AG 013736

AG-013736, a novel inhibitor of VEGF receptor tyrosine kinases, inhibits breast cancer growth and decreases vascular permeability as detected by
dynamic contrast-enhanced magnetic resonance imaging

Lisa J. Wilmesa,*, Maria G. Pallavicinib, Lisa M. Fleminga, Jessica Gibbsa, Donghui Wangc, Ka-Loh Lia, Savannah C. Partridged, Roland G. Henrya, David R. Shalinskye, Dana Hu-Lowee,
John W. Parkb, Teresa M. McShanef, Ying Lua, Robert C. Brascha, Nola M. Hyltona
aDepartment of Radiology, University of California San Francisco, San Francisco, CA 94143-1290, USA
bSchool of Natural Sciences, University of California Merced, Merced, CA 95344, USA
cComprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143-1710, USA
dDepartment of Radiology, University of Washington, Seattle, WA 98109-1023, USA
eResearch Pharmacology, Pfizer Global Research and Development, San Diego, CA 92121, USA
fPfizer Oncology, Pfizer Global Pharmaceuticals, Mystic, CT 06355, USA
Received 28 February 2006; accepted 19 September 2006

Dynamic contrast-enhanced MRI (DCE-MRI) was used to noninvasively evaluate the effects of AG-03736, a novel inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, on tumor microvasculature in a breast cancer model. First, a dose response study was undertaken to determine the responsiveness of the BT474 human breast cancer xenograft to AG-013736. Then, DCE-MRI was used to study the effects of a 7-day treatment regimen on tumor growth and microvasculature. Two DCE-MRI protocols were evaluated: (1) a high molecular weight (MW) contrast agent (albumin-(GdDTPA)30) with pharmacokinetic analysis of the contrast uptake curve and (2) a low MW contrast agent (GdDTPA) with a clinically utilized empirical parametric analysis of the contrast uptake curve, the signal enhancement ratio (SER). AG-013736 significantly inhibited growth of breast tumors in vivo at all doses studied (10–100 mg/kg) and disrupted tumor microvasculature as assessed by DCE-MRI. Tumor endothelial transfer constant (Kps) measured with albumin-(GdDTPA)30 decreased from
0.034F0.005 to 0.003F0.001 ml minti 1 100 mlti 1 tissue ( P b.0022) posttreatment. No treatment-related change in tumor fractional plasma volume (fPV) was detected. Similarly, in the group of mice studied with GdDTPA DCE-MRI, AG-013736-induced decreases in tumor SER measures were observed. Additionally, our data suggest that 3D MRI-based volume measurements are more sensitive than caliper measurements for detecting small changes in tumor volume. Histological staining revealed decreases in tumor cellularity and microvessel density with treatment. These data demonstrate that both high and low MW DCE-MRI protocols can detect AG-013736-induced changes in tumor microvasculature. Furthermore, the correlative relationship between microvasculature changes and tumor growth inhibition supports DCE-MRI methods as a biomarker of VEGF receptor target inhibition with potential clinical utility.
D 2007 Elsevier Inc. All rights reserved.
Keywords: Dynamic contrast-enhanced MRI (DCE-MRI); Vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor; Anti-angiogenic;
Breast cancer

1. Introduction
Growth factor receptor tyrosine kinases are promising targets for cancer therapy [1,2]. Vascular endothelial growth factor (VEGF), a member of the VEGF-platelet-derived
growth factor (PDGF) supergene family, and its signaling pathways have been implicated in vasculogenesis, angio- genesis and vascular permeability [3,4]. VEGF plays an important role in tumor blood vessel formation, which in turn supports tumor growth and metastases [5,6]. The VEGF signaling pathway is activated by ligand-induced phosphor-

