In Vivo Mobilization of Multiple Myeloma Cells Out of the Bone Marrow Using the CXCR4 Inhibitor AMD3100 and Bortezomib: Implications for Sensitization of Myeloma Cells to Apoptosis.

Abstract

Background: MM is characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of myeloma cells. The BM provides protection and resistance of MM to therapeutic agents. Therefore, disruption of the interaction of MM cells with their microenvironment should lead to enhanced sensitivity to therapeutic agents. We hypothesized that disrupting CXCR4/SDF-1 axis will induce mobilization of MM cells from the BM into the circulation.Methods: MM.1S cells were co-cultured with bone marrow stromal cells (BMSCs) in the presence of AMD3100 (50uM, Sigma), bortezomib (0–2.5nM, Millennium) or combination of both; and cell proliferation was measured using [3H]-thymidine uptake. We then tested the in vivo AMD3100-induced mobilization of MM cells after they homed to the BM. MM1.S cells that had been fluorescently labeled with DiD (Invitrogen) were injected into mice through their tail veins. Beginning 24 hours later and for three subsequent days, the mice were treated with 5mg/kg AMD3100 sq daily, injected with fluorescently labeled c-kit antibody for HSC detection and immediately monitored for presence of circulating MM1-S cells or HSCs, using in vivo flow cytometry. To test whether AMD3100 induces mobilization of MM cells in established tumors, a GFP+ and luciferase+ osteotropic MM.1S cell line (Luc+GFP+MM.1S) was developed. This mouse model was used for continuous, real-time quantitation of MM cells mobilization. Bioluminescence imaging was used to determine tumor growth in vivo. Mice were treated with AMD3100 (5mg/kg, daily), bortezomib (1mg/kg biweekly) or the combination.Results: There was a significant increase in proliferation of MM cells in co-culture with BMSCs compared to MM cells alone. Moreover, AMD3100 alone did not inhibit proliferation; however, it significantly enhanced the cytotoxic effect of bortezomib in the presence of stromal cells. In vivo studies revealed that, unlike HSC, no appreciable mobilization of MM1S cells occurred after the first AMD3100 injection; however, the second AMD3100 treatment induced a fourfold increase in circulating MM cell numbers above background, while no further remarkable increase in circulating HSC was observed. The effect of AMD on an established tumor model showed that by the end of the first week of AMD3100 or bortezomib treatment, a 2-fold increase of circulating MM cells was observed compared to control mice, a trend which continued for three weeks. Moreover, compared to AMD3100 or bortezomib treatment alone, a further increase of circulating MM cells was observed in the peripheral blood of mice treated with combination of AMD3100 and bortezomib following the second bortezomib treatment. In the AMD3100 only treated mice, the counts of circulating MM cell continued to increase and tumors continued to progress, while in the AMD3100-bortezomib-treated mice, the circulating MM cell count decreased and the tumors regressed.Conclusion: These data support the hypothesis that disruption of the CXCR4/SDF-1 axis mobilizes MM cells, and that AMD3100 can be used to enhance the effects of therapeutic agents such as bortezomib.