Fig 2: α-SMA+ CAFs correlate with immune infiltration in ESCC. A UMAP visualization of the 13 distinct cell clusters identified from scRNA-seq data (PRJNA777911) from ESCC tumor samples. B Scatter plots showing the correlation between α-SMA+ CAFs and immune cell type enrichment score. The relationship was measured by linear regression and non-parametric Spearman correlation. C The t-SNE plot combined from 10 ESCC tumor tissues and overlaid with 25 differently colored phenograph clusters, with each color representing one cluster. D Heat map of the 25 phenograph clusters were determined by normalized median epitome expression of stained markers using IMC antibodies. E The histogram of α-SMA+ CAFs, CD163+ macrophages (MØs), granzyme B+ immune cells, and CD4+ and CD8+ T cells. Mean intensities for 10 ESCC samples are displayed in different colors. Scatter plots further exhibited the relevance of α-SMA+ CAFs to other immune cell types (CD163+ MØs, granzyme B+ immune cells, CD4+ and CD8+ T cells) based on the mean intensity. The relation was measured by linear regression and non-parametric Spearman correlation. F/G Representative images of multiplex IF staining of ESCC tumor samples. Scale bar = 100 μm. Scatter plots showing the positive correlation between α-SMA+ CAFs and CD163+ MØs (n = 12), or between CD163+ MØs and immunologic effector cells (CTLA4+, FOXP3+, CD4+ and CD8+ cells) (n = 12). P-values were determined by non-parametric Spearman correlation analysis

Fig 3: PTEN deletion or mutation glioma cells promoted M1 macrophage differentiation toward M2 macrophage. (A) Immunoblot analyses of PTEN, total Akt, and phosphor-Akt (Ser473) expression in cell lysates of U87, U251, LN229shCONT, and LN229shPTEN cells. Tubulin served as the loading control. (B) Immunoblot analyses of CD206, CD163, CD86, and iNOS expression of macrophages after co-culture with U87, U251, LN229shCONT, and LN229shPTEN cells. Tubulin served as the loading control. (C) Flow cytometric analysis for pan-macrophage marker Iba1 and M2 macrophage marker CD163 expression in the collected macrophages after co-culture with U87, U251, LN229shCONT, and LN229shPTEN cells. (D) The percentage of M2 macrophages is summarized in the bar chart. (E) Representatives of M2 macrophage migration assays induced by U87, U251, LN229shCONT, and LN229shPTEN cells. Scale bar: 100 µm. (F) Statistical analysis of the percentage of the migrated M2 macrophage cells. The data were calculated as means ± standard error of the mean (SEM) of six independent experiments (right). (G) Immunofluorescent analysis of the pan macrophage marker Iba1 (green) and M2 marker CD163 (red) in GBM xenografts derived from U87 cells or LN229 cells. Frozen sections of these GBM xenografts were immunostained with antibodies against Iba1 and CD163 and counterstained with DAPI (blue). Marked reductions of CD163 and Iba1 signals were detected in the LN229-derived tumors. Scale bars: 100 µm (****p < 0.0001).

Fig 4: HPSE expression between MDA-MB-231-HPSE and MDA-MB-231-mock cells (A). HPSE expression correlates with IL-10 induced M2 macrophage polarization in a mouse breast tumor model: IHC staining of (B) CD163, (C) VSIG4, and (D) IL-10.

Fig 5: Spatial distribution and correlations of characteristic immune cell markers in lymph nodes. The expression of M2 macrophages (CD3-/CD163+), Tregs (CD3+/CD8A-/FoxP3+), and Tfh cells (CD3+/CD8A-/PD1+) is shown in the histogram. ∗∗p < 0.01.