Fig 1: AA inhibits in vitro angiogenesis.a Human angiogenesis antibody arrays of serum-free CM of C4-2 cells identify the ligand-regulated secretion levels of angiogenic factors. Cells treated with 1 nM R1881, 100 µM AA, combination of R1881 and AA, or DMSO as solvent control for 72 h and followed by 2 days in serum-free medium without AR ligands. n = 4, two-tailed student’s t test. b Sprouting assays and invasion analysis of non-immortalised primary HUVEC spheroids embedded in collagen matrix and treated with C4-2 CM. C4-2 cells were treated as described above in (a). In addition, 1 nM DHT is used. n = 3, two-tailed student’s t test. Left panel: representative spheroid pictures, right panel: quantification of sprouts per spheroid. c Non-immortalised primary HUVEC spheroids were treated with CM of C4-2 cells that were immune-depleted from ANGPT2 using antibody coupled beads. n = 3, two-tailed student’s t test. d Non-immortalised primary HUVECs spheroids co-treated with C4-2 CM in combination with the indicated integrin inhibitors verified by two additional independent experiments. Two-tailed student’s t test; *p < 0.05. e Summary picture with the proposed molecular mechanism. Androgen-induced neo-angiogenesis by CRPC can be inhibited at three levels: Through (I) blocking AR by AA treatment, that (Ia) competes for androgen binding, (Ib) inhibits nuclear translocation and (Ic) chromatin binding of AR, thus reducing the expression and secretion of ANGPT2, (II) immune-blocking of ANGPT2 and (III) inhibition of ANGPT2 receptors (integrins) by small molecule inhibitors on primary human endothelial cells.