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Neoplasia. 2005 Sep; 7(9): 847–853.
PMCID: PMC1351316

Imaging of VEGF Receptor Kinase Inhibitor-Induced Antiangiogenic Effects in Drug-Resistant Human Adenocarcinoma Model1


Small molecule vascular endothelial growth factor (VEGF) receptor tyrosinase kinase inhibitors (VEGFR-TKIs) show great promise in inducing antiangiogenic responses in tumors. We investigated whether antiangiogenic tumor responses induced by an experimental VEGFR-TKI (AG013925; Pfizer Global Research and Development) could be reported by magnetic resonance imaging (MRI) during the initial phase of treatment. We used MRI and superparamagnetic nanoparticles for measuring relative vascular volume fraction (rVVF) in a drug-resistant colon carcinoma model. Athymic mice harboring MV522 xenografts were treated with VEGFR-TKI (25 mg/kg, p.o., with a 12-hour interval in between treatments) and were imaged after three consecutive treatments. Relative tumor blood volume fractions were calculated using ΔR2* maps that were scaled by the known VVF value of an in-plane skeletal muscle (1.9%). There was a pronounced and statistically significant (P < .001) decrease of tumor rVVF in treated animals (0.95 ± 0.24%; mean ± SEM, n = 66 slices, eight mice) compared to mice that received a placebo (2.91 ± 0.24%; mean ± SEM, n = 66 slices, nine mice). Tumor histology confirmed a three-fold decrease of vascular density and a concomitant increase of apoptotic cell index. Hence, we demonstrated that: 1) the VEGFR-TKI resulted in antiangiogenic effects that were manifested by a decrease or rVVF; and 2) iron oxide nanoparticles and steady-state MRI enable an early detection of tumor response to antiangiogenic therapies.

Keywords: VEGFR2 inhibitor, angiogenesis, MRI, tumor blood volume, iron oxide


Tumor cells and/or tumor-recruited inflammatory and stromal cells secrete chemokines and growth factors that induce the proliferation of microvascular endothelial cells. Vascular endothelial growth factor (VEGF) is one of the multifunctional cytokines that bind to extracellular domains of at least three different receptor kinases (VEGF receptor [VEGFR]; VEGFR1-VEGFR3) and neuropilins involved in antiapoptotic signaling, mitosis, and cell taxis. Thus, VEGF is an established target of antiangiogenic interventions [1]. In recent years, both receptor-blocking antibodies as well as small molecule inhibitors of receptor kinases have been developed as potential antiangiogenic drugs subject to many clinical trials [1,2]. These molecules are capable of attenuating VEGFR-mediated signaling, thereby causing strong anti-proliferative and antiangiogenic effects. These effects are caused not only by the attenuation of endothelial mitogenic signaling but also by the disruption of VEGF autocrine loop in cancer cells [3–5].

Several experimental VEGFR2 kinase (KDR) inhibitors have been tested in cancer models (e.g., SU5416 and SU6668 showed potent antiangiogenic responses resulting in a significant reduction of tumor vascular density, changes in vessel diameter, higher red blood cell velocity, and blood flow in remnant tumor vessels when compared with the control tumors) [6,7]. Consequently noted were abrogation of metastasis, microvessel formation, cell proliferation, and apoptosis [8]. Another experimental drug (SU11248) selectively inhibited both VEGFR2 and PDGF receptor beta phosphorylation and thereby caused the reduction of VEGF-mediated permeability of blood vessels [9]. Similar findings of reduced microvascular density, as well as blood flow changes, have been reported in renal carcinoma models after treatment with the PTK787/ZK 222584 inhibitor of VEGFR tyrosine kinase [10,11]. Other small molecule angiogenesis inhibitors (CGP 41251 [12] and PKC412 [13]) showed a reduction of neovessel formation in vivo. Another experimental VEGFR inhibitor, AG013736 (Pfizer Global Research and Development, San Diego, CA), has been effective in causing endothelial cell regression in experimental tumors with a 70% decrease in vascular density [14].

Furthermore, antiangiogenic drug research and development prompted the validation of imaging techniques designed for detecting and quantitating physiologic and molecular markers of angiogenesis (reviewed in Refs. [15,16]). The strength of the imaging approach is in the ability to perform survival experiments in intact animals. The above imaging detects changes in vascular endothelial marker expression and physiologic parameters before the changes in tumor volume become apparent.

