Figure 5.

VEGF expression increased the extent of the desmoplastic response observed at both primary and metastatic sites and facilitates metastatic colonization in an experimental metastasis assay. A, cytokeratin immunostaining. Tumor samples collected from tamoxifen-treated mice were immunostained for an epithelial specific cell marker using mixed antibodies against cytokeratin (Keratin Pan Ab-1, NeoMarkers) and counterstained with hematoxylin. Because C9V tumor cells were cytokeratin positive, the cytokeratin-negative areas were considered to be the stromal-enriched compartment within the tumors. The fraction of the cytokeratin-negative stromal compartment within the total area of the microscopic view was analyzed microscopically with the aid of a counting grid and determining the stromal areas over the total areas for 5 primary tumors in each group using three sections per tumor separated by 100 µm. The results are plotted as percentage of stromal area over total counted areas. Increased desmoplasia correlated with doxycycline induction of VEGF expression. B, FGF-2 ELISA assay. Cytosolic tumor FGF-2 was determined by ELISA and normalized by protein concentration. Tumor FGF-2 levels correlated with the induction of tumor VEGF expression. C, experimental metastasis assay. C9V18 cells (0.5 × 10⁶) were directly introduced into the blood circulation by tail vein injection. Doxycycline was provided in the drinking water at 0.2 mg/mL to one of two E₂-supplemented mouse groups starting the day before cell injection. Mouse lung tissues were harvested at 2 h, 4 wk, and 10 wk after the tail vein injection. Paraffin-embedded lung samples were sectioned and immunostained with the cytokeratin antibodies to identify C9V18 cells. Red arrows, disseminated tumor cells; black arrows, fibroblastic stromal cells that surrounded the cytokeratin-positive C9V18 colonies.