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Review
The role of osteoclasts and tumour-associated macrophages in
osteosarcoma metastasis
Liliana Endo-Munoz
a,
⁎, Andreas Evdokiou
b
, Nicholas A. Saunders
a
a
The University of Queensland Diamantina Institute, Brisbane, Australia
b
Basil Hetzel Institute, The Queen Elizabeth Hospital, University of Adelaide, Australia
abstractarticle info
Article history:
Received 17 November 2011
Received in revised form 17 July 2012
Accepted 19 July 2012
Available online 27 July 2012
Keywords:
Osteosarcoma
Metastasis
Osteoclast
Tumour-associated macrophage
Microenvironment
Osteosarcoma (OS) is the most common primary bone tumour in the paediatric age group. Treatment-
refractory pulmonary metastasis continues to be the major complication of OS, reducing the 5-year survival
rate for these patients to 10–20%. The mechanisms underlying the metastatic process in OS are still unclear,
but undoubtedly, a greater understanding of the factors and interactions involved in its regulation will open
new and much needed opportunities for therapeutic intervention. Recent published data have identified a
new role for bone-specific macrophages (osteoclasts) and tumour-associated macrophages (TAMs), in OS
metastasis. In this review we discuss the contribution of TAMs and osteoclasts in the establishment and
maintenance of secondary metastatic lesions, and their novel role in the prevention of metastatic disease
in a primary bone cancer such as osteosarcoma.
© 2012 Elsevier B.V. All rights reserved.
Contents
1. Introduction .............................................................. 434
2. The common origin of osteoclasts and TAMs ............................................... 435
3. Functions of osteoclasts and TAMs in the establishment of secondary tumours of bone ........................... 435
4. Osteosarcoma metastasis ........................................................ 436
4.1. Factors associated with inherent OS metastasis........................................... 437
4.2. The involvement of osteoclasts in OS metastasis .......................................... 437
4.3. TAM involvement in OS metastasis ................................................ 439
5. Conclusion ............................................................... 440
References ................................................................. 440
1. Introduction
Cancers of epithelial or haematological origin frequently leave
their primary niche and metastasise to bone. In contrast, primary tu-
mours of the bone, such as osteosarcoma (OS) frequently metastasise
to non-skeletal elements, in particular, the lung [1]. Despite these dif-
ferences, the mechanisms driving metastasis in these cancers share
many similarities. In metastatic bone disease, such as occurs with
breast cancer, the activation of osteoclasts (OCLs) and the presence
of tumour associated macrophages (TAMs) are irrefutably associated
with the recruitment of circulating tumour cells and the growth and
maintenance of metastatic lesions within the skeletal elements
[2–4]. Similarly, in tumours that arise in the skeletal elements, such
as osteosarcoma, the role of OCLs and TAMs has also been shown to
be intimately associated with metastasis to the lung. In osteosarcoma,
the role of OCLs and TAMs in metastasis has been confounded by
some apparently conflicting published reports. For example, a num-
ber of studies have shown that osteoclast-ablative therapies could re-
duce tumour burden and metastases in OS [5–7]. In contrast, more
recent studies suggest that OCL-ablative therapies increase metastatic
burden in OS [8–11]. On the surface, these data would appear irrecon-
cilable. However, an examination of the function of osteoclasts in pri-
mary and secondary bone tumours would suggest that these effects
are consistent with a model in which OCLs contribute to a niche envi-
ronment within the skeletal elements which in the early stages of OS
Biochimica et Biophysica Acta 1826 (2012) 434–442
⁎Corresponding author at: The University of Queensland Diamantina Institute, Level
4, R Wing, Princess Alexandra Hospital, Brisbane, Qld. 4102, Australia. Tel.: + 61 7 3176
5834; fax: +61 7 3176 5946.
E-mail address: l.munoz@uq.edu.au (L. Endo-Munoz).
0304-419X/$ –see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbcan.2012.07.003
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbacan
acts to nurture the growth and expansion of OS. In later stage disease,
intratumoural heterogeneity drives the acquisition of phenotypes
that inhibit osteoclastogenesis, and the destruction of the niche envi-
ronment, thus permitting tumour cell egress and metastasis.
