Netrin-1 and Its Receptors Unc5b and DCC May be Useful Targets for Preventing Multiple Myeloma Bone Lesions

Purpose: Multiple Myeloma is a hematologic malignancy that involves the malignant proliferation of plasma cells. This type of malignancy is particularly trophic to bone where it induces osteoclastic destruction of bone. We have recently demonstrated that osteoclast differentiation is dependent upon the autocrine/paracrine expression of the laminin-like matrix protein and chemorepulsant netrin1 and binding of netrin1 to the receptor unc5b. We therefore asked whether targeting netrin1 and its receptors unc5b and DCC could regulate myeloma spread in a murine model of myeloma.

Methods: 10⁶ 5TGM1-GFP myeloma cells (GFP expressing) were inoculated through the tail vein in 9 week old female C57Bl/KaLwRijHsd mice. Two weeks after inoculation animals were injected with monoclonal antibodies against Netrin-1, Unc5b and DCC weekly for 4 weeks (n=each). IVIS was performed for in vivo localization of myeloma bone lesions at the end of the experiment. Bone mineral density was measured by DEXA scanner and vertebrae and long bones were collected and prepare for microCT and histology analysis.

Results: IVIS imaging revealed a marked decrease in bone lesions in the anti-Netrin-1 and anti-Unc5b treated groups (n=6 and 9 mice respectively) when compared to control mice (n=6), whereas treatment results with anti-DCC antibody were more heterogeneous (n=7). There was an increase in total bone mineral density in anti-Unc5b and anti-DCC treated mice (0.63±0.09 g/cm³ and 0.8±0.09 g/cm³ respectively vs. 0.42±0.02 g/cm³, p<0.5). microCT analysis revealed no changes in cortical bone parameters (Bone volume/total volume (BV/TV), bone volume (BV), total volume (TV) and Bone Mineral Density (BMD)) for any treatment group, but there was increased in these parameters in Netrin-1 and Unc5b antibody treated groups when trabecular bone was analyzed, consistent with the decrease in myeloma lesions. There was also an increase in Trabecular thickness (Tb.Th), trabecular number (Tb.N.) and a decrease in trabecular separation (Tb.Sp.). TRAP staining revealed decreased osteoclasts in both anti-Netrin-1 and -Unc5b treated mice (7±1 and 6±1 cells/hpf respectively vs. 15±1 cells/hpf for control, p<0.001, n=5) but not for anti-DCC treated mice (13±1 cell/hpf vs. 15±1 cells/hpf for control, p=ns, n=5), and immunofluorescence analysis reveal a decrease in Cathepsin K positive that correlated with the decrease in TRAP-positive osteoclasts.

Conclusions: Netrin-1 and Unc5b treatment decreases osteoclast formation in a murine model of myeloma and decreases myeloma bone lesions. Targeting Netrin-1 or its receptor Unc5b may be a novel therapeutic approach for multiple myeloma.