Identification of MCM4 and PRKDC as new regulators of osteosarcoma cell dormancy based on 3D cell cultures.

Dormancy is a potential way for tumors to develop drug resistance and escape treatment. However, the mechanisms involved in cancer dormancy remain poorly understood. This is mainly because there is no in vitro culture model making it possible to spontaneously induce dormancy. In this context, the present work proposes the use of three-dimensional (3D) spheroids developed from osteosarcoma cell lines as a relevant model for studying cancer dormancy. MNNG-HOS, SaOS-2, 143B, MG-63, U2OS and SJSA-1 cell lines were cultured in 3D using the Liquid Overlay Technique (LOT). Dormancy was studied by staining cancer cells with a lipophilic dye (DiD), and long-term DiD+ cells were considered as dormant cancer cells. The role of the extracellular matrix in inducing dormancy was investigated by embedding cells into methylcellulose or Geltrex™. Gene expression of DiD+ cells was assessed with a Nanostring™ approach and the role of the genes detected in dormancy was validated by a transient down-expression model using siRNA treatment. Proliferation was measured using fluorescence microscopy and the xCELLigence technology. We observed that MNNG-HOS, 143B and MG-G3 cell lines had a reduced proliferation rate in 3D compared to 2D. U2OS cells had an increased proliferation rate when they were cultured in Geltrex™ compared to other 3D culture methods. Using 3D cultures, a transcriptomic signature of dormancy was obtained and showed a decreased expression of 18 genes including ETV4, HELLS, ITGA6, MCM4, PRKDC, RAD21 and UBE2T. The treatment with siRNA targeting these genes showed that cancer cell proliferation was reduced when the expression of ETV4 and MCM4 were decreased, whereas proliferation was increased when the expression of RAD21 was decreased. 3D culture facilitates the maintenance of dormant cancer cells characterized by a reduced proliferation and less differential gene expression as compared to proliferative cells. Further studies of the genes involved has enabled us to envisage their role in regulating cell proliferation.

Roles of inflammatory cell infiltrate in periprosthetic osteolysis.

Classically, particle-induced periprosthetic osteolysis at the implant-bone interface has explained the aseptic loosening of joint replacement. This response is preceded by triggering both the innate and acquired immune response with subsequent activation of osteoclasts, the bone-resorbing cells. Although particle-induced periprosthetic osteolysis has been considered a foreign body chronic inflammation mediated by myelomonocytic-derived cells, current reports describe wide heterogeneous inflammatory cells infiltrating the periprosthetic tissues. This review aims to discuss the role of those non-myelomonocytic cells in periprosthetic tissues exposed to wear particles by showing original data. Specifically, we discuss the role of T cells (CD3+, CD4+, and CD8+) and B cells (CD20+) coexisting with CD68+/TRAP- multinucleated giant cells associated with both polyethylene and metallic particles infiltrating retrieved periprosthetic membranes. This review contributes valuable insight to support the complex cell and molecular mechanisms behind the aseptic loosening theories of orthopedic implants.

Technical report: liquid overlay technique allows the generation of homogeneous osteosarcoma, glioblastoma, lung and prostate adenocarcinoma spheroids that can be used for drug cytotoxicity measurements.

Introduction: The mechanisms involved in cancer initiation, progression, drug resistance, and disease recurrence are traditionally investigated through in vitro adherent monolayer (2D) cell models. However, solid malignant tumor growth is characterized by progression in three dimensions (3D), and an increasing amount of evidence suggests that 3D culture models, such as spheroids, are suitable for mimicking cancer development. The aim of this report was to reaffirm the relevance of simpler 3D culture methods to produce highly reproducible spheroids, especially in the context of drug cytotoxicity measurements. Methods: Human A549 lung adenocarcinoma, LnCaP prostate adenocarcinoma, MNNG/HOS osteosarcoma and U251 glioblastoma cell lines were grown into spheroids for 20 days using either Liquid Overlay Technique (LOT) or Hanging Drop (HD) in various culture plates. Their morphology was examined by microscopy. Sensitivity to doxorubicin was compared between MNNG/HOS cells grown in 2D and 3D. Results: For all cell lines studied, the morphology of spheroids generated in round-bottom multiwell plates was more repeatable than that of those generated in flat-bottom multiwell plates. HD had no significant advantage over LOT when the spheroids were cultured in round-bottom plates. Finally, the IC50 of doxorubicin on MNNG/HOS cultured in 3D was 18.8 times higher than in 2D cultures (3D IC50 = 15.07 ± 0.3 µM; 2D IC50 = 0.8 ± 0.4 µM; *p < 0.05). Discussion: In conclusion, we propose that the LOT method, despite and because of its simplicity, is a relevant 3D model for drug response measurements that could be scaled up for high throughput screening.

