Delta-Like Ligand 4–Notch Blockade and Tumor Radiation Response

1778 Articles | JNCI Vol. 103, Issue 23 | December 7, 2011

DOI: 10.1093/jnci/djr419 © The Author 2011. Published by Oxford University Press. All rights reserved.Advance Access publication on October 18, 2011. For Permissions, please e-mail: [email protected].

Several approaches have been used to target tumor angiogenesis to enhance the effect of ionizing radiation in delaying tumor growth (1–4). For example, vascular endothelial growth factor (VEGF) is an important stimulatory factor for tumor angiogenesis, and target-ing the VEGF pathway reduces tumor vessel density and slows tumor growth in preclinical mouse xenograft models (1–3). The humanized anti-VEGF monoclonal antibody bevacizumab has been incorporated into chemotherapeutic regimens for a variety of solid tumors, resulting in modest improvements in overall and progression-free survival (5). Likewise, the addition of bevacizumab to radiation therapy was shown to enhance tumor growth delay in several preclinical xenograft models (1,2). Further investigations

have shown that because tumor reoxygenation is induced by VEGF pathway blockade, the timing and sequencing of ionizing radiation appear to be important (3,6,7).

However, tumor resistance to VEGF blockade is common (5,8,9) and has been shown to be partly related to another angiogenesis-regulating pathway, the delta-like ligand 4 (DLL4), and Notch signaling pathway (10–15). In mammals, there are four Notch receptors (Notch1, Notch2, Notch3, and Notch4) and five Notch ligands (Jagged-1, Jagged-2, delta-like ligand 1, delta-like ligand 3, and DLL4). Binding of these ligands to the Notch recep-tors leads to a conformational change in the Notch receptor, exposing a cleavage site; cleavage at this site is executed by a

ARTICLE

Delta-Like Ligand 4–Notch Blockade and Tumor Radiation ResponseStanley K. Liu, Saif A. S. Bham, Emmanouil Fokas, John Beech, Jaehong Im, Song Cho, Adrian L. Harris, Ruth J. Muschel

Manuscript received February 15, 2011; revised August 9, 2011; accepted September 6, 2011.

Correspondence to: Adrian L. Harris, MD, DPhil, Molecular Oncology Unit, The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK (e-mail: [email protected]).

Background The microenvironment plays an important role in regulating tumor response to radiotherapy. Ionizing radiation can disrupt tumor vasculature, and Notch pathway inhibition can interfere with functional angiogenesis. We explored the potential cooperativity between Notch inhibition and ionizing radiation in delaying tumor growth.

Methods Human colorectal carcinoma LS174T cells, which express the Notch ligand delta-like ligand 4 (DLL4), and human head and neck cancer FaDu cells, which do not, were grown as subcutaneous xenografts in nude mice. The mice were treated with dibenzazepine (DBZ), a g-secretase inhibitor that blocks all Notch signaling, or a DLL4-specific blocking monoclonal antibody, alone or in combination with ionizing radiation (n = 5–10 mice per group), and response was assessed by tumor growth delay. Microbubble contrast Doppler ultrasound was used to measure tumor blood flow. Tumor Notch activity was monitored by in vivo bioluminescence from a Notch luciferase reporter. Vessel density was assessed using Chalkley vessel counting. All statistical tests were two-sided.

Results In LS174T xenografts, the average time for tumor volumes to reach four times the starting volume was longer for mice treated with the DLL4 monoclonal antibody than for mice treated with DBZ (16.4 vs 9.5 days, difference = 6.9 days, 95% confidence interval [CI] = 3.7 to 10.1 days, P < .001). Both Notch inhibitors suppressed tumor Notch activity within 24 hours of administration compared with vehicle (change in luciferase activity, vehicle vs DBZ: 103% vs 28%, difference = 75%, 95% CI = 39% to 109%, P = .002; vehicle vs DLL4 antibody: 172% vs 26%, difference = 146%, 95% CI = 86% to 205%, P < .001). Administration of the DLL4 antibody or DBZ after ionizing radiation resulted in a supra-additive growth delay compared with vehicle (vehicle vs DLL4 antibody + ionizing radiation: 6.8 vs 44.3 days, difference = 37.5 days, 95% CI = 32 to 43 days, P < .001; vehicle vs DBZ + ionizing radiation: 7.1 vs 24.4 days, difference = 17.3 days, 95% CI = 15.9 to 18.6 days, P < .001). Treatment of mice with the DLL4 antibody alone or in combination with ionizing radiation increased tumor vessel density but reduced tumor blood flow. Combination therapy with DLL4 antibody and ionizing radiation resulted in exten-sive tumor necrosis in LS174T xenografts and enhanced tumor growth delay in FaDu xenografts.

Conclusion The combination of specific DLL4–Notch blockade and ionizing radiation impairs tumor growth by promoting nonfunctional tumor angiogenesis and extensive tumor necrosis, independent of tumor DLL4 expression.

J Natl Cancer Inst 2011;103:1778–1798

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disintegrin and metalloprotease domain (ADAM) family protease that permits an additional cleavage by g-secretase, leading to pro-duction of the Notch intracellular domain (ICD), which subse-quently undergoes translocation into the nucleus, where it transactivates target genes (16). This pathway is an important regulator of tumor angiogenesis, primarily through two Notch li-gands: DLL4 and Jagged-1. During development, DLL4 is essen-tial for arterial development, and its expression is primarily restricted to endothelial cells within normal tissue and is increased in tumor vasculature (17–21).

The Notch pathway is activated in a wide range of human can-cers, including breast and colon carcinomas (22–26), and DLL4 is expressed in malignant epithelial cells of colonic adenomas and adenocarcinomas (20). Overexpression of DLL4 in tumor cells activates the Notch signaling pathway in host stromal and endo-thelial cells, resulting in increased blood vessel size, and improved vascular function within tumors, thereby leading to an increased tumor growth rate (14). Conversely, inhibition of DLL4 function has been shown to cause nonfunctional angiogenesis, which is characterized by increased vessel density and increased tumor hyp-oxia, and suppression of tumor growth (10–15). Furthermore, DLL4 blockade has the potential to overcome tumor resistance to anti-VEGF therapy. Specifically, tumors that continue to grow rapidly in the presence of anti-VEGF therapy experience a marked reduction in growth when DLL4 function is blocked (10,11,14).

In vivo experiments have also revealed an important therapeutic role for DLL4 blockade in combination with systemic therapies. DLL4 neutralizing antibodies given with irinotecan or paclitaxel statistically significantly delayed tumor growth and recurrence in human breast and colon xenograft models compared with irinote-can or paclitaxel alone (P < .05), presumably as the result of its ability to decrease tumor cell proliferation and cancer stem cell frequency, and disrupt functional tumor angiogenesis (15). There is also evidence suggesting that ionizing radiation can affect the Notch pathway in endothelial cells as well as in human breast can-cer and gliomas (27–30). However, to our knowledge there have been no published reports examining the effects of DLL4 blockade with ionizing radiation.

It has long been recognized that ionizing radiation can cause death of tumor cells by inducing lethal double-stranded DNA breaks. However, ionizing radiation also damages endothelial cells (31,32). Irradiation of tissue before tumor cell implantation reduces subsequent tumor growth due to suppression of neovascu-larization (29,33–35), although the extent of this contribution to the tumor radiation response is unclear. It has been reported that ionizing radiation–induced endothelial cell apoptosis primarily determines tumor cell response to radiotherapy (36,37); however, this finding is controversial (38,39). Ionizing radiation is believed to affect Notch signaling by increasing the expression of Notch ligands such as Jagged-1 in tumor cells and in human dermal mi-crovascular endothelial cells (27,28); indeed, a small increase in Notch activity was reported in glioma stem cells following ionizing radiation (30). It was reported that inhibition of Notch signaling in glioblastoma multiforme explants resulted in the loss of endothe-lial cells, reduced tumor cell proliferation, and a decrease in the ability of dissociated explant cells to form neurospheres in vitro, and potentiated the effects of radiation treatment, indicating the

importance of interactions between endothelial cells and tumor cells in modulating the tumor response to radiation (40). In addi-tion, knockdown of Notch1 or Notch2 and ex vivo irradiation of glioma cells before their injection into mice extended tumor latency more than either treatment alone (30). Clearly, there is a strong rationale to examine the effects of combination treatment with Notch inhibition and ionizing radiation in vivo given their separate influences on tumor vasculature and cancer stem cells.

In this study, we used a human colorectal carcinoma xenograft system that endogenously expresses the Notch ligand DLL4 to examine the effects of global Notch blockade (via a g-secretase inhibitor) or selective DLL4–Notch blockade (via a monoclonal antibody against DLL4) with ionizing radiation on tumor blood flow (ie, perfusion) and growth. Because DLL4 blockade increases tumor hypoxia (10,11), which could antagonize the effects of ion-izing radiation, we sequenced the ionizing radiation so that it was given before the blockade was initiated. In addition, we used a human hypopharyngeal squamous cell carcinoma xenograft that

CONTEXT AND CAVEATS

Prior knowledgeThe delta-like ligand 4 and Notch (DLL4–Notch) signaling pathway is an important regulator of tumor angiogenesis that is activated in a wide range of human cancers. Ionizing radiation can affect the Notch pathway in endothelial cells and in some human cancers.

Study designMouse xenograft models using human colorectal carcinoma cells that endogenously express the Notch ligand DLL4 or human hypo-pharyngeal squamous cell carcinoma cells that lack endogenous DLL4 were used to examine the effects of global Notch blockade (via a g-secretase inhibitor) or selective DLL4–Notch blockade (via a monoclonal antibody against DLL4) with ionizing radiation on tumor blood flow and growth.

ContributionIn the DLL4-expressing colorectal carcinoma xenograft model, Notch blockade (with either a global inhibitor or the DLL4 blocking antibody) in combination with ionizing radiation caused tumor growth delay. Treatment of mice with the DLL4 antibody alone or in combination with ionizing radiation increased tumor vessel den-sity but reduced tumor blood flow as early as 2 days after the start of treatment. The DLL4 monoclonal antibody in combination with ionizing radiation also effectively delayed tumor growth in the non–DLL4-expressing xenograft model.

ImplicationsThe fact that combination of specific DLL4–Notch blockade and ionizing radiation impairs tumor growth by promoting nonfunc-tional tumor angiogenesis, and extensive tumor necrosis indepen-dent of tumor DLL4 expression suggests that the host tissues provide the therapeutic target for the DLL4 monoclonal antibody.

LimitationsThe subcutaneous heterotopic xenograft tumor models may not accurately reflect the tumor microenvironment and response to treatment. The use of an immunocompromised mouse model could theoretically mask the contribution of the immune response to treatment.

From the Editors



lacks endogenous DLL4 to investigate the ability of our treatment approach to delay tumor growth independent of tumor DLL4 expression.

