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Abstract and Figures

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.
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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 1020%. 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 identied a
new role for bone-specic 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
[24]. 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 conicting published reports. For example, a num-
ber of studies have shown that osteoclast-ablative therapies could re-
duce tumour burden and metastases in OS [57]. In contrast, more
recent studies suggest that OCL-ablative therapies increase metastatic
burden in OS [811]. 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) 434442
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
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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 identied OCLs and TAMs
as key drivers of tumour behaviour, affecting such processes as tumour
development, progression and metastasis [3,1217]. 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
[1922]. In these cancers TAMs contribute to neoplastic transformation,
progression and metastasis by stimulating inammation, angiogenesis,
tumour cell motility, invasion, intravasation, and tissue remodelling, as
well as by suppressing cytotoxic immune responses and promoting
chemoresistance [17,2226]. 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
[16,2831]. Although the differentiated cells have different biological
functions, it is possible that when it comes to tumour biology, their
common ancestry may reect 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 nally 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-specic 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 sufcient for the activation
and differentiation of these cells into mature OCLs [29,33,3539].
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 [4244]. 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 trafcking, or which act as potent
chemoattractants for monocytes, lymphocytes, eosinophils, dendritic
and natural killer cells [48]. On the other hand, alternative activation,
by interleukins4or13, 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 broblast
proliferation and survival, anti-inammatory responses, as well as the
recruitment of other immune system cells as described above for class
M1 cells [50,51]. Complicating this classication 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 cycleof
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) 434442
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), broblast 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 inltration
begins early and increases signicantly 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 (IL3) and CSF1, and subsequent activa-
tion induced by interleukins-4 and 10 (IL4, IL10), 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 (IL1β)[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 bronectin 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 IL17 [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 inuence the pre-selectionof metastatic sites and ensure
the successful establishment of secondary tumours [7376].
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 6070%, the 5-year survival of patients with metastatic disease
is only 1020% [80]. Unfortunately, little is known about the factors
that inuence 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. Specic 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; IL4, interleukin4; IL13, interleukin13.
436 L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434442
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
mousemodelsofOShaveidentied gene signatures associated with me-
tastasis [9,82]. Other studies have found specic molecules and path-
ways that dene 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 [8688].
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
[9193]. Other molecules include the TNF receptor superfamily, member
6(Fas)[94,95], CXCR4/CXCL2 [96] and CXCR3 [97]. Combined, these
studies have identied 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 signicantly
there was a signicant 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) 434442
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 conicts 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 signicantly 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 [105109]. Indeed,
the direct anticancer activity of bisphosphonates is also thought to con-
tribute to the antimetastatic properties of bisphosphonates in breast
cancer patients [110113].
These seemingly conicting 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 conned 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
signicantly 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 conned 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 signicantly
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-specic 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 signicant 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 identied genes that are essen-
tial to microenvironmental remodelling and osteoclast differentiation
[9,118].Mintzetal.(2005)rst 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) 434442
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
OSOCL 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 inltration 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 inltrated 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 ndings 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 [130135].Mifamurtidehasbe-
come the rst 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) 434442
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 inltrating 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 benecial 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 benetin
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.
References
[1] N. Marina, M. Gebhardt, L. Teot, R. Gorlick, Biology and therapeutic advances for
pediatric osteosarcoma, Oncologist 9 (2004) 422441.
[2] B. Psaila, D. Lyden, The metastatic niche: adapting the foreign soil, Nat. Rev.Cancer
9 (2009) 285293.
[3] J.M. Chirgwin, T.A. Guise, Molecular mechanisms of tumorbone interactions in
osteolytic metastases, Crit. Rev. Eukaryot. Gene Expr. 10 (2000) 159178.
[4] T.A. Guise, The viciouscycle of bone metastases,J. Musculoskelet.Neuronal Interact.
2 (2002) 5 70572.
[5] T. Akiyama, C.R. Dass, P.F. Choong, Novel therapeutic strategy for osteosarcoma
targeting osteoclast differentiation, bone-resorbing activity, andapoptosis pathway,
Mol. Cancer Ther. 7 (2008) 34613469.
[6] T. Akiyama, C.R. Dass, Y. Shinoda, H. Kawano, S. Tanaka, P.F. Choong, Systemic
RANK-Fc protein therapy is efcacious against primary osteosarcoma growth
in a murine model via activity against osteoclasts, J. Pharm. Pharmacol. 62
(2010) 470476.
[7] S. Avnet, A. Longhi, M. Salerno, J.M. Halleen, F. Perut, D. Granchi, S. Ferrari, F.
Bertoni, A. Giunti, N. Baldini, Increased osteoclast activity is associated with
aggressiveness of osteosarcoma, Int. J. Oncol. 33 (2008) 12311238.
[8] L. Endo-Munoz, A. Cumming, D. Rickwood, D. Wilson, C. Cueva, C. Ng, G.
Strutton, A.I. Cassady, A. Evdokiou, S. Sommerville, I. Dickinson, A. Guminski,
N.A. Saunders, Loss of osteoclasts contributes to development of osteosarcoma
pulmonary metastases, Cancer Res. 70 (2010) 70637072.
[9] L. Endo-Munoz, A. Cumming, S. Sommerville, I. Dickinson, N.A.Saunders, Osteosar-
coma is characterised by reduced expression of markers of osteoclastogenesisand
antigen presentation compared with normal bone, Br. J. Cancer 103 (2010) 7381.
[10] A. Labrinidis, S. Hay, V. Liapis, V. Ponomarev, D.M. Findlay, A. Evdokiou,
Zoledronic acid inhibits both the osteolytic and osteoblastic components of
osteosarcoma lesions, Clin. Cancer Res. 15 (2009) 34513461.
[11] A. Labrinidis, S. Hay, V. Liapis, D.M. Findlay, A. Evdokiou, Zoledronic acid protects
against osteosarcoma-induced bone destruction but lacks efcacy against pul-
monary metastases in a syngeneic rat model, Int. J. Cancer 127 (2010) 345354.
[12] G.R. Mundy, Mechanisms of bone metastasis, Cancer 80 (1997) 15461556.
[13] T.A. Guise, K.S. Mohammad, G. Clines, E.G. Stebbins, D.H. Wong, L.S. Higgins, R.
Vessella, E. Corey, S. Padalecki, L. Suva, J.M. Chirgwin, Basic mechanisms respon-
sible for osteolytic and osteoblastic bone metastases, Clin. Cancer Res. 12 (2006)
6213s6216s.
