**1.1 Bone marrow: an overview**

Bones provide both skeletal scaffolding and a unique microenvironment for hematopoiesis and B cell ontogenesis, osteogenesis, and also function as an immunological memory reservoir, in its marrow [1–3]. The bone marrow (BM) is a complex and dynamic structure composed of different and distinct compartments or niches, which accommodate a multitude of cell types, which functionally create an interactive network, critical for BM/bone integrity [2, 4–8]. These niches are composed of different stromal cell types—osteoblasts (OBs), osteocytes, reticular and perivascular cells, endothelial cells, mesenchymal cells (MSCs), smooth muscle cells, macrophages, and dendritic cells (DCs). Disruptions in these compartments can lead to aberrant pathological processes [2, 8–11].

At least three niches can be identified in the BM: (i) the endosteal/subendosteal niche that supports self-renewal and differentiation of hematopoietic stem cells (HSC); (ii) the central niche for multipotent progenitors (MPP); and (iii) the perisinusoidal niche that guarantees the differentiation to the lineage committed progenitors and hematopoietic cells full commitment [4, 11–14]. The endosteal/ subendosteal niche contains OBs, bone-forming cells, and osteoclasts (OCs), boneresorbing cells, as well MSCs, all collaborating to regulate hematopoietic homeostasis and osteogenesis [13]. The central and perisinusoidal niches recruit MSCs, as well as endothelial cells and their progenitors, to promote HSCs proliferation, mobilization, and differentiation,—(i) myelopoiesis, the process in which innate immune cells, such as granulocytes and monocytes, develop from a myeloid progenitor cell; and (ii) lymphopoiesis, the process in which adaptive and innate lymphocytes develop from a lymphoid progenitor cell [5, 15].

More recently, the anatomy of myelopoiesis in the BM was partially mapped *in situ* and the clonal relationships between myeloid progenitors and surrounding cells were assessed [16]. It was demonstrated that colony stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor (M-CSF), produced by perisinusoidal vessels provides a unique niche that regulates, spatially organizes, and controls myeloid differentiation [16]. This type of study provides valuable information on the organization of the various niches inside the BM. Dissection of these processes will allow a better understanding of their influence on bone and/or BM and vice versa, during homeostasis and local and/or systemic diseases, which directly or indirectly affect bone/BM homeostasis.

## **1.2 Bone metabolism and its central players**

Bone tissues in adults are classified as: (i) cortical (long and compact bone) and (ii) trabecular (flat and spongy or cancellous bone) [17, 18]. During fetal development, long bones are modeled by endochondral ossification, in which the cartilage formed by chondrocytes—cells of mesenchymal origin, essential for formation and maintenance of cartilage—is replaced by bone at the edge of the growth plate [19, 20]. The cortical bone is mostly structural, supporting the stability and movement of the body, made up of compactly packed osteons—its key structural unit, formed by layers called lamellae, surrounding the Harvesian canal, which contain small blood vessels responsible for blood supply to osteocytes, former OBs embedded in the bone matrix as differentiated cells [21, 22]. The trabecular bone is highly porous and vascularized and harbors red and white bone marrow [17]. Bone matrix is composed of an organic segment, formed by type I collagen secreted by OBs, and a variety of non-collagenous proteins, such as osteocalcin and osteopontin; and an inorganic segment, also known as bone mineral, formed by calcium, phosphorus, and magnesium, which originates the hydroxyapatite [Ca10(PO4)6(OH)2] [17].

### *Perspective Chapter: Breast-Tumor-Derived Bone Pre-Metastatic Disease – Interplay... DOI: http://dx.doi.org/10.5772/intechopen.107278*

Even after the modeling phase, bone tissue is constantly renewed by a process called bone remodeling. Bone homeostasis is achieved by the performance of bone remodeling system, which is conducted by the synchronized activities of OBs, OCs, and osteocytes [23–25]. Osteocytes are the most abundant cells in bone tissue and play an essential role in bone homeostasis [26]. They translate mechanical—pressure and tension—low oxygen, matrix mineralization, and hormonal stimuli into biochemical signals, due to their extensive long cytoplasmic extensions. The complex network formed by osteocytes in the bone matrix, enables direct communication among them and other effector cells in the bone/BM, including OBs and OCs [21, 22, 26–29]. OBs, derived from mesenchymal progenitors, promote mineralization and bone formation by secreting matrix vesicles containing type I collagen, alkaline phosphatase, and osteocalcin [22]. OCs, derived from myelomonocytic progenitors, otherwise, dissolve and absorb bone matrix by releasing hydrogen ions that acidify the bone interface and secrete lysosomal enzymes—such as tartrate-resistant acid phosphatase and cathepsin K [30–34].