Materials and MethodsThe human colonic adenocarcinoma cell lines LS174T, LS180 (the cell line from which LS174T was derived), COLO 205 (a cell line derived from the same patient as COLO 201), and HCT-15 and the human hypopharyngeal squamous cell carcinoma cell line FaDu were purchased from the American Type Culture Collection (ATCC; distributed by LGC Standards, Middlesex, UK; Catalog numbers: CL-188, CL-187, CCL-222, CCL-225, and HTB-43, respectively). The ATCC authenticates its cell lines on a regular basis (http://www.lgcstandards-atcc.org/ATCCScience/AuthenticationandPreservation/tabid/1019/Default.aspx). We did not independently authenticate the cell lines. Early passage cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g/L glucose (Invitrogen, Paisley, UK) supple-mented with 10% fetal bovine serum (Lonza, Slough, UK) and penicillin–streptomycin (Invitrogen, Paisley, UK) (hereafter referred to as 10% DMEM), and maintained in a 37°C incubator with 5% CO2. Cell lines were passaged when they reached approx-imately 80% confluency and were regularly tested with MycoAlert (Lonza) to ensure the absence of mycoplasma contamination. Cell morphology was regularly checked to ensure the absence of cross-contamination of cell lines. To generate a stable Notch reporter cell line (LS174T Notch-luc cells), LS174T cells were infected with a lentivirus harboring a minimal promoter containing mul-tiple recombination signal-binding protein 1 for J-Kappa (RBP-jκ) Notch response elements (5′-CGTGGGAA-3′) driving expression of the firefly luciferase gene (QIAGEN, Crawley, UK), and a stable pooled line was established by selection in medium contain-ing 2 µg/mL puromycin (Sigma-Aldrich, Dorset, UK).

In Vitro Luciferase ImagingLS174T Notch-luc cells, which stably express a Notch–luciferase reporter, were seeded onto 24-well dishes (100 000 cells per well) that were pre-coated with bovine serum albumin (BSA; Sigma-Aldrich) or recombinant DLL4 (R&D Systems Europe Ltd, Abingdon, UK) as previously described (41), in 10% DMEM con-taining vehicle (dimethyl sulfoxide [DMSO]), mouse isotype im-munoglobulin G1 (IgG1) (1 µg/mL; R&D Systems Europe Ltd), dibenzazepine (DBZ) (20 nM; Axon Medchem BV, Groningen, the Netherlands), or a blocking anti-DLL4 monoclonal antibody (a human IgG1 isotype that cross-reacts with both human and mouse DLL4) (1 µg/mL; a kind gift from MedImmune LLC, Gaithersburg, MD) and incubated for 24 hours. To assess Notch–luciferase activity, D-luciferin (150 µg/mL; Gold BioTechnology, St Louis, MO) was added to the 10% DMEM medium, and the cells were incubated for 2 minutes. Luminescence readings were then captured with the use of a Xenogen IVIS 200 imaging system (Caliper Life Sciences Ltd, Preston Brook, UK) and analyzed with Living Image 3.0 software (Caliper Life Sciences Ltd). The fold change in Notch luciferase activity was calculated by dividing the Notch luciferase activity from the treatment group by the lucif-erase activity from the DMSO BSA control. For the irradiation

experiment (Figure 2, B), Notch luciferase activity was measured 24 hours after the cells were subjected to mock irradiation or irradiation with 2, 4, or 6 Gy of ionizing radiation. For mock irradiation, the cells were treated the same as the irradiated cells, except they were given no dose. The fold change in Notch luciferase activity was calculated by dividing the Notch lucif-erase activity in the irradiated cells by the Notch luciferase ac-tivity in the mock irradiated cells.

Clonogenic Cell Survival AssayLS174T cells were seeded in triplicate at low density (500, 1000, 2000, and 4000 cells per well for irradiation with 0, 2, 4, and 6 Gy ionizing radiation, respectively) in 10% DMEM containing vehicle (DMSO) or phosphate-buffered saline (PBS), DBZ (20 nM; Axon Medchem BV), or the blocking anti-DLL4 monoclonal antibody (1 µg/mL diluted in PBS). The cells were immediately mock irradiated or irradiated with 2, 4, or 6 Gy of ionizing radia-tion at a dose rate of 0.66 Gy/min by using an IBL634 cesium irradiator (CIS Bio International, Gif-Sur-Yvette, France). The cells were incubated at 37°C for 14–21 days to allow colonies to form. Colonies were stained with crystal violet staining solution (0.5% crystal violet [Sigma-Aldrich], 25% methanol) and counted. Survival was expressed as the relative plating efficiencies of the treated cells compared with that of the mock-irradiated (ie, con-trol) cells. The experiments were performed three separate times. Radiation dose–response curves were created by fitting the data to the linear–quadratic equation S = e2aD2bD2 using GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA), where S is the surviving fraction, a and b are inactivation constants, and D is the dose in Gy.

Immunoblotting and AntibodiesLS174T, LS180, HCT-15, COLO 205, or FaDu cells (2 × 106 cells) were rinsed once with PBS, lysed in ice-cold radioimmuno-precipitation assay lysis buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxy-cholate, 0.1% sodium dodecyl sulfate [SDS]; all from Sigma-Aldrich) containing Complete Protease Inhibitor Cocktail (Roche, Burgess Hill, UK) and phosphatase inhibitors (1 mM NaF, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate; all from Sigma-Aldrich), and ultrasonicated for 1 minute. The lysates were clarified by centrifugation at 21 000g at 4°C for 10 minutes. Protein concentrations of the lysates were determined by use of a Bradford protein assay (Bio-Rad, Hemel Hempstead, UK). Each lysate (25–30 µg) was denatured in SDS sample buffer (final con-centration: 40 mM Tris–HCl [pH 6.8], 2% SDS, 2 mM dithioth-reitol, 4% glycerol, 0.01% bromophenol blue) at 100°C for 5 minutes and electrophoretically resolved on Novex 4%–12% Tris–glycine gels (Invitrogen, Renfrew, UK). Resolved proteins were wet transferred onto polyvinylidene fluoride membranes (Millipore, Watford, UK), and the membranes were incubated in 5% nonfat dry milk in Tris-buffered saline Tween-20 (TBST) (10 mM Tris–Base, 150 mM NaCl, 0.05% Tween-20; pH 7.4) for 1 hour at room temperature to block nonspecific antibody binding, followed by incubation with primary antibody in 5% milk in TBST overnight at 4°C with gentle agitation. The membranes were washed three times for 10 minutes each in TBST, then incubated


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with a species-appropriate secondary antibody conjugated to horse-radish peroxidase (HRP; Dako UK Ltd, Ely, UK) in 5% milk in TBST at room temperature for 1 hour, followed by three 10-minute washes with TBST. Protein–antibody binding on the membranes was detected with the use of enhanced chemilumines-cence (ECL) Plus solution (Amersham PLC, Little Chalfont, UK) followed by exposure of the membranes to x-ray film (FujiFilm, Bedfordshire, UK). The following primary antibodies were used: rabbit polyclonal anti-DLL4, rabbit polyclonal anti-Jagged 1, rabbit polyclonal anti-Notch1 ICD (all were used at 1:1000 dilution; all from Cell Signaling Technology Inc. (Danvers, MA) distributed by New England Biolabs, Hitchin, UK), and goat polyclonal anti-b-actin (1:6000 dilution; Santa Cruz Biotechnology Santa Cruz, CA).

Mouse Xenograft StudiesAll experiments involving mice were performed according to University of Oxford institutional guidelines and within the limits of the Project License issued by the Home Office, United Kingdom, as well as in accordance with published guidelines for the welfare and use of animals in cancer research (42). LS174T Notch-luc (1 × 107) or FaDu (5 × 106) cells were mixed in a 1:1 (vol:vol) ratio with Growth Factor Reduced Matrigel (Becton, Dickinson and Company, Oxford, UK), and the mixture was injected subcutaneously into the right flanks of 6- to 7-week-old female BALB/C nude mice (Harlan, Huntingdon, UK or Charles River, Tranent, UK). Tumor volume (in mm3) was determined by caliper measurements performed every 1–3 days and calculated by using the modified ellipse formula (volume = length × width2/2). For the LS174T tumor xenograft experiment investigating DBZ and ionizing radiation, when the xenograft tumor volume reached approximately 100 mm3, the mice were randomly assigned to the vehicle arm (n = 12 mice) or the experimental arms (DBZ [n = 11 mice], 5 Gy ionizing radiation [n = 13 mice], or DBZ and 5 Gy ionizing radiation [n = 12 mice]). Mice in the vehicle arm received intraperitoneal injection with 100 µL of 0.5% hydroxypropyl methylcellulose (Methocel E4M; Sigma-Aldrich) and 0.1% Tween-80 in water (Sigma-Aldrich) every 3 days for a total of five doses beginning on day 1. DBZ was dissolved in 0.5% Methocel E4M (Sigma-Aldrich), 0.1% Tween-80 (Sigma-Aldrich), and water to a final concentration of 1078 µM and injected by intraper-itoneal route at a dose of 8.1 µmol/kg of body weight every 3 days for a total of five doses beginning on day 1 (mice in the DBZ group) or 30 minutes after treatment with ionizing radiation on day 1 (mice in the DBZ and ionizing radiation group). For irradi-ation, mice were gently restrained in a customized brass jig that exposed the tumor while shielding the remainder of the body, and the tumor was irradiated to a dose of 5 Gy with x-rays produced by an Xstrahl RS320 x-ray system (Gulmay Medical Ltd, Chertsey, UK). For the LS174T and FaDu xenograft experiments investi-gating the blocking anti-DLL4 monoclonal antibody and ionizing radiation, when the xenograft tumor volume reached approxi-mately 100 mm3, the mice were randomly assigned to the vehicle arm (n = 13 mice for LS174T; n = 5 mice for FaDu) or the exper-imental arms (DLL4 monoclonal antibody [n = 13 mice for LS174T, n = 5 mice for FaDu], 5 Gy ionizing radiation [n = 14 mice for LS174T, n = 5 mice for FaDu], DLL4 monoclonal anti-body and 5 Gy ionizing radiation [n = 14 mice for LS174T, n = 5

mice for FaDu]). Mice in the vehicle arm received intraperitoneal injection with 100 µL of vehicle (PBS) twice per week for the duration of the experiment. The DLL4 monoclonal antibody was administered twice per week at a dose of 5 mg/kg of body weight beginning on day 1 (mice in the DLL4 monoclonal antibody group) or 30 minutes after treatment with ionizing radiation on day 1 (mice in the DLL4 monoclonal antibody and ionizing radi-ation group) for the duration of the experiment. Tumor irradiation at 5 Gy was performed as described above. For the LS174T xeno-graft experiment investigating DBZ and ionizing radiation, three randomly chosen mice from the vehicle arm and from each treat-ment group were subjected to microbubble Doppler ultrasound (see below) performed on day 1 (before vehicle or treatment was given) and again on day 5 of treatment and then killed by cervical dislocation immediately after ultrasound, and their tumors were excised and processed for immunohistochemical staining (see below). For the LS174T xenograft experiment investigating treat-ment with the DLL4 monoclonal antibody and ionizing radiation, six randomly chosen mice from the vehicle arm and each treatment group were subjected to microbubble Doppler ultrasound on day 1 of treatment; three of these mice from each group were subjected to ultrasound again on day 3 of treatment and then immediately killed by cervical dislocation, and the remaining three mice had ultrasound on day 5 of treatment and were immediately killed by cervical dislocation, and their tumors were excised. The tumors from day 5 of treatment were processed for immunohistochemical staining. For both LS174T xenograft experiments, three addi-tional mice from the vehicle or each treatment arm were randomly chosen and subjected to in vivo luciferase imaging (see below) performed on day 1 (before vehicle or treatment was given) and again on day 2 (ie, 24 hours later). Tumor volumes were deter-mined by caliper measurements performed every 1–3 days. When tumor volumes reached four times the starting volume (to ensure that the final tumor burden remained within the limits of the Project License), the mice were subjected to intraperitoneal injection with the hypoxic cell marker EF5 (10 µL/g of body weight of a 10 mM solution in 0.9% saline; kindly provided by Dr Cameron Koch, Hypoxia-Imaging Service Center, University of Pennsylvania, Pennsylvania, PA). The mice were killed by cer-vical dislocation 2.5 hours later, and their tumors were excised and fixed by incubation overnight in 4% paraformaldehyde (Sigma-Aldrich) at 4°C, then transferred to 25% glucose (in PBS), and incubated for an additional 24 hours at 4°C, followed by snap freezing in isopentane (Sigma-Aldrich) cooled on dry ice and storage at 280°C until cryosectioning. Three fixed tumors were randomly chosen from each vehicle or treatment group and pro-cessed for immunohistochemistry. For confocal vessel imaging, three randomly chosen mice per vehicle and treatment group were also subjected to intravenous injection into the lateral tail vein with phycoerythrin-conjugated anti-CD31 antibody (100 µL of a 0.2 mg/mL solution; BioLegend UK Ltd., Cambridge, UK) and 10 minutes later were killed by cervical dislocation, and their tumors were excised and immediately subjected to confocal im-aging and immunohistochemistry (see below). Analysis of supra-additive effects of the combination treatments of DBZ or the DLL4 monoclonal antibody and ionizing radiation was performed as previously described (43–45). The supra-additive ratio was



