Efficacy of Tie2 Receptor Antagonism in Angiosarcoma

Efficacy of Tie2 Receptor Antagonism in Angiosarcoma

Volume 14 Number 2 February 2012 pp. 131–140 131 www.neoplasia.com Efficacy of Tie2 Receptor Antagonism in Angiosarcoma1,2 Jason R. Hasenstein*, ...

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Volume 14 Number 2

February 2012

pp. 131–140 131

www.neoplasia.com

Efficacy of Tie2 Receptor Antagonism in Angiosarcoma1,2

Jason R. Hasenstein*, Kelsey Kasmerchak*, † † Darya Buehler , Gholam Reza Hafez , Kevin Cleary*, ‡ John S. Moody and Kevin R. Kozak* *Department of Human Oncology, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, WI, USA; †Department of Pathology, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, WI, USA; ‡Division of Radiation Oncology, Moses Cone Regional Cancer Center, Greensboro, NC, USA

Abstract Angiosarcomas are malignant endothelial cell tumors with few effective systemic treatments. Despite a unique endothelial origin, molecular candidates for targeted therapeutic intervention have been elusive. In this study, we explored the tunica internal endothelial cell kinase 2 (Tie2) receptor as a potential therapeutic target in angiosarcoma. Human angiosarcomas from diverse sites were shown to be universally immunoreactive for Tie2. Tie2 and vascular endothelial growth factor receptor (VEGFR) antagonists inhibited SVR and MS1-VEGF angiosarcoma cell survival in vitro. In the high-grade SVR cell line, Tie2 and VEGF antagonists inhibited cell survival synergistically, whereas effects were largely additive in the low-grade MS1-VEGF cell line. Xenograft modeling using these cell lines closely recapitulated the human disease. In vivo, Tie2 and VEGFR inhibition resulted in significant angiosarcoma growth delay. The combination proved more effective than either agent alone. Tie2 inhibition seemed to elicit tumor growth delay through increased tumor cell apoptosis, whereas VEGFR inhibition reduced tumor growth by lowering tumor cell proliferation. These data identify Tie2 antagonism as a potential novel, targeted therapy for angiosarcomas and provide a foundation for further investigation of Tie2 inhibition, alone and in combinations, in the management of this disease. Neoplasia (2012) 14, 131–140

Introduction Angiosarcoma is an uncommon, aggressive subtype of soft tissue sarcoma composed of malignant endothelial cells. Current treatment strategies are woefully inadequate. Despite aggressive therapy, only about half of patients presenting with resectable tumors will be cured of their disease. The median survival of patients presenting with unresectable disease is less than 1 year [1]. Progress in developing novel therapeutic approaches to angiosarcoma has been hindered by the rarity of this disease coupled with a dearth of models to support preclinical investigations. However, two lines of evidence highlight potential roles for antagonists of endothelial growth factor signaling pathways. First, preclinical studies suggest that angiosarcomas share critical molecular features with normal endothelial cells. Specifically, angiosarcomas have been shown to express vascular endothelial growth factor A (VEGF-A) and multiple VEGF receptors (VEGFRs), including the major proangiogenic VEGF-A receptor, VEGFR2 [2–6]. Moreover, a murine endothelial cell line engineered

to overexpress VEGF-A forms invasive angiosarcomas in immunodeficient mice [7]. Evidence addressing the expression of other endothelial growth factor signaling pathway components is scant. Brown et al. [8] examined two cutaneous angiosarcomas and found that tunica internal endothelial cell kinase 2 (Tie2) receptor messenger RNA was

Abbreviations: Ang, angiopoietin; Tie, tunica internal endothelial cell kinase (i.e., TEK); VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor Address all correspondence to: Kevin R. Kozak, MD, PhD, Department of Human Oncology, School of Medicine and Public Health, University of Wisconsin–Madison, 1111 Highland Ave, WIMR 3153, Madison, WI 53705. E-mail: [email protected] 1 This was supported in part by a National Institutes of Health grant (1UL1RR25011). The authors have no conflicting commercial or financial interests. 2 This article refers to supplementary materials, which are designated by Figures W1 and W2 and are available online at www.neoplasia.com. Received 18 December 2011; Revised 25 January 2012; Accepted 26 January 2012 Copyright © 2012 Neoplasia Press, Inc. All rights reserved 1522-8002/12/$25.00 DOI 10.1593/neo.111770