[14] P. Allavena, A. Sica, G. Solinas, C. Porta, A. Mantovani, The inammatory
micro-environment in tumor progression: the role of tumor-associated macro-
phages, Crit. Rev. Oncol. Hematol. 66 (2008) 19.
[15] C.E. Lewis, J.W. Pollard, Distinct role of macrophages in different tumor microen-
vironments, Cancer Res. 66 (2006) 605612.
[16] G. Solinas, G. Germano, A. Mantovani, P. Allavena, Tumor-associated macro-
phages (TAM) as major players of the cancer-related inammation, J. Leukoc.
Biol. 86 (2009) 10651073.
[17] J. Condeelis, J.W. Pollard,Macrophages: obligate partners for tumor cell migration,
invasion, and metastasis, Cell 124 (2006) 263266.
[18] G.R.Mundy,Metastasis to bone: causes, consequencesand therapeuticopportunities,
Nat. Rev. Cancer 2 (2002) 584593.
[19] C. Bailey, R. Negus, A. Morris, P. Ziprin, R. Goldin, P. Allavena, D. Peck, A. Darzi,
Chemokine expression is associated with the accumulation of tumour associated
macrophages (TAMs) and progression in human colorectal cancer, Clin. Exp.
Metastasis 24 (2007) 121130.
[20] N. Nonomura, H. Takayama, M. Nakayama, Y. Nakai, A. Kawashima, M. Mukai, A.
Nagahara, K. Aozasa, A. Tsujimura, Inltration of tumour-associated macro-
phages in prostate biopsy specimens is predictive of disease progression after
hormonal therapy for prostate cancer, BJU Int. 107 (2011) 19181922.
[21] S. Osinsky, L. Bubnovskaya, I. Ganusevich, A. Kovelskaya, L. Gumenyuk, G.
Olijnichenko, S. Merentsev, Hypoxia, tumour-associatedmacrophages,microvessel
density, VEGF and matrix metalloproteinases in human gastric cancer: interaction
and impact on survival, Clin. Transl. Oncol. 13 (2011) 133138.
[22] J.W. Pollard, Macrophages dene the invasive microenvironment in breast cancer,
J. Leukoc. Biol. 84 (2008) 623630.
[23] E.Y. Lin, J.W. Pollard, Tumor-associated macrophages press the angiogenic
switch in breast cancer, Cancer Res. 67 (2007) 50645066.
[24] L.S. Ojalvo, W. King, D. Cox, J.W. Pollard, High-density gene expression analysis
of tumor-associated macrophages from mouse mammary tumors, Am. J. Pathol.
174 (2009) 10481064.
[25] A. Mantovani, A. Sica, Macrophages, innate immunity andcancer: balance, tolerance,
and diversity, Curr. Opin. Immunol. 22 (2010) 231237.
[26] D. DeNardo, D. Brennan,E. Rexhepaj, B.Ruffell, S. Shiao, S.Madden, W. Gallagher,N.
Wadhwani, S. Keil, S. Junaid, H. Rugo, E. Hwang, K. Jirstrom, B. West, L. Coussens,
Leukocytecomplexity predictsbreast cancer survivaland functionallyregulates re-
sponse to chemotherapy, Cancer Discov. 1 (2011) 5467.
[27] E.P. Buddingh, M.L. Kuijjer, R.A. Duim, H. Burger, K. Agelopoulos, O. Myklebost,
M. Serra, F. Mertens, P.C. Hogendoorn, A.C. Lankester, A.M. Cleton-Jansen,
Tumor-inltrating macrophages are associated with metastasis suppression in
high-grade osteosarcoma: a rationale for treatment with macrophage activating
agents, Clin. Cancer Res. 17 (2011) 21102119.
[28] G.D. Roodman, Regulation of osteoclast differentiation, Ann. N. Y. Acad. Sci. 1068
(2006) 100109.
[29] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation,
Nature 423 (2003) 337342.
[30] D.M. Mosser, J.P. Edwards, Exploring the full spectrum of macrophage activation,
Nat. Rev. Immunol. 8 (2008) 958969.
[31] E.R. Stanley, K.L.Berg, D.B. Einstein,P.S. Lee, F.J. Pixley,Y. Wang, Y.G. Yeung,Biology
and action of colonystimulating factor-1, Mol. Reprod. Dev. 46 (1997) 410.
[32] S. Gordon, P.R. Taylor, Monocyte and macrophage heterogeneity, Nat. Rev.
Immunol. 5 (2005) 953964.
[33] T.J. Chambers, Regulation of the differentiation and function of osteoclasts,
J. Pathol. 192 (2000) 413.
[34] T. Negishi-Koga, H. Takayanagi, Ca2+NFATc1 signaling is an essential axis of
osteoclast differentiation, Immunol. Rev. 231 (2009) 241256.
[35] H. Yasuda, N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, A.
Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio,
N. Udagawa, N. Takahashi, T. Suda, Osteoclast differentiation factor is a ligand
for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to
TRANCE/RANKL, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 35973602.
[36] N. Nakagawa, M. Kinosaki, K. Yamaguchi, N. Shima, H. Yasuda, K. Yano, T.
Morinaga, K. Higashio, RANK is the essential signaling receptor for osteoclast
differentiation factor in osteoclastogenesis, Biochem. Biophys. Res. Commun.
253 (1998) 395400.
[37] H. Hsu, D.L. Lacey, C.R. Dunstan, I. Solovyev, A. Colombero, E. Timms, H.L. Tan, G.
Elliott, M.J. Kelley, I. Sarosi, L. Wang, X.Z. Xia, R. Elliott, L. Chiu, T. Black, S. Scully,
C. Capparelli, S. Morony, G. Shimamoto, M.B. Bass, W.J. Boyle, Tumor necrosis
factor receptor family member RANK mediates osteoclast differentiation and
activation induced by osteoprotegerin ligand, Proc. Natl. Acad. Sci. U. S. A. 96
(1999) 35403545.
[38] S.L. Teitelbaum, Bone resorption by osteoclasts, Science 289 (2000) 15041508.
[39] S. Khosla, Minireview: the OPG/RANKL/RANK system, Endocrinology 142 (2001)
50505055.
[40] T. Nakashima, H. Takayanagi, New regulation me chanisms of osteoclast dif-
ferentiation, Ann. N. Y. Acad. Sci. 1240 (2011) E13E18.
[41] J. Xiong, C.A. O'Brien, Oste ocyte RANK L: new insights into the control of bone
remodeling,J.BoneMiner.Res.27(2012)499505.
