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Molecular Cancer Therapeutics 6, 2496-2504, September 1, 2007. doi: 10.1158/1535-7163.MCT-07-0253
© 2007 American Association for Cancer Research
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Research Articles: Therapeutics, Targets, and Development

Hypoxia modulation and radiosensitization by the novel dual EGFR and VEGFR inhibitor AEE788 in spontaneous and related allograft tumor models

Christoph Oehler-Jänne1, Wolfram Jochum2, Oliver Riesterer1, Angela Broggini-Tenzer1, Giorgio Caravatti3, Van Vuong1 and Martin Pruschy1

Departments of 1 Radiation Oncology and 2 Pathology, University Hospital Zurich, Zurich, Switzerland; and 3 Oncology Research, Novartis Institutes for Biomedical Research, Basel, Switzerland

Requests for reprints: Martin Pruschy, Laboratory for Molecular Radiobiology, Department of Radiation Oncology, Raemistr. 100, University Hospital Zurich, CH-8091 Zürich, Switzerland. Phone: 41-44-255-8549; Fax: 41-44-255-4435. E-mail: martin.pruschy{at}usz.ch

Abstract

Concomitant inhibition of ErbB1/2- and VEGF receptor-signaling synergizes when used in combination with DNA-damaging agents. Here, we investigated for the first time the combined treatment modality of the novel dual specific receptor tyrosine kinase inhibitor AEE788 with ionizing radiation and analyzed treatment-induced end points in situ as indicators for a potential sensitizing mechanism. Furthermore, we assessed tumor hypoxia in response to different antiangiogenic and antiproliferative treatment modalities. The combined treatment effect was investigated in a spontaneously growing mammary carcinoma model and against Her-2/neu-overexpressing mammary carcinoma allografts. In tumor allografts derived from murine mammary carcinoma cells of mouse mammary tumor virus/c-neu transgenic mice, a minimal treatment regimen with AEE788 and fractionated irradiation resulted in an at least additive tumor response. Treatment response in the corresponding spontaneous tumor model strongly exceeded the response induced in the isogenic allografts. Treatment-induced changes of tumor proliferation, apoptosis, and microvessel density were similar in the two tumor models. Treatment with AEE788 alone or in combination with IR strongly improved tumor oxygenation in both tumor models as determined by the detection of endogenous and exogenous markers of tumor hypoxia. Specific inhibition of the VEGF-receptor tyrosine kinase versus Erb1/2-receptor tyrosine kinase indicated that it is the antiproliferative and not the antiangiogenic potency of AEE788 that mediates the hypoxia-reducing effect of this dual kinase-specific inhibitor. Overall, we show that concomitant inhibition of ErbB- and VEGF-receptor signaling by AEE788, in combination with ionizing radiation, is a promising treatment approach, especially in hypoxic, oncogenic ErbB-driven tumors. [Mol Cancer Ther 2007;6(9):2496–504]

Introduction

Many strategies have been developed to control tumor angiogenesis using either biomolecular or pharmacologic inhibitors. They either directly target the microvascular endothelial cell, in particular by inhibiting endothelial growth factors and corresponding receptors, or indirectly block the tumor stress response to hypoxia by down-regulating the expression of proangiogenic factors in tumor cells. Vascular endothelial growth factor A (VEGF-A) plays a key role in pathologic angiogenesis by activating the family of VEGF-dependent receptor tyrosine kinases, resulting in enhanced endothelial cell proliferation, migration, and survival. Several preclinical studies showed that abrogation of VEGF signaling is a potent antitumoral strategy whose efficacy can be enhanced by combination with a cytotoxic treatment modality such as ionizing radiation (IR; refs. 1–4). The supra-additive effect in combination with IR has been shown for antibodies specific for VEGF and its corresponding growth factor receptor or multiple small pharmacologic compounds inhibiting the receptor tyrosine kinase activity. The mechanism of the synergistic interaction between inhibitors of angiogenesis and IR is still a matter of debate (5–9). However, a putative reduction of oxygen delivery resulting to inhibition of angiogenesis and destruction of the tumor vasculature could render the tumor more radioresistant and is a major clinical concern. Hypoxia reduces the radiosensitivity of tumor cells; cells irradiated in normoxic conditions are twice to thrice more radiosensitive than cells irradiated under severe hypoxia. The "tumor vascular normalization" concept claims that treatment with low doses of angiogenesis inhibitors preferentially targets immature vessels and by that creates a normalization window with improved functionality of the remaining tumor vasculature. This could avoid tumor hypoxia by improved delivery of oxygen (10). On the other hand, preclinical data from several laboratories, including our own, showed that inhibitors of angiogenesis increase tumor hypoxia during treatment, which is one of the reasons why reservations still exist to fully promote a treatment combination with IR into clinical trials (11–13). However, own results showed that the risk of treatment-induced hypoxia by inhibitors of angiogenesis exists but is kept minimal when combined with a cytotoxic treatment modality, e.g., ionizing radiation (14).

