[CANCER RESEARCH 59, 5012–5016, October 1, 1999]

In Vivo Prediction of Vascular Susceptibility to Vascular Endothelial Growth FactorWithdrawal: Magnetic Resonance Imaging of C6 Rat Glioma in Nude Mice1

Rinat Abramovitch, Hagit Dafni, Eitzik Smouha, Laura E. Benjamin, and Michal Neeman2

Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel [R. A., H. D., E. S., M. N.], and Department of Molecular Biology, Hebrew University,Hadassah Medical School, Jerusalem 91010, Israel [L. E. B.]

ABSTRACT

One of the hallmarks of tumor neovasculature is the prevalence ofimmature vessels manifested by the low degree of recruitment of vascularmural cells such as pericytes and smooth muscle cells. This difference inthe architecture of the vascular bed provides an important therapeuticwindow for inflicting tumor-selective vascular damage. Here we demon-strate the application of gradient echo magnetic resonance imaging (MRI)for noninvasive in vivo mapping of vascular maturation, manifested by theability of mature vessels to dilate in response to elevated levels of CO2.Histological a-actin staining showed a match between dilating vesselsdetected by MRI and vessels coated with smooth muscle cells. Switchable,vascular endothelial growth factor (VEGF)-overexpressing tumors (C6-pTET-VEGF rat glioma s.c. tumors in nude mice) displayed high vascularfunction and significant vascular damage upon VEGF withdrawal. How-ever, damage was restricted to nondilating vessels, whereas mature dilat-ing tumor vessels were resistant to VEGF withdrawal. Thus, MRI pro-vides in vivo visualization of vascular maturity and prognosis of vascularobliteration induced by VEGF withdrawal.

INTRODUCTION

Remodeling of the vascular bed during angiogenesis initiates withthe formation of a fragile endothelial capillary bed. Vascular matura-tion and stabilization is a secondary process, which involves therecruitment of vascular smooth muscle cells and pericytes. The exist-ence of a window of plasticity in the immature vasculature allowsproper adjustment of vessel density to tissue needs during angiogen-esis. This plasticity window provides the possibility for selectiveobliteration of immature tumor neovasculature.

The therapeutic applicability of neovascular obliteration by antian-giogenic therapy depends on the fact that mature blood vessels are notsusceptible to such damage (1–3). One of the hallmarks of vesselmaturation is the recruitment of pericytes and smooth muscle cells (4,5). In the retina as well as in tumors, histological analysis hasestablished that pericyte coating is associated with the resistance ofvessels to VEGF3 withdrawal (2, 3). Interference with the associationbetween endothelial cells and pericytes results in severe vascularmalformations and increased vascular fragility, as detected recently inmice deficient in tissue factor, LKLF, and TIE2 and its ligand, ANG-1(6–9). It was therefore postulated that vascular maturation couldprovide a predictive marker for the sensitivity of neovasculature toVEGF withdrawal in antiangiogenic therapy and for vascular stabili-zation in proangiogenic therapy.

Damage of immature neovasculature was induced here by specificwithdrawal of VEGF using switchable C6-pTET-VEGF glioma cells(3, 10). VEGF, a central growth factor participating in the inductionof normal and pathological angiogenesis (11–16), is also essential forthe maintenance of immature blood vessels (3, 10, 17). In the C6-pTET-VEGF cells, VEGF165 is constitutively overexpressed in theabsence of tetracycline, and overexpression can be switched off by theaddition of antibiotics (10). Previous histological analysis of s.c.C6-pTET-VEGF tumors in nude mice revealed endothelial detach-ment and TUNEL-positive apoptotic endothelial cells 24 h afterVEGF withdrawal (10). Thus, VEGF is an essential survival factor fornewly formed tumor vasculature, as demonstrated previously forretinal angiogenesis (17). After VEGF withdrawal, histologically in-tact vessels were frequently associated with smooth muscle cells, asdetected by staining with a smooth muscle actin (3).

In the study reported here, we used hypercapnia (elevated CO2) andhyperoxia (elevated O2) for in vivo MRI analysis of vascular matu-ration and functionality. The rationale for this experimental approachis that pericyte and smooth muscle cell-coated vessels, in contrast withimmature endothelial capillaries, will dilate in response to hypercap-nia (VD). However, all functional blood vessels will show a change inhemoglobin saturation in response to hyperoxia, reflecting the capac-ity of erythrocytes to deliver inhaled oxygen to the tumor vasculature(VF). Both VD and VF can be detected by gradient echo MRI usingthe intrinsic contrast generated by changes in deoxyhemoglobin,blood volume, and blood flow (18–23).

