Gold nanoparticles as probes in epidermal growth factor receptor clustering and second messenger mediated activation
Embargo Date
2024-01-23
OA Version
Citation
Abstract
The epidermal growth factor receptor (EGFR) is a prototypical receptor tyrosine kinase that plays an important role in cell growth, while its dysregulation is associated with a broad range of cancers. Understanding the mechanisms that underlie EGFR activation are crucial for the development of novel therapeutic targets. In this dissertation, the spatial distribution of EGFR and the role of reactive oxygen species (ROS) in modulating EGFR activation are elucidated.
Spatial clustering of EGFR has been indicated to play a regulatory role in signal initiation, and the distribution of receptors on the cell surface may represent a potential biomarker. To realize its potential for diagnostic purposes, high-throughput assays capable of mapping spatial receptor heterogeneity on the length scale of tens of nanometers are needed. To that end, gold (Au) nanoparticle (NP) labels are used as bright optical probes for detecting large-scale EGFR clustering in cancer cells. The clustering spatial maps obtained from hyperspectral plasmon coupling microscopy (PCM) are compared to those obtained through fluorescence direct stochastic optical reconstruction microscopy (dSTORM). The results revealed that hyperspectral PCM imaging of NP labels identified the same trends in EGFR clustering as dSTORM, but the NP imaging approach provided the information in a fraction of the time.
Au NPs can also be decorated with ligands through nanoconjugation to probe ligand-receptor interactions. Multivalent ligand presentation on NPs is considered a general approach for controlling and amplifying local EGFR activation in receptor clusters. ROS have been indicated to play a role in the regulation of EGFR activation as a second messenger, but the effect of nanoconjugation (NP-EGF) on EGF-mediated ROS formation and ROS-induced EGFR activation is not well established. Au NPs functionalized with two different EGF ligand densities were used to quantify NP-EGF-induced ROS generation and EGFR phosphorylation in breast cancer cell models. In EGFR overexpressing cell lines, nanoconjugated EGF with higher ligand density achieved a multivalent enhancement of ROS that was EGFR and nicotinamide adenine dinucleotide phosphate oxidase (NOX) dependent. This multivalent enhancement is not exclusively related to avidity but also to a stronger stimulation per NP. Importantly, the increase in EGF-induced ROS formation associated with EGF nanoconjugation resulted in a gain in EGFR phosphorylation, confirming that ROS generation contributes to the multivalent enhancement of EGFR activation in response to NP-EGF.