Immunological effects of NSC.CRAd-S-pk7 administration in syngeneic glioma orthograft model. (A,B) Plots depicting mean normalized expression of NanoString analysis pathway signature and cell type differentiation scores obtained using RNA isolated from C57/Bl-6 mice bearing CT-2A glioma seven days after treatment with either sham saline, CRAd-S-pk7, or dose-matched NSC-CRAd-S-pk7 (n = 3 per group). (C,D) Tumors were established using 5.0 × 103 GL261 cells/mouse, then treated using either a Day 7 single dose or three doses of 5.0 × 105 HB1.F3.CD_CRAd-S-pk7 cells on days 7, 14, and 21 (n = 2 per group). Twenty-eight days after the first treatment, brains were cryosectioned and processed using standard immunological techniques. Positive cell quantification was automated using ImagePro segmentation software. Results are expressed normalized with respect to Ki-67 positive cells. (E) Representative brain sections stained with F4/80, then 10× scanned images were tiled to showing the relative sizes of GL261 tumor in mice brains that received either sham, one round of NSC.CRAd-S-pk7, or three rounds of NSC.CRAd-S-pk7. Inset enlarged to aid visualization (10×). Sister brain sections stained with anti-Ki-67, anti-PD1, anti-CD3, anti-CD8, and anti-CD4 are shown.