Figure 1. . In vivo differentiation of 005 GSCs and triple combination therapy in 005 GSC-derived GBM model.

(A) 005 glioblastoma stem-like cells (GSCs) differentiate in vivo into cells expressing neuronal marker (NeuN). Mice implanted with 005 GSCs (2 × 10⁴) on day 0, sacrificed on day 24 and formalin-fixed paraffin-embedded brain tumor sections were double stained for GFP⁺ 005 tumor cells (blue; anti-GFP) and NeuN⁺ neuronal cells (red nuclear; anti-NeuN), as described [17]. A representative image obtained at 20× magnification is presented. Cells positive for both GFP and NeuN (GFP⁺NeuN⁺) are indicated by arrows. *nontumor area. Scale bar = 100 μm. (B) Triple combination therapy in 005 GSC-derived tumor model. Mice implanted with 2 × 10⁴ 005 GSCs on day 0 and treated with G47Δ (5 × 10⁵ pfu) or phosphate-buffered saline (PBS) injected intratumorally on day 8 and anti-CTLA-4 (5 mg/kg) and anti-PD-1 (10 mg/kg) or isotype control IgG intraperitoneally on days 8, 11 and 14 (n = 8/group). Median survival of mock group (PBS+IgG) was significantly different from the dual combination (anti-PD-1+anti-CTLA-4; p = 0.001) or triple combination (anti-PD-1+anti-CTLA-4+G47Δ; p = 0.006). However, the triple combination treatment was no different compared with the dual combination treatment (anti-PD-1+anti-CTLA-4; p = 0.76) (log-rank analysis).