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Primary bone tumors can be divided into two classes, benign and malignant. Among the latter group, osteosarcoma and Ewing sarcoma are the most prevalent malignant primary bone tumors in children and adolescents. Despite intensive efforts to improve treatments, almost 40% of patients succumb to the disease. Specifically, the clinical outcome for metastatic osteosarcoma or Ewing sarcoma remains poor; less than 30% of patients who present metastases will survive 5 years after initial diagnosis. One common and specific point of these bone tumors is their ability to deregulate bone homeostasis and remodeling and divert them to their benefit. Over the past years, considerable interest in the Sonic Hedgehog (SHH) pathway has taken place within the cancer research community. The activation of this SHH cascade can be done through different ways and, schematically, two pathways can be described, the canonical and the non-canonical. This review discusses the current knowledge about the involvement of the SHH signaling pathway in skeletal development, pediatric bone sarcoma progression and the related therapeutic options that may be possible for these tumors.

BackgroundThe poor survival rate of patients with osteosarcoma (OS), specifically with metastases at diagnosis, undergoes the urgency to develop new therapeutic strategies. Although we recently demonstrated the key role of YAP/TEAD signaling in the growth of OS primary tumor, the molecular mechanisms by which YAP regulates metastases development remain poorly understood.MethodsThe molecular mechanisms by which YAP regulates metastases development were studied using an overexpression of mutated forms of YAP able or not able to interact with TEAD. Molecular signatures were identified using RNA-sequencing analysis and gene set enrichment. Interactions between YAP and Smad3 were studied using proximity ligation assay (PLA), immunoprecipitation, and promoter/specific gene assays. The involvement of the TGF-β pathway in the ability of YAP to stimulate metastatic development in vivo was studied using an inhibitor of the TGF-β cascade in a preclinical model of OS and in vitro on the ability of OS cells to migrate and invade.ResultsOur work shows that a high YAP expression is associated with the presence of lung metastases which predicts a poor prognosis. Molecular analysis indicates that TGF-β signaling is involved in YAP-driven osteosarcoma cell pro-migratory phenotype, epithelial mesenchymal transition, cell migration, and in vivo lung metastasis development. Regardless of its ability to bind to TEAD, YAP interacts with Smad3 and stimulates the transcriptional activity of TGF-β/Smad3, thereby enhancing the ability of TGF-β to stimulate lung metastasis development.ConclusionsWe demonstrated the crucial involvement of the TGF-β/Smad3 signaling pathway in YAP-driven lung metastasis development in OS.