Evidence accumulated over the years has identified OCLs and TAMs
as key drivers of tumour behaviour, affecting such processes as tumour
development, progression and metastasis [3,12–17]. Osteoclasts play a
pro-tumourigenic role and are central in the establishment and growth
of skeletal lesions in cancers that metastasise to bone such as breast,
prostate, lung, thyroid, kidney and multiple myeloma[18]. In these can-
cers, osteoclasts participate in what has been termed “the vicious cycle”
of skeletal metastasis, in which primary tumour cells, circulating tu-
mour cells and primary cells present within the bone marrow release
factors that stimulate osteoclastogenesis in the bone. The activated
OCLs then initiate the release of growth factors from bone matrix that,
in turn, promotes further recruitment and growth of tumour cells in
the skeleton [12]. Thus, the presence of OCLs in metastatic tumours of
epithelial origin is generally associated with a poor prognosis. Similarly,
TAMs represent a major component of the stroma in a number of
tumours including those of breast, prostate, colon and stomach
[19–22]. In these cancers TAMs contribute to neoplastic transformation,
progression and metastasis by stimulating inflammation, angiogenesis,
tumour cell motility, invasion, intravasation, and tissue remodelling, as
well as by suppressing cytotoxic immune responses and promoting
chemoresistance [17,22–26]. Recently, however, TAMs have been
shown to play an opposing role in osteosarcoma by impairing metasta-
sis [27]. Osteoclasts (tissue-associated macrophages) and TAMs are de-
rived from a common monocyte precursor in the bone marrow
[16,28–31]. Although the differentiated cells have different biological
functions, it is possible that when it comes to tumour biology, their
common ancestry may reflect common activities, albeit involving
different mechanisms. In this review we will consider the evidence re-
lating to the role of OCLs and TAMs in the development of OS metasta-
ses. Central to this will be an understanding of the role of OCLs and
TAMs in the development and maintenanceof skeletal metastasesof tu-
mours of non-skeletal origin (e.g. breast cancer).
2. The common origin of osteoclasts and TAMs
Osteoclasts and macrophages are derived from a common myeloid
precursor [30] (Fig. 1). Macrophages are phagocytic cells whereas os-
teoclasts are specialist macrophages responsible for bone resorption
[15,29,32,33]. Monocyte differentiation follows a multi-step process
induced and maintained by macrophage colony stimulating factor 1
(CSF1) [31]. Monocyte precursors migrate through the vasculature
and take residence in various tissues where, depending on location
and signal, they differentiate into different classes of macrophages
with various functions [16]. These include macrophages of bone
(osteoclasts), alveoli, central nervous system (microglial cells), connec-
tive tissue (histiocytes), gastrointestinal tract, liver (Kupffer cells),
spleen (white- and red-pulp, marginal-zone and metallophilic) and
peritoneum [30,32] (Fig. 1).
Osteoclast maturation occurs through a series of differentiation
stages that involves the recruitment of haematopoietic stem cells
into the monocyte/macrophage lineage, proliferation of osteoclast
precursor cells, fusion of pre-osteoclasts, and finally differentiation
into mature, multinucleated, bone-resorbing cells [28,29]. Osteoclasts
actively break down proteins in the bone matrix and decalcify bone
by releasing proteolytic enzymes such as cathepsin K (CTSK), matrix
metalloproteinase 9 (MMP9), and hydrochloric acid [34]. They also
secrete a number of osteoclast-specific enzymes such as tartrate-
resistant acid phosphatase (ACP5) and carbonic anhydrase II (CAII)
[28,29]. Key to the regulation of osteoclastogenesis and OCL function
are numerous transcription factors, cytokines and hormones [33],
but CSF1 and receptor activator of nuclear factor kappa B ligand
(RANKL), which bind to their cognate receptors, c-fms and receptor
activator of nuclear factor kappa B (RANK), respectively, on the sur-
face of OCL precursors, are essential and sufficient for the activation
and differentiation of these cells into mature OCLs [29,33,35–39].
RANKL is expressed and secreted by several different cell types
including osteoblasts, osteocytes, and bone marrow stromal cells
[40]. Whilst multiple bone-associated cells have the capacity to regu-
late osteoclastogenesis, recent evidence favours the osteocyte as
the major source of RANKL driving osteoclast differentiation in
the bone [41]. Osteoprotegerin (OPG), the soluble decoy receptor
for RANKL, acts as the major negative regulator of RANKL-induced
osteoclastogenesis [42–44]. An alternate and nonconventional source
of osteoclasts may be via trans-differentiation of TAMs. Earlier
studies have shown that TAMs may trans-differentiate into active
bone-resorbing osteoclasts in the presence of cancer cells, 1,25-
dihydroxyvitamin D3 (1,25(OH)2D3) and CSF1, or in the presence
of CSF1 and RANKL [45,46]. Thus, osteoclastogenesis is regulated by
the opposing actions of pro-osteoclastogenic (RANK, RANKL, CSF1,
macrophage trans-differentiation) and anti-osteoclastogenic factors
(e.g. OPG) [39]. Disruption of these pathways, can lead to metabolic
bone disorders (such as osteoporosis and sporadic Paget's disease),
immune-mediated bone disorders (such as rheumatoid arthritis and
periodontal disease), and inherited skeletal disorders (such as familial
Paget's disease and hyper-phosphatasia) [47].
