Intratumor homing of CD34-TRAIL⁺ cells involves SDF-1 and VCAM-1. (A) Forty-eight hours after a single intravenous injection of CD34-TRAIL⁺ cells, NOD/SCID mice were intravenously injected with 0.2 mL of sulfo-NHS-LC-biotin to biotinylate tumor vasculature. AlexaFluor 488–conjugated streptavidin and Alexa 568–conjugated anti-CD45 double staining of cryosections was used to detect tumor vessels (green) and CD34-TRAIL⁺ cells (red), respectively. CD34-TRAIL⁺ cells (arrowheads) infiltrating tumor tissue were found within tumor tissue near blood vessels. Objective lens, original magnification: 1.0 NA oil objective, 40×. (B) Forty-eight hours after a single intravenous injection of CD34-TRAIL⁺ cells (3 × 10⁶ cells/mouse) alone or in combination with anti–VCAM-1 and/or AMD3100, tumor sections were immunohistochemically stained with anti-CD45 monoclonal antibody. Mean (± SD) number of CD34-TRAIL⁺ cells that had homed to tumors was determined by counting CD45⁺ cells per 10⁵ tumor cells. Results of 1 representative experiment are shown. *P = .001, compared with controls. **P < .001, compared with controls. ***P < .001, compared with controls. ^#P < .001, compared with anti–VCAM-1. (C) Analysis of intratumor recruiting signals was carried out on 4-μm cryosections from in vivo biotinylated tumors. Cryosections were stained with AlexaFluor 488–conjugated streptavidin (green) to detect tumor vasculature (i,iv,vii). Cryosections were also stained with anti–VCAM-1 (ii), anti–SDF-1 (v), or anti–TRAIL-R2 (viii) followed by the appropriate AlexaFluor 568–conjugated secondary antibody for indirect detection of the corresponding antigen (red). Merged images demonstrate VCAM-1 (iii), SDF-1 (vi), or TRAIL-R2 (ix) expression by endothelial cells. Objective lens, original magnification: 1.0 NA oil objective, 40×.