Abstract B02: Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging

The dynamic behavior of proteins in the spatial and temporal context of a cell is critical for dictating downstream signaling and subsequent cellular effects. Human epidermal growth factor receptor 2 (HER2) is a ligand-less receptor tyrosine kinase whose heterodimerization with other HER receptors (EGFR, HER3, HER4) activates potent downstream signaling to modulate cell proliferation and survival. HER2 represents a major therapeutic target as its overexpression is prevalent in a major subset of invasive breast cancers. Yet while current understanding of HER2 signaling entails a broad sequence of signaling events, the spatiotemporal dynamics of how these signaling mechanisms are executed remains undefined. Here, we describe the molecular-scale motions that underlie HER2 signaling behavior in live cells using a bright and photostable anti-HER2 quantum dot (QD) probe designed and validated to track the receptor for long durations with high spatiotemporal resolution. Methods: HER2 was detected using an anti-HER2 affibody that was fused to a fluorogen activating peptide (FAP) which bound with high affinity to a fluorogen (Malachite Green)-QD complex. HER2-QDs were imaged in live unstimulated and stimulated human SKBR3 breast cancer cells. Cells were serum-starved overnight and: 1) received no treatment during the course of imaging (unstimulated) or 2) were chronically treated with the HER3 and HER4 specific ligand, heregulin-beta1 (HRG) during the course of imaging (stimulated). HRG was administered globally across all cells or locally to a specific cellular region. Trajectory information was analyzed using custom single particle tracking MATLAB software to measure key biophysical parameters. Results: The anti-HER2-QD probe showed robust and increased signal-to-noise compared to conventional organic dyes. Validation was measured by sensitive quantitation of single probes in low and high HER2-expressing cell lines with background non-specific binding of less than five probes per cell. HER2 motions were complex and composed of a heterogeneous composite of diffusive, constrained, and stationary motions. These motions were observed in the trajectories of HER2 receptors traveling along cellular protrusions, cell body, and along protrusions between adjacent cells. HRG stimulation produced morphological changes (e.g. membrane ruffling, protrusion growth) as well as changes in HER2 dynamics. HRG-stimulated cells demonstrated an increase in HER2 mobility (as measured by diffusion constant), increased speed (as measured by instantaneous velocity), and wider exploratory area (as measured by maximum trajectory distance) than in unstimulated cells. Interestingly, increased receptor mobility did not correlate strongly with a wider exploratory area, and more mobile receptors showed more constrained motion. Conclusions: In contrast to the simple diffusive motions previously reported for members of major classes of receptors (e.g. GPCRs, tyrosine kinase receptors), our sensitive measurements of HER2 receptor motion demonstrate complex molecular dynamics in both unstimulated and stimulated cells. Ligand stimulation induces changes in cell morphology such as membrane ruffling and protrusion growth that accompany changes in HER2 mobility and range of motion. The diversity of receptor dynamics suggest a heterogeneous environment in which HER2 engages. Future experiments include understanding the dynamic interplay between HER2 with the cytoskeleton and downstream signaling with HER2-targetting therapies using real-time measurements of HER2 molecular motions. Citation Format: Wai Yan Lam, Yi Wang, Mark J. Olah, Keith A. Lidke, Marcel Bruchez, Joe Gray, Tania Q. Vu. Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B02.