cancers

Review
Origin and Therapies of Osteosarcoma
Brice Moukengue 1 , Morgane Lallier 1 , Louise Marchandet 1 , Marc Baud’huin 1,2 , Franck Verrecchia 1 ,
Benjamin Ory 1 and Francois Lamoureux 1, *

1 Nantes Université, INSERM UMR1307, CNRS UMR6075, Université d’Angers, CRCI2NA, Team 9, CHILD,
F-44000 Nantes, France; brice.moukengue@etu.univ-nantes.fr (B.M.); morgane.lallier@univ-nantes.fr (M.L.);
louise.marchandet@univ-nantes.fr (L.M.); marc.baudhuin@univ-nantes.fr (M.B.);
franck.verrecchia@univ-nantes.fr (F.V.); benjamin.ory@univ-nantes.fr (B.O.)
2 CHU de Nantes, F-44300 Nantes, France
* Correspondence: francois.lamoureux@univ-nantes.fr

Simple Summary: Osteosarcoma is the most common malignant bone tumor in children, with a
5-year survival rate ranging from 70% to 20% depending on the aggressiveness of the disease. The
current treatments have not evolved over the past four decades due in part to the genetic complexity
of the disease and its heterogeneity. This review will summarize the current knowledge of OS origin,
diagnosis and therapies.

Abstract: Osteosarcoma (OS) is the most frequent primary bone tumor, mainly affecting children and
young adults. Despite therapeutic advances, the 5-year survival rate is 70% but drastically decreases
to 20–30% for poor responders to therapies or for patients with metastasis. No real evolution of
the survival rates has been observed for four decades, explained by poor knowledge of the origin,
difficulties related to diagnosis and the lack of targeted therapies for this pediatric tumor. This review
will describe a non-exhaustive overview of osteosarcoma disease from a clinical and biological point
of view, describing the origin, diagnosis and therapies.
Citation: Moukengue, B.; Lallier, M.;
Keywords: osteosarcoma; origin; therapy
Marchandet, L.; Baud’huin, M.;
Verrecchia, F.; Ory, B.; Lamoureux, F.
Origin and Therapies of Osteosarcoma.
Cancers 2022, 14, 3503. https://
doi.org/10.3390/cancers14143503 1. Introduction

Academic Editors: Guido Alberto

Osteosarcoma (OS) is the most common primary malignant bone tumor. It is also the
Massimo Tiberio and Jean Jiang
third most common type of cancer affecting children and adolescents after lymphomas and
brain tumors [1]. OS cells derive from the mesenchymal lineage and are able to produce
Received: 1 June 2022 osteoid substances and/or immature bone [2,3].
Accepted: 15 July 2022 OS mainly affects children or young adults. The incidence of OS increases from two to
Published: 19 July 2022 three million per year in the general population to eight to eleven million per year when
Publisher’s Note: MDPI stays neutral only the 15–19-year-old age group is considered. OS represents 3–6% of childhood cancers
with regard to jurisdictional claims in (15% of extra-cranial solid cancers) and 1% of adult cancers. After the peak of incidence at
published maps and institutional affil- 15–19 years of age, another peak of incidence occurs in elderly populations (over 65 years
iations. of age) and occurs as a result of pre-existing bone pathology or fractures. Young males are
1.4 times more likely to develop the disease than young females. However, the risks are
equivalent between the sexes in people older than 65 years [4,5].
The causes of OS are still poorly understood. Its appearance in young populations
Copyright: © 2022 by the authors. during growth and its location at the ends of the long bones suggest the involvement of
Licensee MDPI, Basel, Switzerland.
rapid bone production [6]. This hypothesis is supported by a higher incidence of OS in
This article is an open access article
large dogs compared to small dogs [7]. In older patients, risk factors include radiation and
distributed under the terms and
chemotherapy for treatment of pre-existing malignancies. A history of Paget’s disease is
conditions of the Creative Commons
also present in approximately one third of adult OS cases. OS can also arise from hereditary
Attribution (CC BY) license (https://
genetic disorders, such as Li–Fraumeni syndrome, Rothmund–Thomson syndrome, or
creativecommons.org/licenses/by/
4.0/).
Werner syndrome [8,9].

Cancers 2022, 14, 3503. https://doi.org/10.3390/cancers14143503 https://www.mdpi.com/journal/cancers

Cancers 2022, 14, 3503 2 of 28

2. Clinical Features
OS is often diagnosed after persistent local pains, often mistakenly attributed to growth
in young people, or to physical activity. Patients may also exhibit a palpable mass and
reduced joint range of motion. Systemic symptoms (weight loss, fatigue, fever...) may also
be present. The tumor will most frequently be located in the metaphysis of long bones
(femur, tibia, humerus), with 50% of cases located near the knee [10–12]. The tumor can also
be localized to the axial skeleton, most commonly the pelvis, in adults [12]. Approximately
15–20% of patients develop metastases at the time of diagnosis, particularly in the lungs
(85%) or bone (8–10%) [13]. In rare cases, adenopathy may be indicative of metastasis to
lymph nodes [11,13]. Presence of metastases is a clear sign of poor prognosis and drastically
decreases the survival rate by 70% to 20% at 5 years [1].

3. Diagnosis
Diagnosis is made after palpation to determine the presence of a soft mass near the
bone. Radiographically, calcifications resulting from ectopic bone formation may be seen
within the soft mass, as well as a “sunburst” appearance. Ectopic bone formation is often
associated with osteolysis [14]. Increased lactate dehydrogenase or alkaline phosphatase
in the blood is associated with a poor prognosis [2,15]. Finally, a PET (positron emission
tomography) scan can detect any potential metastases.
Because biopsy can identify OS with an accuracy level of 90%, histological analysis is
often performed to confirm the diagnosis. It allows to determine the level of proliferation
of the tumor cells, to classify the tumor according to the stage of severity.

4. Classification of OS
OS is classified into secondary or primary OS. Secondary OS occurs after pre-existing
events, such as diseases (Paget’s disease, for example) or irradiation [16,17]. Conversely,
primary OS is primitive OS and is subdivided into different categories based on histological
appearance. The conventional intramedullary/central high-grade type is the most common.
It is subdivided into osteoblastic (50%), chondroblastic (25%) or fibroblastic (25%) types
differentiated by the secreted matrix. The osteoblastic type is characterized by the secretion
of a bone matrix, the chondroblastic type secretes a cartilage matrix, and the fibroblastic
type is characterized by collagen-secreting spindle cells. The other types of primary OS are
composed of telangiectatic OS that have blood-filled cysts and multinucleated giant cells.
Parosteal OS show cartilage production in about 50% of cases, but also bone production and
cells with fibroblastic morphology. Central low-grade OS present cells with fibroblast-like
morphology and are organized in bundles. Finally, periosteal OS show mature dense bone
but also atypical hyaline cartilage [17,18].
There are other subtypes of OS, including sclerosing osteoblastic, chondromyxoid
fibroma-like, chondroblastoma-like, clear cell, giant cell, epithelioid, osteoblastoma-like
or malignant fibrous histiocytoma-like OS. However, these types of OS are considered
subtypes of the conventional type due to their similarities and behaviors to the conventional
type [17].
The grade, used to characterize OS, takes into account the severity of the disease and
the presence of metastases. Grade of OS helps to adjust treatment, estimate patient progno-
sis, assess treatment outcomes and facilitate communication between professionals [19,20].
Several disease classification systems exist:
- The Musculoskeletal Tumor Society (MSTS) or Enneking System [19,21]:
Three main pieces of information are given by this system: grade, extension of tumor
and presence or absence of metastasis (Table 1).
Grade (G): indicates the potential of the tumor to grow and spread. It is based on
the histological appearance of the tumors. The G1 grade refers to low-grade tumors
(morphology similar to healthy cells). They are less likely to grow quickly and metastasize.
The G2 grade refers to high-grade tumors that are abnormal in morphology, rapidly
dividing and likely to metastasize.
Cancers 2022, 14, 3503 3 of 28

- Extent of the primary tumor (T): T1 refers to a tumor that is confined to the bone
(intra-compartmental), and T2 refers to a tumor that affects surrounding structures
(extra-compartmental).
- Metastasis (M): M1 indicates that a tumor has spread to nearby lymph nodes. M0
indicates no spread to lymph nodes.