Macrophages undergo activation by either a classical or alternate
mechanism. Classical activation, by Toll-like receptors and interferon-γ,
gives rise to M1 macrophages that play key roles in host defense, tumour
suppression and immune system stimulation. Class M1 macrophages are
characterised by the production of inducible nitric-oxide synthase
and the expression of a suite of cytokines involved in the induction of
T
H
1 cell development and T-cell trafficking, or which act as potent
chemoattractants for monocytes, lymphocytes, eosinophils, dendritic
and natural killer cells [48]. On the other hand, alternative activation,
by interleukins‐4or‐13, gives rise to class M2 macrophages that are in-
volved in tumour angiogenesis, tumour promotion and immune sup-
pression [30,49]. Class M2 macrophages are characterised by the
production of a different suite of cytokines which stimulate fibroblast
proliferation and survival, anti-inflammatory responses, as well as the
recruitment of other immune system cells as described above for class
M1 cells [50,51]. Complicating this classification is the observation that,
macrophages can play different roles depending on the signals received
from the microenvironment, and may switch between M1 and M2 phe-
notypes during different stages of tumour development [52].Thus,there
is evidence that macrophages exist in different functional states and can
switchbetweenanM1andM2phenotype as well as trans-differentiate
intoanOCL.Incontrast,OCLsappeartodifferonlyinthedegreeto
which they have been activated but do not trans-differentiate into alter-
nate cells of the monocyte/macrophage lineage.
3. Functions of osteoclasts and TAMs in the establishment of
secondary tumours of bone
Our understanding of osteoclast function has progressed from a
simplistic view of OCLs as bone resorbing cells to an understanding
that they are major regulators of tumour metastasis through their
critical involvement in what has been termed “the vicious cycle”of
skeletal metastasis [3,12]. Central to this view is the acceptance that
OCLs form a key component of a skeletal niche that contributes to
the recruitment and growth of tumour cells within the bone marrow
compartment. Tumours that metastasise to bone frequently give rise
to circulating tumour cells that are attracted to the bone marrow
environment and are capable of releasing factors that activate
osteoclastogenesis in the bone marrow. In this regard, breast cancer
cells have been shown to release parathyroid hormone-related pro-
tein (PTHrP), RANKL, endothelin-1 (ET-1), platelet-derived growth
factor (PDGF), bone morphogenetic proteins (BMPs), CSF1, activators
of Hedgehog signalling and a number of interleukins (IL-1, IL-6, IL-8,
435L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434–442
IL-11) [13,53,54]. These factors stimulate osteoclastogenesis leading
to increased osteolysis that releases factors from the bone matrix
such as transforming growth factor beta (TGFβ), insulin-like growth
factors (IGFs), fibroblast growth factors (FGFs), BMPs and PDGFs.
These released factors facilitate the establishment and growth of met-
astatic lesions which in turn can induce the expression of matrix
metalloproteinases (MMPs) that further activate osteoclasts and de-
grade components of the extracellular membrane (ECM) thereby re-
leasing more factors and expanding the metastatic niche [13,55,56].
In this model, the formation of the metastatic niche within the bone
marrow cavity is dependent upon theactivationof OCLs by the primary
tumour. This series of events generates a cycle of enhanced tumour
growth and excess bone matrix resorption which results in painful skel-
etal lesions, the major cause of morbidity in patients with cancers such
as breast and prostate [4,57] (Fig. 2). Thus, for these patients, inhibition
of the osteoclast has been the major focus of targeted therapies with
agents such as denosumab, a fully humanised monoclonal antibody to
RANKL [58], and bisphosphonates, such as zoledronic acid [59]. Since
osteoclasts and macrophages are derived from a common precursor,
bisphosphonates have also been shown to directly target TAMs [60,61].
The contribution of macrophages to tumour growth, progression and
metastasis in various epithelial and haematological cancers has also been
the subject of intense investigation in past years. TAM presence and den-
sity have been shown to correlate with tumour cell proliferation, inva-
sion, metastasis and poor prognosis [17,22,62]. Macrophage infiltration
begins early and increases significantly with tumour progression [63].
