CD34+cells expressing membrane-bound tumour necrosis factor-related apoptosis-inducing ligand exert a potent anti-lymphoma effect by targeting tumour vasculature

CD34+ cells transduced with an adenovirus encoding the human TRAIL gene (CD34-TRAIL+) exert a potent anti-lymphoma effect in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. To investigate the mechanism(s) of action of CD34-TRAIL+ cells, in vivo experiments were performed in subcutaneous tumor-bearing mice to analyze (i) the tumor homing capacity of CD34-TRAIL+ cells and (ii) the effects of CD34-TRAIL+ cells on tumor vasculature. Following a single intravenous injection of CD34-TRAIL+ cells, subcutaneous lymphoid tumors were excised at different time-points and immunostained with an anti-human CD45 antibody. Sections were digitally recorded and the total number of CD45+ cells per tissue section was then counted using the software ImageJ (http://rsb.info.nih.gov/ij/). Tumor homing of CD34-TRAIL+ cells peaked 24 hours after injection when a mean of 200 CD34-TRAIL+ cells was recorded per 1 x 105 tumor cells (0.2% CD45+ cells per 1 x 105 tumor cells). To analyze the effects of CD34-TRAIL+ cells on tumor vasculature, tumor-bearing mice were perfused with sulfo-biotin and tumor endotelial cells (TEC) were then revealed by horseradish peroxydase (HRP)-conjugated streptavidin. As compared with CD34-mock- or soluble (s)TRAIL-treated mice, a 24-hours treatment with CD34-TRAIL+ cells significantly (P <=.001) reduced microvessel density (1850 ± 1139 vs 2227 ± 915 vs 757 ± 562 vessel per 1 x 105 tumor cells, respectively) and increased the thickness of the vessel wall (3.7 ± 1 µm vs 3.4 ± 1 µm vs 6 ± 1 µm, respectively), suggesting that CD34-TRAIL+ cells induce an early vascular disruption leading to a progressive disintegration of the vascular bed. Confocal microscopic imaging of tumor sections double-stained with anti-CD31 and anti-TRAIL-R2 showed that this receptor was expressed by 8 - 12% of large tumor vessels. interestingly, upon treatment with CD34-TRAIL+ cells, but not sTRAIL, TUNEL staining revealed an extensive apoptosis of TEC. Forty-eight hours following injection of CD34-TRAIL+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). These data show that: (i) tumor homing of CD34-TRAIL+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction; (ii) the antitumor effect of CD34-TRAIL+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing.