Figure 3 from Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers

IL8 and VEGF signaling sustain AR pathway activation and modulate response to enzalutamide. Cells were treated with anti-IL8 nAb (5 μg/mL), anti-VEGF nAb (10 μg/mL) or the highest concentration of isotype-matched human IgG antibody. A, Effect of anti-IL8 nAb/anti-VEGF nAb on the response of hypoxic or normoxic LNCaP cells to 10 μmol/L Enazlutamide (Enz) over 72 hours. Data shown are mean ± SEM of N = 3 experiments. B, Effect of anti-IL8 nAb and/or anti-VEGF nAb on hypoxia (6 hours)-induced AR and AR-V7 expression in LNCaP and CWRR1 cells. Blots are representative of N = 3 experiments. Equal loading was assessed using GAPDH. Relative expression was determined by densitometry using Image J software. C, Effect of 10 μmol/L E (Enz) with anti-IL8 nAb (5 μg/mL), anti-VEGF nAb (10 μg/mL) or the highest concentration of isotype-matched human IgG antibody on viability of LNCaP cells under normoxia and hypoxia for 72 hours. D, Effect of VEGF (2 ng/mL) or rhIL8 (3 nmol/L) on tubule formation over 10 days. Suramin (20 μmol/L) was included as a negative control. E, Effect of CM harvested from LNCaP cells cultured in hypoxia for 24 hours, in the presence or absence of anti-IL8 nAb and/or anti-VEGF nAb, on tubule formation over 10 days. For both experiments (D and E), number of junctions was measured using AngioSys 2.0 software. Data are mean±SEM of N = 8 fields of view. F, Tumor growth data (N = 5/group), obtained by measuring tumor volume every 2 days for 28 days. Treatment groups were: vehicle-only (VC); Enz (4 mg/kg); Enz (4 mg/kg) + IgG (150 μg/mL); Enz (4 mg/kg) + anti-VEGF nAb (100 μg/mL); and Enz (4 mg/mL) + anti-VEGF (100 μg/mL) and anti-IL8 (50 μg/mL) nAbs. Treatment schematic is shown above graph. Data points represent mean ± SEM. G, Average tumor weights at study completion. Values are mean ± SEM (N = 5/group). For all experiments statistical analysis was carried out using Student two-tailed t test or Mann–Whitney U test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.