Hindawi Publishing Corporation

Stem Cells International

Volume 2016, Article ID 3631764, 13 pages
http://dx.doi.org/10.1155/2016/3631764

Review Article
Osteosarcoma: Cells-of-Origin, Cancer Stem Cells, and
Targeted Therapies

Ander Abarrategi,1 Juan Tornin,2 Lucia Martinez-Cruzado,2 Ashley Hamilton,1

Enrique Martinez-Campos,3 Juan P. Rodrigo,2 M. Victoria González,2 Nicola Baldini,4,5
Javier Garcia-Castro,6 and Rene Rodriguez2
1
Haematopoietic Stem Cell Laboratory, The Francis Crick Institute, London WC2A 3LY, UK
2
Central University Hospital of Asturias (HUCA) and Institute of Oncology of Asturias (IUOPA), 33011 Oviedo, Spain
3
Complutense University of Madrid, 28040 Madrid, Spain
4
Orthopaedic Pathophysiology and Regenerative Medicine Unit, Rizzoli Orthopaedic Institute, 40136 Bologna, Italy
5
Department of Biomedical and Neuromotor Sciences, University of Bologna, 40123 Bologna, Italy
6
Cellular Biotechnology Laboratory, Institute of Health Carlos III (ISCIII), Majadahonda, 28220 Madrid, Spain

Correspondence should be addressed to Rene Rodriguez; renerg@ficyt.es

Received 7 January 2016; Accepted 10 May 2016

Academic Editor: Andrzej Lange

Copyright © 2016 Ander Abarrategi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Osteosarcoma (OS) is the most common type of primary solid tumor that develops in bone. Although standard chemotherapy has
significantly improved long-term survival over the past few decades, the outcome for those patients with metastatic or recurrent
OS remains dismally poor and, therefore, novel agents and treatment regimens are urgently required. A hypothesis to explain the
resistance of OS to chemotherapy is the existence of drug resistant CSCs with progenitor properties that are responsible of tumor
relapses and metastasis. These subpopulations of CSCs commonly emerge during tumor evolution from the cell-of-origin, which
are the normal cells that acquire the first cancer-promoting mutations to initiate tumor formation. In OS, several cell types along the
osteogenic lineage have been proposed as cell-of-origin. Both the cell-of-origin and their derived CSC subpopulations are highly
influenced by environmental and epigenetic factors and, therefore, targeting the OS-CSC environment and niche is the rationale
for many recently postulated therapies. Likewise, some strategies for targeting CSC-associated signaling pathways have already
been tested in both preclinical and clinical settings. This review recapitulates current OS cell-of-origin models, the properties of
the OS-CSC and its niche, and potential new therapies able to target OS-CSCs.

1. Introduction OS associated with several genetic syndromes such as Li-

Fraumeni, hereditary retinoblastoma, and Rothmund Thom-
OS is a malignant neoplasm in which the neoplastic cells son (see below).
produce bone and is the most frequent primary sarcoma Primary OS may arise in any bone, although the vast
of the skeleton. The tumor is primary when the underlying majority originate in the long bones of the extremities, espe-
bone is normal and secondary when the bone is altered cially the distal femur (30%), followed by the proximal tibia
by conditions, such as prior irradiation, coexisting Paget (15%), and proximal humerus (15%), which represent sites
disease, infarction, or other disorders. It has a bimodal age containing the most proliferative growth plates. Within long
distribution with most cases developing between the ages of bones, the tumor is usually (90%) located in the metaphysis
10–16 years and a second smaller peak in older adults (30% of and arises as an enlarging and palpable mass, with progressive
cases in patients over 40 years) [1]. In addition, OS is the most pain [2].
common radiation-induced sarcoma. It has an unknown The hallmark diagnostic feature of OS is the detection
etiology, although there is an increased incidence of primary of osteoid matrix produced by the neoplastic cells. However,
2 Stem Cells International

