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Carcinogenesis Advance Access originally published online on June 16, 2006
Carcinogenesis 2006 27(10):2133-2139; doi:10.1093/carcin/bgl113
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commerical License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer

R.A. Goodlad1,3,*, A.J. Ryan2, S.R. Wedge2, I.T. Pyrah2, D. Alferez1,3, R. Poulsom1, N.R. Smith2, N. Mandir1, A.J. Watkins1 and R.W. Wilkinson2

1 Histopathology Unit, London Research Institute, Cancer Research UK 44 Lincoln's Inn Fields, London WC2A 3PX, UK
2 Department of Cancer and Department of Infection Research and Safety Assessment, AstraZeneca Pharmaceuticals Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
3 Department of Histopathology, Division of Investigative Science, Imperial College, Hammersmith Hospital London W12 0NN, UK

*To whom correspondence should be addressed. Tel: +44 (0) 207 269 3086; Fax: +44 (0) 207 269 3491; Email: r.goodlad{at}cancer.org.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ApcMin/+ mouse model is a clinically relevant model of early intestinal cancer. We used AZD2171, an oral, highly potent and selective vascular endothelial growth factor (VEGF) signaling inhibitor, to investigate the role of VEGF receptor-2 (VEGFR-2) signaling in adenoma development and growth in ApcMin/+ mice. AZD2171 (5 mg/kg body wt/day) was administered once daily for 28 days to 6-week-old (early-intervention) or 10-week-old (late intervention) mice. In the early-intervention study, AZD2171 reduced the number of macroscopic polyps in the small bowel and colon. Macropolyp diameter was lower in the small bowel, but remained unchanged in the colon. In animals receiving AZD2171, microscopic evaluation of the small intestine showed a significant reduction in the number of larger lesions. In the late-intervention study, AZD2171 treatment reduced macropolyp diameter (but not number) in the small intestine. Microscopic analysis revealed that AZD2171 significantly reduced the number of larger micropolyps in the small bowel, with no large micropolyps present in the colon. AZD2171 treatment had no effect on microvessel density or localization of ß-catenin staining in adenomas or non-tumor intestinal tissue, but significantly reduced the number of cells expressing VEGFR-2 mRNA. In conclusion, the effects of AZD2171 in the small intestine of ApcMin/+ mice are consistent with an antiangiogenic mechanism of action, limiting growth of adenomas to ≤1 mm. These data also suggest that an early step in adenoma development may depend on VEGFR-2 signaling. Together, these results indicate that VEGFR-2 signaling may play key roles in the development and progression of intestinal adenomas.

Abbreviations: Apc, adenomatous polyposis coli; Min, multiple intestinal neoplasia; VEGF, vascular endothelial growth factor; VEGFR-1/2/3, VEGF receptor-1/2/3


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Each year around 945 000 people worldwide are diagnosed with cancer of the colon and rectum, and ~492 000 patients die of the disease (1). While there has been success in prevention and treatment of colorectal cancer, the treatment of more advanced disease remains an area of high unmet need. In colon cancer, one of the earliest molecular events is loss of function of the Apc (adenomatous polyposis coli) gene product (2). Germline Apc mutations are found in the autosomal dominant condition, familial adenomatous polyposis (FAP), where patients develop numerous colorectal adenomas and early onset colorectal carcinoma. The importance of the Apc gene product was confirmed by the demonstration that Apc mutates in the majority of sporadic human colorectal cancers (3). APC plays a central role in the regulation of ß-catenin, a key intracellular mediator of the Wnt signaling pathway. A cytoplasmic complex between ß-catenin, APC, axin and GSK3 results in phosphorylation of ß-catenin and its subsequent proteolytic degradation. In the absence of functional APC, ß-catenin is not degraded and consequently translocates to the nucleus where it activates the transcription of genes controlling cell growth, differentiation and survival (4).

A mouse model of spontaneous intestinal cancer has been described that involves loss of the wild-type Apc allele in animals that have inherited a germline inactivating mutation in the Apc gene (5). Multiple intestinal neoplasia (Min) mice are heterozygous at the Apc locus, carrying one mutated, functionally inactive allele, and one wild-type allele (ApcMin/+). Loss of the wild-type allele within the intestinal epithelial cells of ApcMin/+ mice leads to the development of multiple adenomas. Between 60 and 100 adenomas form in the small intestine, and between 3 and 6 adenomas form in the colon of 10-week-old ApcMin/+ mice (5). Intravillus adenomas similar to those in the ApcMin/+ mouse have been observed in the duodenums of FAP patients (6), suggesting that the ApcMin/+ mouse represents a clinically relevant model of intestinal tumorigenesis (7,8).

