Mouse glioblastoma xenograft model

Female CD1-nu/nu mice, at 6 weeks of age, were purchased from Charles River (Milan, Italy) and maintained under the guidelines established by our Institution (University of L’Aquila, Medical School and Science and Technology School Board Regulations, complying with the Italian government regulation n.116 January 27 1992 for the use of laboratory animals). All mice received subcutaneous flank injections of 1 × 10⁶ U87MG, U251, and T98G cells representing models for MGMT negative (U87MG, U251MG) and MGMT positive (T98G) cells. Tumor growth was assessed bi-weekly by measuring tumor diameters with a Vernier caliper (length × width). Tumor weight was calculated according to the formula: TW (mg) = tumor volume (mm³) = d ² × D/2, where d and D are the shortest and longest diameters, respectively. The effects of the treatments were examined as previously described [25]. Mice with tumor volumes of 100–150 mm³ were randomized to receive vehicle, bevacizumab (4 mg/kg iv every 4 days), sunitinib (40 mg/kg po qd), or PRX177561 (50 mg/kg po qd), or combinations of bevacizumab and sunitinib with PRX177561. Vehicle was a mixture of hydroxyl-propyl-β-cyclodextrin (HPβCD) at 10% in water (pH7) and propylene-glycol (PG), 25/75 (w/w). Animals were sacrificed by carbon dioxide inhalation, and tumors were subsequently removed surgically. A part of the tumor was directly frozen in liquid nitrogen for protein analysis, and the other part was fixed in paraformaldehyde overnight for immunohistochemical analyses. Indirect immunoperoxidase staining of tumor xenograft samples was performed on paraffin-embedded tissue sections (4 μm). Briefly, sections were incubated with primary antibodies overnight at 4 °C. Next, avidin–biotin assays was done. Mayer’s hematoxylin was used as nuclear counterstaining. Tumor microvessels were counted at ×400 in five arbitrarily selected fields, and the data were presented as number of CD31⁺ microvessels/×100 microscopic field for each group. Ki67 labeling index was determined by counting 500 cells at ×100 and determining the percentage of cells staining positively for Ki67. Apoptosis was measured as the percentage of tunnel positive cells +/− SD measured on five random fields (×400) on immunofluorescence (IF) images. The presence of red cells in tumor tissue and in blood vessels as well as the presence of microthrombi and bleeding zones was demonstrated by Martius yellow-brilliant crystal scarlet blue technique. Tumor hemoglobin levels were also quantified [25].