br Length Width br Sigma Aldrich St Louis
Length ∗ Width2
Sigma-Aldrich, St. Louis, MO) or 2% ethanol:1× distilled phosphate buﬀered saline (DPBS) vehicle one week before tumor initiation. These injections continued twice a week for the remainder of the study (11 weeks in total). One week after the first 17β-estradiol or vehicle injection, tumors were initiated by injecting 1 million MCF7 Epoprostenol in 50 μL of 1:1 DPBS:BME (Cultrex Pathclear basement membrane extract Type 3, 14.5 mg/mL; #3632; R&D Systems, Minneapolis, MN) solution into the lactiferous duct of the right fourth mammary gland of the mouse [59–61]. Tumors were measured along two perpendicular axes (length and width) with digital calipers beginning at 2 weeks post-cell injection, and tumor volume was calculated using the following for-
against time and comparisons between vehicle and control mice were made using a repeated-measured analysis of variance (ANOVA). P-va-lues < .05 were considered significant. Tumor measurements were taken once a week for 10 weeks until harvest.
Mice were euthanized at 10 weeks via CO2 inhalation. Tumors were harvested and preserved in 10% neutral buﬀered formalin. Fixed tissues were processed, embedded in paraﬃn, and cut into 3-5 μm sections. Sections were stained with H&E to examine cell morphology [64,105].
In a second study, 5-week old female NSG mice were ovar-iectomized . Two weeks post-operatively, mice were injected with 17β-estradiol (N = 6) or vehicle (N = 6) twice a week for 11 weeks as described above. One week after the first 17β-estradiol or vehicle in-jection, mice were implanted with MCF7 cells in the right fourth mammary gland as described above. Tumor size was measured once a week with digital calipers, and tumor volume was calculated using the established system described above. Tumors grown in ovariectomized mice were harvested after 10 weeks, analyzed with μCT, and preserved in formalin. Histologic sections were stained with H&E and analyzed. Tumor volume and percent growth of tumor were graphed vs. time. Animals in both studies were monitored for signs of estrogen toxicity, such as urinary bladder distension and skin rash.
2.2.2. μCT tumor volume measurements Tumors were harvested and immediately scanned with a Bruker Skyscan 1173 μCT at 55 kV and 70 μA, with a resolution of 560 × 560 pixels with an image pixel size of 40.26 μm, an exposure time of 125 ms and a rotation step of 0.8o . A standard re-construction was conducted on the tumors using the NRecon software version 126.96.36.199 (Kontich, Belgium) with a smoothing kernel of and a beam hardening correction of 20%. μCT reconstructions of the tumors were analyzed using the CTAn software version 188.8.131.52 (Kontich, Belgium). Total tumor volume was measured as the total tissue volume. Calculated tumor volumes as measured by digital calipers and total tumor volumes from μCT scans were compared using a 2-way ANOVA with Bonferroni post-tests with α = 0.05.
Two weeks after subcutaneous implantation of zero-order release 17β-estradiol pellets (0.96 mg/45 days) (Innovative Research of America, Sarasota, FL)  at ‘week 0’, female NSG mice (N = 24) were injected with 1 million RFP-MCF7 cells in the right lactiferous duct of the fourth mammary fat pad. MCF7 tumors were allowed to grow for 5 weeks. At that time, mice were divided into 3 groups (n = 8) and each group was injected intraperitoneally 3 times a week with 0, 25 ng, or 100 ng of 24R,25(OH)2D3 (#BML-DM300-0050; Enzo Life Sciences, Farmington, NY) in a 2% ethanol: DPBS vehicle. Mammary fat pad tumors were measured with calipers throughout the study. Tumor volume and tumor accretion rate (tumor volume over time normalized to original tumor volume) were graphed vs. time. Tumor growth rates from weeks 6–10 were analyzed with a linear regression model for each group (0, 25, and 100 ng of 24R,25(OH)2D3). Animal survival was tracked throughout the study and percent surviving mice vs. length of survival was recorded. Animal survival was plotted on a hazard curve