TAMs originate from monocytes which are recruited to the tumour site
by cytokines such as chemokine (C-C motif) ligand 2 (CCL2) and CSF1 se-
creted by cancer and stromal cells [14,64]. Macrophage differentiation is
then induced by interleukin-3 (IL‐3) and CSF1, and subsequent activa-
tion induced by interleukins-4 and ‐10 (IL‐4, IL‐10), TGFβand CSF1
[16]. TAMs comprise up to 50% of the total tumour volume and predom-
inantly display an M2 phenotype [16,50]. However, it is likely that a
number of TAM subtypes exist within the tumour microenvironment,
and that these populations change in function and location as the tumour
progresses [65]. TAMs release a number of cytokines that are known to
favour the metastatic process (Fig. 2). These include epidermal growth
factor (EGF), vascular endothelial growth factor (VEGF), tumour necrosis
factor alpha (TNFα) and interleukin-1 beta (IL‐1β)[66,67],aswell
as various matrix metalloproteinases (particularly MMP7 and 9)
[16,17,68]. These factors may contribute to increased invasiveness by
degrading the basement membrane and exposing fibronectin to tumour
cells, thus maintaining a continuous process of matrix deposition and
remodelling [16,62,69]. For example, TAMs have been shown to regulate
the angiogenic switch that controls microvessel density and which con-
tributes to breast cancer metastasis [23], and to incre ased invasion via se-
cretion of IL‐17 [70]. New evidence suggests that macrophages do not
just facilitate the propagation of tumour cells at the site of the primary
tumour,buttheyalsoactatthedistantpre-metastaticniche,where
they promote extravasation of circulating metastatic cells, and establish
and maintain the growth of new lesions [2,71,72].Thus,theprimary
tumour can influence the “pre-selection”of metastatic sites and ensure
the successful establishment of secondary tumours [73–76].
4. Osteosarcoma metastasis
Osteosarcoma is the most frequent malignant primary bone tu-
mour in children and adolescents and accounts for approximately
20% of all bone cancers [77]. Current treatment for osteosarcoma in-
volves the use of neoadjuvant chemotherapy followed by surgical
resection of the tumour and post-operative chemotherapy [78].
Although the introduction of chemotherapy has contributed to a
reduction in mortality, up to 50% of patients can present with
chemoresistant pulmonary metastatic disease at the time of diagno-
sis, and as a result, have a very poor prognosis [1,79]. Whereas pa-
tients without metastatic disease have a 5-year disease-free survival
rate of 60–70%, the 5-year survival of patients with metastatic disease
is only 10–20% [80]. Unfortunately, little is known about the factors
that influence the movement of OS cells away from bone and the de-
velopment of secondary lesions in the lung. Whilst there is some de-
bate in the literature as to the mechanisms driving the establishment
of metastatic foci of OS in the lung, it is clear that OCL and TAM are
key regulators of this process.
Myeloid
progenitor
Monocyte
Pre-osteoclastsMacrophage
Osteoclast
Classically activated
M1 macrophage
Alternatively activated
M2 macrophage
CSF1
CSF1
CSF1
RANKL
CSF1
RANKL
CSF1
CSF2
IFNγ
TNF
CSF1
IL4
IL13
Monocyte
Fig. 1. Osteoclastsand macrophages derive from a common ancestor. Early differentiationof osteoclasts and macrophages is dependent on CSF1. Specific signalling from RANKL induces
differentiation of monocytes into osteoclasts,while signalling from various cytokines in the microenvironment leads to activation of macrophages through either the classicalor alterna-
tive pathways. CSF1, colonystimulating factor1; RANKL, receptoractivator of nuclearfactor kappa-B ligand,CSF2, colony stimulating factor2 (granulocyte-macrophage);IFNγ,interferon
gamma; TNF, tumour necrosis factor; IL‐4, interleukin‐4; IL‐13, interleukin‐13.
436 L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434–442
4.1. Factors associated with inherent OS metastasis
It is apparent from the clinical data that approximately 80% of all OS
tumours may eventually acquire metastatic potential [81].The
remaining tumours remain localised and never progress to metastasis,
indicating that metastatic potential is an inherent characteristic of OS tu-
mours. Transcriptomic studies in chemo-naïve patient tumours and in
mousemodelsofOShaveidentified gene signatures associated with me-
tastasis [9,82]. Other studies have found specific molecules and path-
ways that define a metastatic phenotype. For example, activation of the
Notch signalling pathway has been shown to modulate epithelial to mes-
enchymal transition (EMT), tumour angiogenesis, and anoikis-resistance
in OS [83,84]. Another study has found overexpression of cysteine-rich,
angiogenic inducer, 61 (CYR61) in metastatic OS, and that silencing of
CYR61 inhibits in vitro OS invasion and migration as well as in vivo
lung metastases in mice [85]. Several Wnt signalling pathway ligands,
receptors and co-receptors are highly expressed in OS cell lines, while
Wnt inhibitors are downregulated, and this aberrant activation of Wnt
signalling is correlated with increased OS tumour metastasis [86–88].
