LAUSR

The extracellular matrix (ECM) is a complex and dynamic meshwork of extracellular macromolecules that plays a key role in cancer progression and metastasis. The matrisome encompasses the genes encoding ECM and ECM-associated secretory proteins, and elucidating the protein-protein interactions (PPIs) of the matrisome is a promising area of study in the quest for a better understanding of cancer pathology and therapeutics. Proximity labeling, a powerful technique for investigating protein interactomes based on enzyme-fusion constructs and affinity chromatography, has considerable potential in detailing the protein-protein interactions of the matrisome. We have recently had success in developing a proximity labeling approach based on fusion constructs containing the biotinylating enzymes BioID2 and TurboID. We apply this approach to gain new insights into the interactome of the matrisome protein tissue inhibitor of metalloproteinase 2 (TIMP2), a protein found to have antitumorigenic potential. At present, we seek to expand this approach to other ECM protein targets, including tissue inhibitor of metalloproteinase 3 (TIMP3) and thrombospondin-1 (THBS-1). TIMP3 has been shown to possess anti-tumor properties, with loss of TIMP3 function associated with poor prognosis in multiple cancers. THBS-1 is a large (129 kDa) ECM protein that is relatively promiscuous with diverse functions in the tumor microenvironment. Expression of THBS-1 is frequently lost during oncogenesis, and such loss is associated with poor prognosis in some cancers. We describe how ePL can be expanded to these matrisome targets in different cellular systems (adherent vs. suspension) and show that ePL assays can be performed with stable and transient transgene expression. We show that ePL is a suitable method for studying PPIs in novel matrisome targets and expect to apply the technique to further targets in the near future. Citation Format: Joshua Rich, Sadeechya Gurung, Sukhbir Kaur, David Roberts, William Stetler-Stevenson, David Peeney. Expansion of the extracellular proximity labeling (ePL) technique to novel matrisome targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4695.