440 L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434442
[42] D.L. Lacey, H.L. Tan,J. Lu, S. Kaufman, G. Van, W. Qiu, A. Rattan, S. Scully,F. Fletcher,
T. Juan, M. Kelley, T.L. Burgess, W.J. Boyle, A.J. Polverino, Osteoprotegerin ligand
modulates murine osteoclast survival in vitro and in vivo, Am. J. Pathol. 157
(2000) 435448.
[43] W.S. Simonet, D.L. Lacey, C.R. Dunstan, M. Kelley, M.S. Chang, R. Luthy, H.Q.
Nguyen, S. Wooden, L. Bennett, T. Boone, G. Shimamoto, M. DeRose, R. Elliott,
A. Colombero, H.L. Tan, G. Trail, J. Sullivan, E. Davy, N. Bucay, L. Renshaw-Gegg,
T.M. Hughes, D. Hill, W. Pattison, P. Campbell, S. Sander, G. Van, J. Tarpley, P.
Derby, R. Lee, W.J. Boyle, Osteoprotegerin: a novel secreted protein involved in
the regulation of bone density, Cell 89 (1997) 309319.
[44] H. Yasuda, N. Shima, N.Nakagawa, S.I. Mochizuki,K. Yano, N. Fujise, Y. Sato, M. Goto,
K.Yamaguchi,M.Kuriyama,T.Kanno,A.Murakami,E.Tsuda,T.Morinaga,K.
Higashio, Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin
(OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro,
Endocrinology 139 (1998) 13291337.
[45] J.M. Quinn, J.O. McGee, N.A. Athanasou, Human tumour-associated macrophages
differentiate into osteoclastic bone-resorbing cells, J. Pathol. 184 (1998) 3136.
[46] T.T. Yang, A. Sabokbar, C.L. Gibbons, N.A. Athanasou, Human mesenchymal
tumour-associated macrophages differentiate into osteoclastic bone-resorbing
cells, J. Bone Joint Surg. Br. 84 (2002) 452456.
[47] L.C.Hofbauer, M. Schoppet,Clinical implications ofthe osteoprotegerin/RANKL/RANK
system for bone and vascular diseases, JAMA 292 (2004) 490495.
[48] F.O. Martinez, A. Sica, A. Mantovani, M. Locati, Macrophage activation and polar-
ization, Front. Biosci. 13 (2008) 453461.
[49] S.Gordon, Alternativeactivation ofmacrophages,Nat. Rev.Immunol.3 (2003)2335.
[50] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, A. Sica, Macrophage polarization:
tumor-associated macrophages as a paradigm for polarized M2 mononuclear
phagocytes, Trends Immunol. 23 (2002) 549555.
[51] F.O. Martinez, S. Gordon, M. Locati, A. Mantovani, Transcriptional proling of the
human monocyte-to-macrophage differentiation and polarization: new mole-
cules and patterns of gene expression, J. Immunol. 177 (2006) 73037311.
[52] R.A. Mukhtar, O. Nseyo, M.J. Campbell, L.J. Esserman, Tumor-associated macro-
phages in breast cancer as potential biomarkers for new treatments and diagnos-
tics, Expert Rev. Mol. Diagn. 11 (2011) 91100.
[53] W.C. Dougall, RANKL signaling in bonephysiology and cancer,Curr. Opin. Support.
Palliat. Care 1 (2007) 317322.
[54] S. Das, R.S. Samant, L.A. Shevde, Hedgehog signaling induced by breast cancer
cells promotes osteoclastogenesis and osteolysis, J. Biol. Chem. 286 (2011)
96129622.
[55] J.J. Yin, K. Selander, J.M. Chirgwin, M. Dallas, B.G. Grubbs, R. Wieser, J. Massague,
G.R. Mundy, T.A. Guise, TGF-beta signaling blockade inhibits PTHrP secretion by
breast cancer cells and bone metastases development, J. Clin. Invest. 103 (1999)
197206.
[56] Y.C. Kuo, C.H. Su, C.Y. Liu, T.H. Chen, C.P. Chen, H.S. Wang, Transforming growth
factor-beta induces CD44 cleavage that promotes migration of MDA-MB-435s
cells through the up-regulation of membrane type 1-matrix metalloproteinase,
Int. J. Cancer 124 (2009) 25682576.
[57] D. Goltzman, Osteolysis and cancer, J. Clin. Invest. 107 (2001) 12191220.
[58] A.T. Stopeck, A. Lipton, J.J. Body, G.G. Steger, K. Tonkin, R.H. de Boer, M.
Lichinitser, Y. Fujiwara, D.A. Yardley, M. Viniegra, M. Fan, Q. Jiang, R. Dansey, S.
Jun, A. Braun, Denosumab compared with zoledronic acid for the treatment of
bone metastases in patients with advanced breast cancer: a randomized,
double-blind study, J. Clin. Oncol. 28 (2010) 51325139.
[59] R. Coleman, The use of bisphosphonates in cancer treatment, Ann. N. Y. Acad. Sci.
1218 (2011) 314.
[60] A.J. Roelofs, K. Thompson, F.H. Ebetino, M.J. Rogers, F.P. Coxon, Bisphosphonates:
molecular mechanisms of action and effects on bone cells, monocytes and
macrophages, Curr. Pharm. Des. 16 (2010) 29502960.
[61] G. Moriceau, B. Ory, B. Gobin, F. Verrecchia, F. Gouin, F. Blanchard, F. Redini, D.
Heymann, Therapeutic approach of primary bone tumours by bisphosphonates,
Curr. Pharm. Des. 16 (2010) 29812987.
[62] J.W. Pollard, Tumour-educated macrophages promote tumour progression and
metastasis, Nat. Rev. Cancer 4 (2004) 7178.
[63] C.E. Clark, S.R. Hingorani, R. Mick, C. Combs, D.A. Tuveson, R.H. Vonderheide,
Dynamics of the immune reaction to pancreatic cancer from inception to invasion,
Cancer Res. 67 (2007) 95189527.
[64] B. Bottazzi, N. Polentarutti, R. Acero, A. Balsari, D. Boraschi, P. Ghezzi, M.
Salmona, A. Mantovani, Regulation of the macrophage content of neoplasms
by chemoattractants, Science 220 (1983) 210212.
[65] J.A. Joyce, J.W. Pollard, Microenvironmental regulation of metastasis, Nat. Rev.
Cancer 9 (2009) 239252.
[66] A.E. Dirkx, M.G. Oude Egbrink, J. Wagstaff, A.W. Grifoen, Monocyte/macrophage
inltration in tumors: modulators of angiogenesis, J. Leukoc. Biol. 80 (2006)
11831196.
