Small and Large Extracellular Vesicles in Circulation of Diffuse Large B‐Cell Lymphoma Patients Originate From Different Cell Types of the Tumor Microenvironment

Malignant and non-malignant cells within the tumor microenvironment (TME) actively secrete extracellular vesicles (EVs) that may mediate intercellular communication or enter the blood stream. Circulating EVs in Diffuse Large B-Cell Lymphoma (DLBCL) patients are a promising source of liquid biopsy biomarkers; however, whether different cellular components of the TME preferentially secrete small (S-) and/or large (L-) EVs is still unknown. With an established density-gradient separation protocol and tunable resistive pulse sensing analysis, we demonstrate that DLBCL cells in culture produce 100-1000-fold higher numbers of S-EVs (50-200 nm) compared with L-EVs (200-1000 nm) and very large EVs (>1000 nm). In contrast, the plasma from DLBCL patients contains comparable concentrations of S- and L-EVs, consistent with various cellular origins. Small RNA sequencing showed minor differences in miRNA content between plasma S- and L-EVs; however, messenger RNA sequencing revealed stark differences in cargo between EV-size subtypes and between healthy donors and patients. Deconvolution analysis with single-cell sequencing data from 17 DLBCL tumor tissues as reference using the Statescope algorithm indicated that circulating S-EVs from malignant cells outnumber the L-EVs. In contrast, TME macrophage-, T cell-, and natural killer-derived L-EVs outnumber S-EVs. Together, these findings suggest that circulating S- and L-EVs can originate from distinct cellular compartments within the DLBCL TME, representing complementary biological information. These observations have important implications for the development of EV-based liquid biopsy strategies.