Inhibition of VEGF-A prevents the angiogenic switch and results in increased survival of Apc^+/min mice

Abstract

Anti-VEGF-A monoclonal antibodies, in combination with chemotherapy, result in a survival benefit in patients with metastatic colorectal and non-small cell lung cancer, but little is known regarding the impact of anti-VEGF-A therapy on benign or premalignant tumors. The Apc^+/min mice have been widely used as a model recapitulating early intestinal adenoma formation. To investigate whether tumor growth in Apc^+/min mice is mediated by VEGF-A-dependent angiogenesis, we used two independent approaches to inhibit VEGF-A: monotherapy with a monoclonal antibody (Mab) targeting VEGF-A and genetic deletion of VEGF-A selectively in intestinal epithelial cells. Short-term (3 or 6 weeks) treatment with anti-VEGF-A Mab G6–31 resulted in a nearly complete suppression of adenoma growth throughout the small intestine. Growth inhibition by Mab G6–31 was associated with a decrease in vascular density. Long-term (up to 52 weeks) treatment with Mab G6–31 led to a substantial increase in median survival. Deletion of VEGF-A in intestinal epithelial cells of Apc^+/min mice yielded a significant inhibition of tumor growth, albeit of lesser magnitude than that resulting from Mab G6–31 administration. These results establish that inhibition of VEGF-A signaling is sufficient for tumor growth cessation and confers a long-term survival benefit in an intestinal adenoma model. Therefore, VEGF-A inhibition may be a previously uncharacterized strategy for the prevention of the angiogenic switch and growth in intestinal adenomas.

Angiogenesis is essential for many physiological processes (1). Several pathological conditions, particularly tumor growth and metastasis, also depend on angiogenesis (2). One of the key positive regulators of angiogenesis is vascular endothelial growth factor (VEGF)-A (reviewed in ref. 3). VEGF-A is part of a gene family that includes VEGF-B, VEGF-C, VEGF-D, and PlGF (4). VEGF-A primarily binds two high affinity receptor tyrosine kinases (RTKs), VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), the latter being the major mediator of mitogenic and angiogenic signals of VEGF-A. Additionally, neuropilin-1 is a coreceptor for heparin-binding VEGF-A isoforms (reviewed in ref. 4).

Early studies showed that an anti-VEGF-A mouse monoclonal antibody (Mab) called A.4.6.1 inhibits the growth of human tumor cell lines transplanted in nude mice (5). Interestingly, the same Mab inhibited tumor angiogenesis in a model of liver metastasis of colorectal cancer (6). Furthermore, several other VEGF inhibitors, including soluble VEGF receptors (7, 8), anti-VEGFR-2 Mabs (9), and small molecule VEGF RTK inhibitors (10), have been shown to inhibit tumor growth.

VEGF-A inhibition with a humanized variant of Mab A.4.6.1 (bevacizumab), in combination with chemotherapy, results in a survival advantage in patients with previously untreated metastatic colorectal cancer (11) and nonsquamous non-small-cell lung carcinoma (12) relative to chemotherapy alone. The small molecule VEGF RTK inhibitors sunitinib (13) and sorafenib (14) have shown efficacy in metastatic renal cell cancer patients and were recently approved by the Food And Drug Administration.

Investigating the mechanisms of tumor angiogenesis in xenografts has limitations, because these models do not recapitulate tumor development in a natural setting. Furthermore, little is known regarding the role of angiogenesis in the growth and progression of benign or premalignant tumors.

The syndrome of Familial Adenomatous Polyposis (FAP) and the majority of sporadic colorectal cancers are caused by mutations in the APC gene (reviewed in ref. 15). FAP patients develop hundreds to thousands of adenomatous polyps in their lower gastrointestinal tract, in addition to extracolonic tumors. APC has been reported to be involved in numerous cellular processes including proliferation, apoptosis, signal transduction, and cell migration, but its best studied function is the regulation of β-catenin in the Wnt signaling pathway (15).

