therapeutic synergy of tnp-470 and ionizing radiation: effects … · tnp-470 has been shown to...

Therapeutic Synergy of TNP-470 and Ionizing Radiation: Effects onTumor Growth, Vessel Morphology, and Angiogenesis in HumanGlioblastoma Multiforme Xenografts1

Eva L. Lund, Lone Bastholm, andPaul E. G. Kristjansen2

Institute of Molecular Pathology, University of Copenhagen, DK-2100 Copenhagen, Denmark

ABSTRACTWe examined the effect on tumor growth, vessel mor-

phology, and expression of angiogenic factors of combiningradiotherapy and antiangiogenesis in the human glioblas-toma line U87 grown in the flank or intracranially in thenude mouse. The antiangiogenic agent TNP-470 was given6.7 mg/kg s.c. daily on day 1–7 starting 1 week after trans-plantation. Irradiation (IR), 10 Gy 3 1, was administered onday 7. A series of tumors were excised 8 and 48 h after theend of treatment. The vascular morphology was evaluated inCD31 immunostained cryosections and by electron micros-copy, and the pattern of expression of angiogenic factors(mRNA and protein) was quantitatively analyzed by phos-phorimaging of Northern blots and Western blots. Signifi-cant inhibition of s.c. flank tumor growth relative to un-treated controls was achieved by monotherapy with bothTNP-470 (P < 0.001) and IR (P < 0.001). A significantenhancement of this effect was obtained by combining TNP-470 and IR (P < 0.05). We saw no effect of TNP-470 eitheralone or in addition to the effect of IR on the survival of micewith intracranial tumors. CD31 immunostaining of s.c. tu-mors showed acute endothelial swelling and luminal protru-sion in irradiated tumor vessels but never in tumors pre-treated with TNP-470, and not in the untreated controls.The vessel density (Chalkley point counts) was unchangedby TNP-470 therapy. In the TNP-470-treated tumors, weobserved a distinct broadening of the endothelial basementmembrane by an approximately 400–700-nm-thick electron-dense yet uncharacterized fibrillar material. TNP-470treated tumors 1/2 IR also had a significantly increasedmRNA expression of angiopoietin-1, whereas angiopoietin-2,vascular endothelial growth factor and basic fibroblast

growth factor mRNA were unchanged by the treatments. Inconclusion, TNP-470 significantly enhanced the tumor effectof ionizing IR, and our findings strongly indicate that acutemicrovascular damage after IR is effectively prevented byconcurrent TNP-470 treatment. A significant up-regulationof angiopoietin-1 seems to play a role in this protectivemechanism, which as yet is not fully elucidated.

INTRODUCTIONAngiogenesis is considered essential for tumors to grow

beyond a few millimeters in size (1). Angiogenic processes areregulated by the balance between proangiogenic and antiangio-genic factors in the microenvironment. VEGF3 and angiopoi-etin-1 and angiopoietin-2 are more specific to endothelium thanbFGF, whose receptors are present in numerous tissues. Glio-blastoma multiforme, the most common primary malignantbrain tumor in humans, is microscopically characterized bycertain vascular patterns produced by endothelial hyperprolif-eration. Cultured glioblastoma cells or tissues also express largeamounts of angiogenic factors, mostly VEGF (2, 3). Therefore,antiangiogenesis is likely to contribute to the outcome of theoverall management of this disease entity, in which cranialradiotherapy plays a pivotal role.

The antiangiogenic compound TNP-470 is an inhibitor ofendothelial cells (4). The mechanism of action is not fullyelucidated, but the compound is believed to act by inhibitingmethionyl aminopeptidase-2, an intracellular enzyme related toprotein myristoylation (5). Accordingly, the effects of TNP-470are mediated by the resulting inhibition of membrane proteins,such as nitric oxide synthase, which are translocated to the cellsurface membrane by myristoylation (6). TNP-470 has beenshown to inhibit the growth of gliomas and other brain tumorsin a number of animal studies (7–11).

