PMC

(A) Tumor volume was significantly decreased in Vav2/3-deficient animals harboring B16 melanomas relative to wild-type control hosts. Data were derived from analysis of 10 independent allografts/condition from 2 independent experiments. (B) There is a significant reduction in tumor cell proliferation in Vav2/3-deficient hosts relative to controls, as assessed by quantification of nuclear PCNA expression (arrowheads indicate PCNA+ nuclei; p<0.05). (C) Tumor cell survival, as scored by TUNEL assay, was not affected in Vav2/3-deficient animals relative to wild-type controls (C, arrowheads indicate TUNEL+ nuclei). (D) Microvascular density, as scored by manual counting von Willebrand factor+ vessels in tumor sections, was reduced in tumors isolated from Vav2/3-deficient host animals relative to controls (arrows indicate vWF+ vessels; p<0.05). Right panel, reduced microvascular density in tumors isolated from Vav2/3-deficient host animals was confirmed by quantification of vWF+ pixel area in tumor sections.

(A) Hematoxylin and eosin stained lung tissue sections confirmed engraftment of LLC cells into the lungs of recipient mice (black arrowheads indicate tumor, red arrowheads indicate adjacent normal lung tissue). Tumor burden, as scored by average lung weight, was significantly decreased in Vav2/3-deficient animals harboring orthotopic LLC tumors relative to wild-type control hosts. Data were derived from analysis of 7 to 8 independent allografts/condition from 2 independent experiments. (B) There is no change in tumor cell proliferation between Vav2/3-deficient hosts and wild-type controls, as assessed by quantification of nuclear PCNA expression (arrowheads indicate PCNA+ nuclei). (C) Tumor cell survival, as scored by TUNEL assay, was significantly reduced in Vav2/3-deficient host animals relative to wild-type hosts (arrowheads indicate TUNEL+ nuclei; p<0.05). (D) Microvascular density, as scored by manual counting von Willebrand factor (vWF)+ vessels in tumor sections, was reduced in tumors isolated from Vav2/3-deficient host animals relative to controls (arrows indicate vWF+ vessels; p<0.05). Right panel, reduced microvascular density in tumors isolated from Vav2/3-deficient host animals was confirmed by quantification of vWF+ pixel area in tumor sections.

B16 melanoma cells were mixed with primary lung microvascular endothelial cells isolated from Vav2/3-deficient mice (KO EC) or wild-type controls (WT EC). Prior to co-transplantation, the endothelial cells were infected with adenoviruses expressing β-galactosidase so as to distinguish them from host endothelium. The cells were suspended in growth factor-reduced Matrigel and injected subcutaneously into the dorsal flank of nude mice. Tumors were harvested after 10 days. (A) Endothelial cells isolated from wild-type or Vav2/3-deficient mice exhibit cobblestone-like morphology in culture and have similar numbers of cells with positive nuclear X-gal staining (arrowheads). (B) There is a significant increase in tumor volume in tumors harboring wild-type endothelial cells relative to tumors harboring no exogenous endothelial cells. Tumor volume was also greater in tumors harboring exogenous wild-type endothelial cells relative to tumors harboring Vav2/3-deficient endothelial cells. (C) Endothelial cells from wild-type mice were abundant and incorporated into tumor blood vessels (arrowheads indicate LacZ+ exogenous endothelial cells; dashed lines indicate tumor mass boundaries). In contrast, Vav2/3-deficient endothelial cells were less abundant, remained isolated and did not incorporate into tumor vessels as efficiently (p<0.05). (D) Co-staining with X-gal (blue nuclei) and CD31 or von Willebrand factor (brown) revealed that exogenous LacZ+ cells retain their endothelial cell character in vivo when incorporating into chimeric vessels (* indicates lumen of chimeric vessel).

(A) Schematic description of co-culture migration assay. Tumor cells harboring a green fluorescent dye are seeded on the lower surface of a transwell membrane and allowed to attach. Primary endothelial cells harboring a red fluorescent dye are placed in the top chamber and migration in response to tumor cells on the lower surface is measured after 5 hours. Loss of Vav2/3 protein expression in Vav2/3-deficient (KO) donor animals versus control (WT) mice was confirmed by immunoblot analysis of whole spleen extracts harvested at the time of primary endothelial cell isolation. (B) Primary lung microvascular endothelial cells isolated from Vav2/3-deficient mice displayed significantly reduced migration in response to Lewis Lung carcinoma and B16 melanoma tumor cells relative to endothelial cells isolated from wild-type control mice (arrowheads indicate endothelial cells that migrated and intercalated into tumor cell monolayer on the lower surface of the chamber). (C) We confirmed ephrin-A1 expression in B16 and LLC tumor cells by immunoblot analysis. Specificity of the antibody was confirmed by probing lysates from ephrin-A1-deficient cardiac tissue (ephrin-A1 KO). In co-culture migration assays, EphA2-deficient endothelial cells (EphA2 KO) significantly reduced migration in response to B16 melanoma cells plated on the lower surface of transwells relative to wild-type control endothelial cells, suggesting that ephrin-A1 actively promotes EphA2-dependent chemotaxis in this tumor cell model.