Correlative Fluorescence and Electron Microscopy of Whole Breast Cancer Cells Reveals Differential Distribution of ErbB2 Dependent on Underlying Actin

Epidermal growth factor receptor 2 (ErbB2) is found overexpressed in many cancers such as gastric or breast cancer and is an important therapeutic target. ErbB2 plays a central role in cancer cell invasiveness and is, as such, closely linked to cytoskeletal reorganization. In order to study the spatial correlation of single ErbB2 proteins and actin filaments, we applied correlative fluorescence microscopy (FM), and scanning transmission electron microscopy (STEM) on specifically labeled SKBR3 breast cancer cells. For this purpose, the breast cancer cells were grown on microchips, transformed to express an actin-GFP fusion protein, and labeled with quantum dot (QD) nanoparticles attached to specific anti-ErbB2 Affibodies. FM was performed to identify the distribution of actin. For STEM of the intact plasma membrane of whole cells, the cells were fixed, and covered with graphene. Spatial distribution patterns of ErbB2 in the peripheral membrane regions were examined at actin-rich structures, and compared to adjacent actin-low regions of the same cell, revealing an association of ErbB2 homodimers with actin-rich regions. ErbB2 homodimers were found absent from actin-low membrane regions, and also after treatment of cells with Cytochalasin D. In both data sets, a significant inter-label distance of 36 nm was identified possibly indicating an association with helical actin filaments. This finding was confirmed by identifying linear QD-chains in actin rich which showed an inter-label distance of 36 nm. In conclusion, single molecule analysis of whole cancer cells by means of STEM revealed different distribution patterns of membrane ErbB2 in dependency of the content of underlying actin filaments.