Table 1. MSTS (Musculoskeletal Tumor Society) staging system for osteosarcoma. Each stage is charac-
terized by a grade (low grade = G1; high grade = G2), the tumor extension (T1 = intra-compartmental;
T2 = extra-compartmental) and presence of metastasis (M0 = no metastasis; M1 = presence of metastasis).

Stage Grade Tumor Metastasis

IA G1 T1 M0
IB G1 T2 M0
IIA G2 T1 M0
IIB G2 T2 M0
III G1 or G2 T1 or T2 M1

- The TNM (Size, Spread, Metastasis) System [19,22]:

The TNM classification system was created by the AJCC (American Joint Committee
on Cancer): the T parameter describes the size of the primary tumor as well as its presence
or absence in different regions of the bone; the N parameter describes the spread to nearby
lymph nodes; the M parameter indicates whether the cancer has metastasized to other
organs in the body (lung, breast, bone, etc.).
The AJCC classification contains 7 stages (IA, IB, IIA, IIB, III, IVA, IVB), which are
subdivided into about 30 stages, each of which accurately describes the tumor. A simplified
version of this classification is shown in Table 2.

Table 2. AJCC (American Joint Committee on Cancer) staging system for osteosarcoma. Each stage
is characterized by a grade (low, high), the primary tumor size and presence of regional lymph
nodes (N0 = no regional lymph nodes metastasis; N1 = regional lymph nodes metastasis) and distant
metastasis (M0 = no distant metastasis; M1a = lung metastasis; any M = lung and other distant sites).
Adapted from Ritter et Bielack [2].

Primary Tumor Lymph Nodes Distant

Stage Grade
Size Metastasis Metastasis
IA Low <8 cm N0 M0
IB Low >8 cm N0 M0
IIA High <8 cm N0 M0
IIB High >8 cm N0 M0
III Any grade Any size N0 M0
IVA Any grade Any size N0 M1a
IVB Any grade Any size N1 Any M

5. Current Therapies for OS

Patients with OS are treated with a multidisciplinary approach established by Rosen et al.
in the 1970s [23]. This protocol combines chemotherapy (neoadjuvant and adjuvant, for
a duration of 6 to 8 months) and surgery with limb preservation. This strategy results
in 5-year survival for approximately 70% of patients [10,24,25]. Radiotherapy is rarely
used for the treatment of OS because of the radioresistance of the tumor [26]. Its use is
reserved for specific situations, such as the impossibility of complete resection of a tumor
located in a high-risk region (head, spine, etc.), or the persistence of small tumor foci after
resection [26,27].
Cancers 2022, 14, 3503 4 of 28

Furthermore, radiotherapy could be used to delay tumor growth and to reduce symp-
toms, such as pain, in the case that surgery is not possible, and could lead to possible
side effects (skin reaction, nausea, diarrhea, slow bone growth in children, lung and
heart function . . . ). The used radioactive drugs are radium-233 or samarium153-EDTMP
(ethylenediaminetetramethylene phosphonic acid), which have high affinity for bone tissue
and, thus, selectively deliver radiation to osteoblastic lesions [28,29]. Samarium153-EDTMP
was approved by the FDA in 1998 for palliative treatment to control pain in patients with
bone metastases and then was used alone or as a combined agent in high-risk OS patients.
Indeed, a high dose of samarium153-EDTMP administered followed by hematopoietic stem
cell rescue was tolerable, with hematological toxicity, and controlled the pain [29–31].

5.1. Chemotherapy
Chemotherapy is used before surgery to reduce the size of the tumor and create the
best conditions for limb salvage surgery and after surgery to eliminate residual lesions and
metastases [32]. The duration of chemotherapy is usually between 6 and 12 months and
combines several agents with high efficacy, including doxorubicin (Adriamycin, ADM),
cisplatin (DDP), ifosfamide (IFO), etoposide (to a lesser extent) or high-dose methotrex-
ate (HDMTX), which are used in varying combinations [33–39]. Indeed, adjuvant MAP
(Methotrexate, Adriamycin and Cisplatine combination) chemotherapy is the most common
treatment for patients with resectable tumors, generally with two to six cycles of preoper-
ative chemotherapy, followed by additional cycles for postsurgical adjuvant chemother-
apy [40,41]. Preoperative chemotherapy is given 8–10 weeks prior to surgery [1,42]. Post-
operative chemotherapy is given up to 21 days after surgery for 12 to 29 weeks of treat-
ment [1,43]. Due to the adverse effects of multidrug therapy (cardiac, atrial dysfunction,
renal and liver toxicity) [44–46], follow-up examinations should be implemented during
and after therapy (echocardiography, audiogram, toxicity tests) [2]. Additional treatments
can limit the side effects of chemotherapy (e.g., antiemetics or opiates) [47].

5.2. Surgery
Surgery for OS aims for the complete removal of the tumor, with a focus on large
resection margins that include the tumor and the surrounding healthy tissue. However, the
local recurrence rate could be up to 25% if the tumor removal is not complete [48]. There is
no consensus on the definition and comparison of these resection margins between surgical
teams, making it difficult to standardize practices [49,50]. Nowadays, the effectiveness of
preoperative chemotherapy prevents amputation of affected limbs [51,52], which conserves
physical patient integrity and motricity [3]. Nevertheless, in some cases, amputation
should sometimes be considered as a superior option to limb salvage [53]. However,
resection of OS involving the axial skeleton remains very difficult, and MRI (magnetic
resonance imaging) of the skeleton can be used to plan surgery. If pulmonary metastases
are suspected, a thoracotomy can be performed to localize them. Moreover, tumor resection
can lead to soft tissue and bone defects that need to be reconstructed. Advances in bone
tissue engineering allow to reconstruct the bone defect with different methods, including
autologous or allogeneic bone transplantation, allograft prosthetic composite reconstruction
or endoprosthetic replacement with restoration of limb function [54].
After preoperative chemotherapy, patients are classified as good or poor responders
based on the number of viable cells remaining in the resection specimen [55]. The method
used is called the Huvos and Rosen score [56]. Good responders have more than 90% necro-
sis (stage III = 91–99% necrosis and grade IV = 100% necrosis). Poor responders have less
than 90% necrosis (grade II = less than 90% necrosis; grade I = less than 50% necrosis) [57,58].
The Huvos score is used to adapt postoperative chemotherapy [59].