The combination of radiotherapy and TNP-470 has beeninvestigated in murine mammary and lung carcinomas but not inhuman glioblastoma multiforme. Murataet al. (12) found thatTNP-470 administered before IR did not induce tumor hypoxia,whereas TNP-470 given during fractionated radiotherapy diddecrease the radiocurability of murine mammary carcinoma. Instudies on murine mammary carcinoma and Lewis lung carci-noma, Teicheret al. (13, 14) found that TNP-470 in combina-tion with other antiangiogenic agents, given during fractionatedradiotherapy, increased the inhibitory effect of radiation ontumor growth. Angiostatin, another potent antiangiogenic agent,has been used in combination with fractionated radiotherapy

Received 9/30/99; revised 12/20/99; accepted 12/20/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by The Danish Cancer Research Foundation, The DanishMedical Research Council (Grant 9700929), and Danish Cancer Society(Grant 9810034).2 To whom requests for reprints should be addressed, at MolecularPathology, University of Copenhagen, Frederik V’s Vej 11, DK-2100Copenhagen, Denmark. Phone: 45-3532-6006; Fax: 45-3532-6081; E-mail: [email protected].

3 The abbreviations used are: VEGF, vascular endothelial growth factor;bFGF, basic fibroblast growth factor; GAPDH, glyceraldehyde-3-phos-phate dehydrogenase.

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against human glioma on nude mice with a greater than additiveeffect on tumor-growth inhibition (15).

On this basis, we evaluated the effect of combinationtherapy with TNP-470 and ionizing radiation on human glio-blastoma xenografts in the s.c. space and intracranially in thenude mouse. In addition to the tumor response evaluation, westudied the effects on vascular morphology and on the expres-sion pattern of a number of relevant angiogenic factors.

By this approach, the present study provides information ofrelevance to the introduction of antiangiogenic therapy in effec-tive combinations with other treatment modalities.

MATERIALS AND METHODSAnimals. A total of 120 male 8-week-old athymic nude

mice (NMRI-nu/nu) obtained from M&B (Ry, Denmark) wereused. The mice were kept in laminar air flow benches. Theyreceived sterile food pellets and waterad libitum. Institutionalguidelines for animal welfare and experimental conduct werefollowed.

Tumor. The human glioblastoma cell line U 87 MG wasoriginally purchased from American Type Culture Collection(Rockville, MD). Xenografts were established by s.c. injectionof cells and maintained by serial transplantation. Prior to trans-plantation, the mice were anesthetized by a s.c. injection ofketamine (10 mg/kg) and xylazine (1 mg/kg) in 0.9% NaClsolution. Through a 1-cm incision in the dorsal skin, 1-mm3

tumor blocks were s.c. implanted into both flanks. For experi-ments, passage 2 and 3 were used. Intracranial tumors wereestablished by injection of 1–23 106 cells intracranially in theright hemisphere. Cells were injected 2 mm anterior to thesulcus coronarius, 2 mm lateral to the midline at 2-mm depth,using a stop device on the injection needle to ensure a repro-ducible implantation in all of the mice.

Treatment. Single-dose 10 Gy tumor X-radiation wasgiven antero-posteriorly to flank tumors and, as two opposinglateral fields including the whole brain, to mice with braintumors. The X-radiation was given during anesthesia with ket-amine/xylazine under aseptic conditions using a Stabilipan (Si-emens) therapeutic unit that yields 4.58 Gy/min at 300 kV.

TNP-470 (O-chloroacetyl carbamoyl fumagillol) was gen-erously provided by Leo Pharmaceutical Products (Ballerup,Denmark). A stock solution of 70% ethanol and TNP-470, 10mg/ml, was prepared and stored at 4°C. Immediately prior totreatment, the stock solution was diluted with 0.9% NaCl toobtain a dosing concentration of 1 mg/ml. TNP-470 was admin-istered as a daily s.c. injection, 6.7 mg/kg/d. This treatmentregimen was found in previous experiments to be efficient, withrelatively low toxicity (11).

Three parallel series of experiments were performed. Ineach experiment the mice were divided into four groups, given:(a) TNP-470 (6.7 mg/kg/d); (b) IR 10-Gy single dose; (c)TNP-4701 IR in the same doses; or (e) no treatment. TNP-470was administered for 1 week immediately before IR. In all of theexperiments, the IR was applied 1–2 h after the last TNP-470dose.

In a separate experiment with s.c. tumor transplants, treat-ment was initiated when an average tumor diameter of 7 mmwas reached, and tumors were excised 8 and 48 h after treat-ment. The excised tumor tissue was processed for morphologystudies and expression of angiogenic factors.

