No. 4
Marie Didion
Identification of a marker panel for extracellular vesicle-based molecular subtyping of diffuse large B cell lymphoma (DLBCL)
Marie Didion1, Marcel Kemper1, Annalen Bleckmann1,2, Georg Lenz1, Stephan Hailfinger1*, Kerstin Menck1* 1 Dept. of Medicine A, Haematology, Oncology, and Pneumology, University of Münster, Münster, Germany 2 West German Cancer Center, University Hospital Münster, Münster, Germany *These authors contributed equally
Based on gene expression profiling, diffuse large B-cell lymphoma (DLBCL) is classified into two major molecular subtypes, activated B-cell-like (ABC) and germinal center B-cell-like (GCB). The subtype influences the prognosis for the patients, with the ABC subtype tending to have a less favourable prognosis. However, DLBCL subtyping currently requires the collection of tissue biopsies and a time-consuming immunohistological workup. This study investigates whether DLBCL-derived extracellular vesicles (EVs) shed into the circulation could be harvested as minimally invasive biomarkers for molecular subtyping of the disease in a liquid biopsy approach.
EVs were isolated from the supernatant of human DLBCL cell lines (GCB: DOHH2, Su-DHL-4, Su-DHL-6; ABC: OciLy3, TMD8) using differential ultracentrifugation, resulting in three EV fractions pelleting at 1,500 g (1.5k EVs), 14,000 g (14k EVs) and 143,000 g (143k EVs). EVs were quantified by nanoparticle tracking analysis (NTA) and characterized for the expression of subtype specific markers by flow cytometry and immunoblots.
Both GCB and ABC cell lines secreted EVs found in different size fractions. Flow cytometric analysis of large EVs showed a consistent expression of the B cell marker CD20 across all DLBCL lines, while CD19 was expressed at much lower levels. A broad surface marker screen (354 antigens) identified a panel of 13 novel antigens on DLBCL cells that could aid in molecular subtyping. Validating their subtype-specific expression pattern, CD39, CD44, and CD70 emerged as promising ABC markers, while CD10 and CD27 were specific for GCB DLBCLs.
In summary, DLBCL cells secrete EVs that carry distinct surface markers reflective of their molecular subtype. These findings highlight the potential of EV-based profiling as a non-invasive approach for DLBCL subtyping. Ongoing work is centered on validating these findings in blood-derived EVs from DLBCL patients.
This work was funded by the Interdisciplinary Center for Clinical Research (IZKF) Münster (project Hai4/007/25).
