**4. Conclusions and perspectives**

Altogether, we assume that the set of our studies are revealing the cellular and molecular dynamic interactions behind breast-cancer-derived pre-metastatic bone niche formation (**Figure 1**). There are still many questions about the factors that determine the chemotaxis of cells, which Ags or ligands are needed, as well as the modulating elements of this distant system. So far, we know, that the immune system is central. Multiple cues need to be investigated to translate our current knowledge toward clinical impact. If these immunophenotypes patterns are confirmed in human disease, this complex network can be used either as prognostic tools or even as therapeutic targets.

### **Figure 1.**

*Left panel: anti-osteoclastogenic cytokines produced by 67NR<sup>+</sup> CD8+ T cells keep bone homeostasis mediated by OBs and OCs cross talk. Right panel: BM pre-metastatic niche formation by 4T1 Th17 RANKL+ CD4+ T cells and RANKL<sup>+</sup> CD8+ T cells activities. DCs loaded with tumor Ags from primary tumor growth site prime naïve CD4+ and CD8+ T cells into draining LNs to differentiate into tumor-activated T cells producing RANKL and IL-17F, which in turn migrate to BM before tumor bone colonization. Migrating DCs can now activate the maintenance of tumor RANKL+ CD4+ Th17 T cells inside BM microenvironment dependent on IL-23 production. Dysregulation of bone homeostasis by an intense activation of BM-OCs by RANKL and M-CSF under breast tumor pre-metastatic osteolytic conditions induced by RANKL+ CD4+ Th17 T cells and RANKL+ CD8+ T cells. Both T cells activities support their osteoclastogenic potential in the establishment of the pre-metastatic niche. Left panel: Bone marrow scenario after subcutaneously 67NR non-metastatic tumor cells challenge. Production of anti-osteoclastogenic cytokines, IFN-γ + , IL-10, and anabolic levels of RANKL expressed by CD8+ T cells keep bone homeostasis mediated by OCs and OBs cross talk.*
