Dissecting the role of CXCR7 in Cell Trafficking of Endothelial-Cells and Endothelial-Progenitor-Cells in Multiple Myeloma,

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis. The BM microenvironment in MM is characterized by an increased micro-vessel density and increased secretion of angiogenic factors. CXCR7 is a G-protein coupled receptor shown to play a major role in the adhesion, migration and angiogenesis of endothelial cells (ECs). Our interest is in the role of CXCR7 in cell trafficking of ECs and EPCs in MM. Thus we characterized ECs and EPCs from MM patients and MM animal models and examined the contribution of CXCR7 to the cell trafficking using in vitro and in vivo assays and using CXCR7-selective compound.

METHODS AND RESULTS: We used flow cytometry to detect the frequency of ECs and EPCs in the BM and peripheral blood (PB) of 5 MM patients and 5 normal subjects. ECs were detected as VEGFR+ CD133- cells, while EPCs were detected as VEGFR+ CD133+ cells. MM patients had significantly higher numbers of ECs and EPCs compared to healthy donors in both the BM and the PB. These results were confirmed in a mouse model of MM in which MM cells or vehicle were injected to SCID mice and the frequency of ECs and EPCs in the BM and the PB was determined 4 weeks after injection. We found that in mice with MM significantly higher numbers of ECs and EPCs could be detected in both the BM and the PB than in control mice. CXCR7 was expressed on both ECs and EPCs isolated from MM patients, healthy donors, and control mice. The expression of CXCR7 on EPCs was higher than the expression on ECs. The expression of CXCR7 on ECs and EPCs isolated from the BM was higher than the expression on ECs and EPCs isolated from the PB, respectively. Therefore, to test the role of CXCR7 in cell-trafficking of ECs and EPCs, we injected 10mg/kg of CXCR7 inhibitor POL6926, a potent and selective CXCR7 antagonist based on the Protein Epitope Mimetics (PEM) Technology (Polyphor, Switzerland), to BALB/c mice and tested the frequency of ECs and EPC in the PB and BM of the mice at 0, 2, 4 and 24 hours after the injection. We found a 3-fold increase in ECs and 6-fold increase in EPCs in the PB; 2 hrs post the injection of the CXCR7 antagonist. The levels of EPCs in the PB returned to baseline at 4 and 24 hrs, while the level of ECs was maintained at 4hrs and went back to baseline at 24 hrs. No significant differences were found in the frequency of ECs and EPCs in the BM after the injection of the CXCR7 antagonist. To investigate the function of CXCR7 in ECs in vitro we used human umbilical vein endothelial cells (HUVECs) as a model for ECs. CXCR7 was highly expressed on HUVECs. We could demonstrate that in vitro tube formation was promoted by either co-culture of MM cells or by conditioned medium from MM cell cultures. Furthermore, migration of HUVEC cells was facilitated by conditioned medium from MM cell cultures. These data suggest that MM cells may secrete factors promoting migration of endothelial cells and pro-angiogenic factors promoting angiogenesis. In addition, we could show that in vitro tube formation is inhibited by POL6926 suggesting that CXCR7 expression on HUVECs is required for tube formation. At the test concentrations POL6926 was not cytotoxic to HUVECs since cell proliferation was unaffected.

We have shown that the level of ECs and EPCs was elevated in the PB and BM of MM patients compared to normal subjects, a finding which was confirmed in a MM mouse model in which CXCR7 was highly expressed on these cells. Injection of PEM CXCR7 antagonist increased the numbers of ECs and EPCs in the PB. These results suggest that CXCR7 may play a role in the cell-trafficking and recruitment of ECs and EPCs in MM. To investigate this hypothesis, using in vitro tube formation and migration assays, we have shown that MM cells secrete factors that promote migration and angiogenesis of HUVECs and the PEM CXCR7 antagonist inhibits these processes. In subsequent studies POL6926 will be tested in vivo in animal models of MM to determine the contribution of CXCR7 in EPC trafficking and its contribution to angiogenesis progression in MM.

Zimmermann:Polyphor: Employment. Patel:Polyphor: Employment. Romagnoli:Polyphor: Employment. Roccaro:Roche:. Ghobrial:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.