The role of small and large extracellular vesicles in lymphoproliferative disorders: the “off the beaten truck” liquid biopsy

Extracellular vesicles (EVs) are particles delimited by a lipid bilayer secreted by all cell types that play a critical role in intercellular communication. EVs are generally classified as small (S-; 50–200 nm) and large (L-; 0.2–10 µm) EVs based on size and biogenesis mechanisms. Distinctions between S- and L-EVs in terms of molecular cargo and functional effect on target cells has been described in various cancers including solid tumors and selected hematologic malignancies, highlighting EV heterogeneity as potential target to develop precision oncology tools. This thesis focuses on the characterization of S- and L-EVs in lymphoproliferative disorders such as Diffuse Large B-cell Lymphoma (DLBCL) and classic Hairy Cell Leukemia (HCL). A special focus on tumor-derived L-EVs is also addressed. In DLBCL, the most common non-Hodgkin lymphoma, RNA sequencing of EV RNA profiles revealed both similarities and differences in mRNA and miRNA content across EV subpopulations, suggesting a selective RNA enrichment within S- and L-EVs. Further deconvolution analysis identified diverse cellular origins for EV RNAs, linking EV mRNA content to the cell of origin, sometimes different according to EV subtype. In HCL, a rare lymphoproliferative B-cell disorder, EV analysis highlighted a distinct vesicle-type and tumor-associated phenotype in both S- and L-EVs. This unique signature was detected at the level of surface proteins (flow cytometry), lipid composition (liquid chromatography-mass spectrometry), and RNA cargo (RNA sequencing), suggesting EVs as potential minimally invasive tool for liquid biopsy biomarkers in rare malignancies. Together, these studies demonstrate EV diagnostic and prognostic potential within lymphoproliferative disorders in order to deepen our knowledge of the pathogenesis of these tumors. The unique profile of S- and L-EVs in cancer offers a significant promise for future liquid biopsy applications, supporting their role in precision oncology.