Effects on tumor growth were determined in two types ofexperiments: (a) in s.c. tumors, in which tumor growth wasmeasured; and (b) with intracranial tumor implants using sur-vival as end point.

Growth Calculations. The s.c. tumor size was measuredfive times per week. Two measurements in two perpendiculardimensions (d1 andd2) were recorded using a sliding gauge, andtumor volume [V(t)] curves were obtained according to thefollowing formula:

V~t! 5 0.35~d1~t! 3 d2~t!!3/2

To evaluate the effect of treatment on total tumor growth, wecalculated the time from the start of TNP-470 treatment until atumor volume of 600 mm3 was reached.

Fig. 1 Effects on s.c. tumor growth. Each curve represents medianvalue of 18–20 tumors in each group.Bars, interquartile range. Mann-Whitney U test was used for comparison.

Table 1 Time from treatment start (day 8) until a total tumorvolumea of 600 mm3 is reached

For each comparison Wilcoxon’s two-sample test was applied.

Treatment groupT600, days

median (range) P

Controls 13 (8–20)Combination (IR1 TNP-470) 28.5 (7–61)

vs.controls ,0.001vs.TNP-470 ,0.01vs. IR ,0.02

TNP-470 23 (12–36)vs.controls ,0.001

IR 23.5 (5–42)vs.controls ,0.01a A tumor volume of 600 mm3 was chosen as a representative size

of a large tumor in exponential growth (see Fig. 1).

972 TNP-470 and Ionizing Radiation



Nonparametric tests were used for statistics because q-qplots of the growth data clearly demonstrated a non-Gaussiandistribution.

Immunohistochemistry. Tissue was frozen in cooledisopenthane, and frozen sections were fixed in formalin andpostfixed in ethanol:acetic acid (2:1). CD31 immunostainingwas performed on sections from tumors in each group. Sectionswere washed in PBS and TBS and incubated with 10% normalrabbit serum for 30 min. They were then incubated with amixture of two monoclonal rat antimouse CD31 antibodies at adilution of 20mg/ml overnight at 4°C. The antibodies used wereclone 390 (Serotec Ltd.) and MEC 13.3 (PharMingen). RatIgG2a (Serotec Ltd.) was used as a negative control. Sectionswere incubated with biotin-conjugated rabbit antirat immuno-globulin (DAKO), at a dilution of 1:600 (2.3mg/ml) for 30 min,washed, and incubated with alkaline phosphatase-conjugatedstreptavidin (DAKO) at a dilution of 1:200 (1.5mg/ml) for 30min. As substrate for the alkaline phosphatase reaction, we usedfreshly prepared Fast Red Substrate System (DAKO), followedby a 10-min wash in tap water. After this, sections were coun-terstained with hematoxylin and mounted with aqueous mount-ing media.

Vessel Density. Vessel density was recorded as the num-ber of point counts of CD31-positive vessels per field, at3200,viewed through an ocular Chalkley Point Array (GraticulesLimited, Tonbridge, United Kingdom). Ten fields per section,randomly selected from nonnecrotic areas of tumors from con-trol and TNP-470 groups, were examined with a Leica DMRBmicroscope.

Preparation for Electron Microscopy. After a brief fix-ation in 70% Karnovsky’s fixative (3% paraformaldehyde and3.5% glutaraldehyde in 0.1M cacodylate buffer) each tumor wascut into tissue slices of 760mm with an EMS AutomaticOscillating Tissue Slicer (Electron Microscopy Sciences, FortWashington, PA). The slices were immersed in Karnovsky’sfixative for 1 h and subsequently washed and stored in 0.1Mcacodylate buffer. Six to ten small tissue blocks were randomlysampled from tumor slices from each group using a biopsyneedle with a diameter of 1 mm. The tissue blocks were furtherfixed in 1% osmium tetraoxide in 0.1M cacodylate buffer,dehydrated in ethanol followed by 1,2-propylenoxide, and em-bedded in Epon.

Ultrathin (70–80 nm) sections were cut on a RMC, MT6000-XL ultramicrotome. Sections were mounted on coppergrids and contrasted with 4% uranyl-acetate (30 min) and Reyn-olds lead-citrate (2.5 min). Ultrathin sections were examined ina JEOL electron microscope.

