Extended Data Fig. 12: Immunogenic circRNA vaccines inhibit mouse tumour growth and metastasis.

(a-f) C57BL/6 mice with B16F10 melanoma and BALB/c mice with 4T1 breast cancer were immunized with the circRNAs and their linear versions encapsulated in liposomal delivery systems. (a) Quantification of the percentage of mice with lung metastases (Met). (b) Quantification of the number of metastatic nodules per lung are shown. ***P = 0.0001. (c, d) Flow cytometric analysis for intracellular IFNγ in the spleen, tumour and lung metastases. Percentages of the stained CD8⁺ or CD4⁺ T cells are shown. (e) Quantitation of the spot count per 2 × 10⁵ T cells isolated from tumours and spleens and restimulated with tumour cells is shown, determined by IFNγ ELISpot. (f) Quantitation of the number of pentamer/tetramer⁺ CD8⁺ T cells in tumours per field is shown, evaluated by immunofluorescent co-staining. (g) C57BL/6 mice inoculated with B16F10 melanoma cells transduced with empty vector (circFam53b WT) and circFam53b shRNA-1 (circFam53b KD) were immunized with liposome-encapsulated circFam53b RNA. Quantification of the number of tumour-infiltrating NK cells (NK1.1⁺CD3⁻), inflammatory myeloid cells (CD11b⁺) and circFam53b(187-196)-specific CTLs (circFam53b(187-196)-pentamer⁺ CD8⁺), determined by immunofluorescent staining. (h) C57BL/6 mice inoculated with B16F10 melanoma transduced with shvec, shGFP, circFam53b shRNA-1 and shRNA-2 (shcirc-1 and shcirc-2, respectively) were immunized with circFam53b RNA encapsulated in liposomal delivery systems. Tumour volumes were monitored every 3 days after tumour inoculation (n =  6 mice per group). (i) BALB/c mice inoculated with 4T1 breast cancer cells transduced with shvec, shGFP, circGigyf2 shRNA-1 and shRNA-2 (shcirc-1 and shcirc-2, respectively) were immunized with circGigyf2 RNA encapsulated in liposomal delivery systems. Tumour volumes were monitored every 3 days after tumour inoculation (n =  6 mice per group). (j, k) NOD/SCID mice bearing B16F10 melanoma (j) or 4T1 breast cancer (k) were immunized with circRNAs encapsulated in liposomal delivery systems and the circRNA-encoded peptides along with adjuvant poly(I:C), respectively. Tumour volumes were monitored every 3 days after tumour inoculation. Results are mean ± s.d. of n = 6 (b, j, k), n = 3 (c-e), n = 4 (f, g) independent experiments producing similar results. ****P < 0.0001. P values, compared with mice bearing shvec-transduced tumour (shvec) (h, i), mice without vaccination ((-) (b-f) or no vaccine (j, k)), indicated treatment (g), were determined by two-tailed one-way ANOVA with Dunnett’s multiple-comparisons test (b-f, h-k) or with Tukey’s multiple-comparisons test (g).

Source Data