defined according to the following formula: (growth delay induced by combination treatment)/(growth delay induced by DBZ or the DLL4 monoclonal antibody + growth delay induced by ionizing radiation). A ratio greater than 1 indicates a greater than additive response. Tumor growth delay was cal-culated by subtracting the average time for control tumors from vehicle-treated mice to grow to four times the starting volume from the time required for treated tumors to grow to four times the starting volume. For LS174T xenograft experiments, to provide a statistical power of at least 80% to detect a twofold difference in mean tumor volume between groups, tumor vol-ume variation of up to 50% within groups, and a statistical sig-nificance level of a = .05, a sample size of seven mice per group was required (46). Similarly, for the FaDu xenograft experi-ment, to provide a statistical power of at least 80% to detect a twofold difference in tumor volume between groups, tumor volume variation of up to 40% within groups, and a statistical significance level of a = .05, a sample size of five mice per group was required (46).

Microbubble Doppler Ultrasound Evaluation of Tumor Blood Flow. Evaluation of tumor blood flow was performed with the use of a Vevo 770 Micro-Ultrasound platform equipped with a RMV-704 probe (VisualSonics, Toronto, ON, Canada) by image enhancement using contrast microbubbles (Vevo MicroMarker Contrast Agent Kit; VisualSonics) according to the manufacturer’s instructions. Mice were kept under anesthesia with inhalational isofluorane (2%) during ultrasound imaging. For each study, a fresh vial of lyophilized microbubble powder was prepared with 0.7 mL of sterile saline according to the manufacturer’s directions. Microbubbles (1 × 108) were injected as a bolus in a volume of 50 µL via a 27-gauge hypodermic needle into a lateral tail vein. The ultrasound transducer probe was placed over the center of the tumor. Before injection of microbubbles, we acquired a dynamic image sequence (contrast mode) of the tumor, in a total of 550 frames over a period of 40 seconds to account for the background intensity. Imaging was recorded on a cine clip. A baseline image sequence of the tumor at a frequency of 40 MHz was also acquired. The same procedure was repeated whereby imaging was recorded on a cine clip beginning immediately before the injection of micro-bubbles. For each tumor, we delineated the region of interest (ROI) using the Vevo770 Ultrasound Platform (VisualSonics), and the image containing the delineated ROI was analyzed using ImageJ software (NIH, Bethesda, MD), to determine the per-centage of green contrast signal (ie, microbubbles) within the ROI area; this percentage represented blood flow within the tumor. We repeated this process for each tumor. We determined relative tumor blood flow by dividing tumor blood flow for the treatment groups by the tumor blood flow for the vehicle group at day 1 and then multiplying by 100%. In addition, we performed contrast kinetics analysis with the use of the Vevo 770 software (v.2.23; VisualSonics) to assess the slope and magnitude of microbubble influx (contrast kinetic curve) on day 5. The ROI (drawn around the tumor) and the acquired cine clip (550 frames; 40 seconds) were evaluated for change in tumor contrast intensity, taking into account the baseline intrinsic contrast to get the contrast kinetic percentage.

In Vivo Luciferase Imaging. Mice were anesthetized with 4% isofluorane (Baxter, Caxton Way, UK) and then placed inside the chamber of a Xenogen IVIS 200 imaging system (Caliper Life Sciences Ltd); anesthesia was maintained with inhalational 2% isofluorane throughout imaging. The mice were subjected to intraperitoneal injection with D-luciferin (150 mg/kg body weight; Gold BioTechnology) 5 minutes before imaging. The parameters for image acquisition were f/stop = 1, bin = Med, and exposure time = 1 minute. Signal intensity was quantified as the total pho-tons per second within an ROI outlining the tumor, using Living Image 3.0 software (Caliper Life Sciences Ltd). The percent change in luciferase activity was calculated by dividing the signal intensity within the ROI measured on day 2 by the signal intensity within the ROI measured on day 1 (baseline) and multiplying by 100%.

Confocal Imaging of Vessels. After mice were killed by cervical dislocation, their tumors were immediately excised, and a central portion of each tumor was directly sectioned (approximately 2-mm-thick sections) and immediately imaged by confocal micros-copy (Leica Microsystems Ltd, Milton Keynes, UK). Confocal z-stacks of 100 µm were taken at 0.5-µm intervals at ×10 magnifi-cation on multiple randomly chosen fields using the 543-nm laser to excite phycoerythrin that was conjugated to the anti-CD31 antibody. Three-dimensional vascular reconstructions were cre-ated using Amira ResolveRT software (Visage Imaging GmbH, Berlin, Germany).

Immunohistochemistry and Image Analysis. After mice were killed by cervical dislocation, excised tumors were immediately placed in 4% paraformaldehyde and incubated overnight at 4°C, then transferred to 25% glucose (in PBS) and incubated for an additional 24 hours at 4°C, followed by snap freezing in isopentane (Sigma-Aldrich) cooled on dry ice and stored at 280°C until cryo-sectioning (10-µm sections). For CD31 staining, we used a rat monoclonal anti-mouse CD31 antibody (1:20 dilution, clone SZ31; Dianova distributed by Stratech Scientific Ltd, Newmarket, UK). Antigen retrieval was performed in S1700 target retrieval solution (Dako) in a Decloaking Chamber (Biocare Medical, Concord, CA). The sections were then pretreated with 0.3% hydrogen peroxide (Sigma-Aldrich) in PBS for 20 minutes, followed by incubation for 30 minutes in blocking buffer (0.1 M Tris–HCl [pH 7.5], 150 mM NaCl, 0.5% Tyramide Signal Amplification Blocking Reagent [PerkinElmer Inc, Seer Green, UK]) and then for 1 hour with primary antibody in blocking buffer. Bound antibody was labeled with HRP-conjugated rabbit anti-rat IgG (1:100 dilution; Dako) and visualized using 3,3′-diaminobenzidine chromogen (Sigma-Aldrich) with hematoxylin (Sigma-Aldrich) counterstaining. Chalkley vessel counts were performed, as previously described by Fox et al. (47), independently by two observers (S. K. Liu and S. A. S. Bham) on a minimum of three-vessel “hot spots” (maximal areas of neovascularization identified by scanning at ×40 magnification) per tumor (n = 3 tumors per group), and vessel counts were esti-mated independently by both observers using a 25-point Chalkley point graticule at ×250 magnification. Any immunoreactive (ie, CD31-positive) endothelial cell(s) that was separate from an adja-cent microvessel was considered a countable vessel. The graticule



was rotated in the eyepiece to a position where the maximum number of graticule points overlay immunohistochemically identi-fied vessels or their lumens. Chalkley vessel counts for individual tumors were generated by averaging the three graticule values.

For Ki-67 staining, we used a mouse monoclonal anti-human Ki-67 antibody (1:100, clone 2531; ABD Serotec, Kidlington, UK). Antigen retrieval was performed using 10 mM sodium cit-rate, 0.05% Tween-20 (pH 6.0) at 90°C for 20 minutes. The sec-tions (n = 3 tumors per group) were then pretreated with 0.3% hydrogen peroxide in PBS for 20 minutes. Endogenous biotin was blocked by using an Avidin/Biotin Blocking Kit (Vector Laboratories Ltd, Peterborough, UK) according to manufacturer’s instructions, followed by incubation with Mouse on Mouse (M.O.M) blocking reagent (Vector Laboratories Ltd) for 1 hour. The sections were incubated with blocking buffer for 5 minutes, followed by incubation for 30 minutes with the primary antibody in blocking buffer. The sections were incubated with biotinylated goat anti-mouse IgG (1:500 dilution; Vector Laboratories Ltd), followed by Vectastain ABC Elite reagent (Vector Laboratories Ltd), and bound antibody was visualized using 3,3′-diaminobenzi-dine chromogen with hematoxylin counterstaining. For Ki-67 scoring, the number of Ki-67-positive nuclei (defined as dark brown staining nuclei) as a proportion of total nuclei was deter-mined while viewing the sections with a high-power objective (×400 magnification) and was performed for a total of six randomly chosen fields. This scoring was only performed in areas of viable tumor; regions of necrosis were avoided.

Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining to detect apoptotic cells was per-formed with the use of an Apoptag Peroxidase In Situ Apoptosis detection kit (Millipore). Antigen retrieval was performed using 10 mM sodium citrate, 0.05% Tween-20 (pH 6.0) at 90°C for 20 minutes. The remainder of the procedure was performed accord-ing to manufacturer’s instructions. Positive cells were visualized using 3,3′-diaminobenzidine chromogen and counterstained with hematoxylin. TUNEL scoring was performed in the same manner as Ki-67 scoring, using six randomly chosen fields per tumor and avoiding regions of necrosis (n = 3 tumors per group).

Immunohistochemistry for EF5 (n = 3 tumors per group) was performed by using a mouse monoclonal anti-EF5 antibody directly conjugated to Cy3 fluorophore (clone Elk3-51, 75 µg/mL; kindly provided by Dr C. Koch, University of Pennsylvania, PA). Antigen retrieval was performed using 10 mM sodium citrate, 0.05% Tween-20 (pH 6.0) at 90°C for 20 minutes, and sections were pre-treated with blocking buffer for 30 minutes, followed by incubation with the primary antibody for 24 hours at 4°C. Stained sections were mounted in VECTASHIELD aqueous mounting media (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole (DAPI). Stained tumor sections were observed field by field simul-taneously by two independent observers (S. K. Liu and S. A. S. Bham) at ×150 magnification. Each field was assigned a hypoxia score that ranged from 0 to 4 based on the percentage of the tumor area positive for EF5 staining (score 0 = 0%; score 1 = 1% to 5%; score 2 = >5% to 15%; score 3 = >15% to 30%; score 4 = >30%); these cut points have been used in other studies to quantitate tumor hypoxia by immunohistochemistry (48–51), and the score for each field was agreed upon by consensus. Areas of necrosis were identified

using DAPI staining and were not included when estimating hyp-oxia scores. Scores obtained for each field were converted to per-centages by using the median values for the corresponding hypoxia scores (median values for each score were as follows: score 0: 0%, score 1: 2.5%, score 2: 10%, score 3: 22.5%, score 4: 40%; because no tumors had >50% hypoxia, we used a median value of 40% for hypoxia score 4). The mean percentage of hypoxic cells for all scored fields was calculated for each tumor. Hematoxylin and eosin staining was performed on these tumor sections, and the estimated percentage of tumor necrosis under low-power magnification was estimated by two independent investigators (S. K. Liu and S. A. S. Bham) and agreed upon by consensus.