[67] S. Goswami, E. Sahai, J.B. Wyckoff, M. Cammer, D. Cox, F.J. Pixley, E.R. Stanley, J.E.
Segall, J.S. Condeelis, Macrophages promote the invasion of breast carcinoma
cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop,
Cancer Res. 65 (2005) 52785283.
[68] L.M. Coussens, C.L. Tinkle, D. Hanahan, Z. Werb, MMP-9 supplied by bone
marrow-derived cellscontributes to skin carcinogenesis, Cell 103 (2000) 481490.
[69] M.J. Pittet, Behavior of immune players in the tumor microenvironment, Curr.
Opin. Oncol. 21 (2009) 5359.
[70] X. Zhu, L.A. Mulcahy, R.A. Mohammed, A.H. Lee, H.A. Franks, L. Kilpatrick, A.
Yilmazer, E.C. Paish, I.O. Ellis, P.M. Patel, A.M. Jackson, IL-17 expression by
breast-cancer-associated macrophages: IL-17 promotes invasiveness of breast
cancer cell lines, Breast Cancer Res. 10 (2008) R95.
[71] D. Lyden, K. Hattori, S. Dias, C. Costa, P. Blaikie, L. Butros, A. Chadburn, B. Heissig,
W. Marks, L. Witte, Y. Wu, D. Hicklin, Z. Zhu, N.R. Hackett, R.G. Crystal, M.A.
Moore, K.A. Hajjar, K. Manova, R. Benezra, S. Rai, Impaired recruitment of
bone-marrow-derived endothelial and hematopoietic precursor cells blocks
tumor angiogenesis and growth, Nat. Med. 7 (2001) 11941201.
[72] S. Hiratsuka, K. Nakamura, S. Iwai, M. Murakami, T. Itoh, H. Kijima, J.M. Shipley,
R.M. Senior, M. Shibuya, MMP9 induction by vascular endothelial growth factor
receptor-1 is involved in lung-specic metastasis, Cancer Cell 2 (2002) 289300.
[73] B. Qian, Y. Deng, J.H. Im, R.J. Muschel, Y. Zou, J. Li, R.A. Lang, J.W. Pollard, A
distinct macrophage population mediates metastatic breast cancer cell extrava-
sation, establishment and growth, PLoS One 4 (2009) e6562.
[74] B.Z. Qian, J. Li, H. Zhang, T. Kitamura, J. Zhang, L.R. Campion, E.A. Kaiser, L.A.
Snyder, J.W. Pollard, CCL2 recruits inammatory monocytes to facilitate
breast-tumour metastasis, Nature 475 (7355) (2011) 222225.
[75] J. Zhang, Y. Lu, K.J. Pienta, Multiple roles of chemokine (C-C motif) ligand 2 in
promoting prostate cancer growth, J. Natl. Cancer Inst. 102 (2010) 522528.
[76] S.W. Kim, J.S. Kim, J. Papadopoulos, H.J. Choi, J. He, M. Maya, R.R. Langley, D. Fan,
I.J. Fidler, S.J. Kim, Consistent interactions between tumor cell IL-6 and macro-
phage TNF-alpha enhance the growth of human prostate cancer cells in the
bone of nude mouse, Int. Immunopharmacol. 11 (2011) 859869.
[77] G. Ottaviani, N. Jaffe, The epidemiology of osteosarcoma, Cancer Treat. Res. 152
(2009) 313.
[78] K. O'Day, R. Gorlick, Novel therapeutic agents for osteosarcoma, Expert Rev.
Anticancer Ther. 9 (2009) 511523.
[79] P.A. Meyers, R. Gorlick, Osteosarcoma, Pediatr. Clin. North Am. 44 (1997)
973989.
[80] P.A. Meyers, C.L. Schwartz, M. Krailo, E.S. Kleinerman, D. Betcher, M.L. Bernstein,
E. Conrad, W. Ferguson, M. Gebhardt, A.M. Goorin, M.B. Harris, J. Healey, A.
Huvos, M. Link, J. Montebello, H. Nadel, M. Nieder, J. Sato, G. Siegal, M. Weiner,
R. Wells, L. Wold, R. Womer, H. Grier, Osteosarcoma: a randomized, prospective
trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin,
doxorubicin, and high-dose methotrexate, J. Clin. Oncol. 23 (2005) 20042011.
[81] J.B. Hayden, B.H. Hoang, Osteosarcoma: basic science and clinical implications,
Orthop. Clin. North Am. 37 (2006) 17.
[82] C. Khanna, J. Khan, P. Nguyen, J. Prehn, J. Caylor, C. Yeung, J. Trepel, P. Meltzer, L.
Helman, Metastasis-associated differences in gene expression in a murine
model of osteosarcoma, Cancer Res. 61 (2001) 37503759.
[83] D.P. Hughes, How the NOTCH pathway contributes to the ability of osteosarcoma
cells to metastasize, Cancer Treat. Res. 152 (2009) 479496.
[84] P. Zhang, Y. Yang, P.A. Zweidler-McKay, D.P. Hughes, Critical role of notch signal-
ing in osteosarcoma invasion and metastasis, Clin. Cancer Res. 14 (2008)
29622969.
[85] O. Fromigue, Z. Hamidouche, P. Vaudin, F. Lecanda, A. Patino, P. Barbry, B. Mari,
P.J. Marie, CYR61 downregulation reduces osteosarcoma cell invasion, migra-
tion, and metastasis, J. Bone Miner. Res. 26 (2011) 15331542.
[86] P. McQueen, S. Ghaffar, Y. Guo, E.M. Rubin, X. Zi, B.H. Hoang, The Wnt signaling
pathway: implications for therapy in osteosarcoma, Expert Rev. Anticancer Ther.
11 (2011) 12231232.
[87] E.M. Rubin, Y. Guo, K. Tu, J. Xie, X. Zi, B.H. Hoang, Wnt inhibitory factor 1 de-
creases tumorigenesis and metastasis in osteosarcoma, Mol. Cancer Ther. 9
(2010) 731741.
[88] P.C. Leow, Q. Tian, Z.Y. Ong, Z. Yang, P.L. Ee, Antitumor activity of natural com-
pounds, curcumin and PKF118-310, as Wnt/beta-catenin antagonists against
human osteosarcoma cells, Invest. New Drugs 28 (2010) 766782.
[89] M. Kaya, T. Wada, T. Akatsuka, S. Kawaguchi, S. Nagoya, M. Shindoh, F.
Higashino, F. Mezawa, F. Okada, S. Ishii, Vascular endothelial growth factor
expression in untreated osteosarcoma is predictive of pulmonary metastasis
and poor prognosis, Clin. Cancer Res. 6 (2000) 572577.
