Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: Magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin

Increased expression of vascular endothelial growth factor (VEGF) has been associated with increased lymph node metastases. The aim of this work was to determine whether VEGF-induced hyperpermeability affects peritumor interstitial convection and lymphatic drain, thus linking this growth factor with lymphatic function. Noninvasive imaging of lymphatic function induced by vascular hyperpermeability was achieved by following the distribution of albumin triple-labeled with biotin, fluorescein, and gadolinium-diethylene triamine pentaacetic acid. This contrast material allowed for multimodality imaging using magnetic resonance imaging (MRI), confocal microscopy, and histology. Overexpression of VEGF in C6-pTET-VEGF165 tumors, inoculated in hind limbs of nude mice, elevated vascular permeability, interstitial convection, and lymphatic drain. These were manifested in dynamic MRI measurements by outward flux of the contrast material, the rate of which correlated with tumor volume followed by directional flow toward the popliteal lymph node. Avidin-chase, namely i.v. administration of avidin, was applied for inducing rapid clearance of the intravascular biotinylated contrast material, thus allowing early experimental separation between vascular leak and lymphatic drain. Repeated MRI measurements of the same mice were conducted 48 h after withdrawal of VEGF by addition of tetracycline to the drinking water. VEGF withdrawal decreased tumor blood-plasma volume fraction by 43%, reduced tumor permeability by 75%, and abolished interstitial convection of the contrast material. Histological sections and whole-mount confocal microscopy confirmed VEGF-induced changes in permeability and interstitial accumulation of the contrast material, as well as uptake of the contrast material into peritumor lymphatic vessels. These results revealed a direct link between expression of VEGF165 and peritumor lymphatic drain, thus suggesting a possible role for tumor-derived VEGF in metastatic spread to sentinel lymph nodes.