Figure 4.
β4 Integrin-Expressing Macrophages and Lymphatic Remodeling Associated with TGF-β Signaling and Adverse Outcome in TNBC Patients
(A) ITGB4 expression in human macrophages. The y axis indicates normalized expression on log2 scale. Red line indicates median expression of all genes. Raw gene counts were obtained from the ARCHS4 database.
(B) Correlation between ITGB4 expression and enrichment of TGF-β signaling in human macrophages (Spearman rho = 0.26; p < 0.001. The x axis indicates normalized expression on the log2 scale. The y axis indicates single sample gene set enrichment analysis (ssGSEA) enrichment scores computed for the TGF-β hallmark gene set obtained from the molecular signatures database (MSigDB). Red curve indicates loess fit. Association strength was quantified using Spearman correlation coefficient. Raw gene counts were obtained from the ARCHS4 database.
(C) Expression of ITGB4 in single cell RNA sequencing (scRNaseq) data of primary breast cancer (GEO: GSE75688). Data are reported as log2(TPM+1). TPM, transcripts per million.
(D) Activation score of TEM gene signature and TGF-β signaling. Red and green dots indicate TNBC with or without distant metastasis, respectively. Enrichment of TNBC with distant metastasis in the top right quadrant, established by hypergeometric testing.
(E) Kaplan-Meier survival curves showing distant metastasis-free survival in TNBC. Stratification based on samples with high TGF-β signaling and TEM gene signature activation score classified as “High TEM-TGFβ signature” versus the remainder (“Low TEM-TGFβ signature”).
(F) Representative breast cancer section (from n = 20) stained with CD14 (red) and podoplanin (brown). Scale bars, 100 μm. Zoomed inset demonstrates CD14+ macrophages associated with podoplanin+ lymphatic vasculature (black arrows). Tissues were selected from 8 patients with or without lymph node positivity. Consecutive sections were stained singly for podoplanin lymphovasculature or doubly using pan-macrophage marker, CD68, and anti-b4 integrin antibody.
(G) Double-stained macrophages per square millimeter shown with patient clinical details (LVI and lymph node positivity).
(H) CD68+ITGB4+ macrophages are indicated in upper right panels (red arrows). CD68 and ITGB4 stainings are indicated below as 2 single panels; CD68+ITGB4+ macrophages are indicated with red arrows. Podoplanin+ vessels shown in upper left images (black arrows). Scale bars, 20 μm.