Abstract
The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the in-situ organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters. Clustering of receptors is an important regulatory mechanism to prime cells to respond to stimuli so, to understand these processes, it is necessary to measure parameters such as numbers of clusters, cluster radii and the number of localizations per cluster for different perturbations. Previously, Fluorescence Localization Imaging with Photobleaching (FLImP), another nanoscale, single-molecule technique, characterized the oligomerization state of HER1 [or Epidermal Growth Factor Receptors (EGFR)] in cell membranes. To achieve an unprecedented resolution (< 5 nm) for inter-molecular separations in EGFR oligomers using FLImP, very few receptors are tagged, and so this method is unsuitable for measurements of whole receptor populations in cancer cells where receptors are frequently upregulated. Here, in order to detect all receptors involved in cluster formation, we saturate endogenous HER2 and HER3 membrane receptors with ligands at a 1:1 dye to protein ratio, in the presence or absence of therapeutic drugs (lapatinib or bosutinib). This is performed in the commonly used breast cancer cell line model SKBR3 cells, where there are ~1.6 million HER2 receptors/cell and 10,000-40,000 HER3 receptors/cell. The basal state of these receptors is studied using HER2- or HER3-specific Affibodies, and likewise, the active state is probed using the natural HER3 ligand, Neuregulin-beta1 (NRGβ1). Stochastic Optical Reconstruction Microscopy (STORM), one form of SMLM, was used here to image cells, which were chemically fixed to minimize image blurring and provide data (x and y coordinates and standard deviation of the measured localizations) for cluster analysis. Further analysis can also determine proportions of receptor colocalizations. Our findings show that lapatinib-bound HER2, complexed with HER3 via a non-canonical kinase dimer structure, induces higher order oligomers. We hypothesized that nucleation of receptors creates signaling platforms that explain the counterintuitive, increase in cell proliferation upon ligand binding, in the presence of the HER2-inhibitor lapatinib.
Highlights
Ligand binding, in the presence of the Human epidermal growth factor receptor 2 (HER2)-inhibitor lapatinib
HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, Human epidermal growth factor receptor 3 (HER3) (Holbro et al, 2003)
It may be possible to use Single-molecule localization microscopy (SMLM) and cluster analysis to study these receptors in live cells this has not been implemented as yet
Summary
Conjugate HER2 and HER3 Affibody ligands to the appropriate Alexa Fluor maleimide dyes according to manufacturer’s instructions. C. Twenty-four hours post-transfection, treat pre-chilled cells with ice-cold 10 nM NRG-Alexa 647 in PBS and incubate for 1 h at 4 °C. d. First bind anti-HER2 pY877 or anti-HER3 pY1289 from Cell Signaling Technology (follow the manufacturer’s instructions) and the species-specific secondary antibody conjugated to HRP (Horse-radish peroxidase) from Jackson ImmunoResearch. 8. Incubate half of the samples in 1 ml of 100 nM HER2Affibody-Alexa[488] and 50 nM HER3Affibody-Alexa[647] diluted in ice-cold PBS and, if required, the appropriate drug (either 14 nM Lapatinib or 41 nM Bosutinib) 9. Incubate the remaining dishes in 1 ml of 100 nM HER2Affibody-Alexa[488] and 10 nM NRGAlexa[647] diluted in ice-cold PBS and, if required, the appropriate drug. Store samples at 4 °C in PBS and prior to imaging supplement fresh PBS with 50 mM cysteamine HCl (total volume 1 ml)
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