The combined treatment modality of IR with inhibitors of epidermal growth factor receptor (EGFR)–signaling has recently been approved for locoregionally advanced head and neck squamous cell carcinoma using cetuximab (C225), a monoclonal antibody against the EGFR, in combination with IR (15). A major rationale for combining inhibitors of EGFR signaling with fractionated radiotherapy is the inhibition of IR-induced cellular proliferation. First results on the preclinical level suggested that combined treatment with IR and cetuximab decreases the repopulation of squamous cell carcinoma (16). Indeed, phase III clinical trials with cetuximab in combination with IR show improved locoregional tumor control and overall survival and minimal normal tissue toxicities (15). However, it has not been shown that this beneficial treatment response is solely due to control of repopulation. On the contrary, multiple mechanisms might contribute to EGFR inhibition–related radiosensitization. Besides controlling accelerated tumor repopulation during repetitive irradiation, EGFR inhibitors can decrease the capacity for DNA-damage repair in overly proliferating cells (17–20), down-regulate endogenous survival pathways leading to enhanced apoptosis (21–23), shift the cells into a more radiosensitive cell cycle phase (24–26), and also indirectly inhibit angiogenesis (17, 27).

Whereas antiangiogenic strategies are directed primarily against the tumor vasculature, irradiation targets both tumor and endothelial cells (7, 28). This strategy may be further improved by the concomitant inhibition of EGFR signaling both in the tumor tissue and endothelial cell compartment. Such an approach may further sensitize tumor cells and endothelial cells on the individual cellular level to IR and at the same time modulate the intercellular network and microenvironment, e.g., by down-regulating the expression of proangiogenic factors such as VEGF from tumor cells (29). Dual specific receptor tyrosine kinase inhibitors, which target two different classes of growth factor receptors, represent an elegant way to avoid triple treatment modalities when used as part of a combined treatment modality with IR (30).

AEE788 is a novel, orally bioavailable, dual specific inhibitor of both EGFRs (ErbB1/2) and VEGF-receptor tyrosine kinase family members (VEGFR1/2; Flt-1 and KDR, respectively; ref. 31) and is already tested in early clinical trials (32). AEE788 displays potent antitumor and antiangiogenic activity tested in various murine tumor model systems alone and in combination with various anti-signaling agents (33–38). Here, we analyze the antitumoral effect of the combined treatment modality of IR in combination with AEE788 in an allograft tumor system, but more important, also in a murine spontaneous tumor model, which closely resembles the clinical situation. In particular, we investigate the treatment response with regard to the dynamics of tumor hypoxia in situ.

Materials and Methods

Reagents, Cell Cultures, Irradiation
AEE788, PTK787/ZK222584, and PKI166 were provided by Novartis Pharma. The murine mammary breast carcinoma cell line NF9006 derived from a spontaneous mammary carcinoma of a mouse mammary tumor virus (MMTV)/c-neu transgenic FVB mouse was kindly provided by Claude G. Gimmi (University Berne, Berne, Switzerland). Cells were maintained in DMEM (Life Technologies) supplemented with 1% nonessential amino acids, 1% HEPES, 10% newborn calf serum (Sigma Chemical Co.), 2% L-glutamine, and 1% penicillin/streptomycin. For in vitro assays, a stock solution (10 mmol/L) of AEE788 was prepared in DMSO and further diluted with H2O/DMSO and serum-containing media. Irradiation was carried out at room temperature using a Pantak Therapax 300-kV X-ray unit at 0.7 Gy/min.