the most common type of OS, conventional OS, has a very [13, 15]. It was reported that RB is needed to promote
broad spectrum of histological appearances and is subclassi- the osteogenic differentiation program of mesenchymal
fied according to the predominant type of stroma (osteoblas- stem/stromal cells (MSCs) [16] and, therefore, it could be
tic, chondroblastic, fibroblastic, giant cell rich, etc.), although speculated that RB mutations synergize with P53 inactivation
this subclassification has no prognostic relevance [1]. in OS formation only when mutations occur in osteogenic-
At present, surgery with chemotherapy is the first-line committed cell types; meanwhile it could favor other sarcoma
treatment for most OS [3]. Almost all patients receive phenotypes when mutated in more immature cell types (see
neoadjuvant intravenous combinational chemotherapy (dox- below).
orubicin and cisplatin with or without methotrexate) as initial Other genes involved in P53 or RB signaling have also
treatment. Surgical resection of the primary tumor with ade- been found to be mutated in sporadic OS [6, 17]. For example,
quate margins is an essential component of the curative strat- the INK4A/ARF locus, which encodes for P16INK4A and
egy for patients with localized OS. If complete surgical resec- P19ARF genes, is deleted in approximately 10% of OS [18, 19].
tion is not feasible or if surgical margins are inadequate, radi- P16INK4A and P19ARF proteins contribute to the stabiliza-
ation therapy may improve the local control rate. The postop- tion of RB and P53 proteins through the inhibition of cyclin-
erative chemotherapy regimen usually depends on the extent dependent kinase 4 (CDK4) and mouse double minute 2
of tumor necrosis observed [1, 3]. homolog (MDM2) repressors, respectively [20]. Interestingly,
Advances in the clinical management of OS have led the region 12q13, containing CDK4 and MDM2, is amplified
to a significant increase in 5-year survival rates, which in in up to 10% of OS [6, 21]. In addition, the absence of
most centers now largely exceed 50%. However, survival rates expression of P16INK4A correlated with a decreased survival
for patients presenting with metastatic and recurrent disease in pediatric OS, while the amplification of MDM2 has been
have historically remained essentially unchanged with a sur- associated with the development of metastases in OS [6, 22].
vival rate below 20%, highlighting the need for a better under- The amplification and/or increased expression of other cell
standing of the disease leading to the development of novel cycle genes, such as Cyclins D1 and E, have also been reported
therapies [4]. in OS, further highlighting an important role of defective cell
cycle regulation in OS development [17, 23].
2. Genomics of OS Several oncogenes like C-FOS, C-JUN, and C-MYC also
play a role in OS development. C-FOS, C-JUN N-terminal
OS is characterized by the presence of complex karyotypes kinase, and C-JUN were found elevated in OS and its expres-
indicative of severe chromosomal instability. This accumula- sion and activation were associated with the progression of
tion of barely recurrent genetic alterations hinders the identi- human OS [24–26]. Transgenic mice overexpressing C-FOS
fication of OS-driver genes. A powerful causal-effect relation developed OS, further suggesting a role in OS pathogenesis
between specific gene alterations and OS initiation came from [27]. C-MYC amplification was found in sporadic OS and OS
studies of human hereditary disorders characterized by a associated with Paget’s disease [28, 29] and, clinically, high
predisposition to the development of OS [5, 6]. The functional C-MYC expression correlates with worse outcome in OS
validation of these genomic alterations as driver events was patients [30].
confirmed in mouse models [5, 7]. The strongest genetic asso- A recent study using a Sleeping Beauty transposon-based
ciation for sporadic and hereditary OS is with the retinoblas- forward genetic screen in mice, with or without somatic
toma (RB) and the P53 tumor suppressor genes; meanwhile loss of P53 restricted to committed osteoblast progenitors,
other relevant alterations include mutations in other cell cycle identified 36 putative protooncogenes and 196 potential
regulators, oncogenes, and DNA helicases [5, 6]. tumor suppressor genes. Among these OS-driver candidates
Li-Fraumeni and hereditary retinoblastoma syndromes the protumorigenic role of PTEN and the axon guidance
are caused by heterozygous germ-line mutations of P53 and genes SEMA4D and SEMA6D were functionally validated.
RB, respectively, and patients presenting with these disorders Moreover, this study highlighted an enrichment of genes
have a higher predisposition to a range of cancers, including involved in PI3K-AKT-mTOR, MAPK, and ERBB signaling
OS [8, 9]. Importantly, mutations in P53 and/or RB genes cascades [51]. Confirming the heterogeneity of OS, an exome
and other components of their pathways are also common in sequencing-based study showed that identified candidate OS-
sporadic OS, suggesting a relevant role for alterations in these driver genes were associated with the development of a small
tumor suppression genes or their related signaling pathways set of tumors, suggesting that multiple oncogenic pathways
in OS development [5, 6, 10, 11]. On this basis, several P53 drive the characteristic chromosomal instability during OS
and/or RB-deficient mouse models have been developed development. However, the overall mutation signatures in
to model sarcomagenesis [5, 12]. The most productive OS these tumors were reminiscent of BRCA1/2 deficient tumors,
models have been developed using conditional mesenchy- a finding with possible therapeutic implications [52]. Com-
mal/osteogenic lineage-restricted mutation of P53 and RB parative genomic hybridization analysis combined with gene
(see below). These models indicate that P53 inactivation is expression data also resulted in the identification of genomic
an initiating event in OS [13–15]. On the other hand, the alterations associated with a small proportion of OS, which
depletion of RB alone was not sufficient to induce sarcoma may play a role in the OS pathogenesis. For instance, the cell
formation in mice. Notably, RB mutation strongly reduced division-related genes MCM4 and LATS2, the antiapoptotic
the latency required for sarcoma formation in P53-deficient genes BIRC2 and BIRC3, and other genes including CCT3,
mice, although it decreased the proportion of OS formed COPS3, and WWP1 were reported to be found as potential
Stem Cells International 3