We describe the case report of an Osteosarcoma patient, with a Li-Fraumeni Syndrome, presenting with a pathological femoral fracture. The patient was treated with a multidisciplinary approach associating neoadjuvant and adjuvant chemotherapy with excisional surgery. The femoral reconstruction consisted of a 'Capasquelet' reconstruction combining an induced membrane and a vascularized fibula allograft allowing a good functional result with an early weight-bearing. We managed to complete our histological analysis in this patient, in order to evaluate the tumor vascularization. Indeed, using the syngeneic osteosarcoma MOS-J mouse model, we highlighted previously that CD31+/\ensuremath{\alpha}-SMA+ vessels may be indicators of vasculature normalization and therefore may be used as specific markers of a good therapeutic response. Thus, we search for its interest in this specific case as preliminary work. The aim was to assess the feasibility and technical validity of the vascularization analysis of a human osteosarcoma tumor specimen. Therefore, we propose an immunohistochemistry methodology with multiplexed immunofluorescence to assess the vascularization as a promising marker in human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumour characterized by osteoid production and/or osteolytic lesions of bone. A lack of response to chemotherapeutic treatments shows the importance of exploring new therapeutic methods. Imatinib mesylate (Gleevec, Novartis Pharma), a tyrosine kinase inhibitor, was originally developed for the treatment of chronic myeloid leukemia. Several studies revealed that imatinib mesylate inhibits osteoclast differentiation through the M-CSFR pathway and activates osteoblast differentiation through PDGFR pathway, two key cells involved in the vicious cycle controlling the tumour development. The present study investigated the in vitro effects of imatinib mesylate on the proliferation, apoptosis, cell cycle, and migration ability of five osteosarcoma cell lines (human: MG-63, HOS; rat: OSRGA; mice: MOS-J, POS-1). Imatinib mesylate was also assessed as a curative and preventive treatment in two syngenic osteosarcoma models: MOS-J (mixed osteoblastic/osteolytic osteosarcoma) and POS-1 (undifferentiated osteosarcoma). Imatinib mesylate exhibited a dose-dependent anti-proliferative effect in all cell lines studied. The drug induced a G0/G1 cell cycle arrest in most cell lines, except for POS-1 and HOS cells that were blocked in the S phase. In addition, imatinib mesylate induced cell death and strongly inhibited osteosarcoma cell migration. In the MOS-J osteosarcoma model, oral administration of imatinib mesylate significantly inhibited the tumour development in both preventive and curative approaches. A phospho-receptor tyrosine kinase array kit revealed that PDGFRα, among 7 other receptors (PDFGFRβ, Axl, RYK, EGFR, EphA2 and 10, IGF1R), appears as one of the main molecular targets for imatinib mesylate. In the light of the present study and the literature, it would be particularly interesting to revisit therapeutic evaluation of imatinib mesylate in osteosarcoma according to the tyrosine-kinase receptor status of patients.

Primary malignant bone tumours, osteosarcomas, and Ewing sarcomas are rare diseases which occur mainly in adolescents and young adults. With the current therapies, some patients remain very difficult to treat, such as tumour with poor histological response to preoperative CT (or large initial tumour volume for Ewing sarcomas not operated), patients with multiple metastases at or those who relapsed. In order to develop new therapies against these rare tumours, we need to unveil the key driving factors and molecular abnormalities behind the malignant characteristics and to broaden our understanding of the phenomena sustaining the metastatic phenotype and treatment resistance in these tumours. In this paper, starting with the biology of these tumours, we will discuss potential therapeutic targets aimed at increasing local tumour control, limiting metastatic spread, and finally improving patient survival.

Our lab developed 99mTc-NTP 15-5 radiotracer as targeting proteoglycans (PGs) for the scintigraphic imaging of joint. This paper reports preclinical results of 99mTc-NTP 15-5 imaging of an orthotopic model of Swarm rat chondrosarcoma (SRC). 99mTc-NTP 15-5 imaging of SRC-bearing and sham-operated animals was performed and quantified at regular intervals after surgery and compared to bone scintigraphy and tumoural volume. Tumours were characterized by histology and PG assay. SRC exhibited a significant 99mTc-NTP 15-5 uptake at very early stage after implant (with tumour/muscle ratio of 1.61 ± 0.14), whereas no measurable tumour was evidenced. As tumour grew, mean tumour/muscle ratio was increased by 2.4, between the early and late stage of pathology. Bone scintigraphy failed to image chondrosarcoma, even at the later stage of study. 99mTc-NTP 15-5 imaging provided a suitable set of quantitative criteria for the in vivo characterization of chondrosarcoma behaviour in bone environment, useful for achieving a greater understanding of the pathology.

We describe the case report of an Osteosarcoma patient, with a Li-Fraumeni Syndrome, presenting with a pathological femoral fracture. The patient was treated with a multidisciplinary approach associating neoadjuvant and adjuvant chemotherapy with excisional surgery. The femoral reconstruction consisted of a 'Capasquelet' reconstruction combining an induced membrane and a vascularized fibula allograft allowing a good functional result with an early weight-bearing. We managed to complete our histological analysis in this patient, in order to evaluate the tumor vascularization. Indeed, using the syngeneic osteosarcoma MOS-J mouse model, we highlighted previously that CD31+/\ensuremath{\alpha }-SMA+ vessels may be indicators of vasculature normalization and therefore may be used as specific markers of a good therapeutic response. Thus, we search for its interest in this specific case as preliminary work. The aim was to assess the feasibility and technical validity of the vascularization analysis of a human osteosarcoma tumor specimen. Therefore, we propose an immunohistochemistry methodology with multiplexed immunofluorescence to assess the vascularization as a promising marker in human osteosarcoma tissue.

