Fig. 2: In vivo efficacy of 1F7 and 4B3 mAbs on tumor growth, survival and metastatic spread in an orthotopic PC-3-luciferase prostate cancer model.

a, b Western blot analysis of EphA receptor activation and AKT inhibition after 20 min-stimulation with ephrin-A1-Fc (efnA1-Fc), clustered ephrin-A1-Fc (cl-efnA1-Fc) and 1F7 and 4B3 alone, in combination and clustered (cl) as indicated. 1F7 blocks efnA1-mediated EphA2 pathway activation (a), while clustered 4B3 induces EphA2 pathway activation (b). β-actin is the loading control. Bar graphs in (b) show the quantification of the relative signal intensities of phospho-EphA and phospho-AKT, with the bars representing the means ± SE (n = 3 independent experiments). c Schematic of experimental design with exemplary histological and macroscopic images (collage of two images to capture the full view) of the orthotopic PC-3 prostate cancer model. NRG mice were orthotopically injected with 2.5 × 10⁵ PC-3-luciferase cells. From day 10, mice received thrice weekly intraperitoneal injections of PBS vehicle control or 1F7 or 4B3 mAbs (8 mg/kg) until endpoint. An IgG isotype control group was included as additional control in later experiments. Tumor burden was imaged weekly by intravital bioluminescence imaging. Tumor (T), bladder (Bl) and seminal vesicles (SV). d Representative intravital bioluminescence images of PC-3-luciferase tumor signals 6 weeks after engraftment showing augmented tumor burden in the 1F7 treatment group. e Chart shows luciferase bioluminescence total flux (pixel/s) over time. Mean ± SE (n = 15 mice per group, pooled from three experiments). Mixed model analysis of log-transformed bioluminescence total flux demonstrated a significant difference between 1F7 and PBS groups over time, which was determined by calculating the fixed effect parameter estimate of treatment (1F7-PBS) × time. f Kaplan–Meier survival analysis (n = 5 mice/group). *p < 0.05 (log-rank (Mantel–Cox) test for pairwise comparisons). g Representative lung ex vivo bioluminescence images of lung metastatic burden 7 weeks after orthotopic PC-3-luciferase engraftment. The box and whisker plot shows the log-scaled median, interquartile range ± maximum/minimum values of ex vivo bioluminescence total flux (pixel/s) of excised lungs; Individual data points represent signals of individual lungs. Data were pooled from three independent animal experiments, except for the isotype control, which had only been included in two of the three experiments. n = 14 lungs (PBS), n = 13 (IgG), n = 12 (1F7), n = 13 (4B3). While lungs from 4B3-treated animals showed a trend towards lower values, one-way ANOVA of log-transformed data with Sidak’s post hoc-test for multiple comparisons found no significant differences due to large variances within each group. h Examples of H&E-stained lung sections comparing metastatic spread from the orthotopic tumor to the lung at 7 weeks after engraftment. Tumor nodules are outlined in blue.