Orthotopic GBM xenograft models

Human GBM8 glioblastoma cells [27] and human E98 glioblastoma cells were lentivirally transduced with a lentivirus vector to stably express the mCherry fluorescent protein and firefly luciferase (Fluc) [28]. GBM8-FM cells were cultured as neurospheres in serum-free medium, supplemented with growth factors (2% of B27 supplement, 1% of N2 supplement, 2 µg/ml heparin, 20 ng/ml recombinant human EGF, 10 ng/ml recombinant human bFGF). E98-FM cells were injected subcutaneously in a donor mouse. When the tumor reached a diameter of 1 cm, the tumor was removed and a single-cell suspension was prepared. The harvested GBM cells were washed once with PBS and concentrated by centrifugation to a concentration of 1 × 10⁵ cells per µl. Mice were stereotactically injected with 5 × 10⁵ tumor cells into the striatum. Intracranial injections were performed under isoflurane anesthesia and systemic and topical analgesia (buprenorphine, 0.1 mg/kg; lidocaine 0.5%). The coordinates used for injections were 0.5 mm X, 2 mm Y, and −3 mm Z from the bregma [29]. Tumor progression was confirmed by Fluc in vivo bioluminescence imaging (BLI) after i.p. injection of d-luciferin (100 mg/kg) and acquiring the photon flux (p/s) using the Xenogen-IVIS Lumina system under isoflurane anesthesia.