Magnetic resonance imaging (MRI) combines excellent anatomic resolution and sensitivity to blood volume and flow changes caused by antiangiogenic treatments. For example, a combination of dynamic contrast-enhanced MRI with kinetic modeling has shown a potential for the detection of drug-induced changes in tumor microvessels (reviewed in Refs. [15,17]). For assessing changes in tumor blood volume, two essentially different MRI approaches were be used: 1) intravascular T1 agents (i.e., predominantly affecting longitudinal relaxation of proton magnetization) enable the quantification of relative as well as absolute blood volume fractions [18,19]; 2) T2 agents (a bolus of paramagnetic chelates [20,21] or an injection of superparamagnetic nanoparticles [11,22–24]) affect the spin-spin relaxation of water proton magnetization and enable relative blood volume change assessment by dephased proton population.

In the current study, we used a 1.5-T clinical MR scanner and steady-state imaging assisted by the injection of nanoparticles for detecting changes in tumor vascular blood volume induced by antiangiogenic therapy. We were specifically interested in determining whether iron oxide-enhanced MRI could assess blood volume changes early after initiating the treatment of MV522 human drug-resistant carcinoma xenografts with an experimental VEGFR2 kinase inhibitor.

Materials and Methods

Cell Culture

Human MV522 carcinoma cells (Pfizer Global Research and Development) were grown in 15% fetal calf serum and RPMI 1640 (Cellgro; Mediatech, Washington, DC).

Fluorescent Probes

Antidigoxigenin F(ab′)2 fragment (Roche Diagnostics, Indianapolis IN) was labeled with Cy3-mono-N-hydroxysuccinimide ester (Amersham Biosciences, Piscataway, NJ) [25]. Tomato lectin was labeled using Alexa Fluor 488 N-hydroxysuccinimide ester (Molecular Probes, Inc., Eugene, OR). Fluorescent conjugates were purified using Biospin P30 minicolumns (Bio-Rad, Hercules, CA).

Tumor Model

All animal experiments were approved and carried out according to the MGH Animal Care and Use Committee. Female nu/nu mice (25 g) were used for ectopic carcinoma xenografting (n = 18 total). Two million MV522 cells were injected in 100 µl of serum-free cell culture medium subcutaneously in the bilateral lower flanks of mice. Approximately 14 days after the implantation of cells, the animals with tumors of an average size of approximately 5 mm in diameter were divided into two groups (treatment and control) that underwent MRI at 1.5 T (see below). Control animals received three doses of 0.5% carboxymethyl cellulose (CMC) placebo (group 1, n = 8), whereas treated animals received three doses of AG013925, 25 mg/kg, p.o., bid, in 0.5% CMC within 36 hours (group 2, n = 9; time delay between dosing, 12 hours). One animal was excluded from the study based on lack of tumor growth. Following treatment, three radii of the tumors were measured using calipers and the volumes were calculated as VT = 4/3πABC, where A, B, and C are radii.

MRI of Mice

Animals were anesthetized using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Custom-made 30-G needle catheters were inserted into the tail vein and attached to a microheparin/saline flush unit. Anesthetized mice were placed prone with tumors located in the center of a custom-built transmit-receive parallel wound solenoid coil (30 mm diameter x 50 mm length; Nova Medical, Wilmington, MA) preheated to 37°C using a water jacket to avoid hypothermia. MRI data were collected using a 1.5-T Signa scanner (General Electric Medical Systems, Milwaukee, WI). After obtaining a fast-spoiled gradient-echo localizer sequence (repetition time TR/echo time TE: 34.0 msec/2.2 msec, 30° flip angle), multiple axial images of bilateral tumors in each animal were obtained (2 of 17 animals developed single tumors). All MRI acquisitions included a conventional gradient-echo sequence: TR/TE: 3000/20, 90° flip angle, and a matrix (frequency x phase = 256 x 128). The field-of-view was set at 6 x 6 cm, and the section thickness was 1.5 mm. All animals were imaged before and after an intravenous injection of 5 mg/kg monocrystalline iron oxide (MION-46L; Center for Molecular Imaging Research, Charlestown, MA) in 100 µl of phosphate-buffered saline. The hydrodynamic diameter of the size of these particles was 27.5 ± 6.8 nm; blood half-life was 11 hours in mice [26].

Steady-state tumoral blood volume maps were calculated from the precontrast and postcontrast monocrystalline iron oxide nanoparticle (MION)-enhanced MR images [24]. We assumed that the change in the transverse relaxation rate (ΔR2*) relative to the preinjection baseline relaxation rate was proportional to the perfused local blood volume per unit tumor volume (BV) multiplied by a function of the plasma concentration of the agent (C): ΔR2* = kt(C)BV.

