Figure 5. Induction of CYR61 protein in vitro and in vivo consequent to oncolytic virus (OV) infection.

(a) U343 human glioma cells were infected with hrR3 at a multiplicity of infection of 0.05. The cell lysate and extracellular matrix were harvested and analyzed for CYR61 expression using western blot. Note the significant induction of CYR61 in U343 cells infected with hrR3 relative to uninfected control cells. Asterisk indicates a proteolytic fragment of secreted CYR61 in U343 cells. Proteolytic processing of CYR61 has been described earlier. (b) Intracranial (human U87ΔEGFR glioma cells) and subcutaneous (LN229) tumors generated in athymic nude mice were treated with HSVQ or phosphate-buffered saline (PBS). The animals were killed 24 hours after treatment, and the harvested tumor lysate was analyzed for CYR61 expression using western blot. Note the substantial induction of CYR61 protein in tumors infected with HSVQ. (c) Representative immunofluorescent tumor sections from U87ΔEGFR tumors treated with OV/PBS in vivo. Seven days after tumor cell implantation, athymic nude mice were treated with PBS or hrR3, and killed 3 days after treatment. Sections from the tumor-containing brains were analyzed by immunofluorescence for CYR61 protein expression (red, top). Nuclear [4′,6-diamidino-2-phenylindole (DAPI)] staining (blue, bottom) shows the tumors containing highly nucleated area in the sections. (d) Rats with intracranial tumors (D74/HveC glioma cells) were treated with hrR3 7 days after tumor implantation. Twenty-four hours after OV treatment, cerebrospinal fluid (CSF) and tumors were harvested from the rats, as described in Materials and Methods. The harvested CSF and tumor lysate were analyzed for CYR61 and β-actin, using western blot. Note the increase in CYR61 protein in the CSF of rats with tumors treated with OV relative to tumor-bearing control rats treated with PBS.