Technical approaches for studying the communications between osteocytes and cancer cells

Osteocyte biology and their role in regulating bone homeostasis

Osteocytes are the most abundant cells in the bone, playing an essential role in mechanosensation, mechanotransduction, and communication with other bone cells. They originated from osteoblasts and are surrounded by self-produced osteoid matrix [ ]. Being entrapped throughout a mineralized matrix, the stellate-shaped processes of osteocytes extend from the cell bodies located in lacunar to all directions throughout canals called canaliculi, forming the lacunar-canalicular system. Such system in the bone allows osteocytes to directly or indirectly cross talk with adjacent and neighboring cells through gap junctions and secreted signaling molecules in the pericellular space [ ]. Osteocytes could also extend their processes onto the bone surface and into bone marrow spaces, controlling the communication between various bone cells. Moreover, it has been proposed that the pericellular matrix surrounding the osteocyte body and processes in canaliculi allows strain amplification with flow-induced drag forces under mechanical loading [ , ], for a major contributor to osteocyte mechanosensation.

A healthy skeletal structure requires lifelong bone remodeling to maintain its quality and integrity, and this process occurs through a balance in osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells) activity [ ]. Osteocytes, acting as major mechanosensory cells in the bone, play an essential role in orchestrating bone remodeling through mechanotransduction signaling cascade [ ]. The mechanical cues applied to the bone are sensed by osteocytes through multiple mechanisms including integrins, primary cilia, mechanosensitive ion channels, and G-protein-coupled receptor (GPCR) [ ], then osteocytes transduce the signal into biological cues that regulate osteoblasts and osteoclasts activity, maintaining balanced bone homeostasis. Several important downstream signaling molecules involved in this process include sclerostin, dickkopf-related protein 1 (DKK-1), receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG). Osteocytes inhibit osteoblasts proliferation and differentiation via sclerostin and DKK-1. Sclerostin and DKK-1 are as Wnt antagonists, blocking the canonical Wnt signaling pathway by binding to low-density lipoprotein–related protein 5/6 (LRP5/6) and Wnt receptors on the surface of osteoblasts [ ]. Osteocytes are also a major source of RANKL, where RANKL and OPG are the key modulators in osteoclasts function. RANKL binds to its receptors, RANK, on the surface of osteoclast precursors and activates downstream pathways to stimulate osteoclasts differentiation and maturation. OPG acts as the decoy receptor binding to RANKL, further blocking osteoclastogenesis [ ].

Along with understanding of osteocytes mechanobiology and its role in regulating bone remodeling, recent studies have started to investigate the potential role osteocytes play in regulating cancer growth in bone, which may disrupt the bone remodeling process and lead to bone tissue destruction.

Cancer in skeletal environment—bone metastasis

Several types of cancers could grow in bone. Metastasized cancer cells, the spread and migration of tumors from the primary site to the bone, is one of the most common bone cancer diseases [ ]. There are estimated 350,000 patients every year die from bone metastasis in the United States [ ]. Breast and prostate cancer account for 70% of the primary site [ ], where 85% of advanced breast cancer patients and 80% of advanced prostate cancer individuals develop bone metastasis [ , ]. Once cancers spread to the bone, they disrupt the bone physiology and bone remodeling process. Currently, bone metastasis is still incurable and usually associated with a wide range of morbidities such as severe pain, high risk of fracture, and hypercalcemia, further reducing the quality of life of patients [ ].

The process of cancer metastasis consists of a series of steps, starting with the intravasation of primary malignancy to the newly promoted blood vessels and travel down in the blood circulation system. Then, tumors are attracted to the capillary bed of distant bone and finally adhere and extravasate across the endothelium to reach the secondary bone microenvironment where they start to proliferate. Cancer cells homing to the bone is critical for bone metastasis [ ]. In 1889, Stephen Paget proposed the “seed and soil” hypothesis [ ], stating that the selected cancer cells (the “seeds”) have affinity to specific fertile microenvironments (the “soils”) and could develop cross talk with the soils to eventually grow into metastasis lesion. In bone-specific metastasis microenvironment, C–X–C motif chemokine 12 (CXCL 12), the key factor of homing hematopoietic stem cells to bone marrow, and its receptor, C–X–C chemokine receptor type 4 (CXCR 4), are considered the key modulators in cancer cell attraction. An in vitro study showed that CXCL 12 enhanced migration and invasion across endothelial for the cells expressing CXCR 4, such as prostate cancer cells [ ].