Advances in Osteosarcoma.

PURPOSE OF REVIEW: This article gives a brief overview of the most recent developments in osteosarcoma treatment, including targeting of signaling pathways, immune checkpoint inhibitors, drug delivery strategies as single or combined approaches, and the identification of new therapeutic targets to face this highly heterogeneous disease. RECENT FINDINGS: Osteosarcoma is one of the most common primary malignant bone tumors in children and young adults, with a high risk of bone and lung metastases and a 5-year survival rate around 70% in the absence of metastases and 30% if metastases are detected at the time of diagnosis. Despite the novel advances in neoadjuvant chemotherapy, the effective treatment for osteosarcoma has not improved in the last 4 decades. The emergence of immunotherapy has transformed the paradigm of treatment, focusing therapeutic strategies on the potential of immune checkpoint inhibitors. However, the most recent clinical trials show a slight improvement over the conventional polychemotherapy scheme. The tumor microenvironment plays a crucial role in the pathogenesis of osteosarcoma by controlling the tumor growth, the metastatic process and the drug resistance and paved the way of new therapeutic options that must be validated by accurate pre-clinical studies and clinical trials.

Three-dimensional in vitro culture models in oncology research.

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.

Lipopolysaccharide-binding protein expression is associated to the metastatic status of osteosarcoma patients.

Osteosarcoma (OS) is a rare malignant primary bone tumours characterized by a high genetic and cell composition heterogeneity. Unfortunately, despite the use of drug combinations and the recent development of immunotherapies, the overall survival has not improved in the last four decades. Due to the key role of the tumour microenvironment in the pathogenesis of OS, a better understanding of its microenvironment is mandatory to develop new therapeutic approaches. From retrospective biological cohorts of OS, we analysed by immunohistochemistry the presence of lipopolysaccharide (LPS)-binding protein (LBP) in diagnostic biopsies with local disease and compared their level of infiltration to patients suffering from metastatic status. LBP is considered as a marker of LPS exposure and can indirectly reflect the presence of Gram-negative microbiota. LBP were detected in the cytoplasm of OS cells as well as in tumour-associated macrophage. Tumour samples of patients with local disease were significantly enriched in LBP compared to tumour tissues of patients with metastatic status. Lung metastatic tissues showed similar level of LBP compared to paired primary tumours. Overall, this study strongly suggests the presence of Gram-negative bacteria in OS tissues and demonstrated their significant differential level according the metastatic status. This tumour-associated microbiome may help in the conceptualisation of new therapeutic approach to trigger efficient therapeutic responses against cancer.

Biological evidence of cancer stem-like cells and recurrent disease in osteosarcoma.

Sarcomas are a large family of cancers originating in the mesenchyme. Composed of more than 100 histological subtypes, soft tissue and bone sarcomas remain clinically challenging, particularly in children and adolescents in whom sarcomas are the second most common malignant entities. Osteosarcoma is the main primary bone tumor in adolescents and young adults and is characterized by a high propensity to induce distant metastatic foci and become multi-drug resistant. The innate and acquired resistance of osteosarcoma can be explained by high histological heterogeneity and genetic/molecular diversity. In the last decade, the notion of cancer stem-like cells (CSCs) has emerged. This subset of cancer cells has been linked to drug resistance properties, recurrence of the disease, and therapeutic failure. Although CSCs remain controversial, many elements are in favor of them playing a role in the development of the drug resistance profile. The present review gives a brief overview of the most recent biological evidence of the presence of CSCs in osteosarcomas and their role in the drug resistance profile of these rare oncological entities. Their use as promising therapeutic targets is discussed.

Inhibiting Endothelin Receptors with Macitentan Strengthens the Bone Protective Action of RANKL Inhibition and Reduces Metastatic Dissemination in Osteosarcoma.