Apc^+/min mice with a heterozygous truncation allele at codon 850 mimic some features of the polyposis of FAP patients with germ-line APC mutation (16, 17). The onset of tumor formation in Apc^+/min mice is in early adulthood, and the animals typically develop 60–150 intestinal polyps in a C57BL/6 genetic background. Tumor development results in a severely compromised longevity of the mice, usually resulting in death from anemia and/or hypoproteinemia (16) at the age of ≈5 months. Whereas humans with FAP typically develop colonic adenomas, the vast majority of polyps in the Apc^+/min mice develop in the small intestine. These polyps reach a size of 1–2 mm in diameter, and larger polyps (up to 4 mm in diameter) arise at a lower frequency.

We sought to determine the role of VEGF-A-dependent angiogenesis in the Apc^+/min model. Tumors were analyzed after short- and long-term treatment with anti-VEGF-A Mab or after genetic deletion of VEGF-A by Cre-LoxP technology in intestinal epithelial cells.

Results

Expression of VEGF-A in the Apc^+/min Intestinal Adenomas.

To investigate the expression pattern of VEGF-A in intestinal tumors of Apc^+/min mouse, we performed in situ hybridization [ISH, see supporting information (SI) Text]. VEGF-A expression was observed in the epithelial cells with varying intensity compared with normal intestinal villus epithelium, whereas a focally prominent signal was observed in stromal cells of the adenomas and the stroma of the normal villi (Fig. 1 A–F).

Fig. 1.

VEGF-A expression in Apc^+/min adenomas and normal villus. In situ hybridization with VEGF-A probe on an intestinal adenoma from large (A, E, I, and L) and small (B, F, J, and M) bowel as well as adjacent normal colon (C and G) and small intestine (D, H, K, and N) of 14-week-old Apc^+/min mice demonstrates increased expression of VEGF-A in adenomas relative to normal intestine. Representative images are shown. (A–D and I–K) Brightfield. (E–H and L–N) Darkfield. VEGF signal in adenomas (E and F) is focally stronger than in adjacent normal intestinal epithelium (G and H). VEGF signal arises from both epithelial cells (I–K, arrows) and stromal cells (I–K, arrowheads). (Scale bars: A–H, 100 μm; I–N, 25 μm.)

VEGF-A in situ hybridization signal in Apc^+/min intestinal adenomas was variable but was focally greater than in the surrounding normal epithelium in all animals examined. In three animals with measured small intestinal adenomas (n = 12), the average adenoma signal intensity was 40–60% above the surrounding normal epithelium [range of individual adenoma signals: 0.9–3.0 times control levels; Student's t test P values (per animal) = 0.02–0.03]. In the single large intestinal adenoma examined, the average epithelial signal intensity was 2.6 times that of the adjacent normal epithelium (Student's t test P value = 0.002).

Inhibition of VEGF-A Lowers Tumor Burden of Apc^+/min Mice.

We sought to determine whether anti-VEGF-A Mab therapy would be effective at lowering the tumor burden in Apc^+/min mice. We chose Mab G6–31 because of its ability to potently block VEGF-A across species (18). This is unlike the well characterized anti-VEGF Mab A.4.6.1, which inhibits human but not mouse VEGF-A (7, 18). To assess the short-term effects of Mab G6–31 on tumor burden, treatment of 10 mice per cohort was started at 13 weeks of age and continued for 3 or 6 weeks. To determine the tumor phenotype at the age of treatment onset, an untreated control group (n = 12) was analyzed at 13 weeks of age (day 0).

Treatment with anti-VEGF-A Mab for either 3 or 6 weeks significantly reduced overall tumor burden in Apc^+/min mice. At day 0, the mean tumor burden of Apc^+/min mice was 39.3 mm³ (ranging from 12.3 mm³ to 97.0 mm³) (Fig. 2A). The mean tumor burden of mice treated with control IgG for 3 weeks was 96.8 mm³ (47.1–299.9 mm³), whereas the mean tumor burden of mice treated for 3 weeks with Mab G6–31 was 23.5 mm³ (4.5–58.2 mm³). This was a statistically significant 76%, or 4-fold reduction in mean tumor burden, with a P < 0.008. After 6 weeks of administration of control IgG, the tumor burden reached a mean of 198.6 mm³ (40.5–315.7 mm³), whereas the tumor burden in mice treated with Mab G6–31 remained at 28.4 mm³ (3.2–75.9 mm³), exhibiting a significant 86%, or 7-fold reduction in mean tumor burden, with a P < 5.3 × 10⁻⁵ (Fig. 2A).

