Protein Profile of Multiple Myeloma-Derived Extracellular Vesicles for the Discovery of Novel Myeloma-Related Biomarkers

Despite therapeutic advances, multiple myeloma (MM) remains characterized by progression and relapse, underscoring the
need for reliable biomarkers for early detection and monitoring. Extracellular vesicles (EV) are cell-derived, lipid bilayer–en-
closed particles that mediate intercellular communication and circulate in body fluids, making them promising biomarkers. This
study compares the proteome of MM-derived EV with parental cells and evaluates whether EV represent a superior source of
MM-specific biomarkers. We used high-resolution mass spectrometry to analyze MM cell lines and their EV. Cell line–derived
EV offer a cleaner, tumor-specific protein profile, minimizing background from nonmalignant cells and improving biomarker
accuracy. MS datasets were subjected to bioinformatic analysis, followed by functional enrichment using gene set libraries
such as Gene Ontology, Jensen Compartments, TRRUST and DisGeNET, to extend interpretation beyond limited protein data-
bases. Compared to cellular proteins, the EV proteome was enriched in functions related to oncogenic signaling, transcriptional
regulation and MM pathophysiology, strengthening its potential as a cancer-related biomarker source. DisGeNET identified
cancer-related proteins that were further evaluated to identify MM-specific candidates analyzing MM patients' gene expression
databases containing clinical data. This led to the identification of macrophage migration inhibitory factor (MIF) and prohibitin.
ELISA on plasma from MM patients detected significantly increased MIF levels in smoldering and active MM compared with
healthy donors. Approximately half of circulating MIF was EV-associated in MM patients, indicating that MIF is distributed be-
tween vesicular and soluble plasma fractions and underscoring its potential as a noninvasive biomarker for MM early detection
and patient monitoring.