Statistical AnalysisAll statistical tests were two-sided, and the statistical analysis was done with the use of the GraphPad Prism program (version 4.0; GraphPad Software). Statistical significance was defined as P less than .05. The Student t test was used to compare the mean values between two groups, and one-way analysis of variance and Bonferroni posttest comparisons were used to compare mean values among multiple groups. Data are presented as mean values with 95% confidence intervals (CIs).

ResultsDLL4 Expression and Notch Activity in Human Colorectal Carcinoma LS174T CellsNotch signaling is known to be active and important in intestinal and colonic cells, both in terms of normal physiology and tumori-genesis (25,52), and thus we were interested in identifying and vali-dating a model colorectal carcinoma cell line with intact Notch signaling. Immunoblot analysis indicated that DLL4 protein is expressed at high levels in COLO 205, LS174T, and LS180 cells, and at a low level in HCT-15 cells (Figure 1, A). LS174T cells also express Notch 1, 3, and 4 receptors (Figure 1, B and data not shown) and thus, these cells have the potential to respond to Notch ligands. Immunoblot analysis also confirmed that LS174T cells express Notch1 ICD, indicating that they can engage in Notch signaling (Figure 1, B, left panel). Treatment of LS174T cells with the g-secretase inhibitor DBZ inhibited production of the Notch1 ICD in a dose-dependent manner (Figure 1, B, left panel). Treatment of LS174T cells with DBZ or the DLL4 blocking monoclonal anti-body caused an increase in DLL4 protein levels compared with cells treated with vehicle (Figure 1, B, right panel). The increase in DLL4 expression with inhibition of Notch signaling suggests the presence of a negative feedback loop in which Notch signaling de-creases DLL4 expression. LS174T cells also express the Notch li-gand, Jagged-1 (Figure 1, B, right panel); however, in contrast to DLL4, expression of this Notch ligand was not affected by treat-ment of the cells with DBZ or the DLL4 monoclonal antibody.

To monitor Notch activation in LS174T cells, we stably expressed a Notch response element (RBP-jk)–luciferase reporter in LS174T cells (LS174T Notch-luc cells) and then assessed luciferase activity as a measure of Notch signaling in cells exposed to recombi-nant DLL4 or BSA (to assess basal Notch activity) in the presence or absence of DBZ or DLL4 monoclonal antibody (Figure 1, C). Cells treated with either the DLL4 antibody or DBZ had statistically



Figure 1. LS174T human colorectal adenocarci-noma as a model Notch signaling cell system. A) Delta-like ligand 4 (DLL4) immunoblotting. Lysates from a panel of human colorectal carci-noma cell lines were immunoblotted for DLL4 expression. B) Immunoblot analysis of Notch signaling proteins. LS174T cells were incubated with dimethylsulfoxide (DMSO), or dibenzaze-pine (DBZ; 2 or 20 nM) for 16 hours, lysed and immunoblotted for Notch1, Notch1 intracellular domain (N1ICD) (left panel). Equal loading was confirmed with immunoblotting for b-actin (left panel). LS174T cells were treated overnight with DMSO (2), DBZ (20 nM), or DLL4 monoclo-nal antibody (mAb) (1 µg/mL), and cell lysates were immunoblotted for DLL4 and Jagged-1 (right panel). C) In vitro Notch luciferase assay. LS174T Notch-luc cells were incubated on bovine serum albumin (BSA) or DLL4-coated plates in triplicates, in the presence of DMSO (vehicle), isotype immunoglobulin G1 (1 µg/mL), DBZ (20 nM), or DLL4 mAb (1 µg/mL) for 24 hours, and Notch luciferase activity measured by the Xenogen IVIS 200. The graph displays the mean fold change in Notch luciferase ac-tivity (DLL4-coated plates normalized to DMSO on BSA-coated plates) from three independent experiments, and error bars correspond to 95% confidence intervals; P values (two-sided Student t test) are for comparisons with the re-spective DMSO control (BSA or DLL4). The bottom panel shows a representative “heat map” of Notch luciferase activity (red repre-sents highest Notch activity and dark blue rep-resents lowest Notch activity). Jag1 = Jagged1.

significantly lower basal and DLL4-induced Notch activity within 24 hours of treatment compared with DMSO-treated cells (DMSO vs DLL4 antibody [BSA]: 1.00- vs 0.56-fold change, difference = 0.44-fold change, 95% CI = 0.31- to 0.56-fold change, P = .004; DMSO vs DLL4 antibody [DLL4]: 2.56- vs 0.53-fold change,

difference = 2.03-fold change, 95% CI = 1.13- to 2.93-fold change, P = .003; DMSO vs DBZ [BSA]: 1.00- vs 0.45-fold change, differ-ence = 0.55-fold change, 95% CI = 0.37- to 0.72-fold change, P = .005; DMSO vs DBZ [DLL4]: 2.56- vs 0.45-fold change, differ-ence = 2.11-fold change, 95% CI = 1.19- to 3.03-fold change,



P = .003). The DLL4 monoclonal antibody inhibited both basal and DLL4-induced Notch activity to the same extent as DBZ (DLL4 antibody vs DBZ [BSA]: 0.55- vs 0.45-fold change, difference = 0.10-fold change, 95% CI = 20.03- to 0.24-fold change, P = .09; DLL4 antibody vs DBZ [DLL4]: 0.53- vs 0.45-fold change, difference = 0.08-fold change, 95% CI = 20.16- to 0.31-fold change, P = .42), indicating that in LS174T colorectal carcinoma cells, Notch sig-naling in vitro occurs primarily through DLL4.

Effect of Notch Blockade by DBZ or DLL4 Monoclonal Antibody on Proliferation and Apoptosis in LS174T Cells In Vitrog-Secretase inhibitors have been reported to decrease proliferation of some tumor cells (53–55) and they sometimes also promote apo-ptosis (53–57). We next examined the effects of DBZ and the DLL4 monoclonal antibody on proliferation and apoptosis in LS174T cells. DBZ did not statistically significantly decrease tumor cell proliferation in vitro compared with control (DBZ fold change vs vehicle control = 0.74, difference = 0.26, 95% CI = 20.04 to 0.55,

P = .06) (Supplementary Figure 1, A, available online). Likewise, the DLL4 monoclonal antibody did not cause a statistically significant reduction in proliferation (DLL4 monoclonal anti-body fold change vs vehicle control = 0.91, difference = 0.09, 95% CI = 20.14 to 0.31, P = .26) (Supplementary Figure 1, A, available online). We did not detect a statistically significant change in apo-ptosis in LS174T cells treated with DBZ or the DLL4 monoclonal antibody in vitro compared with cells treated with vehicle (vehicle vs DBZ: 11 991 vs 11 187 light units, difference = 804 light units, 95% CI = 21141 to 2748, P = .31; vehicle vs DLL4 monoclonal antibody: 11 991 vs 12 488 light units, difference = 497 light units, 95% CI = 21150 to 2144, P = .44) (Supplementary Figure 1, B, available online).

Effects of Notch Blockade by DBZ or DLL4 Monoclonal Antibody on Radiosensitization of LS174T Cells In Vitrog-Secretase inhibitors have been reported to sensitize glioma stem cells to ionizing radiation (30). Thus, we examined whether Notch inhibition with DBZ or the DLL4 monoclonal antibody would

Figure 2. Ionizing radiation and its inter-action with Notch signaling in LS174T colorectal carcinoma cells. A) Radiation clonogenic survival curves. LS174T cells were incubated with vehicle, dibenzaze-pine (DBZ) (left panel), or delta-like ligand 4 (DLL4) monoclonal antibody (mAb) (right panel), mock irradiated or irradi-ated with a 2, 4, or 6 Gy dose of ionizing radiation (IR). The mean survival fractions and 95% confidence intervals (error bars) are plotted on the graph with respect to irradiation dose from three independent experiments; the survival curves were determined by modeling the data to the linear–quadratic equation. B) Notch lucif-erase assay with IR. LS174T Notch-luc cells were mock irradiated or irradiated with a 2, 4, or 6 Gy dose of IR, and Notch luciferase activity was measured 24 hours later; the fold change in Notch luciferase was determined by dividing the Notch luciferase activity in irradiated cells by the Notch luciferase activity in mock irradi-ated cells. P value (two-sided, analysis of variance) is for the comparison with mock irradiation. Data are presented as mean values from three independent experi-ments and 95% confidence intervals (error bars).



radiosensitize LS174T cells in vitro (Figure 2, A). We generated clonogenic survival curves for LS174T cells using a radiation dose range of 0–6 Gy in the presence of vehicle, DBZ, or the DLL4 monoclonal antibody. Neither of these treatments resulted in a statis-tically significant decrease in the surviving fraction of cells compared with vehicle at any radiation dose (surviving fraction, vehicle vs DBZ [2 Gy]: 0.45 vs 0.36, difference = 0.09, 95% CI = 20.27 to 0.46, P = .51; vehicle vs DBZ [4 Gy]: 0.14 vs 0.11, difference = 0.03, 95% CI = 20.07 to 0.13, P = .45; vehicle vs DBZ [6 Gy]: 0.06 vs 0.03, difference = 0.03, 95% CI = 20.03 to 0.08, P = .29; vehicle vs DLL4 monoclonal antibody [2 Gy]: 0.34 vs 0.39, difference = 0.04, 95% CI = 20.05 to 0.14, P = .28; vehicle vs DLL4 monoclonal antibody [4 Gy]: 0.15 vs 0.18, difference = 0.03, 95% CI = 20.12 to 0.16, P = .70; vehicle vs DLL4 monoclonal antibody [6 Gy]: 0.06 vs 0.03, difference = 0.03, 95% CI = 0 to 0.06, P = .07). The plating efficiencies for cells treated with DBZ or the DLL4 monoclonal antibody were 0.05 and 0.04, respectively, and were not statistically significantly different from that of cells treated with vehicle (plating efficiency, vehicle vs DBZ: 0.08 vs 0.05, difference = 0.03, 95% CI = 2.01 to 0.08, P = .11; vehicle vs DLL4 monoclonal antibody: 0.05 vs 0.04, difference = 0.01, 95% CI = 20.12 to 0.14, P = .80). To determine if ionizing radiation could alter Notch activity, we assessed luciferase activity in LS174T-Notch-luc cells immediately before and 24 hours after administration of increasing doses (2, 4, and 6 Gy) of ionizing radiation. We observed a statistically significant reduction in Notch activity after treating LS174T Notch-luc cells with a 6-Gy dose of ionizing radiation compared with mock-irradiated cells (fold change in Notch luciferase, mock irradiation vs 6 Gy: 1.00 vs 0.48, difference = 0.52, 95% CI = 0.15 to 0.87, P = .02) (Figure 2, B); we confirmed that this reduction in Notch activity was not due to a reduction in the number of viable cells (data not shown). We also observed that this dose of ionizing radiation led to reductions in the levels of DLL4 and Notch1 ICD as assessed by immunoblotting (data not shown), which may partly explain the resulting suppression of Notch activity by ion-izing radiation. Thus, the Notch signaling pathway does not appear to play an important role in clonogenic survival following exposure of colorectal carcinoma LS174T cells to ionizing radiation in vitro.