Fig. 2.

Inhibition of VEGF-A lowers tumor burden. (A) Tumor burden is indicated by bars from the largest to the smallest value of tumor burden of individual mice in the group. Black squares indicate group averages. ∗, P < 0.008; ∗∗, P < 5.3 × 10⁻⁵. n, number of animals. (B) Distribution of tumors by diameter and shown as percent of the total number of tumors. n, number of tumors in a group. (C) Overlaid tumor size frequencies after 3 weeks of treatment (Top), after 6 weeks of treatment (Middle), and in comparison to day 0 (Bottom). Vertical bars illustrate the size smaller or equal of which tumor frequency is greater in Mab G6–31-treated animals: 1 mm in 3-week-treatment and 1.2 mm in 6-week-treatment group. (D) Mean tumor diameter plotted against the intestinal location. n, number of tumors per group in the first, second, third, and fourth intestinal quarter, respectively. Day 0 group contained 12 animals; other groups 10. S, stomach; C, caecum; R, rectum. Bars represent SEM. ∗, P < 1.0 × 10⁻¹⁰; ∗∗, P < 0.002 compared with Mab G6–31 3 or 6 weeks. (E) Mean tumor diameter of 14 Apc^+/min mice;Villin-Cre (black columns) and Apc^+/min VEGF^lox;Villin-Cre (gray columns) mice presented in a descending order. Bars represent SD.

After 3 weeks of treatment with control IgG or Mab G6.31, the mean tumor numbers were respectively 116 ± 9 (± SEM) and 107 ± 11 (P < 0.28). After 6 weeks, the mean tumor number was 120 ± 11 in the control IgG group and 100 ± 10 (P < 0.09) in the Mab G6–31 group. At day 0, mice had an average of 100 ± 9 tumors. Thus, the decrease in tumor burden after either 3 or 6 weeks of anti-VEGF-A treatment was due to a decreased adenoma size, rather than to a decrease in the number of adenomas.

There was no evidence of adenoma growth escape during anti-VEGF-A treatment of 3 or 6 weeks. Tumors in mice treated with Mab G6–31 had a more compact size distribution (Fig. 2B, middle and bottom graph) compared with the broader size distribution of tumors from mice treated with control IgG (Fig. 2B, graphs 2 and 4 from the top). The mean polyp diameter in mice treated for 3 weeks with control IgG was 1.28 mm; in the Mab G6–31 group it was 0.85 mm (P < 0.0001). After 6 weeks of treatment, the mean polyp diameters were 1.64 mm in the control IgG group and 0.86 mm in the Mab G6–31 group (P < 0.0001). Mean tumor diameter at day 0 was 0.97 mm.

Interestingly, anti-VEGF-A treatment appeared to inhibit the growth of tumors of all sizes. After a 3-week treatment with Mab G6–31, the frequency of small tumors, 0.3–1.0 mm in diameter (for 6-week treatment 0.3–1.2 mm) was greater than in the control treated group, whereas the frequency of tumors with a diameter >1.0 mm (for 6 weeks >1.2 mm) was decreased (Fig. 2C Top and Middle). A comparison to the tumor size distribution at day 0 (Fig. 2C Bottom) suggested that the growth of the adenomas had virtually arrested after Mab G6–31 administration.

Mab G6–31 was effective at suppressing adenoma growth in all small-intestinal areas as a significantly lower mean tumor diameter was observed after 3 or 6 weeks of therapy (Fig. 2D). Furthermore, the mean adenoma diameter in the first intestinal quarter of mice treated with Mab G6–31 was significantly smaller compared with that observed in mice at day 0 (double asterisk in Fig. 2D). The reduction in mean tumor diameter of the colonic adenomas did not reach statistical significance (Fig. 2D). The mean diameter of the large bowel polyps in mice treated with Mab G6–31 for 3 weeks was 1.3 ± 0.3 mm (± SEM), whereas the mean diameter in the control IgG-treated mice was 2.5 ± 0.4 mm, with a P < 0.064. The mean diameter of large bowel tumors after 6 weeks of treatment with Mab G6–31 was 2.2 ± 0.3 mm and 2.6 ± 0.3 mm after administration with control IgG, with a P < 0.37.

