Fig 5. Bicistronic RCAS virus expressing both Mycn^T58A and ShhN increases tumor incidence and lethality.

(A) Map of the bicistronic RCAS vector. Mycn* denotes Mycn^T58A. Not to scale. (B) Kaplan-Meier curves of time to tumor-related euthanasia of mice infected with the RCASBP(A)ΔF1’-Mycn^T58A/ShhN bicistronic virus (maroon) compared to a mixture of the RCASBP(A)ΔF1’-Mycn^T58A and RCASBP(A)ΔF1’-ShhN single-gene viruses (blue). The inset table shows the incidence of medulloblastomas in BarTeL mice infected with the bicistronic virus (ShhN/Mycn), the separate single-gene viruses and a mixture of the two single-gene viruses (ShhN + Mycn) derived from the bicistronic plasmid. Mice were euthanized upon development of tumor-related symptoms. (C) H&E stain of a BarTeL bicistronic medulloblastoma. Typical field from an H&E stain of a medulloblastoma arising in the cerebellum of a BarTeL mouse that was injected with the ShhN/Mycn-containing bicistronic virus. Right panel is a magnified view. Bar, 10 μm. (D) Immunohistochemical staining of a representative medulloblastoma (ShhN/Mycn bicistronic virus) with antibodies to the indicated proteins. Luciferase staining demonstrates the border between normal cerebellum (nl, negative) and the tumor (MB; positive). MYCN expression is detected by staining with antibody to the human MYC epitope tag and shows that almost all cells in the tumor express nuclear Mycn^T58A. Ki67 staining shown is at the border of the tumor, with the upper left area being adjacent normal brain. SHH staining is shown in medulloblastoma (MB) compared to the normal (NL) cerebellum, the latter of which demonstrates SHH expression in three Purkinje cells at the border between the molecular layer (upper left in image) and the IGL. Negative control of medulloblastoma was stained with nonspecific IgG primary antibody. Bar, 10 μm. (E) Leptomeningeal dissemination and spread in brains from bicistronic virus-infected mice. Shown are an H&E stain (upper left; note the overlying meninges) and staining with antibodies to luciferase (lower left) and the MYC epitope tag of MYCN^T58A (lower right) of areas in the same leptomeningeal dissemination over the hemisphere. In a different mouse, the upper right panel demonstrates leptomeningeal spread over normal cerebellum, which is contiguous to the main tumor mass a few millimeters away, outside the right lower area of the panel. Bar, 10 μm.