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strongly expressed in both lesions. The Tie2 receptor ligands, angiopoietin 1 (Ang1) and Ang2, were also expressed in both angiosarcoma specimens. Stacher et al. [9] observed Tie2 expression in 7 of 10 examined pulmonary epithelioid angiosarcomas. The second line of evidence supporting a potential role for antiangiogenic/antiendothelial therapies in angiosarcoma derives from early clinical experience with VEGF antagonists. Clinical responses have been reported with bevacizumab, a monoclonal antibody directed at VEGF-A, in cutaneous angiosarcomas of the face [10–12]. Interim results of a multicenter phase 2 study of bevacizumab in unresectable angiosarcoma and epithelioid hemangioendothelioma patients showed a 15% partial response rate with disease stabilization in 63% of patients [13]. Results with VEGFR tyrosine kinase inhibitors in unselected soft tissue sarcoma patients, including sorafenib, pazopanib, and sunitinib, are more challenging to interpret because of the small number of angiosarcoma patients in these trials; however, clinical activity has been observed with sorafenib [14–17]. The Ang-Tie2 system plays critical roles in endothelial cell survival and proliferation, vascular plasticity, and angiogenesis [18]. Consequently, there is considerable interest in modulators of the Ang-Tie2 axis as antiangiogenic agents for treatment of solid tumors and several candidate drugs have emerged. Results of the first phase 1 study of an Ang-Tie2 axis-targeted therapy in patients with advanced solid tumors have recently been reported and documented good tolerance and promising antitumor activity [19]. On the basis of a promising molecular rationale, and the growing availability of pharmacophores, we hypothesized that Tie2 inhibition may have therapeutic utility in angiosarcoma. To test this hypothesis, we examined the expression of Tie2 in a panel of human angiosarcomas from diverse sites. We then evaluated a selective Tie2 receptor tyrosine kinase inhibitor, with and without the clinically used VEGFR tyrosine kinase inhibitor sunitinib, in vitro and in vivo using two mouse models of angiosarcoma.

Materials and Methods

Cell Lines and Pharmacologic Agents SVR and MS1-VEGF cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco modified Eagle medium containing 5% fetal bovine serum and 1% penicillin/streptomycin. Human umbilical vein endothelial cells were obtained from Lonza (Basel, Switzerland) and cultured in endothelial growth medium 2. Sunitinib was obtained from Cayman Chemical (Ann Arbor, MI), and purity was confirmed by thin-layer chromatography. The selective Tie2 kinase inhibitor 4-(6-methoxy-2-naphthyl)-2(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H -imidazole was obtained from EMD4Biosciences (Darmstadt, Germany) and Selleck Chemicals (Houston, TX). 1H nuclear magnetic resonance (DMSO-d6) confirmed structure [20]. Purity was shown to be greater than 96% by high-performance liquid chromatography.

Immunoblot Analysis HUVEC, SVR, and MS1-VEGF cells were grown to approximately 70% confluence. Cells were washed twice in cold PBS and protein isolated in cold NP-40 lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na3VO4, 20 mM beta-glycerophosphate,

Neoplasia Vol. 14, No. 2, 2012 10 μg/ml of leupeptin and aprotinin). Proteins were quantified using a standard Bradford absorbance assay. Proteins were separated on 4% to 20% gradient SDS-PAGE gels and transferred to polyvinylidene fluoride membrane by electrophoretic transfer. Membranes were subsequently blocked in 5% nonfat milk or bovine serum albumin in 1 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20. Primary antibodies for Western blot detection of VEGFR2 (1:1000; Santa Cruz Biotechnology, Inc, Santa Cruz, CA) or Tie2 (1:1000; Santa Cruz Biotechnology, Inc) were applied overnight at 4°C. Secondary antibody for detection was either goat antirabbit immunoglobulin G (IgG) HRP (Tie2, 1:4000; Santa Cruz Biotechnology, Inc) or goat antimouse IgG-HRP (VEGFR-2, 1:4000; Santa Cruz Biotechnology, Inc). Proteins were detected through enhanced chemiluminescence detection system (Amersham Biosciences, Buckinghamshire, United Kingdom).