MATERIALS AND METHODS

Animal Protocols. C6-pTET-VEGF cells were derived as reported previ-ously (10). Cells were cultured in DMEM supplemented with 5% FCS (Bio-logical Industries Israel), 50 units/ml penicillin, 50mg/ml streptomycin, and125 mg/ml fungizone (Biolab Ltd.) with the addition of 1mg/ml tetracycline(Sigma). The s.c. tumors were generated by inoculation of cells in the lowerback of male CD1-nude mice (2-month-old mice; body weight, 30 g; 106

cells/mouse). MRI experiments were initiated 16 days after inoculation. VEGFoverexpression was switched off by adding tetracycline (100mg/ml 1 5%sucrose) to the drinking water of the mice, as reported previously (10). Controlmice were given water with 5% sucrose for 48 h. Animal experiments wereapproved by the Animal Committee of The Weizmann Institute of Science.

MRI Measurements. MRI experiments were performed on a horizontal4.7 T Bruker Biospec spectrometer using an actively RF decoupled surface coil(2 cm in diameter) imbedded in a Perspex board and a bird cage transmissioncoil. Mice were anesthetized (75 mg/kg ketamine1 3 mg/kg xylazine, i.p.) andplaced supine with the tumor located at the center of the surface coil. Func-tionality and maturation of the neovasculature were determined from gradientecho images acquired during the inhalation of air, air-CO2 (95% air and 5%CO2), and oxygen-CO2 (95% oxygen and 5% CO2; carbogen). The differentgas mixtures were administered to the mice via a home-built mask. Fourimages were acquired at each gas mixture (117 s/image; slice thickness, 0.5mm; TR5 230 ms; spectral width, 25,000 Hz; field of view, 3 cm; 2563 256pixels; in plane resolution, 110mm; TE 5 10 ms; two averages). Otherexperimental details were as reported previously (18).

Data Analysis. MRI data were analyzed on an Indigo-2 work station(Silicon Graphics) using Paravision software (Bruker) and Matlab (The MathWorks Inc.).

VF was derived as reported previously (24) from images acquired during the

Received 2/16/99; accepted 8/6/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by research grants from the Israel Science Foundation and NIH Grant RO1CA75334-01A1 (to M. N.). R. A. is a recipient of a fellowship from the Charles Clorefoundation. M. N. is incumbent of a Research Career Development Award from the IsraelCancer Research Fund.

2 To whom requests for reprints should be addressed. Phone: 972-8-934-2487; Fax:972-8-934-4116; E-mail: lhneeman@wiccmail.weizmann.ac.il.

3 The abbreviations used are: VEGF, vascular endothelial growth factor; MRI, mag-netic resonance imaging; ANG, angiopoetin; TUNEL, terminal deoxynucleotidyl trans-ferase-mediated nick end labeling; VF, vascular function; VD, vasodilation; TE, echotime; LKLF, lung kruppel-like factor; TR, repetition time; RF, radio frequency; HB-EGF,heparin binding like growth factor.

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Fig. 1. In vivo analysis of VF and VD.a, gra-dient echo image of a C6-pTET-VEGF tumor ac-quired 48 h after VEGF withdrawal;b, photographof the same tumor showing the large vessels clearlyvisible by MRI. Functionality and maturation of theneovasculature were derived from such gradientecho images acquired during inhalation of air, air-CO2, and oxygen-CO2. Representative data of asingle slice from two different mice are presented(first mouse,a, b, and e–l; second mouse,m–p).Data were analyzed by two approaches. Color-coded VF maps (e, i, m,ando) and VD maps (f, j,n, andp) were derived, and values above a thresh-old (VF . 0.006 and VD. 0.01) were overlaid ona gray scale image. A pixel-by-pixelt test analysiswas used to determine functionality (g andk) andmaturation (h and l), and color-codedPs forP , 0.05 were overlaid on the gray scale image.c,values for VD and VF are defined in the codedcolor scale. Theblack barscale of 5 mm applies fore–l. d, color scalePs for the t test analysis ofvascular maturation and functionality.e–h, mapsderived for VEGF overexpression,i.e., before theadministration of tetracycline.i–l, maps derived48 h after switching off VEGF overexpression bythe addition of tetracycline.Arrowspoint to maturevessels, which are resistant to VEGF withdrawal;arrowheadspoint to immature vessels showing lossof function upon VEGF withdrawal.m–p, VF (mando) and VD maps (n andp) acquired before (mandn) and 10 h after (o andp) VEGF withdrawal.The black bar in m is a 5-mm scale form–p.