6. Limitations of Current Treatments

Despite treatment, 30 to 40% of patients will have a recurrence within 2 to 3 years
after the end of treatment. At the time of recurrence, the metastases, mostly pulmonary
Cancers 2022, 14, 3503 5 of 28

(90%), must be surgically removed in order to increase patients’ survival. However, the
5-year survival after the second diagnosis is about 20%. The current treatments cause
severe side effects that diminish patients’ quality of life [13,41]. While surgery has been
improved and allows limb salvage in 80–95% of patients with limb functionality, the surgery
remains invasive and often necessitates reconstruction [60]. The surgical reconstruction
also presents some complications, such as infection, graft fracture, implant reject or local
recurrence, which could compromise the quality of life of the patients [60–62].
Despite the fact that conventional therapies have lowered the frequency of amputa-
tions, the most commonly used drugs date from the 1970s, and the survival rates have
remained relatively constant since then. This lack of progress might be attributed to the
disease’s rarity as well as its wide variability.

7. Genetic Disorders in OS
OS is a complex and heterogeneous tumor characterized by a high level of genomic in-
stability, aneuploidy and genomic rearrangements, with gains of portions of chromosomes
(1p, 1q, 6p, 8q and 17p) or losses of portions of chromosomes (3q, 6q, 9, 10, 13,17p and 18q)
in conventional disease, generally corresponding to regions where oncogenes and tumor
suppressor genes are located, respectively [63]. OS can also arise from inherited genetic
disorders, such as Li–Fraumeni syndrome (p53 mutation), or mutation of the gene encoding
Rb, but also Rothmund–Thomson (RECQL4 gene mutation), Bloom (BLM) and Werner
(WRN) syndromes [8,9]. However, other acquired genetic changes have been described in
pathology. Indeed, with the explosion of high-throughput sequencing methods in recent
years, many studies have attempted to identify “driver gene mutations”, i.e., mutations
that confer a proliferative advantage to cells [64–67]. They can either inhibit/inactivate
tumor suppressor genes or amplify/facilitate oncogene activity [68–71].
Tumor suppressor genes inhibit tumor growth, for example, by regulating the cell
cycle. Many genes belonging to this category are affected in OS. For example, the TP53 gene
is the most mutated gene in human tumors [72], encoding the p53 protein, a transcription
factor regulating cell cycle and apoptosis. In OS, p53 is inactivated by gene mutation
or chromosomal rearrangement [63,64,71,73]. Between 65% and 90% of OS cases have a
mutation in TP53 (point mutation, allelic loss or rearrangement) [9,74,75]. Another cell
cycle regulator Rb (Retinoblastoma protein) is frequently mutated in OS. Rb regulates the
transition from G1 to S phase of the cell cycle by sequestering E2F family transcription
factors. Its loss, therefore, results in the disappearance of this cell cycle checkpoint [70]. Loss-
of-function mutations in Rb occur in approximately 30% of OS [64,73,76–78]. However,
the evidence of the contribution of the Rb mutation in OS is not so clear since several
studies reported that germline Rb mutation did not cause OS development in mice [79–81].
Walkley et al. confirmed this result in an Osx-Cre Rbfl/fl mouse model, in which neither OS
development nor skeletal abnormalities were observed, concluding that Rb mutation is not
sufficient to induce OS development [82].
The functions of p53 and Rb can also be affected by the mutation of CDKN2A (cyclin-
dependent kinase Inhibitor 2A). This gene encodes two proteins, p14Arf and p16INK4,
which activate p53 and Rb, respectively. P14 prevents the degradation of p53 mediated
by the E3 ubiquitin ligase MDM2 (mouse double minute 2 homolog). P16 inhibits CDK4
(Cyclin-dependent kinase 4), a protein capable to inactivate Rb by phosphorylation. The
CDKN2A locus (9p21) is altered in 5–21% of OS [63,83–85]. Hypermethylation of p14ARF
and p16INK4 promoters have also been described in OS, resulting in decreased transcrip-
tion [86,87]. Hypermethylation of promoters of anti-tumor genes is a frequently cited
mechanism in OS. This is the case for the promoters of GADD45 (growth arrest and DNA
damage) or HIC1 (hypermethylated in cancer 1), both involved in the response to DNA
damage signals [88,89].
ATRX is another protein frequently mutated in OS (29% of tumors). It is part of a
multiprotein complex regulating chromatin remodeling and telomere maintenance [78].
ATRX is a known tumor suppressor gene, and its mutations lead to alternative telomere
Cancers 2022, 14, 3503 6 of 28

lengthening (ALT) phenomena [68,90,91]. Other examples of tumor suppressor genes

described as mutated in OS include the β-catenin regulatory protein APC (Adenomatous
polyposis coli), the metalloproteinase inhibitor TIMP3 (Metalloproteinase inhibitor 3) and
the Wnt pathway inhibitor WIF-1 (Wnt inhibitory factor 1) [78,92–97].
Oncogenes confer a proliferative advantage to tumor cells. Their expression or activity
is increased in cancers. In OS, gain-of-function mutations are observed, for example, in
E2F3 (60% of tumors), a transcription factor of the E2F family involved in many cellular
processes, including replication, DNA repair and apoptosis. Its inhibition in cancers notably
decreases tumor growth by disrupting the cell cycle [98]. CDK4 (cyclin-dependent kinase4)
is involved in the transition from G1 to S phase of the cell cycle. It is an inhibitor of the
tumor suppressor Rb. In OS, its gene is mutated in about 10% of tumors [99–101]. CDK4 is
often co-amplified with MDM2 in various cancers [100]. As previously mentioned, MDM2
is a p53 inhibitory ubiquitin ligase. MDM2 is amplified in 3–25% of OS tumors, resulting in
p53 inactivation [102].
C-Myc is an oncogene described in several types of cancers [103–105]. This transcrip-
tion factor induces cell proliferation, particularly through the regulation of CDKs, including
CDK4 [106,107]. Furthermore, via activation of mTOR (mammalian target of rapamycin)
and subsequent phosphorylation of 4EBP1 (eukaryotic translation initiation factor 4E bind-
ing protein 1), c-Myc increases protein synthesis in cancers [108]. C-Myc also regulates
cell death as well as angiogenesis and metastatic processes [109,110]. In OS, the c-Myc
gene is amplified in 7–67% of tumors and is overexpressed in 34% of cases [111–113]. The
17p11.2~p12 region has also been described as amplified in 13–32% of high-grade OS [114].
This region contains, among others, the gene encoding PMP22 (peripheral myelin protein
22) [114], a protein involved in tumor proliferation, migration and invasion, including via
the MAPK (Mitogen-activated protein kinase) pathway [115,116]. Other oncogenes, such as
CDC5L (cell division cycle 5-like involved in G2 cell cycle progression and tumor growth)
or RUNX2 (Runt-related transcription factor 2 in osteoblastic differentiation and known
oncogene in OS), are also recurrently amplified in OS [114,117,118].
Deletions of Methylthioadenosine phophorylase (MTAP) gene coding for the MTAP,
a key enzyme in the salvage of cellular adenine and methionine synthesis, is frequently
observed in OS patients. Indeed, deletion of at least one exon of MTAP gene was observed
in 37.5% of the patients in a cohort of 96 patients with high-grade OS [119]. No expression
of the MTAP protein was observed in 27.5% of the patients in a Japenese OS cohort [120].
These MTAP deletions could be exploited as alternative therapy in MTAP-negative OS
patients with the use of 6-thioguanine (6-TG), showing good results in leukemia or in
metastatic prostate cancer models [121], or other inhibitors of de novo purine synthesis.
However, the use of L-alanosine, a potent inhibitor of adenine biosynthesis, failed to show
anti-cancer efficacy in a phase II clinical trial enrolling 65 patients, including 7 OS (2 OS
patients with stable disease and 5 OS patients with progressive disease) [122].
Beyond proteins, other cellular actors are involved in genetic abnormalities in OS.
Thus, miRNAs (microRNAs), lncRNAs (long non-coding RNAs) and non-protein coding
ribonucleotide sequences also have deregulated expression in OS. For example, Pasic and
colleagues have shown that the lncRNA loc285194, known to be a p53-controlled tumor
suppressor, has its locus deleted in OS. This deletion is associated with poor prognosis
in patients [123,124]. Studies have also shown the involvement of miRNAs in OS. For
example, Zhou and colleagues showed that miR-340 acts as an inhibitor of tumor growth
and metastatic spread by targeting ROCK1 (Rho-associated coil containing protein kinase 1),
while Song et al. showed the involvement of miR-140 in chemoresistance [125,126].
Thus, OS is highly heterogeneous genomically, genetically and epigenetically. As an
example, Poos et al. identified 911 proteins and 81 miRNAs described as associated with
OS [127]. This tumor heterogeneity, coupled with the small number of cases (due to the
rarity of the disease), make it difficult to obtain statistically significant results in clinical
studies [128]. Nevertheless, the use of in vitro or in vivo models allows to evaluate the
efficacy of new therapeutic approaches.
Cancers 2022, 14, 3503 7 of 28