Tumor Growth in Response to Notch Inhibition with DBZ or DLL4 Antibody Blockade and Ionizing RadiationAlthough Notch inhibition had no effect on tumor cell radiosensiti-zation in vitro, it was important to explore this in an in vivo system, where alterations in the tumor microenvironment can be highly relevant on tumor growth and survival (34,58). Other investigators (59,60) have shown that LS174T xenografts have a heterogeneous tumor vascular architecture that closely resembles that in colon carcinoma samples from patients, making this xenograft mouse model a relevant preclinical model. We therefore grew the LS174T Notch-luc cell line as subcutaneous xenografts in BALB/C nude mice and investigated the effect of ionizing radiation and global Notch inhibition by DBZ on tumor growth delay. Specifically, mice bearing LS174T Notch-luc xenografts were randomly assigned to vehicle (n = 9 mice), DBZ (n = 8 mice), 5 Gy ionizing radiation (n = 10 mice), or 5 Gy ionizing radiation and DBZ (n = 9 mice). We administered DBZ after ionizing radiation because Notch inhibition has been reported to increase tumor hypoxia (10,11,61), and the efficacy of ionizing radiation is greatly reduced by hypoxia (62, 63).

To verify inhibition of tumor Notch activity by DBZ, we moni-tored Notch luciferase reporter activity in the xenograft tumors with in vivo bioluminescent imaging immediately before (baseline) and 24 hours after treatment of the mice with vehicle or DBZ alone or in combination with ionizing radiation (Figure 3, top panels). Vehicle-treated mice showed no change in Notch luciferase activity. However, treatment with DBZ or DBZ and ionizing radiation sta-tistically significantly inhibited Notch luciferase activity to 28% and 35%, respectively, of the activity at baseline (luciferase activity, DBZ, baseline vs 24 hours: 100% vs 28%, difference = 72%, 95% CI = 32% to 111%, P = .01; DBZ and ionizing radiation, baseline vs 24 hours: 100% vs 35%, difference = 65%, 95% CI = 48% to 81%, P < .001); both of these decreases in Notch luciferase activity were statistically significant differences compared with the change in Notch luciferase activity for mice treated for 24 hours with vehicle (change in luciferase activity from baseline to 24 hours, vehicle vs DBZ: 103% vs 28%, difference = 75%, 95% CI = 39% to 109%, P = .002; vehicle vs DBZ and ionizing radiation: 103% vs 35%, difference = 68%, 95% CI = 35% to 100%, P = .003). Treatment with ionizing radiation alone also reduced Notch reporter luciferase activity in the tumors to 44% of the activity at baseline (luciferase activity, baseline vs 24 hours after ionizing radiation: 100% vs 44%, difference = 56%, 95% CI = 18.8% to 93%, P < .001), a statistically significant difference compared with the change in Notch luciferase activity for mice treated for 24 hours with vehicle (change in luciferase activity, vehicle vs ionizing radiation: 103% vs 45%, difference = 58%, 95% CI = 3% to 110%, P = .04).

The average time for tumor volumes to reach four times the starting volume was 7.1 days for mice treated with vehicle and 9.5 days for mice treated with DBZ (difference = 2.4 days, 95% CI = 0.1 to 4.9 days, P = .06) (Figure 4, top panels). The average time for tumors to reach four times the starting volume was statistically significantly longer in mice treated with ionizing radiation com-pared with mice treated with vehicle or DBZ (ionizing radiation vs vehicle: 15.1 vs 7.1 days, difference = 8 days, 95% CI = 6.0 to 9.9 days, P < .001; ionizing radiation vs DBZ: 15.1 vs 9.5 days, difference = 5.6 days, 95% CI = 3.3 to 7.8, P < .001). The average time for tumors to reach four times the starting volume was statis-tically significantly longer in mice treated with both DBZ and ionizing radiation compared with mice treated with ionizing radiation or DBZ alone (DBZ and ionizing radiation vs ionizing radiation: 24.4 vs 15.1 days, difference = 9.3 days, 95% CI = 7.0 to 11.6 days, P < .001; DBZ and ionizing radiation vs DBZ: 24.4 vs 9.5 days, difference = 14.9 days, 95% CI = 13.2 to 16.6 days, P < .001). The combined treatment produced a tumor growth delay of 17.3 days compared with vehicle (vehicle vs DBZ and ionizing radiation: 7.1 vs 24.4 days, difference = 17.3 days, 95% CI = 15.9 to 18.6 days, P < .001), which was greater than additive, as defined by a supra-additive ratio of 1.6.

g-Secretases have a broad range of potential substrates in addition to Notch receptors (64); consequently, the use of a global g-secretase inhibitor (ie, DBZ) may have nonspecific effects. Thus, to examine the effects of a more selective inhibitor of Notch signaling, we repeated this xenograft experiment and treated the mice with vehicle, the DLL4 monoclonal antibody, ionizing radiation, or the combina-tion. In agreement with our in vitro data, treatment of mice with the DLL4 monoclonal antibody for 24 hours decreased Notch luciferase



activity in vivo to 26% of the activity at baseline (luciferase activity, baseline vs 24 hours: 100% vs 26%, difference = 74%, 95% CI = 58% to 88%, P < .001), and treatment with the DLL4 monoclonal antibody and ionizing radiation decreased Notch luciferase activity to 36% of the activity at baseline (luciferase activity, baseline vs 24 hours: 100% vs 36%, difference = 64%, 95% CI = 52% to 74%, P < .001) (Figure 3, bottom panels). Treatment with the DLL4 monoclonal antibody alone and in combination with ionizing radia-tion led to a statistically significant reduction in Notch luciferase activity compared with vehicle (change in Notch luciferase activity from baseline to 24 hours, vehicle vs DLL4 antibody: 172% vs 26%, difference = 146%, 95% CI = 86% to 205%, P < .001; vehicle vs DLL4 antibody and ionizing radiation: 172% vs 36%, difference = 136%, 95% CI = 77% to 195%, P < .001). The average time for tumors to reach four times the starting volume was statistically sig-nificantly longer in mice treated with the DLL4 monoclonal anti-body or ionizing radiation compared with mice treated with vehicle (DLL4 monoclonal antibody vs vehicle: 16.4 vs 6.8 days, difference = 9.6 days, 95% CI = 6.3 to 12.8 days, P < .001; ionizing radiation vs vehicle: 17.3 vs 6.8 days, difference = 10.5 days, 95% CI = 9.1 to 11.9 days, P < .001) (Figure 4, bottom panels). The average time for

tumors to reach four times the starting volume was statistically sig-nificantly longer in mice treated with both the DLL4 monoclonal antibody and ionizing radiation compared with mice treated with ionizing radiation or the antibody alone (DLL4 monoclonal anti-body and ionizing radiation vs ionizing radiation: 44.3 vs 17.3 days, difference = 27.0 days, 95% CI = 21.8 to 32.1 days, P < .001; DLL4 monoclonal antibody and ionizing radiation vs DLL4 monoclonal antibody: 44.3 vs 16.4 days, difference = 27.9 days, 95% CI = 221.7 to 34.13 days, P < .001). The tumor growth delay for mice treated with the DLL4 monoclonal antibody and ionizing radiation was 37.5 days (vehicle vs DLL4 monoclonal antibody and ionizing radiation: 6.8 vs 44.3 days, difference = 37.5 days, 95% CI = 32 to 43 days, P < .001), which was greater than additive, as defined by a supra-additive ratio of 1.8. The average time for tumor volumes to reach four times the starting volume was longer for mice treated with the DLL4 monoclonal antibody than for mice treated with DBZ (16.4 vs 9.5 days, difference = 6.9 days, 95% CI = 3.7 to 10.1 days, P < .001). The tumor growth delay provided by DLL4 monoclonal antibody and ionizing radiation was more than double the delay observed with combination treatment with DBZ and ionizing radiation. No statis-tically significant differences in weight loss were observed between

Figure 3. Monitoring tumor Notch activity in vivo. Mice bearing subcu-taneous LS174T Notch-luc xenografts (approximately 100 mm3 volume) were anaesthetized and injected intraperitoneally with luciferin, and tumor bioluminescence was measured before (baseline) and 24 hours after treatment with vehicle, dibenzazepine (DBZ), 5 Gy ionizing radia-tion (IR), or 5 Gy IR and DBZ (top left panel) or before and 24 hours after treatment with vehicle, delta-like ligand 4 (DLL4) monoclonal antibody

(mAb), 5 Gy IR, or 5 Gy IR and DLL4 mAb (bottom left panel); the “heat map” overlying the tumors displays relative Notch activity (red repre-sents highest Notch activity and dark blue represents lowest Notch ac-tivity). The mean percent change in Notch luciferase activity, generated from three mice per group, is shown in graphical form (right panels), with 95% confidence intervals (error bars). P values (two-sided, analysis of variance) are for comparisons with vehicle.



the mice in any of the treatment groups (DBZ, DLL4 monoclonal antibody, ionizing radiation, DBZ and ionizing radiation, or DLL4 monoclonal antibody and ionizing radiation) and mice treated with vehicle (data not shown). Taken together, global Notch inhibition with DBZ in combination with ionizing radiation and selective DLL4–Notch inhibition with the DLL4 monoclonal antibody in combination with ionizing radiation displayed supra-additive effects with respect to delay of tumor growth.

Effect of DBZ or DLL4 Blockade in Combination with Ionizing Radiation on Tumor Vascular Function, Cell Proliferation, and Necrosis In VivoTo monitor the effects of these treatments on tumor vascular function, we assessed changes in rate of tumor blood flow (ie, perfusion) in real time using microbubble contrast ultrasound imaging from just before treatment (day 1) and on day 5 of

treatment (Figure 5 and Supplementary Figure 2, available online). Treatment with DBZ or the DLL4 monoclonal antibody resulted in statistically significant reductions in tumor blood flow compared with vehicle at day 5 (relative blood flow, vehicle vs DBZ: 98.9% vs 38.9%, difference = 60.0%, 95% CI = 29.5% to 90.4%, P < .001; relative blood flow, vehicle vs DLL4 monoclonal antibody: 89.0% vs 29.5%, difference = 59.5%, 95% CI = 36.0% to 82.9%, P < .001). The tumors treated with ionizing radiation alone did not exhibit any statistically significant change in tumor blood flow compared with vehicle-treated tumors at day 5 (DBZ experiment, relative blood flow, vehicle vs ionizing radiation: 98.9% vs 97.8%, difference = 1.1%, 95% CI = 231.4% to 29.3%, P = .52; DLL4 monoclonal antibody experiment, relative blood flow, vehicle vs ionizing radiation: 89.0% vs 77.5%, difference = 11.5%, 95% CI = 211.9% to 34.9%, P = .09). However, ionizing radiation followed by treatment with DBZ or the DLL4 monoclonal

Figure 4. LS174T xenograft tumor growth delay. Mice bearing subcuta-neous LS174T Notch-luc xenografts were randomly assigned to vehicle or treatment (DBZ, 5 Gy ionizing radiation [IR], or 5 Gy IR and dibenzaz-epine [DBZ]; delta-like ligand 4 [DLL4] monoclonal antibody [mAb], 5 Gy ionizing radiation, or 5 Gy IR and DLL4 mAb) groups when tumor volumes were approximately 100 mm3 (ie, day 1), and tumor volumes were measured every 1–3 days. DBZ was administered on day 1, every 3 days thereafter for a total of five doses; DLL4 mAb was given twice per week for the duration of the experiment (black arrows). White arrows indicate IR. Tumor volumes were normalized to the tumor

volumes on day 1; the mean normalized tumor volumes and 95% con-fidence intervals (error bars) are plotted on the tumor growth curves (left panels). Mice were killed when their normalized tumor volumes reached 4 (dotted line). The total number of mice in each group is shown in parentheses. The average time for tumors to reach four times the starting volume (RTV4) and 95% confidence intervals (error bars) are shown in the graphs on the right panels. The dotted line indicates RTV4 for the vehicle-treated tumors; the extent of the respective bars above this line corresponds to tumor growth delay. ns = not statistically significantly different; *P < .001 (two-sided, analysis of variance).



antibody produced statistically significant reductions in tumor blood flow compared with vehicle at day 5 (relative blood flow, vehicle vs ionizing radiation and DBZ: 98.9% vs 31.5%, difference = 67.4%, 95% CI = 36.9% to 97.7%, P < .001; vehicle vs ionizing radiation and DLL4 monoclonal antibody: 89.0% vs 32.0%; dif-ference = 57.0%, 95% CI = 33.5% to 80.4%, P < .001). In mice treated with the DLL4 monoclonal antibody (either alone or in combination with ionizing radiation), we observed a statistically significant reduction in tumor blood flow compared with vehicle-treated mice as early as 48 hours after the start of treatment (ie, day 3) (relative blood flow, vehicle vs DLL4 monoclonal antibody: 103% vs 64.0%, difference = 39.0%, 95% CI = 20.0% to 57.9%, P < .001; vehicle vs DLL4 monoclonal antibody and ionizing radi-ation: 103% vs 54.5%, difference = 48.5%, 95% CI = 29.5% to 67.4%, P < .001) (Supplementary Figure 2, C, available online). Thus, global Notch inhibition with DBZ or disruption of the DLL4–Notch axis profoundly decreased blood flow in both irra-diated and unirradiated tumors in this mouse model.

