Figure 1.

BCG lysate mediates tumor growth inhibition, immune cell-infiltration and a proinflammatory TME. (A) Growth curve of treated and untreated B16F10 melanoma. Data are represented as mean±SEM using two-way ANOVA followed by Šidák’s multiple comparisons test (n=25 per group, four independent experiments). (B–I) Relative frequencies of cell subsets measured by flow cytometry: (B) CD45⁺ cells, (C) CD8⁺ T cells, (D) CD4⁺ T cells, (E) NKT cells, (F) NK cells, (G) Nur77⁺ CD8⁺ T cells, (H) Nur77⁺ CD4⁺ T cells (n=17 per group, three independent experiments) and (I) MΦ (n=8 per group). Data are shown as mean±SD using one-way ANOVA, followed by Tukey’s multiple comparisons test. (J) Donut charts representing mean percentages of M0 MΦ, M1 MΦ and M2 MΦ within treated and untreated tumors. (K, L) BMDM and BMDC were stimulated or left untreated (media) for 48 hours. (K) Mean fluorescence intensity of maturation and activation markers as measured by flow cytometry (n=6–9 per group, three independent experiments). (L) Production of IL-6 and IL-12 measured by ELISA. Data are shown as box-and-whisker plot; the box extends between 25% and 75% and the whiskers extend to the minimum and maximum values, using two way-ANOVA followed by Šidák’s multiple comparisons test (n=4–7 per group, two independent experiments). (M) Protein expression levels of the indicated cytokines and chemokines from tumor lysates on day 12 post tumor injection (24 hours after last treatment). Data are shown as box-and-whisker plots; the box extends between 25% and 75% and the whiskers extend to the minimum and maximum values with a log₂ cut-off >2.5, using two way-ANOVA followed by Šidák’s multiple comparisons test (n=10 per group). (N) Gene expression profiles of tumor lysates shown as ordination plots with 80% CI ellipses (n=8 per group). p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), p≤0.0001 (****). ANOVA, analysis of variance; BMDC, bone marrow dendritic cell; BMDM, bone marrow derived microphages; MΦ, macrophage; TME, tumor microenvironment.