Northern Blotting. Tumor tissue blocks of approxi-mately 0.25 g were frozen in liquid nitrogen. PolyadenylatedRNA was extracted and isolated using the guanidinium thiocy-anate-based QuickPrep mRNA Purification Kit (AmershamPharmacia Biotech). The mRNA concentration was measuredby spectrophotometry. Threemg of denatured mRNA wereseparated on 1% agarose gels containing 6.6% formaldehydeand were subsequently transferred by capillary electrophoresisin 103 SSC buffer to a nylon membrane (GeneScreenPlus,NEN Research Products). The mRNA was cross-linked to themembranes with 1200 J UV light in a UV stratalinker 1800

Fig. 2 Cumulative survival of mice with intracranial tumors. Eachfigure A, B, andC is comprised of all of the mice.A, comparison of allof the groups. Each group represents 8–10 mice.B, all of the irradiatedversusall of the nonirradiated. IR significantly increased survival (me-dian survival, 33 daysversus 25 days; P , 0.002). C, all of theTNP-470-treatedversusall of the non-TNP-470-treated. There was noeffect of TNP-470 on survival.

Table 2 Time from IR (day 15) until triple tumor volume is reachedFor individual tumors the volume on day 15 (V15) was registered,

and time until tumor volume reached three times V15 was noted. Forcomparison, Wilcoxon’s two-sample test was applied.

Treatment groupTtriple, days

median (range)

Combination (IR1 TNP-470) 19 (12–33)P , 0.02

IR 16 (6–33)

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(Stratagene). Membranes were prehybridized for 3 h in 5%dextran sulfate, 50% formamide, 1% SDS, 1M NaCl, and 100mg/ml salmon sperm DNA at 42°C and hybridized with [32P]-labeled cDNA probes overnight at 42°C. The mRNA expressionwas visualized on a phosphorimager (STORM 840, MolecularDynamics Inc.), quantified by the software program ImageQuant version 5.0 (Molecular Dynamics Inc.), and the VEGF,bFGF, angiopoietin-1, and angiopoietin-2 expression was ad-justed to the GAPDH expression. To compare data from differ-ent membranes, mRNA from U87 cellsin vitro was loaded onall of the gels as a common standard. We used cDNA probesencoding VEGF, bFGF, angiopoietin-1 and angiopoietin-2, andGAPDH (Clontech).

Immunoblotting. Tissue blocks were thawed on ice andhomogenized in lysis buffer with four short bursts on a VibraCell 50 (Sonics & Materials Inc.). The protein concentrationwas determined with a BCA Protein Assay (Pierce Inc.) and20/40mg of protein from each tumor were separated on 1.0-mmNuPage 10% Bis-Tris gels (NOVEX) and transferred ontoPVDF membranes (NOVEX) by semi-dry blotting.

Membranes were blocked in Tris-buffered saline (pH 7.4),10% skim milk powder, and 0.1% Tween 20, and washed inTris-buffered saline (pH 7.4). Incubation with primary and

secondary antibodies for 1–16 h was followed by washing inTris-buffered saline (pH 7.4) and 0.1% Tween 20. The proteinexpression was visualized with ECL1 Plus (Amersham) andquantified by chemifluorescence scanning on a STORM 840(Molecular Dynamics Inc.). Expression of VEGF protein wasadjusted to thea-tubulin expression. The following antibodiesand concentrations were used: (a) monoclonal mouse-antihu-man VEGF (1mg/ml, PharMingen); and (b) polyclonal goat-antihuman bFGF (0.2mg/ml; R&D Systems). Monoclonalmouse-anti-a-tubulin (1:10,000) was obtained from SigmaChemical Co. Horseradish peroxidase-conjugated polyclonalgoat-antimouse-immunoglobulin (1:3000 and 1:5000) andhorseradish peroxidase-conjugated polyclonal rabbit-antigoat-immunoglobulin (1:2000) was obtained from DAKO.

RESULTSEffect of Ionizing Radiation and TNP-470 on Tumor

Growth. Both IR and TNP-470 significantly inhibited thegrowth of s.c. U87 tumors, and a significantly enhanced effectwas obtained by the combination of treatments (Fig. 1; Tables 1and 2). All of the tumors eventually regrew, but some tumors inthe combination group started growing very late after treatment.

