In vivo enumeration of bone marrow–derived endothelial cells in TRAMP prostate tumors. (A) Increasing concentrations of isolated TECs were spiked into 1 × 10⁶ CD31⁻ prostate cancer cells (TRAMP C1). CD31⁺ TECs were analyzed using FACS. The minimum number of TECs that could be detected was approximately 100 per 10⁶ tumor cells, or approximately 0.01% of the total cellular pool. (B) FACS analysis of a well-differentiated prostate tumor in TRAMP (24 weeks of age) after bone marrow ablation/engraftment at 8 weeks of age. One-half of each tumor was used for histology and grading; the other half was used for FACS analysis. To enumerate BMDECs, live cells were selected from the forward scatter/side scatter (FSC/SSC) plot. FSC (x-axis) is in the linear scale and all fluorophores are in the log scale. CD45⁺ hematopoietic cells were gated out and the GFP⁺/CD31⁺ population was quantified. Resident or non–bone marrow–derived ECs were defined as CD45⁻/GFP⁻/CD31⁺. A third population (∼ 1%-2%) which was CD45⁻/GFP⁺/CD31⁻ may be bone marrow mesenchymal stem cells. (C) Multiple prostates from wild-type mice and TRAMP mice were analyzed by FACS: WT, 12 to 14 weeks (n = 5); TRAMP, 12 to 14 weeks (n = 5); WT, 24 to 30 weeks (n = 4); TRAMP, 24 to 30 weeks (n = 5). Data were analyzed by analysis of variance (ANOVA) followed by Tukey multiple comparison test. There was no statistical difference in numbers of BMDECs between both groups and both time points. (D) The TRAMP C1 cell line was implanted subcutaneously in C57BL/6J mice 4 weeks after bone marrow ablation/engraftment. After 28 days, individual tumors (n = 5) were analyzed by FACS as described above. (E) Paraffin sections of either prostate tumors (i) or normal prostate (ii) were stained with GFP antibodies. GFP was detected using a horseradish peroxidase–coupled secondary antibody (brown staining). Nuclei were counterstained with hematoxylin (blue). Most GFP⁺ cells had a spherical appearance reminiscent of mononuclear cells, while others were larger and spindle-shaped. Scale bars represent 0.5 mm. (F) A well-differentiated prostate tumor stained with GFP antibodies in addition to antibodies for the blood vessel marker CD31 (i), the pericyte marker αSMA (ii), and the lymphatic endothelial cell marker LYVE-1 (iii). White arrows point to blood vessels. Asterisks mark perivascular GFP⁺ cells and the yellow asterisk marks a LYVE-1⁺/GFP⁺ perivascular cell. Note that erythrocytes autofluoresce within the lumen of some blood vessels. Nuclei were counterstained with DAPI (blue, i-iii). A perivascular positioning of GFP⁺ cells was also observed in a spontaneous lung metastasis in TRAMP mice (iv). The arrow points to a large, erythrocyte-filled tumor blood vessel surrounded by GFP⁺ marrow–derived cells (no counter stain was used). Scale bar represents 100 μm.