to generate OS development in vivo. Thus, the inactivation of

P53 and/or RB in early mesenchymal progenitors of embry-
onic limb buds through PRX1-driven CRE expression (PRX1-
CRE) resulted in sarcoma development, presenting an OS
incidence of 60% in P53−/− mice and 20–30% in P53−/− RB−/−
mice, where most of the alternate tumors formed poorly
differentiated soft tissue sarcomas [14, 16]. Meanwhile,
the inactivation of these cell cycle regulators in osteoblast pre-
Bone
marrow cursors [OSX1 (Osterix)-CRE] resulted in a higher incidence
Bone
of OS formation in both P53−/− (100%) and P53−/− RB−/−
mice (between 53 and 100%) [13, 15, 16]. Similarly, ShRNA-
MSC as cell-of-origin Osteoblast mediated depletion of P53, together with CRE-mediated
inactivation of RB in osteoblast precursors (OSX1 restricted),
OSB as cell-of-origin Osteoclast
resulted in a delayed and consistent formation of OS, present-
Tumor cells Haematopoietic cells ing a higher degree of osteoblastic differentiation than other
CRE-based models [67]. Within the osteogenic differentia-
Osteocyte
MSC and pericytes tion lineage, targeting of P53 in mature osteoblasts, using
COL1A1- (collagen-1-alpha-1-) driven CRE expression to tar-
Endothelial cells
get P53 or OCN- (Osteocalcin-) driven expression of SV40-
Figure 1: Cell-of-origin for OS. The figure shows the most relevant T antigen to inactivate P53 and RB, also resulted in high OS
cell types present in the bone microenvironment. MSCs, repre- formation incidence (85–100%) [14, 68]. Moreover, another
sented in a perivascular niche, and their derived cell types along study using a SV40 immortalized murine osteocyte cell line
the osteogenic lineage, such as the osteoblasts (OSB), are strong suggests that fully differentiated osteocytes may also serve
candidates to acquire the first cancer-promoting mutations and as an OS-initiating cell [69]. Besides P53 deficiency-based
initiate OS formation. OS models, it has been proven that the expression of the
intracellular domain of NOTCH1 (NICD), leading to con-
stitutive NOTCH activation, in osteoblasts (COL1A1-driven
expression) was sufficient to induce the formation of bone
OS drivers [53–55]. Likewise, genomic analysis indicated that tumors, including OS [70]. Moreover, NOTCH activation
ossification factor genes such as MET, TWIST, and APC are combined with loss of P53 synergistically accelerates OS for-
frequently mutated in pediatric high-grade OS and these mation. Notably, the activation of NOTCH in mesenchymal
alterations correlated to a worse outcome, thus suggesting a progenitors or in osteoblast precursors produces embryonic
role in OS development [56]. lethality [70]. Similarly, mice with upregulated Hedgehog
Other genetic and epigenetic alterations likely involved in (HH) signaling in mature osteoblasts with a P53 heterozygous
OS pathogenesis include mutations in RECQL4 DNA helicase background developed OS with high penetrance [71].
associated with the OS-predisposing Rothmund Thomson These results support the concept that OS originates in
syndrome, amplification/mutation in the osteogenic fac- the population of cells that undergoes osteoblast commit-
tor RUNX2, loss of heterozygosity of FGFR2 and BUB3, ment rather than in immature MSC. Nevertheless, these
enhanced telomerase activity, deletion of PRKAR1A, and experiments also show that, although presenting at a lower
reduced expression of WWOX or hypermethylation of HIC1 incidence, early mesenchymal progenitors targeted with
in P53 mutated tumors among others [5, 7, 57–61]. relevant mutations could also initiate OS formation, most
likely influenced by certain microenvironment signals. In this
3. Cell-of-Origin for OS regard, the comparison in a single study of the OS formation
ability of P53/RB-disrupted immature MSC (PRX1-CRE) and
The cell-of-origin concept refers to the normal cell type that osteoblast committed cells (COL1A1-CRE and OCN-CRE)
acquires the first cancer-promoting mutations and initiates confirmed that all types of cells were able to initiate OS forma-
tumor formation [62]. During the last 10 years mounting tion and showed that the level of osteoblastic differentiation
evidence has placed MSCs and/or their immediate lineage of tumors did not correlate with the degree of differentiation
progenitors as the most likely cell-of-origin for many types of the cell-of-origin, suggesting that epigenetic dedifferentia-
of sarcomas including OS [12, 63, 64] (Figure 1). Both tion mechanisms could be active in mature osteoblasts during
translocation- and non-translocation-associated sarcomas osteosarcomagenesis [72]. The fact that early progenitors
have been modeled by introducing relevant mutations into might represent a cell-of-origin for OS is also strengthened
MSC [12, 64–66]. In the case of OS, most of the cell-of-origin by the observation of different histological subtypes, which
models are based on mutated P53, alone or in combination may reflect the ability of these progenitors to undergo other
with RB inactivation, in the mesenchymal/osteogenic lineage differentiation pathways besides osteogenesis.
of mouse models or in murine MSC [63]. By crossing mice Studies using murine MSC containing CRE-inactivated
with conditional (floxed) alleles of P53 and/or RB with mice P53 and/or RB alleles also reveal relevant clues about the
that have the CRE recombinase gene under the control of nature of the OS-initiating cell and the factors condition-
different tissue-restricted promoters, several groups were able ing their sarcomagenic potential. P53−/− and P53−/− RB−/−
4 Stem Cells International