Current treatments for osteosarcoma, combining conventional polychemotherapy and surgery, make it possible to attain a five-year survival rate of 70% in affected individuals. The presence of chemoresistance and metastases significantly shorten the patient's lifespan, making identification of new therapeutic tools essential. Inhibiting bone resorption has been shown to be an efficient adjuvant strategy impacting the metastatic dissemination of osteosarcoma, tumor growth, and associated bone destruction. Unfortunately, over-apposition of mineralized matrix by normal and tumoral osteoblasts was associated with this inhibition. Endothelin signaling is implicated in the functional differentiation of osteoblasts, raising the question of the potential value of inhibiting it alone, or in combination with bone resorption repression. Using mouse models of osteosarcoma, the impact of macitentan, an endothelin receptor inhibitor, was evaluated regarding tumor growth, metastatic dissemination, matrix over-apposition secondary to RANKL blockade, and safety when combined with chemotherapy. The results showed that macitentan has no impact on tumor growth or sensitivity to ifosfamide, but significantly reduces tumoral osteoid tissue formation and the metastatic capacity of the osteosarcoma. To conclude, macitentan appears to be a promising therapeutic adjuvant for osteosarcoma alone or associated with bone resorption inhibitors.

FVIII regulates the molecular profile of endothelial cells: functional impact on the blood barrier and macrophage behavior.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by deficient activity of blood coagulation factor VIII (FVIII). In addition, hemophilia patients show associated diseases including osteopenia, altered inflammation and vascular fragility which may represent the consequence of recurrent bleeding or may be related to the direct FVIII deficiency. Nowadays, recombinant FVIII is proposed to treat hemophilia patients with no circulating FVIII inhibitor. Initially described as a coenzyme to factor IXa for initiating thrombin generation, there is emerging evidence that FVIII is involved in multiple biological systems, including bone, vascular and immune systems. The present study investigated: (i) the functional activities of recombinant human FVIII (rFVIII) on endothelial cells, and (ii) the impact of rFVIII activities on the functional interactions of human monocytes and endothelial cells. We then investigated whether rFVIII had a direct effect on the adhesion of monocytes to the endothelium under physiological flow conditions. We observed that direct biological activities for rFVIII in endothelial cells were characterized by: (i) a decrease in endothelial cell adhesion to the underlying extracellular matrix; (ii) regulation of the transcriptomic and protein profiles of endothelial cells; (iii) an increase in the vascular tubes formed and vascular permeability in vitro; and (iv) an increase in monocyte adhesion activated endothelium and transendothelial migration. By regulating vascular permeability plus leukocyte adhesion and transendothelial migration, the present work highlights new biological functions for FVIII.

FVIII at the crossroad of coagulation, bone and immune biology: Emerging evidence of biological activities beyond hemostasis.

Hemophilia A is an X-linked hereditary disorder that results from deficient coagulation factor VIII (FVIII) activity, leading to spontaneous bleeding episodes, particularly in joints and muscles. FVIII deficiency has been associated with altered bone remodeling, dysregulated macrophage polarization, and inflammatory processes that are associated with the neoformation of abnormal blood vessels. Treatment based on FVIII replacement can lead to the development of inhibitors that render FVIII concentrate infusion ineffective. In this context, hemophilia has entered a new therapeutic era with the development of new drugs, such as emicizumab, that seek to restore the hemostatic balance by bypassing pathologically acquired antibodies. We discuss the potential extrahemostatic functions of FVIII that may be crucial for defining future therapies in hemophilia.

The intratumoral microbiome: Characterization methods and functional impact.

Live-pathogenic bacteria, which were identified inside tumors hundreds year ago, are key elements in modern cancer research. As they have a relatively accessible genome, they offer a multitude of metabolic engineering opportunities, useful in several clinical fields. Better understanding of the tumor microenvironment and its associated microbiome would help conceptualize new metabolically engineered species, triggering efficient therapeutic responses against cancer. Unfortunately, given the low microbial biomass nature of tumors, characterizing the tumor microbiome remains a challenge. Tumors have a high host versus bacterial DNA ratio, making it extremely complex to identify tumor-associated bacteria. Nevertheless, with the improvements in next-generation analytic tools, recent studies demonstrated the existence of intratumor bacteria inside defined tumors. It is now proven that each cancer subtype has a unique microbiome, characterized by bacterial communities with specific metabolic functions. This review provides a brief overview of the main approaches used to characterize the tumor microbiome, and of the recently proposed functions of intracellular bacteria identified in oncological entities. The therapeutic aspects of live-pathogenic microbes are also discussed, regarding the tumor microenvironment of each cancer type.

In vitro three-dimensional cell cultures for bone sarcomas.

