The antiangiogenic peptide VIAN-c4551 inhibits lung melanoma metastasis in mice by reducing pulmonary vascular permeability

Abstract

Introduction: Cancer cells drive the increase in vascular permeability mediating tumor cell extravasation and metastatic seeding. VIAN-c4551, an antiangiogenic peptide analog of vasoinhibin, inhibits the growth and vascularization of melanoma tumors in mice. Because VIAN-c4551 is a potent inhibitor of vascular permeability, we evaluated whether its antitumor action extended to a reduction in metastasis generation.

Methods: Circulating levels of vascular endothelial growth factor (VEGF), lung vascular permeability, melanoma cell extravasation, and melanoma pulmonary nodules were assessed in C57BL/6J mice intravenously inoculated with murine melanoma B16-F10 cells after acute treatment with VIAN-c4551. VEGF levels, transendothelial electrical resistance, and transendothelial migration in cocultures of B16-F10 cells and endothelial cell monolayers supported the findings.

Results: B16-F10 cells increased circulating VEGF levels and elevated lung vascular permeability 2 hours after inoculation. VIAN-c4551 prevented enhanced vascular permeability and reduced melanoma cell extravasation after 2 hours and the number and size of macroscopic and microscopic melanoma tumors in lungs after 17 days. In vitro, VIAN-c4551 suppressed the B16-F10 cell-induced and VEGF mediated increase in endothelial cell monolayer permeability and the transendothelial migration of B16-F10 cells. No detrimental effect of VIAN-c4551 was observed on hematological, biochemical, and histological parameters after its intravenous administration in mice for 14 days.

Conclusions: These findings support the inhibition of distant vascular permeability for the prevention of tumor metastasis and unveil the anti-vascular permeability factor VIAN-c4551 as a potential and safe therapeutic drug able to prevent metastasis generation by lowering the extravasation of melanoma cells.

Fig 1. B16-F10 cells stimulate lung vascular permeability.

(a) Timeline of the evaluation of pulmonary vascular permeability after intravenous inoculation of B16-F10 cells. (b) Photographs of representative lungs showing the accumulation of Evans blue-stained albumin at different times post-B16-F10 cell delivery. (c) Quantification of Evans blue-stained albumin as index of lung vascular permeability. Data from 2 independent experiments (n = 4) is graphed using a box and a whisker plot; the box frames the interquartile range, the horizontal line indicates the median, and the whiskers the min and max values. The mean is indicated by +. *P = 0.02, **P = 0.01, ***P < 0.001 (One-way ANOVA, Tukey’s multiple comparison test).

Fig 2. VIAN-c4551 prevents the increase of vascular permeability stimulated by melanoma cells.

(a) Timeline of the experiment: VIAN-c4551 or vehicle (Veh) were i.v. injected 30 minutes before the i.v. delivery of B16-F10 cells or PBS. Pulmonary vascular permeability or VEGF levels were evaluated 2 hours post-tumor cells or PBS. (b) Photographs of representative lungs showing the accumulation of Evans blue-stained albumin as index of pulmonary vascular permeability. (c) Quantification of lung vascular permeability in Veh- and VIAN-c4551-treated mice after i.v. injection of PBS or B16-F10 cell inoculation. Data from 3 independent experiments (n = 9) are graphed in a box and a whisker plot; the box frames the interquartile range, the horizontal line indicates the median, and the whiskers the min and max values. The mean is indicated by +. ***P < 0.001 (Two-way ANOVA, Sidak’s multiple comparison test). (d) VEGF levels in serum from Veh- and VIAN-c4551-treated mice after injection of PBS or B16-F10 cell inoculation. Values are box and whiskers plot from 2 independent experiments (n = 6), ***P < 0.001 (Two-way ANOVA, Sidak’s multiple comparison test).

Fig 3. A single administration of VIAN-c4551 reduces the number and size of lung melanoma metastases in mice.

(a) Timeline of the experiment: VIAN-c4551 or vehicle (Veh) were injected i.v. 30 minutes before the i.v. inoculation of B16-F10 cells. Lung melanoma nodules were evaluated 17 days post-tumor cell delivery. (b) Representative ventral and dorsal views of the left lung and the four right lobes of a mice treated with Veh or VIAN-c4551. Quantification of the number (c) and size (d) of macroscopic melanoma nodules on the lung surface. (e) Representative lung sections stained with hematoxylin/eosin showing microscopic melanoma nodules (arrows) (scale = 200 μm). Quantification of the number (f) and size (g) of internal microscopic melanoma nodules in lungs. Numbers of nodules are graphed in a box and a whisker plot; the box represents the interquartile range, the horizontal line indicates the median, and the whiskers the min and max values. Size is graphed in a violin plot showing median and quartiles. The mean is indicated by +. Data from 3 independent experiments (n = 13). **P = 0.008, ***P < 0.001 vs Veh (unpaired t-test).

Fig 4. VIAN-c4551 inhibits the melanoma cell-induced permeability of endothelial cell monolayers mediated by VEGF.

(a) Effect of B16-F10 cells on the transendothelial electrical resistance (TEER) of bovine umbilical vein endothelial cell line (BUVEC-E6E7) monolayers in the absence or presence of VIAN-c4551 or anti-VEGF over a 6-hour period. *P = 0.0168, **P = 0.0081 vs Veh. (b) Effect of B16-F10 conditioned media (B16-F10-CM) on TEER of BUVEC-E6E7 monolayers. *P = 0.0182 vs Veh. (Repeated measurements one-way ANOVA, Dunnett’s multiple comparisons test). Values are means ± SD of 3 independent experiments. (c) VEGF levels in the conditioned medium of B16-F10 melanoma cells treated or not with VIAN-c4551 throughout a 24-hour incubation period. Values are means ± SD of 3 independent experiments. (d) Representative images of the actin cytoskeleton distribution in BUVEC-E6E7 monolayers treated or not with B16-F10-CM in the absence or presence of VIAN-c4551 or anti-VEGF (scale = 100 μm). (e) Effect of 3T3 conditioned media (3T3-CM) and B16-F10 conditioned media (B16-F10-CM) on TEER of BUVEC-E6E7 monolayers. *P = 0.0105 vs Veh. (Repeated measurements one-way ANOVA, Dunnett’s multiple comparisons test). (f) Effect of B16-F10 (B16-F10-CM), MDA-MB-231 (MDA-MB-231-CM), and MCF-7 (MCF-7-CM) conditioned media on TEER of human umbilical vein endothelial cell (HUVEC) monolayers. *P < 0.001 vs Veh. (Two-way ANOVA, Dunnett’s multiple comparisons test). Values are means ± SD of 3 independent experiments.

Fig 5. VIAN-c4551 reduces the in vitro transendothelial migration and the lung extravasation of B16-F10 melanoma cells.

(a) Representative images showing the B16-F10 cells expressing GFP that migrated across a confluent endothelial cell monolayer in the presence of vehicle (Veh) or VIAN-c4551. (b) Quantification of the number of migrated B16-F10-GFP cells. Values are box and whiskers plot from 4 independent experiments. The mean is indicated by +. **P = 0.0042 vs Veh (unpaired t-test) (scale = 120 μm). (c) Representative lung sections showing extravasated B16-F10-GFP cells 2 hours after their inoculation (arrows) (scale = 100 μm). (d) GFP mRNA levels in lungs from Veh- and VIAN-c4551-treated mice. Values are box and whiskers plot from 2 independent experiments (n = 6). The mean is indicated by +. **P = 0.0014 vs Veh (unpaired t-test).