Cell Survival Assays SVR and MS1-VEGF cells were plated at 1500 cells per well in 96-well plates and allowed to adhere overnight. The medium was aspirated and replaced with fresh medium containing varying concentrations of sunitinib, Tie2 kinase inhibitor, or DMSO vehicle control. The final concentration of DMSO was 0.1%. Cells were allowed to grow for 72 hours, and cell survival was quantified using the WST-1 cell proliferation reagent (Clontech Laboratories, Mountain View, CA) according to the manufacturer’s instructions. Cell survival assays examining combined treatment were similarly performed at a fixed Tie2 kinase inhibitor-to-sunitinib molar ratio of 25:1 (to reflect relative drug potencies) at total drug concentrations of 26 to 7800 nM. Combination index analyses were conducted using CompuSyn (ComboSyn, Inc, Paramus, NJ).

In Vivo Studies Athymic nude mice (6- to 8-week-old females) were obtained from Harlan Laboratories, Inc (Indianapolis, IN). The care and treatment of experimental animals were in accordance with institutional guidelines. SVR and MS1-VEGF cells (2 × 106 cells) were implanted subcutaneously. For histopathologic confirmation of the angiosarcoma phenotype, SVR tumors were excised at days 3, 6, 9, and 12 after injection, and MS1-VEGF tumors were excised at days 5, 10, 15, and 20 after injection. For tumor growth delay studies, treatment was started when the tumor volume reached 200 mm3 (SVR) or when palpable (MS1-VEGF). Animals were randomized into four treatment groups. The control group and the Tie2 kinase inhibitor alone group were treated daily with 100 μl of vehicle (0.5% carboxymethylcellulose, 0.4% Tween 80, 1.8% NaCl, and 0.9% benzyl alcohol in distilled water, pH 6.0) by oral gavage. The sunitinib-alone and combination treatment groups were treated daily with 100 μl of vehicle containing sunitinib (60 mg/kg for SVR tumors, 30 mg/kg for MS1-VEGF tumors) by oral gavage. The control and sunitinib-alone groups were treated twice weekly with 100 μl of vehicle (5% ethanol, 5% Cremophor, and 90% distilled water) through intraperitoneal injection. The Tie2 kinase inhibitor–alone and combination treatment groups were injected twice weekly with 100 μl of vehicle containing Tie2 kinase inhibitor (50 mg/kg). Tumor volume was determined by direct caliper measurement and calculated by the following formula: volume = 0.5 × (large diameter) × (small diameter)2. For necrosis, apoptosis, and proliferation studies in vivo, SVR tumor-bearing animals (n = 4-6 per group) were randomized and treated as above for 11 days. On

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Figure 1. Specificity of Tie2 immunostaining and Tie2 expression in human angiosarcomas. Immunohistochemical evaluation of Tie2 expression was performed as described in Materials and Methods (magnification, ×400). (A) Ischemic fasciitis (positive control) showing the expected expression of Tie2 in endothelial cells. (B) Uniform expression of Tie2 was observed in a primary small bowel angiosarcoma. (C) Primary small bowel angiosarcoma processed without secondary antibody (negative control). (D-M) Hematoxylin and eosin and Tie2 immunohistochemical evaluation of 10 additional representative human angiosarcomas: extremity soft tissue (D), bone (E), bladder (F), scalp (G), peristernal soft tissue (H), liver (I), small intestine (J), periumbilical soft tissue (K), chest wall (after radiation) (L), and spleen (M).

day 12, animals were killed, and tumors were harvested for histologic and immunohistochemical analyses. Percent tumor necrosis was estimated by a pathologist (D.B.) blinded to treatment using hematoxylin and eosin– stained whole tumor sections. The number of cleaved caspase 3 and PCNA-positive cells per high-power field (n = 5/tumor) was quantified by an observer blinded to treatment.