8. Osteosarcoma Models
The use of experimental models makes it possible to understand the effects of new
therapeutic strategies before their use in humans. Several types of models are currently
used to mimic the pathology. Cellular models from patients with OS have been used
routinely in the laboratory for decades (Table 3). They are used to test potential treatments
or to identify genetic abnormalities in OS. These cellular models are essential, especially
from an ethical point of view, before the use of more complete in vivo models.

Table 3. Most of the used OS cell lines (ND: no details).

Model in
Cells Origin Gender Age Location p53 Status Reference
Mice
MG-63 human male 14 Bone Yes rearranged [129]
Saos-2 human female 11 Bone Yes null [130]
WT
U2OS human female 15 Tibia Yes [131]
inactivated
MMNG-
human female 13 Femur Yes R156P;F270L [132]
HOS
143-B HOS human female 13 Femur Yes R156P;F270L [133]
Knee
CAL-72 human male 10 No WT [134]
recurrence
G-292 human female 9 Bone ND [135]
P53 and
SJSA-1 human male 19 Femur ND MDM2 [136]
amplification
K7M2 BALB/c mice ND ND ND syngenic [137]
POS-1 C3H/He mice ND ND ND syngenic WT [138]
MOS-J C57BL/6J mouse ND ND ND syngenic [139]
Rat Syngenic in
OSRGA ND ND ND WT [140]
(Sprague Dawley) rat
Rat (Sprague Syngenic in
UMR-106 ND ND ND WT [141]
Dawley) rat

The mouse (Mus musculus) is used as an in vivo model in many pathologies. One of
the oldest in vivo xenograft models used in cancer is the transplantation of human OS cell
lines to mice [142–146].
Other OS animal models exist, such as dogs (Canis familiaris), especially large ones,
which spontaneously develop OS with a higher incidence than in humans, although
the disease remains rare [147,148]. As in humans, canine OS produces bone or osteoid
substances. The osteoblastic type is the majority in both species [149,150]. Tumor location
is also similar in both species, with a preponderance in the appendicular skeleton [151].
The similarities between human and canine OS make the latter a good animal model to
complement the previously described in vitro and mouse models [7].
The zebrafish model (Danio rerio) is a model used to study tumor growth, migration
and invasion of tumor cells in cancers [152]. After injection of fluorescent human tumor
cells, a few days are sufficient to observe the processes of proliferation, migration and
invasion without the need for special equipment.
Animal models, although imperfect or restrictive, are essential for the development of
new and effective treatments.
022, 14, x FOR PEER REVIEW 8 of 27
Cancers 2022, 14, 3503 8 of 28

9. The vicious Cycle Hypothesis

9. The in OS
Vicious Cycle Hypothesis in OS
It has been shown thatbeen
It has the tumor
shown is that
able the
to hijack
tumorthe boneto
is able remodeling
hijack the mechanism to
bone remodeling mechanism
ensure its growth.to ensure its growth. This phenomenon, called a vicious cycle, is foundinin OS but also in
This phenomenon, called a vicious cycle, is found in OS but also
other tumors metastasizing
other tumors to bone, such to
metastasizing asbone,
breastsuch
or prostate
as breast cancer [143,153,154].
or prostate In
cancer [143,153,154]. In these
these tumors, the cancer cells synthesize and secrete growth factors, such as PTHrP (par-
tumors, the cancer cells synthesize and secrete growth factors, such as PTHrP (parathyroid
athyroid hormone relatedrelated
hormone protein), IGF, FGF
protein), IGF,(fibroblast growth
FGF (fibroblast factor),
growth VEGF
factor), (vascular
VEGF (vascular endothelial
endothelial growth factor)
growth but also
factor) Wnt,Wnt,
but also which havehave
which activating effects
activating on osteoblasts.
effects Os-Osteoblasts will,
on osteoblasts.
teoblasts will, inin
turn,
turn,promote
promote thethe
differentiation
differentiation andand
resorption activity
resorption of osteoclasts
activity by by producing
of osteoclasts
producing RANKL (receptor activator of NF-kB ligand). The tumor may also activate
RANKL (receptor activator of NF-kB ligand). The tumor may also activate osteoclasts os-
teoclasts through the production
through of IL-6,ofIL-11
the production IL-6,or IL-1β
IL-11 or [117,143,155–157].
IL-1β [117,143,155–157].The combined
The combined action of
action of osteoblasts and tumor cells results in exaggerated bone matrix resorption
osteoblasts and tumor cells results in exaggerated bone matrix resorption by os- by osteoclasts.
teoclasts. This resorption leadsleads
This resorption to theto release
the releaseof BMP,
of BMP,TGF-β,
TGF-β, FGFFGFand
andPDGFPDGF(platelet-
(platelet-derived growth
derived growth factor)
factor) into
into the
the environment,
environment, which which will
will contribute
contribute to to the
the survival
survival and
and proliferation of
proliferation of tumor cells (Figure
(Figure 1).
1). At
At the
the organism
organismlevel,
level,bone
boneresorption
resorptioncan canlead
leadtotohypercalcemia as
hypercalcemia as well
well asas fractures
fractures due
due to to osteolysis
osteolysis [158–160].
[158–160].

