Invasion of breast cancer cells into surrounding tissues arises from alterations of intercellular adhesion and the adhesion between cells and the extracellular matrix (ECM). The role of the cadherin family is prominent in this process (6). E-Cadherin is vital in mediating cell-to-cell adhesion, and the downregulation of E-Cadherin leads to attenuated intracellular adhesion of breast cancer cells, leading to the detachment of cancer cells from the primary lesion (7). Therefore, downregulated E-cadherin is closely correlated with advanced invasion and poor prognosis of breast cancer (8). N-cadherin is closely associated with epithelial-mesenchymal transition (EMT) in breast cancer cells and proved to be another vital factor for tumor invasion (9). High expression of N-cadherin increases the adhesion of tumor cells to stromal cells which facilitates tumor cells to colonize the matrix (10). Cluster of differentiation(CD) 44 is also an important marker of EMT which exerts pivotal role in metastasis of breast cancer. Variant CD44 was detected from the standard isoform during EMT (11). Another EMT marker, α-smooth muscle actin (α-SMA), was highly expressed in carcinoma cells and associated with invasion of tumor invasion (12). EMT induces the production of proteases involved in ECM degradation to enhance the tumor invasion (13). ECM degradation enhances tissue penetration, which is also an essential step in tumor invasion. The degradation of ECM is mainly accomplished by Matrix Metallopeptidase (MMP) and urokinase plasminogen activator (uPA) (14). In breast cancer patients, uPA levels are closely related to the risk of distant metastasis (15). Inhibition of uPA by small interfering RNA (siRNA) can inhibit tumor invasion, and the expressions of MMPs are suppressed simultaneously (16). MMP-mediated degradation of ECM proteins is a prerequisite for breast cancer cell infiltration (17).

Increased expression of heparan sulphate proteoglycans (such as Glypican-1 and syndecan-1) has been observed in the advanced stages of breast cancer (18). Heparan sulphate proteoglycan is the proteoglycan in ECM or cell surface, which helps to maintain the integrity of ECM and mediate the interaction between cell matrix adhesion and growth factor receptor (19). Heparinase (a type of β-glucosidase) can promote ECM degradation by decomposing heparan sulphate proteoglycans (20). tumor cells can synthesize heparinase to degrade heparan sulphate to increase tumor cell invasiveness. Studies have confirmed that overexpression of heparinase in MCF7 cell-lines in vitro and in vivo promote cell proliferation and matrix invasion (21).

The migration of tumor cells can be accomplished individually or in a coordinated manner. Moderately and highly differentiated breast lobular carcinoma cells prefer to coordinate migration whilst poorly differentiated tumors are inclined to undergo single cell migration due to the abnormal structure and function of intercellular adhesion proteins (22). The co-migration of tumor cells requires a firm intracellular connection in case of being scattered. As a result, they usually aggregate as emboli after invasion in blood vessels (23). EMT is a key process in the mesenchymal movement of a single migrating cell. During EMT, tumor cells lose their epithelial phenotype (E-cadherin expression) and express mesenchymal markers, such as N-cadherin, SNAI1, SLUG (SNAI2), TWIST, vimentin, fibronectin (24). Breast tumor cells that undergo EMT are more aggressive. They can remodel their shapes to move through the degraded ECM with the least resistance (25). The transcriptional repressors of E-cadherin include E-box-binding homeobox 1 (ZEB1), zinc finger E-box-binding homeobox 2 (ZEB2), twist related protein (Twist), zinc Finger proteins, Snail and Slug, etc., which initiate EMT through TGF-β, Wnt, and phosphatidylinositol 3’kinase serine/threonine kinase (PI3K/AKT) pathway and indicate poor prognosis of breast cancer (26).

Tumor stromal cells promote tumor cell migration. Most stromal cells in breast cancer are fibroblasts, commonly referred to as cancer-associated fibroblasts (CAF). Conditioned medium collected from CAF can promote breast cancer cell motility and invasion in vitro (25).