Bone sarcomas are rare tumour entities that arise from the mesenchyme most of which are highly heterogeneous at the cellular, genetic and epigenetic levels. The three main types are osteosarcoma, Ewing sarcoma, and chondrosarcoma. These oncological entities are characterised by high morbidity and mortality and an absence of significant therapeutic improvement in the last four decades. In the field of oncology, in vitro cultures of cancer cells have been extensively used for drug screening unfortunately with limited success. Indeed, despite the massive knowledge acquired from conventional 2D culture methods, scientific community has been challenged by the loss of efficacy of drugs when moved to clinical trials. The recent explosion of new 3D culture methods is paving the way to more relevant in vitro models mimicking the in vivo tumour environment (e.g. bone structure) with biological responses close to the in vivo context. The present review gives a brief overview of the latest advances of the 3D culture methods used for studying primary bone sarcomas.

Case Report: Grade 2 Metastatic Pancreatic Neuroendocrine Tumor With Progression of One Metastasis After Pregnancy to Grade 3 Large-Cell Neuroendocrine Carcinoma: One Case Cured by Resection With Genomic Characterization of the Two Components.

Temporal and spatial tumor heterogeneity can be observed in pancreatic neuroendocrine tumor. We report the case of a young woman with long term stabilization of a G2 metastatic pancreatic NET that, after pregnancy, suddenly progressed into one single liver metastasis corresponding to a transformation into G3 large-cell neuroendocrine cancer. The patient underwent liver resection (the progressive and one dormant metastasis). With a 45 months follow-up the patient is without evolutive disease. Exome sequencing of the two metastases revealed completely different genomic signatures and gene alterations: the dormant metastasis was MSS without any gene alteration; the poorly differentiated tumor was MSI, with gain of many mutations including MEN1, BCL2, MLH1 and TP53 corresponding to a mutational signature 11. Could temozolomide play a role in this transformation?

Case Report: Two Cases of Metastatic Pancreatoblastoma in Adults: Efficacy of Folfirinox and Implication of the Wnt/ß-Catenin Pathway in Genomic Analysis.

Pancreatoblastomas are unfrequent tumors usually found in children. We report two cases of metastatic pancreatoblastomas observed in young women. A systemic chemotherapy (FOLFIRINOX regimen) was associated with a disease control in one case and a partial response in the second with an improvement of general status for both. A high-throughput sequencing of the tumor described in both cases alteration in the Wnt/ß-catenin pathway: a mutation in CTNNB1 (exon 3, c.110C>G, p.S37C, reported as a hotspot in COSMIC) in one case and a homozygous loss associated with breakage targeting APC (5q22.2) in the second.

The twin cytokines interleukin-34 and CSF-1: masterful conductors of macrophage homeostasis.

Macrophages are specialized cells that control tissue homeostasis. They include non-resident and tissue-resident macrophage populations which are characterized by the expression of particular cell surface markers and the secretion of molecules with a wide range of biological functions. The differentiation and polarization of macrophages relies on specific growth factors and their receptors. Macrophage-colony stimulating factor (CSF-1) and interleukine-34 (IL-34), also known as "twin" cytokines, are part of this regluatory landscape. CSF-1 and IL-34 share a common receptor, the macrophage-colony stimulating factor receptor (CSF-1R), which is activated in a similar way by both factors and turns on identical signaling pathways. However, there is some discrete differential activation leading to specific activities. In this review, we disscuss recent progress in understanding of the role of the twin cytokines in macrophage differentiation, from their interaction with CSF-1R and the activation of signaling pathways, to their implication in macrophage polarization of non-resident and tissue-resident macrophages. A special focus on IL-34, its involvement in pathophsyiological contexts, and its potential as a theranostic target for macrophage therapy will be proposed.

Bone sarcomas in the immunotherapy era.

Bone sarcomas are primary bone tumours found mainly in children and adolescents, as osteosarcoma and Ewing's sarcoma, and in adults in their 40s as chondrosarcoma. The last four decades the development of therapeutic approaches was based on drug combinations have shown no real improvement in overall survival. Recently oncoimmunology has allowed a better understand of the crucial role played by the immune system in the oncologic process. This led to clinical trials with the aim of reprogramming the immune system to facilitate cancer cell recognition. Immune infiltrates of bone sarcomas have been characterized and their molecular profiling identified as immune therapeutic targets. Unfortunately, the clinical responses in trials remain anecdotal but highlight the necessity to improve the characterization of tumour micro-environment to unlock the immunotherapeutic response, especially in their paediatric forms. Bone sarcomas have entered the immunotherapy era and here we overview the recent developments in immunotherapies in these sarcomas. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Bisphosphonates in common pediatric and adult bone sarcomas.