Caspase-3 Activity Assay
Caspase-3–like activity was determined in cytosolic cell extracts against the colorimetric caspase 3 substrate N-acetyl-Asp-Glu-Val-Asp p-nitroanilide as previously described (39). In brief, 75 µg of protein from the cytosolic S-100 fraction was incubated at 37°C with the colorimetric caspase 3 substrate Ac-DEVD-pNA (100 mmol/L, Calbiochem) and 1 mmol/L dATP in a final volume of 120 µL. Cleavage of the caspase substrate was monitored at 405 nm using a Dynatech MR5000 spectrophotometer.

Tumor Allografts in Nude Mice, Spontaneous Murine Mammary Tumor Model, Administration of Compounds, and Irradiations
Murine mammary carcinoma cells (NF9006) were injected s.c. (4 x 106 cells) on the back of 4- to 8-week-old athymic nude mice. Heterozygous spontaneous mammary tumor–developing mice were generated by crossing female FVB-wild type x male FVB-Tg(MMTV/c-neu) mice (Charles River). These mice develop tumors within 100 days after a first litter, and tumors were allowed to expand to a minimal volume of 200 mm3 ±10% before start of treatment. Tumor volumes were determined from caliper measurements of tumor length (L) and width (l) according to the formula (L x l2)/2. Mice carrying allograft tumors were given a fractionated, locoregional radiotherapy over multiple consecutive days using a customized shielding device. Mice with spontaneous tumors were given upper-half-body radiotherapy with different treatment regimens as indicated under isoflurane anesthesia. A Pantak Therapax 300-kV X-ray unit at 0.7 Gy/min was used for all irradiations. AEE788, PKI166 (both dissolved in N-methylpyrrolidone and further diluted in polyethylene glycol-300) and PTK787 (dissolved in 5% DMSO, 1% Tween-80, and 94% H2O) were applied p.o. (at day 0, 1, 2, and 3) either alone or 1 h before irradiation. Statistical analysis of the in vivo data was done with the Mann-Whitney U test.

Histology, Immunohistochemistry, TUNEL Assay
Tissues were immersion fixed in 4% PBS-buffered formalin and embedded in paraffin. Sections 3 µm thick were mounted on glass slides (SuperFrost Plus; Menzel), deparaffinized, rehydrated, and stained with H&E using standard histologic techniques.

For CD31 staining, a goat polyclonal anti-platelet/endothelial cell adhesion molecule-1 (PECAM1) antibody (M20, Santa Cruz Biotechnology) at a final dilution of 1:50 was used. Detection of primary antibody was done with a Histofine/diaminobenzidine staining kit (Nichirei Corporation). Microvessel density (MVD) was determined in five randomly chosen visual fields (high-power fields, hpf) in each of three similarly treated vital tumor tissues at x100 magnification (0.3 mm2 visual field size). Statistical analysis was done with the Student's t test.

For proliferation marker Ki67 staining, CC1 (antigene retrieval solution; Ventana)-pretreated sections were incubated with monoclonal rabbit Ki67 antibody (clone SP6; NeoMarkers). Detection of primary antibody was done with a biotinylated anti-rabbit immunoglobulin G (IgG) antibody (Jackson ImmunoResearch). For glucose transporter-1 (Glut-1) immunostaining, CC1-pretreated sections were incubated with polyclonal rabbit anti-human Glut-1 antibody (MYM AB 1351; Chemicon International) at a final dilution of 1:1,000 at room temperature. Ki67 and Glut-1 staining procedures were done on a Benchmark immunohistochemistry staining system (Ventana Medical Systems).

For hypoxia detection with the 2-nitroimidazole hypoxia marker, pimonidazole hydrochloride 60 to 80 mg/kg was applied i.v. 6 h after the last treatment and 45 min before mice sacrifice. Staining procedure was conducted according to the protocol of the Hypoxyprobe-1 Plus Kit (Chemicon International). Hypoxyprobe-1 MAb1 (1:50, mouse IgG1) conjugated with FITC, and anti-FITC monoclonal antibody (MAb) conjugated with horseradish peroxidase (HRP; 1:50). Apoptotic cells were identified by the terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) method according to the protocol of the In situ Cell Death Detection Kit (Roche). TUNEL- or Ki67-positive cells were counted in five randomly chosen visual fields in each of at least two similarly treated vital allograft tissues at high power (allograft tumors, 0.12 mm2 field size; spontaneous tumors, 0.0018 mm2 field size). The mean TUNEL- or Ki67-positive cell count from these fields was determined.

