Heterotopic and Orthotopic Mouse Models of Human GBM

6–8 week old female athymic nude mice from NCI were used for developing U251 xenograft and orthotopic brain tumor models.

For flank tumor xenografts, anesthetized (140 mg/kg ketamine, 10 mg/kg xylazine) mice were subcutaneously injected with 2 × 10⁶ U251 cells in 50 μL DMEM in the lower rear flank. Orthotopic models of human GBM were performed as described previously.⁵ In brief, a drill mounted on a stereotactic rig (Stoelting Inc.) was used to create a 0.4 mm hole 2.0 mm posterior and 1.5 mm lateral to the bregma in the skull above the right cerebral hemisphere in anesthetized mice (140 mg/kg ketamine, 10 mg/kg xylazine). A 30 gauge needle attached to the rig was then used to inject 3×10⁵ U251 cells in 6 μL DMEM at a 0.5 μL/min flow rate 3 mm deep to the skull. The hole was then sealed with bone wax (Ethicon) and the incision sealed with sterile veterinary tissue glue. Mice were sacrificed if they exhibited excessive weight loss (>20%), tumor metastasis, lethargy, or other signs of distress consistent with IACUC standards.

U251 cells used in this experiment were genetically engineered to express firefly luciferase, which enables bioluminescent imaging (BLI) of living tumor cells. As shown by our laboratory and by others, measured luminescence is proportional to cell proliferation, and consequently provides a reliable noninvasive indication of tumor size and progression (Fig. 3(c)).^{37, 38} Mice implanted with brain tumors were serially monitored with BLI every 2–3 days to assess tumor size and progression. Anesthetized mice (2% isofluorane in 100% oxygen) were injected subcutaneously with 100 μL of 50 mg/mL solution of D-luciferin (GoldBio Inc.) in PBS. Continuous imaging was performed over 15–30 minutes using the IVIS Lumina II system (Xenogen) until the peak tumor radiance (photons/second/cm²/steradian) was reached (Fig. 3(d)).

GSMs as theranostic agents for GBM.

Notes: (a) GSMs systemically administered to mice bearing orthotopic GBM xenografts can respond to an applied magnetic field (B₀) for MRI imaging and to X-rays for CT imaging and radiotherapy enhancement. (b) Hematoxylin and Eosin stain of normal brain boundary from a GBM mouse tumor. (c) Bioluminescent imaging (BLI) of a mouse orthotopic GBM. Tumor size was monitored via luciferase-expressing U251 cells reacting with injected luciferin, in units of radiance photons emitted. (d) Characterization of radiance emitted per number of luciferase-expressing U251 cells using BLI and Living Image software.