Deletion of VEGF-A in Intestinal Epithelial Cells Reduces Mean Tumor Diameter.

We next sought to dissect the contribution of VEGF-A originating from intestinal epithelial sources to adenoma development. To this end, tumor diameter and number were assessed in 13-week-old Apc^+/min mice that were crossed to mice in which VEGF-A was conditionally deleted in intestinal epithelial cells with Cre/loxP technology (VEGF^lox;Villin-Cre mice).

The expression of Villin, an actin-binding protein and a major structural component of the brush border of specialized absorptive cells, begins during embryogenesis in the intestinal hindgut endoderm and later extends throughout the small- and large-intestinal endoderm (19, 20). In the adult, Villin distribution becomes diffuse with moderate apical polarization in cells of the crypts and polarization in brush borders of fully differentiated cells lining the villi of the small intestine (21). The expression of Cre recombinase driven by Villin promoter (Villin-Cre) has been shown to recapitulate the expression pattern of the Villin gene in every cell of the intestinal epithelium (22).

The mean tumor diameter of control Apc^+/min Villin-Cre mice was 1.02 ± 0.3 mm (±SEM), whereas the mean tumor diameter of Apc^+/min VEGF^lox;Villin-Cre mice was 0.82 ± 0.3 mm (Fig. 2E), demonstrating a 19.8% reduction (P < 0.001). Tumor number was not significantly different between the two groups. Whereas Apc^+/min Villin-Cre mice had 137 ± 11 intestinal adenomas, Apc^+/min VEGF^lox;Villin-Cre mice had 150 ± 17 adenomas (P < 0.27).

These data indicate that deletion of VEGF-A from all intestinal epithelial cells from duodenum through colon, and crypt to villus tip results in a significant inhibition of tumor growth, albeit of a reduced degree compared with that resulting from systemic administration of anti-VEGF-A antibody. These data suggest that extraepithelial sources of VEGF-A contribute to the growth of intestinal adenomas of Apc^+/min mice.

Inhibition of VEGF-A Extends the Median Survival of Apc^+/min Mice.

Given the effectiveness of anti-VEGF-A treatment in tumor growth inhibition, we wished to investigate whether treatment with Mab G6–31 could yield a long-term survival benefit for Apc^+/min mice. To this end, administration with Mab G6–31 or control IgG was continued for up to 52 weeks or until the mice were observed to be moribund. The median survival in the control IgG group was 24.0 weeks. In the Mab G6–31 group it was 33.6 weeks with log-rank P < 2.4 × 10⁻³ (Fig. 3).

Fig. 3.

Extended median survival in mice treated with Mab G6–31. Kaplan–Meier of Mab G6–31 (gray line) or control IgG (black line) -treated mice is shown. Open arrow designates the duration of the treatments. Median survival is indicated with gray arrows. ∗, P < 2.4 × 10⁻³. n, number of mice in a group.

Normal Serum Total Protein, Albumin, and Triglycerides Level and Reduced Splenic Extramedullary Hematopoiesis in Apc^+/min Mice treated with anti-VEGF-A.

Apc^+/min mice treated with Mab G6–31 appeared considerably more alert and responsive than those treated with control IgG. Moreover, pale paws, suggestive of the progressive anemia as initially reported by Moser et al. (16), were observed frequently in animals treated with control IgG, but not in animals treated with Mab G6–31. Consistent with this observation, the mean total serum protein and serum albumin of Apc^+/min mice administered with control IgG was decreased, whereas total protein and albumin levels were within normal range in mice treated with Mab G6–31 (Table 1). As previously reported for Apc^+/min mice (16) and consistent with hypoproteinemia, mean triglyceride level was elevated in animals treated with control IgG, although it was lowered to a level comparable to a reference value upon treatment with Mab G6–31 (Table 1). Although there were no treatment-related differences in body masses after 3 or 6 weeks of treatment, the mean spleen masses were significantly (P < 2.3 × 10⁻³) increased in mice treated with control IgG. After 3 weeks with control IgG, the mice had a mean spleen mass of 0.26 g, or 1.17% of body mass, whereas the mean spleen mass was 0.11 g (0.49% of body mass) in mice treated with Mab G6–31 for the same duration. The increase in mean spleen mass in mice treated with control IgG is consistent with extramedullary hematopoiesis (EMH), possibly secondary to intestinal bleeding. This was confirmed by histologic examination of the spleens (data not shown). Ten of 10 mice treated for 6 weeks with control IgG showed marked EMH, whereas 2 mice treated with Mab G6–31 had moderate EMH, 5 had mild EMH, and 3 had no diagnostic changes in their spleens.

