**1. Introduction**

Cancer induced bone pain (CIBP) is a big accompanying clinical problem of bone tumors with a high unmet medical need [1]. It is a debilitating form of different pain components that severely affects a patients' quality of life. The complex mechanism of CIBP largely involves the nervous system with transmembrane receptors and channels on the nerve fibers. Briefly, the nervous system consists of the central nervous system, i.e. the brain and the spinal cord, and the peripheral nervous system, i.e. the autonomic (unconscious, the para- and sympathetic nervous system) and somatic (conscious/voluntary) nervous system. A neuron is a nerve cell consisting of a cell body (soma), projections receiving input signals (the dendrites) and a single long arm away from the soma (the axon/fiber) that ends with the axon terminal (synapse). Axons contain a sheath of myelin that serves as isolation in a similar way as plastic around an electrical wire. Regarding the somatic nervous system, neurons with projections towards the spinal cord (afferent) respond to stimuli and are the sensory neurons. The neurons that respond to the brain and the signals from the spinal cord (efferent) are the motor neurons [2].

Pain is the defense mechanism against external factors that could cause tissue damage (a noxious stimuli) and nociception is detecting such stimulus. The somatosensory nervous system contains the sensory neurons that respond to noxious stimuli (nociceptors). There are three types of nociceptors, receptors that sense 1) thermal, 2) mechanical and 3) chemical stimulants. When a threshold of either one of those three properties is exceeded, the nociceptor is activated – *the neuron depolarizes and an action potential occurs –* and an electrical signal follows through the nociceptive pathway. Two major nociceptive fibers are reasonably fast-conducting A-δ fibers, containing a thin layer of myelin and the unmyelinated slow-conducting C-fibers. Finally, there are thickly myelinated fast conducting A-β fibers, faster than A-δ fibers, primarily for the normal sensation of touch [2].

Pain can be acute, serving a biological purpose, e.g. protection, and chronic, without a biological purpose, becoming an own medical disease more than a symptom [3]. A workgroup from the international association for the study of pain (IASP) has defined chronic pain as a pain that persists for more than 3 months. They defined a subgroup in 2018 where it has been considered that pain can be the primary disease, i.e. in low-back pain. Moreover, they have made subgroups and considered conditions with chronic secondary pain, such as chronic cancer-related pain [4]. The transition to chronic pain involves neuronal plasticity – *the ability of the nervous system to adapt the composition, signaling and structure* – represented by the enhancement of neurons and pain pathways, entitled as central and peripheral sensitization [3]. A very detailed elaboration on the molecular mechanism of sensitization is described by Latremoliere and Woolf (2009). Here, it is important to know that central and peripheral sensitization is a mechanistic explanation for mechanical allodynia (*non-noxious stimuli become painful*), hyperalgesia (*painful, noxious stimuli are prolonged in response and exaggerated*) and secondary hyperalgesia (*pain spreads beyond the site of injury*) [3]. The definition of pain by IASP is: *"An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage"* and for nociception: "*The neural processes of encoding noxious stimuli*" [5]*.* Pain can be distinguished between injury to the peripheral tissue, nociceptive pain – *Immunologic response –* and pain directly to the nervous system, neuropathic pain. IASP has defined neuropathic pain as: "*Pain caused by a lesion or disease of the somatosensory nervous system*". No specific definition is mentioned for nociceptive pain, however, chronic inflammation is a particular pain-related event, recognized by chemical and inflammatory mediators, affecting nociceptive axons and resulting in lowered thresholds of neuronal excitation [6]. CIBP is a unique type of pain with nociceptive and neuropathic components but the exact mechanism remains unclear.

This chapter elaborates on the mechanism of action of bone cancer pain. Next, a brief subsection of the bone anatomy & physiology. Finally, treatment options used for CIBP and bone metastases are described, including CIBP models to assess novel compounds and the mechanism of action.

