Proximity Labeling and SILAC-Based Proteomic Approach Identifies Proteins at the Interface of Homotypic and Heterotypic Cancer Cell Interactions

Cell-cell interactions are critical for the growth of organisms and maintaining homeostasis. In the tumor microenvironment, these interactions promote cancer progression. Given their importance in healthy and diseased conditions, we have developed a method to analyze the cell-to-cell interactome. Our approach uses enzyme-catalyzed proximity labeling and SILAC-based proteomics to identify the proteins involved in cancer cell interactions. By targeting HRP to the outer leaflet of the plasma membrane in bait cells, we were able to label the neighboring prey cells and distinguish between the proteomes of bait and prey cells using SILAC labeling in a coculture system. We mapped both the homotypic and heterotypic interactomes of epithelial and mesenchymal breast cancer cells. The enrichment of cell surface and extracellular proteins confirms the specificity of our methodology. We further verified selected hits from different cell-cell interactomes in cocultures using microscopy. This method revealed prominent signaling pathways orchestrating homotypic and heterotypic interactions of epithelial and mesenchymal cells. It also highlights the importance of exosomes in these interactions. Our methodology can be applied to any type of cell-cell interaction in 2D coculture or 3D tumor models.