Table 1.

Serum chemistry

Group (n)	Total protein, g/dl*	Albumin, g/dl†	Triglycerides, mg/dl‡
Control IgG 3 weeks (10)	3.7 ± 0.2	1.9 ± 0.1	268.9 ± 82.5
G6–31 3 weeks (10)	4.9 ± 0.2	2.6 ± 0.1	75.5 ± 4.9
control IgG 6 weeks (10)	3.0 ± 0.3	1.6 ± 0.2	591.1 ± 81.3
G6–31 6 weeks (10)	4.9 ± 0.1	2.7 ± 0.1	71.1 ± 4.3

Data are ± SEM.

*Reference value 3.9–5.5 g/dl.

^†Reference value 2.3–3.2 g/dl.

^‡Reference value 35–244 mg/dl.

The lower degree of EMH in the spleens of mice treated with Mab G6–31 short-term suggests that anti-VEGF-A therapy reduces intestinal bleeding.

Kidney Changes After Long-Term Treatment with Mab G6–31.

To investigate potential toxicities related to administering high-affinity anti-VEGF-A Mab G6–31, pancreas, liver, and kidney were analyzed histologically after short- (3–6 weeks) and long-term (18–53 weeks) treatment. No significant toxicity was noted in animals treated for 3–6 weeks. After long-term treatment with Mab G6–31, mice showed variable (mild to severe) diffuse global glomerulosclerosis and moderate stromal edema of the pancreas (reflecting hypoproteinemia). These observations are consistent with previously observed toxicity resulting from long-term administration of Mab G6–31 (23). Importantly, the adverse effects were outweighed by the overall improvement of health reflected by the increased median survival. Four of five mice treated with Mab G6–31 for 18–53 weeks were diagnosed with mild to extensive EMH in the spleen.

Altered Tumor Morphology upon Mab G6–31 Treatment Was Not Accompanied by a Change in Proliferative Index.

To further characterize intestinal polyps in Apc^+/min mice, macroscopic and histologic analyses were performed (see SI Text). The gross morphology of polyps treated with Mab G6–31 differed noticeably from that of the polyps treated with control IgG (SI Fig. 5 A and B). Whereas tumors from control mice typically had a relatively unbroken, smooth surface, tumors from animals treated with Mab G6–31 appeared with deep invaginations on their surface. Histologic analysis confirmed that tumors from Mab G6–31 and control IgG-treated mice are tubular adenomas (SI Fig. 5C–F). Adenomas from control IgG-treated mice had marked intravillous epithelial proliferation, with vertical and lateral expansion, and were typically widened >2-fold from their base to luminal surface. There was minimal fibrous stroma. Adenomas from mice treated with Mab G6–31 characteristically had fewer intravillous epithelial cells, were less broad at the luminal surface, shallower, and involved fewer adjacent villi. Histologic analysis of the colonic polyps in both treatment groups showed pendunculated tubular adenomas with abundant fibrovascular stroma and a variable amount (up to 100%) of dysplastic epithelium (data not shown).

To assess the extent of proliferation in the tumor tissue and in normal mucosa, an indirect immunohistochemical staining with Ki-67 antibody was performed (SI Fig. 5 G–J). Quantitative analysis revealed similar amounts of Ki-67-positive cells in tumors from mice treated with either control IgG or with Mab G6–31. Likewise, the proliferative index of the normal adjacent mucosa was comparable between both treatments (SI Fig. 5K).

Reduced Vascular Density in Mab G6–31 Treated Tumors.