### **2. Bone cancer pain**

#### **2.1 Bone anatomy, physiology and innervation**

Bones can be classified by their shapes, i.e. flat, short, long and irregular bones [7]. The most common bones that encounter metastasis of tumor cells are long bones [8, 9], i.e. the tibia, femur and humerus, characterized by an extended tubular diaphysis and round-shaped distal and proximal epiphyses [10]. The outer part is covered with a fibrous layer and an inner osteogenic layer, the periosteum and cambium layer, respectively [11]. The latter contains progenitor cells for the bone building cells, osteoblasts [11]. Briefly, mesenchymal-derived cells are the progenitors which are stimulated by the transcription factors core binding factor

**5**

*Bone Cancer Pain, Mechanism and Treatment DOI: http://dx.doi.org/10.5772/intechopen.95910*

space, the resorption lacuna. Activation of H<sup>+</sup>

α1 (Cbfa1), Osterix (Osx) and activating transcription factor 4 (ATF4) to initiate osteoblastogenesis [12]. Matured osteoblasts secrete bone matrix until they become resting osteoblastic cells (bone-lining cells) [7, 12, 13]. Behind the periosteum are densely packed tube-like structures called osteons (Haversian system). One osteon consists of several layers (lamella) with small gaps (lacunae) in between, containing nutrient transportation cells, osteocytes, constituting 90 to 95% of the bone cells present in the mature human skeleton [7, 13]. Osteocytes originate from differentiated bone-linings cells after they are encapsulated by secreted bone matrix and are suggested to coordinate the location of bone formation or resorption [12]. The packed osteons is the bone matrix, surrounding and protecting the medullary cavity of the diaphysis, containing bone marrow, with a thin connective tissue membrane separating both. The hematopoietic lineage in the bone marrow is responsible for pre-osteoclastogenesis [14, 15]. The macrophage colony-stimulating factor (M-CSF) stimulates the progenitor bone marrow cell for differentiation into a pre-osteoclast, initiating the expression of the receptor activator of NF-κB (RANK) receptor [16, 17]. The osteoblasts express the opposite part of the RANK receptor, necessary for activation, the RANK ligand (RANKL). Upon activation of RANK by RANKL the osteoblasts ensure that several activated pre-osteoclasts fuse together, forming a larger multinucleated mature osteoclast [16]. A mature osteoclast is a specialized macrophage with multiple mitochondria and lysosomes, prepared for bone degradation [14, 15]. In addition, the cell–cell fusion process of pre-osteoclasts forming a mature osteoclast has a checkpoint, the stromal cells, which have the ability to interfere by secretion of Osteoprotegerin (OPG). This is a decoy receptor able to bind excessive levels of RANKL, preventing over-population of osteoclasts [9, 16, 18, 19]. Subsequently, the degradation of bone is initiated after maturation of osteoclasts and their allocation to the site-of-destruction, where they form a closed

exchanger by osteoclasts follows, in combination with the secretion of lysosomal enzymes and active protease Cathepsin K into the lacuna [15]. The net effect of this cascade is an acidic environment of pH ± 4.5 to degrade the nearby bone cells [9, 15]. This triad of RANK/RANKL/OPG that regulates osteoclast activation is an important process in healthy bone physiology and plays an important role during bone cancer pain development [16–20]. Finally, At the level where the diaphysis reaches the proximal epiphysis, the medullary cavity is more spongy-like and is called trabecular or cancellous bone. Both epiphyses are composed primarily of spongy bone and a small quantity of compact bone, surrounded by cartilage [7]. Nociceptors are necessary to let the brain perceive CIBP, however, very little is known regarding the innervation of bone with sensory nerve fibers. Immunoreactivity studies have shown that sensory neurons are present in periosteum, cambium, bone matrix, Haversian canals and in bone marrow in the medullary cavity, and no detection was found in the articular cartilage of the epiphysis [21–29]. The density (nerves per unit area) of sensory fibers is largest in the periosteum, followed by bone marrow, mineralized bone and articular cartilage consisting in a ratio of 100:2:0.1:0, respectively [9, 10, 28]. Up to 80% of the nerve fibers innervating the bone have been shown TrkA positive [22], suggesting innervation of mostly thin myelinated Aδ-fibers and unmyelinated C-fibers [9, 10, 29, 30]. It seems that the fast conducting, highly myelinated Aβ-fibers do not contribute, or

very scarcely, to the innervation of sensory neurons in the bone [29].