Figure 1. The vicious cycle

Figure 1. in
Theosteosarcoma:
vicious cyclethe in cancer cell secretes
osteosarcoma: the growth factors
cancer cell that activate
secretes growth os-factors that activate
teoblasts. These, together withThese,
osteoblasts. the tumor cell,with
together promote osteoclastic
the tumor differentiation
cell, promote anddifferentiation
osteoclastic exaggerated and exaggerated
resorption of the resorption
bone matrix. Thisbone
of the resorption
matrix.leadsThisto the release
resorption intotothe
leads themicroenvironment of
release into the microenvironment of
factors involved in tumor survival and proliferation. PTH/rP: parathyroid hormone-related protein,
factors involved in tumor survival and proliferation. PTH/rP: parathyroid hormone-related protein,
IGF: insulin-like growth factor, FGF: fibroblast growth factor, VEGF: vascular endothelial growth
IGF: insulin-like growth factor, FGF: fibroblast growth factor, VEGF: vascular endothelial growth
factor, IL-(1, 6, 11): interleukin-(1, 6, 11, RANKL: receptor activator of NF-kB ligand, c-fms: colony
stimulating factorfactor, IL-(1,TGF-ß:
1 receptor, 6, 11): transforming
interleukin-(1,growth
6, 11, RANKL:
factor ß; receptor
BMP: bone activator of NF-kBpro-
morphogenetic ligand, c-fms: colony
stimulating factor 1 receptor, TGF-ß: transforming growth factor
tein, PDGF: platelet-derived growth factor, M-CSF: macrophage colony-stimulating factor, Ca : cal- ß; BMP: bone2+morphogenetic protein,
PDGF: platelet-derived growth factor, M-CSF: macrophage colony-stimulating factor, Ca 2+ : calcium
cium ions.
ions.
10. New Therapeutic Approaches in OS
10. New Therapeutic Approaches in OS
10.1. Immunotherapy
10.1. Immunotherapy
Immunomodulation is the modification
Immunomodulation of modification
is the immunity or of theimmunity
immune response, with response, with
or the immune
the use of interferons (IFN) as an example. These are glycoproteins of the cytokine family
the use of interferons (IFN) as an example. These are glycoproteins of the cytokine family
that are producedthatbyareimmune cells by
produced in response
immune to viral
cells ininfections,
response to showing some efficacy
viral infections, showing some effi-
in cancers [161].cacy
In OS, IFN-α-2b inhibits growth of tumor cells and PDX
in cancers [161]. In OS, IFN-α-2b inhibits growth of tumor tumors (Table
cells 4)
and PDX tumors
[162]. The efficacy of pegylated
(Table recombinant
4) [162]. The human IFN-α
efficacy of pegylated 2b (IFN-α2b)
recombinant human was studied
IFN-α in
2b (IFN-α2b) was stud-
patients with OSiedin an
in international
patients with randomized controlledrandomized
OS in an international trial. The patients were trial.
controlled treatedThe patients were
with combination chemotherapy
treated (methotrexate,
with combination doxorubicin
chemotherapy and cisplatin:
(methotrexate, MAP) with
doxorubicin andor cisplatin: MAP)
without pegylated IFN-α-2b and the overall survival rate was not significantly different
with or without pegylated IFN-α-2b and the overall survival rate was not significantly
different between MAP + IFN-α2b and MAP [25]. Activation of lymphocytes and their
Cancers 2022, 14, 3503 9 of 28

differentiation into lymphokine-activated killer (LAK) cells can be induced by interleukin-2

(IL-2). LAK recognize and eliminate various tumor cells [163]. The addition of IL-2 to the
standard treatment of OS has been studied with LAK reinfusion and surgery in children
with metastatic OS, showing 3-year event survival rates of 34% and overall survival rates
of 45%, highlighting the interest of IL2 and LAK cells in immunotherapy [164]. However,
severe side effects were observed after treatment with a high-dose of IL2, such as fever and
influenza-like symptoms in all patients. In some of them, increases in white blood cells,
creatinine, gamma-glutamyltransferase, C-reactive protein, glucose and body weight and
decreases in red blood cells, platelets, protein, albumin and cholinesterase were observed.
Nevertheless, two of the four patients with OS, included in a study of high-dose IL-2
treatment, showed a complete response [165]. Stimulation of the immune system can also
be achieved by synthetic molecules, such as mifamurtide (immune modulator liposomal
muramyl tripeptide), which is a synthetic derivative of a peptidoglycan that makes up
the plasma membrane of bacillus Calmette–Guerin. In cancer, mifamurtide induces the
activation of monocytes and macrophages against tumors via the secretion of IL-6, TNFα
and increased anti-tumor activity of infiltrating immune cells [166]. The addition of mi-
famurtide showed an increase in event-free life span and overall survival [167,168] but
without a significant difference between non metastatic and metastatic patients [169]. The
European Medicine Agency approved the use of mifamurtide in treatment of OS patients
between 2 and 30 years of age [170]. Regarding other immunotherapy approaches based
on the infusion of antitumor immune-effectors, recent studies reported cytokine-induced
killer cells (CIK) are able to kill sarcoma cancer stem cells (sCSC) in vitro [171,172]. CIK
are an ex vivo expanded mix of T lymphocytes and T-NK cell-like phenotype that showed
therapeutic efficacy in sarcoma including OS, even in chemo-resistant sCSC [173], which
provides a therapeutic alternative in the clinic, such as the phase 1 clinical trial CRX100
in patients with refractory solid tumors, including OS patients, which is a dose-escalation
study combining CIK cells with an oncolytic virus (NCT04282044).

10.1.1. Targeting Cell Surface Proteins

One of the immunotherapy strategies is to target the cell surface proteins using mon-
oclonal antibodies, which bind specific antigens on the surface of tumor cells, and then
activate NK cells and macrophages (Table 4). A phase II clinical trial investigated the effi-
cacy of monoclonal antibody trastuzumab, targeting the human epidermal growth factor
receptor 2 (HER2) protein, in metastatic OS patients. While trastuzumab was well tolerated,
it failed to significantly show any difference in the outcome between the HER2-positive
and HER2-negative patients [174]. A new phase II trial, which started in February 2021,
studies the effects of trastuzumab deruxtecan in treating new diagnosed or recurrent HER2
positive OS patients (NCT04616560). The monoclonal antibody trastuzumab is linked
to deruxtecan, a chemotherapeutic drug. High HER2 expression in OS was also used to
evaluate the efficacy of HER2-specific chimeric antigen receptor modified T cells (CAR T
cells) in a clinical trial with 16 enrolled HER2-positive tumor in-patients with recurrent
or refractory OS [175]. Infusion of CAR T cells was well tolerated but showed modest
anti-tumor activity. Indeed, only four of sixteen patients had stable disease for 12 weeks to
14 months [175].
Monoclonal antibodies have also been used to target the growth hormone receptor
insulin-like growth factor-1 (IGF-1). A first phase II clinical trial including 38 OS patients
showed that R1507, a recombinant human monoclonal antibody, is well tolerated but
has limited efficacy in patients with relapsed or refractory bone and soft tissue sarcomas
(NCT00642941) [176]. The efficacy of the monoclonal antibody cixutumumab, which targets
IGF1R, was evaluated in a phase II clinical trial in children with relapsed and refractory
solid tumors, showing that cixutumumab was well tolerated but with limited efficacy as a
single agent [177,178].
Disialoganglioside (GD2) is highly expressed in more than 95% of OS and is involved
in cell proliferation, motility, migration, adhesion, and invasion, amounting tumor develop-
Cancers 2022, 14, 3503 10 of 28

