Analysis of GFP expression in tumor endothelial cells and BMD-ECs by fluorescence flow cytometry and microscopy in 2 GFP-transgenic mouse models: Tie2-2Gfp and Actb-Gfp. (A) Identification of tumor endothelial cells as CD31^brightCD45^– in single-cell suspensions of lung carcinoma tissue from WT/Actb-Gfp-BMT mice (left plot). The surface phenotype of these lung tumor endothelial cells was Tie2⁺Sca1⁺CD133^–, and the vast majority of them were GFP^– (ie, local vessel–derived). In red, isotype-matched IgG staining; for GFP, the control represents cell suspensions of tumors grown in wild-type mice. (B) Identification of tumor endothelial cells (CD31^brightCD45^–) in single-cell suspensions of mammary carcinoma tissue from Tie2-Gfp mice (left plot). All the Tie2⁺ cells (identified by detecting the Gfp reporter gene using flow cytometry) were also positive for CD31 (right plot). (C-F) Representative images of histochemical analysis of GFP⁺ cells in functional tumor vessels after perfusion staining using tomato lectin (shown in red; blue represents DAPI nuclear staining). (C) Mammary carcinoma vessels in Tie2-Gfp mice after BMT from nontransgenic FVB donors; most of the blood-perfused endothelial lining was composed of locally derived GFP⁺ cells. (D) Spontaneous metastasis of lung carcinoma after primary tumor resection in a WT/Tie2-Gfp-BMT mouse; BMD-ECs were detected in larger diameter-size blood vessels. (E) The new vessels formed shortly after mammary tumor-cell implantation under the pial surface of the brain in a WT/Tie2-Gfp-BMT mouse contained a high proportion of BMD-ECs. (F) Spontaneous squamous cell carcinoma vessels in a WT/Tie2-Gfp-BMT mouse; BMD-ECs were occasionally detected in perfused blood vessels (arrow). Functional endothelial cells in C-F were identified by Texas-Red–labeled streptavidin (Molecular Probes, Eugene, OR) staining of biotinylated-lypopersicon lectin (Vector Labs, Burlingame, CA) infused in the mice prior to sacrifice, and were imaged using a BX61WI confocal microscope (Olympus, Tokyo, Japan). The images were captured using Olympus FluoView software and were analyzed using Adobe Photoshop CS2 software (Adobe Systems, San Jose, CA). Images are 423 μm across in panels C, E, and F and 310 μm across in panel D, using Olympus 40×/1.00 NA (panels C, E, and F) and 60×/1.35 NA (panel D) oil objectives.