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

Diana B Peckys,Daniel Gaa,Niels De Jonge

doi:10.3390/cells10113244

Diana B Peckys, Daniel Gaa + Show 1 more

Open Access

https://doi.org/10.3390/cells10113244

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Journal: Cells	Publication Date: Nov 19, 2021
Citations: 5	License type: CC BY 4.0

Affiliation: Saarland University, Leibniz-Institute for New Materials

Abstract

Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.

Highlights

EGFR and human epidermal growth factor receptor 2 (HER2) are overexpressed membrane proteins and therapeutic targets in many different types of cancer, for instance, in breast and gastric cancers [1,2]
The used quantum dot (QD) differ in size, shape, and fluorescence emission so that the receptors are distinguishable in both light microscopy (LM) and EM
To analyze the interaction between EGFR and HER2, five functionally different protein conformations were detected in an experiment, (i) EGFR monomer, (ii) EGFR homodimer, (iii) HER2 monomer, (iv) HER2 homodimer, and (v) EGFR-HER2 heterodimer (Figure 1)

Summary

Introduction

EGFR and HER2 are overexpressed membrane proteins and therapeutic targets in many different types of cancer, for instance, in breast and gastric cancers [1,2]. These receptors can form homodimers, as well as heterodimers, resulting in the activation of their intracellular tyrosine kinase and the subsequent initiation of downstream signaling cascades, promoting cell proliferation, survival, and dissemination [3,4]. Cancer cells exhibit a striking deregulation between the spatial distribution, interaction, and signaling strength of membrane receptors, including EGFR [14] and HER2 [15,16,17,18], which correlates with an increased risk of resistance against targeted therapeutics [19]. We aimed to develop a method for the absolute quantification of EGFR and HER2 ρR, the HER2/EGFR ρR ratio, the corresponding dimerization profile, and the associated heterogeneity thereof, including the specific, subcellular localization in distinct plasma membrane regions

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