Figure 3.

Induction of VEGF expression results in tamoxifen-resistant tumor growth and spontaneous lung metastases. A, tumor growth in tamoxifen-treated mice. Four groups of E₂-supplemented mice were inoculated with 10⁷ C9V18 cells on day 0. The day before inoculation, doxycycline was supplied at 0.2 mg/mL in the drinking water for groups 3 and 4. On day 11, when the average tumor size in all groups reached ~200 mm³, the animals in groups 1 and 3 had their E₂ pellet replaced with a tamoxifen pellet (5-mg 60-day release; Innovative Research). B, Ki-67 immunostaining. Paraffin-embedded tumor samples collected from tamoxifen (Tam)-treated xenograft mice were sectioned and immunostained with antibody against Ki-67 (Santa Cruz), a cell proliferation marker. The number of Ki-67–positive cells per microscopic view is approximately doubled when VEGF was induced by doxycycline. Quantitative analysis, as shown in the histogram, was done by counting the number of Ki-67–positive cells per microscopic field. Three microscopic fields (×20 magnification) per tumor and six tumors per group were analyzed. C, macroscopic metastases. X-gal staining of the lungs (top) showed metastatic deposits of LacZ-tagged tumor cells in the tamoxifen-treated tumor-bearing mice that also received doxycycline (group 3). Top left, a mouse lung with a number of metastatic deposits. Top right, an image with higher magnification. Bottom, H&E staining of lung samples confirming the presence of metastatic lesions.