Immunohistochemical Analyses Formalin-fixed, paraffin-embedded angiosarcoma specimens were obtained from the University of Wisconsin’s surgical pathology archives on an institutional review board–approved protocol, and the diagnosis was confirmed by a pathologist with expertise in soft tissue sarcomas. For Tie2, tissue sections (5 μm thick) were cut using traditional water bath technique and dried overnight at room temperature. Slides

were deparaffinized in subsequent xylene and ethanol incubations, followed by heat-induced epitope retrieval using the Lab Vision PT module (Thermo Fisher Scientific, Fremont, CA) with Lab Vision citrate buffer pH 6.0 at 98°C for 20 minutes without boiling. Immunolabeling was performed at room temperature using the Lab Vision 360 automated staining system following a standard biotin-avidin protocol. Biocare Medical (Concord, CA) were used except where noted. Endogenous peroxidase was blocked for 5 minutes with Peroxidazed 1. Nonspecific protein binding was eliminated through a 30-minute block with Sniper, and nonspecific avidin was blocked using the Avidin Biotin kit, with a 15-minute incubation for each reagent. Slides were incubated with 1:50 dilution of goat anti-Tie2 IgG (R&D Systems, Minneapolis, MN) for 60 minutes followed by incubation with biotinylated swine antigoat IgG 1:50 (Invitrogen, Carlsbad, CA) and 4plus Streptavidin-HRP treatment. Betazoid DAB and Mayer hematoxylin

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were each incubated for 1 minute. All antibodies were diluted using Renaissance Background Reducing Diluent. Slides were washed with Tris-buffered saline–Tween after each reagent. Tie2 immunoreactivity was scored by two authors (K.R.K. and D.B.): 0 indicates negative; 1+, weak; 2+, moderate; and 3+, strong staining. For PCNA and cleaved caspase 3 immunohistochemistry, tissue was fixed for 24 hours in 10% neutral buffered formalin, dehydrated through graded ethyl alcohols, paraffin infiltrated, and embedded. Tissue sections were cut at 5 μm and mounted on slides. Slides were deparaffinized in xylene and hydrated through graded ethyl alcohols to water. Slides were washed with PBS three times. Nonspecific binding was blocked with 10% goat serum in PBS for 1 hour, and endogenous peroxidase was blocked with 0.3% hydrogen peroxide in PBS for 10 minutes. Endogenous biotin was blocked with 0.001% avidin in PBS for 10 minutes, and avidin was quenched with 0.001% biotin in PBS for 10 minutes. The slides were then incubated with either anti-PCNA (Novus, Littleton, CO) at a 1:200 dilution in PBS with 1% goat serum and 0.001% Triton X-100 or anti–cleaved caspase 3 (Asp175) (Cell Signaling Technology, Inc, Danvers, MA) at a 1:800 dilution in PBS with 1% goat serum overnight at 4°C. After washing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:200 in PBS for one hour at room temperature. Slides were washed in PBS and then incubated 30 minutes at room temperature with Vectastain ABC Elite (Vector Laboratories). After PBS wash, slides were developed with DAB (Vector Laboratories), counterstained with Mayer hematoxylin. All reagents and chemicals were from Sigma (St Louis, MO), unless otherwise noted.

Statistical Analyses

Neoplasia Vol. 14, No. 2, 2012 MS1-VEGF cells express Tie2 and VEGFR2. On the basis of its clinical availability, promise in this disease setting and lack of activity against the Tie2 receptor, the VEGFR inhibitor, sunitinib, was selected as a model VEGFR antagonist [22]. Tie2 antagonists have not yet reached the clinic. However, Semones et al. [20], using a combination of library screening and in silico design, developed a highly selective Tie2 kinase inhibitor, 4-(6-methoxy-2-naphthyl)-2-(4-methylsulfinylphenyl)-5-(4pyridyl)-1H-imidazole (Tie2 kinase inhibitor) with in vivo activity in mouse. This inhibitor was used as the model Tie2 antagonist in vitro and in vivo. The effects of VEGFR and Tie2 antagonism on cell survival were then assessed in vitro. As shown in Figure 3, both sunitinib and Tie2 kinase inhibitor modestly reduced SVR and MS1-VEGF cell survival in a dose-dependent manner. Unsurprisingly, MS1-VEGF cells were more sensitive to sunitinib than SVR cells were. In contrast, SVR cells were modestly more sensitive to Tie2 kinase inhibition than MS1VEGF cells were. To test for potential cytotoxic synergy of dual Tie2 and VEGFR inhibition, SVR and MS1-VEGF cells were treated with the combination of sunitinib and Tie2 kinase inhibitor, and a combination index analysis was conducted. The combined treatment was synergistic against SVR cells throughout the fractional affect range, whereas the combination was generally additive in MS1-VEGF cells (Figure 3C). To confirm the angiosarcomagenic potential of SVR and MS1VEGF cells, time course experiments were performed to examine the temporal evolution of gross and microscopic tumor phenotype. Subcutaneous implantation of SVR cells into athymic nude mice resulted in rapidly progressive, purpuric nodules reminiscent of the human disease (Figure 4A). Implantation of MS1-VEGF cells resulted in

