Fig. 2

Intranasal circRNA vaccine mediates potent anti-tumor response with limited adverse effects. a Timeline of the experiment designed to evaluate the anti-tumor ability of different routes of circRNA vaccine. b Images of lung metastasis tumors at the endpoint in different groups. NT, not-treated group. I.m., intramuscular injection group. I.v., intravenous injection group. I.n., intranasal administration group. n = 4 for NT group. n = 5 for other groups. c The percentage of area occupied by metastatic tumor among various groups at the end point. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. d Timeline of the experiment designed to evaluate the systemic adverse effects of different routes of circRNA vaccine. (n = 4) e, f Serum cytokine release after administrated with the vaccine tested by mouse IFN-α Elisa kit (e) and mouse inflammation kit (f). Data were analyzed by Student’s t test to compare i.n. group with i.v. or i.m. group in e. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test in f. g Blood routine analysis 24 h after the third vaccination. Data were analyzed by Student’s t test to compare i.n. group with i.v. group. h–j Timeline of the experiments to evaluate the anti-tumor ability of intranasal in various models. Mice received with LNP without RNA were considered as mock vaccine group. h Mice were immunized with SIINFEKL-coding circRNA twice, followed by rechallenging B16-OVA-luciferase cells 60 days after vaccination. i Mice were challenged with B16-luciferase cells and received three doses of B16-antigen-coding circRNA vaccine. j Mice were challenged with LL/2 cells and then received three doses of LL/2-antigen-coding circRNA vaccine. k–m Survival curves of the experiments related to h, i and j, respectively. n = 5 for each group. Data were analyzed via Kaplan-Meier analysis. For mice received with vaccination, each dose contained 2.5 μg circRNA. All data are represented as mean ± SEM