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. 2022 Apr 21;10(6):680–697. doi: 10.1158/2326-6066.CIR-21-0804

Figure 2.

Figure 2. The immune microenvironment of NMU-induced mammary tumors. A, Leukocyte composition of the indicated tissues from tumor-bearing (TB) or tumor-free (TF) animals from the characterization cohort (see Fig. 1) determined by polychromatic flow cytometry. LN, mammary gland-draining lymph node. BM, bone marrow. MG, mammary gland. *, P < 0.05 on Mann–Whitney–Wilcoxon test. B, Immunofluorescence analysis for CD244 (NK cells) and granzyme b (GZMB). C, Immunofluorescence analysis for CD8 (cytotoxic T cells), CD44, and Ki67. Yellow arrows, Ki68+CD44+CD8+ cells. D, Immunofluorescence analysis of CD3 T-cell and SMA myoepithelial markers in normal, DCIS-like, and IDC-like regions. E, Immunofluorescence analysis of Ki67+CD8+ T cells (yellow arrow) and SMA+ myoepithelial cells in small and large tumors. Graph depicts frequency of Ki67+CD8+ T cells in small and large tumors. Error bars, SD. P value calculated using a Mann–Whitney Wilcoxon test. F, Immune cell type frequency predicted by CIBERSORT based on RNA-seq data of CD45+ cells from 10 NMU-induced tumors. P values calculated by Mann–Whitney Wilcoxon test and difference between means between small and large tumors shown. *, P < 0.05. G, Heatmap of row-normalized Z-scores of DEGs in CD45+ cells from large and small tumors, defined as having an adjusted p value of ≤ 0.05 and absolute fold change ≥ 1.5. Red and blue indicates HRlow and HRhigh tumors, respectively. Green and orange marks large and small tumor, respectively. H, Enriched pathways in CD45+ cells from large and small tumors. Green and orange bars depict pathways enriched in stable and growing tumors, respectively. Node size indicates the number of genes within the pathway, line width indicates the number of shared genes between two gene sets. I, Gini index measure of BCR heterogeneity in tumor and normal mammary gland (MG) infiltrating B cells and the number of unique BCR clonotypes. Box and whisker plot, quartiles. P values were calculated using Mann–Whitney Wilcoxon test. J, Immunofluorescence analysis of NMU-induced tumor organoid culture stained for EpCAM (red) and SMA (green). K, Representative phase-contrast images of rat mammary tumor organoid or autologous normal kidney organoids incubated with or without opT for indicated times. L, INFγ levels in the medium of rat tumor organoids co-cultured for 2 days with opT under mIgG1 mAb, PD-L1 mAb, LY, or PD-L1+LY treatment measured by ELISA. P value calculated using Mann–Whitney Wilcoxon test. Error bars, SD. B–E, J and K, Scale bar, 100 μm. All tests for significance used Mann–Whitney–Wilcoxon test using a threshold of P = 0.05 unless otherwise specified; error bars representative of SD; box-whisker plots indicate 0th, 25th, 50th, 75th, and 100th percentiles.

The immune microenvironment of NMU-induced mammary tumors. A, Leukocyte composition of the indicated tissues from tumor-bearing (TB) or tumor-free (TF) animals from the characterization cohort (see Fig. 1) determined by polychromatic flow cytometry. LN, mammary gland-draining lymph node. BM, bone marrow. MG, mammary gland. *, P < 0.05 on Mann–Whitney–Wilcoxon test. B, Immunofluorescence analysis for CD244 (NK cells) and granzyme b (GZMB). C, Immunofluorescence analysis for CD8 (cytotoxic T cells), CD44, and Ki67. Yellow arrows, Ki68+CD44+CD8+ cells. D, Immunofluorescence analysis of CD3 T-cell and SMA myoepithelial markers in normal, DCIS-like, and IDC-like regions. E, Immunofluorescence analysis of Ki67+CD8+ T cells (yellow arrow) and SMA+ myoepithelial cells in small and large tumors. Graph depicts frequency of Ki67+CD8+ T cells in small and large tumors. Error bars, SD. P value calculated using a Mann–Whitney Wilcoxon test. F, Immune cell type frequency predicted by CIBERSORT based on RNA-seq data of CD45+ cells from 10 NMU-induced tumors. P values calculated by Mann–Whitney Wilcoxon test and difference between means between small and large tumors shown. *, P < 0.05. G, Heat map of row-normalized Z-scores of DEGs in CD45+ cells from large and small tumors, defined as having an adjusted p value of ≤ 0.05 and absolute fold change ≥ 1.5. Red and blue indicates HRlow and HRhigh tumors, respectively. Green and orange marks large and small tumor, respectively. H, Enriched pathways in CD45+ cells from large and small tumors. Green and orange bars depict pathways enriched in stable and growing tumors, respectively. Node size indicates the number of genes within the pathway, line width indicates the number of shared genes between two gene sets. I, Gini index measure of BCR heterogeneity in tumor and normal mammary gland (MG) infiltrating B cells and the number of unique BCR clonotypes. Box and whisker plot, quartiles. P values were calculated using Mann–Whitney Wilcoxon test. J, Immunofluorescence analysis of NMU-induced tumor organoid culture stained for EpCAM (red) and SMA (green). K, Representative phase-contrast images of rat mammary tumor organoid or autologous normal kidney organoids incubated with or without opT for indicated times. L, INFγ levels in the medium of rat tumor organoids cocultured for 2 days with opT under mIgG1 mAb, PD-L1 mAb, LY, or PD-L1+LY treatment measured by ELISA. P value calculated using Mann–Whitney Wilcoxon test. Error bars, SD. B–E, J and K, Scale bar, 100 μm. All tests for significance used Mann–Whitney–Wilcoxon test using a threshold of P = 0.05 unless otherwise specified; error bars representative of SD; box-whisker plots indicate 0th, 25th, 50th, 75th, and 100th percentiles.