To examine the effects of the treatments on the tumor vascula-ture, we performed immunohistochemistry with an anti-CD31 antibody to detect vessels on tumor sections obtained from mice that were killed when their tumor volume reached four times the starting volume and conducted Chalkley vessel counts, which pro-vide a measure of the area occupied by blood vessels (47). Tumors from mice treated with the DLL4 monoclonal antibody alone or in combination with ionizing radiation had higher Chalkley vessel counts compared with tumors from vehicle-treated mice (Chalkley vessel count, vehicle vs DLL4 monoclonal antibody: 6.1 vs 9.9, difference = 3.8, 95% CI = 2.4 to 5.2, P < .001; vehicle vs DLL4 monoclonal antibody and ionizing radiation: 6.1 vs 7.6, difference = 1.5, 95% CI = 0.7 to 2.2, P = .04) (Figure 6, A). The Chalkley vessel counts were statistically significantly higher in tumors treated with the DLL4 monoclonal antibody vs those treated with DBZ (DLL4 monoclonal antibody vs DBZ: 9.9 vs 5.9 counts; dif-ference = 4.0 counts, 95% CI = 3.3 to 4.6 counts, P < .001). Confocal microscopy and three-dimensional reconstruction of the

Figure 5. Effect of Notch inhibition on tumor blood flow. Microbubble contrast ultrasound was performed on mice bearing LS174T Notch-luc xenografts when the tumors were approximately 100 mm3 before treatment (day 1) and on day 5 following initiation of treatments, as indicated (n = 3 tumors per group). Representative images show tumor blood flow as depicted by microbubble contrast agent (green signal); the amount of green signal is representative of the number of microbubbles within the out-lined tumor (top panels); the region of interest outlining each tumor is shown in blue. Relative tumor blood flow values were normalized to the vehicle (control) group, day 1, and the mean values and 95% confidence intervals (error bars) derived from three mice per group are plotted in the lower panel. *P < .001 com-pared with vehicle day 1 (two-sided, analysis of variance). IR = ionizing radiation.



tumor vasculature provided qualitative evidence of many more smaller, highly branched, and torturous vessels in tumors treated with the DLL4 monoclonal antibody compared with tumors treated with vehicle (Figure 6, B).

We next examined the effect of the same treatments on tumor hypoxia by immunostaining xenograft tumors for the hypoxia marker EF5. Tumors from mice treated with the DLL4 monoclo-nal antibody alone or in combination with ionizing radiation had a higher percentage of EF5-positive (ie, hypoxic) cells compared with tumors treated with vehicle (vehicle vs DLL4 monoclonal antibody: 5.2% vs 31.9%, difference = 26.7%, 95% CI = 20.2 % vs 33.1%, P < .001; vehicle vs DLL4 monoclonal antibody and

ionizing radiation: 5.2% vs 37.0%, difference = 31.8%, 95% CI = 25.3% to 38.2%, P < .001). Taken together, these observa-tions indicate that treatment with the DLL4 monoclonal antibody alone or in combination with ionizing radiation causes increased angiogenesis but worse vasculature function with reduced tumor blood flow and increased tumor hypoxia (Figure 7, A). Tumors from mice treated with DBZ displayed no statistically significant increase in the percentage of hypoxic cells compared with tumors from vehicle-treated mice (data not shown).

We also performed immunostaining of the xenograft tumors for Ki-67 to assess the effects of the treatments on tumor prolifer-ation. Compared with vehicle-treated tumors, tumors from mice

Figure 6. Immunohistochemistry and con-focal imaging of LS714T xenograft tumor vasculature. Mice bearing LS174T xenografts were treated with vehicle, Notch inhibitors (dibenzazepine [DBZ] or the delta-like ligand 4 [DLL4] monoclonal antibody [mAb]), ionizing radiation (IR), or IR and DBZ or the DLL4 mAb and then killed when tumor volume reached four times the starting volume and the tumors were excised and processed for immunohis-tochemistry. A) Immunohistochemistry on fixed tumor sections using anti-CD31 anti-body. Representative images at ×200 are shown in the left panels; scale bar indicates 100 µm. Chalkley vessel counts, which pro-vide a measure of the area occupied by blood vessels, were performed on three tumors per group (a minimum of three regions of max-imal neovascularization examined per tumor) to quantify tumor microvasculature (dark brown staining identifies endothelial cells, and nuclei are counterstained blue); mean values and 95% confidence intervals (error bars) are shown (right panels). P values (two-sided, analysis of variance) are for com-parisons with vehicle. B) Representative three-dimensional tumor vasculature recon-structions generated from confocal micros-copy z-stack images of tumor sections obtained from mice (n = 3 per group) fol-lowing intravenous injection of a phycoery-thrin-conjugated anti-CD31 antibody. Scale bar indicates 100 µm.



treated with DBZ alone or in combination with ionizing radiation and from mice treated with the DLL4 monoclonal antibody alone had a statistically significantly lower proportion of Ki-67-positive (ie, proliferating) cells (DBZ vs vehicle: 0.36 vs 0.65, difference = 0.29, 95% CI = 0.11 to 0.46, P = .002; DBZ and ionizing radiation vs vehicle: 0.40 vs 0.65, difference = 0.25, 95% CI = 0.07 to 0.41, P = .007; DLL4 monoclonal antibody vs vehicle: 0.54 vs 0.71, dif-ference = 0.17, 95% CI = 0.05 to 0.27, P = .01) (Figure 7, B). To examine treatment effects on tumor necrosis, we also stained tumor sections with hematoxylin and eosin. Compared with vehicle-treated tumors, tumors treated with DBZ, ionizing radia-tion, or the combination displayed increasingly higher percentage of necrosis (vehicle vs DBZ: 6.5% vs 16.2%, difference = 9.7%, 95% CI = 22.1% to 21.6%, P = .09; vehicle vs ionizing radiation: 6.5% vs 22.5%, difference = 16.0%, 95% CI = 3.9% to 28.0%, P = .08; vehicle vs DBZ and ionizing radiation: 6.5% vs 28.3%, difference = 21.8%, 95% CI = 211.6% to 31.9%, P = .04) (Figure 7, C). Similarly, compared with tumors from vehicle-treated mice, tumors from mice treated with the DLL4 monoclonal antibody displayed a statistically significantly higher percentage of necrosis (vehicle vs DLL4 monoclonal antibody: 4.6% vs 45.0%, difference = 40.4% necrosis, 95% CI = 18.2% to 62.4%, P = .015), which was further increased in tumors from mice treated with the DLL4 monoclonal antibody in combination with ionizing radiation (vehicle vs DLL4 antibody and ionizing radiation: 4.6% vs 66.6%, difference = 62.0%, 95% CI = 39.9% to 84.0%, P < .001). Treatment with ionizing radiation alone did not statistically significantly increase tumor necrosis compared with vehicle (vehicle vs ionizing radia-tion: 4.6% vs 26.6%, difference = 22.0%, 95% CI = 9.3% to 44.1%, P = .07) (Figure 7, C). The tumor necrosis observed in tumors treated with ionizing radiation in combination with the DLL4 monoclonal antibody was located predominantly in the center of the tumor, which is reminiscent of histological findings typically seen with vascular disrupting agents (4). The proportions of Ki-67-positive cells were not statistically significantly different for combination treatment with the DLL4 monoclonal antibody and ionizing radiation compared with vehicle or ionizing radiation (vehicle vs DLL4 monoclonal antibody and ionizing radiation: 0.71 vs 0.66, difference = 0.05, 95% CI = 20.13 to 0.22,

P = .55; ionizing radiation vs DLL4 monoclonal antibody and ionizing radiation: 0.63 vs 0.66, difference = 0.03, 95% CI = 20.22 to 0.14, P = .59); however, the overall amount of remaining viable tumor was considerably less in the combination-treated tumors compared with tumors treated with vehicle or ionizing radiation, consistent with the observed tumor growth delay (Figure 4).

To assess the effects of the treatments on apoptosis of tumor cells, we performed TUNEL staining of tumor sections. TUNEL staining revealed no changes in apoptosis associated with any of the treatments (<1% TUNEL-positive cells for vehicle or treat-ments) (Supplementary Figure 3, A, available online). This finding was in agreement with our in vitro findings, suggesting that an increase in apoptosis was not responsible for the observed tumor growth delays and that another mechanism, such as necrosis or mitotic catastrophe (65), may be a predominant mode of cell death in these tumors.

