Fig. 1: OV delivered as a second dose does not infect the tumor yet enhances the infection of the initial OV treatment.

a Tumor measurements for CT26^LacZ-bearing untreated animals (n = 6) and mice injected with either one (n = 6) or two doses (n = 8) of VSV (10⁶ PFU, 48 h between treatments). Results are shown as mean ± SEM; two-way ANOVA followed by Sidak’s multiple comparisons test. b Kaplan–Meier survival plots for CT26^LacZ-bearing untreated animals (n = 5) and mice injected with either one (n = 8) or two doses (n = 9) of VSV; log-rank test. c Luminescence intensity for CT26^LacZ tumors after single or repeated VSV-FLUC dosing (10⁶ PFU, 48 h between doses, n = 5). Results are shown as mean with individual values; two-way ANOVA. d. Representative IVM image of infection (green) in CT26^LacZ tumors (red) 24 h after a second VSV-GFP dose (10⁶ PFU, 48 h between treatments). Scale bar, 200 µm. e Virus titers in CT26^LacZ tumors at 72 h post single dose treatment (n = 8) or 24 h post second VSV-GFP dose (10⁶ PFU; n = 5) measured as TCID₅₀/g tissue. Results are shown as mean ± SEM; Mann–Whitney test. f Representative bioluminescence images for the groups shown in (g) at 24 h post first or second VSV-FLUC dose. g Luminescence intensity for CT26^LacZ tumors after first or second VSV-FLUC i.v. injection (10⁶ PFU, 48 h between doses, n = 4). To discriminate between first and second dose infection, VSV-NR was used as the second or first treatment, respectively. Results are shown as mean with individual values; two-way ANOVA. h Representative IVM images of infection (green) in CT26^LacZ tumors (red) 24 h after first or second VSV-GFP dose (10⁶ PFU, 48 h between treatments). To evaluate the impact of the second dose in overall infection, VSV-NR was used as the first treatment in the 2-dose schedule. Scale bar, 200 µm. i Representative bioluminescence images for the groups shown in j at 96/48 h post first/second OV treatment. j Luminescence intensity for CT26^LacZ tumors after VSV-FLUC treatment (10⁶ PFU) with or without VSV-NR injection (10⁶ PFU) coming 48 h later (n = 6). Luminescence intensities for individual tumors are normalized to the mean luminescence intensity in each group at the time of second dosing. Results are shown as mean with individual values; two-way ANOVA.