The CAM assay for local cell invasion, intravasation, metastasis and tumor (or xenograft) formation was performed as described previously.37, 48, 75 After 3 days of implanting the cells in each egg, lower CAM was harvested and extraembryonic tumors were isolated and weighed. For metastasis assay, the embryonic livers were harvested on day 18 of embryonic growth and analyzed for the presence of tumor cells by quantitative human Alu-specific PCR. Genomic DNA from lower CAM and livers were prepared using Puregene DNA purification system (Qiagen) and quantification of human Alu was performed as described.37, 48, 75 An average of eight eggs per group was used in each experiment.