All i.v. injections were performed in the heat dilated tail vein; the day of tumour implantation was day 0. On the basis of the biodistribution Imatinib molecular weight studies of TNF�� and BAb�CTNF�� complexes (Robert et al, 1996), we decided to inject TNF�� 3h prior to RT (group 6) and BAb�CTNF�� complexes 24h prior to RT (group 7). The mice were weighed twice a week and routinely observed for signs of toxicity throughout the study particularly digestive toxicity because of the local flank irradiation. Statistical analyses The nonparametric Wilcoxon’s signed-rank test was used to compare the surviving fraction between the two groups (RT alone and RT+TNF��). For in vivo experiments, the results were expressed in terms of the time taken for the tumour to reach a volume of 1500mm3.

The Kaplan�CMeier method was used to estimate the median time taken to reach a tumour volume of at least 1500mm3. Differences among treatment groups were tested by the log-rank test. All statistical tests were two-sided with an �� level of 0.05. Data were analysed with software STATA 7.0 (Stata Corporation, College Station, TX, USA). RESULTS TNF�� inhibits BxPC-3 proliferation The cytotoxic effects of increasing concentrations of TNF�� (0.3�C5000Uml?1) on asynchronous, exponentially growing BxPC-3 cells were determined in colony-forming assays. Cell survival followed a dose�Cresponse curve fitted to a four-parameter logistic model as described in Materials and Methods. Cells were killed by concentrations of TNF�� as low as 10Uml?1 (Figure 1A). The LC50, defined as concentration of drug that reduced the cell survival rate to 50% of that of the controls, was 625Uml?1.

Next, BxPC-3 cells were treated with TNF�� (625Uml?1)+BAb (molar ratio of 100:1, 1:1, or 1:100) and plating efficiencies were compared with that obtained with TNF�� alone. No difference in the surviving fraction was observed when BAb was added to TNF�� at the same or lower molar ratio. In contrast, when BAb was added in a 100-fold excess, the surviving fraction of cells exposed to 2Gy was 30% greater than that observed with TNF�� alone, probably due to competition between the anti-TNF�� arm of the BAb in solution and the TNF�� receptor on the cell surface. Figure 1 Dose�Cresponse curves of the effects of TNF�� and irradiation treatment of BxPC-3 cells. (A) Response of BxPC-3 cells to TNF��. Cells were grown in the presence of increasing concentrations of TNF�� (0.

3�C5000Uml … TNF�� enhances radiosensitivity Cell survival following irradiation (Figure 1B) in aerated medium fit a linear quadratic model as described in Materials and Methods. The surviving fraction at 2Gy (SF2) was 0.73 and a D0 (dose of radiation giving 37% survival rate) of 4Gy when irradiation was used alone. Carfilzomib As shown in Figure 2, TNF�� added 12h before RT (H-12) led to a significant decrease of the surviving fractions as compared with those obtained when TNF�� was added at H-1 or H+12 (P=0.02) or when RT was delivered alone.