The tumor microenvironment (TME) is composed of fibroblasts, immune cells, blood vessels and the extracellular matrix (ECM) (27, 28) and exerts crucial effect in tumor metastasis. The complexity of the TME means that tumor development and progression rely not only on the tumor cells themselves but also on stromal and immune cells. CAFs can provide both the physical support and direct the intracellular communications (29). CAFs undergo the Reverse Warburg effect and provide cancer cells with glycolytic metabolites (30), and CAF-derived exosomes can reprogram the metabolic pathway of cancer cells (31). ECM also provide the architectural support to faciliate the cell adhesion、water and growth factors preservation for the cancer cells. During the tumor progression, cancer cells lead to the stiffness of ECM, and the stiffen ECM contributes to abnormal proliferation、enhanced metastasis、immuno-suppression、resistance to theraputics in return (32). Macrophages in the tumor environment can interact with breast cancer cells and endothelial cells to form a niche to facilitate tumor colonization, proliferation, and escape from immune surveillance (33). T cells, neutrophils, and other immune cells also play crucial roles in breast cancer metastasis, influencing both the progression of the disease and the response to treatment. T cells are a critical component of the adaptive immune system and can infiltrate the TME where they exert anti-tumor effects. However, their function can be impaired within the TME due to various immunosuppressive mechanisms. Induced by IL1β, γδ T cells were capable of producing IL17, which leads to the systemic expansion and polarization of neutrophils dependent on G-CSF(Colony Stimulating Factor) in mice with mammary tumors. These tumor-generated neutrophils gain the capacity to suppress cytotoxic CD8+ T cells, which are crucial for controlling metastasis. Neutralizing IL17 or G-CSF, as well as the absence of γδ T cells, prevented the accumulation of neutrophils and reduced their T cell-suppressive characteristics. Furthermore, the lack of γδ T cells or neutrophils significantly diminished metastases in the lungs and lymph nodes without affecting the progression of the primary tumor. These findings suggest that targeting the newly identified immune pathway involving γδ T cells, IL17, and neutrophils could be a promising strategy to prevent metastatic disease (34). IL-22 induced by T cell helps to elevate CD155 expression by cancer cells, which interrupts NK cell function and activates immunosuppressive circuit to enhance lung metastasis (35). Increased CD8+ T cell infiltration with promoted T cell immunity infiltration was associated with reduced breast cancer distant recurrence (36). Activation of GM-CSF-JAK/STAT5-C/EBPβ pathway helps TINs to defense against ferroptosis via the Acod1-dependent immunometabolism, which inflicts antitumor T cell immunity and enhances metastasis (37). MHCII^hi neutrophils were shown to facilitate the metastasis of breast cancer to the lung, which were recruited by C-C Motif Chemokine Ligand 2 (CCL2) from lung tissues with chronic pulmonary infection, acts as the vital bacterial-immune mediator to bridge chronic infection and lung metastasis of breast cancer in a cell-intrinsic manner in a mouse model (38). Co-cultured BC cells with mesenchymal stem cells increased the expression of the receptor activator of nuclear factor κB (RANK) and epidermal growth factor receptor (EGFR) to facilitate osteoclastogenesis, which also indicated EGFR signalling could be a promising strategy to intercept bone metastasis (39). However, involvement of immunity and therapeutic opportunities in bone metastasis of breast cancer provoke more investment and intensive research.

Tumor cells themselves may affect the microenvironment of the secondary site before metastasis, establishing a “pre-metastasis niche” (40). Vascular endothelial growth factor receptor 1 (VEGFR-1)-positive clusters of hematopoietic progenitor cells are observed in the pre-metastatic lymph nodes of breast cancer patients before the tumor cells spread to the distant site (40). Chemokines are involved in the colonization of tumor cells to target organs. Chemokine receptor 4 (CXCR4) is highly expressed by breast cancer tissues, and its ligand, chemokine ligand 12 (CXCL12), is mainly in the lymph nodes. Organs with high CXCL12 expression are associated with some sites of metastatic breast cancer, such as the lung, bones and lymph nodes (41). The interaction of CXCR4-CXCL12 promotes the migration of breast cancer cells to the common site (41). Another important aspect of metastasis is neovascularisation, which provides nutrition and oxygen for metastases (42). Tumors grow faster than normal tissues and this easily leads to hypoxia in the lesions. Hypoxia stimulates production of pro-angiogenic factors in tumor cells and promote the formation of new blood vessels. For example, hypoxia-inducible factor-1 (HIF-1) triggers the production of an angiogenic protein vascular endothelial growth factor (VEGF) (42, 43). Through binding with specific VEGF receptors, VEGF can enhance the proliferation of vascular endothelial cells and increased the permeability of micro-vessels to induce neovascularisation (44). However, the new rapidly formed tumor vessels are excessively branched with varying shunts and diameters, which are different from the normal vessel both in structure and function. Abnormal blood vessels do not provide sufficient oxygen for the tumor, leading to a vicious cycle of tumor hypoxia (45). In breast cancer, the expression of VEGF indicates a poor prognosis and the tumor is prone to metastasis (46).