Sustained angiogenesis is one of the hallmarks in the development of cancer (9). Pre-neoplastic lesions initially lack angiogenic ability, but at some point early in development they can gain the ability to activate host endothelial and perivascular cells in order to form new vascular capillaries (10). Targeting new vessel growth is therefore an attractive therapeutic approach given that, with the exception of the female reproductive system, the vasculature of the normal adult is quiescent.

Vascular endothelial growth factor (VEGF) signaling is considered pivotal in regulating endothelial cell proliferation and migration, neovascular survival and vascular permeability by binding to the specific transmembrane receptors, VEGF receptor-1 (VEGFR-1, Flt-1) and VEGF receptor-2 (VEGFR-2, KDR, Flk-1), on endothelial cells (11,12). In particular, VEGF binding to VEGFR-2 and activation of the intrinsic VEGFR-2 tyrosine kinase activity is required to propagate an angiogenic response (13).

A number of approaches to inhibiting VEGF signaling are under investigation, including sequestration of VEGF-A with an antibody, use of a blocking antibody to VEGFR-2 to prevent ligand binding and inhibition of VEGFR-2 tyrosine kinase activity with small molecules (14). AZD2171 is an oral, highly potent and selective VEGF signaling inhibitor that inhibits all known VEGFR tyrosine kinases (VEGFR-1, VEGFR-2 and VEGFR-3) (15), and it is currently under investigation in a broad range of tumors, including colorectal cancer. Data obtained with AZD2171 are consistent with potent inhibition of VEGF signaling, angiogenesis, neovascular survival and tumor growth. In human endothelial cells in vitro, AZD2171 inhibits VEGF-induced phosphorylation of VEGFR-2 and VEGF-induced proliferation with IC50 values of 0.5 and 0.4 nM, respectively (15). Once-daily oral dosing of AZD2171 ablated VEGF-induced angiogenesis in vivo, and significantly inhibited the growth of established subcutaneous human tumor xenografts (colon, lung, prostate, breast and ovary) (15). In this study, we have used AZD2171 to evaluate the effects of inhibiting VEGF signaling on adenoma development in ApcMin/+ mice.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
All procedures, including breeding of ApcMin/+ mice, were approved by the Cancer Research UK Animal Ethics Committee and covered by the appropriate licenses under the UK Home Office Animal Procedures Act, 1986. ApcMin/+ heterozygote mice were originally obtained as a gift from Amy R. Moser (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI). Male mice were back-crossed to female C57BL/6J and the resultant embryos were transferred by aseptic hysterectomy to foster mothers in specific pathogen-free isolators. All breeding was subsequently by brother-(C57BL/6J-ApcMin/+)/sister-(C57BL/6J) mating. The general PCR conditions, including primer pairs and amplification, have been described previously (16) for detection of the Apc mutation.

Study design
For macroscopic polyp analysis, 6-week-old (early-intervention study) or 10-week-old (late-intervention study) ApcMin/+ mice were given drug vehicle only (1% polysorbate 80 in de-ionized water; control) or AZD2171 (5 mg/kg body wt/day) by once-daily oral gavage (0.1 ml/10 g body weight, n = 12 mixed male/female animals per group) for 28 days. For each treatment, additional satellite groups (n = 4–6 animals) were set up for analysis of micropolyps. Mice were humanely killed 24 h after the last dose of AZD2171 or vehicle.

Macroscopic assessment of tumor burden
Macroscopic assessment of tumor burden was performed as described previously (17). A dissecting microscope (x20 magnification) was used to assess the number and size (mean of two largest diameters measured with digital calipers) of polyps in the small and large intestines. Polyp volume was derived from polyp diameter; consistent with their histological appearance, a hemispherical shape was assumed for the small bowel polyps and a spherical shape for colon polyps. Tumor burden was calculated as the product of polyp number and polyp volume (18).