adipose-derived-MSC (ASC) or BM-derived-MSC (BM- of self-renewing cells that can generate the full repertoire of
MSC) give rise to leiomyosarcoma-like tumors when injected tumor cells and display tumor reinitiating properties [12, 81–
subcutaneously into immunodeficient mice [72–74]. Oth- 83]. In the most likely scenario, these CSC subpopulations
erwise, when BM-MSCs were differentiated along the emerge after the accumulation of further epigenetic and/or
osteoblastic lineage before CRE-mediated deletion of P53 genetic alterations in a cell within the aberrant population,
and RB, they generated OS-like tumors upon subcutaneous initially generated by the cell-of-origin [62], that is, MSC-
injection into immunodeficient mice, whereas P53−/− RB−/− derived cell types.
ASC-derived osteogenic progenitors did not display tumori- Several methods have been developed to isolate and/or
genic potential [74]. These data highlight the differences in enrich subpopulations with stem cell properties within
the sarcomagenic potential of MSC from different tissues the tumors [82–85]. The isolation of OS-CSC was first
and indicate that a certain level of osteogenic differentiation achieved on the basis of their ability to form spherical
of BM-MSC is needed for the development of the OS phe- and clonal expanding colonies (sarcospheres) in anchorage-
notype. Nevertheless, orthotopic (intrabone or periosteal) independent and serum-starved conditions [86–88]. This
inoculation of P53−/− and P53−/− RB−/− BM-MSC and ASC sarcosphere formation may be further improved by repro-
undifferentiated MSC consistently generated osteoblastic OS ducing the hypoxic conditions of tumor microenvironment
displaying human OS radiographic and histological features [89]. In addition, OS-CSCs are commonly isolated by sorting
alongside metastatic potential. Importantly, all the histologi- cells according to the expression of specific surface markers
cal and radiological OS-related features were less evident or
associated with stemness and/or tumorigenesis. For example,
completely lost in the areas of the tumor distant from the
CD133+ [90–92], STRO1+ CD117+ [93], and CD271+ popula-
recipient bone, thus demonstrating that bone microenviron-
tions [94] sorted from OS cell lines demonstrated CSC-like
mental signals play a role in osteogenic differentiation and
sarcomagenesis by defining the sarcoma phenotype [75]. In features. Other methods used to isolate OS-CSCs include the
addition, an ectopic model to assay osteosarcomagenesis that identification of a “side population” of cells able to exclude
fluorescent dyes, alone or in combination with surface mark-
relies on the use of P53−/− RB−/− MSC embedded in hydroxya-
ers like CD248 [95, 96]; the sorting of cells with aldehyde
patite/tricalcium phosphate ceramics also demonstrates a rel-
evant contribution of bone microenvironmental factors, like dehydrogenase 1 (ALDH1) activity [97, 98]; the tracking of
bone morphogenic protein 2 (BMP2) and calcified substrates, subpopulations that express pluripotency-associated genes,
in the acquisition of the OS phenotype [75]. such as OCT-4 [99]; the enrichment of subpopulations
Furthermore, evidence of undifferentiated MSC as cell- with high telomerase activity [100, 101]; or the long-term
of-origin for OS comes from the introduction of other treatment with chemotherapy [102, 103]. Reinforcing their
oncogenic events into undifferentiated BM-MSC, like the expected mesenchymal progenitor origin, many of these
sarcoma-initiating cells express MSC markers [86, 93, 99, 104]
expression of C-MYC in a P16INK4A−/− P19ARF −/− genetic and retain in vitro differentiation properties, giving rise to
background or the aneuploidization accompanied by the loss adipogenic, chondrogenic, and osteogenic lineages [86, 104].
of the INKAA locus, resulting in OS development [76, 77]. These CSC commonly show increased expression of the
Additionally, similar gene expression signatures were found pluripotent stem cell markers OCT3/4, NANOG, and SOX2
between human OS samples and undifferentiated MSC or [87, 89, 105, 106]. Remarkably, SOX2 has been reported to
osteogenic-committed MSC [78], suggesting that OS may identify a population of CSC in OS required for self-renewal
develop from both osteogenic progenitors and undifferenti- and tumorigenesis [107]. Importantly, CSCs isolated from OS
ated MSC. are able to self-renew and sustain tumor generation in serial
Finally, extraskeletal OS is a very rare type of soft tissue transplantation experiments and are associated with metasta-
mesenchymal neoplasm that produces osteoid. It could be sis and drug resistance [89, 93, 96, 98, 105, 107]. This increased
speculated that, rather than BM-MSC, extraskeletal OS could chemoresistance of CSC subpopulations has been associated
be initiated by MSCs from soft tissues (muscle-derived MSC,
with an increase in the DNA repair ability, with an inhibition
ASC, etc.) presenting mutations that favor osteogenic differ-
of the apoptotic signaling, with increased levels of lysosomal
entiation and/or influenced by pathologically osteogenic sig-
nals from the microenvironment [79]. In this regard this type activity due to the overexpression of vacuolar ATPse [108],
of tumors could be related to fibrodysplasia ossificans pro- and, specially, with a gain in the drug efflux capacity due to
gressiva, a rare genetic disorder of connective tissue charac- the overexpression of the ABC transporters [96, 102, 105, 109–
terized by the presence of activating mutations in the ACVR1 111]. In this line, the inhibition of the ABC transporters is
gene, which encode a BMP type I receptor [80]. able to sensitize OS-derived sarcosphere to doxorubicin [112].
Overall, the most likely situation is that either MSC- Therefore, it is clear that OS-CSCs possess specific properties,
derived osteogenic progenitors or undifferentiated MSC may which make them more resistant to therapies.
represent the cell-of-origin for OS under the influence of Similar to normal stem cells, microenvironmental niches
proper microenvironmental or epigenetic signals. may play a role in OS-CSC regulation [113]. In this regard,
many bone microenvironment signals, including those medi-
4. Osteosarcoma Cancer Stem Cell ated by fibroblastic growth factor (FGF), transforming
growth factor 𝛽 (TGF-𝛽), insulin-like growth factor 1 (IGF1),
Experimental evidence supports the notion that sarcomas are BMP, vascular endothelial growth factors (VEGF), hypoxia
hierarchically organized and sustained by a subpopulation inducible factors (HIF1), wingless-type MMTV integration
Stem Cells International 5

