Fig 1.
VCN-01 exerts a potent antitumor effect in established and brain tumor stem cell lines.
(A) Cell viability analyses of VCN-01 infected glioma cell lines. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays five days after infection. Data are shown as the percentage (mean ±SD) of cells alive after infection with VCN-01 at the indicated multiplicities of infection (MOIs) relative to cells infected with UV-inactivated VCN-01 (control, equal to 100%).
Fig 2.
VCN-01 replicates efficiently in glioma cell lines in vitro.
(A) The expression amounts of Fiber and E1A proteins in human glioblastoma were determined by Western blot analysis. Note that the levels were viral dose-dependent in all the cell lines. (B) The expression of Fiber and PH20 mRNA in all the cell lines were determined by real time PCR; expression of both genes was correlated, suggesting similar kinetics and dose-dependency. The cell lines used were SNB19, T98G, U87 MG, A172, U373 MG, U251 MG, GSC23 and GSC11. (C) The quantification of VCN-01 viral replication in all the cell lines. Human glioblastoma cells were infected with two dilutions of VCN-01 (1 MOI and 10 MOIs). The virus replicated up to ten-times better in the cells infected with 10 MOIs, which demonstrates that the virus infected and replicated efficiently in glioblastoma cell lines in vitro. (D) The quantification of VCN-01 viral replication in GL261 murine cell line. GL261 cells were infected with two dilutions of VCN-01 (100 MOI and 300 MOIs). As a control for the replication we used cells infected with VCN-01 at 300 MOIs and collected 4 hours after infection, during this time the virus does not have time to replicate indicating the initial viral particles.
Fig 3.
VCN-01 treatment results in a significant antitumor effect in two different in vivo glioma models.
(A) Kaplan-Meier survival curves for overall survival in VCN-01 (107 pfu), VCN (108 pfu) and control (PBS)-treated athymic mice bearing U87 MG and GSC23 intracranial xenografts. Intracranial implantation of U87 MG or GSC23 cells (day 0) was followed by one intratumoral injection (on day 3) of VCN-01 (107 pfu; n = 10), VCN-01 (108 pfu; n = 10), VCN-01 108 pfu UV inactivated (VCN01-UVi; n = 10) or PBS (control; n = 10). The P values were determined by the log-rank test and represent a comparison of survival of VCN-01–treated mice with that of mice treated with PBS. (B) Hematoxilin and eosin staining of cross sections of U87 MG or GSC23 xenografts treated with PBS or VCN-01. (C) Hexon immunostaining of the brains of animals treated with VCN-01 (107 pfu), VCN (108 pfu) or control (PBS). The tissue sections were incubated with anti-hexon antibodies. The magnification of histo-chemical images is ×20. (D) Representative Hyaluronic acid and hexon immunostaining of the brains of animals bearing GSC23 cell line treated with VCN-01 (107 pfu), or control (PBS). The tissue sections were incubated with anti-hyaluronic acid and hexon antibodies. The magnification of histo-chemical images is ×20.