxia and thus limit the effective-ness of radiation, there is accumulating experimental evidenceto refute this expectation. Preclinical evidence suggests thatantiangiogenic agents reduce tumor hypoxia and provides arationale for combining these agents with ionizing radiation.The combination of antiangiogenic/vascular targeting agentsand radiation therapy has led to additive or greater than additivetumor responses. The basis for the enhanced tumor responseslies in the complex interactions between the tumor microenvi-ronment (tumor stroma and vasculature) and the tumor cellsthemselves. Although many factors such as VEGF expression,survivin down-regulation, endothelial sensitization to radiation,and many other complex interactions may be affected by anti-angiogenic/vascular targeting agents, these molecules and inter-actions ultimately affect tumor size and pO2, which are primaryfactors affecting tumor response to radiation (67).

Most tumors exist in a hypoxic and low pH environment byvirtue of abnormal tumor vasculature giving rise to poor tumorblood perfusion and high IFP. Hypoxic conditions stimulateup-regulation of angiogenic and tumor cell survival factors bytumor and tumor stromal cells that promote tumor and endothe-lial cell proliferation and high IFP, all of which give rise toradioresistance. In response to radiation alone, oxic cells arekilled, leaving behind a hypoxic, radioresistant fraction that canproduce protective cytokines giving rise to a second wave ofangiogenesis and tumor cell proliferation. Antiangiogenicagents, by targeting both tumor cells and endothelial cells, canreduce tumor proliferation and improve tumor oxygenation byinhibiting angiogenesis and high IFP through VEGF blockade.Thus, antiangiogenic agents can decrease overall tumor resist-ance to radiation by targeting both the tumor stroma and thetumor cell compartments, thereby eliminating the codependentcycle of tumor growth and angiogenesis (see Fig. 4 for summaryof possible mechanisms of interaction between radiation andantiangiogenic agents).




A close examination of this area of research indicates thereis great promise for future clinical treatment protocols. Becauseangiogenesis is one of the most fundamental processes of de-velopment, the complexity and redundancy of the process arenot surprising. Whereas complexity and redundancy may maketreatment and interpretation of the results difficult, they alsoprovide for many opportunities to interfere with the angiogenicprocess. Exploring which compounds and combinations of com-pounds will work best with radiation and how to optimize theiruse will be a long and difficult task. The potential to signifi-cantly benefit patients, however, warrants further investigations.

In conclusion:(a) Antiangiogenic agents appear to target tumor cells and

tumor vasculature by both indirect and direct mechanisms.(b) Antiangiogenic agents can decrease overall tumor re-

sistance to radiation by targeting both tumor cells and tumorvasculature.

(c) Tumor growth and angiogenesis are part of a codepen-dent cycle. Antivascular treatments can break the cycle andprevent revascularization after radiation.

(d) The differential effects observed with combined radia-tion and antivascular treatments on tumor growth among varioustumor models may arise from differences in the intrinsic radi-osensitivities of endothelial cells and tumor cells. The intrinsicradiosensitivity of the endothelial cell compartment in a tumormay be the result of differences in expression of survival factorssuch as VEGF, survivin, and BCL2 that come about in responseto changes in the tumor microenvironment. The intrinsic radio-sensitivity of the tumor cell compartment likewise depends onmicroenvironmental factors influencing expression of growth

factors and receptors controlling tumor cell growth, prolifera-tion, and capacity to repair DNA damage.

(e) Many factors are involved in the response to radiationand antiangiogenic/vascular targeting agents, but ultimatelythese factors affect tumor size and pO2, which are primaryfactors affecting tumor response to radiation.

(f) Hypoxia not only participates in resistance to radiother-apy but can also affect the efficacy of antivascular therapy byup-regulating angiogenic and survival pathways. Understandinghow tumor oxygenation changes during tumor regression andregrowth during exposure to antivascular agents and radiation iscritical for the optimal scheduling of combined treatments.

AcknowledgmentsWe acknowledge the expert administrative assistance of Stephanie

Lavorgna and Nichol Marrero in preparation of the manuscript.

References1. Owen, J. B., Coia, L. R., and Hanks, G. E. Recent patterns of growthin radiation therapy facilities in the United States: a patterns of carestudy report. Int. J. Radiat. Oncol. Biol. Phys., 24: 983–986, 1992.2. McGinn, C. J., Shewach, D. S., and Lawrence, T. S. Radiosensitizingnucleosides. J. Natl. Cancer Inst. (Bethesda), 88: 1193–1203, 1996.3. Stratford, I. J. Concepts and developments in radiosensitization ofmammalian cells. Int. J. Radiat. Oncol. Biol. Phys., 22: 529–532, 1992.4. Kerbel, R. S. Inhibition of tumor angiogenesis as a strategy tocircumvent acquired resistance to anti-cancer therapeutic agents. Bioes-says, 13: 31–36, 1991.5. Folkman, J. Tumor angiogenesis: therapeutic implications. N. Engl.J. Med., 285: 1182–1186, 1971.