strategies included the targeting of the signaling mediated by

receptor tyrosine kinases (EGFR, VEGFR, IGF1R, HER2, or
Perivascular PDGFR), mTOR, or WNT/𝛽-catenin. In addition, since OS-
niche
CSCs reside within the bone microenvironment and this a
key factor in the regulation of tumor homeostasis, therapies

e l
ch ea
directed against microenvironmental niche factors could

ni dost
Hypoxic

En
niche
contribute to the improvement of clinical response [2]. There-
fore, several therapeutic strategies have been developed to
target the role of the tumor-promoting osteoclast activity [31–
33, 121], to reduce the vascularization of tumors [34, 122] and
to enhance the immune response against tumors [123–126]
(Table 1).
As seen before, OS-CSCs are resistant to most con-
ventional treatments like radiation and chemotherapy and
Vas
Vascular structure are, therefore, responsible for tumor relapses and metastasis.
CSC
Hence, in addition to the proposed therapies directed against
Ost
O
Osteoblast
Tumor cells
specific signaling and/or tumor niches, there is a need for
Osteoclast
O developing and testing therapies able to target CSC subpopu-
lations in OS. Below, we reviewed current work reporting spe-
Osteocyte
cific antitumoral activity on OS-CSC subpopulations or CSC-
related features (Table 2).
Figure 2: OS-CSC niches. The figure represents possible niches for Broad genomic, metabolomic, and proteomic analyses
CSCs in OS. Suggested locations for CSCs are the perivascular niche, have been useful to better characterize OS-CSC and therefore
the endosteal niche, and areas of poor vascularization (hypoxic define potential OS-CSC-specific therapeutic targets with
niche). molecular rationale [58, 127, 128]. Among the reported altered
signaling pathways with therapeutic implications, nuclear
factor 𝜅B (NF-𝜅B) is activated in radioresistant subpopula-
tions of OS cell lines, and these subpopulations could be
site family (WNT), HH, or NOTCH among others, are dereg- sensitized to radiation by parthenolide, an inactivator of
ulated in OS and seem to be involved in the regulation of their NF-𝜅B [129]. Another NF-𝜅B inhibitor, BRM270, specifically
self-renewal, differentiation, growth, drug resistance, and/or targeted a multidrug resistant OS stem-like cell population
metastatic potential of OS-CSC subpopulations (reviewed in by increasing their apoptosis rate and thereby reducing
[2]). Three types of niches within the bone microenvironment tumorigenic potential [130]. Phosphatidylinositol-3-kinase
where these signaling pathways are particularly active could (PI3K) is also an interesting therapeutic target due to its high
be inferred to be the location OS-CSCs: the perivascular mutation frequency and its role in regulation of proliferation,
niche, the hypoxic niche, and the endosteal niche (Figure 2). cell cycling, survival, and apoptosis. BYL719, a specific PI3K𝛼
OS is a highly vascularized tumor and as OS-CSCs seem to inhibitor, induces cell cycle arrest and inhibition of cell
arise from MSC it is likely that OS-CSCs may be located in the migration in OS cells, and, therefore, has been postulated to
same perivascular niche already well-described for normal be useful for multidrug therapeutic approaches [35]. More-
MSCs. Besides providing stemness signals, this perivascular over, another PI3K inhibitor, LY294002, induces cell cycle
location would favor migration and metastasis of CSC [84, arrest and apoptosis in OS-CSC [131].
114]. On the other hand, the bone is a hypoxic environment Developmental signaling pathways like WNT and
and hypoxia-induced signaling is a key environmental factor NOTCH, which are highly involved in the regulation of