Microscopic assessment of tumor burden
The small intestine was sectioned, as described previously (17), and, together with the colon, the tissues were rolled and processed to paraffin blocks in a standard manner. Five 4 µm ‘step sections’, at least 100 µm apart, were cut and stained with hematoxylin and eosin (H&E) and scored morphologically for micropolyps. The total numbers of each category of tumor in the five-step sections were recorded for each region (proximal, middle and distal) of the small bowel and also for the colon. Micropolyps were assigned to a category from 1 to 4: polyps that remained within the limits of a single villus were classified as category 1; those within the space of >1–5 villi were classified as category 2; >6–10 villi were classified as category 3; and >10 villi were classified as category 4 (19).

Immunohistochemistry
Formalin-fixed paraffin-embedded small bowel samples from control and AZD2171-treated ApcMin/+ mice were sectioned at 4 µm. ß-catenin was detected using a mouse anti-ß-catenin monoclonal primary antibody (1 : 100; Dako, Ely, UK). After counterstaining with hematoxylin, the slides were assessed in a blinded semi-quantitative manner. Ten high-power fields (x40) per slide were assigned a score for the proportion and intensity of staining in both the cytoplasm and nuclei. For proportion: 0 = no staining; 1 = 0–33% stained; 2 = >33–66% stained; 3 = >66–100% stained. For intensity: 0 = no staining; 1 = 0–33% intensely stained (i.e., remainder are weakly stained or not stained); 2 = >33–66% intensely stained; 3 = >66–100% intensely stained.

Blood vessels were detected using a goat anti-mouse CD31 polyclonal primary antibody (1 : 50; Santa Cruz, sc-1506) and a rabbit anti-human von Willebrand factor (vWF) polyclonal primary antibody (1 : 200; Dako). A quantitative ‘point counting’ method was used. Ten high-power fields (five from both normal and tumor tissue) from each of the 4–6 mice per group (control and treated) were scored blinded for ‘hits’ using a 50-point Weibel 2 graticule, with the number of hits being proportional to the volume fraction of the vasculature (20,21).

In situ hybridization for VEGFR-2 mRNA
Formalin-fixed paraffin-embedded small bowel samples were stained for specific localization of VEGFR-2 mRNA by in situ hybridization using an antisense riboprobe synthesized with T7 RNA polymerase using 35S-UTP (~800 Ci/mmol; Amersham-GE, Amersham, UK) and template prepared from IMAGE clone 5359101 (MRC Geneservice, Babraham, Cambridge, UK), linearized with EcoRI to yield antisense probe that was used without hydrolysis. The methods for pre-treatment, hybridization, washing and dipping of slides in Ilford K5 for autoradiography were as described by Senior et al. (22) for formalin-fixed paraffin-embedded tissue, with modifications (23). The presence of hybridizable mRNA in all compartments of the tissues studied was established in near-serial sections using an antisense ß-actin probe. Autoradiography was performed at 4°C (two exposures per section at 7 and 14 days for VEGFR-2 mRNA, 7 days for ß-actin mRNA), before developing in Kodak D19 and counterstaining with Giemsa. Sections were examined under conventional or reflected light (dark field) conditions (Nikon ME600 with epi-illumination) that allowed individual autoradiographic silver grains to be seen as bright objects on a dark background. The total number of positive cells per villus was counted for all the villi in the section. Occasional positive cells not associated with vessel structures were excluded from the analyses. Signal above the muscular layer (i.e. mucosa, muscularis mucosae and sub-mucosa) was scored in a blinded fashion and semi-quantitative analysis was used to rank sections in order of staining intensity.

Statistics
All results are presented as group mean ± standard error of the mean. Data were analyzed by a two-tailed t-test with pooled estimates of variance or by the non-parametric Mann–Whitney U-test. All statistics were performed using Minitab Statistical Software, Release 10.5 Xtra (Minitab, Coventry, UK).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Microscopic and histological characteristics of the C57BL/6J-ApcMin/+ mouse
C57BL/6J-ApcMin/+ mice developed numerous spontaneous adenomas, predominantly located in the small intestine, which were visible macroscopically (Figure 1A). In non-tumor intestine, there was strong immunohistochemical staining for blood vessel markers (CD31 and vWF) in large thick-walled vessels at the junction of the muscularis and mucosa, with single thin-walled vessels passing through the central core of the villus (data not shown). In polyps, staining indicated an increased number of small, thin-walled vessels throughout the mucosa that were most obvious immediately underlying the luminal epithelium and overlying neoplastic glandular structures (Figure 1B and C).


Figure 1
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  		Fig. 1 Characteristics of polyps in the ApcMin/+ mouse. Macroscopic polyps in the small intestine of an ApcMin/+ mouse (A; the division between each red mark is 1 mm). Microscopic analysis of vessels in large polyps stained for vWF (B) or CD31 (C).