Similarly, expression of VEGF also correlates with the development of
metastasis [89,90]. The cytoskeleton linker protein, ezrin, has been
linked to increased metastasis in OS, via interaction with beta4 integrin
[91–93]. Other molecules include the TNF receptor superfamily, member
6(Fas)[94,95], CXCR4/CXCL2 [96] and CXCR3 [97]. Combined, these
studies have identified key molecules that have the capacity to contrib-
ute to the inherent metastatic potential of OS. However, these studies
have not demonstrated how these molecules may modulate metastasis.
In particular, it is still unclear whether these molecules act directly to en-
hance tumour cell migration or adhesion, or whether these molecules act
indirectly by stimulating other cells to establish a pre-metastatic niche or
to enhance OS cell migration or extravasation. Regardless of whether the
effects are direct or indirect, targeting these molecules represents a po-
tentially novel therapeutic opportunity. However, for OS tumours that
have inherent metastatic potential, there is also evidence to suggest
that the development of metastasis also has an acquired component.
For example, a correlation between metastatic potential and OCL num-
bers in primary OS has been recently reported [9]. More significantly
there was a significant inverse relationship between the number of
OCLs in primary OS and the time to the manifestation of clinical signs
of metastasis [9]. These studies would suggest that a major regulator of
metastatic potential is dictated by elements of the tumour/bone micro-
environment, in particular OCLs and TAMs. In this regard, the metastatic
potential of OS may share many similarities with the metastatic drivers
of tumours derived from epithelial tissues such as breast.
4.2. The involvement of osteoclasts in OS metastasis
Pathologically, OS is characterised by the formation of immature
bone (osteoid) by the tumour cells [98]. Radiologically, OS bone tu-
mours are characterised according to their appearance which may be
either osteolytic (30% of cases), osteoblastic (45% of cases), or most
commonly, mixed [99]. As established for secondary bone metastases
arising from primary cancers, a vicious cycle between osteoclasts,
bone stromal cells/osteoblasts and cancer cells has been hypothesised
to drive the progression of osteosarcoma and there is clear radiological
evidence of osteolysis and bone remodelling in primary OS [3,100].
However, it is not known whether osteolytic activity and bone
remodelling in early disease has the same implications for tumour
growth and metastasis as osteolytic activity and bone remodelling in
late stage disease. This is an important consideration in light of recent
TAM s
Osteoclast
activation
Bone -derived
tumour growth
factors
Osteoclast -
activating
factors
Bone
resorption
Angiogenic factors
Proteases
Growth factors
Chemokines
Metastatic
epithelial
tumour
Metastasis TO bone
Fig. 2. Role of TAMs and osteoclasts in metastasis to bone. Tumours of epithelial origin, such as those of breast, have a tendency to metastasise to bone. TAMs secrete angiogenic and
growth factors, as well as proteases and chemokines that stimulate the process of metastasis in epithelial tumours. Metastatic cancer cells release a number of factors that lead to
increased activation of osteoclasts in the bone marrow niche and as a consequence, enhanced bone resorption. The latter releases a number of factors from the bone matrix that
further promote the growth of metastatic epithelial cancer cells.
437L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434–442
data showing that the presence of OCLs and/or TAMs at the site of pri-
mary OS lesions may suppress metastasis [8,27]. These more recent
data appear to contradict earlier reports that osteoclasts may enhance
OS metastasis [6,7,101,102]. This has important clinical implications.
For example, if OCLs contribute to the development of fatal pulmonary
metastases, then the use of osteoclast ablative therapies such as
bisphosphonates or RANK-Fc would be effective therapeutic strategies.
However, if OCLs and TAMs suppressed metastatic potential then the
use of osteoclast ablative therapies would be contra-indicated. It is
therefore important to develop a model which accommodates these
seemingly disparate observations whilst maintaining relevance to the
clinical presentation and outcome. In particular, the major conflicts in
the literature relate to the role of OCLs in the development of metasta-
sis. On the one hand, there are clinical and experimental data showing
that the presence of OCLs in OS is associated with poor outcomes
[6,7,101,102], and on the other hand, there is clinical and experimental
evidence to suggest that the presence of OCLs and TAMs at the primary
site of OS lesions prevents metastasis [8,9,11,27,103,104].Similarly,
studies in which OCLs are selectively ablated have shown con-
trasting effects. For example, recent data show that targeted ablation
of OCLs significantly enhances OS metastasis, whilst stimulation of
osteoclastogenesis inhibits OS metastasis [8,11]. In contrast, earlier
studies had reported that high dose bisphosphonate treatments could
ablate OCLs and reduce OS tumour growth and metastasis [101,102].
