0.1 uM in 1.5% DMSO). The plates were returned to 37uC, 5% CO2 for 6 hours. Wells were photographed and tube formation was quantified by counting the number of branch points. The experiment was repeated using human recombinant VEGF (60 ng/ml) (Cat# 293-VE/CF, R&D Systems, Minneapolis, MN) in place of SFRP2. Endothelial cell and breast tumor cell migration assay The migration properties of tacrolimus on MMTV-neu breast tumor cells and 2H11 endothelial cells were determined using a scratch wound assay. MMTV-neu cells were seeded at a concentration of 80,000 cells/well in 96 well-plates in a complete media (RPMI-1640, 20%FBS, 2 mM L-glutamine, 12 mM HEPES, 0.1 mM NEAA, 1 mM Sodium pyruvate, 1% Pen Strep, 50 uM 2-ME, 0.2 U/ml Regular insulin). After 24 hours, cells were starved in RPMI with 1% FBS. 2H11 endothelial cells were seeded at a concentration of 10,000 cells/well in a 96 well plate in DMEM with 10%FBS and mouse recombinant SFRP2 (700 pM). After 24 hours, 2H11 endothelial cells were starved in DMEM with 2% FBS. For both cell lines, 20 hours into starvation a scratch wound was made using a 1 mL pipette tip and the media was changed to DMEM with 5% FBS. Cells were treated with 1.5% DMSO (control), or tacrolimus at a concentration of 10 uM, 1 uM, and 0.1 uM in 1.5% DMSO. The distance of the wound was measured with an ocular micrometer at 0, 12, 18 and 24 hr into treatment. Statistical analyses Statistical differences between treated and control were calculated using a two-tailed student’s T-test, with a p#0.05 being significant. Results are expressed as means 6 standard error of the mean (SEM). Results Tacrolimus inhibits breast tumor growth in vivo To evaluate whether tacrolimus inhibits breast carcinoma growth, we compared MMTV-neu transgenic mice treated with tacrolimus in 20% intralipid (n = 19) at 3 mg/kg/day i.p. for 21 days to control mice that received no treatment (n = 9) or control with 20% intralipid i.p. (n = 6). There was no statistically significant difference in the growth rate between control with no treatment (227651) and control with 20% intralipid (2796101, p = 0.6), demonstrating that the control vehicle did not affect tumor growth. Therefore the control groups were combined for analyses. The mean growth rate (% change in tumor volume) after 21 days was 68625 in tacrolimus treated mice, compared to the combined controls (n = 15) 248648, which is a reduction of 73% (p = 0.003, Fig. 1). When tacrolimus is compared to the no treatment controls the p = 0.01, and to the 20% intralipid controls the p = 0.01. There were no signs of toxicity (i.e., diarrhea, infection, lethargy, or weight loss) after treatment. This demon- strates that tacrolimus inhibits the growth rate of breast carcinoma in vivo. Microvessel density is decreased in tacrolimus treated tumors To show whether decreased tumor growth in tacrolimus treated mice correlates with a decrease in tumor angiogenesis, we performed IHC on paraffin embedded tumors from control (20% intralipid, n = 4) mice and tacrolimus treated mice (n = 4). The number of microvessels per high power field for control tumors was 104613, compared to tacrolimus tumors 2061, Tacrolimus Inhibits Breast Cancer Growth In Vivo p,0.001 (Fig. 2), indicating that tacrolimus decreased tumor angiogenesis in vivo. FKBP12, the binding partner of tacrolimus, is expressed in human and mouse breast tumor endothelium by immunohistochemistry We evaluated the vascular staining of FKBP12 in paraffin embedded human and mouse breast tumors using IHC with antibodies to FKBP12, and found that FKBP12 localized to human invasive ductal carcinomas in 8 of 11 tumors; and mouse MMTV-neu breast tumor endothelium in 8 of 8 tumors (Figure S1). Since the binding partner for tacrolimus is expressed in Figure 3. Beta catenin was not required for SFRP2 induced endothelial tube formation: 2H11 endothelial cells were transfected with siRNA to beta catenin or sham transfected. A) Western blot results of siRNA to beta catenin showed silencing of beta catenin by 80%. The loading control was TATA binding protein TBP antibodies (a nuclear marker). B) 2H11 endothelial cell tube formation assay. Cells were plated in Matrigel as described in ‘‘Material and Methods’’. After 6 hours the number of branch points were counted. There was no significant decrease in de novo or SFRP2 (7 nm) induced tube formation following the silencing of beta catenin. Full-length blots/gels are presented in Figure S2A. doi:10.1371/journal.pone.0020412.g003 PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20412