ment and malignant phenotypes [179,180]. The efficacy of anti-GD2 antibodies (monoclonal
or BiTE antibodies) are evaluated in several clinical trials. Indeed, in a phase II clinical, the
combination of the monoclonal antibody dinutuximab with sargramostim (recombinant
granulocyte-macrophage colony-stimulating factor (GM-CSF)) is evaluated in the treatment
of 39 patients with recurrent OS. While dinutuximab is well tolerated, it failed to demon-
strate sufficient efficacy in the disease control rate (NCT02484443) [181]. Other antibodies
composed of two single-chain variable fragments linked by a flexible coupler can be used,
such as bispecific T cell activators (BiTE). The use of bispecific antibodies recognizing an
antigen on the tumor target close to CD3 receptor of T cells, leading to T cells activation
and thus cytolysis of tumor cells [182,183]. The efficacy of activated T cells armed with a
bispecific GD2 antibody is evaluated in cancer cell lines, PDX models [184], and in a phase
I/II clinical trial in children and young adults with neuroblastoma and OS (NCT02173093).
A Phase I/II clinical trials is investigating the safety and efficacy of the humanized anti-
GD2 and anti-CD3 bispecific antibody 3F8 (Hu3F8-BsAb) in patients with relapsed and
refractory neuroblastoma, osteosarcoma and other solid tumors (NCT03860207).
Leucine rich repeat containing 15 (LRRC15) is a membrane protein highly expressed on
the cell surface of stromal fibroblasts in many solid tumors including OS and its expression
is induced by TGFß. This protein has a role in cell–cell and cell–extracellular matrix
interaction. It is a novel mesenchymal protein and stromal target for monoclonal antibody-
drug conjugates [185,186]. A phase I clinical trial was designed to evaluate the efficacy,
the safety and pharmacokinetics of ABBV-085 (Samrotamab vedotin), an antibody drug
conjugate, in solid tumors, especially sarcomas (NCT02565758). ABBV-085 was safe and
well tolerable with promising antitumor activity in OS patients [187].
B7 Homolog 3 (B7-H3), also known as CD276, is a protein whose main role is to inhibit
adaptive immunity by suppressing T cell activation and proliferation [188]. A phase I
clinical trial is still active since 2004 and evaluates the effects of Omburtamab (antibody
8H9), a radiolabeled monoclonal antibody in patients with sarcoma and other cancers
(NCT00089245). A multicenter phase I/II clinical trial is currently recruiting patients with
advanced solid malignant tumors and is investigating the effect of DS-7300a, a novel
B7-H3-targeting antibody–drug conjugate with a DNA topoisomerase I inhibitor DXd
(NCT04145622).
Other immunoconjugates have been tested in OS using toxin or radionuclide as a linker
joined with a carrier antibody and have shown promising results [189]. Indeed, an anti-
gp72 mAb 791T/36 conjugated with methotrexate and ricin toxin A chain (RTA) showed
encouraging results by inhibiting OS cells proliferation associated with immunotoxin
internalization [190,191]. In the same way, TP-3 mAb, recognizing an antigen present
on the surface of OS cells, was conjugated with pokeweed antiviral protein (PAP) [192].
TP-3-PAP showed promising results by reducing tumor cell growth and the number of
lung metastases in an OS model [192].
CD146, overexpressed in OS, was used to develop an anti-CD-146 murine antibody
named OI-3 coupled with Iodine-125 or Lutetium-177 and was evaluated in an OS xenograft
model, showing promising results with therapeutically relevant biodistribution of the
radionuclide [193]. Insulin-like growth factor 2 receptor (IGF2R) is overexpressed in
OS [194,195], which has been used to develop radiolabeled antibody targeting IGF2R with
Indium-111, Lutetium-177 and Bismuth-213 and showing delayed tumor growth in vivo in
an OS model [196].

10.1.2. Checkpoint Inhibitors

Checkpoints restrict overly aggressive immune responses and, in some cases, block
T cells from killing tumor cells (Table 4). T lymphocytes are better able to kill tumor
cells when these checkpoints are blocked [197]. The checkpoint inhibitors targeting anti-
cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death receptor1 (PD-1) and its
ligand (PD-L1) are the most studied in OS [182]. CTLA-4 is a transmembrane glycoprotein
receptor, also known as CD152 (cluster of differentiation 152), and overexpressed in patients
Cancers 2022, 14, 3503 11 of 28

with OS [198]. CTLA-4 receptor is expressed by activated T cells and regulatory T cells
(Tregs) [199] and able to bind CD80 (B7-1) and CD86 (B7-2) expressed by dendritic cells,
leading to functional inhibition [199]. Most checkpoint inhibitors targeting CTLA-4 are not
living up to expectations and are indeed less effective in treating solid tumors, including
OS, compared with other malignant tumors, such as melanoma [200,201], without clear
explanations. Ipilimumab, a CTLA-4 inhibitor, was evaluated in a phase I clinical trial in
children with relapsed solid tumors, including patients with OS. No anti-tumor response
was reported, while ipilimumab increased activation and levels of cycling of CTLs but not
Tregs [202].
PD-1, also known as CD279, is a transmembrane member of the immunoglobulin
family expressed on activated cytotoxic CD8+ T cells and natural killer cells [203]. PD-
1 ligand (PD-L1) binds to PD-1 receptor, leading to inactivation of T cells [204]. The
expression level PD-L1, also called B7-H1 or CD274, is expressed in OS cells and immune
cells, especially tumor-infiltrating lymphocytes (TILs) [205,206]. In addition, high PD-
L1 expression is associated with poorer 5-year event-free survival in OS compared with
patients with low PD-L1 expression [207]. It was reported that PD-L1 was more highly
expressed in pediatric metastatic OS tissues compared with primary OS samples [208]. PD-1
and PD-L1 checkpoint inhibitors have shown promising results in basic and preclinical
research [209,210]. PD1/PD-L1 inhibitor pembrolizumab was evaluated in a multicenter
phase 2 clinical in advanced soft-tissue sarcoma and bone sarcoma including 22 patients
with OS and showed a poor response in OS [211]. Indeed, one patient out of 22 (5%) with
recurrent OS treated with pembrolizumab had a partial response, 6 patients (27%) had
stable disease and 15 patients (68%) had disease progression [211]. The efficacy of avelumab,
an anti-PD-L1 antibody, was evaluated in a phase II clinical trial in patients with recurrent
OS and did not demonstrate any activity (NCT03006848) [212]. In the SARC038 phase II
clinical trial, the efficacy of nivolumab combined with regorafenib, a receptor of tyrosine
kinase inhibitor, is currently being evaluated in patients with refractory or recurrent OS
(NCT04803877).
However, the clinical evaluation of the checkpoint inhibitors in sarcoma was not a
success, as observed in melanoma as an example, probably due to the immunosuppressive
role of the microenvironment [213,214].

10.2. Bone Resorption Inhibition

Some therapeutic strategies are based on the inhibition of osteoclasts (Table 4). By
doing so, they reduce pathological bone remodeling and its consequences (osteolysis,
hypercalcemia, etc.) and directly impact the vicious cycle. In this category, we find the
RANK/RANKL inhibitors. The RANK receptor and its ligand RANKL, as well as the
decoy receptor OPG, are closely associated with bone remodeling. They control osteoclast
differentiation via activation of NF-kB and JNK (Jun N-terminal kinase). RANK is expressed
by OS cells, and this expression negatively affects treatment response and survival [215,216].
Several studies have described beneficial effects to targeting the RANK/RANKL/OPG triad
in OS [143,217–221]. Denosumab, for example, is a monoclonal antibody directed against
RANKL. It inhibits the binding of RANKL to RANK, thus mimicking the physiological
decoy receptor role of OPG, inhibiting osteoclastic differentiation [217,222]. Denosumab is
currently in a phase II clinical trial in OS, in patients resistant to conventional therapies or
in relapse (NCT02470091).
Other bone resorption inhibitors have shown promising results. Indeed, bisphos-
phonates (e.g., zoledronate) are molecules capable of inhibiting bone resorption. They
are pyrophosphate analogues capable of binding to the hydroxyapatite constituting the
bone matrix and of inducing apoptosis of osteoclasts [158]. They have been used for
the treatment of osteoporosis for 40 years, but also in Paget’s disease, breast cancer and
prostate cancer [223–226]. In OS, zoledronate has shown encouraging results. In vitro, it
inhibits OS cell proliferation and induces cell death [227,228]. In addition, zoledronate
sensitizes OS cells to radiation treatment by increasing oxidative stress and inhibiting DNA
Cancers 2022, 14, 3503 12 of 28

repair mechanisms [229]. In vivo, zoledronate reduces tumor growth, osteolysis, angiogen-
esis, tumor cell invasion and lung metastasis [230–233]. A phase III clinical trial (OS2006,
NCT00470223) using zoledronate in combination with chemotherapy, however, did not
show an improvement in survival (overall or event-free) of OS patients [55]. A phase I
clinical trial (METZOLIMOS, NCT02517918) is ongoing in OS, combining zoledronate with
methotrexate, cyclophosphamide and the mTOR inhibitor sirolimus.