involved in stemness maintenance, OS progression, and drug stemness and differentiation, have also been reported to
resistance and therefore could constitute a suitable niche play a role in OS development [2, 37, 70]. In OS cell lines,
for OS-CSCs [2, 115]. Finally, the endosteal niche is a rich the inactivation of NOTCH and WNT pathways resulted in
environment where tumor cells interfere with the bone sensitization to chemotherapeutic drugs [36]. In addition,
remodeling process, establishing a “vicious cycle” that favors aberrant active WNT/𝛽-catenin signaling has been described
osteoclast-mediated osteolysis and the subsequent release of in the OS-CSC population and has been associated with
calcium and growth factors (FGF, TGF-𝛽, IGF1, BMP, etc.), SOX2 overexpression and tumorigenicity [38, 132]. On the
which support stem and tumorigenic properties [2, 116, 117]. other hand, the WNT-antagonist Dickkopf proteins 1 (DKK1)
has been proposed to enhance protumorigenic properties in
5. Cancer Stem Cell Targeted Therapies in OS OS, in part, through the upregulation of the stress response
enzyme and CSC marker ALDH1A1 [133]. In this case, the
Studies concerning the molecular biology of cancer are now inhibition of the canonical WNT pathway by DKK1 leads
promoting the identification of new potential therapeutic to the activation of noncanonical JUN-mediated WNT
targets with molecular rationale able to target OS. As a result, pathways, which mediate the induced tumorigenic effects.
therapies targeting altered signaling are being thoroughly Likewise, NOTCH signaling has been associated with ALDH
tested in several clinical trials [58, 118–120] (Table 1). These activity and increased metastatic potential in OS cells [39].
6 Stem Cells International

Table 1: Selected clinical trials targeting altered signaling and tumor environment in OS∗ .

Target Drug Type of drug Clinical trial reference number (NCT number)
Cell membrane receptors
ERBB2 Trastuzumab Monoclonal antibody NCT00023998
Cixutumumab Monoclonal antibody NCT01016015/NCT00831844/NCT01614795/NCT00720174
IGF1R
RG1507 Monoclonal antibody NCT00642941
EGFR ZD1839 Inhibitor NCT00132158
Erlotinib Inhibitor NCT00077454
PDGFR
Imatinib Inhibitor NCT00031915/NCT00030667
NCT01804374/NCT00889057/NCT00880542/NCT00330421/
PRGFR/VEGFR Sorafenib Inhibitor
NCT01518413
NCT01759303/NCT02357810/NCT01130623/NCT02180867/
Pazopanib Inhibitor
NCT01532687/NCT01956669
VEGFR Bevacizumab Monoclonal antibody NCT00667342
Endostar
Inhibitor NCT01002092
(rh-endostatin)
Intracellular signaling
Everolimus Inhibitor NCT01216826
mTOR
Ridaforolimus Inhibitor NCT00093080/NCT00538239
WNT/𝛽-catenin Curcumin Inhibitor NCT00689195
Niche cells and their signaling
Zoledronic acid Bisphosphonate NCT00691236
Osteoclasts
Pamidronate Bisphosphonate NCT00586846
RANKL Denosumab Monoclonal antibody NCT02470091
T cells expressing
Cells NCT02107963
GD2
GD2Bi-armed T
Cells NCT02173093
cells
Immune system Anti-GD2 Monoclonal antibody NCT00743496/NCT02502786
Stem and natural
Cells NCT02409576/NCT01807468
killer cells
Monocyte/macrophage
Mifamurtide NCT02441309/NCT00631631
activator glycopeptide
∗
Source: https://clinicaltrials.gov/. Osteosarcoma clinical trials: total: 363, open: 122.
ERBB2: Erb-B2 receptor tyrosine kinase 2; IGF1R: insulin-like growth factor 1 receptor; EGFR: epidermal growth factor receptor; PDGFR: platelet-derived
growth factor receptor; VEGFR: vascular endothelial growth factor receptor; mTOR: mechanistic target of rapamycin; WNT: wingless-type MMTV integration
site family; RANKL: receptor activator of nuclear factor kappa-B ligand.