However, these latter studies may have been confounded by the direct
anticancer activity of bisphosphonates on OS cells [105–109]. Indeed,
the direct anticancer activity of bisphosphonates is also thought to con-
tribute to the antimetastatic properties of bisphosphonates in breast
cancer patients [110–113].
These seemingly conflicting observations may be resolved if one
considers that the impact of OCLs and TAMs on OS may differ during
the various phases of disease progression. For example, during initia-
tion and early OS growth, OCLs may function to promote tumour
growth by the release of growth factors from the bone matrix during
the bone resorption that accompanies OS-induced bone remodelling.
During this period, the OS lesion may remain confined to the primary
site, and may be discouraged from leaving the primary site due to the
abundance of growth factors released by the lytic activity of OCLs.
Thus, the persistence of OCLs at this stage would be predicted to
suppress metastasis by the continuing production of growth factors
released into the local tumour environment. During this time, it
would be predicted that OS growth and development could be
inhibited by targeted ablation of OCLs. However, as with all tumours,
persistent growth is often accompanied by the acquisition of further
genetic/epigenetic lesions [114], that eventually lead to the expansion/
selection of OS cells with inherent metastatic activity and an acquired
ability to suppress osteoclastogenesis [8]. In this regard, it was recently
shown that metastatic OS was associated with loss of OCLs in patients
and it was shown, in several independent OS cell lines, that the loss of
OCLs was due to the acquisition of an ability to secrete factors that
inhibit osteoclastogenesis in vitro and in vivo [8]. Thus, in late stage
metastatic OS we would predict that ablation of OCLs would stimulate
metastases and stimulation of osteoclastogenesis would inhibit metas-
tases. Both these predictions have been proven experimentally [8,11].
This model would appear to accommodate published clinical and
experimental data.
Supporting this model of OS progression and metastasis, data from
independent laboratories demonstrate that the effect of the OCL in
regulating OS metastasis is restricted to the bone marrow environ-
ment. For example, OPG, the soluble decoy receptor for RANKL and
a major inhibitor of osteoclastogenesis, reduced osteoclast numbers
but did not prevent the formation of metastatic lesions in mice bear-
ing sub-cutaneous OS tumours, or in rats with radiation-induced OS
[100]. Similarly, OCL ablation by zoledronate did not inhibit the ability
of OS cells injected subcutaneously to metastasise to the lung [8].In
the same model, it was shown that OCL ablation with zoledronate
significantly reduced the ability of metastatic OS cells injected intra-
femorally to metastasise to the lung [8]. These data clearly indicate
that the effect of the OCL on OS metastatic capacity is confined to
the OCL bone niche at the site of the primary OS lesion.
In support of the role of OCLs in the promotion of OS development
and growth in early disease are several pieces of experimental and
clinical evidence. Firstly, a recent study has shown that patient OS le-
sions that fail to develop metastases are associated with significantly
more OCLs than are patient lesions that do develop metastases [8]. In-
deed it was shown that the time to the appearance of clinical signs of
metastasis was a direct function of the OCL number within the prima-
ry lesion [8]. This suggests that there may be a progressive loss of
OCLs in OS as the disease progresses. This is validated by independent
studies showing high levels of RANKL protein and OCL-specific gene
expression in OS [115,116]. In addition, another study has reported
increased osteoclast numbers and increased expression of osteoclast
markers in the biopsies of 16 OS patients which was associated
with OS primary tumour growth and aggressiveness in 6/16 (37%)
of the patients [7]. More recently, in a mouse model of genomically
unstable OS that phenocopies the human condition, a bone tumour
suppressor, c-AMP-dependent protein kinase type I, αregulatory
subunit (PRKAR1A), was found to drive RANKL overexpression during
OS tumourigenesis which would be predicted to increase OCL num-
bers [117]. Thus, there appears to be a relationship between OCL
numbers and early stage disease and OS growth and progression. In
this regard the growth and maintenance of primary OS may share a
similar dependence on the OCL niche as does the growth of breast
cancer metastases within the skeleton.
In late stage disease the picture appears to change as OS progressive-
ly loses OCLs until a threshold is reached that permits OS cell metastasis.