Data are presented as means ± SE. Multigroup comparisons were conducted by analysis of variance (GraphPad Prism; GraphPad Software, La Jolla, CA) with Bonferroni posttest corrections for multiple comparisons unless otherwise specified. Results To assess Tie2 expression in a diverse set of human angiosarcomas, we first confirmed the specificity of Tie2 immunohistochemistry in normal intestinal wall and in ischemic fasciitis. As expected, Tie2 immunoreactivity was identified in the vascular endothelium (Figure 1A). We then tested Tie2 immunoreactivity in a case of human angiosarcoma (small bowel primary) and found uniform, moderate Tie2 expression in malignant cells (Figure 1B). Ten additional specimens from diverse primary sites were then examined. As shown in Figure 1D to M, strong Tie2 expression was identified in 50%, moderate expression in 40%, and weak expression in 10% of the specimens. These results raise the possibility that the Tie2 receptor may represent a therapeutic target in angiosarcoma. To test the hypothesis that Tie2 receptor antagonism may have therapeutic potential in angiosarcoma, models that recapitulate the human disease are required. Arbiser et al. [7,21] have reported the generation of two cell lines that form angiosarcomas in mice. Using an immortalized, murine endothelial cell line (MS1), they showed that introduction of activated H-ras yields a cell line (SVR) that forms rapidly growing angiosarcomas in vivo. Similarly, overexpression of primate VEGF 121 in the parental MS1 cell line results in a cell line (MS1-VEGF) capable of forming slow-growing angiosarcomas in mice. To confirm the utility of these models for assessing vascular targeted therapies in angiosarcoma, we first examined the expression of Tie2 and VEGFR2 in these cells. As shown in Figure 2, both SVR and

Figure 2. Expression of VEGFR2 and Tie2 in SVR and MS1-VEGF cells. SVR and MS1-VEGF cells express both the Tie2 and VEGFR2 receptors; human umbilical vein endothelial cells are shown as a positive control.

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Figure 3. Inhibition of SVR (solid line) and MS1-VEGF (dashed line) cell survival by sunitinib (A) and Tie2 kinase inhibitor (B) and dose effect analysis of combined sunitinib and Tie2 kinase inhibitor (C). Cell survival was determined as described in Materials and Methods. Combination indices <1, =1, and >1 denote synergy, additivity, and antagonism, respectively. Values represent mean ± SE (n = 4).

slow-growing, faintly violaceous nodules (Figure 4B). Histomorphologically, both SVR and MS1-VEGF lesions very closely resemble human angiosarcoma. As seen in Figure 4C to D, both early SVR tumors and MS1-VEGF tumors display the irregular, anastomosing vascular channels characteristic of angiosarcoma. Lesional cells are epithelioid or spindled with significant nuclear pleiomorphism, high nuclear-to-cytoplasmic ratios, and hyperchromatic nuclei. The malignant, infiltrating character of the lesions is best observed in day 12 SVR tumors where frank skeletal muscle invasion is evident. Both tumors retained expression of Tie2 as well as diagnostic expression of CD31 (Figures W1 and W2). The sensitivity of SVR and MS1-VEGF angiosarcomas to VEGFR and Tie2 kinase inhibition was then tested in vivo. In SVR tumors (Figure 5A), sunitinib (60 mg/kg, orally everyday), Tie2 kinase inhibitor (50 mg/kg, intraperitoneally, twice a week), and the combination resulted in statistically significant reductions in tumor volume by day 15. By day 20, sunitinib treatment resulted in a 44% reduction in tumor volume compared with control-treated animals (P < .01). Tie2 kinase inhibitor treatment resulted in a 61% reduction in tumor volume (P < .01). The combination of sunitinib and Tie2 kinase inhibitor resulted in the greatest tumor volume reduction (70%; P < .01). The combination was more potent than sunitinib alone (P < .01) and Tie2 kinase inhibitor alone, although the latter did not reach statistical significance after correction for multiple comparisons (P > .05). No differences in animal weights were observed between groups at any time point (P > .3).