The world health organization (WHO) report from 2014 predicted that a total of 19 million cancer cases exist globally in 2025 [18] and in 2018 a WHO press release

**2.2 Epidemiology and primary vs. secondary tumors**


#### *Bone Cancer Pain, Mechanism and Treatment DOI: http://dx.doi.org/10.5772/intechopen.95910*

*Recent Advances in Bone Tumours and Osteoarthritis*

somatosensory nervous system contains the sensory neurons that respond to noxious stimuli (nociceptors). There are three types of nociceptors, receptors that sense 1) thermal, 2) mechanical and 3) chemical stimulants. When a threshold of either one of those three properties is exceeded, the nociceptor is activated – *the neuron depolarizes and an action potential occurs –* and an electrical signal follows through the nociceptive pathway. Two major nociceptive fibers are reasonably fast-conducting A-δ fibers, containing a thin layer of myelin and the unmyelinated slow-conducting C-fibers. Finally, there are thickly myelinated fast conducting A-β fibers, faster than A-δ fibers, primarily for the normal sensation of touch [2]. Pain can be acute, serving a biological purpose, e.g. protection, and chronic, without a biological purpose, becoming an own medical disease more than a symptom [3]. A workgroup from the international association for the study of pain (IASP) has defined chronic pain as a pain that persists for more than 3 months. They defined a subgroup in 2018 where it has been considered that pain can be the primary disease, i.e. in low-back pain. Moreover, they have made subgroups and considered conditions with chronic secondary pain, such as chronic cancer-related pain [4]. The transition to chronic pain involves neuronal plasticity – *the ability of the nervous system to adapt the composition, signaling and structure* – represented by the enhancement of neurons and pain pathways, entitled as central and peripheral sensitization [3]. A very detailed elaboration on the molecular mechanism of sensitization is described by Latremoliere and Woolf (2009). Here, it is important to know that central and peripheral sensitization is a mechanistic explanation for mechanical allodynia (*non-noxious stimuli become painful*), hyperalgesia (*painful, noxious stimuli are prolonged in response and exaggerated*) and secondary hyperalgesia (*pain spreads beyond the site of injury*) [3]. The definition of pain by IASP is: *"An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage"* and for nociception: "*The neural processes of encoding noxious stimuli*" [5]*.* Pain can be distinguished between injury to the peripheral tissue, nociceptive pain – *Immunologic response –* and pain directly to the nervous system, neuropathic pain. IASP has defined neuropathic pain as: "*Pain caused by a lesion or disease of the somatosensory nervous system*". No specific definition is mentioned for nociceptive pain, however, chronic inflammation is a particular pain-related event, recognized by chemical and inflammatory mediators, affecting nociceptive axons and resulting in lowered thresholds of neuronal excitation [6]. CIBP is a unique type of pain with nociceptive and neuro-

pathic components but the exact mechanism remains unclear.

compounds and the mechanism of action.

**2.1 Bone anatomy, physiology and innervation**

**2. Bone cancer pain**

This chapter elaborates on the mechanism of action of bone cancer pain. Next, a brief subsection of the bone anatomy & physiology. Finally, treatment options used for CIBP and bone metastases are described, including CIBP models to assess novel

Bones can be classified by their shapes, i.e. flat, short, long and irregular bones [7]. The most common bones that encounter metastasis of tumor cells are long bones [8, 9], i.e. the tibia, femur and humerus, characterized by an extended tubular diaphysis and round-shaped distal and proximal epiphyses [10]. The outer part is covered with a fibrous layer and an inner osteogenic layer, the periosteum and cambium layer, respectively [11]. The latter contains progenitor cells for the bone building cells, osteoblasts [11]. Briefly, mesenchymal-derived cells are the progenitors which are stimulated by the transcription factors core binding factor

