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. Author manuscript; available in PMC: 2024 May 28.
Published in final edited form as: ACS Nano. 2024 Mar 21;18(13):9584–9604. doi: 10.1021/acsnano.3c13038

Figure 6.

Figure 6.

ACNVax remodeled the tumor immune microenvironment. Control (PBS), IONPVax (14.6 nmol HER2 epitope with 13.9 nmol 2′3′-cGAMP), ACNVax (14.6 nmol HER2 epitope with 13.9 nmol 2′3′-cGAMP), and ACNVax/LIGHT (14.6 nmol antigen, 13.9 nmol 2′3′-cGAMP, 50 ng LIGHT). Vaccines were given on days 4, 11, and 18 in combination with 100 μg of anti-PD-1 antibody biweekly for 3 weeks in D2F2/E2 tumor-bearing mice. (a) Uniform manifold approximation and projection (UMAP) plot of tumor-infiltrating immune cells. (b) Stacked bar charts show the quantified ratio of different phenotypes of immune cells in different treatment groups. (c) Boxplot of selected gene expression levels in tumor infiltrated B and T cells from single-cell RNA sequencing. Statistical comparisons were conducted between ACNVax&LIGHT and ACNVax with IONPVax and control. (d and e) Uniform manifold approximation and projection (UMAP) plot of CD4 T cell subclusters (d) and CD8 T cell subclusters (e) from tumor-infiltrating immune cells. (f) Heatmap of selected gene expression levels related to organized aggregates of immune cells in total tumor infiltrated immune cells from single-cell RNA sequencing. (g) Immunofluorescent staining of tumor lymphoid follicle from mouse tumor samples in the ACNVax&LIGHT + anti-PD-1 antibody group (14.6 nmol HER2 B/CD4 epitope, 13.9 nmol 2′3′-cGAMP, 50 ng of LIGHT, 3 times every 7 days, 100 μg of anti-PD-1 antibody biweekly for 3 weeks). Primary antibodies used are rabbit antimouse CD20 antibody (Invivogen), goat antimouse CD3 antibody (R&D Systems), mouse antimouse CD23 antibody (Invivogen), and rat antimouse PNAd antibody (Biolegend). Secondary antibodies (Abcam) used are donkey antirabbit antibody-AF488, donkey antigoat antibody-AFS94, donkey antimouse antibody-AF647, and donkey antirat antibody-AB405. Scale bar is 100 μm. Statistical comparisons are based on one-way ANOVA, followed by post hoc Tukey’s painwise comparisons or by Student’s unpaired t test. The asterisks denote statistical significance at the level of **** p < 0.0001. ANOVA, analysis of variance; SD, standard deviation.