Fig. 4 Possisble mechanismsfor enhanced tumor response toradiation with antiangiogenesisand vascular targeting therapy.Antiangiogenic agents maywork synergistically or addi-tively with ionizing radiationby improving tumor oxygen-ation through inhibition of neo-vascularization processes. Anti-angiogenic agents may alsoimprove radiosensitivity by di-rectly and/or indirectly inhibit-ing protective cell survivalsignaling pathways in both en-dothelial and tumor cells, re-sulting in increased apoptosis inboth the tumor cell compart-ment and the tumor stromal(i.e., endothelial cell) compart-ment. Vascular targeting agentsmay act in an additive fashionto improve tumor response toradiation by decreasing overalltumor burden through vascularshutdown and tumor necrosisand by creating a tumor envi-ronment whereby remaining vi-able, well-oxygenated tumorcells present an ideal target forradiation therapy.




6. Camphausen, K., and Menard, C. Angiogenesis inhibitors and radio-therapy of primary tumours. Expert Opin. Biol. Ther., 2: 477–481,2002.

7. Scappaticci, F. A. Mechanisms and future directions for angiogene-sis-based cancer therapies. J. Clin. Oncol., 20: 3906–3927, 2002.

8. Kerbel, R., and Folkman, J. Clinical translation of angiogenesisinhibitors. Nat. Rev. Cancer, 2: 727–739, 2002.

9. Gorski, D. H., Mauceri, H. J., Salloum, R. M., Gately, S., Hellman,S., Beckett, M. A., Sukhatme, V. P., Soff, G. A., Kufe, D. W., andWeichselbaum, R. R. Potentiation of the antitumor effect of ionizingradiation by brief concomitant exposures to angiostatin. Cancer Res.,58: 5686–5689, 1998.

10. Gorski, D. H., Beckett, M. A., Jaskowiak, N. T., Calvin, D. P.,Mauceri, H. J., Salloum, R. M., Seetharam, S., Koons, A., Hari, D. M.,Kufe, D. W., and Weichselbaum, R. R. Blockage of the vascularendothelial growth factor stress response increases the antitumor effectsof ionizing radiation. Cancer Res., 59: 3374–3378, 1999.

11. Mauceri, H. J., Hanna, N. N., Wayne, J. D., Hallahan, D. E.,Hellman, S., and Weichselbaum, R. R. Tumor necrosis factor � (TNF-�)gene therapy targeted by ionizing radiation selectively damages tumorvasculature. Cancer Res., 56: 4311–4314, 1996.

12. Mauceri, H. J., Hanna, N. N., Beckett, M. A., Gorski, D. H., Staba,M. J., Stellato, K. A., Bigelow, K., Heimann, R., Gately, S., Dhanabal,M., Soff, G. A., Sukhatme, V. P., Kufe, D. W., and Weichselbaum, R. R.Combined effects of angiostatin and ionizing radiation in antitumourtherapy. Nature (Lond.), 394: 287–291, 1998.

13. Griffin, R. J., Williams, B. W., Wild, R., Cherrington, J. M., Park,H., and Song, C. W. Simultaneous inhibition of the receptor kinaseactivity of vascular endothelial, fibroblast, and platelet-derived growthfactors suppresses tumor growth and enhances tumor radiation response.Cancer Res., 62: 1702–1706, 2002.

14. Lee, C. G., Heijn, M., di Tomaso, E., Griffon-Etienne, G.,Ancukiewicz, M., Koike, C., Park, K. R., Ferrara, N., Jain, R. K., Suit,H. D., and Boucher, Y. Anti-vascular endothelial growth factor treat-ment augments tumor radiation response under normoxic or hypoxicconditions. Cancer Res., 60: 5565–5570, 2000.15. Teicher, B. A., Holden, S. A., Ara, G., Dupuis, N. P., Liu, F., Yuan,J., Ikebe, M., and Kakeji, Y. Influence of an anti-angiogenic treatmenton 9L gliosarcoma: oxygenation and response to cytotoxic therapy. Int.J. Cancer, 61: 732–737, 1995.16. Teicher, B. A., Williams, J. I., Takeuchi, H., Ara, G., Herbst, R. S.,and Buxton, D. Potential of the aminosterol, squalamine in combinationtherapy in the rat 13,762 mammary carcinoma and the murine Lewislung carcinoma. Anticancer Res., 18: 2567–2573, 1998.17. Folkman, J. The vascularization of tumors. Sci. Am., 234: 58–64,70–73, 1976.18. Thomlinson, R., and Gray, L. The histological structure of somehuman lung cancers and the possible implications for radiotherapy.Br. J. Cancer, 9: 539–549, 1955.19. Hanahan, D., and Folkman, J. Patterns and emerging mechanisms ofthe angiogenic switch during tumorigenesis. Cell, 86: 353–364, 1996.20. Los, M., and Voest, E. E. The potential role of antivascular therapyin the adjuvant and neoadjuvant treatment of cancer. Semin. Oncol., 28:93–105, 2001.21. Carmeliet, P., and Jain, R. K. Angiogenesis in cancer and otherdiseases. Nature (Lond.), 407: 249–257, 2000.22. Ferrara, N. Role of vascular endothelial growth factor in regulationof physiological angiogenesis. Am. J. Physiol. Cell Physiol., 280:C1358–C1366, 2001.23. Asano, M., Yukita, A., Matsumoto, T., Kondo, S., and Suzuki, H.Inhibition of tumor growth and metastasis by an immunoneutralizingmonoclonal antibody to human vascular endothelial growth factor/vascular permeability factor 121. Cancer Res., 55: 5296–5301, 1995.24. Benjamin, L. E., Golijanin, D., Itin, A., Pode, D., and Keshet, E.Selective ablation of immature blood vessels in established humantumors follows vascular endothelial growth factor withdrawal. J. Clin.Investig., 103: 159–165, 1999.

25. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., andFerrara, N. Vascular endothelial growth factor is a secreted angiogenicmitogen. Science (Wash. DC), 246: 1306–1309, 1989.

26. Bellamy, W. T., Richter, L., Frutiger, Y., and Grogan, T. M.Expression of vascular endothelial growth factor and its receptors inhematopoietic malignancies. Cancer Res., 59: 728–733, 1999.

27. Bertolini, F., Mancuso, P., Gobbi, A., and Pruneri, G. The thin redline: angiogenesis in normal and malignant hematopoiesis. Exp. Hema-tol., 28: 993–1000, 2000.

28. Fiedler, W., Graeven, U., Ergun, S., Verago, S., Kilic, N., Stock-schlader, M., and Hossfeld, D. K. Vascular endothelial growth factor, apossible paracrine growth factor in human acute myeloid leukemia.Blood, 89: 1870–1875, 1997.

29. Levy, A. P., Levy, N. S., Wegner, S., and Goldberg, M. A. Tran-scriptional regulation of the rat vascular endothelial growth factor geneby hypoxia. J. Biol. Chem., 270: 13333–13340, 1995.

30. Shweiki, D., Neeman, M., Itin, A., and Keshet, E. Induction ofvascular endothelial growth factor expression by hypoxia and by glu-cose deficiency in multicell spheroids: implications for tumor angiogen-esis. Proc. Natl. Acad. Sci. USA, 92: 768–772, 1995.

31. Ellis, L. M., Staley, C. A., Liu, W., Fleming, R. Y., Parikh, N. U.,Bucana, C. D., and Gallick, G. E. Down-regulation of vascular endo-thelial growth factor in a human colon carcinoma cell line transfectedwith an antisense expression vector specific for c-src. J. Biol. Chem.,273: 1052–1057, 1998.

32. Mukhopadhyay, D., Tsiokas, L., Zhou, X. M., Foster, D., Brugge,J. S., and Sukhatme, V. P. Hypoxic induction of human vascularendothelial growth factor expression through c-Src activation. Nature(Lond.), 375: 577–581, 1995.

33. Maxwell, P. H., Dachs, G. U., Gleadle, J. M., Nicholls, L. G.,Harris, A. L., Stratford, I. J., Hankinson, O., Pugh, C. W., and Ratcliffe,P. J. Hypoxia-inducible factor-1 modulates gene expression in solidtumors and influences both angiogenesis and tumor growth. Proc. Natl.Acad. Sci. USA, 94: 8104–8109, 1997.34. Mazure, N. M., Chen, E. Y., Laderoute, K. R., and Giaccia, A. J.Induction of vascular endothelial growth factor by hypoxia is modulatedby a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptionalelement. Blood, 90: 3322–3331, 1997.35. Jung, Y. D., Nakano, K., Liu, W., Gallick, G. E., and Ellis, L. M.Extracellular signal-regulated kinase activation is required for up-regu-lation of vascular endothelial growth factor by serum starvation inhuman colon carcinoma cells. Cancer Res., 59: 4804–4807, 1999.36. Rak, J., and Kerbel, R. S. Ras regulation of vascular endothelialgrowth factor and angiogenesis. Methods Enzymol., 333: 267–283,2001.37. Takahashi, Y., Bucana, C. D., Cleary, K. R., and Ellis, L. M. p53,vessel count, and vascular endothelial growth factor expression inhuman colon cancer. Int. J. Cancer, 79: 34–38, 1998.38. Koukourakis, M. I. Tumour angiogenesis and response to radiother-apy. Anticancer Res., 21: 4285–4300, 2001.39. Vaupel, P. W. The influence of tumor blood flow and microenvi-ronmental factors on the efficacy of radiation, drugs and localizedhyperthermia. Klin. Paediatr., 209: 243–249, 1997.40. St Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K. E.,Montgomery, E., Lal, A., Riggins, G. J., Lengauer, C., Vogelstein, B.,and Kinzler, K. W. Genes expressed in human tumor endothelium.Science (Wash. DC), 289: 1197–1202, 2000.41. Eberhard, A., Kahlert, S., Goede, V., Hemmerlein, B., Plate, K. H.,and Augustin, H. G. Heterogeneity of angiogenesis and blood vesselmaturation in human tumors: implications for antiangiogenic tumortherapies. Cancer Res., 60: 1388–1393, 2000.42. Jain, R. K. Determinants of tumor blood flow: a review. CancerRes., 48: 2641–2658, 1988.43. Less, J. R., Skalak, T. C., Sevick, E. M., and Jain, R. K. Microvas-cular architecture in a mammary carcinoma: branching patterns andvessel dimensions. Cancer Res., 51: 265–273, 1991.




