Fig. 4

miR-CVB3 + CpGMel increases immune cell infiltration into the tumor microenvironment and reduces distant tumor growth and tumor metastasis into the lungs. A Immunostaining of CRT, IFN-γ, TNF-α, IL-6, and granzyme B in tumor tissues obtained at 14 days after indicated treatments. The purple, white, green, and blue fluorescences represent CRT, cytokines, granzyme B, and nucleus, respectively (left). Scale bar = 50 μm. The relative fluorescence intensity from 3 to 5 slides/treatment was quantified (right). B IHC staining of immune cell markers, F4/80, NK1.1, and CD8 in tumor tissues collected at day 14 post-treatment (top). Scale bar = 150 μm. Relative optical densities for immune cell markers are presented (bottom, n = 3–5 slides/treatment). C, D Average tumor size (untreated tumor) (C) and weight (D) of mice after various treatments (n = 5 mice for each group). E H&E staining of lungs harvested from mice treated with various formulations at the endpoint (left, n = 5 mice/group, scale bar = 500 μm), along with number of nodules in the lungs (right). The concentrations of each agent for animal treatment were as follows: miR-CVB3 at 10.⁵ pfu/mouse, CpG at 50 μg/mouse, and melittin at 100 μg/mouse. Data in this figure were analyzed by one-way ANOVA followed by Tukey’s test to determine differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001)