Therefore, a number of different therapies have been assayed [43, 44]. Moreover, TGF-𝛽 signaling activation has been
to target WNT or NOTCH pathways via downregulation, involved in the induction of stemness, tumorigenicity,
inactivation, or silencing techniques [2, 37, 40] (Table 1). metastatic potential, and chemoresistance in nonstem OS
Interestingly, curcumin, a natural product that shows high cells, and, conversely, the blocking of this signaling resulted
antitumoral activity against OS cells, is a WNT/𝛽-catenin in the inhibition of this dedifferentiation process of nonstem
antagonist whose antitumor activity seems to be mediated cell populations, thereby highlighting TGF-𝛽 as a potential
through the inactivation of NOTCH1 signaling, thereby therapeutic target [45].
linking both signaling pathways [41]. In addition, TGF-𝛽 Not surprisingly, microRNAs (miRNAs) are extensively
is also a well-known regulator of bone biology that plays related to OS development [46]. In a CSC context, a list
a relevant role in OS development [42]. The blocking of of 189 miRNAs that are differentially expressed in OS-CSC
TGF-𝛽 signaling using the natural TGF-𝛽/SMAD signaling has been reported [127]. Some of these miRNAs, such as
inhibitor SMAD7, the inhibitor of TGF-𝛽 receptor complexes miR-382 and miR-29b-1, were significantly decreased in
SD-208, or the natural alkaloid halofuginone hindered OS human OS and their overexpression resulted in a decrease
progression. These treatments reduced in vivo tumorigenic in CSC properties, metastatic potential, or chemoresistance,
potential of OS cell lines, repressed tumor-associated bone thus suggesting that these miRNAs could constitute novel
remodeling, and inhibited the development of metastasis therapeutic strategies to target OS-CSC [47, 48]. On the
Stem Cells International 7

Table 2: Therapeutic agents with reported activity on OS-CSCs subpopulations or related properties.

Therapeutic agent Proposed mechanisms of action Effect on CSC/CSC properties Reference

NF-𝜅B inhibition/oxidative stress Sensitizes to ionizing radiation reducing the viability of [31]
Parthenolide
induction CD133+ CSCs
NF-𝜅B/CDK6/IL6 [32]
BRM270 Induces programmed cell death
downregulation
BYL719 PI3K inhibition Induces cell cycle arrest and inhibits migration [33]
Induces cell cycle arrest and apoptosis in [34]
LY294002 PI3K inhibition
OS-sarcospheres
Reduces self-renewal and differentiation and increases [35]
SB431542 TGF-𝛽 inhibition
chemosensitivity of OS-sarcospheres
Decreases OS-CSCs, reduces metastatic potential, and [36]
miR-382 expression YB-1 inhibition
inhibits tumor formation from CD133+ OS cells
Reduces sarcosphere formation and induces [37]
miR-29b-1 expression —
chemosensitization of OS cells
Reduces cell invasion of CD133+ OS cells and suppresses [38]
miR-133a inhibition —
metastasis in combination with chemotherapy
Reduces CD133+ cells and impairs sphere-forming in [39]
lncRNA HIF2PUT HIF-2𝛼
OS cells
AMPK/mTOR signaling Reduces sphere-forming ability and sensitizes OS cells [40, 41]
Metformin
alteration to chemotherapeutic agents
Inhibits differentiation and proliferation of [42, 43]
Bufalin miR-148a/DNMT1/CDKN1B
OS-sarcospheres
Reduces sphere-formation and tumor-initiation ability
Salinomycin WNT signaling downregulation of OS cells and sensitizes them to chemotherapeutic [44]
drugs
Salinomycin-loaded When combined with CD133 aptamers selectively [45]
—
nanoparticles targets OS-CD133+ cells
Upregulation of
tumor-suppressive Prevents invasion, angiogenesis, and drug resistance in
Diallyl trisulfide miRNAs/inhibition of NOTCH1 OS cells and in combination with methotrexate reduces [46, 47]
signaling/downregulation of OS-CD133+ cells
ABCB1
Induces apoptosis and promotes differentiation of [48]
MC1742/MC2625 Histone deacetylase inhibition
sarcoma CSCs
Vorinostat Histone deacetylase inhibition Reduces metastatic potential of OS cells [49]
Increased macrophage [50]
Anti-CD47 antibody Inhibits invasion and metastasis of OS cells
phagocytosis
CDK6: cyclin-dependent kinase 6; IL6: interleukin 6; TGF-𝛽: transforming growth factor 𝛽; YB-1: Y box-binding protein 1; HIF-2𝛼: hypoxia inducible factors
2𝛼; AMPK: AMP-activated protein kinase; mTOR: mechanistic target of rapamycin; DNMT1: DNA (cytosine-5-)-methyltransferase 1; CDKN1B: cyclin-
dependent kinase inhibitor 1B; WNT: wingless-type MMTV integration site family; ABCB1: ATP-binding cassette subfamily B member 1.