Fig. 2. MRI-detected vascular maturation coincides with resistance to VEGF withdrawal. VF and VD maps were acquired before and after VEGF withdrawal(6 tetracycline). Themean6 SE (n 5 6) values of VD and VF of four mice (one to two slices/mouse) are plotted for regions of interest selected at three locations:a, normal tissue, aproximately 7 mmfrom the border of the tumor;b, the tumor periphery; andc, the tumor center. Pairedt test analysis was applied to evaluate the effects of VEGF withdrawal. A highly significant lossof VF after the administration of tetracycline was measured at the tumor center (c; ppp, P 5 0.005, paired Studentt test;n 5 6). This loss of function correlated with vascular immaturitymanifested by the significantly reduced mean VD in the tumor center (c) relative to the tumor periphery (b; pp, P 5 0.01). Surprisingly, VEGF withdrawal resulted in elevated VDin the tumor center as well as in normal tissue (a andc; p, P 5 0.05).

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inhalation of oxygen-CO2 (95% oxygen and 5% CO2) and air-CO2 (95% airand 5% CO2) using the following equation:

VF 5 bDY 5 ln~Ioxygen-CO2/I air-CO2!/~TE p CMRI! (1)

WhereIoxygen-CO2andIair-CO2 are the mean signal intensity during the inhala-tion of oxygen-CO2 and air-CO2, respectively; Y is the fraction of oxyhemo-globin; b is the volume fraction of blood; and CMRI 5 599 s21 at 4.7 T (18).This parameter measures the capacity of erythrocyte-mediated oxygen deliveryfrom the lungs to each pixel in the image (18). The sensitivity for detection offunctional vessels will be reduced for highly oxygenated blood and mighttherefore differ between arterial and venous vessels. It is important to note,however, that oxygenation of s.c. blood in anesthetized mice is relatively low(17.7 and 15.7 mmHg in s.c. arterioles and venules, respectively; Ref. 25).

VD was derived from air and air-CO2 images using the following equation:

VD 5 ln~Iair-CO2/I air!/~TE p CMRI! (2)

in which Iair is the mean signal intensity during inhalation of air. Positive VDcorresponds to increased signal intensity by hypercapnia due to increasedblood flow.

In addition to this analysis, a pixel-by-pixelt test analysis of VD and VFwas performed showing a map of significance of change in signal intensitybetween images acquired during inhalation of air and air-CO2 and betweenimages acquired during inhalation of air-CO2 and oxygen-CO2, respectively.Results analyzed using both approaches yielded a similar distribution formature and functional vessels (Fig. 1). Data are presented in color overlayed onthe gray scale baseline image for values of VD. 0.01, VF . 0.006, andP , 0.05.

The change in signal intensity due to hypercapnia (VD) was found to bepredominantly a result of a change in the apparent T1 relaxation due to achange in blood flow. On the other hand, the signal change due to hyperoxia(VF) was due to a change in T2* due to a change in blood oxygenation.4

RESULTS

Vascular functionality and maturation in C6-pTET-VEGF tumorswere determined by MRI at three time points, immediately before and10 and 48 h after the administration of tetracycline (Figs. 1–3; Tables1 and 2). Control C6-pTET-VEGF tumors were imaged at two timepoints (48 h apart) without the addition of tetracycline (Table 2). Thetumors were then fixed for histological analysis. Mice were imagedduring inhalation of air, air-CO2, or oxygen-CO2, and the images wereused for derivation of the VF and VD maps (see “Materials andMethods”).

Overexpression of VEGF (i.e., before the addition of tetracycline)led to the generation of fully functional blood vessels, as evidenced bythe significant signal enhancement (P 5 0.0014;n 5 4) detected uponinhalation of oxygen-CO2 relative to inhalation of air-CO2 (Fig. 1, eandg; Fig. 2,b andc; Table 1). The extent of VF, which was highestat the tumor periphery and lowest in normal tissue, was in agreementwith the spatial distribution of blood vessels (Fig. 1b).

Images acquired during inhalation of air and air-CO2 (VD) showeda significant change in signal intensity for normal vessels and vesselsin the tumor periphery (P 5 0.04;n 5 4; Table 1). VD in response tohypercapnia was significantly lower in the tumor center relative to thetumor periphery (Fig. 1,f andh; Fig. 2, b andc). Maturation of thetumor vasculature appeared to proceed from the margins of the tumorinward and could represent secondary migration of supporting muralcells such as pericytes and vascular smooth muscle cells. The lack ofresponse to CO2 (low VD) within the tumor was not due to hypovas-cularity, as evidenced by the high density of functional vasculature(high VF; Fig. 1,e andg). The bimodal response in the VF and VD

maps enabled us to define two distinct populations of functional tumorblood vessels that differ in their degree of maturation.

