Viral spread in epithelial tumors in vivo.

A) Scheme of experiment. Immunodeficient CB17-SCID beige mice with pre-established subcutaneous T84 or A549 tumors (∼100 mm3 diameter) were intravenously injected with 2×109 IU of wt-Ad3GFP or mu-Ad3GFP. Fourteen days later mice were sacrificed and tumors harvested. B) Tumor sections were analyzed for GFP and DSG2. Representative sections are shown. The scale bar of all images is 20 µm. C) Tumors were digested with collagenase and dispase, and single cell suspensions were then analyzed for GFP expression by flow cytometry. N = 5. Shown is the percentage of GFP expressing cells and the mean GFP fluorescence intensity. The differences between the two viruses were significant (p<0.01). D) Immunofluorescence analysis of A549 tumor sections. The first (left) panels show GFP expression (green) and staining for the junction marker E-cadherin (red). The second and third panels show E-cadherin and GFP staining individually. The 4th panel shows nuclei stained with DAPI. Representative sections are shown. The staining pattern suggests that junctions around wt-Ad3GFP infected cells are absent while they are visibly present in areas of mu-Ad3GFP transduced cells. For the immunofluorescence experiments, cellular nuclei were counterstained with DAPI (blue).