 
Treatment with AZD2171 reduces macroscopic polyp burden in the small bowel of ApcMin/+mice
In an early-intervention study, 6-week-old ApcMin/+ mice were treated for 28 days with either vehicle or AZD2171 (5 mg/kg body wt/day). In vehicle-treated animals, macroscopic analysis showed a large number of polyps in the small bowel, with fewer polyps in the colon (Figure 2). Animals treated with AZD2171 had significantly fewer polyps compared with vehicle-treated controls in both the small bowel and in the colon (Figure 2). The mean polyp diameter in the small bowel was also lower in AZD2171-treated mice, but was unchanged in the colon (Figure 2). The effects of AZD2171 on polyp number and diameter resulted in a significant decrease in polyp burden (product of polyp diameter and number) in the small bowel (P < 0.01), and a strong trend toward a decrease in polyp burden in the colon (P = 0.06; Figure 2). The effects of AZD2171 treatment were similar throughout the small bowel, with 77, 75 and 91% reductions in polyp burden compared with control in distal, middle and proximal regions, respectively (P < 0.01, data not shown).


Figure 2
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  		Fig. 2 Macroscopic analysis of polyps in the early-intervention study. AZD2171 (5 mg/kg body wt/day) decreases polyp number, diameter and tumor burden in the small intestine and reduces colon polyp number. AZD2171-treated ApcMin/+ mice had significantly fewer polyps in the small intestine and in the colon. In addition, the polyp diameter and the tumor burden were significantly reduced in the small intestine. Individual data points are shown with horizontal bar representing mean value. n = 12 per group. *P < 0.05; **P < 0.01; ***P < 0.001.

 
In a separate late-intervention study, 10-week-old ApcMin/+ mice were treated for 28 days with vehicle or AZD2171 (5 mg/kg body wt/day). Although treatment with AZD2171 did not significantly reduce polyp number, it significantly reduced polyp diameter in the small bowel (P < 0.01, Figure 3), producing a 46% reduction in overall tumor burden in the small bowel (P < 0.06). No significant effects of AZD2171 treatment on macroscopic polyps were observed in the colon in this study.


Figure 3
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  		Fig. 3 Macroscopic analysis of polyps in the late-intervention study. AZD2171 (5 mg/kg body wt/day) decreases polyp diameter in the small intestine. Comparison between control and AZD2171-treated ApcMin/+ mice. Individual data points are shown with horizontal bar representing mean value. n = 12 per group. **P < 0.01.

 
Treatment with AZD2171 also reduces microscopic polyp burden in the small bowel of ApcMin/+mice
In control animals, all sizes of polyps ranging from category 1–4 were observed within the small bowel (Figure 4). In the early-intervention study, AZD2171-treated animals had fewer micropolyps in the small bowel, though this did not reach statistical significance. Notably, significantly fewer larger micropolyps (categories 3 and 4; P = 0.003 and 0.01, respectively) were observed following AZD2171 treatment. (Figure 4A). Few polyps were detected in the colons of control or AZD2171-treated mice in the early-intervention study, with no significant difference between groups (Figure 4B).


Figure 4
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  		Fig. 4 Effects of AZD2171 (5 mg/kg body wt/day) on micropolyp counts in the early and late-intervention studies. Size category and micropolyp counts in the small intestine (15 sections per animal) and colon (five sections per animal) in the early-intervention (A and B) and the late-intervention studies (C and D). Each group consisted of 4–6 ApcMin/+ mice.

 
Control animals in the late-intervention study had a higher number of micropolyps than the early-intervention control group (Figure 4A and C). In contrast to the early-intervention study, the majority of micropolyps in the colon were category 4 (Figure 4D). In the small bowel, AZD2171 treatment significantly reduced the number of large polyps in categories 3 and 4 (P = 0.026 and 0.006, respectively), but had no significant effect on the smaller polyps (categories 1 and 2; Figure 4C). A marked effect was observed in the colons of the late-intervention animals, with large polyps (category 4) seen in vehicle-treated, but not AZD2171-treated, mice (Figure 4D). The histopathological appearance of colonic polyps from the late-intervention study is shown in Figure 5. It may be noted that there are two large polyps (category 4) in the lumen of the control animal, which was typical of this group. For the AZD2171-treated group, an image of the largest polyp seen in this group is shown. This animal had a single medium-sized (grade 3) polyp in the lumen.


