Proapoptotic effects of CD34-TRAIL⁺ cells. NOD/SCID mice bearing subcutaneous tumor nodules 10 mm in diameter were randomly assigned to receive CD34-TRAIL⁺ cells, mock-transduced CD34⁺ cells (3 × 10⁶ cells/mouse, intravenously), recombinant sTRAIL (500 μg/mouse, intraperitoneally), or control vehicle. Forty-eight hours after treatment, NOD/SCID mice were intravenously injected with sulfo-NHS-LC-biotin to biotinylate tumor vasculature. (A) Representative confocal images of tumors from untreated and treated animals processed by triple immunofluorescence staining. (i-iv) Cell nuclei were detected in blue by TO-PRO-3. (v-viii) Apoptotic cells were detected in green by TUNEL staining. (ix-xii) Tumor endothelial cells were detected in red by Alexa 568–conjugated streptavidin. (xiii-xvi) After merging of single-color images, apoptotic nuclei (green) were detectable throughout tumor parenchyma after treatment with either sTRAIL or CD34-TRAIL⁺ cells, whereas endothelial cells with apoptotic nuclei (yellow) could be detected only in CD34-TRAIL⁺ cell-treated animals. Objective lens, original magnification: 1.0 NA oil objective, 40×. (B) Percentages of apoptotic cells in tumors from untreated or treated animals were computationally calculated on digitally acquired images (objective lens, original magnification 20×) using ImageJ. At least 3 sections from different animals were analyzed. The boxes extend from the 25th to the 75th percentiles, the lines indicate the median values, and the whiskers indicate the range of values. *P < .001, compared with controls. #P < .001, compared with sTRAIL. (C) Tissue sections were processed using the Proteome Profiler Human Apoptosis array kit to detect apoptosis-related proteins. Array images were collected on x-ray films and scanned for analysis with ImageJ. Data are shown as fold increase over controls. A representative experiment is shown. cf indicates cleaved fragment.