This is supported by a recent study which reported a significant inverse
relationship between the loss of OCLs and OCL markers in primary OS
lesions and the time to presentation of clinical signs of pulmonary
metastasis [8]. This observation is further supported by several inde-
pendent studies. For example, transcriptomic studies performed on
chemo-naïve OS patient biopsies have identified genes that are essen-
tial to microenvironmental remodelling and osteoclast differentiation
[9,118].Mintzetal.(2005)first reported that genes involved in osteo-
clast differentiation and function were associated with a poor response
to chemotherapy in OS patients [118]. In a more recent study,
Patino-Garcia et al. (2009) found upregulation of early B-cell factor 2
(EBF2), a negative regulator of osteoclastogenesis, in OS tumour biop-
sies [119]. Overexpression of EBF2 led to an increase in OPG, the
major inhibitor of osteoclast differentiation. Silencing of EBF2 led to a
decrease in OPG and increased sensitivity of OS to TRAIL-mediated ap-
optosis [119]. In addition, a transcriptomic study comparing chemo-
naïve OS patient biopsies from patients who did (n=16) and did not
(n=6) go on to develop metastatic disease, and non-malignant bone
(n=5), found overall decreased expression of more than 15 genes in-
volved in osteoclast development and function in OS biopsies [9].This
included a profound downregulation of the S100 calcium binding pro-
tein A8 (S100A8), which is highly expressed in osteoclasts and func-
tions in the coupling of osteoclast and osteoblast activity [120].
Among the other downregulated genes were cathepsin G (CTSG), nec-
essary for the recruitment of osteoclast precursors [121],TYROprotein
tyrosine kinase binding protein (TYROBP/DAP12), essential for RANK
signalling and osteoclast differentiation [122,123], and tartrate-
resistant acid phosphatase 5 (ACP5/TRAP), a classic marker of active
and mature osteoclasts [124]. Moreover, the study also found that the
inhibitor of DNA binding 1 (ID1) was the most highly overexpressed
gene in OS tumours. ID1 has been shown to be a negative regulator of
osteoclastogenesis [125] and may provide an explanation for the de-
creased expression of osteoclast markers in OS. It is of interest to note
that both transcriptomic studies reported above, independently
showed thatdysregulation of osteoclastogenesis wasa feature of osteo-
sarcoma, and thus pointed to the involvement of the microenvironment
438 L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434–442
in OS chemoresistance and metastasis. These data also suggest that the
loss of osteoclasts in OS is an activeprocess and is linked tothe changing
nature of the disease and the OCL niche.
The mechanism by which osteoclasts may prevent metastasis in OS
tumours remains unclear. As already discussed, osteoclasts play an ac-
tive role in the establishment and maintenance of breast cancer metas-
tases in bone by secreting factors that promote the growth of cancer
cells [12,126]. Therefore, it would seem reasonable to suggest that oste-
oclasts contribute to a niche environment that retains and nurtures
OS cell growth whilst discouraging the migration of OS cells away
from the bone. Thus, a key difference between a metastasising and
non-metastasising OS may be attributable to their inherent ability to
modulate osteoclastogenesis at the site of the primary lesion (Fig. 3).
Metastasising OS has been shown to secrete factors that can inhibit
osteoclastogenesis [8]. As tumour volume increases, the growing abun-
dance of these secreted factors may lower osteoclast numbers and
activity in the lesion below a threshold required to maintain a microen-
vironment that is attractive for OScells. Thus, metastatic OS cellsmay be
able to tip the balance towards an environment in which the OCL niche
is progressively ablated. The impact of the loss of OCLs may not
simply be mediated via the loss of local growth factors. For example,
in vitro migration assays showed that conditioned medium from bone
marrow cell cultures increases the migration of OS cells, but that condi-
tioned medium from osteoclast cultures inhibits this migration [8].In
addition, metastasis-competent OS cells secrete factors that inhibit
osteoclastogenesis [8]. Thus, in early disease, OCLs may nurture the
growth and expansion of OS cells. However, as the OS cells progress
and acquire new mutations, they secrete factors that reduce
osteoclastogenesis. In turn, the loss of OCLs increases cytokines in the
bone microenvironment that restrain OS cell migration. Thus, the loss
of OCLs that accompanies OS progression leads to the loss of a niche
and the derepression of OS migration. The potential importance of the
OS–OCL relationship is clearly shown in two independent orthotopic
rat and mouse models of OS in which, the ablation of osteoclasts with
a clinically-relevant dose of a bisphosphonate (i.e. zoledronic acid)
increased the number and severity of lung metastases [8,10,11].
Conversely, treatment with fulvestrant, an oestrogen receptor blocker,
increased osteoclast numbers in the bones of OS tumour-bearing
animals and decreased lung metastasis [8].