Abstract Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient’s tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Pediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and treatments development in advanced pediatric malignancies.

In Europe, with an incidence of 7.5 cases per million, Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children, adolescents and young adults, after osteosarcoma. Since the 1980s, conventional treatment has been based on the use of neoadjuvant and adjuvant chemotherapeutic agents combined with surgical resection of the tumor when possible. These treatments have increased the patient survival rate to 70% for localized forms, which drops drastically to less than 30% when patients are resistant to chemotherapy or when pulmonary metastases are present at diagnosis. However, the lack of improvement in these survival rates over the last decades points to the urgent need for new therapies. Genetically, ES is characterized by a chromosomal translocation between a member of the FET family and a member of the ETS family. In 85% of cases, the chromosomal translocation found is (11; 22) (q24; q12), between the EWS RNA-binding protein and the FLI1 transcription factor, leading to the EWS-FLI1 fusion protein. This chimeric protein acts as an oncogenic factor playing a crucial role in the development of ES. This review provides a non-exhaustive overview of ES from a clinical and biological point of view, describing its main clinical, cellular and molecular aspects.

Chondrosarcomas and osteosarcomas are malignant bone tumors with a poor prognosis when unresectable or metastasized. Moreover, radiotherapy and chemotherapy could be ineffective. MiRNAs represent an alternative therapeutic approach. Based on high-throughput functional screening, we identified four miRNAs with a potential antiproliferative effect on SW1353 chondrosarcoma cells. Individual functional validations were then performed in SW1353 cells, as well as in three osteosarcoma cell lines. The antiproliferative and cytotoxic effects of miRNAs were evaluated in comparison with a positive control, miR-342-5p. The cytotoxic effect of four selected miRNAs was not confirmed on SW1353 cells, but we unambiguously revealed that miR-4270 had a potent cytotoxic effect on HOS and MG-63 osteosarcoma cell lines, but not on SaOS-2 cell line. Furthermore, like miR-342-5p, miR-4270 induced apoptosis in these two cell lines. In addition, we provided the first report of Bcl-xL as a direct target of miR-4270. MiR-4270 also decreased the expression of the anti-apoptotic protein Mcl-1, and increased the expression of the pro-apoptotic protein Bak. Our findings demonstrated that miR-4270 has tumor suppressive activity in osteosarcoma cells, particularly through Bcl-xL downregulation.

Chondrosarcomas are heterogenous tumours of the bone cartilage and have highly variable prognoses. Here, the authors perform a multi-omics analysis, revealing molecular features that can stratify clinical outcomes.

Abstract Background Osteosarcoma (OS) is the most common cancer of bone. Jaw osteosarcoma (JOS) is rare and it differs from other OS in terms of the time of occurrence (two decades later) and better survival. The aim of our work was to develop and characterize specific mouse models of JOS. Methods Syngenic and xenogenic models of JOS were developed in mice using mouse (MOS-J) and human (HOS1544) osteosarcoma cell lines, respectively. An orthotopic patient-derived xenograft model (PDX) was also developed from a mandibular biopsy. These models were characterized at the histological and micro-CT imaging levels, as well as in terms of tumor growth and metastatic spread. Results Homogeneous tumor growth was observed in both the HOS1544 and the MOS-J JOS models by injection of 0.25 × 106 and 0.50 × 106 tumor cells, respectively, at perimandibular sites. Histological characterization of the tumors revealed features consistent with high grade conventional osteosarcoma, and the micro-CT analysis revealed both osteogenic and osteolytic lesions. Early metastasis was encountered at day 14 in the xenogenic model, while there were no metastatic lesions in the syngenic model and in the PDX models. Conclusion We describe the first animal model of JOS and its potential use for therapeutic applications. This model needs to be compared with the usual long-bone osteosarcoma models to investigate potential differences in the bone microenvironment.