4 Corresponding author. Tel.: +1 415 476 1950; fax: +1 415 476 8809. E-mail address: [email protected] (L.J. Wilmes).
0730-725X/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mri.2006.09.041
ylation of the VEGF receptors (VEGFRs). The blocking of VEGFR phosphorylation by a kinase inhibitor is expected to

disrupt VEGF signaling pathways resulting in changes in tumor vascular characteristics and growth.
AG-013736 is a small molecule RTK inhibitor with picomolar potency against VEGFR 1, 2, 3 and nanomolar potency against PDGFR-h and c-Kit. AG-013736 has demonstrated antitumor activity in cancer xenograft models, including melanoma, lung, colon and breast [7,8]; however, its antitumor or effects in the HER2-overexpressing BT474 human breast cancer xenograft have not been established. This agent has also been evaluated in a Phase I clinical trial in solid tumors including breast cancer [9].
In this work, we investigated the dose dependence of AG-013736 antitumor effects on BT474 breast cancer xenografts in mice. Furthermore, since changes in tumor microvasculature induced by a VEGFR inhibitor are likely to accompany tumor growth inhibition, we sought to characterize these changes using dynamic contrast-enhanced magnetic resonance imaging. The ability to monitor changes in tumor microvasculature may provide indicators of early treatment response and may also increase the understanding of the basis of the response.
Dynamic contrast-enhanced MRI (DCE-MRI) is a noninvasive method used both preclinically and clinically to assess vascular changes associated with antitumor therapies [10–13]. DCE-MRI techniques employ contrast agents and provide information about tumor microvascula- ture structure and function. Our group and others have used DCE-MRI to evaluate tumor response to neoadjuvant chemotherapy in patients with breast cancer [14–17].
DCE-MRI using high molecular weight (MW) contrast agents, also known as macromolecular contrast media, has been used to generate quantitative measures of tumor microvasculature such as endothelial transfer constant and fractional plasma volume in animal models [18]. Because high MW contrast agents have a low first-pass extraction from normal vessels, they are ideal for characterizing tumor microvasculature, which is typically hyper-permeable to macromolecules. However, these high MW contrast agents are not clinically approved, thus limiting the clinical translatability of associated imaging techniques.
DCE-MRI using low MW, clinically approved contrast agents has also been used to evaluate tumor microvascula- ture. However, it is more difficult to calculate quantitative measures of perfusion and permeability from DCE-MRI data acquired with low MW contrast agents for a number of reasons, including the high first-pass extraction of low MW contrast agents, complex data acquisition requirements and limitations of the pharmacokinetic models employed [12,13]. Therefore, empirical parametric methods are commonly used to analyze DCE-MRI data in the clinical setting.
After establishing the dose response of the BT474 breast tumor xenografts to AG-013736, DCE-MRI was used to directly study the relationship between potential drug- induced perturbation of tumor microvasculature and tumor growth inhibition. Two DCE-MRI strategies were evaluated for their ability to detect AG-013736-induced changes after 7

days of treatment. The first method utilized the prototype high MW contrast agent albumin-(GdDTPA)30 [19] with pharmacokinetic modeling of the contrast uptake data. The second method used the clinically approved low MW contrast agent gadopentetate dimeglumine (GdDTPA) with an empirical parametric analysis of the contrast uptake data. All MRI studies were performed on a 1.5-T clinical scanner to facilitate translation of the results to future clinical studies.
The overall goal of this study was to evaluate the antitumor effects of AG-013736 in the BT474 model of human breast cancer using (1) a dose response study of tumor growth inhibition; (2) a high MW DCE-MRI technique; (3) a low MW, clinical DCE-MRI technique and (4) histological assessment of tumor tissue.

2.1.Tumor model and treatments
Immune-deficient female mice (Nu/nu; age 8–12 weeks; Charles Rivers, Hollister, CA, USA) were implanted subcu- taneously with 17h estradiol pellets (0.1 mg/pellet, Innova- tive Research of America, Sarasota, FL, USA) 24–48 h prior to tumor cell injection. Human BT474 breast cancer cells, optimized for growth in vivo, were obtained from the UCSF Cancer Center and were implanted subcutaneously into the axillary flank (2ti l07 cells/injection). The study was per- formed with approval of our institution’s Committee for Animal Research and in accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals. Tumor volumes were estimated using caliper measurements. Caliper volume was calculated as 0.4tiLtiW2, where L and W are the geometric length and width of the tumor, respectively.
In order to establish the dose-dependent tumor growth inhibition by AG-013736 in the BT474 breast cancer model, tumor-bearing mice were randomized into four groups (10–12 mice/group), when tumors approximated 250 mm3. Animals received daily oral (po) dosing of AG-013736 (10, 30 or 100 mg/kg in 0.5% carboxymethylcellulose; CMC) or an equivalent volume of the vehicle for 3 weeks. For subsequent DCE-MRI studies, tumors were allowed to grow to an average size of approximately 400 mm3 prior to baseline imaging. Tumor-bearing animals received AG-013736 (25 mg/kg), dissolved in polyethylene glycol/
H2O (30:70), or an equivalent volume of vehicle intra- peritoneally twice daily for 7 days.
2.2.MR image acquisition
MR images were acquired on a 1.5-T Signa whole-body MRI scanner (General Electric Medical Systems, Milwau- kee, WI, USA). Mice were imaged in pairs (one treated and one control animal) using a wrist radiofrequency coil (Medical Advances, Milwaukee, WI, USA) and a custom- ized animal holder. Axial images were obtained for tumor localization. Precontrast tumor T1 was measured using a 3D