**4**

α1 (Cbfa1), Osterix (Osx) and activating transcription factor 4 (ATF4) to initiate osteoblastogenesis [12]. Matured osteoblasts secrete bone matrix until they become resting osteoblastic cells (bone-lining cells) [7, 12, 13]. Behind the periosteum are densely packed tube-like structures called osteons (Haversian system). One osteon consists of several layers (lamella) with small gaps (lacunae) in between, containing nutrient transportation cells, osteocytes, constituting 90 to 95% of the bone cells present in the mature human skeleton [7, 13]. Osteocytes originate from differentiated bone-linings cells after they are encapsulated by secreted bone matrix and are suggested to coordinate the location of bone formation or resorption [12]. The packed osteons is the bone matrix, surrounding and protecting the medullary cavity of the diaphysis, containing bone marrow, with a thin connective tissue membrane separating both. The hematopoietic lineage in the bone marrow is responsible for pre-osteoclastogenesis [14, 15]. The macrophage colony-stimulating factor (M-CSF) stimulates the progenitor bone marrow cell for differentiation into a pre-osteoclast, initiating the expression of the receptor activator of NF-κB (RANK) receptor [16, 17]. The osteoblasts express the opposite part of the RANK receptor, necessary for activation, the RANK ligand (RANKL). Upon activation of RANK by RANKL the osteoblasts ensure that several activated pre-osteoclasts fuse together, forming a larger multinucleated mature osteoclast [16]. A mature osteoclast is a specialized macrophage with multiple mitochondria and lysosomes, prepared for bone degradation [14, 15]. In addition, the cell–cell fusion process of pre-osteoclasts forming a mature osteoclast has a checkpoint, the stromal cells, which have the ability to interfere by secretion of Osteoprotegerin (OPG). This is a decoy receptor able to bind excessive levels of RANKL, preventing over-population of osteoclasts [9, 16, 18, 19]. Subsequently, the degradation of bone is initiated after maturation of osteoclasts and their allocation to the site-of-destruction, where they form a closed space, the resorption lacuna. Activation of H<sup>+</sup> -ATPase proton pump and Cl/HCO3 exchanger by osteoclasts follows, in combination with the secretion of lysosomal enzymes and active protease Cathepsin K into the lacuna [15]. The net effect of this cascade is an acidic environment of pH ± 4.5 to degrade the nearby bone cells [9, 15]. This triad of RANK/RANKL/OPG that regulates osteoclast activation is an important process in healthy bone physiology and plays an important role during bone cancer pain development [16–20]. Finally, At the level where the diaphysis reaches the proximal epiphysis, the medullary cavity is more spongy-like and is called trabecular or cancellous bone. Both epiphyses are composed primarily of spongy bone and a small quantity of compact bone, surrounded by cartilage [7].

Nociceptors are necessary to let the brain perceive CIBP, however, very little is known regarding the innervation of bone with sensory nerve fibers. Immunoreactivity studies have shown that sensory neurons are present in periosteum, cambium, bone matrix, Haversian canals and in bone marrow in the medullary cavity, and no detection was found in the articular cartilage of the epiphysis [21–29]. The density (nerves per unit area) of sensory fibers is largest in the periosteum, followed by bone marrow, mineralized bone and articular cartilage consisting in a ratio of 100:2:0.1:0, respectively [9, 10, 28]. Up to 80% of the nerve fibers innervating the bone have been shown TrkA positive [22], suggesting innervation of mostly thin myelinated Aδ-fibers and unmyelinated C-fibers [9, 10, 29, 30]. It seems that the fast conducting, highly myelinated Aβ-fibers do not contribute, or very scarcely, to the innervation of sensory neurons in the bone [29].

#### **2.2 Epidemiology and primary vs. secondary tumors**

The world health organization (WHO) report from 2014 predicted that a total of 19 million cancer cases exist globally in 2025 [18] and in 2018 a WHO press release

announced that lung (2.09 million cases), breast (2.09 million cases), colorectal (1.08 million cases) and prostate (1.28 million cases) are the most common [31]. All of these, except for colorectal, follow a high pattern of bone metastasis in 60 to 84% of the cases [9, 32]. In breast and prostate cancer patients particularly, it is expected that 90% develop bone metastases [33, 34]. Additionally, there are primary bone tumors that have their origin within the bone and the most common type is an osteosarcoma with a worldwide incidence of 3.4 cases per million people per year [35]. In pediatrics it accounts for 3 to 5% of the cancers and in adults less than 1% [8]. Tumors can affect osteoblasts, resulting in osteoblastic lesions and in contrast affect osteoclasts, causing osteolytic lesions [36]. Primary bone tumors, e.g. osteosarcoma, are more osteolytic [37], prostate cancer seems more osteoblastic and breast cancer osteolytic [38]. The latter two have been observed in 1/4th of the cases to be mixed [39]. A specific group of well-known signaling proteins, the Wnt pathway, is suggested to shift tumors towards an osteoblastic phenotype as blockage showed a highly osteolytic tumor [40]. This pathway has been observed to directly enhance osteoblast differentiation and bone formation, whereas indirectly inhibits osteoclast differentiation and bone resorption by OPG production from osteoblasts and osteocytes [41].