Given that VEGF-A is known to be a mitogen for vascular endothelial cells through VEGFR-2 signaling, we examined the tumor vascular networks in mice treated with Mab G6–31 and control IgG by immunohistochemical staining of thick tissue sections with antibodies for three independent vascular markers, CD31, CD105, and von Willebrand Factor (vWF) (Fig. 4 A–H) (see SI Text). Quantification of the vessel density indicated that the vascular component of tumors from Mab G6–31-treated mice was significantly reduced compared with that seen in control IgG-treated mice, both at the 3 and 6 weeks time points (Fig. 4I). A similar reduction was obtained with all three markers used.

Fig. 4.

Distribution of vascular markers and quantification of blood vessel density after Mab G6–31 treatment. (A and B) Fluorescence micrographs comparing the distribution of vascular endothelial cells positively stained for CD105 (red, isolated in C and D), CD31 (green, isolated in E and F), or von Willebrand Factor (blue, isolated in G and H) in tumors from Apc^+/min mice after 6 weeks of treatment with G6–31 or control IgG. (Scale bar: 100 μm, applies to all panels). (I) After 3 or 6 weeks of G6–31 treatment, vascular density expressed as the percent area positive for vascular markers CD31, CD105, or von Willebrand Factor (vWF) relative to total tumor area is significantly reduced in Apc^+/min mice when compared with IgG control. Columns represent mean vessels area density (n = 3–6 tumors per mouse, 2 mice per group); bars represent SEM. ∗, P < 0.01; ∗∗, P < 0.005 significant difference compared with IgG control treatment.

Discussion

We used Apc^+/min mice to investigate the role of VEGF-A in benign intestinal tumorigenesis. Our in situ analysis documented an up-regulation of VEGF-A in adenomas compared with the normal villi. Our experiments were designed to measure the effects of short- and long-term anti-VEGF-A treatment on established intestinal adenomas undergoing robust growth. We show that treatment with anti-VEGF-A Mab G6–31 significantly lowers the tumor burden and extends the survival of the Apc^+/min mice.

Several studies have been conducted on the effect of dietary and chemopreventive agents on tumor burden of Apc^+/min mice (reviewed in ref. 24), of which an updated list exists at http://corpet.net/min. Many of these studies report a significant decrease in tumor number. Nonsteroidal antiinflammatory drugs such as piroxicam and sulindac, which target both COX-1 and COX-2, have been among the most potent agents in suppressing tumor formation in Apc^+/min mice (25–28). More specifically, the important role of COX-2 in intestinal polyposis was demonstrated by using different selective COX-2 inhibitors (29–31). Recently, Goodlad et al. (32) reported that short-term administration of the RTK inhibitor AZD2171 resulted in reduced tumor burden in the Apc^+/min model. AZD2171 inhibits several RTKs including, but not limited to, VEGFR-1, -2, and -3 (33). The authors (32) noted that earlier treatment onset (at 6 weeks) with AZD2171 was able to reduce tumor number, whereas later intervention (at 10 weeks) only reduced tumor size. That anti-VEGF-A Mab G6–31 did not reduce the number of tumors potentially reflects its unique mechanism of tumor inhibition, by antiangiogenesis. Moreover, it is possible that a tumor prevention approach (with an earlier treatment onset) is more effective at reducing tumor number than a tumor intervention approach (with a later treatment onset) that was used in our study.

Our study demonstrates that targeting VEGF-A is sufficient to achieve profound therapeutic effects in the Apc^+/min model. Comparing systemic VEGF-A inhibition by Mab G6–31 to genetic deletion of VEGF-A in the intestinal epithelial compartment suggests that, in addition to epithelial cells, other cellular sources of VEGF-A play an important role in Apc^+/min adenoma growth. These additional sources of VEGF-A potentially include mononuclear cells (34) and stromal fibroblasts (35, 36). Our in situ analysis indicates extraepithelial VEGF-A expression within the adenomas and normal villi, supporting this notion.

It is conceivable that much of the observed antitumor effects of Mab G6–31 is mediated by suppression of VEGFR-2-dependent angiogenesis (4, 37). Indeed, a reduced vascular supply in response to anti-VEGF-A monoclonal antibody has been observed in several tumor xenograft studies (38). In agreement with these observations, using three independent vascular markers, we were able to demonstrate a significant reduction in vessel area density of the Apc^+/min intestinal adenomas after 3 or 6 weeks of administration of Mab G6–31 compared with control IgG treatment. However, in contrast to our study, treatment with AZD2171 was reported to have no effect on vascular density (32). Whether such lack of effects of AZD2171 on blood vessel density reflects qualitative and/or quantitative differences in the mechanism of tumor suppression compared with the anti-VEGF Mab, or other experimental variables, remains to be established.

