Figure 6
AdLCY targeted more CD44- and CD133-positive bladder tumor cells than Ad9OC. (a, b) Detection of cells expressing CD44 and CD133 after viral infection. TCC-SUP cells were infected with AdLCY, Ad9OC or AdLacZ at an MOI of 1 for 5 days. Intensities of CD44 (a) and CD133 (b) expression on surviving cells after viral infection were analyzed by flow cytometry. Black, green and blue lines denote cell percentages after AdLacZ, Ad9OC and AdLCY infection, respectively; red zones represent background fluorescence on the cells. (c) Self-renewal capability of bladder cancer cells after viral infection under hypoxic or normoxic conditions. TCC-SUP cells infected with various adenoviruses at an MOI of 1 were cultured under hypoxic or normoxic conditions for 48 h. Cells were then grown on soft agar, and cell colonies were observed and counted after 24 days. Representative images for colonies grown in soft agar in each group are shown ( × 40 magnification; scale bar=500 μm, upper panel). The number of colonies in each group was quantified (lower panel). Each group was performed in triplicate. Results are representative of three independent experiments. Values are the mean±s.e.m. of the mean. ***P<0.001; **P<0.01; *P<0.05. (d) Immunofluorescence double staining showing targeting of AdLCY to CD133-positive cells in bladder tumors. NOD/SCID mice bearing TCC-SUP tumors (n=5) were intratumorally treated with 5 × 108 PFU of AdLCY, Ad9OC or AdLacZ on days 21, 23 and 25, and then killed on day 31. Representative images in each group are shown ( × 400 magnification; scale bar=50 μm). Magnified images correspond to the boxed areas in the merged images. Colocalization of Ad5 hexon proteins with CD133 is indicated by arrows. The nucleus was counterstained with DAPI.