Assuming that the MION distribution remains constant (steady-state distribution in blood) during the time of MRI experiment, the equation was simplified to a linear relationship between ΔR2* and the perfused blood volume fraction: ΔR2* = k′BV or BV = ΔR2* / k′, where ΔR2* is the change in the transverse relaxation rate of the tumor and BV is the tumor blood volume. The constant k′ = 4/3πγΔχB0 includes susceptibility change Δχ. The latter is blood pool agent concentration-dependent and, therefore, is dose-dependent.

Further, the change of relaxivity could be calculated as ΔR2* = [(1/T2* post) - (1/T2* pre)] = (-1/TE)[ln(Spost/Spre)], where Spost and Spre are MR signal intensities after and before MION administration, respectively (TE, echo time; T2*, transverse relaxation time). The maps depicting the change in transverse relaxation rate were calculated by using the CMIR-Image IDL-based software (courtesy of Dr. Ed Graves; Center for Molecular Imaging Research). On average, seven to nine MRI slices per tumor per animal were used in calculations (i.e., 14–18 measurements per animal were made due to bilateral tumors). Relative vascular volume fractions (rVVFs) were obtained by scaling measurements to a skeletal muscle with a known VVF of 1.9% [27]. Obtained data were analyzed by determining statistics per tumor and per group (by analyzing combined data obtained in each measured tumor slice; n = 66 per group). For statistical analysis of significance between obtained mean values, both rigorous (Student's t test) and less rigorous nonparametric (Mann-Whitney U test) tests gave comparable results.


Three randomly chosen animals from each group were injected with fluorescent labeled tomato (Lycopersicon esculentum) lectin approximately 10 minutes before the euthanasia. The above lectin specifically binds to fucose residues on the surface of mouse endothelial cells [17]. Three to five midline sections and two sections obtained from tumor edges (total: 5–7 sections per tumor, 10–14 sections per animal, and 30–36 sections per group) were stained for apoptotic cells and images were analyzed using fluorescence microscopy in two channels that enabled simultaneous analysis of vascular density, morphology, and occurrence of apoptotic tumor cells. Tissue collection and analysis were performed as in Ref. [25] with modifications: Alexa Fluor 488-labeled tomato lectin (50–80 µg per animal in 100 µl of saline) was injected 15 minutes before euthanasia (n = 2 in each group, four mice in total, eight tumor samples); tumors were excised immediately after the euthanasia, frozen in liquid nitrogen, and cut into 8-µm sections around the center (equatorial) part and the edges (10 sections per tumor sample). To determine DNA fragmentation in apoptotic cells, we used terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL) assay (ApopTag kit; Chemicon, Temecula, CA) in combination with a Cy3-labeled antidigoxigenin F(ab′)2 fragment (Roche Diagnostics). Fluorescence was observed and recorded in two channels using a Zeiss Axiovert TV100 microscope equipped with a CoolSnap HQ CCD camera (Photometrics, Tucson, AZ). Color coding and fusion of two fluorescence channels were achieved by using the IPLab Spectrum software (Scanalytics, Inc., Fairfax, VA).


Blood Volume Measurements in Tumors

The accuracy of the ΔR2*-based method of rVVF measurements depends on contrast agent distribution in vivo. The hydrodynamic diameter of MION particles is in the range of 20 to 30 nm [28], and their blood half-life is dose-dependent and species-dependent [24,26,29]. Thus, we initially determined whether the contrast agent (iron oxide nanoparticles) was restricted to MV522 tumor vascular compartment after systemic administration. Because the total time required for the MRI study did not exceed 1 hour, we tested whether iron oxide showed any extravasation into the tumor interstitium at the above time point. We addressed this question by using the histology of tumor tissue samples obtained from animals injected intravenously with 5 mg/ml iron oxide (MION) and euthanized approximately 1 hour postinjection (i.e., at a time point corresponding to the end of the MRI experiment). The histology of frozen tumor tissue sections stained with Prussian blue for iron and counterstained with eosin showed highly iron-positive blood vessels (Figure 1A). The study revealed no detectable iron staining outside the vessels within or outside the tumor boundaries.

Figure 1
Iron oxide-mediated imaging of relative tumor blood volume fractions in MV522 human drug-resistant colon carcinoma. (A) Prussian blue staining of iron oxides in tumor blood vessels (counterstaining with eosin) at 1 hour postinjection. Bar = 250 µm. ...

