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.
Highlights
The tyrosine kinase human epidermal growth factor receptor 2 (HER2, ErbB2) belongs to the epidermal growth factor receptor (EGFR) family, consisting of EGFR, HER2, HER3 and HER4 [1]
HER2 was labeled to EGFR, first by attachment of an anti-HER2 specific Affibody carrying a single biotin, by addition of streptavidin conjugated nanoparticles of a different size. This resulted in a maximum 1:1:1 labeling ratio for HER2:anti-HER2 Affibody biotin:streptQD565 (HER2-QD565)
Aberrant growth factor receptor signaling by members of the EGFR family is linked to cancer and all four members (EGFR, HER2, HER3 and HER4) were shown to contribute to cancer cell growth, migration and metastasis formation [5]
Summary
The tyrosine kinase human epidermal growth factor receptor 2 (HER2, ErbB2) belongs to the epidermal growth factor receptor (EGFR) family, consisting of EGFR, HER2, HER3 and HER4 [1]. Dimerization of receptors leads to the activation and phosphorylation of the carboxyl tail of the “activator” kinase. This will in turn phosphorylate and activate the “receiver” kinase of the dimerization partner, leading to full activation of downstream signaling pathways. Preventing dimerization of kinases and inhibiting their kinase activity is a therapeutic strategy to block HER2 signaling in breast cancer [3]. Current therapeutic strategies for HER2 driven breast cancer include I) blocking receptor dimerization with antibodies, such as trastuzumab and pertuzumab, designed to bind to the extracellular domain of HER2 or II) blocking the kinase activity of the receptors with small molecule compounds like lapatinib and neratinib [6,7,8,9]. Antibody drug conjugates like trastuzumab-emtansine (T-DM1) are used to kill HER2 positive cancer cells with the conjugated cytotoxic drug emtansine (DM1), while bispecific antibodies are used to attract and activate components of the immune system against HER2 positive tumor cells [10]
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