Results

Treatment of Her2/c-neu Overexpressing Mammary Carcinoma Cell Allografts and Spontaneous Carcinomas with IR and AEE788
In vitro experiments with the combined treatment modality of IR and the dual specific ErbB1/2- and VEGF-receptor tyrosine kinase inhibitor AEE788 resulted in a dose-dependent and at least additive antiproliferative and cytotoxic effect in primary endothelial and multiple established tumor cell lines (A431, MDA-MB-468, SW480, E1A/ras-transformed p53-def. mouse embryo fibroblasts, murine c-neu–overexpressing NF9006 mammary tumor cell). Interestingly, a strong antiproliferative effect of AEE788 against the different tumor cells was already observed in the submicromolar concentration range (IC50, 0.1–0.6 µmol/L), in comparison with the treatment response determined in endothelial cells (IC50, 1 µmol/L; data not shown). This is most probably due to the dominant growth-controlling effect of ErbB1/2 in these tumor cells and the more potent inhibitory effect of AEE788 against ErbB1/2 than against the KDR VEGF receptor of endothelial cells (31).

Due to the concomitant antiangiogenic and ErbB1/2-inhibitory effect of the AEE788 compound, we were specifically interested to assess the treatment response of AEE788 alone and in combination with IR in in vivo tumor models, which are driven by a high ErbB signaling status. In addition to an allograft tumor model in nude mice, we have evaluated the treatment responses in a spontaneously growing, orthotopic mammary carcinoma model using MMTV–c-neu transgenic mice. These mice spontaneously develop slow-growing mammary carcinomas 10 to 12 weeks after their first litter, and thus, the microenvironment and the neovascularization in these orthotopic tumors represent more closely the clinical situation. The NF9006 tumor cell line used for the allograft experiments was originally established from such a spontaneous mammary carcinoma. This also enabled to directly compare the treatment response in these related tumor models.

We first determined the in vivo response in the allograft tumor model to treatment with increasing doses of AEE788 alone. Treatment with AEE788 for 4 consecutive days was started when tumors reached a minimal size of 200 mm3 ± 10% (approximately days 10–14 after cell injection). A dose-dependent absolute growth delay (AGD) for doubling the initial tumor volume was achieved with a minimal treatment regimen of 4x30 mg/kg and was further extended by a treatment with 4 x 50, 4 x 70, and 4 x 100 mg/kg (AGD > 20 days), respectively (Fig. 1A ). Treatment with 4 x 25 mg/kg did not delay tumor growth in this allograft system (data not shown).


Figure 1
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  		Figure 1. The effect of AEE788 and IR alone or combined on the growth of NF9006-derived allografts in nude mice. A, mice were treated with increasing doses of AEE788 alone on 4 consecutive days. B and C, mice were treated with AEE788 alone or in combination with IR [30 mg/kg AEE788; 2 Gy (B); or 50 mg/kg AEE788; 3 Gy (C)] on 4 consecutive days. Points, mean tumor volume per group; bars, SE.

 
Combined treatment of AEE788 with IR was done with locoregional application of IR using a shielding device and fractionated single doses of either 2 or 3 Gy. These daily doses are also applied when fractionated radiotherapy is used for the treatment of human malignancies. For practical reasons, only four fractions were chosen as treatment regimen, but the response to such a regimen was previously found to be useful for treatment evaluation. AEE788 was applied 0.5 to 1 h before irradiation. Tumor growth delay in response to 4 x 3 Gy alone was similar in extent to treatment with 4 x 30 mg/kg AEE788 alone, and combined treatment resulted in an at least additive absolute growth delay to double or triplicate the initial treatment volume at the start of treatment. Combined treatment resulted in a significantly extended tumor growth delay (P < 0.05; RT versus combined treatment; Fig. 1B). Next, a treatment regimen with a reduced dose of irradiation (4 x 2 Gy) and an increased dose of AEE788 (4 x 50 mg/kg) was tested. We observed that tumor growth delay was again extended on treatment with 4 x 50 mg/kg (versus 4 x 30 mg/kg) and also resulted in an at least additive absolute growth delay when combined with irradiation (4 x 2 Gy; P = 0.01 RT versus combined treatment; Fig. 1C).Unfortunately, a direct comparison of the treatment responses to the different irradiation schedules was inconclusive, which is most probably due to the slightly more aggressive tumor growth of the NF9006-derived tumors in the experimental groups of Fig. 1C versus Fig. 1B, as already observed in the direct comparison of the respective control groups. No significant treatment-related body weight (<10%) and skin changes were observed during treatment and in the follow-up period, independent of the treatment modality.

