Proteomic and Functional Comparison of Extracellular Vesicles from Wild-Type and Lyn-Deficient Stromal Cells.

Chronic lymphocytic leukemia (CLL) progression critically depends on bidirectional communication between malignant B cells and non-malignant bystander cells within the tumor microenvironment, including stromal cells, macrophages, and T lymphocytes. Among the key regulators of this cross talk is the Src-family kinase Lyn, whose activity in stromal cells controls extracellular matrix remodeling, fibrotic niche formation, and the release of tumor-supportive extracellular vesicles (EVs). This chapter presents a comprehensive proteomics-based strategy to identify Lyn-dependent effector molecules conveyed by stromal-cell-derived EVs and to evaluate their functional relevance for leukemic cell survival. State-of-the-art EV purification methods compliant with the Minimal information for studies of extracellular vesicles (MISEV) guidelines, nanoparticle tracking analysis, and high-resolution Orbitrap mass spectrometry are integrated with CRISPR-mediated Lyn knockout models and functional co-culture assays using primary CLL cells. Comparative metaproteomic profiling of EVs from Lyn-proficient and Lyn-deficient stromal cells reveals profound alterations in extracellular matrix proteins, adhesion molecules, redox regulators, and signaling mediators, highlighting downstream pathways through which Lyn programs a tumor-supportive microenvironment. Bioinformatic network analysis and pathway enrichment further identify druggable candidate proteins, including matricellular components and integrin-associated anchors such as CD248, as potential therapeutic nodes. Collectively, this chapter illustrates how EV-focused proteomics enables the systematic dissection of microenvironment-driven oncogenic signaling and provides a translational framework for discovering actionable stromal targets in CLL and other Lyn-dependent malignancies.