Enzalutamide treatment directly modulates tumor vasculature. LNCaP in vivo tumors (N = 4/group) treated with enzalutamide (Enz) in the presence or absence of anti-IL8 nAb (50 μg/mL) and/or anti-VEGF nAb (100 μg/mL) for 28 days and measured time-dependent changes in (A) intratumoral oxygenation concentration (mmHg) and (B) tumor vessel density. Values shown are mean±SD. Treatment schematic is illustrated above the graph. Stereological methods were used to analyze the change in vessel density over time in all treatment groups and values were used to calculate percentage area covered by vessels. C, qRT-PCR data demonstrating detectable basal AR expression in LNCaP and HUVEC cells. Data shown are mean±SEM; N = 4 experiments. D, Representative images of prostate tumor stained for (i, ii) AR (20X and 40X), (iii, iv) CD31 (20X and 40X), and (v) hematoxylin and eosin (20X). Endothelial cells (EC) and vessels are marked by black arrows in the prostate tumor. E, Effect of 10 μmol/L Enz on in vitro tubule formation over 10 days. The number of junctions was measured using AngioSys 2.0 software. Data presented are mean±SEM of N = 8 fields of view; N = 4 experiments (F) Effect of 10 μmol/L Enz on viability of HUVEC cells following either 72-hour normoxic or hypoxic conditions. Data shown are mean±SEM; N = 3 experiments. G, Effect of 10 μmol/L Enz on apoptosis in HUVEC cells following either 72-hour normoxic or hypoxic conditions. Data shown are mean±SEM; N = 3 experiments. H, Effect of 10 μmol/L Enz on the colonization of PC3 cells over 5 days in the in ovo assay. Data shown are mean ± SEM of N = 17 embryos for DMSO and N = 23 embryos for the Enz. For all data, control cells were treated with equivalent volume of DMSO and statistically significant differences were determined using a Student two-tailed t test or Mann–Whitney U test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.