Based on the results obtained with allografts, we then evaluated treatment responses in the spontaneously growing orthotopic mammary carcinoma model using MMTV-rat c-neu transgenic mice. Interestingly, these orthotopic mammary carcinomas were more sensitive to fractionated radiation treatment than the respective allograft tumors in nude mice, with almost complete regression using our standard IR treatment regimen of 4 x 3 Gy (data not shown). We therefore reduced the treatment regimen to 2 Gy single doses of IR on 3 consecutive days. The dosage of 3 x 50 mg/kg AEE788 alone induced a strong tumor regression during treatment, followed by an extended growth delay, but tumor growth consistently resumed 7 to 10 days after treatment start. On the other hand, IR alone was more effective in the spontaneous tumor model than AEE788 alone. Combined treatment with IR and AEE788 after a minimal treatment regimen resulted in extended tumor regression and growth delay over 20 days with an additive absolute growth delay for triplicating the initial tumor size [Fig. 2 ; combined treatment resulted in a significantly extended tumor growth delay (P < 0.04; RT versus combined treatment)].


Figure 2
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  		Figure 2. The effect of AEE788 and IR alone or in combination on the growth of spontaneous, mammary carcinomas in MMTV–c-neu transgenic mice. Mice were treated with AEE788 alone (50 mg/kg), IR alone (2 Gy), and in combination applied on 3 consecutive days. Points, mean tumor volume per group; bars, SE.

 
Tumor Hypoxia after Treatment with AEE788 and IR in the Allograft and Orthotopic Tumor Model
IR and AEE788 target not only tumor cells, but also the tumor vasculature. Both target structures affect tumor oxygenation, which is a relevant determinant for the tumor response. We therefore determined treatment-dependent changes of oxygenation in allograft and orthotopic tumors of equal sizes at the start of treatment using Glut-1 as an endogenous marker of chronic tumor hypoxia (Fig. 3 ). We observed a typical speckled staining pattern in the control untreated allografts and orthotopic tumors. Treatment with IR alone had only a minimal effect on Glut-1 staining in both tumor models, whereas treatment with AEE788 dramatically reduced Glut-1 staining to nearly undetectable levels, indicative of a drastic AEE788-induced reduction of tumor hypoxia. Combined treatment of IR with AEE788 also resulted in strongly reduced Glut-1 staining in both tumor models.


Figure 3
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  		Figure 3. Immunohistochemical detection of tumor hypoxia with antibodies against endogenous Glut-1 in NF9006-derived allografts and orthotopic ErbB/neu-overexpressing mammary carcinomas. Mice with NF9006-derived allografts and orthotopic tumors were treated with vehicle, AEE788 (50 mg/kg), IR (2 Gy), or in combination for 4 d (allografts) or 3 d (orthotopic tumors). Bars, 200 µm.

 
The level of endogenous Glut-1 may also be influenced by the activity status of specific signal transduction cascades, including the EGFR/AKT/HIF-1{alpha} pathway, which is also targeted by AEE788. We therefore determined treatment-dependent changes of tumor hypoxia also by coinjection of the hypoxia probe pimonidazole, which is injected 45 min before mice sacrifice and specifically accumulates in hypoxic tissue areas. Two adjacent tumor sections were then probed either with antibodies specific for Glut-1 or pimonidazole (see Fig. 6). Strongly reduced accumulation of pimonidazole in tumors of AEE788-treated animals also showed AEE788-dependent tumor hypoxia reduction. These results also support that endogenous Glut-1 is an adequate marker for tumor hypoxia in these tumor models.


Figure 6
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  		Figure 6. Immunohistochemical detection of tumor hypoxia with antibodies against endogenous Glut-1 or the exogenous 2-nitroimidazole hypoxia marker pimonidazole hydrochloride. Mice with NF9006-derived allografts and spontaneous tumors were treated with vehicle, AEE788 (50 mg/kg), PTK787 (100 mg/kg), or PKI166 (100 mg/kg) for 4 d (allografts) or 3 d (spontaneous tumors). Bars, 200 µm.

