Figure 2
Long-term treatment with cabozantinib induces minor resistance. (a) Scheme of long-term treatment of BxPc-3 cells with cabozantinib and establishment of subclones XL-1 to XL-7. BxPc-3 cells were treated seven times with increasing concentrations of cabozantinib (5–10 μ M) over 14 weeks. After each treatment, cells were allowed to recover for 2 weeks before the next treatment with increased concentration was applied. Two weeks after each treatment respective subclones were obtained that were either left untreated for examination of resistance or used for the next treatment round. (b) Parental BxPc-3 cells or the derived subclone XL-7 were treated with 10 μ M cabozantinib. Seventy-two hours later cells were analyzed with a Nikon Eclipse TS100 microscope and × 100 magnification and photographed. (c) Parental BxPc-3 cells or the derived subclones XL-1 to XL-7 were treated with 10 μ M cabozantinib. Seventy-two hours later viability was analyzed by MTT-assay and controls for each cell line were set to 100%. Apoptosis was analyzed by staining with annexinV-FITC and FACS-analysis. The percentage of specific apoptosis was calculated by the formula: 100 × (((apoptosis of treated cells)-(spontaneous apoptosis of control cells))/(100-spontaneous apoptosis of control)) (*p<0.05 compared with control; calculated using a two-sided Student's t test). (d) BxPc-3, XL-5 and XL-7 cells were treated with 10 μ M cabozantinib. Seventy-two hours later cells were cytospinned to glass slides and expression of the proliferation marker Ki67 was detected by immunohistochemistry and photographs are shown. The number of positive cells was quantified in 10 vision fields under × 400 magnification and the means±S.D. are shown in a diagram. (*p<0.05 compared with control) (e) Likewise, c-Met expression was analyzed by western blot analysis or by evaluation of positively-stained cells in immunohistochemistry as described in point (d)