Osteosarcomas are the most common type of malignant bone tumor. These tumors are characterized by the synthesis of an osteoid matrix. Current treatments are based on surgery and combination chemotherapy. However, for metastatic or recurrent tumors, chemotherapy is generally ineffective, and osteosarcomas are sometimes unresectable. Thus, the use of microRNAs (miRNAs) may represent an attractive alternative for the development of new therapies. Using high-throughput functional screening based on impedancemetry, we previously selected five miRNAs with potential chemosensitizing or antiproliferative effects on chondrosarcoma cells. We validated the tumor-suppressive activity of miR-491-5p and miR-342-5p in three chondrosarcoma cell lines. Here, we carried out individual functional validation of these five miRNAs in three osteosarcoma cell lines used as controls to evaluate their specificity of action on another type of bone sarcoma. The cytotoxic effects of miR-491-5p and miR-342-5p were also confirmed in osteosarcoma cells. Both miRNAs induced apoptosis. They increased Bcl-2 homologous antagonist killer (Bak) protein expression and directly targeted Bcl-2 lymphoma-extra large (Bcl-xL). MiR-342-5p also decreased B-cell lymphoma-2 (Bcl-2) protein expression, and miR-491-5p decreased that of Epidermal Growth Factor Receptor (EGFR). MiR-342-5p and miR-491-5p show tumor-suppressive activity in osteosarcomas. This study also confirms the potential of Bcl-xL as a therapeutic target in osteosarcomas.

Tumorigenesis is a long-term and multistage process that often leads to the formation of metastases. During this pathological course, two major events appear to be crucial: primary tumour growth and metastatic expansion. In this context, despite research and clinical advances during the past decades, bone cancers remain a leading cause of death worldwide among paediatric cancer patients. Osteosarcomas are the most common malignant bone tumours in children and adolescents. Notwithstanding advances in therapeutic treatments, many patients succumb to these diseases. In particular, less than 30% of patients who demonstrate metastases at diagnosis or are poor responders to chemotherapy survive 5 years after initial diagnosis. LIM kinases (LIMKs), comprising LIMK1 and LIMK2, are common downstream effectors of several signalization pathways, and function as a signalling node that controls cytoskeleton dynamics through the phosphorylation of the cofilin family proteins. In recent decades, several reports have indicated that the functions of LIMKs are mainly implicated in the regulation of actin microfilament and the control of microtubule dynamics. Previous studies have thus identified LIMKs as cancer-promoting regulators in multiple organ cancers, such as breast cancer or prostate cancer. This review updates the current understanding of LIMK involvement in osteosarcoma progression.

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan–Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

High-grade osteosarcomas are the most frequent malignant bone tumors in the pediatric population, with 150 patients diagnosed every year in France. Osteosarcomas are associated with low survival rates for high risk patients (metastatic and relapsed diseases). Knowing that the canonical Wnt signaling pathway (Wnt/β-catenin) plays a complex but a key role in primary and metastatic development of osteosarcoma, the aim of this work was to analyze the effects of ICG-001, a CBP/β-catenin inhibitor blocking the β-catenin dependent gene transcription, in three human osteosarcoma cell lines (KHOS, MG63 and 143B). The cell proliferation and migration were first evaluated in vitro after ICG-001 treatment. Secondly, a mouse model of osteosarcoma was used to establish the in vivo biological effect of ICG-001 on osteosarcoma growth and metastatic dissemination. In vitro, ICG-001 treatment strongly inhibits osteosarcoma cell proliferation through a cell cycle blockade in the G0/G1 phase, but surprisingly, increases cell migration of the three cell lines. Moreover, ICG-001 does not modulate tumor growth in the osteosarcoma mouse model but, rather significantly increases the metastatic dissemination to lungs. Taken together, these results highlight, despite an anti-proliferative effect, a deleterious pro-migratory role of ICG-001 in osteosarcoma.

