Multiplex Immunofluorescence Reveals Therapeutic Targets EGFR, EpCAM, Tissue Factor, and TROP2 in Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is a clinically and molecularly heterogeneous subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. In this study, tumor specimens from 104 TNBC patients were analyzed to characterize molecular and clinicopathological features and to assess the expression and therapeutic potential of four key surface markers: epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), tissue factor (TF), and trophoblast cell surface antigen (TROP2). Multiplex immunofluorescence (mIF) demonstrated elevated EGFR and TROP2 expression in the majority of samples. Significant positive correlations were observed between EGFR and TF, as well as between TROP2 and both TF and EpCAM. Expression analyses revealed increased EGFR and TF levels with advancing tumor stage, whereas EpCAM expression declined in advanced-stage tumors. TROP2 and TF expression were significantly elevated in higher-grade tumors. Additionally, EGFR and EpCAM levels were significantly higher in patients with elevated Ki-67 indices. Binding specificity assays using single-chain variable fragment (scFv-SNAP) fusion proteins confirmed robust targeting efficacy, particularly for EGFR and TROP2. These findings underscore the therapeutic relevance of EGFR and TROP2 as potential biomarkers and targets in TNBC.

Keywords: EGFR; EpCAM; TROP2; multiplex immunofluorescence; tissue factor; triple-negative breast cancer.

Figure 1

Expression profiles of EGFR, TF, EpCAM, and TROP2 in TNBC tissues. (a) Representative mIF images showing the expression of EGFR, TF, EpCAM, and TROP2 in TNBC issues (TMA BR1102). Images included full core scans of 110 samples and corresponding high-magnification (100×) views from selected regions, with each marker visualized in a distinct fluorescent channels. (b) Heatmap depicting the distribution and relative expression levels of EGFR, TF, EpCAM, and TROP2 across 110 different tissues cores, enabling comparative visualization of marker-positive cell proportion. (c) Boxplot illustrating the median, the interquartile range, and the individual percentage of cells positive for each marker across the 104 different TNBC tissues cores. **, p ≤ 0.01; ****, p ≤ 0.0001.

Figure 2

Correlation of the EGFR, TF, EpCAM, and TROP2 expressions in TNBC. Pearson correlation plots illustrate pairwise associations between the surface marker expression levels in TNBC tissue cores (n = 104). (a) EGFR vs. TF, (b) EGFR vs. EpCAM, (c) EGFR vs. TROP2, (d) TF vs. EpCAM, (e) TF vs. TROP2, and (f) EpCAM vs. TROP2. The blue line indicates the fitted linear regression line between the two markers.

Figure 3

Differential expression of EGFR, TF, EpCAM, and TROP2 across TNBC stage, grade, and patient age. (a) Comparison of EGFR, TF, EpCAM, and TROP2 expression levels between early-stage (stage I–II) and advanced-stage (stage III) TNBC tissues. (b) Expression differences of the four markers between lower-grade tumors (grade 1−2) and high-grade tumors (grade 3). (c) Expression patterns of EGFR, TF, EpCAM, and TROP2 across five patient age groups. Boxplots display the distribution of marker-positive cell (%), and statistical significance was assessed using one-way ANOVA with post hoc Tukey’s test. Asterisks indicate levels of significance: *, p < 0.05.

Figure 4

Expression patterns of EGFR, TF, EpCAM, and TROP2 in relation to HER2 and Ki-67 status in TNBC. (a) Comparison of EGFR, TF, EpCAM, and TROP2 expression levels across TNBC samples stratified by HER2 status: HER2-0, Her2-1+, and Her2-2+. (b) Expression analysis of the same four markers in TNBC samples grouped by Ki-67 proliferation index: Ki-67 < 2%, Ki-67 low (2–49%), and Ki-67 high (≥50%). Boxplots represent the distribution of marker-positive cells (%). Statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test. *, p < 0.05.

Figure 5

Binding specificity scFv-SNAP fusion proteins in TNBC tissue. (a) Microphotographs from mIF showing the binding specificities of four scFv-SNAP fusion proteins—scFv-Erbitux-SNAP, scFv-Tisotumab-SNAP, scFv-anti EpCAM-SNAP, and scFv-Sacituzumab-SNAP—across 110 tissue samples. Representative 100× images from a single core are shown for each fusion protein, with visualization across four fluorescence channels. (b) A heatmap depicting the percentage of cells bound by each scFv-SNAP fusion proteins in all 104TNBC tissues. (c) Boxplot illustrating the median, interquartile range, and individual percentage of cells binding for each scFv-SNAP fusion proteins across the tissue samples for each marker. ****, p ≤ 0.0001.