Evaluating the Expression of Angiogenic Mediators in a Mouse Model of Tumor Metastasis

Solid tumors typically require angiogenesis, the development of new blood vessels, for growth and metastasis. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating receptors on host endothelial cells. One such receptor, Flt-1, occurs as either a membrane bound or a secreted form (sFlt-1) that can inhibit angiogenic signaling. Previous studies have shown that variation in mRNA expression of VEGF and its receptors KDR, sFlt-1 and Flt-1 occurs in pathological angiogenesis, i.e. metastatic tumorigenesis. We hypothesize that the ratio of sFlt-1:Flt-1 mRNA will be altered in the presence of solid tumors. The objective of this study was to evaluate the expression of sFlt-1 and Flt-1 mRNAs in a mouse metastatic tumor model using CT26.CL25 cells. CT26.CL25 cells are VEGF-producing murine colon carcinoma cells transfected with the lacZ gene, which expresses B-galactosidase activity. These cells, injected intravenously, form tumor nodules in the lung. A pilot study revealed development of lung nodules in mice nine days after intravenous injection with 105 cells. In a second study, twenty-five 10- week-old female Balb/c mice were injected intravenously, via tail vein, with 2 x 105cells, and fifteen with vehicle control. Lung nodules developed in all mice injected with cells. Tissues were harvested by routine necropsy and either formalin-fixed for routine histology/histochemistry or stored for quantitative RT-PCR (QPCR) analysis of gene expression. Under microscopic evaluation, sections of lungs stained with Hematoxylin & Eosin (H&E) revealed nodules composed of polygonal neoplastic cells. cDNA from lungs (14 tumor-bearing, 10 controls) and cultured CT26.CL25 cells was analyzed by QPCR using primers and TaqMan probes directed against sFlt-1, Flt-1, KDR, VEGFA, PlGF (Placental Growth Factor), Angiotensin Converting Enzyme (ACE), 18S ribosomal RNA and neoR (neomycin phosphotransferase). We observed an increased sFlt-1:Flt-1 ratio in tumor-bearing versus control lungs, suggesting that tumor-derived signals may influence sFlt-1 and Flt-1 expression differentially. Additionally, there was increased expression of Flt-1, sFlt-1 and KDR in tumors versus controls, but not in VEGF expression in tumors versus controls. Interestingly, expression of PlGF was increased in tumors versus controls, suggesting its role as an enhancer of tumor progression in the presence of other angiogenic factors. Together, these findings indicate that solid tumor angiogenesis results from an intricate balance of various angiogenic factors.