Bone Marrow Mobilization Of Endothelial Progenitor Cells Represents An Early Pathogenic Event During Multiple Myeloma Progression
Authors: Moschetta Michele , Mishima Yuji , Aljawai Yosra , Sahin Ilyas , Calcinotto Arianna , Manier Salomon , Maiso Patricia , Sacco Antonio , Bellone Matteo , Roccaro Aldo M , Ghobrial Irene M. 
Published: October 2019

Abstract

Background
Increased bone marrow (BM) microvessel density (MVD) has been associated with progression of multiple myeloma (MM). Endothelial progenitor cells (EPCs) are circulating precursors with the capacity to differentiate into endothelial lineage cells through a process known as “vasculogenesis” thus contributing to vessel formation. The role of these cells in MM pathogenesis remains largely unexplored. We studied EPC BM mobilization in several MM mouse models (5TGM1, MM1S and Vk*MYC) during disease progression and quantified these cells in peripheral blood (PB) from patients at different stages of MM disease.

Methods
EPCs were quantified using flow cytometry (circulating CD34+ VEGFR2+ cells) in PB from mice injected intravenously (i.v.) with either murine MM 5TGM1-turboRFP+ cells or human MM1S-GFP+/luc+ cells. Circulating EPCs were also enumerated in PB from mice previously transplanted with BM from SCID/GFP mice (GFP-BM SCID mice) after the i.v. injection of 5TGM1-turboRFP+ cells as circulating GFP+ CD34+ VEGFR2+ cells. Peripheral blood samples were obtained from transgenic Vk*MYC mice affected by smoldering (s) MM (M-spike less than 6% of total protein on SPEP); active (a) MM (M-spike higher than 6% of total protein on SPEP); or healthy syngeneic mice, and examined for EPC levels through flow-cytometry (circulating CD34+ VEGFR2+ cells). Finally, the level of EPCs was evaluated in PB from healthy controls, smoldering (s) MM patients, in remission (r) and active (a) MM patients by using flow-cytometry (CD34+VEGFR2+ cells) and in vitro by performing endothelial colony forming assays [endothelial cells colony forming units (EC-CFUs) and endothelial colony forming cells (ECFCs)].

Results
An increase in EPCs was evident starting one week after i.v. injection of tumor cells in both murine 5TGM1 and human MM1S orthotopic models. Compared to control mice, this EPC increase became significant (P<.05) two week after injection of 5TGM1 cells (8.2 times increased, p=0.04) and three weeks after MM1S cell injection (4.1 times increased, p=0.04). The baseline percentage of CD34+VEGFR2+ cells in normal SCID mice, and of GFP+CD34+VEGFR2+ cells in GFP-BM SCID mice was comparable (0.0176% and 0.0156% of PBMCs respectively). A significant increase of EPCs to 0.059% (p=0.04) and 0.049% (p=0.02) was observed in SCID or SCID-GFP-BM mice after 2 week of tumor engraftment of 5TGM1-turboRFP+ murine myeloma cells. This, suggest that the origin of EPCs is from the bone marrow and that the number of EPC increases with MM progression. In transgenic Vk*MYC mice, a significant increase (P<.05) of circulating CD34+VEGFR2+ cells was observed in both sMM (2.88%) and aMM (1.706%) compared to healthy syngenic mice (0.38%). This further suggests that an increase of vasculogenic activity takes place at the early stage of MM development and persists during MM progression. We next corroborated our findings in MM patients. Smoldering MM patients as well as in remission and active MM patients presented in PB a significant increase in circulating CD34+VEGFR2+ cells compared to healthy donors (0.039% and 0.042% and 0.078% respectively vs 0.005%, P<.05)
Peripheral blood mononuclear cells (PBMCs) from sMM, rMM and aMM showed an increase ability to form both EC-CFUs (10.05 and 12.85 and 16.05 respectively vs 6.4 colonies per 20x106 total PBMCs) and ECFCs compared to healthy controls (0.75 and 0.51 and 0.75 respectively compared to 0.33 colonies per 15 ml blood).

Conclusion
Together, these results demonstrate that vasculogenesis may represent an early pathogenic event driving MM progression. Vasculogenesis mediated by BM-derived EPC could contribute to the “angiogenic switch” described during the transition MGUS/sMM to overt MM. Therefore, EPCs represent a new cell target in MM that could potentially halt MM progression.

Disclosures:
Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.