The receptor activator of nuclear factor-κB (RANK)/receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) molecular system is the most important pathway activated during bone remodeling process [35–38]. Notably, BM stromal cells are responsible to initiate osteoclastogenesis, being the main sources of M-CSF and RANKL [39]. Osteocytes first release M-CSF causing myelomonocytic progenitors to commit to the OC line [26, 39]. M-CSF stimulates RANK expression in the late stages of OCs development, which interact with RANKL expressed on or secreted by OBs and osteocytes [26]. This interaction leads to the activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways, through tumor necrosis factor receptor–associated factor 6 (TRAF-6) and c-Fos molecules [30, 31, 33, 34, 40], giving rise to large multinucleated differentiated mature OCs [40]. RANKL activation also induces the expression of nuclear factor of activated T cells c1 (NFATc1), the master transcription factor for osteoclastogenesis [41]. B-lymphocyte-induced maturation protein 1 (Blimp1), which can be induced by NFATc1, downregulates the expression of the transcriptional factors interferon (IFN) regulatory factor 8 (IRF-8) [42, 43] and B-cell lymphoma 6 (Bcl6), in turn promoting osteoclastogenesis [43].

Notably, mice that lack RANKL or its receptor RANK develop severe osteopetrosis accompanied by a defect in tooth eruption due to a complete lack of OCs [42, 43]. Conditional deletion of RANKL in chondrocytes [44, 45] and OBs led to a severe osteopetrosis [22, 43, 45–47], whereas osteocytes-specific RANKL-deficient mice displayed a high bone mass phenotype at the adult stage [43, 45]. Thus, chondrocytes and OBs are the major source of RANKL in supporting osteoclastogenesis during skeletal development, whereas osteocyte-derived RANKL contributes to bone remodeling at the adult stage [44, 45] . In humans, loss-of-function mutations in *Tnfrsf11a* (gene encoding RANK) and *Tnfsf11* (gene encoding RANKL) genes cause autosomal recessive osteopetrosis with a complete lack of OCs [43, 48].

OBs and osteocytes also settle the termination of osteoclastogenesis [21, 27, 29, 49]. This step initiates through the secretion of OPG—the RANKL decoy receptor, the main counter-regulator of osteoclastogenesis, which attenuates bone resorption by binding to RANKL with higher affinity than RANK and blocking RANKL osteoclastogenic effects [50]. Of note, mice lacking *Tnfrsf11b* (gene encoding OPG) exhibited severe osteoporosis due to an increased OC number and severe bone resorption [43, 50–52]. The same cells control the beginning

of osteoblastogenesis, and several molecules regulate this next step, including parathyroid hormone (PTH), the RUNX Family Transcription Factor 2 (RUNX2), osterix transcription factor, bone morphogenetic protein (BMP), and the Wnt pathway [40, 53]. The Wnt signaling pathway is the most important player in osteoblastogenesis, preventing apoptosis of OBs and accelerating its cell cycle progression and proliferation, leading to inhibition of adipogenesis [54]. Wnt molecules activate G-protein-coupled receptors and coreceptors of the low-density lipoprotein receptor (Lrp) family, resulting in β-catenin activation, effectively upregulating aerobic glycolysis, β oxidation, and other anabolic mechanisms, through activation of the RUNX2 gene [54]. Moreover, binding of BMP to BMP receptors leads to their dimerization followed by phosphorylation of Smad proteins (main signal transducers for receptors of the TGF-β superfamily), which in turn also activate RUNX2, upregulating OB activity and differentiation [54, 55].

More recently, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) was reported to be another receptor for RANKL, which negatively regulates osteoclastogenesis by not only competing with RANK for RANKL binding, but also inhibiting NFATc1 activation via Gq protein alpha subunit (Gαq) [56]. Interestingly, OCs can also regulate the activity of OBs, by secreting bone morphogenetic protein-6 and sphingosine-1-phosphate, which function as coupling factors promoting OBs proliferation and bone formation [57]. Finally, osteocytes negatively regulate osteoblastogenesis by secretion of Dickkopf-1 (DKK-1) and sclerostin molecules, both antagonists of the Wnt pathway [58, 59]. Sclerostin is a marker for mature osteocytes, and its expression increases with age [60, 61], and mice deficient in this molecule show an increase in osteoblastogenesis and a decrease in the shape of BM cavities, resulting in impairment of hematopoiesis and B cells ontogenesis [62].

Taking together, we conclude that intra and intercellular and molecular interactions between osteocytes, OBs, OCs, and chondrocytes are crucial for maintaining the BM/bone niches, under physiological conditions. Currently, we know that bone remodeling process is also regulated by immune cells, residing at, or migrating to BM, such as T and B cells, innate lymphoid cells, macrophages, DCs, and other hematopoietic cells [63]. Any imbalance in one of these connections can lead to several bone pathologies, including, among others, breast-cancer-derived bone metastases [64].
