Figure 3 from Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers
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posted on 2025-11-26, 13:40 authored by Pamela J. Maxwell, Melanie McKechnie, Christopher W. Armstrong, Judith M. Manley, Chee Wee Ong, Jenny Worthington, Ian G. Mills, Daniel B. Longley, James P. Quigley, Amina Zoubeidi, Johann S. de Bono, Elena Deryugina, Melissa J. LaBonte, David J.J. Waugh<p>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. <b>A,</b> 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 <i>N</i> = 3 experiments. <b>B,</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 <i>N</i> = 3 experiments. Equal loading was assessed using GAPDH. Relative expression was determined by densitometry using Image J software. <b>C,</b> 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. <b>D,</b> 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. <b>E,</b> 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 (<b>D</b> and <b>E</b>), number of junctions was measured using AngioSys 2.0 software. Data are mean±SEM of <i>N</i> = 8 fields of view. <b>F,</b> Tumor growth data (<i>N</i> = 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. <b>G,</b> Average tumor weights at study completion. Values are mean ± SEM (<i>N</i> = 5/group). For all experiments statistical analysis was carried out using Student two-tailed <i>t</i> test or Mann–Whitney U test: *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001.</p>
