Abstract 998: Extracellular proximity labeling (ePL) as a tool to identify protein-protein interactions in the tumor microenvironment

Abstract The extracellular matrix (ECM) is a dynamic niche that is extensively reshaped in the development of the tumor microenvironment (TME). Our current understanding of ECM function and dynamics is largely informed by identification of protein-protein interactions (PPIs) using co-immunoprecipitation (co-IP) techniques that may miss transient and weak/unstable interactions. Recent advances in proximity labeling techniques have greatly expanded the interactome networks of numerous intracellular proteins, however these tools have yet to be extended to study PPIs in the ECM. We have recently optimized a systematic approach to identify PPIs in the ECM using fusion constructs of the biotinylating enzymes, BioID2 and TurboID, with the widely expressed matrix regulator TIMP2. BioID2 and TurboID offer differing reaction kinetics that may provide complimentary information on extracellular PPIs (ePPIs). Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs) are crucial regulators of ECM structure and composition. TIMPs are widely expressed multifunctional proteins that serve to promote ECM homeostasis that is often perturbed in many cancers and chronic disorders. Although biochemical data suggests that TIMPs are promiscuous proteins, the TIMP interactome is poorly defined. We have optimized a protocol for the identification of ePPIs for the TIMP family of proteins. Fusion constructs equipped with a promiscuous biotin ligase (BioID2/TurboID) fused to the N- or C-terminal of full length TIMP2 were packaged into retroviral vectors for cellular delivery. Cells were exposed to the extracellular proximity labelling (ePL) fusion proteins and processed in our optimized analysis pipeline. ePPIs were identified via streptavidin pulldown and proteomic techniques. We present our optimized ePL pipeline and show that this technique is an effective tool for the identification of novel ePPIs for multiple extracellular targets. Citation Format: Sadeechya Gurung. Extracellular proximity labeling (ePL) as a tool to identify protein-protein interactions in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 998.