other hand, high levels of miR-133a and CD133 correlated inhibiting proliferation, invasion, and metastatic potential in
with poor prognosis in OS and the inhibition of miR-133a OS cells, the hypoglycemic agent metformin also induces
associated with chemotherapy suppressed lung metastasis a marked reduction of the self-renewal and differentiation
and prolonged survival in preclinical models of OS [49]. potential of CSC subpopulations and sensitizes OS cell to
Moreover, other miRNAs like miR-215 and miR-140 have also cisplatin [136, 137]. In a similar way, bufalin inhibits the
been related to OS chemoresistance [50, 134]. In addition, a differentiation and proliferation of OS-CSC through a mech-
recent report shows that the overexpression of the novel long anism regulated by miR-148a [138, 139]. Also, the polyether
noncoding RNA HIF2PUT, involved in the regulation of HIF- ionophore antibiotic salinomycin has demonstrated spe-
2𝛼 expression, markedly inhibited proliferation, migration, cific antitumoral activity against OS-CSC [140]. Moreover,
and stem cell features in OS cells, thus providing a proof of salinomycin-loaded nanoparticles conjugated with CD133
principle for testing HIF2PUT in future therapeutic strategies aptamers highly increase the therapeutic effect of the drug
[135]. on CD133+ OS-CSC [141]. Another natural derivative with
Several natural compounds with reported antitumoral reported antitumoral activity in OS is diallyl trisulfide, which
activity in OS have recently been shown to demonstrate can reverse drug resistance through the downregulation of
specific inhibitory effects in OS-CSC (Table 2). Thus, besides ABCB1, and, in combination with methotrexate, is able to
8 Stem Cells International

reduce the CD133+ subpopulation of drug resistant human should be proposed. These strategies may include com-
OS cells [142]. Antitumoral effects of diallyl trisulfide seem bined targeted therapies, immune-based treatments, and/or
to be mediated by the upregulation of tumor-suppressive therapy targeting tumor microenvironment. Recent studies
miRNAs associated with an inhibition of NOTCH1 signaling have highlighted the importance of OS-CSCs, which have
[143]. Additionally, several histone deacetylase inhibitors been associated with chemoresistance, relapse, and metastasis
have demonstrated antitumoral activity in OS, including the events. More research aimed towards the characterization
induction of differentiation in OS-CSC and the reduction of of CSC biology and evolution during tumor progression is
the metastatic potential [144, 145]. needed to develop powerful methods of detection and effi-
Finally, immunotherapy is an attractive option to target cient therapies. Targeting the tumor OS-CSCs or disrupting
CSC subpopulations. Thus, the treatment of human OS cell the interaction between OS-CSCs and their bone niche also
lines with T cells expressing a specific chimeric antigen to constitutes a valuable approach, with promising clinical trials
target the human epidermal growth factor receptor ERBB2 ongoing that could yield exciting new therapies for the future.
was able to efficiently decrease the sarcosphere formation
capacity and the ability to generate OS in vivo, suggesting
that this immune-based strategy is able to target CSC sub-
Competing Interests
populations [125]. In addition, the membrane receptor CD47, The authors declare they have no competing interests.
which plays an important role in the mechanisms of tumor
immune escape, has been found overexpressed in OS samples
and highly expressed in cell subpopulations expressing the Authors’ Contributions
CSC marker CD44. Notably, the blockade of CD47 by specific
antibodies suppressed the invasive ability and the metastatic Ander Abarrategi, Juan Tornin, Lucia Martinez-Cruzado,
potential of OS cells, suggesting a potential use for these anti- Ashley Hamilton, and Enrique Martinez-Campos con-
CD47 antibodies in the treatment of OS [146]. tributed equally to this work.
It is important to mention that CSC subpopulations are
heterogeneous and different subpopulations may exist within Acknowledgments
a tumor with different genetic alterations. Moreover, these
subpopulations are highly dynamic and there are processes of This work was supported by the Plan Nacional 2008–2011
dedifferentiation and phenotype switching which may render (ISC III/FEDER (Miguel Servet Program CP11/00024) and
CSC resistant to a specific CSC therapy [147]. In this regard, RTICC (RD12/0036/0015)), the Plan Nacional 2013–2016
future therapies should combine different treatments to target (MINECO/FEDER (SAF-2013-42946-R)), and the Plan de
both non-CSCs and CSCs, and those CSC-specific treatments Ciencia Tecnologı́a e Innovación del Principado de Asturias
should target multiple pathways altered in different subsets (GRUPIN14-003), to Rene Rodriguez, and the Plan Nacional
of CSCs within the tumor. These broader spectrum thera- 2008–2011 (ISC III/FEDER (PI11/00377) and RTICC (RD12/
peutic approaches include immune-based treatments and/or 0036/0027)), to Javier Garcia-Castro.
therapy targeting tumor microenvironment. In addition,
inhibition of transcription factors presenting altered activity
offers a promising choice since they are pivotal points in
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