**3.2 "The vicious cycle"**

Bone metastases are not established randomly, instead they request a complex reciprocal interplay between primary cancer cells and BM microenvironment stroma. Indeed, BM stroma provides an advantageous architecture for bone colonization, playing critical roles for breast cancer cells initial seeding, dormancy, and outgrowth [137]. Circulating breast cancer cells enter BM by the sinusoids—small blood vessels lined with fenestrated endothelial cells, more permissive than other types of capillaries [155, 156]. After extravasation into BM, they migrate to the perisinusoidal or to the endosteal/subendosteal niche, where OBs and other stromal cells secrete a variety of chemo-attracting factors, such as CXCL12, RANKL, osteopontin, and BMPs [155, 156]. Breast cancer cells express high levels of CXCR4—the receptor for CXCL12, which increase their ability to survive in BM and the establishment of overt metastases in this microenvironment [155, 156]. Moreover, CXCL12 stimulates PI3K-AKT signaling pathway and Src activity, which enhance cancer cell survival in challenging environments [41]. These results obtained from animal model studies were validated in clinical datasets, in which Src and CXCR4 expression in tumor cells was associated with breast cancer bone relapse [155]. In BM niches, metastatic cells adapt, survive, and reside for a prolonged period of time—possibly years or even decades [137].

When invading breast cancer cells escape from dormancy, they disrupt the normal bone remodeling process in order to promote their outgrowth, eventually leading to the development of overt bone metastases [137]. Metastatic breast tumor cells express and secrete a series of molecules, such as parathyroid hormone-related protein (PTHrP), IL-11, and TNF-α, vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), lysyl oxidase (LOX), RANK, RANKL, and IL-6, which in turn mobilize and activate OCs to resorb bone matrix and release chemotactic stimuli and additional growth factors attached to

the bone matrix [126, 130, 157]. Bone matrix degradation by the hyperactivated OCs releases TGF-β, which in turn is activated due to pH changes in the local environment and proteolytic cleavage from latent peptides [128]. In sequence, this molecule triggers the production of osteolytic factors, such as PTHrP, IL-11, IL-1β, and Jagged1 from breast cancer cells [156, 158– 165]. Jagged1 promotes osteoclastogenesis via Notch signaling in pre-OCs, while PTHrP induces the production of RANKL by OBs [32]. Activated OCs then degrade the bone matrix on cortical and trabecular surfaces, leading to the release of numerous growth factors, including more TGF-β [158]. Consequently, TGF-β-induced Jagged1 enhances a vicious cycle between bone and tumor cells, by stimulating the expression of IL-6 from stromal cells and OBs, promoting tumor growth [158]. VCAM1 also stimulates the outgrowth of metastases, through the recruitment of OCs progenitors via expression of integrin α4β1 [166]. Importantly, several therapies using denosumab—monoclonal antibody against RANKL [167]; and monoclonal against human Jagged1 [168] or small-molecule inhibitors—have already been approved for clinical use or are under development to treat osteolytic bone metastases, by preventing progression of the vicious cycle [169, 170].

In the last few years, accumulating evidences suggest that breast cancer bone colonization is preceded by changes in BM microenvironment [171–173]. In this context, a pre-metastatic niche is established by cellular and molecular mechanisms, mostly educated by the primary breast cancer cells [64, 171, 174]. Therefore, tumor cells prepare BM microenvironment to host them, before "switching homes" and moving to bone [171]. Importantly, the pre-metastatic niche formation also leads to the disruption of bone remodeling system, in favor of osteoclastogenesis and bone consumption, but prior to metastatic cells arrival [64, 157, 174]. Accordingly, the pre-metastatic osteolytic lesions facilitate subsequent bone tumor colonization [175].

## **3.3 Bone pre-metastatic niche formation**