10.3. Targeting Receptor Tyrosine Kinases and Intracellular Signaling

Receptor tyrosine kinases (RTKs) are transmembrane receptors with an extracellular
domain that binds the ligand and an intracellular domain with kinase activity (e.g., IGF-R:
insulin-like growth factor 1 receptor, PDGFR: platelet-derived growth factor receptor...).
RTKs are involved in cell growth, proliferation and survival, in particular through their in-
volvement in signaling pathways, such as the PI3K (phosphoinositide 3-kinase) pathway or
the MAPK pathway. About 30% of RTKs are mutated or overexpressed in cancer, including
osteosarcoma. As a result, the expression of certain RTKs is associated with poor prognosis
for patients [234]. Six subfamilies, among the 20 RTK subfamilies, are particularly associ-
ated with cancer pathologies. These are the EGFR/ErbB (epidermal growth factor receptor)
families, PDGF, FGF, VEGF, HGF (hepatocyte growth factor), and IGF receptors [235].
Indeed, it has been observed that high expression of VEGF, IGF1R or AXL is associated
with a decreased overall survival in osteosarcoma [236–238]. For example, IGF expression
is associated with higher OS aggressiveness [239]. The AXL receptor is overexpressed in
OS, and its expression is associated to a poor prognostic factor for patients [237], while AXL
inactivation induces apoptosis of OS cells [240]. Dysfunction of RTKs is generally related to
neovascularization, invasion, metastasis and chemotherapy resistance of tumors [241,242].
In OS, it has been shown that RTK KIT is expressed in 46.15% of patients with a poor
response to chemotherapy [243]. RET RTK overexpression is also associated with resistance
to cisplatin and bortezomib [244,245]. FGFR1 amplification, meanwhile, is observed in 18%
of patients resistant to neo-adjuvant chemotherapy [246]. Regarding cell proliferation and
metastasis development, it has been shown that deregulation of FGFR found in OS plays
an important role in its formation as well as in the development of lung metastasis [247].
The PDGFs/PDGFRs signaling pathway also plays a central role in OS proliferation and
migration [248,249]. Inhibition of signaling pathways from aberrantly activated MET and
AXL RTKs increases the rate of apoptosis and suppresses migration, proliferation and
invasion of OS cells [240,250–252]. IGF-1R expression is also related to the presence of
distant metastasis in patients [253]. In addition, RET and MET RTKs are described for their
involvement in the development of malignancy in OS cells. Indeed, it has been shown that
MET overexpression leads to the malignant transformation of primary osteoblastic cells
and that RET overexpression increases the stem-cell-like properties of OS cells [254,255].
RTKs are, therefore, therapeutic targets, which are of major interest in the treatment of OS.
Since the FDA approved imatinib for the treatment of chronic myeloid leukemia in
2001, many potent and well-tolerated tyrosine kinase inhibitors have been developed and
have contributed significantly to advances in cancer treatment [256]. Therefore, several ther-
apeutic approaches targeting RTKs have thus been developed but have proven relatively
ineffective in light of the emergence of resistance mechanisms. This is notably the case of
the VEGFRs inhibitor bevacizumab, tested in addition to conventional MAP chemotherapy
(NCT00667342), which showed no progression regarding the histological response or the
outcome of patients with localized OS [257]. More recent approaches based on RTKi (RTK
inhibitors) favor simultaneous targeting of multiple RTKs or constitutive RTK activation
pathways to circumvent resistance mechanisms [258]. Imatinib (STI571), for example, is
an RTK inhibitor that targets multiple receptors (PDGFRα and β, cKIT, AXL, RYK (related
to receptor tyrosine kinase), EGFR, EphA (ephrin type-A receptor) 2 and 10, IGF1R) [259].
Imatinib induces apoptosis of OS cells in vitro and reduces tumor growth in vivo in mouse
OS models [259,260]. However, Imatinib failed to show effective anticancer activity in phase
II clinical trials (in OS, Ewing’s sarcoma, neuroblastoma and desmoplastic small round
Cancers 2022, 14, 3503 13 of 28

cell tumors) [261–263]. The same finding resulted for cixutumumab, an IGF-1R inhibitor,
which showed very limited effects in young patients with refractory solid tumors [177].
Dasatinib (c-KIT, Epha2 and PDGFRβ RTK inhibitor) has shown similar results. It is used
for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia [162].
In OS, dasatinib has shown anti-metastatic effects but failed to inhibit primary tumor
growth [264,265]. Two studies are ongoing (NCT00464620, NCT00788125) with dasatinib
alone or in combination with other molecules. An inhibitor of Met (HGF receptor) activity
called PF-2341066 reduces tumor growth in a mouse model of OS [266].
The intertwining of different signaling pathways downstream of RTKs is responsible
for the rapid emergence of compensatory mechanisms that negate the effect of molecules
targeting a limited number of RTKs. Thus, multi-tyrosine kinase inhibitors (mTKIs) have
emerged and have shown effects on patients in relapse and/or with unresectable OS. Five
molecules stood out for their major effects: Sorafenib, Regorafenib, Cabozantib, Lenvatinib
and Pazopanib. Sorafenib, an inhibitor of VEGFR, PDGFR, RET and c-Kit, has shown
antiangiogenic and anti-metastatic effects in preclinical models [267]. In the clinical trial
NCT00889057, sorafenib showed encouraging results in 35 patients with a PFS of 46% at
4 months and a PR of 9%, although treatment had to be reduced or briefly discontinued
in 46% of patients due to toxicity [268]. The results from another phase II clinical trial
in OS in patients with advanced or metastatic OS after failure of initial therapy indicate
that sorafenib inhibits tumor progression at 6 months in half of the patients [269]. Two
clinical trials also demonstrated the efficacy and safety of Regorafenib (targeting VEGFR,
PDGFR, KIT, FGFR and RET) in patients with advanced or metastatic OS after failure
of prior therapy, showing a PFS of 62% at 12 weeks and a PR of 8% (NCT02048371 and
NCT02389244) [270,271]. Cabozantinib is an inhibitor of the VEGFR, KIT, RET, AXL and
PDGFR RTKs that was studied in a multicenter phase II clinical trial of 42 patients with
advanced or metastatic OS after failure of other systemic therapy (NCT02243605). Further,
12% of the patients developed a PR, and the PFS of 33.3% at 6 months was the best result
obtained by RTKi in the treatment of OS to date [272]. A clinical trial is also underway to
test the activity of Lenvatinib (targeting VEGFR, PDGFR, KIT, FGFR and RET) in relapsed
patients (NCT02432274). The first published results from 31 patients show a 4-month PFS
of 29% [273]. Finally, Pazopanib, a second-generation MTKi targeting VEGF, PDGFR and
KIT, showed positive effects in 3 patients with relapsed OS for the second time, appearing
to stabilize disease progression and thus prolong patient survival [274].
Inhibition of RTKs is promising and actively studied, but other approaches aim to
inhibit intracellular signaling downstream of RTKs (Table 4). Indeed, given the importance
of signaling pathways in cellular processes and in the development of cancers, including
OS, many inhibitors targeting members of these pathways have been developed. For
example, there are inhibitors of members of the SFK (Src family kinase), proteins that
integrate and regulate the signaling of many RTKs (EGFR, PDGFR, IGF1R, VEGRF, HER2...).
Through their targets or partners, SFK family members regulate cell survival, angiogenesis,
cell mobility... [275]. Src (steroid receptor co-activator) kinase belonging to this family is
notably involved in the activation of osteoclasts under physiological conditions [276]. Src
is overexpressed in OS and other types of cancers, and this overexpression correlates with
lower patient survival [277]. Saracatinib (AZD0530), a selective Src kinase inhibitor, has
been tested in 18 subjects with recurrent OS localized to the lung. The study demonstrated
an increase in the median PFS in the treated group from 8.6 months in the placebo treatment
group to 19.4 months [278].
Inhibitors of mTOR have also been developed. mTOR is a serine/threonine kinase in-
volved in the deregulated PI3K/Akt pathway in most cancers [279,280]. mTOR is involved
in protein synthesis, cell cycle or survival and is overexpressed in OS [281–283]. In addition
to the sirolimus, the class of mTOR inhibitors also includes ridaforolimus, a rapamycin
analog tested in a phase III clinical trial in metastatic bone sarcoma. It showed weak but
statistically significant inhibition of tumor progression in patients [284,285]. CC-115 is
an analog of thalidomide [286] inhibiting mTOR, but also the serine/threonine kinase
Cancers 2022, 14, 3503 14 of 28

