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.

[1]  S. Eccles,et al.  Expression of vascular endothelial growth factor family members in head and neck squamous cell carcinoma correlates with lymph node metastasis , 2001, Cancer.

[2]  R K Jain,et al.  Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Folkman.  What is the evidence that tumors are angiogenesis dependent? , 1990, Journal of the National Cancer Institute.

[4]  H. Dvorak,et al.  Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. , 1983, Science.

[5]  S. Hirohashi,et al.  Expression of vascular endothelial growth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[6]  Hisataka Kobayashi,et al.  Comparison of the Chase Effects of Avidin, Streptavidin, Neutravidin, and Avidin‐Ferritin on a Radiolabeled Biotinylated Anti‐tumor Monoclonal Antibody , 1995, Japanese journal of cancer research : Gann.

[7]  Michal Neeman,et al.  In Vivo Prediction of Vascular Susceptibility to Vascular Endothelial Growth Factor Withdrawal Magnetic Resonance Imaging of C6 Rat Glioma in Nude Mice , 1999 .

[8]  E. Rofstad,et al.  Pulmonary and lymph node metastasis is associated with primary tumor interstitial fluid pressure in human melanoma xenografts. , 2002, Cancer research.

[9]  A. Nakagawara,et al.  Expression of vascular endothelial growth factors (VEGF-A/VEGF-1 and VEGF-C/VEGF-2) in postmenopausal uterine endometrial carcinoma. , 2001, Gynecologic oncology.

[10]  G. Martiny-Baron,et al.  Active interaction of human A375 melanoma cells with the lymphatics in vivo , 2000, Histochemistry and Cell Biology.

[11]  Rakesh K Jain,et al.  Lymphatic Metastasis in the Absence of Functional Intratumor Lymphatics , 2002, Science.

[12]  N. van Bruggen,et al.  Assessing tumor angiogenesis using macromolecular MR imaging contrast media , 1997, Journal of magnetic resonance imaging : JMRI.

[13]  Thomas Hawighorst,et al.  Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis , 2001, Nature Medicine.

[14]  N. van Bruggen,et al.  Magnetic resonance imaging detects suppression of tumor vascular permeability after administration of antibody to vascular endothelial growth factor. , 1998, Cancer investigation.

[15]  K. Alitalo,et al.  Lymphatic Vessels as Targets of Tumor Therapy? , 2001, The Journal of experimental medicine.

[16]  R. B. Campbell,et al.  In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy , 2001, Nature Medicine.

[17]  R. Jain,et al.  Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation. , 2000, Cancer research.

[18]  R. Gillies,et al.  Applications of magnetic resonance in model systems: tumor biology and physiology. , 2000, Neoplasia.

[19]  M. Oda,et al.  Increased vascular endothelial growth factor and vascular endothelial growth factor-c and decreased nm23 expression associated with microdissemination in the lymph nodes in stage I non-small cell lung cancer. , 2000, The Journal of thoracic and cardiovascular surgery.

[20]  P. Caliceti,et al.  Pre-targeted locoregional radioimmunotherapy with 90Y-biotin in glioma patients: phase I study and preliminary therapeutic results. , 2001, Cancer biotherapy & radiopharmaceuticals.

[21]  P. Caliceti,et al.  Pretargeted adjuvant radioimmunotherapy with Yttrium-90-biotin in malignant glioma patients: A pilot study , 2002, British Journal of Cancer.

[22]  K. Alitalo,et al.  Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling. , 2002, Journal of the National Cancer Institute.

[23]  R K Jain,et al.  Tumor angiogenesis and interstitial hypertension. , 1996, Cancer research.

[24]  E. Keshet,et al.  Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis , 1992, Nature.

[25]  M. Karkkainen,et al.  Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. , 2001, Cancer research.

[26]  E. Keshet,et al.  Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Michal Neeman,et al.  MRI and fluorescence microscopy of the acute vascular response to VEGF165: vasodilation, hyper‐permeability and lymphatic uptake, followed by rapid inactivation of the growth factor , 2002, NMR in biomedicine.

[28]  D. Pode,et al.  Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. , 1999, The Journal of clinical investigation.

[29]  N. Ferrara,et al.  Analysis of Biological Effects and Signaling Properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2) , 2001, The Journal of Biological Chemistry.

[30]  M. Witte,et al.  Lymphangiogenesis: mechanisms, significance and clinical implications. , 1997, EXS.