 
Tumor Cell Proliferation, Apoptosis, and Microvessel Density in Response to AEE788 and IR
Reduced tumor hypoxia could be due to improved oxygen diffusion or perfusion in the tumor tissue or a result of reduced tumor oxygen consumption. We therefore investigated in situ the proliferative status, tumor cell apoptosis, and tumor microvessel density in response to the different treatment modalities.

Tumor cell proliferation was determined using immunohistochemistry for the Ki-67 protein, which is expressed during all phases of the cell cycle except G0 (Fig. 4A ). In allograft tumors, treatment with AEE788 (4 x 50 mg/kg), and irradiation (4 x 3 Gy) alone reduced the proliferative activity to 28% and 58% of control tumors, respectively, at the end of treatment (P < 0.005 control versus AEE788, IR, combined). Combined treatment of NF9006 allografts resulted in a further reduction of the proliferative activity to 12% of the proliferative activity in control tumors (P < 0.05 AEE788, IR versus combined).


Figure 4
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  		Figure 4. Tumor cell proliferation, apoptosis, and microvessel density in response to AEE788 and IR. Mice with NF9006-derived allografts and orthotopic mammary carcinomas were treated with vehicle, AEE788 (50 mg/kg), IR (2 Gy), or in combination for 4 d (allografts) or 3 d (spontaneous tumors). Six hours after the last treatment, mice were sacrificed, and tumors were harvested, formalin fixed, and stained for the Ki-67 (A), TUNEL (B), and CD31 (C) as marker of tumor cell proliferation, apoptosis, and microvessel density, respectively. Columns, mean value per group; bars, SD.

 
At day 4 of treatment, AEE788 alone and in combination with IR resulted in a 6- and 10-fold increase of TUNEL-positive cells per visual field, respectively, whereas irradiation alone doubled the amount of this apoptotic marker (P < 0.005 control versus all treatment modalities; AEE788 versus combined). Thus, the initial tumor regression, especially in response to AEE788 alone and in combination with IR, is associated with high apoptotic indices in allograft tumors (Fig. 4B).

To examine the effect of the different treatment modalities on the tumor vasculature, microvessel density was determined by CD31 immunohistochemistry at the end of treatment. IR alone did not affect microvessel density, whereas AEE787 alone and in combination with IR resulted in a 21% reduction of microvessel density and a slightly enhanced reduction of 26% when used in combination with IR (26%; Fig. 4C).

The same histologic end points were also determined in the mitotically less active orthotopic tumor model. Combined treatment with AEE788/IR resulted in the strongest reduction of the proliferative activity down to 19% of control tumors. Likewise, combined treatment induced an at least additive 12-fold increase of the apoptotic index when compared with the control tumor. Irradiation did not affect microvessel density, which was only reduced on treatment with AEE788 alone or in combination with IR (P < 0.05, IR versus AEE788 or combined). Of note, the orthotopic carcinoma tissue contained large lake-like cavities or vessels, but the response on the level of microvessel density was similar to the allograft tissue (Fig. 4). Thus, on the level of tumor proliferation, apoptosis, and microvessel density, the qualitative and quantitative response in the spontaneous tumor model was overall similar to the response in the related allograft tumor model.

In this study, we did not directly determine the inhibitory effect of AEE788 at the level of ErbB and VEGFR receptor tyrosine kinases in situ. However, we could directly determine a cellular response on the level of tumor cell proliferation and apoptosis and changes at the level of the tumor vasculature. Furthermore, other groups showed that the activity of these receptors is specifically inhibited upon treatment of AEE788, applying even lower AEE788 treatment regimens (31, 33, 34, 38).

We have previously determined a strong antiproliferative and cytotoxic effect of AEE788, IR, and in combination on the cellular level against different tumor cells. In situ TUNEL-positive cells indicated that these treatment modalities also induce apoptosis. To confirm this treatment response in vitro, we did a caspase-3 activity assay on NF9006 cells. Caspase activity could be determined both in response to submicromolar concentrations of AEE788 and irradiation alone. Combined treatment with AEE788 and IR significantly enhanced this apoptosis-related effector protease activity (Fig. 5 ; 0.05 AEE788 versus combined; IR versus combined).