DNA-PK (DNA-dependent protein kinase) involved in DNA repair. Treatment of OS cells

with CC-115 increases their sensitivity to cisplatin and etoposide chemotherapy [287]. A
phase I clinical trial is underway in several tumors, including OS (NCT01353625).
The YAP/TAZ (yes-associated protein/paralog transcriptional coactivator with PDZ-
binding motif) signaling pathway was recently described to be involved in OS with aberra-
tions in the Hippo signaling pathway [288,289]. Indeed, several immunohistochemistry
studies demonstrated that 60% to 85% of OS exhibit high YAP expression, with only 30%
to 46% of cases showing YAP nuclear expression, with no more frequency of YAP nuclear
expression in metastatic patients [290–294]. However, Morice et al. demonstrated that
high YAP expression in primary tumors in OS is higher in metastatic patients versus non-
metastatic patients at diagnosis and associated with poor prognosis [295]. Inhibition of
YAP using shRNA or chemical inhibitors (verteporfin or CA3) in OS cell lines delays tumor
proliferation in vitro and in vivo and blocks migration and invasion [292,294,296]. Indeed,
inhibition of the primary tumor growth is explained by the ability of YAP to bind TEAD,
while inhibition of lung metastasis is due to the inhibition of YAP/smad3 interaction in the
TGF-ß signaling pathway [295,297–299].
Thus, many therapeutic targets are currently being studied for the treatment of OS
(Table 4). Often very targeted, they may come up against tumor heterogeneity. Indeed,
the tumor is composed of a set of clonogenic populations that have evolved and present
different characteristics and gene expressions. The use of therapeutic approaches targeted
to a restricted tumor population can lead to recurrences due to the proliferation of cell
populations not affected by the treatment. In this context, the strategy of targeting multiple
mechanisms shared only by tumor cells has a clear advantage: impacting all tumor cells
while sparing healthy cells.

Table 4. New therapeutic approaches in OS.

Target Cell, Gene or Protein Agent Used Reference

Targeting Receptor Tyrosine Kinases and Intracellular Signaling
PDGFRα and β, cKIT, Axl, RYK, EGFR, EphA 2
Imatinib (STI571) [259,261–263]
and 10, IGF1R
c-KIT, Epha2 and PDGFRβ RTK inhibitor Dasatinib [264,265]
Met (HGFr) PF-2341066 [266]
VEGFR, PDGFR, RET and c-Kit Sorafenib [267–269]
VEGFRs Bevacizumab [257]
IGF-1R Cixutumumab [177]
VEGFR, PDGFR, KIT, FGFR and RET Regorafenib [270,271]
VEGFR, KIT, RET, AXL and PDGFR Cabozantinib [272]
VEGFR, PDGFR, KIT, FGFR and RET Lenvatinib [273]
VEGF, PDGFR and KIT Pazopanib [274]
Src Saracatinib (AZD0530) [278]
mTOR Ridaforolimus [284,285]
mTOR and DNA-PK CC-115 [286,287]
Immunomodulation
immune system IFN-α-2b [25,162]
lymphocytes IL-2 [164,165]
Monocytes and macrophages Mifamurtide [167,168]
Cancers 2022, 14, 3503 15 of 28

Table 4. Cont.

Target Cell, Gene or Protein Agent Used Reference

Targeting surface proteins
Trastuzumab [174]
HER2
Trastuzumab deruxtecan NCT04616560
NCT00642941
R1507
IGF-1/IGF-1R [176]
Cixutumumab [177,178]
Dinutuximab + Sargramostim NCT02484443 [181]
humanized bispecific anti-GD2 antibody
NCT03860207
GD2 3F8 (Hu3F8-BsAb)
activated T cells armed with a bispecific
NCT02173093 [184]
GD2 antibody
LRRC15 ABBV-085 NCT02565758 [187]
Omburtanab NCT00089245
B7-H3
DS-7300a NCT04145622
Checkpoint inhibitors
CTLA-4 Ipilimumab [202]
Pembrolizumab [211]
PD-1
Nivolumab NCT04803877 [210]
PD-L1 Avelumab NCT03006848 [212]
Bone resorption inhibition
RANKL Denosumab [217,222]
Zoledronate [227,228,230,232,233,281]
Hydroxyapatite Zoledronate + chemotherapy [55]
Zoledronate + sirolimus NCT02517918 (METZOLIMOS)

11. Conclusions
Development of therapeutic targets in OS is very complex due to the heterogeneity of
this pathology, which limits the effectiveness of treatments and favors tumor recurrence
and the emergence of drug resistance. Indeed, in the last few decades, only a few new
therapies have shown a clinically significant impact for patients with OS. To supplement the
current knowledge and uncover possible ways to improve patient outcomes, fundamental,
translational and clinical research must cooperate, thus allowing the development of new
prognostic markers and new therapeutic targets in OS.

Author Contributions: B.M., M.L. and L.M. were responsible for the literature review, the draft of
the manuscript and reviewed edits. B.M., M.L., L.M., B.O., M.B. and F.V. reviewed edits and F.L.
provided mentorship for the manuscript project and reviewed the final edits. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: We thank La ligue contre le cancer (comités 44, 49), la Fédération Enfants Cancer
Santé et la Société Française de lutte contre les Cancers et leucémies de l’enfant et de l’adolescent for
their support.
Conflicts of Interest: The authors declare no conflict of interest.
Cancers 2022, 14, 3503 16 of 28

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