Figure 2

DU145 LN tumors show increased growth, angiogenesis, lymphangiogenesis, and metastasis. (A) Metastasis was measured by K18 staining of lymph nodes from mice bearing DU145 tumor (top left panel) and DU145-LN4 tumor (top right panel). K18-positive (brown color) tumor foci (arrow) were counted as positive incidence of metastasis. Scale bars = 0.5 mm. Tumor vascularization was assessed by CD31 IHC (brown color) of DU145 (middle left panel) and DU145-LN4 (middle right panel) prostate tumors. Increased vascularization was observed in DU145-LN4 tumors relative to DU145 tumors. Scale bars = 100 μm. Lymphangiogenesis was measured by podoplanin staining. Enlarged podoplanin-positive vessels (brown color) were observed in DU145-LN4 orthotopic tumors (lower right panel), compared to DU145 tumors (lower left panel). All sections were counterstained with hematoxylin (blue color). Scale bars = 100 μm. (B) In vitro proliferation assays of the DU145LN sublines indicate similar proliferation rates with slight reduced proliferation of DU145-LN4. 2.5x10³ cells plated/well, absorbance measured using Cyquant dye (Ex = 485 nm). Data in arbitrary fluorescence units x1000. Filled circles: parental DU145, filled triangles: DU145-LN2, empty squares: DU145-LN4. Error bars indicate S.D. of triplicate wells. (C) DU145-LN2 shows increased tumor growth compared to parental DU145 when injected subcutaneously into nude mice. 5x10⁶ cells were injected into the flank. Symbols as above.