Fig. 3 CD31 immunostaining of tumor tissue.Top, magnification3650. A, untreated controls.B, 8 h after irradiation;arrows, swelling andprotrusion of endothelial cells.C, 8 h after IR in tumors pretreated with 1 week of TNP-470. Vessel walls are slightly increased in thickness.Endothelial cells otherwise resembles controls.Bottom, magnification3100.D, untreated controls.E, TNP-470-treated after 1 week’s treatment withTNP-470. No systematic difference in vessel density is seen.




The time for tumors in the combination group to reach a volumeof 600 mm3 (T600, Table 1) was significant longer than in all ofthe other tumor groups. Intracranially implanted U87 tumorswere treated with TNP-470, IR, or combination therapy in thesame doses. In these mice, TNP-470 treatment did not affectsurvival (Fig. 2), whereas the expected effect of single-dose IRwas observed.

Effect of Ionizing Radiation and TNP-470 on TumorEndothelium. In tumors treated with ionizing radiation alone,a large fraction of the CD31-positive tumor vessels appearedirregular with swelling and luminal protrusion of endothelialcells at light microscopic examination 8 h after treatment. Thesechanges were not found in tumors pretreated with TNP-470 norin untreated controls (Fig. 3,A, B, andC).

Similar changes were found by electron microscopy.Here, a large fraction of the irradiated endothelial cells wereseen to be rounded, protruding into the lumen of the vessel,and with loss of contact between cells and loss of normalcellular organelles (Fig. 4A). At the ultramorphological level,untreated tumor vessel endothelium in nonnecrotic areas wasgenerally found to be continuous with tight junctions betweenendothelial cells (Fig. 4B). With light microscopy, an in-creased thickness of vessel walls was observed in TNP-470-treated tumors (Fig. 3C) and, correspondingly, by electronmicroscopy, an approximately 400 –700-nm thick layer ofelectron-dense material was ubiquitously observed along theendothelial basement membrane (Fig. 4C). This material wasfound in TNP-470-treated tumors with and without additionalradiotherapy but not in untreated controls nor in tumorstreated with ionizing radiation only.

Effect of TNP-470 Treatment on Vessel Density. After1 week of TNP-470 treatment, no significant difference invascular density was seen between treated and control tumors(Fig. 3,D andE), despite a pronounced growth-retarding effectof TNP-470. The mean number of vessel counts (Chalkley pointcounts) per field in TNP-470-treated tumors was 5.0 (4.5- 5.5)compared with 4.6 (4.2–5.0) in control tumors (parenthesesindicate 95% confidence intervals). At test for equality ofmeans yielded no significant difference (P 5 0.13).

Effect of Treatment on Expression of Angiogenic Fac-tors. The expression of VEGF, bFGF, angiopoietin-1, andangiopoietin-2 at mRNA level in different treatment groups wasexamined using Northern blots. Four membranes, each compris-ing tumor-tissue mRNA from one of the mice from each treat-ment group, were analyzed. We found no significant differencein GAPDH-adjusted expression of VEGF, angiopoietin-2, andbFGF between groups (Table 3 and Fig. 5), whereas a signifi-cantly greater expression of angiopoietin-1 was observed at 48 hafter withdrawal of the 1-week TNP-470 treatment (Table 3).

At the protein level we examined the expression of VEGFand bFGF with Western blotting. No difference in VEGF pro-tein was found between groups. The bFGF expression was verylow in all of the groups at both the mRNA and protein level.

The mRNA expression of VEGF, bFGF, angiopoietin-1,and angiopoietin-2 per total amount of mRNA was, withoutexception, greater in cultured U87 cellsin vitro than in thecorresponding U87 tumors grownin vivo (Fig. 5).

DiscussionOur findings document that pretreatment with TNP-470

significantly enhances the growth-retarding effect of ionizingIR on a s.c. transplanted human glioblastoma tumor line innude mice. TNP-470 therapy alone also produced a signifi-cant delay relative to untreated controls. As a result ofthe pretreatment with TNP-470, the tumors in the combina-tion-treatment group were smaller than the tumors in the IRgroup at the day of radiation. But this difference in size couldnot alone explain the difference in growth curves betweenthese two groups because the time for each tumor to reach a3-fold increase of its volume on the day of IR was longerin the combination-treatment group than in the IR group(Table 2).

