Tamoxifen

TamR tumors were implanted orthotopically (8 mm³ fragment inserted sc into the mammary fat pad) into 65 ovariectomized tamoxifen treated (5 mg/60 days pellet, Innovative Research of America) female Nu/J mice (~6 weeks of age). Tumor volume and body weight were measured 3X weekly until tumors reached ~0.1-0.15 cm³ volume (l x w² x 0.5). Mice were then randomized (n = 8-9) to 4 weekly treatments with 0, 25, 50, 100 or 200 mg/kg fulvestrant injected sc (5% DMSO/95% com oil) with continued tumor measurement and weight monitoring. After 28 days of treatment, animals were euthanized by CO₂ exposure, followed immediately by cardiac puncture for blood collection. Plasma and tumor tissues were cryopreserved for future analysis.

All procedures were approved by the Duke University Institute for Animal Care and Use Committee. tamoxifen stimulated TamR tumors were initiated orthotopically by serial transfer into female NU/NU mice (~6 weeks age, Duke breeding colony). Briefly, ovariectomized recipient mice received no treatment or tamoxifen (tam) treatment via a timed release pellet (5 mg tamoxifen/60 days – Innovative Research of America) implanted subcutaneously. Two days later, TamR tumors (~0.8cm³ volume) were sterilely excised from euthanized tam treated donor mice, diced to ~2mm³ sections and implanted into the axial mammary gland of recipient mice under anesthesia (10g trochar). Tumor growth was measured three times weekly by caliper (tumor volume = (A² × B)/2, where A is the longer axis). When tumor volume reached ~0.15cm³ (~20 days), mice (n = 9–13) were randomized to vehicle or RO 4929097 treatment (10 mg/kg oral gavage in 0.1cc 1% hydroxypropyl cellulose/0.2% tween 80/2.5% DMSO). Tumor growth was monitored over 4 weeks of daily treatment.

When PyMT mice were 4-weeks-old, a 60-day release pellet containing tamoxifen (5 mg/pellet, Innovative Research of America) was subcutaneously implanted on the left dorsal side of the mouse just below the rib cage. Tumors were resected at 12 weeks of age and weighed as a measurement of tumor size.

For establishment of tumors with the TamR subline cells, approximately 5 × 10⁶ MCF7-derived cells were suspended in 100 μL PBS/Matrigel (1:1) and injected s.c. into overiectomized, female BALB/c nu/nu athymic mice (4 week-old, Sprague–Dawley, Harlan). For establishment of tumors with NSD2-overexpressing or control cells, approximately 5 × 10⁶ MCF7-derived cells were suspended in 100 μL PBS/Matrigel (1:1) and injected s.c. into overiectomized, female BALB/c nu/nu athymic mice (4 week-old, Sprague–Dawley, Harlan), which simultaneously received a 60-day, slow release pellet containing 0.72 mg of 17β-estradiol (Innovative Research of America). The mice were ovariectomized before the injection. When the tumor volume was approximately 100 mm³, the estrogen source was removed, and either a tamoxifen-containing pellet or a placebo (Innovative Research of America) was implanted. Six mice were used for each treatment. Tumor growth was monitored every week by calipers with volume calculated using the equation: [½ (length × width2)]. The procedures were approved by the Institutional Animal Care and Use Committee of University of California Davis (protocol No. 17789).