The initiation of PDGF-driven gliomas (PDG) with RCAS-hPDGF-B-HA in adult mice has been previously described (8 (link), 13 (link), 58 (link)). Briefly, Ntv-a;Ink4a/Arf−/− mice were fully anesthetized with ketamine/xylazine prior to surgery. Pain management included a 50 μl subcutaneous injection of bupivacaine (0.25%) at the surgical site prior to surgery, and an intraperitoneal injection of buprenorphine immediately following surgery. Mice were intracranially injected with DF-1:RCAS-hPDGF-B-HA cells (200,000 cells/1 μl) between 5–6 weeks of age using a fixed stereotactic apparatus (Stoelting). Injections were made into the right frontal cortex, approximately 1.5 mm lateral and 1 mm caudal from bregma, and at a depth of 2 mm into the subventricular zone (SVZ). In this model, injection into the SVZ induces tumors with low latency (4–5 weeks), 100% penetrance, and histological features characteristic of patient GBM including microvascular proliferation and pseudopalisading necrosis (13 (link)). The incision was sealed using Vetbond tissue adhesive (3M). Tumors were imaged by MRI after 5 weeks, and drug intervention was initiated for tumors ≥2 mm3. A total of 90 animals were treated in long-term experiments with BLZ945 alone, which represented 5 independent cohorts. These data are compiled and presented in Fig. 1D , 2E , and 7G (red lines).
Fixed stereotactic apparatus
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The Fixed Stereotactic Apparatus is a precision instrument designed for positioning and stabilizing biological samples during research and experimentation. It provides a stable, adjustable platform to securely hold specimens in a fixed orientation. The apparatus enables accurate and reproducible measurements and observations.
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4 protocols using fixed stereotactic apparatus
1
PDGF-driven Glioma Induction in Mice
2
BBB Permeability Assessment in Mice
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The alteration of the BBB was determined by following procedures previously described by Manaenko et al. [40 (link)]. 6–8 weeks old athymic/nude mice were intracraneally injected with U251-TGL and TS543-TGL cells using a fixed stereotactic apparatus (Stoelting) and monitored for brain tumor development as described above for PET imaging. Littermates were mock injected with 1.5μl of PBS as controls and sacrificed at the same time. Non injected athymic/nude littermates were included as intact brain controls.
To evaluate the BBB status, mice were injected IP with 800 ul of 2% Evan's blue dye (Sigma). One hour after injection, mice were anesthetized and perfused with acidified fixative (1% PFA in 0.05 mM citrate buffer, pH 3.5). Thirty mg of brain tissue were incubated in 500 μl formamide (Sigma) to extract Evan's blue at 60°C overnight. Absorbance was measured at 610 nm and 740 nm on a plate reader (Sinergy 4, BioTek Instruments, USA).
To evaluate the BBB status, mice were injected IP with 800 ul of 2% Evan's blue dye (Sigma). One hour after injection, mice were anesthetized and perfused with acidified fixative (1% PFA in 0.05 mM citrate buffer, pH 3.5). Thirty mg of brain tissue were incubated in 500 μl formamide (Sigma) to extract Evan's blue at 60°C overnight. Absorbance was measured at 610 nm and 740 nm on a plate reader (Sinergy 4, BioTek Instruments, USA).
3
Glioma Induction in Nestin-Tv-a Ink4a/Arf-/- Mice
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The Nestin-Tv-a Ink4a/Arf−/− mouse line has been described previously27 (link) and was generously provided by Dr Eric Holland. Briefly, to initiate gliomas, mice at 5-6 weeks of age (males and females) were intracranially injected with DF1 cells harboring the replication-competent avian retrovirus-hPDGF-B-HA virus, using a fixed stereotactic apparatus (Stoelting, Wood Dale, IL, USA) under full anesthesia with ketamine and xylazine. The injection was performed at a depth of 2 mm into the right front cortex, using a coordinate that was 1.5 mm lateral and 1 mm caudal from bregma, as previously described.22 (link), 36
4
Orthotopic Brain Tumor Xenograft Model
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Cell lines for use in vivo were transduced with pHIV-Luc-ZsGreen (a gift from Bryan Welm, Addgene plasmid #39196). Labeled cells were sorted for ZsGreen expression. Cells were implanted into the brains of 6-to-8-week-old female NOD-scid IL2Rγnull mice (Jackson Laboratory), using a fixed stereotactic apparatus (Stoelting). A 1-mm burr hole was drilled into the skull using a microdrill (Harvard Apparatus) at 1.5 mm lateral and 1 mm caudal to bregma, and injections were made at a depth of 2 mm. Mice receiving injections of cells harboring inducible shCD24 or shNTC were fed dox-containing chow (doxycycline hyclate diet formulated at 625 mg/kg; Envigo) to induce expression of the hairpin.
All mouse experiments were approved by the Institutional Animal Care and Use Committee at MSKCC. Tumor-bearing mice were monitored for symptoms such as hunching, deteriorating body condition, hydrocephaly, lethargy, and neurological symptoms such as head tilt, rolling, and seizures. Symptomatic animals were sacrificed, and tissues were obtained at this time. Animals that died of unrelated causes were excluded from the study. Pathologists aided in the examination and scoring of slides: Dr. Sebastien Monette (general assessment), Dr. Tejus Bale (tumor scoring).
All mouse experiments were approved by the Institutional Animal Care and Use Committee at MSKCC. Tumor-bearing mice were monitored for symptoms such as hunching, deteriorating body condition, hydrocephaly, lethargy, and neurological symptoms such as head tilt, rolling, and seizures. Symptomatic animals were sacrificed, and tissues were obtained at this time. Animals that died of unrelated causes were excluded from the study. Pathologists aided in the examination and scoring of slides: Dr. Sebastien Monette (general assessment), Dr. Tejus Bale (tumor scoring).
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