Figure 7.

SINGER-mediated immune activation for clearance of tumors

(A) Schematic illustration of SINGER-mediated antitumor therapy. C57BL/6 mice bearing H22 tumors received an intravenous injection of PBS, VNP20009, US, VNP20009+US, SINGER-azurin+US, SINGER-PD-L1+US, an equal mixture of SINGER-azurin and SINGER-PD-L1 (SINGER-2), or SINGER-2+US.

(B) Tumor volume measurement in the indicated mouse groups.

(C) Kaplan-Meier curves for mouse survival in (B).

(D) The number of visible metastatic nodules in each group.

(E) Schematic diagram illustrating the experimental procedure for the analysis of immune cells as well as cytokines using flow cytometry and ELISAs. C57BL/6 mice bearing H22 tumors were treated with PBS, VNP20009+US, SINGER-azurin+US, SINGER-PD-L1+US, SINGER-2, and SINGER-2+US.

(F–L) Splenocytes isolated from tumor-bearing mice on day 11 after the indicated treatments were analyzed by flow cytometry for the presence of CD8⁺ T cells (F), IFNγ⁺CD8⁺ T cells (G), IFNγ⁺Foxp3⁻CD4⁺ T cells (H), Foxp3⁺CD4⁺ T cells (I), LAG3⁺CD8⁺ T cells (J), TIM3⁺CD8⁺ T cells (K), and NK cells (L).

(M and N) Tumor tissues isolated from tumor-bearing mice on day 11 after the indicated treatments were analyzed by ELISA for the levels of TNF-α (M) and IL-10 (N).

Data in (B)–(D) and (F)–(N) are represented as means ± SEM; n = 4–6 mice. p values were calculated by one-way ANOVA with Tukey’s post-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, n.s., no significance).

See also Figures S12–S23.