Fig. 4 Electron microscopy.A, 8 h after irradiation. An endothelial cellis seen protruding into the lumen of the vessel.Arrow head, a gapbetween endothelial cells.B, untreated control. The endothelium iscontinuous with tight junctions between endothelial cells (arrowhead)and a normal basal membrane (arrow).C, TNP-470-treated tumor.Arrow, an approximately 400–700-nm endothelial basement membrane-related, electron-dense material;l, leukocyte in vessel lumen;t, tumorcell; e, endothelial cell.




In contrast, similar treatment schedules with TNP-470 hadno effect on the survival of mice with intracranial tumors, givenalone or in combination with IR. This discrepancy may reflectdifferences in tumor vessels due to differences in tumor micro-environment. In the present study, the TNP-470 treatment ofintracranial tumors was applied while tumors were relativelysmall, i.e., from day 7 to 14 after the injection of tumor cells.

Recently, Holashet al. (16) proposed a preangiogenic phase, inwhich small tumor implants are predominantly supported bycooption of existing brain vessels. They observed that intracra-nially injected glioma cells grew around existing vessels in thefirst 2 weeks, and thatde novoangiogenesis did not start untillater. This could explain the presumable lack of effect on theintracranial formation of tumor vessels in the present study

Fig. 5 Representative mem-brane showing expression of an-giogenic factors at mRNA level8 h and 48 h after treatment. An-giopoietin-1 mRNA expressionis up-regulated 48 h after thewithdrawal of TNP-470, bothwith and without IR. No signifi-cant difference in expression ofVEGF and angiopoietin-2 is seenbetween different treatmentgroups. bFGF expression is lowin both treated and untreated tu-mors, compared within vitro ex-pression. GAPDH is used asloading control.Bottom,no dif-ference in VEGF protein expres-sion is found.

Table 3 mRNA expression of angiogenic factorsEach value was corrected for background and GAPDH expression.

1IRa 2IR

1TNP-470 2TNP-470 1TNP-470 2TNP-470

8 h 48 h 8 h 48 h 8 h 48 h 8 h 48 h

VEGF 0.21 0.23 0.18 0.14 0.18 0.26 0.11 0.09(0.11–0.24) (0.20–0.27) (0.09–0.77) (0.04–0.17) (0.05–0.24) (0.07–0.47) (0.03–0.44) (0.03–0.10)

bFGF 0.02 0.03 0.01 0 0.02 0.02 0 0(0–0.03) (0–0.07) (0–0.04) (0–0.03) (0.01–0.05) (0.01–0.03) (0–0.004) (0–0.43)

Ang-1b 0.38 0.65c 0.14 0.26 0.22 0.49c 0.24 0.21(0–0.83) (0.43–1.08) (0.06–0.49) (0.16–0.38) (0–0.40) (0.35–0.72) (0.10–0.75) (0.01–0.58)

Ang-2 0.77 1.03 0.77 0.65 0.47 0.68 0.77 0.53(0.62–1.63) (0.64–1.27) (0.59–1.32) (0.64–1.26) (0.41–1.58) (0.62–1.21) (0.46–1.43) (0.39–1.76)

a Median values from 4 membranes; numbers in parentheses, range. Arbitrary units; mRNA from U87in vitro was loaded on each membraneand the U87in vitro value was set to 1.

b Ang-1, angiopoietin 1; Ang-2, angiopoietin 2.c Significant increased value compared to non-TNP-470 treated tumors.




because a much smaller tumor volume is lethal in the brain. Towhat extent the blood-brain barrier also plays a role for thedelivery of TNP-470 to endothelial cells in brain tumors isuncertain. The microenvironment of the brain stimulates forma-tion of a blood-brain barrier, and the U87 tumor environmentmay disrupt the vascular barriers through release of VEGF andother cytokines. According to the competitive environment hy-pothesis, the outcome of the competition between these envi-ronmental factors determines the intracranial tumor vessel per-meability (17). Previous reports on TNP-470 treatment ofintracranial gliomas are equivocal. Takamiyaet al. (18) treatedmice with U87 tumors with TNP-470 from day 5 after implan-tation until death and found prolonged survival compared withcontrols. Wilson and Penar (19) found no effect of TNP-470 onsurvival in mice with intracranial gliosarcomas.

