Transgenic marking before the initiation of tumor angiogenesis resulted in LacZ-positive tumor endothelium. (A) Overview of the experimental strategy: endothelial cells were marked by inducing recombination in end-SCL-Cre-ER^T;R26R (n = 3) mice by 2 weeks of tamoxifen (TAM) treatment (2 mg every 48 hours). In order to ensure the absence of any residual tamoxifen effect during tumor angiogenesis, mice were left untreated for 3 weeks before LLC tumor cells were implanted. (B-C) Tumors were dissected after 2 weeks and the contribution of marked endothelium to tumor vasculature was assessed by LacZ/CD31 costaining: the majority of vessels were LacZ⁺CD31⁺. (C) The arrow points at a CD31⁺ tumor vessel, which is negative for LacZ. (D) Outline of possible options for the origin of tumor endothelium. (Left) Tumor ECs, which are derived from transplanted EPCs of end-SCL-Cre-ER^T;R26R mice, should become LacZ-positive upon differentiation and tamoxifen treatment. (Right) If tumor ECs are derived from pre-existing wild-type recipient endothelium and not from transplanted EPCs, these cells should not be positive for LacZ upon tamoxifen treatment. Original magnifications × 20 (B) and × 40 (C).