Identification of Protein Cargo in Extracellular Vesicles from Macrophages in Progressing and Regressing Tumors

Exosomes are extracellular vesicles (EVs) that carry bioactive molecules from a cell of origin and may alter gene expression of the acceptor cell via binding to cell surface receptors and/or delivering their cargo into the cell. Tumor-associated macrophages (TAMs) can increase or decrease tumor growth through various mechanisms, but the impact of TAM-EVs on tumors has been difficult to elucidate due to challenges in isolating and culturing TAMs for EV purification. In this study, we set out to uncover the protein identities within EVs from TAMs in progressing and regressing tumors, thereby uncovering EV proteins roles in TAM-tumor crosstalk. TAMs were purified from tumors using magnetic bead isolation and cultured for up to 9 days. TAMs from regressing tumors were found to be more M1-like by high expression of major histocompatibility complex (MHC) class II, while TAMs from progressing tumors are more M2-like (low expression of MHC class II). Long-term in vitro culture of TAMs resulted in reduced expression of MHC class II. EVs were harvested from plated TAMs, and EV proteins were identified by mass spectrometry and compared to EV proteins from bone marrow-derived macrophages (BMDMs). Unsupervised hierarchical clustering studies revealed that proteins from TAM-EVs converge based on their in vitro culture duration rather than their tumor origin. Signature proteins in TAM-EVs were characterized, with the identification of galectin-3-binding protein as a signature protein present in EVs from M1-like BMDMs and regressor macrophages. Finally, immunostimulatory pathways were identified in progressor-TAM-EVs through protein-protein interaction network analysis.