Figure 3.

The intratumoral treatment with BO-112 and the STING agonist DMXAA increases the presence of effector CD8 T cells in the tumor microenvironment in both directly treated and untreated tumors. MC38 tumor-bearing mice received two doses of BO-112 and/or DMXXA following the dose regimen described in figure 1A and tumors and tumor draining lymph nodes (dLN) were collected 24 hours after the last treatment. Experimental group results are color coded as indicated. (A) The weight of tumors is shown for each treatment on day +11. (B) Total number of CD8 T cells per gram of tissue. (C) The ratio between CD8 and Tregs in the tumors is shown. (D) The number of gp70⁺ CD8 T cells is shown per gram of tumor. (E–F) Number of CD4 (D) and CD4 Tregs (E) per gram of tissue. (G) Representative microphotograph of H&E-stained sections from inflamed tissue that surrounds the area where intratumoral BO-112/DMXAA-treated tumors had been located and rejected, with magnification of the indicated region of interest. Scale bars represent 100 µm and 25 µm. (H) Representative images of multiplexed immunofluorescence of treated tumors stained for CD8, FOXP3, Ki67 and diamidino-2-phenylindole with magnifications of the indicated regions of interest. Scale bars represent 500 µm and 100 µm. (I–N) In separate experiments in which untreated contralateral tumors were excised 72 hours following the two-treatment schedule, tumor weight (I), density of CD8 T cells (J), ratio CD8/Tregs (K), content of gp70 antigen-specific CD8 T cells (L), CD4 T cell density (M) and Treg density (N) were assessed. Data represent an experiment with six or five mice per group for flow cytometry or tissue immunofluorescence analyses, respectively (mean±SEM). A one-way analysis of variance (ANOVA) was used to assess significance. Significant differences are displayed for comparisons of each single-treatment group with the BO-112 +DMXAA group (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). DMXAA, 5,6-dimethylxanthenone-4-acetic acid.