EGFR Expression in HER2-Driven Breast Cancer Cells

Abstract

The epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5–5) × 101 QDs/µm2, whereas labeled HER2 expression was higher ranging from (2–10) × 102 QDs/µm2. Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells.

Critical roles for EGFR and EGFR–HER2 clusters in EGF binding of SW620 human carcinoma cells

“…A number of previous findings have inferred indirectly that EGFR may form heterocomplexes with other RTKs [14,20,[22][23][24], and recent evidence shows that HER2 inhibitor lapatinib induces HER2/HER3 heterocomplex formation in breast cancer cells [53].…”

Met–HER3 crosstalk supports proliferation via MPZL3 in MET-amplified cancer cells

“…To understand the crosstalk signaling axis between Met and EGFR family RTKs, we assembled a panel of MET -amplified cancer cell lines, derived from esophageal (OE33), gastric (KatoII, Okajima, Snu5, MKN45) and lung (EBC1, H1993) adenocarcinomas. SkBr3 cells were included as a control, in which EGFR, HER2 and HER3 phosphorylation is driven by ERBB2 amplification and dependent on HER2 kinase activity [ 30 ] (Fig. 1 c and Supplementary Fig.…”

Quantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy

“…The procedures applied for fixation and growth factor labeling of the cells were performed as described earlier [32]. In brief, the applied two-step fixation started rinsing the cells 1× with serum-free medium, and 1× with CB (0.1 M cacodylate buffer, 0.1 M sucrose, pH 7.4), followed by a 5 min incubation with 3% formaldehyde (in CB), and another 5 min in 3% formaldehyde and 0.2% glutaraldehyde (in CB.)…”

“…The maximal electron dose was calculated to be 280 e − Å −2 , which lies under the limit of radiation damage for these samples. A detailed demonstration of how correlative microscopy was used to identify and image the same membrane regions with LM and LPEM, can be found in our earlier publication [32].…”

“…A two-step labeling protocol prevented label-induced receptor clustering. Prior to labeling, the cells were chemically fixed with formaldehyde and glutaraldehyde, to prevent any ligand-induced dimerization or clustering that can occur during labeling of unfixed samples or with samples fixed using only formaldehyde, a detailed discussion of this topic can be found in our earlier publication [32]. We performed correlative LM and LPEM [33], using STEM, of intact, chemically fixed SKBR3 cells.…”