Figure 5
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  		Figure 5. Apoptosis induction determined in NF9006 cells. Caspase 3–like activity was determined 15 h after treatment in cytosolic extracts using Ac-DEVD-pNA as a colorimetric caspase-3 substrate. Caspase activity was determined at least thrice, and pooled data are shown as percentage of caspase activity in the control samples.

 
Tumor Hypoxia after Treatment with AEE788 and IR in the Allograft and Orthotopic Tumor Model
To attribute the contribution of the antiangiogenic or antitumor cell–directed inhibitory effect of AEE788 to the observed tumor tissue–related end points, we treated mice carrying tumor allografts or spontaneously growing tumors of equal sizes at the start of treatment with the specific clinically relevant VEGF-receptor tyrosine kinase inhibitor PTK787/ZK22584 or the specific ErbB1/2/c-neu receptor tyrosine kinase inhibitor PKI166, which is structurally related to AEE788. For these comparative studies, we used dosages of the (single specific) receptor tyrosine kinase inhibitors PKI166 and PTK787 (100 mg/kg each), which when used as part of a combined treatment modality, are as potent as AEE788, at a p.o. dose of 30 to 50 mg/kg (31). We previously determined that the proliferative index in these tumors is reduced in response to PKI-166 treatment alone, whereas PTK787 alone has no effect on their proliferative status (data not shown). Interestingly, treatment with PTK787 induced a slight increase of tumor hypoxia both in the allograft and spontaneous mammary carcinomas, whereas treatment with PKI-166 reduced Glut-1 expression and pimonidazole accumulation as strongly as AEE788 (Fig. 6 ). These results suggest that it is the ErbB-directed activity of AEE788 that mediates the hypoxia-reducing effect of this dual kinase-specific inhibitor.

Discussion

The combined treatment of ionizing radiation with inhibitors of either ErbB-receptor or VEGF-receptor signaling is of increasing clinical interest. Here, we have examined the effects of a combined treatment modality using IR and the novel, dual specific VEGF/ErbB-receptor tyrosine kinase inhibitor AEE788 in a spontaneously growing murine mammary carcinoma model and against ErbB/neu-overexpressing mammary carcinoma allografts. Treatment-induced changes of tumor cell proliferation, apoptosis, and microvessel density were similar in both tumor models, and interestingly, treatment with AEE788 alone and in combination with IR strongly improved tumor oxygenation in both the allograft and the orthotopic tumor model, suggesting a putative radiosensitizing potential.

AEE788 is a dual specific receptor tyrosine kinase inhibitor targeting ErbB1/2- and VEGF-receptor signaling and has been tested alone and in combination with other cytotoxic or specific anti-signaling agents (e.g., paclitaxel, imatinib mesylate) in xenografts and various orthotopic murine tumor models (e.g., prostate and colon carcinoma, non–small cell lung cancer; adenoid cystic carcinoma, etc.; refs. 34, 36–38). In our study, we used primary mammary carcinomas of MMTV-ErbB-2/neu transgenic mice and allografts derived from the tumor cell line NF9006, which was originally established from such a mammary carcinoma. Combined treatment with IR in vitro resulted in an additive antiproliferative and cytotoxic effect in all different tumor cell types tested. This suggested that combined treatment with AEE788 and IR in vivo will induce a strong supra-additive treatment response based on the multilayered synergistic effects on tumor cells and tumor vasculature. However, only an at least additive absolute tumor growth delay could be determined in both murine tumor models in response to the combined treatment modality. Nevertheless, this treatment combination will be of clinical interest taking the clinical applicability of AEE788 into consideration (32).

As an important functional end point in the analysis of novel treatment modalities, we determined AEE788-induced changes of tumor hypoxia using the endogenous hypoxia marker Glut-1 and the exogenous hypoxia marker pimonidazole, which accumulates in hypoxic tissue at pO2 levels below 5 to 10 mm Hg. Treatment with AEE788 alone or in combination with IR strongly improved tumor oxygenation in both tumor models, which was also observed in mice treated with the specific ErbB1/2 receptor tyrosine kinase inhibitor PKI-166 at equipotent in vivo concentrations. On the other hand, treatment with the specific VEGF-receptor tyrosine kinase inhibitor PTK787/ZK222584 reduced tumor oxygenation in both tumor models. Using serial high-resolution [18F]-fluoromisonidazole positron emission tomography, we previously showed that treatment with PTK787/ZK222584 alone increases overall and local tumor hypoxia in these allografts (14). We cannot exclude an additional mechanism, but these results show also on the functional in vivo level a more potent antiproliferative effect of AEE788, leading to improved tumor oxygenation. This ErbB tumor cell–directed effect of AEE788 is dominant over its VEGFR-directed, probably hypoxia-increasing antiangiogenic effect.

