Figure 3.

Induction of apoptosis and anti-angiogenic effects of rapamycin in HNSCC xenograft model in vivo. A, Apoptosis was determined in tumor tissues by the activation of caspase cascade as detected by IF against the cleaved form of caspase-3. Double IF labeling with anti-CD31 and anti-E-cadherin were performed to discriminate between endothelial cells and tumor cells, respectively. Note the overlapping immunodetection of cleaved caspase-3 and tumor cells (lower panels) but not with the endothelial cells (upper panels). B, Quantitative analysis of blood vessel density in tumor tissues obtained from rapamycin treated animals during the course of the treatment. Tissues were collected at the indicated time after initiating the rapamycin treatment and processed for fluorescent labeling of endothelial cells with CD31 antibody. The images from different 8 microscopic fields were taken and the blood vessel density was analyzed by Metamorph 4.0 as described in detail in Materials and Methods. Column, means blood vessel density in arbitrary unit; bars, SE