variable flip angle fast gradient echo technique [20]. Contrast-enhanced imaging was performed using a coronal T1-weighted 3D gradient echo sequence (TR=10.2 ms, TE=4.2 ms, flip angle=208, FOV=10ti 10 cm, imaging matrix=256ti192, slice thickness=1.0 mm, 1 NEX, acqui- sition time=63 s).
For DCE-MRI studies, five baseline scans were acquired, then mice were injected via the tail vein with either 0.03 mmol/kg albumin-(GdDTPA)30 or 0.2 mmol/kg GdDTPA (Magnevist; Berlex Laboratories, Wayne, NJ, USA) fol- lowed by postcontrast imaging of 40 or 20 min, respectively. Mice were anesthetized with 1.5% isoflurane administered via an MR-compatible mobile inhalation anesthesia system (Vet Equip, Pleasanton, CA, USA). The tail vein was cannulated before placing mice in the magnet. DCE-MR images were acquired prior to initiating treatment with AG-013736 and again after 7 days of treatment.
2.3.MRI image analysis
Image analysis was performed on SUN workstations (SUN Microsystems, Santa Clara, CA, USA) using in-house software developed in Interactive Data Language (Research Systems, Boulder, CO, USA). Total MRI tumor volume was calculated from T1-weighted 3D images by manually delineating tumor boundary regions of interest (ROIs) on sequential MR images over the whole tumor and summing the volumes.

2.3.1.DCE-MRI data analysis: albumin-(GdDTPA)30
Quantitative analysis of dynamic contrast-enhanced images from the albumin-(GdDTPA)30 study was performed using a two-compartment unidirectional pharmacokinetic model incorporating precontrast tissue T1 values to estimate tumor endothelial transfer coefficient (Kps) and fractional plasma volume (fPV) for the tumors [21,22]:

compares early to late enhancement levels to characterize rates of contrast washout in the tumor and has previously been shown to correlate with markers of tumor aggressive- ness in clinical studies [25].
First, the initial percent enhancement (PE) over the whole tumor was calculated on a voxel-by-voxel basis to determine the level of contrast uptake. PE is defined as
0)/S0, where S0 is the precontrast signal intensity and Searly is the early postcontrast signal intensity measured at t =0.5 min. SER was then computed for tumor voxels
0), and Slate is the late postcontrast signal intensity measured at t =5 min. Peak SER and peak PE values were determined by taking the highest eight-contiguous-pixel average over the entire tumor. The percentage of total tumor volume with an SER N 1.3 (%Vol SERhigh) was also computed. An additional measure of tumor volume was evaluated by subdividing total MRI-ROI tumor volume into (1) early GdDTPA enhancing voxels (VolCE), having PE N 25%; and (2) nonenhancing voxels (Volnon-CE), having PE b 25%. The following variables — peak PE, peak SER, %Vol SERhigh and VolCE — were computed for treated and control tumors.
Mice were euthanized immediately after the day 7 imaging, and tumor tissues were collected for histological analysis. Tumors were sectioned along the plane corresponding to the DCE-MRI. Half of each tumor was fixed in formalin for hematoxylin and eosin (H&E) staining using standard protocols, and the remainder was frozen in OCT for CD-31 staining to evaluate microvessel density. OCT tumor samples were sectioned and fixed in acetone. Endogenous peroxidase activity was quenched by incubat- ing the section in 3% H2O2. The sections were blocked with 5% rabbit serum (Vector Laboratories, Burlingame, CA, USA) and incubated with antibody against CD-31 (Mec

