S644 I. J. Radiation Oncology d Biology d Physics Volume 78, Number 3, Supplement, 2010

Conclusions: Targeting Bcl-2 prosurvival family members sensitizes breast cancer cells to radiation through activating Bak-ap-optotic pathway. These results support the combination of radiation and ABT 737 as a potential novel therapeutic strategy in breastcancer treatment.Supported by the Breast Cancer Research Foundation.

Author Disclosure: H. Wu, None; D.S. Schiff, None; S. Goyal, None; B.G. Haffty, None.

2986 Proton Therapy Targets Cancer Stem Cells in Treatment-resistant Non-small Cell Lung Cancer

J. Y. Chang, X. Zhang, O. Vassiliev, M. Gillin, R. Mohan

M. D. Anderson Cancer Center, Houston, TX

Purpose/Objective(s): Treatment resistance caused by cancer stem cells (CSCs) is a challenging clinical issue. Targeting CSCsmay improve cancer cure. The relative biological effectiveness (RBE) of protons is assumed to be close to that of photons at 1.1.However, the tissue- and cell-specific RBEs and the molecular mechanisms of proton therapy in treatment-resistant cancer cellssuch as CSCs are not well understood. We hypothesized that protons may be more effective than photons in eliminating CSCs,thereby reducing the probability of recurrence and metastasis.

Materials/Methods: We established 2 chemotherapy-resistant (CR) non-small cell lung cancer (NSCLC) cell lines (H460/CR andA549/CR) by repeatedly treating parental H460 and A549 cells with paclitaxel and 3 radiation-resistant (RR) NSCLC cell lines(H460/RR, A549/RR, and Seg-1/RR) by repeatedly irradiating parental cells to up to 60 Gy in 2-Gy fractions. Both CR andRR cell lines expressed higher percentages of ‘side population’ (SP) cells (i.e., those showing CSC phenotype) than did parentalcells (H460/RR, 15% ± 0.7%; H460/CR, 35% ± 1.1%; H460, 3% ± 0.4%; p\0.05). Enrichment was further enhanced after serialsorting and reanalysis (H460/RR, 35%; H460/CR, 52%) and confirmed by the tumorigenicity of the cells in nude mice after serialdilution. CR or RR CSCs and normal bronchial epithelial (NHBE) cells were then irradiated with the same doses (RBE) of protonsor photons and analyzed for growth, apoptosis, and invasion (metastatic potential). We further measured intracellular concentra-tions of reactive oxygen species (ROS), critical mediators of radiation-induced cell killing, by 2’-7’-dichlorofluorescein diacetatestaining and flow cytometry in parental cells and CSCs before and after photon or proton irradiation.

Results: At the same dose (RBE), protons killed significantly more than did photons (p\0.05), but this effect was not seen inNHBE cells. Protons induced significant increases in apoptosis compared with photon (p\0.05). Protons reduced metastatic po-tential in all cell lines tested, particularly in CSCs, in a dose-dependent manner; by contrast, photon-induced apoptosis was notdose-dependent and photons increased cellular invasion at lower doses (0.5 or 1 Gy). CSCs contained less than half the ROS levelsof the parental cancer cells or the NHBE cells. Notably, protons induced higher ROS levels than photons in CR/RR CSCs (3.3-foldincrease from protons vs. 2.1-fold increase from photons in H460/CR/SP cells), but protons and photons produced equivalent,smaller increases in ROS levels in NHBE cells.

Conclusions: Protons preferentially target CSCs and increase ROS levels in treatment-resistant CSCs to a greater extent than pho-tons for the same dose (RBE), but protons and photons produced equivalent effects in NHBE cells.

Author Disclosure: J.Y. Chang, None; X. Zhang, None; O. Vassiliev, None; M. Gillin, None; R. Mohan, None.

2987 Combining Integrin Inhibition (Cilengitide), EGFR Inhibition (Cetuximab) and Radiation in a Pancreatic

Cancer Model

C. Timke1,2, E. Fritz2, S. Schoelch3,2, F. Roeder1,2, A. Abdollahi1,2, J. Debus1, M. Koch3, S. Goodmann4, P. E. Huber1,2

1University of Heidelberg, Dept. Radiationoncology, Heidelberg, Germany, 2DKFZ German Cancer Research Centre,Heidelberg, Germany, 3University of Heidelberg Dept. Surgery, Heidelberg, Germany, 4Merck KG, Darmstadt, Germany

Purpose/Objective(s): Combination of targeted drugs is considered as one possibility for improving the therapeutic index in can-cer therapy. However, it is far from obvious which targeted drugs can favorably be combined. Combining radiotherapy with eitherthe EGFR antibody Cetuximab or the small molecule integrin inhibitor Cilengitide in dual combinations has shown promise inpreclinical and clinical settings. We analyzed the therapeutic potential of combining all three modalities in a human pancreatic can-cer model in vitro and in vivo.

Materials/Methods: Human dermal microvascular endothelial (HDMEC) and BXPC3 human pancreatic cancer cells were ex-posed to Cilengitide, Cetuximab and radiation alone and in dual and triple combinations with various concentrations and doses.In vitro, proliferation, migration/invasion, clonogenic and tube formation assays were performed and apoptosis was measuredby FACS. Genome wide transcriptomics (44k Agilent platform) was performed along with qRT-PCR and Western for protein anal-ysis. In vivo, in a s.c. BXPC3 model in balb c nude mice the effects of mono, dual and triple treatments with concurrent radiation(5x2Gy) were analyzed in 8 groups by tumor growth, histology and imaging using MRI, CT, and US.

Results: We found that in vitro and in vivo (e.g. tumor growth delay), the anti-endothelial and anti-tumor effects of the triple com-bination consisting of Cetuximab, Cilengitide and radiation was superior to dual combinations, while dual combinations were su-perior to monotherapies. The superiority was evident in cell proliferation, migration, tube formation, clonogenic survival, andapoptosis assays. Supra-additivity was found in isobologram analyses for the Cilengitide and Cetuximab combination in prolifer-ation and clonogenic assays as well as for reducing tumor growth in vivo. Exploring the underlying signaling mechanisms, wefound by transcriptomics in vitro that the Cilengitide drug target (integrinbetaV) is upregulated by radiation and Cetuximab, whichwas confirmed by IHC in vivo. Moreover, Cetuximab suppressed the Cilengitide and radiation induced upregulation of VEGF sig-naling. The strong antiangiogenic effects of the triple combination with a short period of transient vessel normalization in vivo wasevident in SMA/CD31 IHC along with corresponding perfusion analyses of contrast enhanced MRI/CT and US imaging.

Conclusions: Our data suggest that Cilengitide and Cetuximab can favorably be combined in dual combinations and in a triple combi-nation with radiotherapy in a human pancreatic cancer model. The combinatorial benefits can be explained in part in terms of the under-lying signaling mechanisms. Clinical translation for pancreatic carcinoma seems feasible due to the clinical availability of all modalities.

Author Disclosure: C. Timke, None; E. Fritz, None; S. Schoelch, None; F. Roeder, None; A. Abdollahi, None; J. Debus, None; M.Koch, None; S. Goodmann, at Merck KG, A. Employment; P.E. Huber, None.