Mice bearing MS1-VEGF tumors were similarly treated with sunitinib (30 mg/kg, orally everyday), Tie2 kinase inhibitor (50 mg/kg, intraperitoneally, twice a week), or the combination (Figure 5B). Persistent, statistically significant tumor growth delay was observed for all treatment groups by day 33. Compared with control-treated mice, sunitinib treatment reduced tumor volume by 41% (P < .01) and Tie2 kinase inhibitor treatment reduced tumor volume by 45% (P < .01) by 6 weeks. Combined treatment resulted in the greatest tumor volume reduction (74% vs control, P < .01). As in the case of SVR tumors, combined treatment displayed greater activity than monotherapy with either sunitinib or Tie2 kinase inhibitor, and these differences reached statistical significance by 6 weeks (P < .05). No differences in animal weights were observed between groups at any time point (P > .15). To identify potential mechanisms of tumor growth delay, SVR tumorbearing mice (n = 4-6 per group) were treated as above for 11 days and killed at day 12 for tumor histologic and immunohistochemical analysis. Tumors from control-treated animals displayed 27% ± 7% necrosis. Sunitinib treatment, Tie2 kinase inhibitor treatment and combination treatment resulted in 48% ± 9% (P = .1 vs control, Student’s t test), 41% ± 8% (P = .2, Student’s t test) and 39 ± 8% (P = .3, Student’s t test) necrosis. Apoptosis was assessed by cleaved caspase 3 immunohistochemistry (Figure 6A). Consistent with its activity in vivo, Tie2 kinase inhibitor treatment led to a three-fold increase in cleaved caspase 3–positive cells compared with tumors from control-treated animals (P < .05). Sunitinib had no significant effect on the number of cleaved caspase 3–positive

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cells compared with control-treated animals (P > .05) and the combination of sunitinib and Tie2 kinase inhibitor yielded intermediate levels of apoptosis (P < .05). In contrast, sunitinib markedly reduced the number of proliferating (PCNA-positive) tumor cells compared with control-treated animals (Figure 6B), although this did not achieve statistical significance after correction for multiple comparisons (P > .05). Unexpectedly, Tie2 kinase inhibitor treatment, alone or in combination with sunitinib, led to a dramatic increase in PCNA-positive tumor cells compared with both control- and sunitinib-treated animals (P < .05 for each comparison).

Neoplasia Vol. 14, No. 2, 2012 Discussion This study presents evidence supporting a role for Tie2 targeting in the treatment of angiosarcoma. After confirming the specificity of Tie2 immunostaining, we demonstrated expression of this vascular tyrosine kinase receptor in a panel of human angiosarcomas from diverse sites (Figure 1). This finding extends the observations of Brown et al. [8] who found strong Tie2 messenger RNA expression in two human angiosarcomas and is largely consistent with the findings of Stacher et al. [9] who identified Tie2 expression in most pulmonary epithelioid angiosarcomas. Intriguingly, the intensity of Tie2 immunoreactivity

Figure 4. SVR (A, C) and MS1-VEGF (B, D) tumor xenografts recapitulate the gross and microscopic phenotype of angiosarcoma. Representative gross appearance of SVR (A) and MSI-VEGF (B) tumors at the indicated day after implantation in mice. Note the bruise-like appearance consistent with human cutaneous angiosarcomas. Representative histologic (hematoxylin and eosin) appearance of SVR (C) and MSI-VEGF (D) tumors at the indicated day after implantation in mice. Note the hypercellularity and irregular vascular channels consistent with angiosarcoma. Skeletal muscle invasion is seen most prominently in day 12 SVR tumors.