To directly determine whether changes in tumor blood flow corresponded to changes in tumor histology, we killed the mice that underwent contrast ultrasound on day 5, excised and sectioned their tumors, and examined tumor Chalkley vessel counts, hypoxia, and proliferation (Supplementary Figure 3, B, available online). Compared with vehicle, the DLL4 monoclonal antibody statisti-cally significantly increased Chalkley vessel counts (vehicle vs DLL4 monoclonal antibody: 4.3 vs 8.3 counts, difference = 4.0 counts, 95% CI = 1.0 to 6.9 counts, P = .003), reduced tumor blood flow (relative blood flow, vehicle vs DLL4 monoclonal antibody: 89.0% vs 29.5%, difference = 59.5%, 95% CI = 36.0% to 82.9%, P < .001) (Figure 5), and increased hypoxia (vehicle vs DLL4 monoclonal antibody: 5.6% vs 34.9% hypoxic cells; difference = 29.3% hypoxic cells, 95% CI = 4.0% to 54.5% hypoxic cells, P = .03). However, DLL4 monoclonal antibody did not statistically significantly decrease tumor cell proliferation compared with ve-hicle at this time point (proportion of Ki-67-positive cells, vehicle vs DLL4 monoclonal antibody: 0.65 vs 0.56, difference = 0.09, 95% CI = 20.08 to 0.26, P = .23) (Supplementary Figure 3, B, available online). In contrast, compared with vehicle, combination treatment with the DLL4 monoclonal antibody and ionizing radiation resulted in increased Chalkley vessel counts (vehicle vs DLL4 monoclonal antibody and ionizing radiation: 4.3 vs 8.0

(continued)



Figure 7. Immunohistochemistry of LS714T xenograft tumors to assess hypoxia, proliferation, and necrosis. Mice bearing LS174T xenografts were treated with vehicle, Notch inhibitors (dibenzazepine [DBZ] or the delta-like ligand 4 [DLL4] monoclonal antibody [mAb]), ionizing radiation (IR), or IR and DBZ or the DLL4 mAb and then killed when tumor volumes reached four times the starting volume and the tumors were excised and processed for immunohistochemistry. A) Hypoxia. Left panels show images of representa-tive staining with an antibody for the hypoxic marker, EF5, in tumor sections at ×150 (scale bar indicates 200 µm); red indicates hypoxic cells. Whole tumor sections were scored on a field-by-field basis to estimate the area of EF5 immunostained tumor cells, and each field assigned a hypoxic score; the mean hypoxic score for each tumor was then determined and converted to the percentage of hypoxic cells as described in “Materials and Methods”. The mean percentage of hypoxic cells for the vehicle and treatment groups are plotted with 95% confidence inter-vals (error bars) in the right panel. *P < .001 compared with vehicle (two-sided, analysis of variance). Three tumors were examined per group, and an entire tumor section was scored for each tumor. B) Proliferation. Left panels show images of representative staining with an antibody to the cell prolifera-tion marker Ki-67, in tumor sections at ×400 (scale bar indicates 50 µm); prolif-erating cells are identified by the brown nuclei. The proportion of Ki-67 positive cells to total number of cells was deter-mined from six high-power fields from each tumor, and the mean values are plotted for vehicle and treatment groups with 95% confidence intervals (error bars) (right panels). P values (two-sided, analysis of variance) are for comparisons with vehicle. Three tumors were examined per group, with six high-power fields counted per tumor. C) Necrosis. Left panels show images of representative staining with hematoxylin–eosin in tumor sections at× 20; scale bar indicates 1000 µm. Regions of necrosis are outlined in black. The percentage of tumor necro-sis was estimated from a single section of the entire tumor viewed at low power, and the mean values for the vehicle and treatment groups are plotted with 95% confidence intervals (error bars) (right panels). P values (two-sided, analysis of variance) are for comparisons with vehicle. Three tumors per group were examined.



counts, difference = 3.7 counts, 95% CI = 0.73 to 6.6 counts, P = .02), reduced tumor blood flow (vehicle vs DLL4 monoclonal antibody and ionizing radiation: 100% vs 32.0% relative blood flow; difference = 68.0%, 95% CI = 33.5% to 80.4%, P < .001) (Figure 5), and led to a statistically significant reduction in the proportion of Ki-67-positive tumor cells (vehicle vs DLL4 mono-clonal antibody and ionizing radiation: 0.65 vs 0.20, difference = 0.45, 95% CI = 0.15 to 0.74, P = .006) but did not increase tumor hypoxia (vehicle vs monoclonal antibody and ionizing radiation: 5.6% vs 11.8% hypoxic cells, difference = 6.2%, 95% CI = 217.4% to 29.8%, P = .50). The lack of a statistically significant increase in the percentage of hypoxic cells in combination (ie, DLL4 mono-clonal antibody and ionizing radiation)-treated tumors compared with vehicle-treated tumors may be partly due to decreased oxygen consumption resulting from the lower proportion of proliferating tumor cells and hence smaller tumor burden (ie, note the lower mean tumor volume at day 5 with the combination treatment

compared with vehicle-treated tumors) (Figure 4). We did not observe a statistically significant increase in tumor cell necrosis with the combination treatment compared with vehicle at this early time point (data not shown), suggesting that prolonged DLL4 blockade (>5 days) is required to induce necrosis.

It is interesting that immunohistochemical staining of tumor sections for DLL4 revealed increased DLL4 expression following Notch inhibition with DBZ or the DLL4 monoclonal antibody (data not shown), which is consistent with our in vitro data (Supplementary Figure 1, A, available online).

Tumor Growth Delay and Tumor DLL4 ExpressionTo examine whether tumor expression of DLL4 is a critical target for anti-DLL4 therapy, we tested the effects of using the DLL4 monoclonal antibody to treat mice bearing xenograft tumors derived from human hypopharyngeal squamous cell carcinoma FaDu cells, which do not express DLL4 (Figure 8, A). Nude mice

Figure 8. Tumor growth delay and tumor DLL4 expression. A) Immunoblot analysis. Lysates of untreated human hypopharyngeal squamous cell carcinoma FaDu cells were immunoblotted with anti-bodies against the Notch ligands delta-like ligand 4 (DLL4) and Jagged1 (Jag1). Equal loading was confirmed with an antibody against b-actin. B) FaDu xenograft tumor growth. Mice bearing FaDu xenograft tumor were randomly assigned to vehicle or treatment (DLL4 monoclonal antibody [mAb], 5 Gy ionizing radiation [IR], or 5 Gy IR and DLL4 mAb) groups when tumor volumes reached approx-imately 100 mm3 (ie, day 1), and the tumor volumes were measured every 1–3 days. DLL4 mAb was given twice per week for the duration of the experiment (black arrows). White arrow indicates ionizing

radiation. The tumor volumes were normalized relative to tumor vo-lumes on day 1; the mean normalized tumor volumes and 95% con-fidence intervals (error bars) are plotted on the survival curve (left panel). Mice were killed when their normalized tumor volumes reached 4 (dotted line). The total number of mice in each group is shown in parentheses. The mean number of days for tumors to reach four times the starting volume (RTV4) and 95% confidence intervals (error bars) are shown in the graphs on the right panels. The dotted line indicates RTV4 for the vehicle-treated tumors; the extent of the respective bars above this line corresponds to tumor growth delay. *P < .001 (two-sided, analysis of variance); ns = not statistically sig-nificantly different.



bearing these tumors were treated with vehicle, the DLL4 mono-clonal antibody, a 5-Gy dose of ionizing radiation, or the DLL4 monoclonal antibody and ionizing radiation (n = 5 mice per group). The average time to reach four times the starting tumor volume for mice treated with vehicle was 9.8 days (95% CI = 7.1 to 12.4 days), and for mice treated with ionizing radiation, it was 14.0 days (95% CI = 12.0 to 15.9 days), yielding a tumor growth delay for ionizing radiation of 4.2 days (95% CI = 1.4 to 9.7 days, P = .08) (Figure 8, B). The average time to reach four times the starting tumor volume for mice treated with the DLL4 monoclo-nal antibody was 28.8 days (95% CI = 25.5 to 32.0 days), resulting in a tumor growth delay of 19.0 days (vehicle vs DLL4 monoclonal antibody: 9.8 days vs 28.8 days, difference = 19.0 days, 95% CI = 13.4 to 24.5 days, P < .001). Combination treatment with the DLL4 monoclonal antibody and ionizing radiation yielded an average time to reach four times the starting tumor volume of 42.6 days (95% CI = 37.0 to 48.1 days), corresponding to a supra-additive tumor growth delay of 32.8 days (vehicle vs DLL4 mono-clonal antibody and ionizing radiation: 9.8 days vs 42.6 days, difference = 32.8 days, 95% CI = 27.6 to 38.3 days, P < .001); the supra-additive ratio was 1.4. No statistically significant differences in weight loss were observed between any of the treatment groups and the vehicle-treated group (data not shown). These results sug-gest that tumor growth delay resulting from combination treat-ment with the DLL4 monoclonal antibody and ionizing radiation does not require DLL4 expression by the tumor and support the broad relevance for this treatment approach in different tumor types, independent of tumor DLL4 expression.

DiscussionWe found that Notch blockade with either a global inhibitor or with a blocking antibody to the critical Notch ligand, DLL4, in combination with ionizing radiation had no effect on clonogenic tumor cell survival in vitro; however, in the context of a colorectal carcinoma xenograft model, Notch blockade by these same means in combination with ionizing radiation caused tumor growth delay. By using FaDu xenografts, which do not express DLL4, we dem-onstrated that the DLL4 monoclonal antibody in combination with ionizing radiation was still able to effectively delay tumor growth. This finding suggests that the host tissues must be pro-viding the therapeutic target for the DLL4 monoclonal antibody, highlighting the relevance of targeting the vasculature.

Targeting the tumor vasculature through inhibition of Notch signaling could be a useful strategy to enhance the efficacy of radi-ation. At the moderate dose of ionizing radiation that was used in this preclinical study—5 Gy—we did not observe any functional impairment of the tumor vasculature after treatment with ionizing radiation alone, as evidenced by the absence of changes in tumor blood flow or vessel counts compared with vehicle-treated tumors, although morphological effects were seen on three-dimensional confocal reconstructions and there was evidence of tumor hypoxia. We hypothesize that although the existing tumor vasculature fol-lowing combination treatment with DLL4 monoclonal antibody and ionizing radiation was sufficient to maintain some persisting tumor cells, as the tumor cells proliferated, they overwhelmed their blood supply and were unable to develop an effective tumor

vasculature because of DLL4 blockade, causing them to undergo necrosis as a result. By contrast, in the absence of DLL4 blockade, tumor angiogenesis would proceed following ionizing radiation, and hence support the continued growth of the tumor. In tumors of mice that received combination treatment with the DLL4 monoclonal antibody and ionizing radiation, we observed exten-sive tumor hypoxia and necrosis predominantly at the center of the tumor, which implies that the proliferating tumor cells were mainly confined to the outer rim of the tumor. Thus, a further improvement in tumor control might be achieved by the delivery of additional radiation treatment or chemotherapy to the tumor at a later time, which could lower the burden of proliferating cells in the viable (and better oxygenated) tumor rim. We believe that ionizing radiation and DLL4–Notch inhibition are complemen-tary treatments. Ionizing radiation can decrease the number of clonogenic cancer cells and may induce endothelial cell dysfunc-tion (43,66,67), whereas DLL4 blockade has been reported to decrease the frequency of cancer stem cells (15) and can disrupt tumor vasculature function by promoting nonfunctional angiogen-esis (10,11). The use of the two treatment modalities together may thus result in a double-pronged attack against tumor growth.

The possibility of vasculature targeting after ionizing radiation has been elegantly demonstrated by Martin Brown’s group (34,35). They used a high single dose of ionizing (15 Gy) that was sufficient to ablate endothelial cells within the irradiated field and thereby prevent subsequent tumor angiogenesis. In this case, vasculogen-esis mediated by the recruitment of bone marrow–derived cells (mainly CD11b-positive cells) was required before tumor regrowth could occur (34,35). They also showed that administration of a CD11b blocking monoclonal antibody to mice interferes with recruitment of CD11b-positive myelomonocytes, which delay tumor regrowth following an ablative dose of tumor irradiation (34). Recently, two groups have shown that bone marrow–derived cells are recruited via stromal cell–derived factor-1 (SDF-1) and that pharmacological blockade of the SDF-1 receptor C-X-C che-mokine receptor type 4 interferes with tumor regrowth following tumor irradiation (68,69). It is not yet known whether a lower dose of ionizing radiation, such as 5 Gy, also induces recruitment of CD11b-positive cells to the tumor because tumor angiogenesis is not ablated at this dose of ionizing radiation (and hence, vasculo-genesis is not essential for tumor growth). It is interesting that there is evidence that DLL4–Notch signaling is involved in regu-lating the infiltration of immune cells in nonmalignant inflamma-tory states, such as macrophages in atherosclerotic plaques (70), leukocytes in ischemic muscle (71), and mononuclear cells in spinal cord following viral-induced demyelination (72). Thus, it will be important to determine if DLL4–Notch blockade influences CD11b-positive myelomonocyte infiltration into tumors, and if so, the relative contribution of these cells to the tumor vasculature in this context.