Some cancer patients encounter bone tumors without the presence of pain. Unfortunately, 30 to 50% of the patients will experience mild to moderate pain and in advanced cancer patients 75 to 90% have life-altering pain [37, 42]. The most prevalent type of pain experienced is bone cancer pain [9, 17, 33], which patients describe as a persistent presence of a dull ache that increases in intensity over time [32]. They start noticing mechanical allodynia during normal activities, such as coughing, turning in bed or gentle limb movements [43]. Furthermore, there is incident pain, that occurs when the pain spontaneously intensifies as a result of weight-bearing or during movement. Finally, there are breakthrough events of very sharp intense pain that can happen during rest [9, 32]. These breakthrough pain episodes occur in 40 to 80% of the patients with a median of 4 episodes per day, lasting up to 30 minutes [44]. Particularly the incident and breakthrough pain events are devastating for the quality of life and are considered as most difficult pain conditions to treat [9, 33].

#### **2.3 Mechanism of action of bone cancer pain**

The Aδ-fibers are recognized to be important in acute pain, whereas C-fibers are the slower conducting sensors that account for physiological changes such as "second pain" [9]. It has been observed during chronic pain that these start sprouting and show enhanced spontaneous activity, ectopic firing, resulting in allodynia and hyperalgesia [45–48]. Important surface channels and receptors of Aδ- and C-fibers involved in nociceptive signaling are TrkA, acid sensing ion channels (ASIC), Transient receptor vanilloid-1 (TRPV1), P2X receptors, endothelin receptor (ET-1), bradykinin receptor (B2R), prostaglandin (PGE2) receptor, the voltagegated sodium channels Na.v1.7–1.9 and cytokine receptors [9, 18, 29, 49].

The mechanism of CIBP in osteoblastic lesions is poorly understood and the most influential factors described are bone morphogenetic factors and endothelin-1. The mechanisms in osteolytic lesions have been better elucidated [36]. First, the infiltrating tumor cells start an interaction with the stromal cells, resulting in a cascade of different pathways, shown in **Figure 1**. A primary effect on sensory nerve fibers occurs as the secreted mediators, e.g. NGF, PGE2, transforming growth factor-β (TGF-β), bradykinin, endothelin, cytokines (e.g. IL-1, IL-6, IL-8, IL-11 and IL-17) are ligands for the receptors and cause excitation of the nerve fibers [17, 22, 29, 50–53]. It has been shown in a rat CIBP model that IL-6 plays a pivotal

**7**

rapid Na+

**Figure 1.**

*growth factor.*

*Bone Cancer Pain, Mechanism and Treatment DOI: http://dx.doi.org/10.5772/intechopen.95910*

role by sensitizing nociceptive fibers, mediating peripheral and spinal sensitization [54] by upregulation of TRPV1 receptors via JAK/PI3K signaling in dorsal root ganglia neurons [55]. In addition, PGE2, TGF-β, IL-1, IL-6, IL-8, IL-11 and IL-17 showed to be involved in a secondary effect, namely the ability to increase the expression of RANKL and decrease OPG [17, 19, 52, 56]. TGF-β is also released by the bone matrix and stimulates osteolytic bone destruction of cells close to the tumor cells [56]. The normally present OPG that serves as a peace-keeper between osteoclasts and osteoblasts is overwhelmed by the excessive amounts of RANKL, resulting in exaggerated activity of osteoclasts [19]. Consequently, osteoclastogenesis is initiated resulting in many resorption lacunae creating an acidic environment [20]. Additional pro-inflammatory cells become active, secreted cytokines bind

*The cascade of events responsible after infiltration of a tumor cell, resulting in CIBP with a nociceptive and neuropathic component. First, disturbance of the RANK/RANKL/OPG triad. Next, the nociceptive component; an acidic environment occurs, directly activating sensory nerve fibers and secreted mediators contribute to the upregulation of RANKL. In addition, the neuro-inflammatory mediator upregulates TRPV1 channels. The neuropathic component; nerves are damaged and denervate, resulting in ectopic firing and sprouting and an enlarged tumor activates mechano-sensitive nociceptors. The NGF/TrkA is pivotal in the process of sprouting and thereby for hypersensitivity. RANK = receptor activator of NF-*κ*B, RANKL = RANK ligand, OPG = osteoprotegerin, Na.v1.7–1.9 = sodium channels, P2X = purinergic receptor, TrkA = Tromomyocin receptor kinase a, NGF = nerve growth factor, ET1 = endothelin receptor, B2R = bradykinin receptor, ATP = Adenosinetriphosphate, IL-6 = interleukin-6, ASIC = acid-sensing ion channel, TRPV1 = transient receptor vanilloid-1, TGF-*β *= transforming growth factor-*β*, TNF = tumor* 

