Figure 7.
Combination treatment of mice intracerebrally implanted with GSCs. A) Athymic mice were stereotactically implanted with GBM8 on day 0 and PBS or TMZ (5 mg/kg/d) administered intraperitoneally on days 7–9. Mock (PBS) or 2 × 106 pfu of G47Δ were intratumorally injected on day 8 (n = 8 per group). Combination vs G47Δ only, HR of survival = 7.1, 95% CI = 1.9 to 26.1, P = .003; combination vs TMZ only, TMZ vs Mock, and G47Δ alone vs Mock, P < .001 (two-sided log-rank test). B) γH2AX induction by the combination treatment in vivo. Mice were treated for 3 consecutive days with TMZ (5 mg/kg/d) starting 23 days after GSC (GBM8) implantation (1 × 105 per mouse). G47Δ (2 × 106 pfu) was intratumorally injected on the second day of chemotherapy, and mice were killed 10 hours after the last chemotherapy. Brains were fixed, sectioned, and examined for immunofluorescence (DAPI, blue; γH2AX, green; HSV, red; merge, yellow). Scale bar = 100 μm. C) Athymic mice were stereotactically implanted with BT74 on day 0, and treated as in (A), except for TMZ dose (50 mg/kg/d) and BG (0.3 mg/d) administration on days 7–9 (Mock: n = 8, TMZ only: n = 6, TMZ + BG: n = 6, G47Δ only: n = 8, TMZ + G47Δ: n = 6, TMZ + BG + G47Δ: n = 6). P < .001 (G47Δ only vs Mock, TMZ + BG vs Mock, TMZ + BG + G47Δ vs TMZ + BG), P = .002 (TMZ + BG + G47Δ vs G47Δ only) (two-sided log-rank test). D) γH2AX induction by the combination treatment in vivo (BT74 model: MGMT positive). Mice were treated for 3 consecutive days with TMZ (50 mg/kg/d) + BG (0.3 mg/kg/d) starting 23 days after GSC (BT74) implantation (1 × 105 per mouse). G47Δ (2 × 106 pfu) was intratumorally injected on the second day of chemotherapy, and mice were treated as in (B). Scale bar = 100 μm. BG = O6-benzylguanine; DAPI = 4’,6-diamidino-2-phenylindole; GSC = GBM stem cell; HSV = herpes simplex virus; MGMT = O6-methylguanine methyltransferase; PBS = phosphate buffered saline; TMZ = temozolomide.