Metastasized cancer cells demonstrate tight cross talk with bone cells and disrupt the normal balance between bone resorption and formation, resulting in different patterns of bone lesions, either osteolytic lesion, osteoblastic lesion, or a combination of both [ ]. Over 80% of breast cancer bone metastasis patients suffer from osteolytic metastasis due to increased osteoclasts activity and bone resorption. In osteolytic metastasis, malignancy tumors not only induce bone destruction but also form a “vicious cycle” to promote the aggressive growth of the tumor itself [ , ]. Metastasized tumors first secrete osteoclasts-activating factors, parathyroid-hormone related peptide (PTHrP), to osteoblasts, and regulate RANKL and OPG secretion to activate osteoclasts precursors, leading to bone destruction. While bone undergoes the degradation, the embedded growth factors in the bone matrix, including transforming growth factor-β (TGF-β), insulin-like growth factors (IGFs), platelet-derived growth factors (PDGFs), and bone morphogenetic proteins (BMPs), are released to further proliferate and attract breast cancer cells, completing the vicious cycle.

On the other hand, prostate cancer bone metastasis patients usually experience osteoblastic lesion [ ], due to the upregulated osteoblasts bone formation. In osteoblastic lesion, the increased bone matrix with mostly low strength woven tissue formed by osteoblasts is poorly organized, resulting in thickened, rigid, and inflexible bone [ ]. Several tumor-secreted factors are important in mediating osteoblastic response during prostate cancer metastasis, including endothelin-1 (ET-1), fibroblast growth factors (FGFs), PDGFs, and BMPs. These factors stimulate osteoblasts bone formation and promote tumor metastasis directly. ET-1, secreted by cancer cells, binds to ET _A receptor on osteoblasts, promoting the deposition activity [ ]. FGFs expressed by prostate cancers mediate osteoblasts proliferation [ ]. Moreover, BMP is responsible for osteoblast differentiation and matrix production. Other than that, proteases such as prostate-specific antigen (PSA), serine protease urokinase (uPA), and cathepsin D could activate osteoblastic-stimulating factors by activating latent TGF-β, cleaving IGFs from its inhibitory binding proteins (IGF-BPs), or inactivating the osteolytic factor PTHrP to further advance bone formation [ , , ].

Cancer in skeletal environment—multiple myeloma

Multiple myeloma is another cancer that could lead to osteolytic bone lesion due to an unbalanced and uncoupled bone remodeling process. It is characterized by the expansion and accumulation of monoclonal plasma cells in the bone marrow. Multiple myeloma is the most frequent cancer in the skeleton diseases, with up to 90% of patients developing bone lesion [ ]. The bone lesion of multiple myeloma patients results in pathological bone fractures, hypercalcemia, and spinal cord compressions which significantly affect their survival and well-being. Unlike other metastasized cancer in the bone, multiple myeloma cells originating from bone marrow grow on surrounding bone without distanced migration. They affect the bone microenvironment via soluble factors that are directly released by myeloma cells or released by stromal and osteoprogenitor cells after cell–cell interactions [ ]. Myeloma cells upregulate RANKL and downregulate OPG in stromal cells, and directly secrete osteoclastogenic factors, chemokine (C-C motif) ligand 3 (CCL3), leading to osteoclast formation and survival [ ]. In contrast, they suppress osteoblast differentiation through inhibiting Runx2 [ ], the main pro-osteoblastogenic transcription factor, and through secreting Wnt signaling inhibitors such as Dkk-1 [ ], where Wnt signaling promotes osteogenesis by directly stimulating Runx2 gene expression. The formation of osteoclasts and inhibition of osteoblasts results in dysregulated bone homeostasis, leading to osteolytic bone lesion.

Current findings in osteocyte mediating bone cancers

Despite the understanding of secretive factors and the interactions between tumors, osteoblasts, and osteoclasts in bone cancer, the implication of osteocytes in bone cancers remains largely unknown. In recent years, increasing interests in the role of osteocytes in bone metastasis and multiple myeloma bone disease, as well as their potential role as a therapeutic target, led to intense investigation in the field.