& Na+

influx is associated with ASICs and a second slow current activated at

pH and thereby triggering P2X7 and TRPV1 receptors, and ASICs [1, 20, 49]. The

pH < 6.2 is typical for TRPV1 [20]. Subsequently, tumor cells release NGF, tumor necrosis factor (TNF), IL-1 and IL-6, chemokines and endothelins which contribute to further develop an acidic environment [32]. This could be the explanation

Next to the nociceptive component of CIBP is the neuropathic component, caused by damage or denervation of nerves, pressure of tumors on the nerves

) amounts increase, lowering the

their designated receptors and proton (H+

regarding the difficulty of treating CIBP [29].

#### *Bone Cancer Pain, Mechanism and Treatment DOI: http://dx.doi.org/10.5772/intechopen.95910*

#### **Figure 1.**

*Recent Advances in Bone Tumours and Osteoarthritis*

and osteocytes [41].

pain conditions to treat [9, 33].

**2.3 Mechanism of action of bone cancer pain**

announced that lung (2.09 million cases), breast (2.09 million cases), colorectal (1.08 million cases) and prostate (1.28 million cases) are the most common [31]. All of these, except for colorectal, follow a high pattern of bone metastasis in 60 to 84% of the cases [9, 32]. In breast and prostate cancer patients particularly, it is expected that 90% develop bone metastases [33, 34]. Additionally, there are primary bone tumors that have their origin within the bone and the most common type is an osteosarcoma with a worldwide incidence of 3.4 cases per million people per year [35]. In pediatrics it accounts for 3 to 5% of the cancers and in adults less than 1% [8]. Tumors can affect osteoblasts, resulting in osteoblastic lesions and in contrast affect osteoclasts, causing osteolytic lesions [36]. Primary bone tumors, e.g. osteosarcoma, are more osteolytic [37], prostate cancer seems more osteoblastic and breast cancer osteolytic [38]. The latter two have been observed in 1/4th of the cases to be mixed [39]. A specific group of well-known signaling proteins, the Wnt pathway, is suggested to shift tumors towards an osteoblastic phenotype as blockage showed a highly osteolytic tumor [40]. This pathway has been observed to directly enhance osteoblast differentiation and bone formation, whereas indirectly inhibits osteoclast differentiation and bone resorption by OPG production from osteoblasts

Some cancer patients encounter bone tumors without the presence of pain. Unfortunately, 30 to 50% of the patients will experience mild to moderate pain and in advanced cancer patients 75 to 90% have life-altering pain [37, 42]. The most prevalent type of pain experienced is bone cancer pain [9, 17, 33], which patients describe as a persistent presence of a dull ache that increases in intensity over time [32]. They start noticing mechanical allodynia during normal activities, such as coughing, turning in bed or gentle limb movements [43]. Furthermore, there is incident pain, that occurs when the pain spontaneously intensifies as a result of weight-bearing or during movement. Finally, there are breakthrough events of very sharp intense pain that can happen during rest [9, 32]. These breakthrough pain episodes occur in 40 to 80% of the patients with a median of 4 episodes per day, lasting up to 30 minutes [44]. Particularly the incident and breakthrough pain events are devastating for the quality of life and are considered as most difficult

The Aδ-fibers are recognized to be important in acute pain, whereas C-fibers are the slower conducting sensors that account for physiological changes such as "second pain" [9]. It has been observed during chronic pain that these start sprouting and show enhanced spontaneous activity, ectopic firing, resulting in allodynia and hyperalgesia [45–48]. Important surface channels and receptors of Aδ- and C-fibers involved in nociceptive signaling are TrkA, acid sensing ion channels (ASIC), Transient receptor vanilloid-1 (TRPV1), P2X receptors, endothelin receptor (ET-1), bradykinin receptor (B2R), prostaglandin (PGE2) receptor, the voltage-

The mechanism of CIBP in osteoblastic lesions is poorly understood and the most influential factors described are bone morphogenetic factors and endothelin-1. The mechanisms in osteolytic lesions have been better elucidated [36]. First, the infiltrating tumor cells start an interaction with the stromal cells, resulting in a cascade of different pathways, shown in **Figure 1**. A primary effect on sensory nerve fibers occurs as the secreted mediators, e.g. NGF, PGE2, transforming growth factor-β (TGF-β), bradykinin, endothelin, cytokines (e.g. IL-1, IL-6, IL-8, IL-11 and IL-17) are ligands for the receptors and cause excitation of the nerve fibers [17, 22, 29, 50–53]. It has been shown in a rat CIBP model that IL-6 plays a pivotal

gated sodium channels Na.v1.7–1.9 and cytokine receptors [9, 18, 29, 49].

