we investigated the effects of the new irreversible EGFR/HER2 TKIs BIBW 2992 36 and BIBW 2669 in combination with irradiation on cell proliferation and clonogenic cell survival in vitro and on tumor growth and tumor growth delay in FaDu xenografts. For the experiments in vitro, BIBW 2992 or BIBW 2669 were dissolved in DMSO at 5 mM and CI-1040 diluted with cell culture medium to the final concentrations of 3, 30, and 300 nM, respectively. Control cultures received DMSO. For experiments in mice, 30 mg BIBW 2992 or 6 mg BIBW 2669 were dissolved in 14.25 ml aqueous 0.5% Natrosol, 0.75 ml 10% acetic acid and 270 mg hydroxypropyl-β-cyclodextrin solution, to the final concentrations

2 mg BIBW 2992 ml–1 and 0.4 mg BIBW 2669 ml–1. BIBW 2992 (20 mg kg–1 body weight) and BIBW 2669 (4 mg kg–1 body weight, later 3 mg kg–1 body weight) were Dasatinib BMS-354825 applied orally. The dose for the in vivo experiments was determined in dose-finding studies (maximum tolerable dose in mice) performed by Boehringer Ingelheim. The dose reduction of BIBW 2669 was performed because median body weight decreased more pronouncedly in the treated animals. The initial weight loss was associated with a reduction in the performance status of the treated animals. At this time, 15 out of 45 animals had already been treated with the higher dose of BIBW 2669 for up to 12 days.
The experiments were performed using 7- to 14-week-old female and male NMRI (nu/nu) mice from the specific pathogen- free breeding facility of the Experimental Center of the Dasatinib 302962-49-8 Medical Faculty Carl Gustav Carus, University of Dresden, Germany. Experiments were approved in accordance with institutional guidelines and the German animal welfare regulations. Animals were kept as described previously [19]. For further

immunosuppression, whole-body irradiation with 4-Gy X-rays (200 kV, 0.5 mm Cu, ~1 Gy min–1) was performed 2 days before tumor transplantation. FaDu (ATCC HTB-43), an undifferentiated human hypopharyngeal cell line [29], was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). FaDuDD is a subline of ATCC HTB-43 [12], which has been used in different laboratories for radiobiological experiments in nude mice and in vitro since the 1980s [40]. In extensive series of quantitative tumor transplantation and of radiation tumor control assays, FaDu tumors evoke no or only a very low level of residual immune reactivity in nude mice [4, 48]. For the experiments, source tumors were cut into small pieces and transplanted subcutaneously into the right

hind leg of anesthetized mice [20]. Histology, lactate dehydrogenase (LDH) isoenzymes, DNA flow cytometry, and microsatellite analysis confirmed human origin of the tumor and constancy of basic biological characteristics. 0.5 mm Cu, dose rate ~1 Gy min–1). Up to five animals were irradiated simultaneously in specially designed jigs. For order Dasatinib treatment, the mice were immobilized in a plastic tube fixed on a Lucite plate. The tumor-bearing leg was held positioned in the irradiation field by a foot holder distal to the tumor. Treatments were started at a tumor diameter of 6 mm. Animals were randomly allocated in the treatment arms. Arm (a): daily oral application of carrier, BIBW 2669 (4 mg kg–1; later 3 mg kg–1) or BIBW 2992