FIGURE 3.

Primary tumor development and angiogenesis are susceptible to the effects of cotargeting endothelial NRP1 and NRP2 in multiple cancer models. Inducible, endothelial specific deletion of NRPs, either individually, or in combination was achieved by crossing mice expressing the PDGFb.iCreER promoter of Cre-recombinase to those floxed for NRP1/NRP2. A, Experimental schematic: tamoxifen-induced activation of Cre-recombinase and thus deletion of targets was employed via the following regime. Cre-positive and Cre-negative littermate control mice received intraperitoneal injections of tamoxifen (75 mg/kg bodyweight, 2 mg/mL stock) thrice weekly (Monday, Wednesday, Friday) from D7 to induce Cre-recombinase activity. B16F10 melanoma cells (4 × 10⁵) were implanted subcutaneously into the flank of mice at D0 and allowed to grow until D18. B, B16F10 tumors harvested on D18 removed from Cre-negative and Cre-positive mice. Scale bar, 5 mm. C, Raw tumor volume growth kinetics from 10 days after B16F10 injection to harvest. Tumor volume calculated using the formula: length × width² × 0.52. Error bars show mean ± SEM; N = 2 (n ≥ 10). D, Quantification of tumor volume (mm³; left axis) and weight (g; right axis) measured on D18. Data presented as percentages of the average tumor volume and weight observed in respective littermate controls. Error bars show mean ± SEM; N = 2 (n ≥ 10). E, Raw tumor volume growth change measured between D10 and D18, n ≥ 10. F, Quantification of mean animal weight measured at point of harvest. Data presented as a percentage of the average animal weight observed in respective littermate controls. Error bars show mean ± SEM, N = 2 (n ≥ 10). G, Experimental schematic: Cre-positive and Cre-negative littermate control mice received intraperitoneal injections of tamoxifen (75 mg/kg bodyweight, 2 mg/mL stock) thrice weekly (Monday, Wednesday, Friday) from D7 to induce Cre-recombinase activity. PyMT-BO1 breast cancer cells (1 × 10⁵) were implanted orthotopically into the flank of mice at D0 and allowed to grow until D15. H, PyMT-BO1 tumors harvested on D15 removed from Cre-negative and Cre-positive mice. Scale bar, 5 mm. I, Raw tumor volume growth kinetics from 10 days after PyMT-BO1 injection to harvest. Tumor volume calculated using the formula: length × width² × 0.52. Error bars show mean ± SEM; n ≥ 10. J, Quantification of tumor volume (mm³; left axis) and weight (g; right axis) measured on D15. Data presented as percentages of the average tumor volume and weight observed in respective littermate controls. Error bars show mean ± SEM; n ≥ 10. K, Raw tumor volume growth change measured between D7 and D15, n ≥ 10. L, Quantification of mean animal weight measured at point of harvest. Data presented as a percentage of the average animal weight observed in respective littermate controls. Error bars show mean ± SEM, n ≥ 10. M, Left, Representative tumor sections from Cre-negative and Cre-positive tumors showing endomucin⁺ blood vessels. Right, Confirmation of endothelial-specific target depletion in tumor sections from Cre-negative and Cre-positive tumors. Scale bar = 100 μm. N, Quantification of % blood vessel density per mm² from PyMT-BO1 tumors. Mean quantification performed on 3× ROIs per tumor section, from 1 to 3 sections per tumor. Data presented as a percentage of the average % vessel density observed in their Cre-negative littermate controls. Error bars show mean ± SEM; n ≥ 10. Asterixis indicate significance.