[90] S.Y. Yang, H. Yu, J.E. Krygier, P.H. Wooley, M.P. Mott, High VEGF with rapid
growth and early metastasis in a mouse osteosarcoma model, Sarcoma 2007
(2007) 95628.
[91] C. Khanna, X. Wan, S. Bose, R. Cassaday, O. Olomu, A. Mendoza, C. Yeung, R.
Gorlick, S.M. Hewitt, L.J. Helman, The membrane-cytoskeleton linker ezrin is
necessary for osteosarcoma metastasis, Nat. Med. 10 (2004) 182186.
[92] X. Wan, S.Y. Kim, L.M. Guenther, A. Mendoza, J. Briggs, C. Yeung, D. Currier, H.
Zhang, C. Mackall, W.J. Li, R.S. Tuan, A.T. Deyrup, C. Khanna, L. Helman, Beta4
integrin promotes osteosarcoma metastasis and interacts with ezrin, Oncogene
28 (2009) 34013411.
[93] L. Ren, S.H. Hong, J. Cassavaugh, T. Osborne, A.J. Chou, S.Y. Kim, R. Gorlick, S.M.
Hewitt, C. Khanna, The actin-cytoskeleton linker protein ezrin is regulated during
osteosarcoma metastasis by PKC, Oncogene 28 (2009) 792802.
[94] L.L. Worth, E.A. Laeur, S.F. Jia, E.S. Kleinerman, Fas expression inversely corre-
lates with metastatic potential in osteosarcoma cells, Oncol. Rep. 9 (2002)
823827.
[95] N. Gordon, N.V. Koshkina, S.F. Jia, C. Khanna, A. Mendoza, L.L. Worth, E.S.
Kleinerman, Corruption of the Fas pathway delays the pulmonary clearance of
murine osteosarcoma cells, enhances their metastatic potential, and reduces
the effect of aerosol gemcitabine, Clin. Cancer Res. 13 (2007) 45034510.
[96] S.Y. Kim,C.H. Lee, B.V. Midura,C. Yeung, A. Mendoza,S.H. Hong, L. Ren, D. Wong,W.
Korz, A. Merzouk, H. Salari, H. Zhang, S.T. Hwang, C. Khanna, L.J. Helman, Inhibition
of the CXCR4/CXCL12 chemokine pathway reduces the developme nt of murine
pulmonary metastases, Clin. Exp. Metastasis 25 (2008) 201211.
[97] E. Pradelli, B. Karimdjee-Soilihi, J.F. Michiels, J.E. Ricci, M.A. Millet, F. Vandenbos,
T.J. Sullivan, T.L. Collins, M.G. Johnson, J.C. Medina, E.S. Kleinerman,A. Schmid-Alliana,
H. Schmid-Antomarchi, Antagonism of chemokine receptor CXCR3 inhibits osteosar-
coma metastasis to lungs, Int. J. Cancer 125 (2009) 25862594.
441L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434442
[98] P.A. Meyers, Malignant bone tumors in children: osteosarcoma, Hematol. Oncol.
Clin. North Am. 1 (1987) 655665.
[99] F.O. Kesselring, W. Penn, Radiological aspects of classicprimary osteosarcoma:
value of some radiological investigations: A review, Diagn. Imaging 51 (1982)
7892.
[100] F. Lamoureux, P. Richard, Y. Wittrant, S. Battaglia, P. Pilet, V. Trichet, F.
Blanchard, F. Gouin, B. Pitard, D. Heymann, F. Redini, Therapeutic relevance of
osteoprotegerin gene therapy in osteosarcoma: blockade of the vicious cycle
between tumor cell proliferation and bone resorption, Cancer Res. 67 (2007)
73087318.
[101] C.R. Dass, P.F. Choong, Zoledronic acid inhibits osteosarcoma growth in an
orthotopic model, Mol. Cancer Ther. 6 (2007) 32633270.
[102] B. Ory, M.F. Heymann, A. Kamijo, F. Gouin, D. Heymann, F. Redini, Zoledronic acid
suppresses lung metastases and prolongs overall survival of osteosarcoma-bearing
mice, Cancer 104 (2005) 25222529.
[103] P.A. Meyers, Muramyl tripeptide (mifamurtide) for thetreatment of osteosarcoma,
Expert Rev. Anticancer Ther. 9 (2009) 10351049.
[104] J.E. Frampton, Mifamurtide: a review of its use in the treatment of osteosarcoma,
Paediatr. Drugs 12 (2010) 141153.
[105] M.H. Moon, J.K. Jeong, J.S. Seo, J.W. Seol, Y.J. Lee, M. Xue, C.J. Jackson, S.Y. Park,
Bisphosphonate enhances TRAIL sensitivity to human osteosarcoma cells via
death receptor 5 upregulation, Exp. Mol. Med. 43 (2011) 138145.
[106] K. Koto, H. Murata, S. Kimura, N. Horie, T. Matsui, Y. Nishigaki, K. Ryu, T. Sakabe,
M. Itoi, E. Ashihara, T. Maekawa, S. Fushiki, T. Kubo, Zoledronic acid inhibits pro-
liferation of human brosarcoma cells with induction of apoptosis, and shows
combined effects with other anticancer agents, Oncol. Rep. 24 (2010) 233239.
[107] M.S. Molinuevo, L. Bruzzone, A.M. Cortizo, Alendronate induces anti-migratory
effects and inhibition of neutral phosphatases in UMR106 osteosarcoma cells,
Eur. J. Pharmacol. 562 (2007) 2833.
[108] B. Kubista, K. Trieb, F. Sevelda, C. Toma, F. Arrich, P. Heffeter, L. Elbling, H.
Sutterluty, K. Scotlandi, R. Kotz, M. Micksche, W. Berger, Anticancer effects of
zoledronic acid against human osteosarcoma cells, J. Orthop. Res. 24 (2006)
11451152.
[109] A. Evdokiou, A. Labrinidis, S. Bouralexis, S. Hay, D.M. Findlay, Induction of cell
death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis,
Bone 33 (2003) 216228.
[110] E. Hamilton, T.M. Clay, K.L. Blackwell, New perspectives on zoledronic acid in
breast cancer: potential augmentation of anticancer immune response, Cancer
Invest. 29 (2011) 533541.
[111] J.R. Berenson, Antitumor effects of bisphosphonates: from the laboratory to the
clinic, Curr. Opin. Support. Palliat. Care 5 (2011) 233240.