While excluding complicated factors and looking at the direct interactions between osteocytes and tumors, osteocytes demonstrate prometastasis properties. Osteocytes compact tumor formation and stimulate the proliferation of specific breast and prostate cancer cell-line in vitro [ , ]. These results might be due to the chemoattraction of bone matrix protein in the osteocyte-conditioned medium, such as type I collagen. CXCL 12 produced by osteocytes acting as chemoattractant might also contribute to cancer cells homing to bone [ ]. In multiple myeloma, the direct contact of osteocytes and multiple myeloma cells trigger osteocytes apoptosis and autophagy through activating bidirectional Notch signaling in both osteocytes and multiple myeloma cells [ ]. Such activation results in the increased activity of osteoclasts and the enhanced production of pro-resorption proteins, RANKL and sclerostin. The Notch signaling in osteocytes leads to cell apoptosis, while, in turn, supports the growth and proliferation of multiple myeloma cells. Also, the increase in sclerostin expression in osteocytes inhibits osteoblast differentiation. Although there are prometastasis properties mentioned above, adverse effects toward cancer cells were also observed in osteocyte–tumor interactions in vitro , showing the possibility of osteocytes in preventing the migration and arrival of cancer cells to bone. For example, direct and indirect interactions between tumors and osteocytes downregulate the expression of Snail, a transcription factor important in promoting epithelial-to-mesenchymal transition and tumor metastasis, and Src activity to suppress cancer migration behavior [ ].

Since osteocytes are the major mechanosensory cells in bone, emerging studies have been focusing on the effect of mechanically stimulated osteocytes on bone cancers. Dynamic mechanical loading on osteocytes was shown to be antimetastatic, inhibiting breast cancer transendothelial migration and increasing cancer apoptosis through indirect cross talk with osteoclasts and endothelial cells [ ]. Mechanical stimulation also opens the endogenous Cx43 hemichannels in osteocytes and triggers the downstream release of ATP, which inhibits the migration, invasion, and growth of breast cancer cells [ ]. Osteocytes were also shown to reduce extravasation distance and breast cancer cells extravasation percentage under oscillatory fluid flow [ ]. Interestingly, a recent study showed that mechanically stimulated osteocytes demonstrate an intensity-dependent biphasic effect in modulating breast cancer migration. Stimulated osteocytes were shown to be prometastatic under 5N level loading, reducing osteopontin (OPN) expression, while osteocytes under 1N mechanical loading elevated OPN expression and inhibited tumor growth [ ]. Moreover, osteocytes were shown to be capable at responding to mechanical load despite the presence of the soluble factors secreted by tumor cells [ ].

Despite osteocytes showing their potential in regulating bone cancers, their role in the process of metastasis and homing of cancers remain largely unknown. One of the limitations while elucidating the role of osteocytes in bone cancer is the lack of appropriate technical approaches. Different cell types and techniques among the studies could lead to divergent results. Hence appropriate technical approaches are required for future studies to further help to identify osteocyte-related therapeutic targets.

Important factors to consider while looking at osteocyte–cancer microenvironment

There are several limitations in traditional approaches for investigating tumor–osteocytes communication. While osteocytes are embedded in bone matrix forming the 3D lacuna-canalicular system, it remains challenging to isolate osteocytes and at the same time maintain real-time signaling with their surroundings. For instance, conventional 2D in vitro models allow the isolation of osteocytes, however, sacrificing their significant properties in the 3D microenvironment and distinctive phenotype. In a physically relevant in vivo model, it is difficult to isolate osteocytes and control specific factors in key signaling events, especially that osteocytes are embedded in complex bone matrices. Thus, developing appropriate technical approaches that successfully meet the above requirements is important and would provide researchers with platforms to better understand the role of osteocytes in bone cancers.

The unique characteristics of the bone niche support the homing and growth of cancers with both biological secreting factors and the physical properties [ ]. Biochemical factors including hormone and osteocyte/cancer cell–derived factors play a significant role in cell–cell interactions. From early metastasis events to a vicious cycle and bone lesions, biochemical cues remain important regulators to mediate bone microenvironment between cells. On the other hand, important physical properties that need to be considered in the osteocyte–cancer microenvironment include extracellular matrix (ECM) mechanical factors, low PH, and hypoxia. Due to the lack of expansibility in bone ECM, tumors were shown to induce pressure and further impel osteocytes to promote prostate cancer bone metastasis [ ]. In turn, osteocytes could express matrix metalloproteinases (MMPs) that modulate ECM degradation and are capable of generating low PH environment by proton pumps to remove matrix in lacunar space. Osteocytes were also shown to increase cell viability in acidic conditions compared to osteoblasts and fibroblasts [ ]. Moreover, residing deeply in the bone matrix with restricted oxygen supply where oxygen tensions are below 5%, osteocytes are enriched in hypoxia markers. Hypoxia environment was shown to induce osteocytes with prolonged dendrites comparing with normoxic environment [ ]. Hypoxia also inhibits osteoblast differentiation and increases osteolytic bone metastasis. These physical properties need to be taken into account while looking into osteocytes–cancer interactions.