Despite research and clinical advances during recent decades, bone cancers remain a leading cause of death worldwide. There is a low survival rate for patients with primary bone tumors such as osteosarcoma and Ewing’s sarcoma or secondary bone tumors such as bone metastases from prostate carcinoma. Gap junctions are specialized plasma membrane structures consisting of transmembrane channels that directly link the cytoplasm of adjacent cells, thereby enabling the direct exchange of small signaling molecules between cells. Discoveries of human genetic disorders due to genetic mutations in gap junction proteins (connexins) and experimental data using connexin knockout mice have provided significant evidence that gap-junctional intercellular communication (Gj) is crucial for tissue function. Thus, the dysfunction of Gj may be responsible for the development of some diseases. Gj is thus a main mechanism for tumor cells to communicate with other tumor cells and their surrounding microenvironment to survive and proliferate. If it is well accepted that a low level of connexin expression favors cancer cell proliferation and therefore primary tumor development, more evidence is suggesting that a high level of connexin expression stimulates various cellular process such as intravasation, extravasation, or migration of metastatic cells. If so, connexin expression would facilitate secondary tumor dissemination. This paper discusses evidence that suggests that connexin 43 plays an antagonistic role in the development of primary bone tumors as a tumor suppressor and secondary bone tumors as a tumor promoter.

Osteosarcomas are the most frequent primary bone sarcomas, affecting mainly children, adolescents, and young adults, and with a second peak of incidence in elderly individuals. The current therapeutic management, a combined regimen of poly-chemotherapy and surgery, still remains largely insufficient, as patient survival has not improved in recent decades. Osteosarcomas are very heterogeneous tumors, both at the intra- and inter-tumor level, with no identified driver mutation. Consequently, efforts to improve treatments using targeted therapies have faced this lack of specific osteosarcoma targets. Nevertheless, these tumors are inextricably linked to their local microenvironment, composed of bone, stromal, vascular and immune cells and the osteosarcoma microenvironment is now considered to be essential and supportive for growth and dissemination. This review describes the different actors of the osteosarcoma microenvironment and gives an overview of the past, current, and future strategies of therapy targeting this complex ecosystem, with a focus on the role of extracellular vesicles and on the emergence of multi-kinase inhibitors.

Osteosarcomas are the most frequent form of primary bone tumors and mainly affect children, adolescents, and young adults. Despite encouraging progress in therapeutic management, including the advent of multidrug chemotherapy, the survival rates have remained unchanged for more than four decades: 75% at 5 years for localized disease, but two groups of patients are still at high risk: metastatic at diagnosis (overall survival around 40% at 5 years) and/or poor responders to chemotherapy (20% at 5 years). Because these tumors are classified as “complex genomic,” it is extremely difficult to determine the signaling pathways that might be targeted by specific therapies. A hypothesis has thus emerged, stating that the particular microenvironment of these tumors may interfere with the tumor cells that promote chemoresistance and the dissemination of metastases. The stroma is composed of a large number of cell types (immune cells, endothelial cells, mesenchymal stromal cells, etc.) which secrete growth factors, such as transforming growth factor-β (TGF-β), which favors the development of primary tumors and dissemination of metastases by constituting a permissive niche at primary and distant sites. Rather than targeting the tumor cells themselves, which are very heterogeneous in osteosarcoma, the hypothesis is instead to target the key actors secreted in the microenvironment, such as TGF-βs, which play a part in tumor progression. In the last decade, numerous studies have shown that overexpression of TGF-β is a hallmark of many cancers, including primary bone tumors. In this context, TGF-β signaling has emerged as a crucial factor in the cross talk between tumor cells and stroma cells in poor-prognosis cancers. Secretion of TGF-β by tumor cells or stroma cells can effectively act in a paracrine manner to regulate the phenotype and functions of the microenvironment to stimulate protumorigenic microenvironmental changes. TGF-β can thus exert its protumorigenic function in primary bone tumors by promoting angiogenesis, bone remodeling and cell migration, and by inhibiting immunosurveillance. This review focuses on the involvement of TGF-β signaling in primary bone tumor development, and the related therapeutic options that may be possible for these tumors.