[112] L. Costa, P. Harper, R.E. Coleman, A. Lipton, Anticancer evidence for zoledronic
acid across the cancer continuum, Crit. Rev. Oncol. Hematol. 77 (Suppl. 1)
(2011) S31S37.
[113] G. Daniele, P. Giordano, A. De Luca, M.C. Piccirillo, M. Di Maio, A. Del Giudice, G.
De Feo, J. Bryce, L. Lamura, A. Vecchione, N. Normanno, F. Perrone, Anticancer
effectof bisphosphonates: new insightsfrom clinical trialsand preclinicalevidence,
Expert Rev. Anticancer Ther. 11 (2011) 299307.
[114] F.S. Nicholas, A. Saunders, ErikW. Thompson, MichelleM. Hill, LilianaEndo-Munoz,
Graham Leggatt, Rodney F. Minchin, Alexander Guminski, Role of intratumoral
heterogeneity in cancer drug resistance: molecular and clinical perspectives,
EMBO, Mol. Med. (Jun 25 2012), http://dx.doi.org/10.1002/emmm.201101131.
[115] A.M. Barger, T.M. Fan, L.P. de Lorimier, I.T. Sprandel, K. O'Dell-Anderson,Expression
of receptor activator of nuclear factor kappa-B ligand (RANKL) in neoplasms of
dogs and cats, J. Vet. Intern. Med. 21 (2007) 133140.
[116] K. Mori, B. Le Goff, M. Berreur, A. Riet, A. Moreau, F. Blanchard, C. Chevalier, I.
Guisle-Marsollier, J. Leger, J. Guicheux, M. Masson, F. Gouin, F. Redini, D.
Heymann, Human osteosarcoma cells express functional receptor activator of
nuclear factor-kappa B, J. Pathol. 211 (2007) 555562.
[117] S.D. Molyneux, M.A. Di Grappa, A.G. Beristain, T.D. McKee, D.H. Wai, J. Paderova,
M. Kashyap, P. Hu, T. Maiuri, S.R. Narala, V. Stambolic, J. Squire, J. Penninger, O.
Sanchez, T.J. Triche, G.A. Wood, L.S. Kirschner, R. Khokha, Prkar1a is an osteosarcoma
tumor suppressor that denes a molecular subclass in mice, J. Clin. Invest. 120
(2010) 33103325.
[118] M.B. Mintz, R. Sowers, K.M. Brown, S.C. Hilmer, B. Mazza, A.G. Huvos, P.A.
Meyers, B. Laeur, W.S. McDonough, M.M. Henry, K.E. Ramsey, C.R. Antonescu,
W. Chen, J.H. Healey, A. Daluski, M.E. Berens, T.J. Macdonald, R. Gorlick, D.A.
Stephan, An expression signature classies chemotherapy-resistant pediatric
osteosarcoma, Cancer Res. 65 (2005) 17481754.
[119] A. Patino-Garcia,M. Zalacain, C. Folio, C.Zandueta, L. Sierrasesumaga, M. SanJulian,
G. Toledo, J. De Las Rivas, F. Lecanda, Proling of chemonaive osteosarcoma and
paired-normal cells identies EBF2 as a mediator of osteoprotegerin inhibition to
tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis, Clin.
Cancer Res. 15 (2009) 50825091.
[120] H. Zreiqat, C.R. Howlett, S. Gronthos, D. Hume, C.L. Geczy, S100A8/S100A9 and
their association with cartilage and bone, J. Mol. Histol. 38 (2007) 381391.
[121] T.J. Wilson, K.C. Nannuru, R.K. Singh, Cathepsin G recruits osteoclast precursors
via proteolytic activation of protease-activated receptor-1, Cancer Res. 69
(2009) 31883195.
[122] M.B. Humphrey, K. Ogasawara, W.Yao, S.C. Spusta, M.R.Daws, N.E. Lane, L.L.Lanier,
M.C. Nakamura, The signaling adapter protein DAP12 regulates multinucleation
during osteoclast development, J. Bone Miner. Res. 19 (2004) 224234.
[123] A. Mocsai, M.B. Humphrey, J.A. Van Zife, Y. Hu, A. Burghardt, S.C. Spusta, S.
Majumdar, L.L. Lanier, C.A. Lowell, M.C. Nakamura, The immunomodulatory
adapter proteins DAP12 and Fc receptor gamma-chain (FcRgamma) regulate
development of functional osteoclasts through the Syk tyrosine kinase, Proc.
Natl. Acad. Sci. U. S. A. 101 (2004) 61586163.
[124] A.R. Hayman, Tartrat e-resistant acid phosphatas e (TRAP) and the osteoclast/
immune cell dichotomy, Autoimmunity 41 (20 08) 218223.
[125] J. Lee, K. Kim, J.H. Kim, H.M. Jin, H.K. Choi, S.H. Lee, H. Kook, K.K. Kim, Y. Yokota,
S.Y. Lee, Y. Choi, N. Kim, Id helix-loop-helix proteins negatively regulate
TRANCE-mediated osteoclast differentiation, Blood 107 (2006) 26862693.
[126] J.M. Chirgwin, T.A. Guise, Skeletal metastases: decreasing tumor burden by
targeting the bone microenvironment, J. Cell. Biochem. 102 (2007) 13331342.
[127] P.A. Meyers, C.L. Schwartz, M.D. Krailo, J.H. Healey, M.L. Bernstein, D. Betcher,
W.S. Ferguson, M.C. Gebhardt, A.M. Goorin, M. Harris, E. Kleinerman, M.P. Link,
H. Nadel, M. Nieder, G.P. Siegal, M.A. Weiner, R.J. Wells, R.B. Womer, H.E.
Grier, Osteosarcoma: the addition of muramyl tripeptide to chemotherapy
improves overall survivala report from the Children's Oncology Group, J. Clin.
Oncol. 26 (2008) 633638.
[128] A. Nardin, M.L. Lefebvre, K. Labroquere, O. Faure, J.P. Abastado, Liposomal
muramyl tripeptide phosphatidylethanolamine: targeting and activating macro-
phages for adjuvant treatment of osteosarcoma, Curr. Cancer Drug Targets 6
(2006) 123133.
[129] E.S. Kleinerman, P.A. Meyers, A.K. Raymond, J.B. Gano, S.F. Jia, N. Jaffe, Combination
therapy with ifosfamide and lipo some-encapsulated muramyl tripeptide: tolerability,
toxicity, and immune stimulation, J. Immunother. Emphasis Tumor Immunol. 17
(1995) 181193.