Tumor hypoxia results from limited diffusion and intermittent blood flow due to an imbalance of rapid tumor growth, insufficient tumor angiogenesis, and supply of oxygen. This can directly reduce the efficacy of chemo- and radiotherapy, but also cause the selection of more aggressive tumor cell populations. Thus, tumor hypoxia is a relevant factor in the treatment strategy, and it is important to understand treatment-induced changes of tumor oxygenation. These may be affected by treatment-dependent changes of tumor vascularization (supply) or by the reduction of oxygen consumption in response to antiproliferative or cytotoxic agents targeting tumor cells (demand). Solomon et al. (40) previously showed in human EGFR-expressing A431 squamous cell carcinoma xenografts that EGFR inhibitor Iressa reduces intratumoral hypoxia but without investigating the mechanism leading to Iressa-modulated tumor hypoxia. Here, we show that AEE788-enhanced tumor oxygenation correlates with strongly reduced tumor cell proliferation and reduced microvessel density. On the other hand, treatment with the VEGFR inhibitor PTK787 alone also reduces microvessel density in both tumor models, but has no effect on tumor proliferation (ref. 14 and data not shown). Although changes in microvessel density are only one functional end point of an antiangiogenic treatment modality, our results with AEE788 indicate that treatment with AEE788 primarily reduces the proliferative status and concomitant oxygen consumption in the tumor, which eventually translates into a reduced tumor hypoxia status.

In comparison to allograft tumors, we observed an increased sensitivity of orthotopic mammary carcinomas to all treatment modalities, especially to IR. This required a reduction of the AEE788 and IR dosages to investigate a potential additive effect. The increased sensitivity of the orthotopic carcinomas may be linked to differences in the tumor vasculature and the local microenvironment as well as immunomodulatory effects in the immunocompetent host. On the other hand, the NF9006 cell line, which is derived from a respective spontaneous tumor, might have developed additional treatment resistances during the in vitro establishment, which translates into a more aggressive growth and treatment resistance.

A combined treatment modality with IR and ZD6474, another dual specific inhibitor of both VEGF and EGFR signaling, has previously been tested in xenografts of non–small cell lung cancer and glioblastoma (30, 41). These studies focused on the antiangiogenic aspect of ZD6474, demonstrating reduced perfusion in the lung cancer model and reduced expression of VEGF in the glioblastoma model. These responses were discussed as part of a mechanistic explanation for the (supra-)additive tumor growth delay when used in combination with irradiation and at the same time suggested an even improved effect when the combination is used in an adjuvant treatment regimen. We have also tested various scheduling regimens, but in contrast to the results obtained with ZD6474, we did not observe a schedule-dependent effect [AEE788 (50 mg/kg)/IR (2 Gy), three fractions, neoadjuvant, concomitant, adjuvant] on the treatment response in the spontaneous tumor model (data not shown).

Overall, we show that the combined treatment modality of the novel dual specific inhibitor AEE788 in combination with IR is a promising approach, especially in hypoxic, oncogenic ErbB-driven tumors and show that AEE788-induced changes on tumor hypoxia mainly depend on its antiproliferative effect and not on its antiangiogenic potency. Detailed kinetic analysis will be additionally required to dissect the individual steps of the treatment response to AEE788 to further exploit its effect on tumor hypoxia.

Acknowledgments

We thank Jeanette Wood and Peter Traxler for the PKI166 and PTK787/ZK222584 reagents and acknowledge Marion Bawohl for excellent technical support.

Footnotes

Grant support: Oncosuisse, the Radiumfonds, the Sassella- and the Swiss National Foundations (to C. Oehler-Jänne, O. Riesterer, and M. Pruschy).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 9/07; revised 7/ 2/07; accepted 7/26/07.

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