44. Jain, R. K. Transport of molecules, particles, and cells in solidtumors. Annu. Rev. Biomed. Eng., 1: 241–263, 1999.

45. Jain, R. K. Normalizing tumor vasculature with anti-angiogenictherapy: a new paradigm for combination therapy. Nat. Med., 7: 987–989, 2001.

46. Movsas, B., Chapman, J. D., Hanlon, A. L., Horwitz, E. M.,Greenberg, R. E., Stobbe, C., Hanks, G. E., and Pollack, A. Hypoxicprostate/muscle pO2 ratio predicts for biochemical failure in patientswith prostate cancer: preliminary findings. Urology, 60: 634–639,2002.

47. Reinmuth, N., Stoeltzing, O., Liu, W., Ahmad, S. A., Jung, Y. D.,Fan, F., Parikh, A., and Ellis, L. M. Endothelial survival factors astargets for antineoplastic therapy. Cancer J., 7 (Suppl. 3): S109–S119,2001.

48. Koukourakis, M. I., Giatromanolaki, A., Sivridis, E., Simopoulos,K., Pissakas, G., Gatter, K. C., and Harris, A. L. Squamous cell head andneck cancer: evidence of angiogenic regeneration during radiotherapy.Anticancer Res., 21: 4301–4309, 2001.

49. Chapman, J. D., Dugle, D. L., Reuvers, A. P., Meeker, B. E., andBorsa, J. Studies on the radiosensitizing effect of oxygen in Chinesehamster cells. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med., 26:383–389, 1974.

50. Moore, J. V. Re: Tumor cure studies on the rat sarcoma BA1112using continuous low-dose-rate radiation. Radiology, 155: 837–838,1985.

51. Tannock, I. F., and Hill, R. P. The Basic Science of Oncology, 3rded. McGraw-Hill, 1998.

52. Evans, S. M., Hahn, S. M., Magarelli, D. P., and Koch, C. J.Hypoxic heterogeneity in human tumors: EF5 binding, vasculature,necrosis, and proliferation. Am. J. Clin. Oncol., 24: 467–472, 2001.

53. Rockwell, S., and Moulder, J. E. Hypoxic fractions of humantumors xenografted into mice: a review. Int. J. Radiat. Oncol. Biol.Phys., 19: 197–202, 1990.

54. Tannock, I. F. Oxygen diffusion and the distribution of cellularradiosensitivity in tumours. Br. J. Radiol., 45: 515–524, 1972.

55. Wouters, B. G., and Brown, J. M. Cells at intermediate oxygenlevels can be more important than the “hypoxic fraction” in determiningtumor response to fractionated radiotherapy. Radiat. Res., 147: 541–550, 1997.

56. Brown, J. M. Evidence for acutely hypoxic cells in mouse tumours,and a possible mechanism of reoxygenation. Br. J. Radiol., 52: 650–656, 1979.

57. Chaplin, D. J., and Hill, S. A. Temporal heterogeneity in microre-gional erythrocyte flux in experimental solid tumours. Br. J. Cancer, 71:1210–1213, 1995.

58. Sutherland, R. M., and Franko, A. J. On the nature of the radiobi-ologically hypoxic fraction in tumors. Int. J. Radiat. Oncol. Biol. Phys.,6: 117–120, 1980.

59. Giaccia, A. J. Hypoxic stress proteins: survival of the fittest. Semin.Radiat. Oncol., 6: 46–58, 1996.

60. Jung, Y. D., Ahmad, S. A., Liu, W., Reinmuth, N., Parikh, A.,Stoeltzing, O., Fan, F., and Ellis, L. M. The role of the microenviron-ment and intercellular cross-talk in tumor angiogenesis. Semin. CancerBiol., 12: 105–112, 2002.

61. Rak, J., Yu, J. L., Kerbel, R. S., and Coomber, B. L. What dooncogenic mutations have to do with angiogenesis/vascular dependenceof tumors? Cancer Res., 62: 1931–1934, 2002.

62. Kung, A. L., Wang, S., Klco, J. M., Kaelin, W. G., and Livingston,D. M. Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nat. Med., 6: 1335–1340, 2000.

63. Bowers, G., Reardon, D., Hewitt, T., Dent, P., Mikkelsen, R. B.,Valerie, K., Lammering, G., Amir, C., and Schmidt-Ullrich, R. K. Therelative role of ErbB1–4 receptor tyrosine kinases in radiation signaltransduction responses of human carcinoma cells. Oncogene, 20: 1388–1397, 2001.

64. Hendry, J. H. Treatment acceleration in radiotherapy: the relativetime factors and dose-response slopes for tumours and normal tissues.Radiother. Oncol., 25: 308–312, 1992.