[130] I.J. Fidler, S. Sone, W.E. Fogler, Z.L. Barnes, Eradication of spontaneous metastases
and activation of alveolar macrophages by intravenous injection of liposomes
containing muramyl dipeptide, Proc. Natl. Acad. Sci. U. S. A. 78 (1981)
16801684.
[131] E.G. MacEwen, I.D. Kurzman,R.C. Rosenthal, B.W.Smith, P.A. Manley, J.K. Roush,P.E.
Howard, Therapy for osteosarcoma in dogs with intravenous injection of
liposome-encapsulated muramyl tripeptide, J. Natl. Cancer Inst. 81 (1989) 935938.
[132] P.J. Creaven, J.W. Cowens, D.E. Brenner, B.M. Dadey, T. Han, R. Huben, C.
Karakousis, H. Frost, D. LeSher, J. Hanagan, et al., Initial clinical trial of the mac-
rophage activator muramyl tripeptide-phosphatidylethanolamine encapsulated
in liposomes in patients with advanced cancer, J. Biol. Response Mod. 9 (1990)
492498.
[133] I.D. Kurzman, E.G. MacEwen, R.C. Rosenthal, L.E. Fox, E.T. Keller, S.C. Helfand, D.M.
Vail, R.R. Dubielzig, B.R. Madewell, C.O. Rodriguez Jr., et al., Adjuvant therapy for
osteosarcoma in dogs: results of randomized clinical trials using combined
liposome-encapsulated muramyl tripeptide and cisplatin, Clin. Cancer Res. 1
(1995) 15951601.
[134] E.S. Kleinerman, S.F. Jia, J. Grifn,N.L. Seibel, R.S.Benjamin, N. Jaffe, Phase II study of
liposomal muramyl tripeptide in osteosarcoma: the cytokine cascade and mono-
cyte activation following administration, J. Clin. Oncol. 10 (1992) 13101316.
[135] B.W. Smith, I.D. Kurzman, K.T. Schultz, C.J. Czuprynski, E.G. MacEwen, Muramyl
peptides augment the in vitro and in vivo cytostatic activity of canine
plastic-adherent mononuclear cells against canine osteosarcoma cells, Cancer
Biother. 8 (1993) 137144.
[136] S. Bielack, D. Carrle, P.G. Casali,Osteosarcoma: ESMO clinical recommendationsfor
diagnosis, treatment and follow-up, Ann. Oncol. 20 (Suppl. 4) (2009) 137139.
442 L. Endo-Munoz et al. / Biochimica et Biophysica Acta 1826 (2012) 434442
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Objectives Investigation of the therapeutic effect of zoledronic acid (ZA) in a preclinical model of jaw osteosarcoma (JO). Materials and Methods The effect of 100 μg/kg ZA administered twice a week was assessed in a xenogenic mouse model of JO. The clinical (tumor growth, development of lung metastasis), radiological (bone microarchitecture by micro‐CT analysis), and molecular and immunohistochemical (TRAP, RANK/RANKL, VEGF, and CD146) parameters were investigated. Results Animals receiving ZA exhibited an increased tumor volume compared with nontreated animals (71.3 ± 14.3 mm ³ vs. 51.9 ± 19.9 mm ³ at D14, respectively; p = 0.06) as well as increased numbers of lung metastases (mean 4.88 ± 4.45 vs. 0.50 ± 1.07 metastases, respectively; p = 0.02). ZA protected mandibular bone against tumor osteolysis (mean bone volume of 12.81 ± 0.53 mm ³ in the ZA group vs. 11.55 ± 1.18 mm ³ in the control group; p = 0.01). ZA induced a nonsignificant decrease in mRNA expression of the osteoclastic marker TRAP and an increase in RANK/RANKL bone remodeling markers. Conclusion The use of bisphosphonates in the therapeutic strategy for JO should be further explored, as should the role of bone resorption in the pathophysiology of the disease.
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Background: Osteosarcoma (OS) is a common primary malignant bone tumor that mainly occurs in children and adolescents. The use of IL-8 inhibitor compounds has been reported in patents, which can be used to treat and/or prevent osteosarcoma, but the pathogenesis of osteosarcoma remains to be investigated. At present, osteoblasts and osteoclasts play an important role in the occurrence and development of OS. However, the relationship between osteoblasts and osteoclasts in the specific participation mechanism and inflammatory response of OS patients has not been further studied. Methods: The transcriptome, clinical data, and other data related to OS were downloaded from the GEO database to analyze them with 200 known inflammatory response genes. We set the screening conditions as p < 0.05 and | log2FC|>0.50, screened the differentially expressed genes (DEGs) related to OS, tested the correlation coefficient between the OS INF gene and clinical risk, and analyzed the survival prognosis. We further enriched and analyzed the DEGs and inflammatory response genes of OS with GO/KEGG to explore the potential biological function and signal pathway mechanism of OS inflammatory response genes. Moreover, the virtual screening of drug sensitivity of OS based on the FDA drug library was also carried out to explore potential therapeutic drugs targeted to regulate OS osteogenesis and osteoclast inflammation, and finally, the molecular dynamics simulation verification of OS core protein and potential drugs was carried out to explore the binding stability and mechanism between potential drugs and core protein. Results: Through differential analysis of GSE39058, GSE36001, GSE87624, and three other data sets closely related to OS osteoblasts and osteoclasts, we found that there was one upregulated gene (CADM1) and one down-regulated gene (PHF15) related to OS. In addition, GSEA enrichment analysis of the DEGs of OS showed that it was mainly involved in the progress of OS through biological functions, such as oxidative photosynthesis, acute junction, and epithelial-mesenchymal transition. The enrichment analysis of OS DEGs revealed that they mainly affect the occurrence and progress of OS by participating in the regulation of the actin skeleton, PI3K Akt signal pathway, complement and coagulation cascade. According to the expression of CSF3R in OS patients, a risk coefficient model and a diagnostic model were established. It was found that the more significant the difference in the CSF3R gene in OS patients, the greater the risk coefficient of disease (p <0.05). The AUC under the curve of the CSF3R gene was greater than 0.65, which had a good diagnostic significance for OS. The above results showed that the prognosis risk gene CSF3R related to OS inflammation was closely related to the survival status of OS patients. Finally, through the virtual screening of the ZINC drug library and molecular dynamics simulation, it was found that the docking model formed by the core protein CSF3R and the compounds, Leucovorin and Methotrexate, were the most stable, which revealed that the compounds Leucovorin and Methotrexate might play a role in the treatment of OS by combining with the inflammatory response related factor CSF3R of OS. Conclusion: CSF3R participates in the occurrence and development of OS bone destruction by regulating the inflammatory response of osteoblasts and osteoclasts and can affect the survival prognosis of OS patients.