The initial histologic validation of MION in the MV522 human carcinoma model was followed by a MION-assisted T2-weighted MRI of bilateral flank tumors (Figure 1, B and C) performed in animals that were treated either with a placebo (Figure 1B) or with an inhibitor of VEGF type 2 receptor kinase (Figure 1C), in three doses within 36 hours in each group. Tumor volumes measured using calipers were 148 ± 36 mm3 (n = 16) in the placebo group vs 191 ± 38 mm3 (n = 16) in the VEGFR tyrosinase kinase inhibitor (TKI)-treated group. The differences between tumor volumes in these groups were statistically insignificant (P = .48). The use of bilateral tumors allowed imaging of two tumors at the same time in the same tomographic slice. This was feasible because MV522 did not show any substantial suppression of a contralateral tumor growth as only 2 of 17 mice developed a single tumor instead of two contralateral tumors. The following assumptions were made to calculate the blood volume: 1) under identical imaging conditions, the blood volume of the nearby skeletal muscle was steady; and 2) at the dose used in the study, MION injection resulted in image darkening (signal loss), which was proportional to the regional vascular volume. Sixteen-bit grey-scale tumor region-of-interest areas were then converted into pseudo-colored rVVF maps (Figure 1, B and C, color-coded scale) using skeletal muscle signal intensity as an intrinsic standard corresponding to 1.9% blood volume (VVF). The resultant assessment of the average rVVF values determined in the experimental group (n = 8 animals) and in the control group (n = 9 animals) showed that, on the average, rVVF was more than three times lower in animals treated with VEGFR2-TKI tumor than in the control group (Table 1). The differences in measured rVVF were highly significant (Figure 2, Table 1): in the VEGFR-TKI treatment group, rVVF was in the range of 0.95 ± 0.24% (mean ± SEM, n = 66 slices, eight mice) compared to the untreated group (2.91 ± 0.24%; mean ± SEM, n = 66 slices, nine mice). By performing a t-test of the means, assuming unequal variances within the groups or by using a nonparametric test, we obtained similar P factor values (P < .0001), suggesting a high statistical significance of differences between the mean measured rVVF values. In a separate experiment, mice were treated with VEGFR-TKI for a period of 6 days. In these animals, caliper-measured tumor volumes were three times lower in AG013925-treated groups (tumor volume 286 ± 69 mm3) than in control animals (tumor volume 753 ± 151 mm3). The above differences were statistically significant (P = .03).

Figure 2
The distribution of relative VVF values measured in each of the tumoral MRI tomographic slices in VEGFR-TKI-treated and placebo-treated groups. Mean ± SD is plotted for each group (total: 66 measurements per group). Skeletal muscle rVVF values ...
Table 1
Relative Blood Volume Fraction Measurements in MV522 Xenografts Using MION-Assisted MRI.

Correlative Histology

Histology of frozen sections was performed to establish the number and morphology of perfused tumor blood vessels, as well as the number of apoptotic TUNEL-positive cells. Imaging of tumor sections under blue excitation light showed markedly dilated blood vessels in control tumors (Figure 3A). Illumination under green excitation light revealed a relatively low frequency of Cy3 (TUNEL)-positive cells within the same sections (i.e., 6 ± 2 positive cells/mm2 section, on average). Overall, after the treatment, we observed an increase of the above number to 20 ± 4 cell/mm2 (P < .05). TUNEL-positive cells were unevenly distributed throughout tumor sections and were frequently assembled in clusters. In addition, in treated tumor sections, histology revealed an overall three-fold decrease of blood vessel densities from 60 ± 20 to 21 ± 8 vessels/mm2). Remarkably, lectin-positive vessels showed a drastic decrease in diameter and no longer appeared dilated (Figure 3C). In CMC placebo group, vascular diameter was in the range of 20 to 90 µm. After the treatment, the median diameter of tumor vessels did not exceed 20 µm. Standard hematoxylin-eosin stain also revealed the presence of cell death in treated tumors that had characteristic clusters of condensed nuclei (cf. Figure 3, D [shown with arrowheads] and B).

Figure 3
Correlative histology of MV522 frozen sections. VEGFR2 kinase inhibitor-treated tumors (panels A and B); control tumors (panels C and D). (A and C) Fluorescence histology showing Alexa Fluor 488-positive tumor blood vessels (green) and TUNEL-positive ...