65. Hansen-Algenstaedt, N., Stoll, B. R., Padera, T. P., Dolmans, D. E.,Hicklin, D. J., Fukumura, D., and Jain, R. K. Tumor oxygenation inhormone-dependent tumors during vascular endothelial growth factorreceptor-2 blockade, hormone ablation, and chemotherapy. Cancer Res.,60: 4556–4560, 2000.66. Teicher, B. A. Hypoxia and drug resistance. Cancer MetastasisRev., 13: 139–168, 1994.67. Vaupel, P., Kallinowski, F., and Okunieff, P. Blood flow, oxygenand nutrient supply, and metabolic microenvironment of human tumors:a review. Cancer Res., 49: 6449–6465, 1989.68. Geng, L., Donnelly, E., McMahon, G., Lin, P. C., Sierra-Rivera, E.,Oshinka, H., and Hallahan, D. E. Inhibition of vascular endothelialgrowth factor receptor signaling leads to reversal of tumor resistance toradiotherapy. Cancer Res., 61: 2413–2419, 2001.69. Griscelli, F., Li, H., Cheong, C., Opolon, P., Bennaceur-Griscelli,A., Vassal, G., Soria, J., Soria, C., Lu, H., Perricaudet, M., and Yeh, P.Combined effects of radiotherapy and angiostatin gene therapy in gli-oma tumor model. Proc. Natl. Acad. Sci. USA, 97: 6698–6703, 2000.70. Hess, C., Vuong, V., Hegyi, I., Riesterer, O., Wood, J., Fabbro, D.,Glanzmann, C., Bodis, S., and Pruschy, M. Effect of VEGF receptorinhibitor PTK787/ZK222584 [correction of ZK222548] combined withionizing radiation on endothelial cells and tumour growth. Br. J. Cancer,85: 2010–2016, 2001.71. Huang, S. M., Li, J., Armstrong, E. A., and Harari, P. M. Modula-tion of radiation response and tumor-induced angiogenesis after epider-mal growth factor receptor inhibition by ZD1839 (Iressa). Cancer Res.,62: 4300–4306, 2002.72. Kozin, S. V., Boucher, Y., Hicklin, D. J., Bohlen, P., Jain, R. K.,and Suit, H. D. Vascular endothelial growth factor receptor-2-blockingantibody potentiates radiation-induced long-term control of human tu-mor xenografts. Cancer Res., 61: 39–44, 2001.73. Lund, E. L., Bastholm, L., and Kristjansen, P. E. Therapeuticsynergy of TNP-470 and ionizing radiation: effects on tumor growth,vessel morphology, and angiogenesis in human glioblastoma multi-forme xenografts. Clin. Cancer Res., 6: 971–978, 2000.74. Milas, L., Kishi, K., Hunter, N., Mason, K., Masferrer, J. L., andTofilon, P. J. Enhancement of tumor response to �-radiation by aninhibitor of cyclooxygenase-2 enzyme. J. Natl. Cancer Inst. (Bethesda),91: 1501–1504, 1999.75. Murata, R., Nishimura, Y., and Hiraoka, M. An antiangiogenicagent (TNP-470) inhibited reoxygenation during fractionated radiother-apy of murine mammary carcinoma. Int. J. Radiat. Oncol. Biol. Phys.,37: 1107–1113, 1997.76. Asano, M., Yukita, A., and Suzuki, H. Wide spectrum of antitumoractivity of a neutralizing monoclonal antibody to human vascular en-dothelial growth factor. Jpn. J. Cancer Res., 90: 93–100, 1999.77. Prewett, M., Huber, J., Li, Y., Santiago, A., O’Connor, W., King,K., Overholser, J., Hooper, A., Pytowski, B., Witte, L., Bohlen, P., andHicklin, D. J. Antivascular endothelial growth factor receptor (fetal liverkinase 1) monoclonal antibody inhibits tumor angiogenesis and growthof several mouse and human tumors. Cancer Res., 59: 5209–5218,1999.78. Hill, S. A., Smith, K. A., Williams, K. B., and Denekamp, J. Thefractionated response of mouse stroma after X-rays and neutrons: influ-ence of early versus late expression of damage. Radiother. Oncol., 16:129–137, 1989.79. Teicher, B. A., Emi, Y., Kakeji, Y., and Northey, D. TNP-470/minocycline/cytotoxic therapy: a systems approach to cancer therapy.Eur. J. Cancer, 32A: 2461–2466, 1996.80. Dicker, A. P., Williams, T. L., and Grant, D. S. Targeting angio-genic processes by combination rofecoxib and ionizing radiation. Am. J.Clin. Oncol., 24: 438–442, 2001.81. Kishi, K., Milas, L., Hunter, N., and Sato, M. Recent studies onanti-angiogenesis in cancer therapy. Nippon Rinsho, 58: 1747–1762,2000.




82. Milas, L. Cyclooxygenase-2 (COX-2) enzyme inhibitors as poten-tial enhancers of tumor radioresponse. Semin. Radiat. Oncol., 11: 290–299, 2001.