Article
Objectives Osteosarcoma (OS) is the most common primary malignant bone tumour, and mainly affects adolescents and young adults. Although there has been substantial improvement in management of OS with surgery and chemotherapy, further survival increase has not been achieved over the past two decades. Methods We focused on the receptor activator of nuclear factor κB ligand (RANKL)–osteoclast (OCL) system as a biological target for OS. RANKL is a critical factor for OCL formation and bone resorption activity. The primary lesion in bone and ensuing metastasis in OS both require the induction of OCLs. RANK-Fc is a potent RANKL antagonist and inhibitor of OCL formation and activity. Key findings In an orthotopic model in Balb/c nu/nu mice, a twice weekly dosing regimen of 350 μg of RANK-Fc per mouse subcutaneously (n= 5) reduced lung metastasis (P > 0.05), preserved bone structure and reduced tartrate-resistant acid phosphatase (TRAP)+ OCLs (P < 0.005) in OS-bearing bone. In vitro, RANK-Fc suppressed OCL formation (P < 0.005), bone resorption activity (P < 0.005) and RANKL-induced anti-apoptosis (P < 0.5) of OCLs.
Article
PURPOSEA phase II trial that uses liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) in patients with relapsed osteosarcoma is underway. To determine if in vivo cytokine induction plays a role in the mechanism of action of L-MTP-PE, we investigated the circulating cytokine levels of 16 patients who were undergoing therapy.PATIENTS AND METHODS Patients had histologically proven osteosarcoma and pulmonary metastases that developed either during adjuvant chemotherapy or that were present at diagnosis and persisted despite chemotherapy. Patients were rendered disease-free by surgery. The major goal of the study was to improve the disease-free interval in this high-risk group. L-MTP-PE 2 mg/m2 was infused during a 1-hour period twice a week for 12 weeks, then once a week for 12 weeks. Serial blood samples were collected after L-MTP-PE administration and were assayed for cytokine levels (tumor necrosis factor-alpha [TNF alpha] interleukin-1 alpha [IL-1 alpha], IL-1 beta, IL-6, interfero...
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Solid cancers metastasize to bone by a multistep process that involves interactions between tumor cells and normal host cells. Some tumors, most notably breast and prostate carcinomas, grow avidly in bone because the bone microenvironment provides a favorable soil. In the case of breast carcinoma, the final step in bone metastasis (namely bone destruction) is mediated by osteoclasts that are stimulated by local production of the tumor peptide parathyroid hormone-related peptide (PTH-rP), whereas prostate carcinomas stimulate osteoblasts to make new bone. Production of PTH-rP by breast carcinoma cells in bone is enhanced by growth factors produced as a consequence of normal bone remodeling, particularly activated transforming growth factor-β (TGF-β). Thus, a vicious cycle exists in bone between production by the tumor cells of mediators such as PTH-rP and subsequent production by bone of growth factors such as TGF-β, which enhance PTH-rP production. The metastatic process can be interrupted either by neutralization of PTH-rP or by rendering the tumor cells unresponsive to TGF-β, both of which can be accomplished experimentally. The osteoclast is another available site for therapeutic intervention in the bone metastatic process. Osteoclasts can be inhibited by drugs such as the new-generation bisphosphonates; as a consequence of this inhibition, there is a marked reduction in the skeletal events associated with metastatic cancer to bone, such as pain, fracture, and hypercalcemia. However and possibly even more importantly, there is also a reduction of tumor burden in bone. In experimental situations, this has clearly been shown to affect not only morbidity but also survival. The precise mechanism by which bisphosphonates inhibit osteoclasts is still unclear and may represent a combination of inhibition of osteoclast formation as well as increased apoptosis in mature osteoclasts. However, studies with potent bisphosphonates such as ibandronate, pamidronate, and risedronate have clearly documented that reduction of bone turnover and osteoclast activity leads to beneficial effects not only on skeletal complications associated with metastatic cancer, but also on tumor burden in bone. Cancer 1997; 80:1546-56.
Article
The osteoclast is the cell that resorbs bone. It has been known for many years that its formation and function are regulated by cells of the osteoblastic lineage. Recently the molecular basis for this regulation was identified; osteoblastic cells induce osteoclastic differentiation and resorptive activity through expression of tumour necrosis factor (TNF) activation-induced cytokine (TRANCE) (also known as RANKL, ODF, OPGL, and TNFSF11), a novel membrane-inserted member of the TNF superfamily. Osteoclastic regulation is assisted through secretion of an inhibitor, osteoprotegerin (OPG) (OCIF, TNFRSF11B), a soluble (decoy) receptor for TRANCE. Osteoclast formation and survival also depend on and are substantially enhanced by transforming growth factor-β (TGF-β), which is abundant in bone matrix. Surprisingly, not only TRANCE but also TNF-α can induce osteoclast formation in vitro from bone marrow-derived mononuclear phagocytes, especially in the presence of TGF-β. Whether or not TNF-α does the same in vivo, its ability to generate osteoclasts in vitro has significant implications regarding the nature of osteoclasts and their relationship to other mononuclear phagocytes, and a possible wider role for TRANCE in macrophage pathobiology. A hypothesis is presented in which the osteoclast is a mononuclear phagocyte directed towards a debriding function by TGF-β, activated for this function by TRANCE, and induced to become specifically osteoclastic by the characteristics of the substrate or signals from bone cells that betoken such characteristics. Copyright
Article
Macrophages are widely distributed immune system cells that play an indispensable role in homeostasis and defense. They can be phenotypically polarized by the microenvironment to mount specific functional programs. Polarized macrophages can be broadly classified in two main groups: classically activated macrophages (or M1), whose prototypical activating stimuli are IFNgamma and LPS, and alternatively activated macrophages (or M2), further subdivided in M2a (after exposure to IL-4 or IL-13), M2b (immune complexes in combination with IL-1beta or LPS) and M2c (IL-10, TGFbeta or glucocorticoids). M1 exhibit potent microbicidal properties and promote strong IL-12-mediated Th1 responses, whilst M2 support Th2-associated effector functions. Beyond infection M2 polarized macrophages play a role in resolution of inflammation through high endocytic clearance capacities and trophic factor synthesis, accompanied by reduced pro-inflammatory cytokine secretion. Similar functions are also exerted by tumor-associated macrophages (TAM), which also display an alternative-like activation phenotype and play a detrimental pro-tumoral role. Here we review the main functions of polarized macrophages and discuss the perspectives of this field.