Normal bone metabolism is the process within dynamic balance of bone remodeling which is well orchestrated by osteoblasts, osteoclasts and osteocytes. Bone remodeling is a continuous process where old bone is removed (bone resorption) and new bone is formed (bone formation). Bone remodeling, a process in which osteoclasts and osteoblasts coordinate with each other, can regulate calcium homeostasis, repair bone damage to resist stress and maintain skeletal system function. The remodeling process is initiated by various signals, including mechanical stress and hormonal changes (51). Osteoclasts are large, multinucleated cells responsible for the resorption of bone. Once the pre-osteoclasts are stimulated and differentiate into mature osteoclasts, osteoclasts attach to the bone matrix and form resorption lacunae by secreting enzymes and acids to dissolve the mineral matrix and collagen fibers.This process releases minerals like calcium and phosphate into the bloodstream (52). After resorption, the area undergoes a transitional phase where the resorbed bone surface is prepared for new bone formation. Osteoblasts, known as mesenchymal stem cells differentiate from precursor cells, are responsible for the formation of new bone. They produce new bone matrix and initiate its mineralization (53). Osteocytes are mature bone cells that originate from osteoblasts. They become embedded in the bone matrix and help maintain the bone tissue. Osteocytes communicate with other bone cells to regulate the remodeling process (54). Studies revealed that several molecular mechanisms promote the bone resorption process, in which the receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) are critical in regulating osteoclast function (55). Osteoclast precursors (monocytes/macrophages lineage) express a receptor called RANK (Receptor Activator of Nuclear factor Kappa-B). RANKL binds to RANK on the surface of these osteoclast precursors The binding of RANKL to RANK initiates a cascade of intracellular signaling events within the osteoclast precursor, primarily involving the activation of the NF-κB (Nuclear Factor Kappa-B) pathway and other downstream signaling pathways like MAPK (Mitogen-Activated Protein Kinase) (55, 56). These signaling pathways promote the differentiation and maturation of osteoclast precursors into fully functional multinucleated osteoclasts. Mature osteoclasts attach to the bone matrix and secrete hydrochloric acid (HCl) and lysosomal enzymes, like cathepsin K, which degrade the organic matrix and dissolve the mineral components of bone (57).This activity creates small pits or resorption lacunae on the bone surface, effectively re-absorbing bone tissue. Osteoblasts and other cells also produce OPG (osteoprotegerin), a decoy receptor that binds to RANKL, preventing it from interacting with RANK. This serves as a natural inhibitor of RANKL-mediated osteoclastogenesis and bone resorption (58). Besides RANKL pathway, hormones, cytokines and growth factors influence the proliferation of osteoclasts and osteoblast progenitor cells. Parathyroid hormone (PTH) induced the generation of osteoclast to enhance osteoclast mediated bone resorption (59). IL-6, IL-8, TGF-β, and other molecules are also involved in stimulating osteoclast activity and supporting cancer cell survival and growth in bone ( Table 1 ). Sex steroids inhibited maturation of osteoclasts, and reduced secretion of sex hormone enhanced the activity of osteoclasts, which may lead to apoptosis of bone cells to promote bone resorption (60).

The process of bone metastasis of breast cancer is a multiple-step cascade which contains four main steps: (1) invasion, proliferation and dissociation of cancer cells from the primary lesion, (2) intravasation and dissemination in the circulation (3) extravasation of cancer cells (4) colonization in the bone, disseminated tumor cells settle down in the bone niche, where it is normally hosted by hematopoietic stem cells (HSCs), followed by a survival under dormancy, reactivation and ultimate outgrowth (61, 62).

The bone matrix itself provides a physical barrier which may impede the colonization of cancer cells. Bone marrow is an immunological niche where immune cells like T-cells and natural killer (NK) cells can survey and eliminate aberrant cells (63), including cancer cells. Osteoblasts and other bone marrow stromal cells can out-compete cancer cells for space and nutrients, limiting their growth. The bone matrix releases anti-angiogenic factors including angiostatin, interferons (α, β and γ), endostatin, interleukin-12 and retinoids that can suppress the formation of new blood vessels that are required during tumor growth (64). However, once the dynamic balance is destroyed, it may lead to osteolytic lesions, presenting lower bone density or osteoblastic lesions, with excessive bone deposition. After menopause, due to the rapid decline in oestrogen levels, osteoclasts are active and bone loss is accelerated. After breast cancer patients received chemotherapy or hormone adjuvant therapy, the risk of low bone density and osteoporosis has been found to be increased (65). When breast cancer cells spread to the bones, they will gradually adapt to the bone microenvironment, destroy the bone homeostasis, then, start a vicious cycle of bone metastasis under various mechanisms.