83. Pyo, H., Choy, H., Amorino, G. P., Kim, J. S., Cao, Q., Hercules,S. K., and DuBois, R. N. A selective cyclooxygenase-2 inhibitor,NS-398, enhances the effect of radiation in vitro and in vivo preferen-tially on the cells that express cyclooxygenase-2. Clin. Cancer Res., 7:2998–3005, 2001.84. Nasu, S., Ang, K. K., Fan, Z., and Milas, L. C225 antiepidermalgrowth factor receptor antibody enhances tumor radiocurability. Int. J.Radiat. Oncol. Biol. Phys., 51: 474–477, 2001.85. Folkman, J., and Camphausen, K. Cancer. What does radiotherapydo to endothelial cells? Science (Wash. DC), 293: 227–228, 2001.86. Gupta, V. K., Jaskowiak, N. T., Beckett, M. A., Mauceri, H. J.,Grunstein, J., Johnson, R. S., Calvin, D. A., Nodzenski, E., Pejovic, M.,Kufe, D. W., Posner, M. C., and Weichselbaum, R. R. Vascular endo-thelial growth factor enhances endothelial cell survival and tumor ra-dioresistance. Cancer J., 8: 47–54, 2002.87. O’Reilly, M. S., Holmgren, L., Chen, C., and Folkman, J. Angiosta-tin induces and sustains dormancy of human primary tumors in mice.Nat. Med., 2: 689–692, 1996.88. Teicher, B. A., Holden, S. A., Dupuis, N. P., Kakeji, Y., Ikebe, M.,Emi, Y., and Goff, D. Potentiation of cytotoxic therapies by TNP-470and minocycline in mice bearing EMT-6 mammary carcinoma. BreastCancer Res. Treat., 36: 227–236, 1995.89. Shaheen, R. M., Tseng, W. W., Davis, D. W., Liu, W., Reinmuth,N., Vellagas, R., Wieczorek, A. A., Ogura, Y., McConkey, D. J.,Drazan, K. E., Bucana, C. D., McMahon, G., and Ellis, L. M. Tyrosinekinase inhibition of multiple angiogenic growth factor receptors im-proves survival in mice bearing colon cancer liver metastases by inhi-bition of endothelial cell survival mechanisms. Cancer Res., 61: 1464–1468, 2001.90. Wahl, M. L., and Grant, D. S. Effects of microenvironmentalextracellular pH and extracellular matrix proteins on angiostatin’s ac-tivity and on intracellular pH. Gen. Pharmacol., 35: 277–285, 2000.91. Moser, T. L., Stack, M. S., Wahl, M. L., and Pizzo, S. V. Themechanism of action of angiostatin: can you teach an old dog newtricks? Thromb. Haemostasis, 87: 394–401, 2002.92. Milas, L., and Hanson, W. R. Eicosanoids and radiation. Eur. J.Cancer, 31A: 1580–1585, 1995.93. Mehrabi, M. R., Serbecic, N., Tamaddon, F., Kaun, C., Huber, K.,Pacher, R., Wild, T., Mall, G., Wojta, J., and Glogar, H. D. Clinical andexperimental evidence of prostaglandin E1-induced angiogenesis in themyocardium of patients with ischemic heart disease. Cardiovasc. Res.,56: 214–224, 2002.94. Furuta, Y., Hunter, N., Barkley, T., Jr., Hall, E., and Milas, L.Increase in radioresponse of murine tumors by treatment with indo-methacin. Cancer Res., 48: 3008–3013, 1988.95. Kishi, K., Petersen, S., Petersen, C., Hunter, N., Mason, K., Mas-ferrer, J. L., Tofilon, P. J., and Milas, L. Preferential enhancement oftumor radioresponse by a cyclooxygenase-2 inhibitor. Cancer Res., 60:1326–1331, 2000.96. Petersen, C., Petersen, S., Milas, L., Lang, F. F., and Tofilon, P. J.Enhancement of intrinsic tumor cell radiosensitivity induced by a se-lective cyclooxygenase-2 inhibitor. Clin. Cancer Res., 6: 2513–2520,2000.97. Milas, L., Akimoto, T., Hunter, N. R., Mason, K. A., Buchmiller,L., Yamakawa, M., Muramatsu, H., and Ang, K. K. Relationship be-tween cyclin D1 expression and poor radioresponse of murine carcino-mas. Int. J. Radiat. Oncol. Biol. Phys., 52: 514–521, 2002.98. Emoto, M., Iwasaki, H., Mimura, K., Kawarabayashi, T., and Kiku-chi, M. Differences in the angiogenesis of benign and malignant ovariantumors, demonstrated by analyses of color Doppler ultrasound, immu-nohistochemistry, and microvessel density. Cancer (Phila.), 80: 899–907, 1997.99. Kakolyris, S., Giatromanolaki, A., Koukourakis, M., Leigh, I. M.,Georgoulias, V., Kanavaros, P., Sivridis, E., Gatter, K. C., and Harris,A. L. Assessment of vascular maturation in non-small cell lung cancer

using a novel basement membrane component, LH39: correlation withp53 and angiogenic factor expression. Cancer Res., 59: 5602–5607,1999.

100. Kakolyris, S., Fox, S. B., Koukourakis, M., Giatromanolaki, A.,Brown, N., Leek, R. D., Taylor, M., Leigh, I. M., Gatter, K. C., andHarris, A. L. Relationship of vascular maturation in breast cancer bloodvessels to vascular density and metastasis, assessed by expression of anovel basement membrane component, LH39. Br. J. Cancer, 82: 844–851, 2000.

101. Milosevic, M. F., Fyles, A. W., Wong, R., Pintilie, M., Kavanagh,M. C., Levin, W., Manchul, L. A., Keane, T. J., and Hill, R. P.Interstitial fluid pressure in cervical carcinoma: within tumor heteroge-neity, and relation to oxygen tension. Cancer (Phila.), 82: 2418–2426,1998.

