Immortalized variant human mammary epithelialcells expressing oncogenic ras are capable of anchorage-independent growth but are not tumorigenic in vivo. (a) Soft agar colony assay. Parental vHMEC (par), vHMEC-vector (vec), vHMEC-ras (ras), vHMEC-ras0.5 (ras0.5), clone 1 (cl-1), and clone 2 (cl-2), which were isolated from the vHMEC-ras0.5 cells, were plated in 35 mm dishes at a concentration of 50,000 cells per dish, in triplicate. After 14 days, colonies were counted manually in eight different fields. The data are presented as the average of the sum of eight different fields counted. (b) Bioluminescence imaging of SCID-Beige mice following orthotopic injection of 1 × 106 (top panel), 4 × 106 (middle panel), or 10 × 106 (bottom panel) vHMEC-ras clone 1 cells expressing GFP-luciferase into the left and right #4 mammary fat pad. Cell growth and survival was monitored weekly by bioluminescence imaging utilizing the Xenogen IVIS imaging system. Representative images of each experiment are shown.