Figure 5
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  		Fig. 5 Histopathological appearance of polyps in the late-intervention study from control (A) and AZD2171-treated animals (B). Colon from ApcMin/+ mice were stained with H&E. The tissue was rolled to maximize the amount of tissue visible in a single wax block. Rolls were prepared starting with the proximal end toward the center of the roll. Arrows indicate polyps. Scale bars = 500 µm (A) or 1 mm (B).

 
AZD2171 treatment reduces disease-related splenomegaly
ApcMin/+ mice with a large polyp burden develop anemia owing to excessive blood loss from the intestine. Onset of anemia in this model is indicated by splenomegaly (24). As a consequence, spleen weight is a useful surrogate marker of polyp load. The spleens of vehicle-treated mice were enlarged and 28 days of treatment with AZD2171 in 6- or 10-week-old mice reduced spleen weight by 48 (P < 0.003) and 34% (P < 0.02), respectively, compared with controls.

Pharmacodynamic analyses
Tissue samples from normal crypts and adenomas in the small bowel of mice from the late-intervention study were assessed for expression of ß-catenin and vWF protein by immunohistochemistry, and VEGFR-2 mRNA by in situ hybridization. ß-catenin immunostaining was present in both the cytoplasm/cell membrane and nucleus of small bowel sections. In control mice, the proportion of nuclear ß-catenin staining was higher in the adenomas compared with the normal surrounding intestinal epithelium. In addition, the intensity of nuclear ß-catenin staining was higher in adenomas compared with surrounding non-tumor epithelium. A similar staining pattern was observed in both control and AZD2171-treated animals. This indicates that AZD2171 did not affect translocation of ß-catenin to the nucleus.

Vessel density in both small bowel normal crypts and adenoma tissue was assessed by vWF staining. No significant difference in vessel density was observed between control and AZD2171-treated animals in either adenoma or non-tumor tissue. In addition, no significant difference between control and AZD2171 treatment was found when vessel density was assessed by CD31 staining (data not shown). AZD2171 treatment significantly reduced the number of endothelial cells per villus expressing VEGFR-2 mRNA (P = 0.022; Figure 6). However, there was no apparent difference between the groups in the intensity of signal in VEGFR-2 mRNA-expressing cells (data not shown).


Figure 6
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  		Fig. 6 Effects of AZD2171 on VEGFR-2 mRNA expression. Small intestine showing localization of VEGFR-2 mRNA using in situ hybridization (black grains in bright field, white areas in dark-field illumination) from control (A) and AZD2171-treated (B) animals; C and D show the corresponding dark field images. Scale bar = 100 µm. The marked reduction in the signal (silver grains) in the treated mice and the location of the signal in the lamina propria of the villi may be noted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The growth of human tumor xenografts in immunocompromised mice is an extremely useful preclinical tool (25). However, such models lack the ability to model early or spontaneous disease. Mutant and transgenic murine models have been developed in which spontaneous tumor growth and staged tumorigenesis more closely resemble human disease (26). For example, the ApcMin/+ mouse has been used to examine the early stages of intestinal cancer (27).

Evidence for the role of neovascularization in the development of colorectal cancer has been obtained from animal models (28) and from clinical observations (29). Hanrahan et al. (30) observed a correlation between tumor grade and expression of VEGF family members, suggesting that VEGF may have an important role at the stage of adenoma formation. The offspring from a cross between the ApcMin/+ mouse and a mouse null for the endogenous antiangiogenic protein thrombospondin 1 (TSP-1–/–) developed more polyps, with larger diameters, compared with controls (31), indicating a possible role for angiogenesis in both polyp development and polyp growth. In Apc716 mice, VEGF levels were elevated in polyps compared with epithelial cells from controls; this is consistent with an association between expression of VEGF and polyp development (32).

The method of preparation of the mouse guts used in the present studies was highly effective at revealing macroscopic polyps (17,18). Macropolyps <0.2 mm in diameter could be reliably detected. For microscopic analysis, there was a risk that polyps >100 µm in diameter could be scored more than once, perhaps overestimating the number of larger polyps. Control ApcMin/+ mice from both the early- and the late-intervention studies had similar levels of macroscopically visible polyps in the small bowel and the colon. In contrast, there were significantly more microscopic polyps in the small bowel and the colon of the control mice in the late-intervention study, compared with the early-intervention study, indicating that there may be some accumulation of micropolyps in older mice.