102. Boucher, Y., Lee, I., and Jain, R. K. Lack of general correlationbetween interstitial fluid pressure and oxygen partial pressure in solidtumors. Microvasc. Res., 50: 175–182, 1995.

103. Papetti, M., and Herman, I. M. Mechanisms of normal and tumor-derived angiogenesis. Am. J. Physiol Cell Physiol., 282: C947–C970,2002.

104. Dvorak, H. F., Nagy, J. A., Feng, D., Brown, L. F., and Dvorak,A. M. Vascular permeability factor/vascular endothelial growth factorand the significance of microvascular hyperpermeability in angiogene-sis. Curr. Top. Microbiol. Immunol., 237: 97–132, 1999.

105. Dunst, J., Hansgen, G., Lautenschlager, C., Fuchsel, G., andBecker, A. Oxygenation of cervical cancers during radiotherapy andradiotherapy � cis-retinoic acid/interferon. Int. J. Radiat. Oncol. Biol.Phys., 43: 367–373, 1999.

106. Busse, M., and Vaupel, P. W. The role of tumor volume in“reoxygenation” upon cyclophosphamide treatment. Acta Oncol., 34:405–408, 1995.

107. Rofstad, E. K. Hypoxia and reoxygenation in human melanomaxenografts. Int. J. Radiat. Oncol. Biol. Phys., 17: 81–89, 1989.

108. Olive, P. L. Radiation-induced reoxygenation in the SCCVII mu-rine tumour: evidence for a decrease in oxygen consumption and anincrease in tumour perfusion. Radiother. Oncol., 32: 37–46, 1994.

109. Mayr, N. A., Yuh, W. T., Magnotta, V. A., Ehrhardt, J. C.,Wheeler, J. A., Sorosky, J. I., Davis, C. S., Wen, B. C., Martin, D. D.,Pelsang, R. E., Buller, R. E., Oberley, L. W., Mellenberg, D. E., andHussey, D. H. Tumor perfusion studies using fast magnetic resonanceimaging technique in advanced cervical cancer: a new noninvasivepredictive assay. Int. J. Radiat. Oncol. Biol. Phys., 36: 623–633, 1996.110. Wartenberg, M., Donmez, F., Ling, F. C., Acker, H., Hescheler, J.,and Sauer, H. Tumor-induced angiogenesis studied in confrontationcultures of multicellular tumor spheroids and embryoid bodies grownfrom pluripotent embryonic stem cells. FASEB J., 15: 995–1005, 2001.111. Yuan, F., Chen, Y., Dellian, M., Safabakhsh, N., Ferrara, N., andJain, R. K. Time-dependent vascular regression and permeabilitychanges in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor anti-body. Proc. Natl. Acad. Sci. USA, 93: 14765–14770, 1996.112. Holmgren, L., O’Reilly, M. S., and Folkman, J. Dormancy ofmicrometastases: balanced proliferation and apoptosis in the presence ofangiogenesis suppression. Nat. Med., 1: 149–153, 1995.113. Kirsch, M., Strasser, J., Allende, R., Bello, L., Zhang, J., andBlack, P. M. Angiostatin suppresses malignant glioma growth in vivo.Cancer Res., 58: 4654–4659, 1998.114. Wassberg, E., Hedborg, F., Skoldenberg, E., Stridsberg, M., andChristofferson, R. Inhibition of angiogenesis induces chromaffin differ-entiation and apoptosis in neuroblastoma. Am. J. Pathol., 154: 395–403,1999.115. Masood, R., Cai, J., Zheng, T., Smith, D. L., Hinton, D. R., andGill, P. S. Vascular endothelial growth factor (VEGF) is an autocrinegrowth factor for VEGF receptor-positive human tumors. Blood, 98:1904–1913, 2001.116. Denekamp, J. Vascular attack as a therapeutic strategy for cancer.Cancer Metastasis Rev., 9: 267–282, 1990.




