GLV-1h68 does not infect endothelial cells. (a) Confocal image showing the morphological intact tumor vasculature (arrowheads) in the GLV-1h68-infected (i.v.) GI-101A tumor tissue 42 days p.i.; the tumor vasculature was labelled with anti-CD31 antibody (red), GLV-1h68-infection was indicated by GFP (green), and nuclei were stained with Hoechst (blue). (b) Systemic perfusion of the vasculature with lectin revealed colocalization (arrowhead) of CD31-positive tumor blood vessels (blue) and biotinylated-Lycopersicum esculentum lectin (red). (c) Viral replication in GI-101A tumor cells, human endothelial (HUVEC) and murine endothelial (2H-11, bEnd.3) cells inoculated with GLV-1h68 at m.o.i. of 0.01. Infected cells were harvested 6, 24, and 48 h after infection. Shown are viral titers of cell monolayers as p.f.u./cell in triplicate samples +/- standard deviations determined by plaque assay. (d) Infection of co-cultures of GI-101A-RFP (red) and HUVEC cells (unlabelled) on Matrigel matrix with GLV-1h68 (green) with m.o.i. 0.5 confirmed the specific infection of tumor cells. The illustration visualized the formation of tube-like structures composed of endothelial cells surrounded by tumor cells on Matrigel matrices. The asterisk (c) indicate a significant difference between experimental groups (** P < 0.01; Student's t test). All images are representative examples. Scale bars represent 75 μm (a) and 300 μm (b).