Use of αv Integrin Linked to Green Fluorescent Protein in Osteosarcoma Cells and Confocal Microscopy to Image Molecular Dynamics During Lung Metastasis in Nude Mice.

BACKGROUND: We report here imaging of the behavior of αv integrin linked to green fluorescent protein (GFP) in human osteosarcoma cells colonizing the lung of nude mice. MATERIALS AND METHODS: 143B osteosarcoma cells expressing αv integrin-GFP were generated by transfection of an αv integrin-GFP fusion-gene vector pCMV-AC- ITGAV-GFP. In order to generate experimental lung metastases, 143B osteosarcoma cells (1×10(6)), stably expressing αv integrin-GFP, were injected intravenously via the tail vein. The osteosarcoma cells were transplanted orthotopically in the tibia of nude mice in order to generate spontaneous metastases. Lungs were harvested and imaged by confocal microscopy within 1 hour. RESULTS: In the experimental lung-metastasis model, extravasating and deformed osteosarcoma cells expressing αv integrin-GFP were observed. Pseudopodia of the osteosarcoma cells contained small puncta of αv integrin-GFP. In early-stage spontaneous lung metastasis, tumor emboli were observed in pulmonary vessels. At high magnification, small αv integrin-GFP puncta were observed in the tumor embolus. In late-stage spontaneous metastasis, tumor emboli were also observed in pulmonary vessels. Invading cancer cells with strong expression of αv integrin-GFP were observed at the margin of the tumor emboli. CONCLUSION: The results of this study demonstrate that molecular dynamics of αv integrin-GFP can be imaged in lung metastasis, which will allow further understanding of the role of αv integrin in this process. The results also suggest a general concept for imaging molecular behavior in vivo.

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins.

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.