Ct ðtÞ ¼ Kps
Cp t V


13.3, Pharmingen, San Diego, CA, USA). The sections were exposed to biotinylated rabbit antirat IgG (BA-400, Vector

where Ct and Cp are the concentration of contrast medium in tissue and plasma, respectively.
The model was fit to the average signal intensity uptake curve for all pixels in the tumor volume and assumes no reflux of the contrast agent from the tumor extracellular extravascular space into the vascular compartment occurs during the time course of the experiment [23]. Individual vascular input functions were determined from enhancing voxels in the abdominal aorta of each mouse and were assumed to have mono-exponential decay.

2.3.2. DCE-MRI data analysis: GdDTPA
GdDTPA contrast-enhanced images were assessed using an empirical parametric three-time-point method, the signal enhancement ratio (SER), to characterize enhancement kinetics in the tumors. The SER method was developed at our institution for clinical breast cancer studies [24]. SER
Laboratories) followed by incubation in horseradish perox- idase conjugated streptavidin (Vector Laboratories). The immune complexes were visualized using DAB substrate.
2.5.Statistical analyses
Statistical analysis was performed using JMP V5.1 (SAS Institute, Cary, NC, USA) and Excel functions. Mean and standard deviations of tumor volume for each dose group on different follow-up days were calculated. A mixed-effects model of tumor volumes as a function of dose and longitudinal follow-up time was used to assess the treatment effect on tumor volumes. The random effect in such a model is the animal identity. The tumor volume was transformed using a natural logarithm transformation to improve the goodness of model fit. Linear contrasts were used to assess the relationship between tumor volume and treated doses. The mixed-effects model included data from days 22 to 34. For imaging studies of AG-013736 treatment effects on

tumor volume and microvasculature parameters, the pre- treatment values were first compared using a two-tailed t test. A t test was also used to evaluate the significance of changes after 7 days as well as the differences in changes between treatment groups.

3.1.Dose response study in BT474 human breast tumor xenograft model
The relationship between AG-013736 dose (0, 30, 50 and 100 mg/kg) and tumor growth inhibition was assessed using caliper measurements to estimate tumor volume. Treatment with AG-013736 resulted in significantly decreased tumor growth for all treated groups ( P b.0001) (Fig. 1). However, the treatment effect of AG-013736 was not significantly dose dependent ( P N.3441). Tumors treated by AG-013736 grew 4% slower per day than untreated tumors with a 95% confidence interval between 3% and 6%. Toxicity, as evaluated by treatment-related deaths or reduced body weight, was minimal at all dose levels.
3.2.AG-013736-induced tumor volume changes measured by caliper and MRI
In the group of mice evaluated with albumin-(GdDTPA)30 DCE-MRI, tumor volume changes were calculated using both caliper measurements and 3D-MRI images. Caliper- based calculations of tumor volume showed a treatment- related tumor growth inhibition after 7 days, with an average volume increase of 52 mm3 in the AG-013736-treated group (n =5) vs. a 214-mm3 increase in the control group (n =5; P =.0449). MRI tumor ROI measurements in the same animals showed a larger post-treatment difference between the two groups, measuring an average tumor volume decrease of 147 mm3 in the treated group and an average increase of 257 mm3 in the control group ( P =.0006).

Fig. 1. Tumor volume dose response curves (meanFS.E.) as measured by caliper for groups of mice (n =10–12) treated for 21 days with 10, 30 or 100 mg/kg of AG-013736 or control vehicle (0 mg/kg). Treatment was initiated 25 days post-tumor cell implantation. A significant reduction in tumor growth was seen for all AG-013736 doses vs. controls ( P b.05).

Fig. 2. Mean change in tumor volume after 7 days of AG-013736 treatment for the cohort of mice studies with GdDTPA DCE-MRI, treated (n =3) and control (n =3) caliper-measured volume change (A); MRI-measured volume change calculated from tumor ROIs defined on 3D images (B); MRI volume change calculated from early GdDTPA-enhancing voxels (VolCE) within the tumor (C). Significant differences in 7-day tumor volume change for treated vs. control groups were found for MRI ROI volume (*P =.034) and MRI enhancing volume (yP =.009).