Recent studies have raised the possibility that VEGF-A also may have direct effects on intestinal epithelial cells through VEGFR-1 (39, 40). Future studies are required to determine whether such a mechanism plays a role in the growth of Apc^+/min adenomas.

A long-standing concept postulates that a tumor requires a vascular support to grow larger than 1 mm (41). Recent studies also indicate that tumors <1 mm in diameter remain in a nonangiogenic state for 100 days or more (42). In the present study, however, we observed significant accumulation of adenomas smaller than 1 mm upon inhibition of VEGF-A. This suggests that, in the intestinal adenomas of the Apc^+/min mice, the angiogenic switch (43) may occur earlier than generally believed for tumor development, as has been seen in the Apc^Δ716 model (35).

An important and unexpected conclusion of our study is that a monotherapy targeting a single angiogenic factor may be highly effective at suppressing tumor growth and yield a survival benefit. In contrast, in advanced malignant tumors antiangiogenic therapy seems to be most useful when combined with cytotoxic chemotherapy (11, 12, 44). Therefore, our data suggest the possibility of a nonsurgical treatment for benign tumors, even without the need of chemotherapeutic agents. However, it should be noted that a systemic VEGF-A blockade can be associated with significant side effects in some patients, including hypertension, proteinuria, and arterial thromboembolism (45). Therefore, further understanding of the epidemiology and mechanism of such side effects will be needed to better define the risk/benefit ratio before clinical trials with VEGF-A inhibitors in benign tumors can be considered. Finally, selection of an anti-VEGF agent with the appropriate balance of efficacy/toxicity may be critical for such long-term treatments (23).

Materials and Methods

Animal Acquisition and Husbandry.

Apc^+/min mice (stock number 002020; ref. 16) and 12.4KbVilCre mice (stock number 004586), hereafter VillinCre (22), were obtained from The Jackson Laboratory (Bar Harbor, ME). VEGF^lox/lox mice (hereafter VEGF^lox) have been described in ref. 46. Mice were housed in micro isolator cages in a barrier facility and fed ad libitum. Maintenance of animals and experimental protocols were conducted by following federal regulations and approved by Institutional Animal Care and Use Committee.

Treatment of Mice with anti-VEGF-A or Control IgG Antibodies.

The anti-VEGF-A Mab G6–31 was derived from human Fab phage libraries as described in ref. 18. To generate an antibody suitable for long-term administration in mice, the variable domains were grafted into murine IgG2a constant domain. Mab G6–31 (18) or isotype matched control murine IgG2a (anti-gp120), both at the dose of 5 mg/kg, was administered i.p. once a week in a 90- to 140-μl volume in PBS. Treatment durations were 3 weeks, 6 weeks, up to 1 year, or until the mice were found moribund. Treatment of 10–14 mice per each group was started at 91 ± 3 days of age.

Analysis of Tumor Size and Number.

The gastrointestinal tract, from glandular stomach to rectum, was opened longitudinally, rinsed, and spread flat on a filter paper. After overnight fixation with Notox Histo Fixative (Scientific Design Laboratory, Inc., Des Plaines, IL) and staining with methylene blue 0.1% aqueous solution, the number, location, and diameter of each intestinal adenoma of the small and large bowel was scored by a single observer, blinded to the treatment, through an ocular scale under ×20 magnification on a Leica dissection microscope. By this method, polyps with a diameter 0.3 mm or greater were recorded reliably. Tumor volumes were calculated as hemispheres. Tumor burden for each mouse was calculated as a sum of its tumor volumes. P values were calculated by a two-tailed Student's t test. A nontreated group of mice (day 0) was analyzed at the age of treatment onset (13 weeks) as a control to the antibody treated mice. Apc^+/min;Villin-Cre and Apc^+/min;VEGF^lox;Villin-Cre mice were likewise analyzed at 13 weeks of age.

Abbreviations

EMH

extramedullary hematopoiesis

FAP

Familial Adenomatous Polyposis

RTK

receptor tyrosine kinase.