The therapeutic strategies proposed currently for bone sarcomas are based on neo-adjuvant chemotherapy, delayed en-bloc wide resection, and adjuvant chemotherapy. Unfortunately, bone sarcomas are characterized by high rates of poor drug response, with a high risk of drug resistance, local recurrence and/or a high propensity for induced metastases. The pathogenesis of bone sarcomas is strongly associated with dysregulation of local bone remodeling and increased osteolysis that plays a part in tumor development. In this context, bisphosphonates (BPs) have been proposed as a single agent or in combination with conventional drugs to block bone resorption and the vicious cycle established between bone and sarcoma cells. Pre-clinical in vitro studies revealed the potential "anti-tumor" activities of nitrogen-bisphosphonates (N-BPs). In pre-clinical models, N-BPs reduced significantly primary tumor growth in osteosarcoma and Ewing sarcoma, and the installation of lung metastases. In chondrosarcoma, N-BPs reduced the recurrence of local tumors after intralesional curettage, and increased overall survival. In pediatric and adult osteosarcoma patients, N-BPs have been assessed in combination with conventional chemotherapy and surgery in randomized phase 3 studies with no improvement in clinical outcome. The lack of benefit may potentially be explained by the biological impact of N-BPs on macrophage differentiation/recruitment which may alter CD8+-T lymphocyte infiltration. Thanks to their considerable affinity for the mineralized extracellular matrix, BPs are an excellent platform for drug delivery in malignant bone sites with reduced systemic toxicity, which opens up new opportunities for their future use.

Circulating Tumor Cells as a Tool for Assessing Tumor Heterogeneity.

Tumor heterogeneity is the major cause of failure in cancer prognosis and prediction. Accurately detecting heterogeneity for the development of biomarkers and the detection of the clones resistant to therapy is one of the main goals of contemporary medicine. Metastases belong to the natural history of cancer. The present review gives an overview on the origin of tumor heterogeneity. Recent progress has made it possible to isolate and characterize circulating tumor cells (CTCs), which are the drivers of the disease between the primary sites and metastatic foci. The most recent methods for characterizing CTCs are summarized and we discuss the power of CTC profiling for analyzing tumor heterogeneity in early and advanced diseases.

The Heterogeneity of Osteosarcoma: The Role Played by Cancer Stem Cells.

Osteosarcoma is the most common bone sarcoma and is one of the cancer entities characterized by the highest level of heterogeneity in humans. This heterogeneity takes place not only at the macroscopic and microscopic levels, with heterogeneous micro-environmental components, but also at the genomic, transcriptomic and epigenetic levels. Recent investigations have revealed the existence in osteosarcoma of cancer cells with stemness properties. Cancer stem cells are characterized by their specific phenotype and low cycling capacity, and are linked to drug resistance, tumour growth and the metastatic process. In addition, cancer stem cells contribute to the enrichment of tumour heterogeneity. The present manuscript will describe the main characteristic features of cancer stem cells in osteosarcoma and will discuss their impact on maintaining tumour heterogeneity. Their clinical implications will also be briefly addressed.

Survival and prognostic factors analysis of 151 intestinal and pancreatic neuroendocrine tumors: a single center experience.

BACKGROUND: Intestinal and pancreatic neuroendocrine tumors (IP-NETs) are rare tumors with heterogeneous outcomes. The aim of our study was to determine the clinical, therapeutic and pathological factors which impact the overall survival (OS) in IP-NETs. METHODS: All the patients diagnosed with IP-NETs at the Nantes University Hospital between October 1994 and October 2013 were retrospectively analysed. Patients with MEN-1 (Type 1 Multiple Endocrine Neoplasia) or Von Hippel-Lindau syndrome were excluded. Additionally, a prospective analysis of tumor grade (mitotic index and Ki67 index) was performed on tumor samples. OS was evaluated by Kaplan-Meier method and prognostic factors by log-rank test and Cox model. RESULTS: The study included 151 patients. Median age was 60 (range, 14-81). Primary tumor was pancreatic in 86 patients (56.95%) and intestinal in 65 patients (43.05%). Tumors were metastatic (synchronous or metachronous) in 72 patients (47.7%). The median OS was 157 months. For all IP-NETs, age >65 years (P<0.0001), Ki67 >5% (P=0.03), synchronous metastases (P=0.016), primary tumor size >25 mm (P=0.03) and emergency surgery (P=0.007) were independent poor prognostic factors. CONCLUSIONS: In this large series of patients with IP-NET, age >65 years, Ki67 >5%, primary tumor size >25 mm, synchronous metastases and emergency surgery for acute complications have been identified as independent poor prognostic factors.