Figure 5. Systemic I.V. administration of CMP_CDA eliminates established tumors.

a–i) Therapeutic effects of CMP_CDA on CT26 tumors after I.V. administration. a) CT26 tumor-bearing BALB/c mice were treated with CDA+ Mn²⁺ or CMP_CDA, I.V., containing 20 μg CDA and 10 μg Mn²⁺, on days 9, 12, and 15. b) Serum cytokines were measured by ELISA at 6 h post the second dose. c) Antigen-specific T-cell response was analyzed on day 21 by re-stimulating PBMCs with AH1 peptide, followed by IFN-γ ELISPOT assay. d–f) Tumor growth (d–e) and animal survival (f) were monitored over time. g) Survivors re-challenged with CT26 tumor cells on day 145 were monitored for tumor growth and survival. h) CDG-Dy547 either in free form or CMP_CDA was administrated I.V., and after 24 h, CDG-Dy547 signal among immune cells within the TME was analyzed by flow cytometry. i–k) CT26 tumor-bearing mice were treated as in a) and analyzed on day 17 by flow cytometry for the frequency of granulocytic and monocytic MDSCs (i), M1-like and M2-like macrophages (j) within the TME and CD86 expression on DCs in TDLNs (k). l–n) Therapeutic effects of CMP_CDA on B16F10 tumors after I.V. administration. B16F10 tumor-bearing C57BL/6 mice were treated with CDA+ Mn²⁺ or CMP_CDA, I.V., containing 20 μg CDA and 10 μg Mn²⁺, on days 6, 9, and 13 (l), and tumor growth was monitored over time (o–p). Data represent mean ± SEM, from a representative experiment from 2 independent experiments with n = 5 (b–c, g–k), n = 5–7 (m–n), and n = 10 (e–f). Data were analyzed by (b, c, j, k) one-way ANOVA or (f, g, i, n) two-way ANOVA with Bonferroni’s multiple comparisons test, or (h) two-tailed multiple t-tests with Bonferroni-Dunn correction, or (f, g) log-rank (Mantel-Cox) test. Figure 5a and 5l were created using BioRender.com.