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Figure 5. In vivo SVR (A) and MS1-VEGF (B) tumor growth inhibition with sunitinib, Tie2 kinase inhibitor, and combination. Tumor-bearing mice were treated as described in Materials and Methods (SVR n = 5, MS1-VEGF n = 4). *P < 0.05, control versus each treatment group.

varied considerably among the 11 human angiosarcomas examined. This observation raises the possibility that Tie2 expression, defined immunohistochemically, might serve as a predictive biomarker for Tie2-directed therapies and warrants further exploration. To facilitate testing the hypotheses that VEGFR and Tie2 kinase inhibitors have activity in angiosarcoma, models of both welldifferentiated, slow-growing (MS1-VEGF), and poorly differentiated, rapidly progressive (SVR) angiosarcomas were used. In line with prior observations, VEGFR2 was expressed in both cell lines [7,21]. Consistent with our immunohistochemical observations in human angiosarcomas, both cell lines also retained Tie2 expression (Figures 2 and W1). Trials of VEGFR-directed therapies provide hints of clinical activity in angiosarcoma [10–13,15–17], and SVR xenografts have been used to screen for novel anti-VEGFR compounds [23–27]. However, few data are available on the ability of VEGFR antagonists to directly inhibit SVR tumor cell survival in vitro. Moreover, the activity of clinically available VEGFR antagonists (e.g., sunitinib, sorafenib, pazopanib) against SVR cells is largely undefined. Similarly, the cytotoxic potential of Tie2 antagonists in the SVR cell line, or the related MS1-VEGF cell line, is undefined. In this study, we found that both sunitinib and the selective Tie2 kinase inhibitor, 4-(6-methoxy-2-naphthyl)-2-(4-

methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole, reduced cell survival in both SVR and MS1-VEGF cell lines in vitro (Figure 3). Concurrent inhibition of the VEGF-VEGFR and Ang-Tie2 axes in solid tumor antiangiogenic therapy has been explored based on the hypothesis that such dual inhibition may more effectively ablate tumor-driven angiogenesis compared with monotherapy [23,28]. To assess potential antineoplastic synergy between VEGFR and Tie2 kinase inhibition in SVR and MS1-VEGF cells, formal combination index analyses were performed. Intriguingly, dual inhibition proved potently synergistic in the rapidly proliferating SVR cell line and largely additive in the slowly proliferating MS1-VEGF line (Figure 3C). In addition to demonstrating the mechanistic independence of sunitinib and the Tie2 kinase inhibitor, these data raise the possibility that dual inhibition of VEGFR and Tie2 may be uniquely efficacious in poorly differentiated angiosarcoma. Because poorly differentiated variants are the most common angiosarcoma histology observed clinically, this hypothesis merits further investigation. After validation of SVR and MS1-VEGF cell models of angiosarcoma in vivo (Figure 4), cell culture results were extended to mouse models of the disease. In both tumors, sunitinib and Tie2 kinase inhibitor displayed significant tumor growth delay. Combined therapy was more potent

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than monotherapy in both models. It is interesting to note that treatment, either monotherapy or combined therapy, did not result in durable tumor control. This observation is consistent with early clinical results with VEGFR-directed therapies in angiosarcoma patients. Clinical benefit is almost exclusively due to disease stabilization; confirmed responses (i.e., reductions in tumor size) are uncommon. For example, in combined analysis of phase 2 studies of sorafenib in this setting, only 5 of 45 vascular sarcoma patients had confirmed responses as their best response. In contrast, stable disease was observed in 26 patients [15,16]. The consistency between our preclinical findings and early clinical observations lend support for the use of SVR and MS1-VEGF angiosarcoma models in assessing the therapeutic utility of vascular-targeted agents in this disease. Potentially more importantly, our observation that VEGFR and Tie2 kinase inhibition results in angiosarcoma growth delay rather than tumor

Neoplasia Vol. 14, No. 2, 2012 regression suggests that such therapies may best be used in combination with other agents. Straightforward mechanistic studies were performed to identify potential mechanisms of tumor growth delay with VEGFR and Tie2 kinase inhibition. As expected given the pro-proliferative effects of VEGF on endothelial cells, sunitinib tended to increase tumoral necrosis and reduce tumor cell proliferation in SVR tumors (Figure 6). Because sunitinib did not modify tumor cell apoptosis in vivo, tumor delays seem to be a simple function of reduced proliferation. This finding likely explains why sunitinib monotherapy results in tumor growth delay rather than tumor regression/cure. In stark contrast, Tie2 kinase inhibition and the drug combination paradoxically resulted in increased tumor cell proliferation. Tumor growth delay seems to be achieved through increased tumor cell apoptosis that more than offsets the observed

Figure 6. Apoptosis (A) and proliferation (B) of SVR cells in vivo in response to vehicle, sunitinib, Tie2 kinase inhibitor, and combination treatment. Quantification of apoptotic (cleaved caspase 3 positive) and proliferating (PCNA positive) cells was as described in Materials and Methods. n = 4-6 per group; values represent mean ± SE.