Forty-eight h after switching off VEGF overexpression by addingtetracycline to the drinking water, VF within the treated tumors was4 H. Dafni and M. Neeman, unpublished observations.

Fig. 3. Vascular dilation detected by MRI corresponds to histologically mature vessels.a, MRI analysis of VF 48 h after tetracycline administration.b, VD of the same tumor.c,H&E staining of a section in the same plane as the MRIs shown ina and b. d,magnification of theboxed regionin c shown with thearrowheadsin a andb. This regionmaintains VD and VF and contains large peripheral blood vessels leading into the tumor.e, anti-smooth muscle actin staining confirms that these blood vessels (arrows in d ande)that retain VD are covered by smooth muscle.f, lectin staining of endothelium shows thatthe peripheral and functional blood vessels are intact (black arrows), whereas thoseimmediately adjacent but inside the tumor are not intact (white arrow). g, a highermagnification of lectin staining inside the tumor demonstrates that staining is primarily ofvessel remnants damaged by VEGF withdrawal, and not intact blood vessels.p, necroticregion of the tumor aids in orienting the histology with the MRI (a, b, c). h, TUNELanalysis of programmed endothelial cell death (arrows) in the same region as shown ing.i, magnification of one capillary fromh showing TUNEL-positive endothelial cellssurrounding a lumen with a RBC (arrowhead). Lectin, anti-smooth muscle actin, andTUNEL staining were done as reported previously (3).

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significantly reduced (P 5 0.005; Fig. 1,i and k, arrowheads; Fig.2c). Damage to the vascular bed detected by MRI was not immediate,and, accordingly, no reduction in VF was detected 10 h after theadministration of tetracycline (Fig. 1,m ando; Table 2), a time pointat which no damage to endothelial cells could be detected in histo-logical sections. The results at 48 h are consistent with impairedoxygen delivery and massive vascular damage associated with VEGFwithdrawal for tumor blood vessels. TUNEL-positive staining ofendothelial cells was observed in tumor regions in which MRI meas-urements showed a loss of VF (Fig. 3). However, 48 h after VEGFwithdrawal, no TUNEL staining was detected in the tumor cells, thusshowing the direct and selective effect of VEGF withdrawal onendothelial cell survival (Fig. 3).

VEGF withdrawal did not lead to complete collapse of the entiretumor-induced vasculature. Vessels at the immediate periphery oftumors and a few vessels within the tumors effectively managed toescape destruction by VEGF withdrawal and continued to showsimilar oxygen-dependent signal modulation and significant VF (Fig.1, i andk). We therefore examined the VD maps to test the hypothesisthat vascular maturation is correlated with vessel resistance. Indeed, inall cases, tumor and normal vessels showing CO2-induced dilation(high VD) were refractory to vascular occlusion induced by VEGFwithdrawal (Fig. 1,arrows). Furthermore, vascular maturation (VD)detected by MRI matched the maturation defined by the smoothmuscle cell coating of vessels, as revealed in corresponding histolog-ical sections of the same tumors (Fig. 3).

DISCUSSION

The induction of angiogenesis in solid tumors initiates with thestimulation of proliferation and migration of endothelial cells. Apivotal role has been implicated for VEGF and its receptors in theseearly stages of vascular sprouting (26). However, recent studies sug-gested that the role of VEGF is not completed with the establishmentof a primary endothelial plexus and the initiation of perfusion. In fact,VEGF is essential for maintaining the viability and functionality ofendothelial cells in these immature vessels, and acute withdrawal of

VEGF results in endothelial programmed cell death and vascularcollapse, as reported previously (2, 3, 10, 17) and shown here.

Stabilization of the vascular bed is achieved by recruitment ofpericytes and smooth muscle cells. Vascular maturation has long beenrecognized as an integral component of angiogenesis (5, 27). Some ofthe molecular signals that can promote vascular maturation have beenrevealed recently. For example, platelet-derived growth factor hasbeen implicated in the proliferation and migration of pericytes (28),and HB-EGF has been demonstrated to induce VEGF secretion byvascular smooth muscle cells (29). TIE2 and its ligands, ANG-1 andANG-2, were recently shown to control vascular maturation (30, 31).ANG-1 promotes the association of vessels with pericytes and appearsin the later stages of angiogenesis, whereas its inhibitor, ANG-2,induces dissociation of pericytes. ANG-2 is thus elevated in the earlystages of angiogenesis and in immature neovasculature.

