Delta-like ligand 4-notch blockade and tumor radiation response.
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 γ-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 extensive tumor necrosis in LS174T xenografts and enhanced tumor growth delay in FaDu xenografts.


CONCLUSIONS
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.
DPhil, Molecular Oncology Unit, The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
Stanley K
SK
Liu
Saif A S
SA
Bham
Emmanouil
E
Fokas
John
J
Beech
Jaehong
J
Im
Song
S
Cho
Adrian L
AL
Harris
Ruth J
RJ
Muschel
D048868:Adaptor Proteins, Signal Transducing; D000230:Adenocarcinoma / Q000098:blood supply / Q000378:metabolism / Q000532:radiotherapy; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001783:Blood Flow Velocity; D002135:Calcium-Binding Proteins; D002294:Carcinoma, Squamous Cell / Q000098:blood supply / Q000378:metabolism / Q000532:radiotherapy; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000532:radiotherapy; D003131:Combined Modality Therapy; D003984:Dibenzazepines / Q000494:pharmacology*; D006801:Humans; D007012:Hypopharyngeal Neoplasms / Q000378:metabolism / Q000532:radiotherapy; D015151:Immunoblotting; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology*; D008163:Luminescent Measurements; D051379:Mice; D008819:Mice, Nude; D009336:Necrosis; D009369:Neoplasms / Q000098:blood supply* / Q000378:metabolism / Q000532:radiotherapy*; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000517:prevention & control / Q000532:radiotherapy*; D011839:Radiation, Ionizing; D051880:Receptors, Notch / Q000037:antagonists & inhibitors*; D015398:Signal Transduction; D014183:Transplantation, Heterologous; D018608:Ultrasonography, Doppler
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D002135:Calcium-Binding Proteins; C424126:DLL4 protein, human; D003984:Dibenzazepines; D036341:Intercellular Signaling Peptides and Proteins; D051880:Receptors, Notch; D053829:Amyloid Precursor Protein Secretases; C527817:dibenzazepine