Roundabout receptors regulate axon guidance. A comparative transcriptomic analysis demonstrated that Robo1 and Robo4 receptors were overexpressed in a subpopulation of MDA-MB-231 breast cancer cells that only metastasise to bone (referred to as B02). Interestingly, high Robo4 expression in primary tumours from patients with breast cancer correlated with poor prognosis and increased risk of relapse to bone. In vivo, a 50% reduction in tumour burden was observed when Robo4-depleted cells were inoculated in the mammary fat pad of mice. Moreover, the extent of osteolytic lesions after injection of tumour cells into the caudal artery of mice was decreased in animals bearing Robo4-depleted tumours compared with animals bearing Robo1-depleted tumours. In vitro, the treatment of parental B02 cells with anti-Robo4 antibody dose-dependently inhibited B02 cell invasion. Altogether these results let us hypothesise that Robo4 might facilitate the engraftment of tumour cells in the bone marrow. To address this question, B02 cells were incubated with an anti-Robo4 antibody 1h before intra-tibial inoculation. The bone marrow was flushed from the hind limbs of animals 14 days after tumour cell inoculation. Flushed bone marrow cells were then cultured under antibiotic selection, enabling the selective growth of antibiotic-resistant tumour cells. The incidence of tumour cell colony formation was reduced by 50% when B02 cells were incubated with the anti-Robo4 antibody. In vitro, the co-culture of B02 cells with MC3T3-E1 osteoblastic cells generated an increase in Slit2 production, the Robo receptor ligand, by MC3T3 cells. Further, the treatment with anti-Robo4 antibody dramatically reduced B02 cell adhesion to MC3T3-E1 cells. These results provide strong evidence that axon guidance receptors Robo1 and Robo4 are involved in bone metastasis formation and that the use of an antibody directed against Robo4 receptor could lead to the development of innovative therapies to prevent metastatic niche formation in the bone marrow.
Disclosure: The authors declared no competing interests.