**6**

*The cascade of events responsible after infiltration of a tumor cell, resulting in CIBP with a nociceptive and neuropathic component. First, disturbance of the RANK/RANKL/OPG triad. Next, the nociceptive component; an acidic environment occurs, directly activating sensory nerve fibers and secreted mediators contribute to the upregulation of RANKL. In addition, the neuro-inflammatory mediator upregulates TRPV1 channels. The neuropathic component; nerves are damaged and denervate, resulting in ectopic firing and sprouting and an enlarged tumor activates mechano-sensitive nociceptors. The NGF/TrkA is pivotal in the process of sprouting and thereby for hypersensitivity. RANK = receptor activator of NF-*κ*B, RANKL = RANK ligand, OPG = osteoprotegerin, Na.v1.7–1.9 = sodium channels, P2X = purinergic receptor, TrkA = Tromomyocin receptor kinase a, NGF = nerve growth factor, ET1 = endothelin receptor, B2R = bradykinin receptor, ATP = Adenosinetriphosphate, IL-6 = interleukin-6, ASIC = acid-sensing ion channel, TRPV1 = transient receptor vanilloid-1, TGF-*β *= transforming growth factor-*β*, TNF = tumor growth factor.*

role by sensitizing nociceptive fibers, mediating peripheral and spinal sensitization [54] by upregulation of TRPV1 receptors via JAK/PI3K signaling in dorsal root ganglia neurons [55]. In addition, PGE2, TGF-β, IL-1, IL-6, IL-8, IL-11 and IL-17 showed to be involved in a secondary effect, namely the ability to increase the expression of RANKL and decrease OPG [17, 19, 52, 56]. TGF-β is also released by the bone matrix and stimulates osteolytic bone destruction of cells close to the tumor cells [56]. The normally present OPG that serves as a peace-keeper between osteoclasts and osteoblasts is overwhelmed by the excessive amounts of RANKL, resulting in exaggerated activity of osteoclasts [19]. Consequently, osteoclastogenesis is initiated resulting in many resorption lacunae creating an acidic environment [20]. Additional pro-inflammatory cells become active, secreted cytokines bind their designated receptors and proton (H+ & Na+ ) amounts increase, lowering the pH and thereby triggering P2X7 and TRPV1 receptors, and ASICs [1, 20, 49]. The rapid Na+ influx is associated with ASICs and a second slow current activated at pH < 6.2 is typical for TRPV1 [20]. Subsequently, tumor cells release NGF, tumor necrosis factor (TNF), IL-1 and IL-6, chemokines and endothelins which contribute to further develop an acidic environment [32]. This could be the explanation regarding the difficulty of treating CIBP [29].

Next to the nociceptive component of CIBP is the neuropathic component, caused by damage or denervation of nerves, pressure of tumors on the nerves

and sprouting. The degradation of bone and the damage that occurs can activate mechanosensitive ion channels, e.g. TRPV, ASIC and P2X7 [29, 57, 58]. Activated NGF regulates the maintenance of the peripheral sensory neuron system and initiates sprouting of adjacent non-injured afferents upon injury or denervation, resulting in collateral sprouting [59, 60]. Random sprouting of sensory neurons co-expressing TrkA was shown in prostate cancer metastases [9, 48] and similar in breast cancer metastases [47]. Hypersensitivity occurs as a result of sprouting, causing sensitization of sensory nerves, which in its turn induces mechanonociception (by Aδ-fibers) [59]. Changes also have been shown to occur in the central nervous system in the spinal cord where the excitatory synaptic transmission mediated through A-δ and C-fibers was enhanced [61].

On the one hand, it is suggested that the increase in activated osteoclasts causes the development of CIBP while on the other hand the secreted mediators directly exciting sensory nerve fibers is suggested to be the primary explanation [17, 51]. Nevertheless, all these multidisciplinary factors – *neurological, oncological and immunological* – contribute to CIBP and while they are described extensively, the exact mechanism remains to be elucidated.