Recent advances in iron oxide-mediated MRI staging of lymph nodes in cancer patients [30], as well as novel areas of iron oxide use in vascular and interventional radiology [31,32], accentuated the long-standing interest in this class of MR contrast agents. One of the potential applications of iron oxides in the clinical setting includes the measurement of blood volume fractions. The sensitivity of T2-type agents to subtle changes in blood volume makes them invaluable for assessing blood volume dynamics as a result of antiangiogenic treatments of cancer. The advantage of T2-type MIONs is in their negligible extravasation into the tumor interstitium during a typical MRI study (i.e., 45–60 minutes [24]; Figure 1A). In contrast, low-molecular-mass chelated gadolinium easily permeates through the leaky tumor vascular endothelium [33]. Rapid extravasation of contrast agents may present a problem for accurate measurements of VVFs in tumors [34]. Low-molecular-weight gadolinium-based agents enable measurements of vascular permeability changes (i.e., permeability-surface area product reflecting a decrease of vascular “leak”), which serve as an alternative to blood volume in many MRI studies of angiogenesis [35]. However, the above compound parameter depends on a vascular area that usually decreases as a result of vascular diameter change and vascular disintegration induced by the therapy [14,36], thereby introducing potential ambiguities when treated and nontreated tumors are compared. Therefore, in characterizing antiangiogenic therapies, blood volume MRI measurements with iron oxides, or, alternatively, T1 macromolecular agents [34,37–40] appear more suitable.

In the current study, we performed MION-mediated MR assessment of VVF changes in tumors treated with an experimental antiangiogenic drug that blocks signal transduction through a VEGFR2 receptor kinase. A potent and selective small molecule VEGF/PDGF RTK inhibitor, AG013925, demonstrated antiangiogenesis, antivascular permeability, and antitumor efficacy in preclinical studies. The compound dose dependently (25–100 mg/kg) induced tumor stasis in many tumor types, including syngeneic Lewis lung carcinoma and xenogeneic human drug-resistant MV522 carcinoma, in mice [41,42]. At the dose of 25 mg/kg, p.o., twice daily, the concentration of the drug in the plasma of mice surpassed the IC50 value required to stop VEGF-mediated proliferation of endothelial cells [41]. Thus, the above concentration was deemed sufficient to induce potent antiangiogenic effects in mice.

A combination of a steady-state phase MRI and the ultralong blood half-life of MION in rodents [22] (682 ± 34 minutes in mice [26]) enabled us to perform a quantitative analysis of vascular volume changes occurring at a very early stage of antiangiogenic treatment. Whereas tumor volumes measured after three doses of the VEGR-TKI were similar in the experimental and control groups, a drastic change of tumor vascular signal in response to the treatment was apparent by visual examination of MR images. The signal change translated into a three-fold drug-mediated decrease of rVVF. The above highly significant changes observed in treated groups of animals were confirmed by fluorescence histology showing a narrowing of lectin-perfused tumor blood vessels—a typical effect induced by various antiangiogenic drugs [43] with a moderate concomitant increase of TUNEL-positive (apoptotic) cell frequency. As demonstrated by continuing treatments for six more days, apoptosis in tumors of animals treated with the VEGFR-TKI becomes more prevalent and results in tumor growth inhibition. It remains to be determined whether reinjections of MION in the same animal would be useful for tracking blood volume changes in tumors over time. First, due to prolonged circulation in the blood and relatively slow biodegradation [29], and due to a high tumor vascular permeability, MION particles extravasate into the tumor interstitium 12 to 24 hours postinjection. Iron oxides do not extravasate into the normal muscle. Therefore, the accuracy of relative blood volume measurements could be low if performed several hours after intravenous administration. Second, rat tumor models should be used with caution because dextran-coated iron oxides could cause anaphylactoid responses [44,45]. Thus, as a general rule, tumor blood volume MRI measurements with dextrancoated iron oxides should be carefully timed and are most suitable for single (early assessment) or dual time point studies (i.e., early and late time points) due to the long half-life in the bloodstream.

In conclusion, the results of our study suggest that iron oxides at relatively low doses serve as excellent tracers of perfused vascular volume in human carcinoma xenografts in mice. We demonstrated that MION-assisted MRI could enable the monitoring of early tumor blood volume responses to therapy with VEGF receptor kinase inhibitors with a potential inclusion of other antiangiogenic drugs and translational studies.


The authors are grateful to David Shalinsky (Pfizer Global Research and Development) for useful discussions.


VEGF receptor tyrosinase kinase inhibitor
relative vascular volume fraction


1This work has been supported in part by National Institutes of Health (NIH) grants (1P50CA86355-01 (Project 2) and 5RO1 CA74424-01). W.R. received support from the German National Academy Foundation (Studienstiftung). D.T. was supported by the NIH Research Supplement for Underrepresented Minorities.


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