Figure 3. Endothelial cell-specific expression of ER_His6 attenuates syngeneic E0771 breast tumor allograft volume, vasculature and hyaluronan levels in Tie2Cre^ERT2;ER^Ki transgenic mice.

(A) Schematic diagram of the Tie2Cre^ERT2;ER^Ki transgene construct before and after tamoxifen-induced Cre recombination. (B) Confocal images of ex vivo aortic rings of Tie2Cre^ERT2;ER^Ki mice injected with i.p. vehicle or tamoxifen and labeled for the endothelial cell marker IB4 (red) and ER_His6, (green). Scale bar, 500 μm (top row) and 200 μm (bottom row, inset). (C) Quantification of mean fluorescence intensity of ER_His6 in the sprouted area of vehicle- or tamoxifen-treated rings; n = 3 mice and 3 biological replicates. (D) Schematic diagram of the time course treatment of i.p. tamoxifen (2 mg per injection) before E0771 implantation. (E) Growth of E0771 murine orthotopic breast tumor allografts in Tie2Cre^ERT2;ER^Ki transgenic mice treated with vehicle (corn oil) or tamoxifen; mean ±SEM, n = 7 and 10 biological replicates for vehicle- and tamoxifen-treated allografts. (F) Immunofluorescence of tumor allografts treated with i.p. injections of vehicle or tamoxifen and labeled for Domain V, ER_His6, HABP and CD31 (all red). Nuclei are blue; scale bar, 10 μm. (G) Quantification of fluorescence intensity of Domain V, ER_His6, HABP and CD31 in vehicle- or tamoxifen-treated allografts. Mean ±SEM, n = 8–10 biological replicates each representing an average of 5–20 technical replicates. Statistical analyses were calculated via two-tailed Student’s t test (*p <0.05, **p <0.01, ***p<0.001).