Although it is lacking in understanding, characteristics of bone environment and properties of breast cancer cells certainly bear traits at levels of tissues, cells and genes for the predisposition of bone metastasis from breast cancer. The inorganic phase of bone is mainly composed of the mineral hydroxyapatite nanocrystals (HA). High HA induced the secretion of pro-osteoclastic interleukin-8 (IL-8) by MDA-MB-231 cells to facilitate bone colonization (66). The extracellular bone matrix is enriched with type-I collagen, osteopontin (OPN), and bone sialoprotein (BSP). Elevated expression of OPN and BSP can facilitate tumor cell adhesion to collagen and increase metastatic propensity to bone (67, 68). The skeletal microenvironment is known to be a highly hypoxic environment and the pressure of oxygen (pO2) in mouse bone marrow is significantly lower than other tissues or organs (69). Hypoxia is known to be involved in various steps of bone metastasis, including the premetastatic niches, dormancy and osteolytic vicious cycles (70, 71). Bone marrow hypoxia can promote the expression of HIF-1 (72), which subsequently induce the secretion of C-X-C motif chemokine 12 (CXCL12) (73). Upon binding with Ca²⁺ and chemokine receptor 4 (CXCR-4), CXCL12 activate multiple signalling pathways such as PI3K/Akt, ERK/MAPK pathway to facilitate the colonisation of disseminated tumor cells in bone tissue (74). Hypoxia could also enhance the activity of osteoclasts and suppress the differentiation of osteoblasts (70). The bone environment contains a lot of alkaline minerals (hydroxyapatite) and the buffer system to maintain a normal pH value. In the early stage of bone metastasis, due to the hypoxia and excess secretion of H+ both inside and outside of the cell membrane, caused by a high glycolysis status in the tumor cells, the bone microenvironment is maintained in a state of acidosis. Among this process, vacuolar H⁺-ATPase (V-ATPase) performed actively for bone microenvironment acidosis, which was expressed in both tumor cells and osteoclasts (75). Acidosis significantly enhanced the activity of osteoclasts with elevated secretion of cytokines, leading to bone loss, such as activated T-cell nuclear factor 1 in activated osteoclasts (76). Acidosis could also inhibit the biological functions of osteoblasts, leading to impaired trabecular bone formation and promoted the expression of osteoclast RANKL (77). In addition, the acidosis environment activated NF-κB signal transduction pathway in mesenchymal stromal cell can promote the secretion of inflammatory factors, chemokines and growth factors, such as IL-1, IL-6 and CXCL2, which can subsequently induce tumor-induced nociception and hyperalgesia to facilitate invasion and immune escape (75).

Trabecular of cancellous bone is fenestrated which contains rich blood vessels with slow blood flow and is suitable for breast cancer cells to colonise after successfully spreading through blood circulation (78).

Various kinds of tumor cells including ovarian, gastric and colorectal cancers can be detected in the bone marrow (79–81), which indicates that the bone metastasis of breast cancer at the initial stage is passive. However, only a few kinds of cancer cells including breast cancer cells can form overt metastatic bone lesions (49), which indicates that passive dissemination of breast tumor cells to the bone marrow is an early step in forming bone metastasis, but it is not the critical driving event of bone metastasis. Apart from the passive transportation, the properties of breast cancer cells are essential in bone metastasis. The bone environment is a reservoir for minerals, especially for calcium ions. Breast cancer cells highly express calcium-sensing receptor (CaSR), which could bind with Ca²⁺, and promote breast tumor cell spread to the bone tissue with high Ca²⁺ concentration. In vitro studies have shown that extracellular Ca²⁺ combined with CaSR expressed by tumor cells activate AKT and MAPK pathways to enhance migration and proliferation of cancer cells, whilst application of CaSR antagonists to interfere with renal cancer in mice significantly reduced the incidence of bone metastasis (82).

RANK was highly expressed on the surface of breast cancer cells, while RANKL was overexpressed in bone tissues (83, 84). In addition, the chemokine receptor CXCR4 was highly expressed in breast cancer tissues, and its ligand CXCL12 was overexpressed in common metastatic sites of breast cancer including bone marrow (41). Interaction mediated by these molecules between cancer cells and bone microenvironment may account for at least partially for the predisposition of breast cancer to metastasize to the bone ( Figure 1 ).

Bone metastasis is preferential in poorly differentiated and ER-positive breast tumors (85), upregulated trefoil factor-1 (pS2 or TFF1) was reported as the potential biomarker for the predilection of bone metastasis in early stage of ER-positive breast carcinoma rather than the advanced stage (86). Notably, although the early incidence of bone metastasis in ER-negative cancers is higher, the frequency of bone recurrence is higher in ER-positive cancers (87), which may caused by the reduced ER expression and activities by osteogenic niche in bone micrometastases, leading to the endocrine resistance (88). Lymph circulation plays a vital role in the invasion of breast cancer and the lymph node status is very important in predicting the prognosis of breast cancers. Generally, the assessment of the risk of developing bone metastasis in breast cancer includes the number of positive lymph nodes, the size of the tumor, and age of the patient. If the numbers of positive lymph nodes are more than 4, and the size of the tumor is larger than 2cm and the patient is younger than 35 years, the patients are usually associated with higher risk of bone metastasis (87).