Neoplasia Vol. 14, No. 2, 2012 increase in proliferation. Tie2 signaling has been implicated in both endothelial cell proliferation and endothelial cell survival [29,30]. Several hypotheses can be offered to explain the observed increases in both apoptosis and proliferation in SVR tumors after Tie2 kinase (and dual) inhibition. Because a single, low-dose regimen of Tie2 kinase inhibition was examined in vivo, the relative effects of treatment on apoptosis and proliferation may vary with dose intensity. Similarly, there may be temporal changes in the balance between apoptosis and proliferation not captured by the time point examined in this study. Increased tumor cell proliferation after Tie2 kinase inhibition may simply reflect accelerated repopulation similar to that observed in solid tumors treated with potent cytotoxins (e.g., ionizing radiation). Finally, the apparent failure of Tie2 kinase inhibition to reduce tumor cell proliferation may also be a consequence of the model used. Tie2-mediated increases in nonresting endothelial cell proliferation are, at least in part, mediated by activation of the Ras/Raf/MAPK pathway [29–31]. As SVR cells contain activated H-ras, constitutive activation of this pathway may limit the impact of Tie2 signaling disruption on cell proliferation. Defining the relative contribution of various Tie2 functions on angiosarcoma development and progression will be critical to the refined clinical application of Tie2 kinase inhibitors in this disease. As discussed above, findings from these experiments as well as early clinical experience with VEGF antagonists suggest that VEGFR- or Tie2-directed therapies, alone or together, will be insufficient to achieve durable tumor control. Consequently, the rational combination of these therapies with other treatments active in angiosarcoma may offer the best chance for genuine clinical progress. Few nonsurgical therapies with well-evidenced activity in angiosarcoma are available and include ionizing radiation and paclitaxel [32–34]. Both of these cytotoxic modalities are dependent on cell proliferation for efficacy. Our results suggest that concurrent use of either paclitaxel or ionizing radiation with sunitinib may be suboptimal because the latter reduces tumor cell proliferation rate and thus may blunt the efficacy of cytotoxic therapy. In contrast, the observation that Tie2 kinase inhibition dramatically increases tumor cell proliferation suggests this treatment may complement cytotoxic therapies exceedingly well. Further investigation is needed to define the potential roles for Tie2 kinase inhibitors in multimodality angiosarcoma regimens. Taken together, the results of this study highlight a potential role for Tie2 kinase inhibition in angiosarcoma treatment. The Tie2 receptor is expressed in human angiosarcomas. In two models of angiosarcoma, Tie2 kinase inhibition reduced cell survival in vitro and delayed tumor growth in vivo. Mechanistically, in vivo tumor growth delay seems to be mediated by an increase in lesional cell apoptosis. Given the dearth of effective therapies for this disease and its poor prognosis, further preclinical and clinical investigations of Tie2 kinase inhibition are warranted. More generally, the experimental approach used here may prove valuable in the identification and testing of other potential molecular targets in angiosarcoma. References [1] Young RJ, Brown NJ, Reed MW, Hughes D, and Woll PJ (2010). Angiosarcoma. Lancet Oncol 11, 983–991. [2] Dim D, Ravi V, Tan J, Hicks D, and Wong M (2007). The actin-bundling motility protein fascin and vascular endothelial growth factor (VEGF) are universally over-expressed in human angiosarcoma. J Clin Oncol 25(18S). Abstract 10068. [3] Itakura E, Yamamoto H, Oda Y, and Tsuneyoshi M (2008). Detection and characterization of vascular endothelial growth factors and their receptors in a series of angiosarcomas. J Surg Oncol 97, 74–81.

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Figure W1. Tie2 expression in day 12 SVR (A) and day 20 MS1-VEGF (B) tumors. (C and D) The same tumors processed without primary antibody (negative control).

Figure W2. CD31 expression in day 12 SVR (A) and day 20 MS1-VEGF (B) tumors. (C and D) The same tumors processed without primary antibody (negative control).