Ncrnu

Female athymic nude mice (NCr-nu) were purchased from Taconic Farms (Hudson, NY). All mouse studies were approved by the Institutional Animal Care and Use Committee and the animals were cared for in accordance with guidelines set by the American Association for Accreditation of Laboratory Animal Care and the US Public Health Service Policy on Human Care and Use of Laboratory Animals. For therapeutic experiments, ISHIKAWA uterine cancer cells were directly injected into the uterine horn and to create an orthotopic mouse model of uterine cancer as previously described (20 (link)). Forty mice were randomly allocated into 4 treatment groups: control, onapristone (10 mg/kg subcutaneously, dissolving the drug in castor oil containing 10% benzyl benzoate, daily) (22 (link)), trametinib (1 mg/kg orally, dissolving the drug in 30% PEG400/0.5% Tween80/5% propylene glycol, daily) (23 (link)), or onapristone plus trametinib. Treatment was initiated 2 weeks after cancer cell injection into the uterine horn. After 6 weeks of treatment, the mice were sacrificed, and total body weight, tumor location and weight, and number of tumor nodules were recorded. Tumor specimens were preserved in either optimum cutting temperature medium (Miles Inc., Elkhart, IN) (for frozen slides), or fixed in formalin (for paraffin slides) for further analysis.

In compliance with all relevant ethical regulations for animal research under a protocol approved by the Henry Ford Hospital Institutional Animal Care and Use Committee (IACUC)GBM neurosphere cell suspensions were implanted into 8-week old female nude mice (NCRNU, Taconic Farms) as described^{41 (link)}. A minimum of 8 mice were implanted with each neurosphere line. Animals were anesthetized with a mixture of ketamine and xylazine. Dissociated neurosphere cells (3×10⁵) were injected using a Hamilton syringe at a defined intracranial location: AP+1.0, ML+2.5, DV-3.0. Animals were monitored daily by an observer blinded to the group allocation and sacrificed upon first signs of neurological deficit or weight loss greater than 20%. Brains were harvested, placed in a coronal matrix for 2 mm sections, with the first cut across the implant site. Brain sections were alternately frozen in dry ice and embedded in OCT for storage at −80°C, or formalin fixed and paraffin embedded (FFPE).

Athymic nude mice (NCRNU) were obtained from Taconic Biosciences and used when 6–7 weeks old. For intracranial tumor grafts, mice were anesthetized and placed into a stereotactic frame. A stereotactic device with a UltraMicroPump (UMP3) (World Precision Instruments) was used to inject 5 μl of melanoma cell suspension (10 × 10⁴ cells for A375P control and PLEKHA5 overexpressing clone, and 4 × 10⁴ cells for all other cell lines) into the striatum (2 mm lateral and 0.5 mm posterior to the bregma and 3 mm below the dura). Tumor growth was monitored weekly by bioluminescence imaging using IVIS Spectrum. Mice were euthanized at the respective end points in a carbon dioxide chamber and brains were dissected for analysis. Subcutaneous tumors were established by inoculation of cells in 100μl of PBS:Matrigel mixture (1:1 ratio) into the flanks of mice (4×10⁵ cells/flank, n = 4–8 per group). Mice received subcutaneous flank xenografts at 2 different sites. Palpable tumors formed within 3 weeks. Tumor growth was measured three times a week using a digital caliper and tumor size was estimated using the ellipsoid volume calculation formula. Tumor growth experiments were repeated two times. All work was done in accordance with the Yale Institutional Animal Care and Use Committee.

Female athymic nude (NCr-nu) were purchased from Taconic Farms, Inc. (Rockville, MD). The development and characterization of the orthotopic mouse model of ovarian cancer has been previously described. (34 (link)) SKOV3-IP1 (1 × 10⁶ cells/mouse), A2780 (1 × 10⁶ cells/mouse), or HeyA8 (0.25 × 10⁶ cells/mouse) were lifted with trypsin/EDTA, washed with PBS, and re-suspended in 200 μL of Hank’s balanced salt solution (HBSS, Mediatech, Inc. Manassas, VA) and were injected into the peritoneal cavity of female nude mice.