We observed specific morphological effects of IR ontumor endothelium that apparently were avoided by pretreat-ment with TNP-470. Single-dose radiation in the same dosesas used here has been shown to induce an immediate increasein vascular permeability (20). On this basis, we suggest thatTNP-470 inhibits the acute endothelial cell membrane dam-age and edema that are normally seen after irradiation. We,therefore, propose a potential clinical synergy of the combi-nation of TNP-470 and radiotherapy because TNP-470 ap-parently prevents an acute side effect of ionizing IR whileadding to its beneficial effects.

The extensive accumulation of basement membrane mate-rial in vessels of tumors treated with TNP-470 is, to our knowl-edge, a new observation. Perivascular amorphous matrices notorganized as a conventional basal lamina have been observedafter gentamicin and cisplatin treatment in normal brain and insome malignant tumors and benign lesions4 (21). The compo-sition and nature of the basement membrane-related materialfound in TNP-470-treated tumors are not defined at present.Angiopoietin-1 is considered a regulator of vascular remodelingsubsequent to the formation of proliferating vascular sprouts byinteraction with pericytes through pathways distinct from thoseof VEGF (22, 23) and also in concerted action with VEGF (16).The increased expression of this proangiogenic factor aftertermination of TNP-470 treatment may be related to the mor-phological changes discussed above. This potential relationshipdeserves further experimental validation.

The tumor vessel endothelium in the nonirradiated tumorsin this study was generally continuous with tight junctionsbetween endothelial cells. Fenestrations were rarely seen. Oth-ers have found that U87 tumors on nude mice have fenestrationsin 37% of vessels, with 1–44 fenestrations per vessel profile(24). The reason for this difference is not clear. One explanationmay be the passage number used because they (24) used U87tumors passed more than 7 times on nude mice to select foraggressive, faster growing, and more angiogenic tumors. Weused passage 3 for our morphological studies.

There is a theoretical problem with combining radiotherapyand antiangiogenesis because the biological effect of ionizingirradiation is sensitive to hypoxia. Antiangiogenic agents, bytheir inhibition of vessel formation, are sometimes believed todecrease vessel density, and, as a consequence, tumor hypoxiawould increase. Assuming that angiogenesis is a growth-limit-ing step in tumor growth—which is why antiangiogenic therapyworks—the ratio of tumor cells to vessels,i.e., vessel density,must remain relatively constant in an individual tumor unless itsnutritive demands are changed. Concordantly, we observed nosignificant change in Chalkley counts of tumor vessels after 1week of TNP-470 treatment despite a significant growth inhi-bition. Also, with a 6-week TNP-470 schedule in human glio-blastoma xenografts, no changes in vascular density were re-ported (7), whereas others have actually found a decrease invessel density after TNP-470 treatment of brain tumors (8–10).

TNP-470 in combination with another antiangiogenic com-pound, minocycline, has been shown to actually decrease tumorhypoxia (25). We found no change in the VEGF expression,and, because VEGF is known to be up-regulated by hypoxia(26–28), this further supports our notion that TNP-470 does notinduce hypoxia.

In summary, pretreatment with TNP-470 significantly en-hanced the growth-retarding effect of ionizing IR, while pre-venting radiation-induced microvascular damage. A broadeningof the endothelial basement membrane and an increased expres-sion of angiopoietin-1 seem to be involved in this protectivemechanism. The vessel density was found unaltered by theantiangiogenic treatment, which further adds to the evidencethat vessel counts are not reliable parameters for the evaluationof antiangiogenic effect.

ACKNOWLEDGMENTSWe thank G. D. Yancopoulos (Regeneron Pharmaceuticals Inc.,

Tarrytown, NY) for generously providing angiopoietin-1 and angiopoi-etin-2 cDNA’s. We also thank H. A.Weich (Gesellschaft für Biotech-nologische Forchung, Braunschweig, Germany) for providing VEGFcDNA, and D. B. Rifkin, (New York University Medical Center, NewYork, NY) for providing bFGF cDNA. Finally, we thank AnnetteFischer, Ursula Hellhammer, and Jette Christiansen for excellent tech-nical assistance.

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2000;6:971-978. Clin Cancer Res Eva L. Lund, Lone Bastholm and Paul E. G. Kristjansen Angiogenesis in Human Glioblastoma Multiforme XenograftsEffects on Tumor Growth, Vessel Morphology, and Therapeutic Synergy of TNP-470 and Ionizing Radiation:

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