Background: Multiple myeloma (MM) is characterized by widespread involvement of the bone marrow (BM) at diagnosis, implying a continuous (re) circulation of the MM cells in the peripheral blood and (re) entrance into the BM. The normal process of B cell homing is regulated by cytokines and receptors such as SDF-1, CXCR4, VLA4, LFA-1, VCAM-1 and ICAM-1. In order to better understand the role of homing in MM, we developed an in vivo model which allows the continuous real-time imaging of MM cells as they home and adhere to the BM, as well as quantifying the numbers of cells in the circulation.

Methods: MM.1S (2 ×106/ml) were fluorescently labeled by incubation with the dialkylcarbocyanine membrane dye “DiD” (Molecular Probes) 1uM dye for 15 min at 37°C. Cells were i.v injected in Balb/c mice. Appropriate arterioles in the ear pinnae of the mice were chosen for obtaining measurements, and the fluorescence signal on the MM cells was excited as the labeled cells passed through a slit of light (from a 632 nm He:Ne laser) focused across the vessel. Cell counts were obtained every 5 min from the time of injection. MM cell homing to bone marrow vasculature of the skull was analyzed using fluorescence confocal microscopy. A small incision was made in the scalp so as to expose the underlying dorsal skull surface. The mouse was placed on a warmed microscope stage. Imaging duration was 1–3 hours per session. DiD was excited with a 635 nm diode laser. High-resolution images with cellular details were obtained through the intact mouse skull at depths of up to 250 um from the surface of the skull. Images from several depths were obtained and z-stacking was performed to merge the images. Quantitative evaluation was made by dividing the bone marrow into pre-determined quadrants (areas 1 to 4) and counting numbers of fluorescent cells per field. To demonstrate that that this new model identifies changes in homing of MM cells, we used the CXCR4 inhibitor AMD3100 and anti-VLA-4 antibody to inhibit homing to the BM. MM cells were pre-incubated with AMD3100 (50 uM overnight, Sigma, MO) or anti-VLA-4 antibody (1hr incubation with 10 ug/ml, BD Pharmingen, CA) or control PBS under the same conditions. In vivo flow cytometry and confocal imaging were then performed on control and AMD3100 treated mice.

Results: The number of cells in the control group decreased dramatically (86% decrease) after 1 hour indicating homing, whereas there was only a 47% reduction in the cells at 1 hour in the AMD3100 treated cohort, (p=0.002). Similarly, we demonstrated that the number of cells present in the perivascular bone marrow niches of the skull was significantly higher in the control mice as compared to the AMD3100-treated group at 1 hour after injection. The mean cell count in the AMD3100 treated mice decreased to 38% as compared to controls, p=0.01 Likewise, the use of anti-VLA-4 antibody demonstrated significant retention of MM cells in the circulation 1 hr after injection (4% reduction in circulating cell count vs 82% for control).

Conclusion: We describe a new model that detects in vivo real-time homing of MM cells from the peripheral circulation into BM niches, which can be used to study the trafficking of MM cells into and out of the BM, as well as the effect of novel agents on this dynamic process.

Disclosure: No relevant conflicts of interest to declare.

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