Angiogenesis in the normal ovarian cycle is characterized by tightregulation of pericyte migration, which correlates well with the pat-terns of expression of ANG-1 and ANG-2 (32). In contrast, in tumors,the vascular bed is frequently immature, with many vessels devoid ofpericytes or smooth muscle cells. Recent studies showed that hyper-vascularized tumors were characterized by elevated expression ofANG-2 (33, 34). Thus, the low degree of maturation of the tumorvasculature provides an attractive window for specific obliteration ofthe tumor neovasculature (3). This finding calls for efficient nonin-vasive methods that would enable us to classify the degree of vascularmaturation in tumors for patient selection and follow-up of treatment.

Vascular functionality and maturation were differentially mappedhere by the change in MRI signal intensity in response to hyperoxia,which changed hemoglobin saturation (VF), and hypercapnia, whichaffected signal intensity due to the change in blood flow (VD). VFmapping by MRI using hyperoxia is a valid approach for s.c. tumorsin anesthetized mice, in which hemoglobin in arterial blood is notfully oxygenated. It must be noted, however, that in well-oxygenatedorgans, VF can be assessed by alternative MRI methods, includingsignal changes in response to hypoxia, as demonstrated previously forthe brain (35), and arterial spin labeling (36) or by tracking exog-enously administered contrast material. Mapping of vascular matura-tion by signal changes associated with increased flow in response tohypercapnia, as reported here, is not sensitive to blood oxygenationand should therefore be applicable in any organ.

We show here that tumor vascular maturation, which is manifestedby smooth muscle cell recruitment, can be detected by MRI throughthe response to CO2-induced VD. Moreover, we show that the mat-uration detected by MRI marks the window of susceptibility of tumorvessels to VEGF withdrawal. Thus, noninvasive high-resolution MRIprovides anin vivo approach for three-dimensional assessment of VFand maturation (VD). MRI could provide a tool for prediction ofvascular response to anti-VEGF treatment of tumors as well as fornoninvasive assessment of vascular maturation during proangiogenictherapy.

Table 1 Effects of hyperoxia and hypercapnia on signal intensity inC6-pTET-VEGF tumors

Gas mixture

Tumor Normal vessel

Signal intensitya P Signal intensitya P

Air 767,0006 11,000 830,0006 20,000Air-CO2 772,0006 2,500 0.35b 881,0006 15,000 0.04b

Oxygen-CO2 832,0006 12,000 0.0014c 976,0006 15,000 0.002c

a Data are presented for two characteristic regions: a tumor with immature, nondilatingfunctional vasculature and a normal region with mature vessel (mean6 SE;n 5 4). Fourimages were acquired at each gas mixture (117 s/image; slice thickness, 0.5 mm;TR 5 230 ms; spectral width, 25,000 Hz; field of view, 3 cm; 2563 256 pixels; in planeresolution, 110mm; TE 5 10 ms; two averages).

b Student’st test, airversusair-CO2.c Student’st test, air-CO2 versusoxygen-CO2.

Table 2 Reproducibility of the assessment of VD and VF in C6-pTET VEGF tumors

Control mice (n 5 8; two slices/mouse) were imaged, and VD and VF maps were derived (t 5 0). The mice were then given 5% sucrose in the drinking water without (n 5 6;two slices/mouse) or with tetracyclin for the last 10 h (n 5 2; two slices/mouse). No significant changes in VD or VF were observed under these conditions, in which there was novascular damage (two-tailed unpairedt test). Some local effects were observed in VD maps at 10 h after the administration of tetracycline as shown in Fig. 1,n andp.

VF VD

Normal tissue Tumor rim Tumor center Normal tissue Tumor rim Tumor center

t 5 0 0.0036 0.002 0.0066 0.006 0.0066 0.003 0.0016 0.001 0.0056 0.005 0.0026 0.001548 h sucrose 0.0036 0.0015 0.00756 0.006 0.00536 0.002 0.00246 0.0016 0.0086 0.006 0.0026 0.02

P 0.89 0.55 0.47 0.15 0.26 0.69

t 5 0 0.0046 0.0008 0.0076 0.003 0.0086 0.002 0.0016 0.001 0.0136 0.005 0.0046 0.000810 h tetracycline 0.0016 0.0015 0.0056 0.002 0.0066 0.0009 0.00256 0.0006 0.0156 0.001 0.0056 0.0006

P 0.67 0.65 0.6 0.3 0.79 0.4

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ACKNOWLEDGMENTS

We thank Prof. Eli Keshet, Prof. Yoram Salomon, and Dr. Peter Bendel forhelpful suggestions.

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