117. Siemann, D. W. Vascular targeting agents. Horiz. Cancer Ther., 3:4–15, 2002.118. Ching, L. M., Joseph, W. R., and Baguley, B. C. Antitumourresponses to flavone-8-acetic acid and 5,6-dimethylxanthenone-4-aceticacid in immune deficient mice. Br. J. Cancer, 66: 128–130, 1992.119. Cliffe, S., Taylor, M. L., Rutland, M., Baguley, B. C., Hill, R. P.,and Wilson, W. R. Combining bioreductive drugs (SR 4233 or SN23862) with the vasoactive agents flavone acetic acid or 5,6-dimethyl-xanthenone acetic acid. Int. J. Radiat. Oncol. Biol. Phys., 29: 373–377,1994.120. Hill, S. A., Sampson, L. E., and Chaplin, D. J. Anti-vascularapproaches to solid tumour therapy: evaluation of vinblastine and fla-vone acetic acid. Int. J. Cancer, 63: 119–123, 1995.121. Laws, A. L., Matthew, A. M., Double, J. A., and Bibby, M. C.Preclinical in vitro and in vivo activity of 5,6-dimethylxanthenone-4-acetic acid. Br. J. Cancer, 71: 1204–1209, 1995.122. Dark, G. G., Hill, S. A., Prise, V. E., Tozer, G. M., Pettit, G. R.,and Chaplin, D. J. Combretastatin A-4, an agent that displays potent andselective toxicity toward tumor vasculature. Cancer Res, 57: 1829–1834, 1997.123. Li, L., Rojiani, A., and Siemann, D. W. Targeting the tumorvasculature with combretastatin A-4 disodium phosphate: effects onradiation therapy. Int. J. Radiat. Oncol. Biol. Phys., 42: 899–903, 1998.124. Murata, R., Siemann, D. W., Overgaard, J., and Horsman, M. R.Interaction between combretastatin A-4 disodium phosphate and radia-tion in murine tumors. Radiother. Oncol., 60: 155–161, 2001.125. Siemann, D. W., and Rojiani, A. M. Enhancement of radiationtherapy by the novel vascular targeting agent ZD6126. Int. J. Radiat.Oncol. Biol. Phys., 53: 164–171, 2002.126. Tozer, G. M., Prise, V. E., Wilson, J., Locke, R. J., Vojnovic, B.,Stratford, M. R., Dennis, M. F., and Chaplin, D. J. Combretastatin A-4phosphate as a tumor vascular-targeting agent: early effects in tumorsand normal tissues. Cancer Res., 59: 1626–1634, 1999.127. Horsman, M. R., Ehrnrooth, E., Ladekarl, M., and Overgaard, J.The effect of combretastatin A-4 disodium phosphate in a C3H mousemammary carcinoma and a variety of murine spontaneous tumors. Int.J. Radiat. Oncol. Biol. Phys., 42: 895–898, 1998.

128. Ching, L. M., Joseph, W. R., Crosier, K. E., and Baguley, B. C.Induction of tumor necrosis factor-� messenger RNA in human andmurine cells by the flavone acetic acid analogue 5,6-dimethylxanthe-none-4-acetic acid (NSC 640488). Cancer Res., 54: 870–872, 1994.

129. Mahadevan, V., Malik, S. T., Meager, A., Fiers, W., Lewis, G. P.,and Hart, I. R. Role of tumor necrosis factor in flavone acetic acid-induced tumor vasculature shutdown. Cancer Res., 50: 5537–5542,1990.

130. Philpott, M., Baguley, B. C., and Ching, L. M. Induction of tumournecrosis factor-� by single and repeated doses of the antitumour agent5,6-dimethylxanthenone-4-acetic acid. Cancer Chemother. Pharmacol.,36: 143–148, 1995.131. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., andWilliamson, B. An endotoxin-induced serum factor that causes necrosisof tumors. Proc. Natl. Acad. Sci. USA, 72: 3666–3670, 1975.132. Galbraith, S. M., Chaplin, D. J., Lee, F., Stratford, M. R., Locke, R. J.,Vojnovic, B., and Tozer, G. M. Effects of combretastatin A4 phosphate onendothelial cell morphology in vitro and relationship to tumour vasculartargeting activity in vivo. Anticancer Res., 21: 93–102, 2001.133. Chaplin, D. J., Pettit, G. R., and Hill, S. A. Anti-vascular ap-proaches to solid tumour therapy: evaluation of combretastatin A4phosphate. Anticancer Res., 19: 189–195, 1999.134. Paris, F., Fuks, Z., Kang, A., Capodieci, P., Juan, G., Ehleiter, D.,Haimovitz-Friedman, A., Cordon-Cardo, C., and Kolesnick, R. Endo-thelial apoptosis as the primary lesion initiating intestinal radiationdamage in mice. Science (Wash. DC), 293: 293–297, 2001.135. Pena, L. A., Fuks, Z., and Kolesnick, R. N. Radiation-inducedapoptosis of endothelial cells in the murine central nervous system:protection by fibroblast growth factor and sphingomyelinase deficiency.Cancer Res., 60: 321–327, 2000.136. Hall, E. J. Radiobiology for the Radiologist, 5th ed., Philadelphia:LWW, 2000.137. Dicker, A. P., Anne, R., Bonanni, R., Sidor, C., Gubish, E., andCurran, W. J., Jr. Phase I trial results of recombinant hluman angiostatinprotein (rhA) and external beam radiation therapy (EBRT). In: 38thAnnual Meeting of the American Association of Clinical Oncology.Alexandria, VA: ASCO Publications, 2002.




2003;9:1957-1971. Clin Cancer Res Phyllis Wachsberger, Randy Burd and Adam P. Dicker Mechanisms of InteractionAntiangiogenesis or Vascular Targeting Agents: Exploring Tumor Response to Ionizing Radiation Combined with

Updated version

http://clincancerres.aacrjournals.org/content/9/6/1957

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/9/6/1957.full#ref-list-1

This article cites 130 articles, 48 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/9/6/1957.full#related-urls

This article has been cited by 32 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/9/6/1957To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/content/9/6/1957.full#ref-list-1

http://clincancerres.aacrjournals.org/content/9/6/1957.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



tumor response to ionizing radiation combined with antiangiogenesis or … · review tumor response...

Documents