A similar result was found in the group of mice evaluated with GdDTPA DCE-MRI. Caliper measurements of tumor volume showed a small AG-013736-induced inhibition of tumor growth after 7 days, with an average tumor volume increase of 282 mm3 in the treated group compared to an average increase of 370 mm3 in the control group ( P =.5043) (Fig. 2A). MRI tumor ROI measurements in the same animals showed an average 30-mm3 decrease in tumor volume after 7 days in the treated group and an average increase of 270 mm3 in the control group, a significant difference ( P =.0335) (Fig. 2B). The early GdDTPA contrast-enhancing tumor volume (VolCE) was also calculat- ed for this group and showed the greatest treatment-related effect of the three volume measurement techniques. The average VolCE in treated animals decreased 160 mm3 after 7 days, while the control animals showed an average increase in VolCE of 213 mm3 ( P =.0090) (Fig. 2C).
3.3.AG-013736-induced changes in tumor microvascular parameters: DCE-MRI with albumin-(GdDTPA)30
Kps and fPV values were used to evaluate the effects of AG-013736 on permeability and fractional plasma volume, respectively, of the tumor microvasculature. Fig. 3A shows representative color maps of baseline and 7-day post- treatment distribution of tumor Kps values in a pair of mice (one control, one treated). Prior to treatment, both control and treated tumors show voxels with high Kps values. After 7 days, a large decrease in voxels with high Kps values was seen in the AG-013736-treated tumor, but not in the control. These changes are also reflected in Fig. 3B, showing plots derived from Eq. (1), in which the slope corresponds to the fitted Kps value and the y-intercept corresponds to fPV.
Average tumor Kps values were measured for a group of 12 tumor-bearing mice (seven control, five treated). No significant difference in mean Kps between control and

Fig. 3. Color maps of Kps pixel values in the central tumor slice for a pair of mice (one control and one AG-013736 treated), shown prior to and after 7 days of treatment (A). Kps values for the control tumor (left) remained fairly constant after 7 days, while the Kps values in the treated tumor (right) were greatly decreased, with few pixels having Kps values N 0.02. The slopes of the contrast uptake curve fitting (B) also reveal the decrease in permeability, as seen by the decreased slope at 7 days for the AG-013736-treated tumor relative to the control tumor.

treated groups was found prior to AG-013736 treatment. After 7 days, the control group Kps values decreased from 0.036F0.005 to 0.024F0.004 ml minti 1 100 mlti 1 tissue ( P =.018) (Fig. 4). A much larger 7-day decrease was observed in the AG-013736-treated group, with K ps decreasing from 0.034F0.005 to 0.003F0.001 ml minti 1 100 mlti 1 tissue ( P b.0022). Although a reduction in vessel permeability to albumin-(GdDTPA)30 after 7 days was observed in both groups, the extent of the decrease was significantly greater in the AG-013736-treated group than in the control group ( P =.0021).
Pretreatment tumor fPVs were similar for both treated and control groups with no significant change measured for either group post-treatment. In the AG-013736-treated group, the average post-treatment tumor fPV value was 1.0F0.1%, compared to a baseline average fPV of 1.2F0.2%. In the control group, the average post-treatment tumor fPV value was 1.2F0.1%, compared to a baseline average fPV of 1.4F0.1%.
3.4.AG-013736-induced changes in tumor microvascular parameters: DCE-MRI with GdDTPA
SER analysis was used to evaluate tumor contrast uptake curves in a smaller cohort of mice studied with GdDTPA DCE-MRI. Representative tumor SER color maps for a pair of mice (treated and control) show a decline in high SER values 7 days post-treatment (Fig. 5A) that is not seen in the control mouse. In the treated group, mean peak SER values prior to treatment (2.40F0.03) decreased signifi- cantly to 2.23F0.05 after 7 days ( P =.042). Post-treatment
%Vol SERhigh was also decreased, changing from 41.8% to 26.5% ( P =.015) (Fig. 5B). For the control group, the 7-day mean tumor peak SER remained constant at 2.36 and the mean tumor %Vol SERhigh decreased slightly from 42.6% to 41.8%. No clear trend was seen for peak PE in either group.