The French phase 3 trial (OS 2006) testing zoledronic acid, an osteoclast inhibitor, with chemotherapy and surgery did not improve the outcome of patients with osteosarcoma (OS). To understand this unexpected result, the presence of infiltrating immune cells was investigated in 124 pre-therapeutic biopsies of patients enrolled in the trial. The percentage of CD68/CD163 tumor-infiltrating macrophages (TAMs), CD8+ lymphocytes, osteoclasts, and the PD1/PDL-1 checkpoint were assessed by immunohistochemistry. M1/M2 macrophage polarization was characterized by pSTAT1/CMAF staining. The expression of these biomarkers was correlated with clinical outcome. No statistical correlations were found with response to chemotherapy. High CD163 levels (>50% of cells per core; 43.8% of patients) were associated with CMAF nuclear expression and significantly correlated with better overall survival (p = 0.0025) and longer metastasis progression-free survival (MPFS, p = 0.0315) independently of metastatic status (p = 0.002). Only a trend was observed for patients with high CD68-positive cells (p = 0.0582). CD8+ staining was positive in >50% of cases with a median staining of 1%. Lower CD8+ levels were associated with metastatic disease at diagnosis and the presence of CD8-positive cells significantly correlated with improved overall survival in zoledronate-treated patients (p = 0.0415). PD1/PDL-1 staining was negative in >80% of cases and was not correlated with outcome. Finally, CD163-positive TAMs and CD8 positive cells are crucial prognostic biomarkers in OS, whereas PD1/PDL-1 checkpoint plays a minor role. For the first time, we described a correlation between CD8 positive cells and survival in zoledronate-treated patients. The immunohistochemical analysis of the microenvironment in biopsies may represent a novel tool for therapeutic stratification.

Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases which is the main cause of death. Unfortunately, the conventional chemotherapy is not fully effective on osteosarcoma metastases. The progression of a primary tumor to metastasis requires multiple processes, which are neovascularization, proliferation, invasion, survival in the bloodstream, apoptosis resistance, arrest at a distant organ, and outgrowth in secondary sites. Consequently, recent studies have revealed new insights into the molecular mechanisms of metastasis development. The understanding of the mechanism of molecular alterations can provide the identification of novel therapeutic targets and/or prognostic markers for osteosarcoma treatment to improve the clinical outcome.

BACKGROUND: Osteosarcoma is the most common malignant primary bone tumour in young adult treated by neo adjuvant chemotherapy, surgical tumor removal and adjuvant multidrug chemotherapy. For correction of soft tissue defect consecutive to surgery and/or tumor treatment, autologous fat graft has been proposed in plastic and reconstructive surgery. PRINCIPAL FINDINGS: We report here a case of a late local recurrence of osteosarcoma which occurred 13 years after the initial pathology and 18 months after a lipofilling procedure. Because such recurrence was highly unexpected, we investigated the possible relationship of tumor growth with fat injections and with mesenchymal stem/stromal cell like cells which are largely found in fatty tissue. Results obtained in osteosarcoma pre-clinical models show that fat grafts or progenitor cells promoted tumor growth. SIGNIFICANCE: These observations and results raise the question of whether autologous fat grafting is a safe reconstructive procedure in a known post neoplasic context.