In considering the clinical implications of our findings, we note that in clinical radiotherapy, single-dose ionizing radiation is not commonly given at high enough doses to cause ablation of tumor angiogenesis, with the exception being stereotactic radiosurgery. Thus, in the majority of clinical situations, targeting tumor angiogen-esis could be a useful strategy to improve the efficacy of radiation therapy. This preclinical study used a 5-Gy dose of ionizing radiation,



which is well within the dose range that is commonly used in clinical radiotherapy. For example, single doses of irradiation in the range of 8–10 Gy are often used for treatment of malignant spinal cord compression, bone metastases, or bulky compressive lesions, and our findings suggest that administering Notch inhibitors after irra-diation may improve symptom and tumor control. In addition, we noted no statistically significant weight loss in mice treated with the DLL4 monoclonal antibody alone or in combination with ionizing radiation. In future preclinical studies, we will investigate Notch inhibitors in combination with a fractionated radiotherapy schedule and determine the clinically effective scheduling of the inhibitors in relation to radiotherapy fractions.

This study has several limitations. First, we used subcutaneous heterotopic xenograft tumor models, which may not fully account for the tumor microenvironment and response to treatment (73). Second, our use of an immunocompromised tumor host could theoretically mask the contribution of the immune response to treatment. However, previous investigators have used heterotopic xenografts in immunocompromised hosts and shown the effective-ness of DLL4 blockade therapy on tumor growth and angiogenesis (10,11). In this study, we observed a supra-additive tumor growth delay with the combination treatment in two clinically distinct xenograft tumor models. However, it remains to be determined whether the same combination treatment with the DLL4 mono-clonal antibody and ionizing radiation will be effective in other common tumor types, such as breast and lung cancer. It will also be important to establish the optimal threshold of DLL4 mono-clonal antibody concentration and radiation dose required to max-imize tumor control while ensuring the safety of nearby critical tissues.

The exact mechanisms underlying the ability of ionizing radia-tion and DLL4 monoclonal antibody combination therapy to delay tumor growth remain to be elucidated. It is important to consider that Notch signaling between endothelial cells and tumor cells can alter the tumor microenvironment. For example, expres-sion of the Notch ligands DLL4 or Jagged-1 on tumor cells can trigger Notch signaling on adjacent endothelial cells, thereby al-tering tumor angiogenesis and enhancing tumor growth (13,74,75). Likewise, stromal cell induction of Notch signaling in tumor cells has been implicated in tumor escape from dormancy (76) as well as in tumor intravasation and metastasis (77). Calabrese et al. (78) reported that a perivascular niche is important for maintaining brain tumor stem cells and that disruption of this niche with antiangiogenic agents impairs tumor growth. In addition, tumor explants from glio-blastoma multiforme patients grown in a transwell system showed decreased tumor cell proliferation and self-renewal after treatment with the g-secretase inhibitor N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), which potentiated the effects of radiation; it was postulated that this effect is due to an antiangiogenic effect of Notch blockade with disruption of the vascular niche (40). However, a limitation of an explant model is that important contributors and properties of the tumor microvasculature, such as infiltrating bone marrow–derived cells or cyclical tumor hypoxia, are not present, unlike an in vivo xenograft system. In this study, neither DBZ nor the DLL4 monoclonal antibody modified the response of tumor cells to radiotherapy in vitro; however, in vivo, there was statistically

significant tumor growth delay with Notch blockade plus ion-izing radiation. It is interesting that two groups (79,80) recently demonstrated a role for glioblastoma cancer stem cell differentia-tion into tumor vasculature and that a g-secretase inhibitor blocked differentiation to endothelial cell progenitors (80). Our future work will be directed at using in vivo markers for cancer stem cells (ie, CD133 or aldehyde dehydrogenase 1) and Notch activity (ie, nuclear localized Notch ICD) to explore the effect of Notch inhibitors and ionizing radiation on cancer stem cell fate within the context of glioblastoma and breast cancer.

This study revealed that a DLL4 monoclonal antibody, either alone or in combination with ionizing radiation, was considerably more effective than DBZ in delaying tumor growth. There are several explanations for this finding. Although we observed that inhibition of tumor Notch activity occurred within 24 hours after treatment of mice with DBZ (in the Notch luciferase biolumines-cence experiment), we would not expect Notch inhibition to be sustained throughout the entire dosing interval of 3 days because DBZ has been reported to have low plasma levels due to poor ab-sorption and/or rapid elimination when administered by intraper-itoneal injection (plasma half-life <12 hours) (81). By contrast, the DLL4 monoclonal antibody has a high affinity (Kd in subnanomo-lar range) for both mouse and human DLL4 (S. Cho, unpublished data) and is thus expected to have a substantially longer plasma half-life than DBZ, as the average half-life for human IgG1 anti-bodies in mice has been reported to be about 6–10 days (82–84); indeed, at twice per week dosing, the DLL4 monoclonal antibody remained physiologically active in this study, as evidenced by tumor growth delay. Thus, we would expect the DLL4 monoclo-nal antibody to provide continuous effective blockade of DLL4–Notch signaling, whereas Notch inhibition resulting from treatment with DBZ would be expected to wane between dosing intervals. In addition, global blockade of Notch signaling with a g-secretase inhibitor might be predicted to result in conflicting and opposing effects on angiogenesis, given that Benedito et al. (12) observed opposing effects of Jagged-1 and DLL4 on angiogenesis. The opposing effects of these Notch ligands on vessel density may explain our findings that DBZ did not demonstrate a statistically significant increase in Chalkley vessel counts. This finding is in contrast to the increase in vessel counts found with specific block-ade of DLL4. It is interesting that we noted an increase in tumor cell expression of DLL4 following Notch blockade both in vitro and in vivo; the increased levels of DLL4 could theoretically be blocked more effectively with the DLL4 monoclonal antibody, to prevent DLL4 from participating in Notch signaling. Thus, a shorter plasma half-life as well as potential opposing angiogenic effects from global blockade of Notch signaling by DBZ may explain why DBZ was less effective than DLL4 blockade in pro-moting tumor growth delay. Another advantage of using the DLL4 monoclonal antibody instead of a g-secretase inhibitor is that it avoids dose-limiting gastrointestinal toxicity caused by the conver-sion of proliferative crypt cells into goblet cells (52,81); although we did not observe any statistically significant effects of DBZ on weight loss in this study, we only administered it every third day for a maximum of five doses as this schedule has previously been shown to be safe (61). If more frequent and prolonged dosing had been possible [it would not have been possible because of



gastrointestinal toxicity (52)], we may have observed improved tumor growth delay with DBZ. Successful attempts to reduce gas-trointestinal toxicity resulting from g-secretase inhibitor treatment include concurrent administration of glucocorticoids (85).

The toxicity of g-secretase inhibitors may be avoided by blockade of individual Notch receptors and ligands with antibodies. The use of monoclonal blocking antibodies specific for Notch1 or Notch2 receptors has successfully avoided the dose-limiting gastrointestinal toxicity (goblet cell metaplasia appears to require both Notch1 and Notch2 blockade) while retaining potent antitumor activity (86). Thus, future experiments examining additional specific Notch path-way inhibitors including blocking monoclonal antibodies against Notch1, Notch2, or the Notch ligands Jagged-1 or DLL1 in com-bination with ionizing radiation in these preclinical models will be important. Notably, the xenografts used in this study expressed Jagged-1; thus, the DLL4 monoclonal antibody was not expected to inhibit Jagged-1-triggered Notch signaling. We expected that a more comprehensive inhibition of Notch signaling via DBZ treat-ment would be more effective than the DLL4 monoclonal antibody in delaying tumor growth. However, the antibody was so well toler-ated and could be given to produce continuous blockade vs intermit-tent blockade with tolerable doses of DBZ, which may explain the greater effectiveness of the DLL4 monoclonal antibody in combina-tion with ionizing radiation for delaying tumor growth.

Other studies have investigated the combination of antiangio-genic agents with radiotherapy. Lee et al. (3) treated normoxic and hypoxic LS174T xenograft tumors with an anti-VEGF monoclonal antibody in combination with ionizing radiation (20–40 Gy) and found that the effect on tumor growth delay was additive at best; the exception was seen only with the highest dose of ionizing radiation (40 Gy) in hypoxic tumors. By contrast, using the same xenograft system, we observed supra-additive tumor growth delay following DLL4 blockade and ionizing radi-ation, and this delay occurred at a lower more clinically relevant radiation dose. Given the previously reported findings that DLL4 blockade is able to overcome tumor resistance to anti-VEGF therapy in preclinical models (11,14), it will be interesting to explore this approach of combining Notch inhibition, VEGF in-hibition, and radiotherapy.

To our knowledge, this is the first study to report in vivo find-ings for combination therapy with Notch inhibitors and ionizing radiation in tumor xenograft models. We showed that this combi-nation is an effective and well-tolerated strategy for prolonging tumor growth delay, and we foresee clinically relevant situations where this approach could be used (eg, locally recurrent breast or colorectal cancer, glioblastoma, and head and neck cancer). In conclusion, the use of Notch inhibitors following ionizing radia-tion generates changes in vasculature that result in reduced tumor blood flow and profound tumor necrosis and may be an effective approach to reduce tumor growth or recurrence in patients.

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FundingThis work was supported by the Cancer Research UK/EPSRC Cancer Imaging Centre (BPRSPUO), and Metoxia, EU Framework 7 Grant (RQPJ1). S.K.L.

was supported through a Research Fellowship from the Ontario Institute for Cancer Research through funding provided by the Government of Ontario. This work was also supported by an ASCO Cancer Foundation Young Investigator Award to S.K.L.

NotesThe authors would like to thank Sabira Yameen for her technical assis-tance in tumor sectioning, Dr Sally Hill for tumor cell injections and valuable insight and advice with the xenograft experimental design, Karla Watson and Magda Flieger for their outstanding help with animal work, and MedImmune LLC for kindly providing the anti-DLL4 monoclonal antibody.

S. K. Liu, R. J. Muschel, and A. L. Harris conceived and designed the ex-periments. S. K. Liu performed the experiments, analyzed data, and wrote the article. R. J. Muschel and A. L. Harris interpreted data, provided invaluable intellectual input, oversaw this study, and provided valuable revisions for the article. E. Fokas performed the ultrasound contrast microbubble experiments and analyzed the associated data. J. Beech performed the FaDu xenograft exper-iment. S. A. S. Bham performed the immunohistochemistry and some associated data analysis. J. Im provided technical assistance and expertise with the confocal imaging. The study sponsors did not have any role in the design of the study, the collection, analysis or interpretation of the data, the writing of the article, or the decision to submit the article for publication.

R. J. Muschel and A. L. Harris shared senior authorship.S. K. Liu is a Research Fellow of The Terry Fox Foundation through

an award from the National Cancer Institute of Canada (award #353). Any opinions, findings, and conclusions expressed in this material are those of the authors and do not necessarily reflect those of the American Society of Clinical Oncology or The ASCO Cancer Foundation.

Affiliations of authors: Gray Institute for Radiation Oncology & Biology, Radiobiology Research Institute, University of Oxford, Oxford, UK (SKL, SASB, EF, JB, JI, RJM); Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK (SKL, SASB, ALH); Department of Pre-Clinical Oncology, MedImmune, Gaithersburg, MD (SC).


Delta-Like Ligand 4–Notch Blockade and Tumor Radiation Response

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