4.3. TAM involvement in OS metastasis
In most cancers, the presence of TAMs is rarely associated with a good
prognosis. However, Buddingh et al. (2011) recently reported that the
expression of TAM-associated genes in pretreatment biopsies of osteo-
sarcoma patients correlated with a lower risk of metastatic disease, a
good response to chemotherapy and better patient survival [27].The
study found an association between macrophage infiltration and higher
microvessel density, suggesting that, as in epithelial cancers, TAMs may
support the growth of a tumour of mesenchymal origin such as OS. How-
ever, in OS this activity appears not to override their anti-metastatic role.
The mechanism by which TAMs inhibit metastasis in OS remains unclear,
but the observation that OS tumours were infiltrated with a heteroge-
neous population of TAMs with M1 and M2 phenotypes may suggest
that in osteosarcoma, the constitutive presence of M1 macrophages
may have an anti-metastatic rather than a pro-metastatic effect [27].
These findings are supported by a randomised clinical trial of 662 osteo-
sarcoma patients using the macrophage-activating agent, muramyl
tripeptide (MTP) [127,128]. The addition of a liposomal formulation of
MTP, MTP-PE (mifamurtide), to a standard chemotherapy regimen of
cisplatin, doxorubicin and methotrexate resulted in a statistically signif-
icant improvement in 6-year overall survival, from 70% to 78%, without
an increase in toxicity [127,129]. Similar results had been reported in
earlier studies of human and canine OS [130–135].Mifamurtidehasbe-
come the first new therapeutic drug for the treatment of OS in more than
20 years [103]. In the EU, the drug is approved for the treatment of OS
patients [136], while in the US it remains investigational [104]. Thus, it
is possible that the ratio of M1 to M2 macrophages may regulate the po-
tential of OS to metastasise, as in the osteoclast model, by changing the
tumour microenvironment to one that is metastasis-permissive once a
threshold number of either phenotype is reached. Further investigation
will be needed in order to show whether or not this is the mechanism
operating in OS.
TAM s
Osteoclasts
Metastatic
osteosarcoma
Metastasis
AWAY FROM
bone
Fig. 3. Role of TAMs and osteoclasts in osteosarcoma metastasis away from bone. Metastatic osteosarcomas have a propensity to metastasise to the lung. The constitutive presence
of TAMs in osteosarcomas has an anti-metastatic effect. Similarly, the presence of osteoclasts in the tumour microenvironment prevents metastasis, while their loss increases the
metastatic potential of osteosarcomas.
439L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434–442
5. Conclusion
Recent data from independent labs suggest that the growth and
metastasis of primary osteosarcoma could be dependent upon a func-
tional osteoclast niche and on the presence of TAMs within the bone
marrow compartment and the tumour. While much work has been
done to elucidate the mechanisms that regulate and mediate the
pro-tumourigenic and pro-metastatic role of osteoclasts and TAMs
in a number of tumours of epithelial and haematological origin,
their potential anti-tumourigenic and anti-metastatic role in primary
bone tumours such as osteosarcoma has only recently been observed.
Furthermore, the mechanism by which OCLs and TAMs may inhibit
OS metastasis still requires further elucidation. The combined body
of evidence accumulated so far would suggest that in the early stages
of development, OS tumour cells can activate the osteoclast to disrupt
normal bone remodelling, resulting in osteolysis. Continued break-
down of the mineralized bone matrix by OS-activated osteoclasts re-
leases factors that provide OS cells with an attracting, nurturing and
retaining bone environment, similar to that observed in secondary
bone tumours of epithelial and haematological origin. However, as
OS tumours progress, they may acquire more genetic/epigenetic le-
sions which may lead to their capacity to inhibit osteoclastogenesis.
The resulting reduction in osteoclast numbers leads to a decrease in
bone matrix resorption and a concomitant loss of the growth factors
that create a nurturing and retaining environment for OS tumour
cells. The loss of this niche may result in the derepression of OS mi-
gratory activity. TAM populations can change in function and location
as tumours progress. Therefore, OS development may also be accom-
panied by a switch in the phenotype of infiltrating TAMs, from
anti-metastatic M1 macrophages, to pro-metastatic M2 macrophages,
which may further facilitate OS cell metastasis. Combined, these data
suggest that targeted ablation of OCLs and TAMs in early stage OS
may be beneficial due to their ability to reduce tumour growth. How-
ever, in late stage OS, strategies that promote osteoclastogenesis or
the retention of TAMs of the M1 phenotype may be of benefitin
inhibiting OS metastasis. A focused investigation into the function of
osteoclasts and TAMs, and a better understanding of their regulation
by osteosarcoma cells and the bone microenvironment, is essential
if osteoclasts and TAMs are to be exploited as therapeutic targets
that may ultimately translate into more effective therapies for
inhibiting OS metastatic disease.
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