Clinical Implications of Plasma-Derived Extracellular Vesicles in Myeloproliferative Neoplasms: Unveiling Inflammatory Potential and Biomarkers for Cancer Treatment

Introduction. Along with driver genes, the inflammatory microenvironment plays a strong role in nurturing the malignant hemopoietic clone of Myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET) and Myelofibrosis (MF). Therefore, strategies to counteract the inflammatory niche are worth investigating. Extracellular vesicles (EVs) play a role in intercellular signaling due to their proteins, lipids and nucleic acids cargo and exert a role in the (tumor) inflammatory network. However, the EV cargo has been poorly investigated and their functional role remains unexplored in MPN. In this study, we profiled EV surface proteins and lipidomic to understand mechanisms in MPN pathogenesis and discover novel therapeutic targets. Our focuses are on whether and how EVs modulate the inflammatory microenvironment in MPN, Of note, Lemon-derived EVs are gaining interest due to their Vitamin-C-related anti-inflammatory activity. Thus, we investigated the ability of lemon-derived EVs, to regulate the inflammatory microenvironment of MPN.

Methods. EVs were isolated from platelet-poor plasma of PV (n=10) , ET (n=10) and MF (n=20) patients at diagnosis and sex/age-matched healthy donors (HD; n=30) by size-exclusion chromatography and characterized for the expression of EV-related markers (western blot), size and concentration (nanoparticle tracking analysis), morphology (transmission electron microscopy) and phenotype for a panel of 37 EV surface proteins including immune/inflammatory-, platelet-, cancer, and stemness-related markers (flow cytometry). Semi-quantitative lipidomic profiling of MPN/HD EVs was performed by liquid chromatography/mass spectrometry. Lemon-EVs were isolated from Citrus limon L. juice by differential ultracentrifugation. Through EV functional analysis, we investigated the pro/anti-inflammatory properties of PV, ET and HD EVs, with /without Lemon-EVs, via measuring the in vitro nitric oxide (NO) production (Griess Test) by mouse macrophages and the in vitro pro-fibrotic activity of MPN/HD EVs by measuring alpha-smooth muscle actin (αSMA) in human dermal fibroblasts assay.

Results. Surface protein profiling showed that PV-EVs had a significant enrichment of immune/inflammatory- (CD8, CD29), EV- (CD9, CD63, CD81) and platelet-associated markers (CD41a, CD42b, CD62P) compared to those from HD. Of note, the expression of CD9, CD41b and CD42a on the surface of PV-EVs positively correlated with platelet number. The expression of CD9, CD29, and CD63 was found elevated in ET-EVs. Also, MF-EVs displayed significant higher expression of immune/inflammatory-(CD86, CD20), stem cell/tumor-associated markers (CD133-1, CD24, CD146, ROR1) vs HD and significantly lower expression of CD326, an epithelial/tumor-associated marker. Focusing on lipid class analysis, we discovered that PV-EVs were significantly enriched in ether phospholipids (plasmenyl-linked phosphatidylcholine PC-P and ether-linked PC-O) in comparison to the HD. When comparing MF vs HD-EVs, MF-EVs exhibit significant enrichment in ether-linked PC, diacylglycerol, ceramide, and phosphatidylethanolamine. Interestingly, ether phospholipids take part of oxidative stress response. Conversely, no significant differences were observed in lipid class cargo between ET and HD-EVs. Functional bioassays indicated that PV- and ET-EVs induce a pro-inflammatory response by promoting in vitro NO production in murine macrophages. Notably, Lemon-EVs were able to reduce the pro-inflammatory activity of ET- or PV-derived EVs. Moreover, only MF-EVs were able to promote the fibrotic activity of fibroblasts by elevating the protein expressions of α-SMA.

Conclusion. The integrated analysis of protein/lipid cargo of MPN EVs reveals that circulating EVs are enriched of immune/inflammatory and platelet biomarkers with a potential diagnostic role in MPN liquid biopsy. We also, demonstrated that circulating EVs can promote the inflammatory environment of PV and ET. Lemon-EVs may counteract this effect. Furthermore, MF-EVs have the potential to promote fibrosis, resembling the bone marrow fibrotic pattern seen in patients. Our work highlights the potential therapeutic implications of modulating EV-mediated inflammatory pathways in PV/ET microenvironment and provides new insights into the pathophysiology of MPN.