AZD2171 is an oral, highly potent and selective VEGF signaling inhibitor of all known VEGFR tyrosine kinases (VEGFR-1, VEGFR-2 and VEGFR-3) (15). It has demonstrated highly significant tumor growth inhibition in a diverse panel of human tumor xenograft models, including colorectal cancer (15). The present study utilized AZD2171 to investigate the effects of inhibiting VEGFR-2 signaling on the development and growth of gastrointestinal adenomas in ApcMin/+ mice.

AZD2171, when given daily for 28 days to 6-week-old ApcMin/+ mice, reduced macropolyp number, size and burden in the small bowel. In the same animals, micropolyps were also decreased in number and size by AZD2171 treatment. However, in 10-week-old mice, AZD2171 treatment did not affect macropolyp number, but did reduce polyp diameter and tumor burden in the small bowel. Fewer large polyps were present in the colons of 10-week old mice, following AZD2171 treatment compared with vehicle-treated controls. In Apc716 mice, 2 months treatment with the cyclooxygenase-2 (COX-2) inhibitor, rofecoxib, resulted in fewer and smaller intestinal polyps (33). One of the downstream consequences of COX-2 expression is thought to be induction of VEGF. The results of the present study suggest that VEGFR-2 signaling plays a key role in the development of early intestinal adenomas in ApcMin/+ mice, and that inhibiting this pathway can reduce tumor burden.

Data obtained from Apc716 mice have indicated that the angiogenic switch occurs at an early stage in intestinal tumorigenesis (33) Interestingly, microvessel density and VEGF expression in polyps increased in a size-dependent manner, but only when polyps expanded >1 mm in diameter (33). Notably, in the present study, the small bowel polyp diameter in AZD2171-treated groups did not exceed 1 mm. Recent studies using novel optical technology revealed that increases in blood supply can occur within 2 weeks of chemical carcinogen treatment in a rat tumor model, well before the appearance of aberrant crypt foci (34). Increased blood supply was also seen in the small bowel of 6-week-old ApcMin/+ mice (34). In the present study, 28 days of AZD2171 treatment did not produce significant effects on the vessel density in adenomas, despite the marked effects on polyp size and number. However, it is recognized that there are considerable limitations to using changes in vessel density during treatment to determine the effectiveness of angiogenesis inhibitors, particularly where there is an effect of treatment on tumor size (35).

Easwaran et al. (36) reported a direct correlation between activation of ß-catenin signaling and upregulation of VEGF in colon cancer. Clear differences in ß-catenin staining intensity and localization could be observed between normal crypt and adenomatous tissue in both control and AZD2171-treated mice. There was no apparent difference in nuclear localization following the AZD2171 treatment, indicating that enterocytes were similar to vehicle-treated controls in terms of ß-catenin expression.

Interestingly, AZD2171 treatment significantly reduced the number of endothelial cells positive for VEGFR-2 mRNA expression in vessels of normal crypt tissue. Recent studies have demonstrated that inhibition of VEGFR signaling can reduce the intensity of VEGFR-2 immunofluorescence in tumor blood vessels, and in certain normal blood capillaries in the mouse (37,38). Together with the present study, these data support the concept that reduced expression of VEGFR-2 mRNA and protein in tumor vessels, or in certain normal capillary beds, may be an indicator of vascular response to VEGFR signaling inhibition (37).

In conclusion, the present study has shown that AZD2171 treatment significantly inhibits adenoma development and growth in the ApcMin/+ mouse model, suggesting that VEGFR-2 signaling plays a critical role in pre-invasive early tumor growth. This study also provides a scientific rationale for studying the antitumor effects of VEGFR-2 signaling inhibitors in earlier disease in the clinic. AZD2171 is currently undergoing clinical evaluation as a once-daily oral therapy in a variety of tumor types.


   Acknowledgments
 
We would like to thank the staff at the BSU Clare Hall for all their help; George Elia of the Histopathology Service; the staff of the ISH Service at Cancer Research UK; and Kerry Ratcliffe at AstraZeneca Safety Assessment for histological support. We would also like to thank Juliane Jürgensmeier for helpful comments on the preparation of the manuscript. Financial support for this study was provided by AstraZeneca. D.A. is funded by a Biology and Biotechnology Science Research Council (BBSRC) CASE research studentship in conjunction with AstraZeneca.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 18, 2006; revised June 7, 2006; accepted June 9, 2006.


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