(370e) Award Submission: Detection and Visualization of HER2 Clustering Using an Aggregation Induced Emission Based Fluorescent Probe Molecule

The spatio-temporal organization of proteins within the cell membrane can influence basic cell behaviors including signaling and proliferation which play a role in disease initiation and progression. Understanding these control mechanisms can motivate new therapeutic designs. Several methods exist for studying the organization of membrane proteins including electron microscopy, super-resolution optical microscopy methods, and proximity-based assays. However, these methods are either not compatible with living cells or require access to specialized instrumentation. Furthermore, the highest-resolution approaches that can detect the spatial organization of proteins rely on single-molecule imaging methods and cannot be utilized in a high-throughput format.

To overcome many of the limitations of the current methods, we have developed a fluorescent microscopy tool based on a family of fluorophores that exhibit Aggregation Induced Emission (AIE) behavior for studying the spatial organization of membrane-bound proteins. Fluorophores with the AIE property can go through a fluorescence turn-on process when their intramolecular motions are restricted. For example, when AIE molecules form solid aggregates, the emission increases, and this increase in signal can be easily detected using conventional fluorescence microscopy. When proteins cluster or are in close proximity, they behave as a biological mimic for aggregation. Therefore, a tool based on an AIE fluorophore can overcome many limitations of previous methods.

In this work, we demonstrate the development and utilization of an AIE-based tool to study the response of the breast cancer biomarker HER2 to a conventional targeted cancer therapeutic (Trastuzumab). Trastuzumab (Herceptin), an FDA-approved monoclonal antibody therapeutic, is hypothesized to change the biophysical distribution of HER2 in the membrane, disrupting clusters or decreasing densities. The dependence of AIE luminogen’s (AIEgen’s) fluorescent response on its proximity to other AIEgens can provide insight into this mechanism.

Our AIE-based tool is developed by designing, synthesizing, and characterizing a molecule based on Tetraphenylethylene (TPE) (an AIE fluorophore) and binding it to an antibody specific to the extracellular domain of HER2 through the non-selective NHS Ester-Amine bioconjugation reaction. The target specificity of the AIE-based tool is confirmed through a competing colocalization assay using a HER2+ cell line. Then, the AIE-based tool was utilized for visualizing HER2 clusters in Her2+ cells with the presence of HER2 clusters in comparison to HER2- cells with a more uniform distribution of HER2. Since the molecular motions of the TPE domain of the tool become restricted in the cellular regions where receptors are in proximity of each other, a significantly higher fluorescent signal was observed in the HER2+ cells compared to HER2- cells.

Furthermore, the developed AIE-based tool was utilized in an assay for detecting the effect of Trastuzumab on HER2 clusters. Fluorescein-HER2 antibody, a control that is not sensitive to proximity, and the AIE-based tool are applied to HER2+ cells post-treatment with a range of Trastuzumab concentrations for different time points. The results determine that, unlike the control Fluorescein-HER2 antibody, the AIE-based tool demonstrates a significant reduction in the average fluorescent intensity of the cells treated with higher Trastuzumab concentrations and for longer. This reduction can directly correlate with the disruption of HER2 clusters or the decrease of HER2 densities.

Current results confirm the applicability of the AIE-based tool for visualization of spatial organization of HER2 and set the stage for generalizability of the tool for studying other biological systems with applications in developing improved therapeutics in the future.