Fig. 4. Tumor Kps values (meanFS.E.) prior to (Day 0) and after 7 days of treatment for AG-013736-treated (n =5) and control (n =7) groups. A highly significant decrease in Kps is seen for the treated group (*P b.0022) after 7 days, with a smaller but significant decrease also found for the control group (§P =.018). Tumor Kps in the treated group was significantly lower than in the control group (yP =.0021) on Day 7.

Fig. 5. (A) Color maps of SER pixel values in the central tumor slice for a control and AG-013736-treated mouse prior to (Day 0) and after 7 days of treatment. Post-treatment, the number of high SER values (red pixels) in- creased in the control tumor, while the number of high SER pixels markedly decreased in the treated tumor. (B) Percent of tumor pixels (meanFS.E.) with high ( N 1.3) SER values in tumor prior to and after 7 days of treatment with AG-013736 for a group of treated (n =3) and control (n =3) mice. A marked decrease in %Vol SERhigh was seen for treated mice after 7 days.

Histological analysis was used to validate that AG- 013736 perturbed tumor microvasculature. Tumor micro- vessels, visualized with an anti-CD31 antibody, were markedly reduced in number in AG-013736-treated tumors as compared to control tumors (Fig. 6A and B). Large vacuolated areas devoid of cells in AG-013736-treated tumors seen on H&E staining are consistent with drug- induced necrosis and cell death (Fig. 6C and D).

In this study, AG-013736 was administered in a breast cancer xenograft model in nude mice. This model features highly tumorigenic BT474M1-6 human breast cancer cells, derived by in vivo selection from the HER2/neu-over-

Fig. 6. Representative histologic findings in a control tumor (A,C) and an AG-013736-treated tumor (B,D). CD31 staining of tumor sections reveals a decrease in microvessel density (brown staining, indicated by arrows) in the treated tumor (B) as compared to control tumor (A). H&E staining shows an increase in necrosis and vacuolated areas (indicated by arrows) with treatment (D) not seen in the control tumor (C). Magnification, ti10.

expressing BT474 cell line. A number of anticancer agents, including standard chemotherapy, anti-HER2 monoclonal antibody trastuzumab and novel antibody-targeted liposo- mal agents (immuno-liposomes), have been tested previ- ously in the BT474 and related tumor models [26]. This is the first time an anti-angiogenic agent has been tested for antitumor efficacy in this model.
The results of the dose response study demonstrated that daily administration of AG-013736 for 3 weeks significant- ly inhibited the growth of BT474 breast cancer xenografts at doses in the range shown to be effective in other xenograft tumors [7]. These data are further evidence of AG-013736’s broad spectrum of antitumor activity in preclinical models.
An intermediate effective AG-013736 dose was chosen for subsequent DCE-MRI studies to assess tumor response after a shorter 7-day treatment. While the tumor growth inhibition at this early treatment time point was less than that observed with the 3-week schedule, tumor volume changes calculated from 3D-MRI regions of interest revealed signif- icant differences between the treated and control tumors for both the albumin-(GdDTPA)30 and GdDTPA DCE-MRI groups. In comparison, the post-treatment differences detected for the same groups using tumor volumes calculated from 2D caliper measurements were found to be less significant and not significant, respectively.
These data illustrate the capability of MRI to detect early and relatively small changes in tumor volume and in this study suggest that MRI measurements are more sensitive
than two-dimensional caliper volume estimates. The in- creased sensitivity of MRI-based measurements may result from a more accurate approximation of tumor volume, particularly in cases of irregular tumor shapes that are not well approximated by the ellipsoid equation of the caliper measurement. The sensitivity of MRI to changes in tumor volume may have the potential utility of reducing the numbers of animals needed for assessing antitumor efficacy in preclinical trials of novel therapies.
In DCE-MRI of tumor xenografts and many human tumors, it is generally accepted that early GdDTPA enhance- ment reflects well-vascularized tumor tissue, while slowly enhancing regions are more likely to reflect necrotic tissue. Our data demonstrated that the tumor volume with early GdDTPA contrast enhancement was dramatically reduced after AG-013736 treatment. This measure of tumor volume also showed the greatest decrease with treatment when compared to caliper and 3D-MRI measured volumes.
VEGFR and PDFGR are critical for tumor angiogenesis [27]. We therefore used DCE-MRI with the high MW contrast agent albumin-(GdDTPA)30 to quantify changes in tumor microvasculature permeability or volume that might be associated with the observed AG-013736-induced tumor growth inhibition. After 7 days of AG-013736 treatment, the average tumor endothelial transfer coefficient, Kps, a measure of the exchange between the vascular and extracellular space, decreased by 92%. The smaller decrease in tumor Kps observed for the control group at 7 days may

be related to tumor growth and accompanying maturation of vessels, resulting in decreased permeability. In a DCE-MRI study of MCF7 breast tumor xenografts, Bogin et al. [28]
found that GdDTPA permeability rate constants were significantly higher in small tumors than in large tumors. The large decrease in tumor Kps we observed in the treated group is consistent with AG-013736-induced reductions in microvessel permeability observed using dye exclusion assays [7] and loss of endothelial cell fenestrations in tumors detected with microscopy [29] in other animal models. DCE-MRI studies of other anti-angiogenic agents have reported decreases in tumor Kps values in conjunction with reduced microvessel density staining [30,31]. Our data showed a similar relationship between a decrease in Kps and reductions in CD31-labeled microvasculature.
No reduction in tumor fPV was observed in association with the decreasing microvessel density resulting from AG- 013736 treatment. Treated tumors displayed similar fPV values as control tumors 7 days post-treatment. Consistent with this result, Inai et al. [29] utilized fluorescence microscopy to study tumor vessels in AG-013736-treated and control RIP-Tag2 tumors, and found no significant difference in vessel diameter after 7 days. Other DCE-MRI studies of anti-angiogenic agents have reported decreased tumor Kps values in conjunction with decreased microvessel density, with no change in fPV [30]. A limitation of microvessel density measurements is that they indicate the number of microvessels, but not the diameter. Indeed, Drevs et al. [32] observed decreases in tumor Kps and microvessel density accompanied by an increase in fPV after treatment with an angiogenesis inhibitor. In that study, microvascular corrosion casting revealed an increased diameter in the remaining tumor vessels in the treated group relative to controls. Thus, MRI measurements complement histological analyses and lend insight into mechanisms potentially contributing to decreased tumor growth.
We performed DCE-MRI studies with the high MW contrast agent albumin-(GdDTPA)30 to obtain quantitative measures of potential changes in microvascular permeability and fractional plasma volume following AG-013736 treat- ment. However, since high MW contrast agents are not approved for clinical MRI studies, we additionally sought to determine whether AG-013736 microvasculature changes could be detected using DCE-MRI with a low MW contrast agent. We used the clinically approved contrast agent, GdDTPA, with SER analysis that closely paralleled the contrast-enhanced breast MRI performed clinically at our institution [25].
DCE-MRI with GdDTPA detected a significant decrease in the parameters of peak SER and %SERhigh in tumors after 7 days of AG-013736 treatment. SER measurements characterize the GdDTPA uptake curve, particularly the washout component, in tissue. High SER values have been shown to correlate with high microvessel density and tumor grade in human breast cancer [25]. Computer simulation has shown that SER can be highly correlated to a physiokinetic

parameter (kep), the flux rate between the extravascular extracellular space and plasma.1 Kep has been reported to be closely correlated with tissue VEGF expression in breast tumors [10].
In conclusion, this study showed that AG-013736 treatment resulted in tumor growth inhibition and perturba- tion of tumor microvasculature in the BT474 model of human breast cancer. Both albumin-(GdDTPA)30 and GdDTPA DCE-MRI strategies were able to noninvasively detect AG-013736-induced changes in tumor microvascula- ture that occurred in conjunction with histologically verified decreases in microvessel density and cellularity. This work supports the potential role of DCE-MRI for preclinical and clinical evaluation of VEGF target inhibition.

The authors thank Maren Grazzini and Young Kim (Pfizer, La Jolla) for technical assistance in the performance of the MRI experiments and Marlene Wiart and Viktor Novikov (UCSF) for helpful discussions. We are grateful to Yanjun Fu (UCSF) for providing the albumin-(GdDTPA)30. This work was supported by Pfizer Global Research and Development and NIH grants CA069587 and CA82923.

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