Influence of growth modulation on the effective permeability of the vertebral end plate. A porcine experimental scoliosis model

Background: Abnormal mechanical loading occurs in scoliosis as compared to normal spines. Intervertebral disc degeneration has been correlated with alteration of bone density in adjacent vertebral bodies. How vertebral end plate remodels in scoliosis and the consequences on disc homeostasis are not well understood. Permeability is a relevant physical measure to quantify mass transport in porous media. We hypothesized that effective permeability of the vertebral end plate was modified by growth modulation in a scoliosis animal model. Methods: Flexible asymmetric posterior instrumentation was undertaken on six healthy four-week-old pigs. Two sets of left pedicle screws were inserted and connected with a stainless steel cable. After two months, the apical intervertebral unit and three units located cranially and caudally, were harvested. One central and two lateral specimens were investigated using a previously validated method for measuring permeability. Findings: A three-dimensional deformity was obtained in all six animals with an average of 42 degrees right thoracic curve. 44 degrees lordosis and 21 degrees rotation. Permeability was significantly greater in the center of the end plates than in the periphery and it was decreased by -45% towards the apex of the deformity. Fluid flow direction did not play a significant role. No significant difference was found between the convex side and the concave side. Interpretation: The end plate is a crucial zone for diffusive and convective transport and we showed in a scoliosis animal model that a growth modulation may decrease its effective permeability. The proposed methodology and associated results could help to understand degenerative changes in human spine

Whole-exome sequencing in osteosarcoma reveals important heterogeneity of genetic alterations

BACKGROUND: Whole-genome sequencing studies have recently shown that osteosarcomas (OSs) display high rates of structural variation, i.e. they contain many somatic mutations and copy number alterations. TP53 and RB1 show recurrent somatic alterations in concordant studies, suggesting that they could be key players in bone oncogenesis. PATIENTS AND METHODS: we carried out whole-genome sequencing of DNA from seven high-grade OS samples matched with normal tissue from the same patients. RESULTS: We confirmed the presence of genetic alterations of the TP53 (including novel unreported mutations) and RB1 genes. Most interestingly, we identified a total of 84 point mutations and 4 deletions related to 82 different genes in OS samples, of which only 15 have been previously reported. Interestingly, the number of mutated genes (ranging from 4 to 8) was lower in TP53mut cases compared with TP53wt cases (ranging from 14 to 45). This was also true for the mutated RB1 case. We also observed that a dedifferentiated OS harboring MDM2 amplification did not carry any other mutations. CONCLUSION: This study suggests that bone oncogenesis driven by TP53 or RB1 mutations occurs on a background of relative genetic stability and that the dedifferentiated OS subtype represents a clinico-pathological entity with distinct oncogenic mechanisms and thus requires different therapeutic managemen

Quantification of bone tissue heterogeneity and cell distributionpatterns from digital histology: application to osteosarcoma

Like most sarcomas with complex genomics,or more generally bone tissues, osteosarcoma isa type of tumors exhibiting a strong spatialheterogeneity of the micro-environment. Thisheterogeneity makes the diagnostic complex andcan induce strong spatial variability in theresponse to treatments. New researchstrategies are consequently needed tounderstand the impact of spatial heterogeneity onthe diagnostic accuracy and on the treatmentefficiency, and more generally to understand thelinks between tissue scale bone matrix structuresand underlying biology occurring at the cell scale.The aim of this interdisciplinary work is to obtain the quantification of correlations between clinicaldata, heterogeneity of bone tissues and mechanobiological parameters. To this purpose, original numerical developments were initiated in our group to study the intratumoral and healthy bone tissue heterogeneity from histological and immunohistological sections. The code aimed at obtaining quantitative metrics of the cell population distribution, of the bone matrix micro-architecture (porosity) and of the transport properties (such as effective diffusivity). Because tissues exhibit naturally a complex spatial scales cascade, it can be modeled, at the tissue scale, as a three phases porous medium (fluid, solid, cell populations). Using methodologies related to porous media analysis, characteristic lengths were extracted and correlations of phenomena occurring cell and tissue scale examined. Further developments permitted the calculation of effective mechanical properties. The methodology used successive algorithms of machine learning for the histological image segmentation and a combination of iterative algorithms and filters for the correlation calculations. Results put forward the strength of this approach for the identification of new markers in the study of pathological bone tissues

Upscaling of fluid flow in spatially heterogeneous bone tumors

Osteosarcoma is a malignant bone tumor that preferentially arises in adolescents and young adults. Like many sarcomas with complex genomics, this type of tumors exhibits strong spatial heterogeneities in terms of micro-architecture or differentiated response to treatments due to localized effect of chemotherapy. Clinical images at a tissue scale such as histological and immunohistological sections, exhibit three phases: fluid, solid, cells populations. Therefore the tumor can be considered as a porous medium. The objective of this work was to develop a mechanical approach based on upscaling methods to study the interstitial flow within the tumor at the tissue scale. The statistical study of the micro-architecture of the media shows that the identification of characteristic lengths is complex and that a separation of spatial scales is not necessarily identified. We therefore chose a special sequential upscaling technique, named Grid-Block approach to solve this problem

Planning for Bone Excision in Ewing Sarcoma: Post-Chemotherapy MRI More Accurate Than Pre-Chemotherapy MRI Assessment

International audienceBACKGROUND: In determining the level of bone resection in Ewing sarcoma, the most suitable time at which to perform magnetic resonance imaging (MRI) remains controversial. Current guidelines recommend that surgical planning be based on MRI performed prior to neoadjuvant chemotherapy. The goal of this study was to determine whether pre-chemotherapy or post-chemotherapy MRI provides greater accuracy of tumor limits for planning bone excision in the management of Ewing sarcoma.METHODS: This was a single-center, retrospective study. MRI was performed using 3 sequences: T1-weighted, T1-weighted with contrast enhancement by gadolinium injection, and a fluid-sensitive sequence (STIR [short tau inversion recovery] or proton-density-weighted with fat saturation). The tumor extent as assessed on pre-chemotherapy and post-chemotherapy MRI was compared with histological measurement of the resected specimen.RESULTS: Twenty patients with Ewing sarcoma of a long bone were included. In 6 cases, the tumor was located on the femur, in 5, the tibia; in 5, the fibula; and in 4, the humerus. The median patient age at diagnosis was 9.7 years. We found greater accuracy of measurements from MRI scans acquired after chemotherapy than from those acquired before chemotherapy. For both pre-chemotherapy and post-chemotherapy MRI, the greatest accuracy was achieved with the nonenhanced T1 sequence. There was no benefit to gadolinium enhancement. The median difference between T1 MRI and histological measurements was 19.0 mm (interquartile range [IQR], 4.3 to 32.8 mm) before chemotherapy and 5.0 mm (IQR, 2.0 to 13.0 mm) after chemotherapy. Adding a minimum margin of 20 mm to the limit of the tumor on post-chemotherapy T1 MRI always led to safe histological margin.CONCLUSIONS: Post-chemotherapy MRI provided a more accurate assessment of the limits of Ewing sarcoma. Surgical planning can therefore be based on post-chemotherapy MRI. Surgical cuts can be, at minimum, 20 mm from the limits as seen on MRI

Hypoxia Induces VEGF-C Expression in Metastatic Tumor Cells via a HIF-1α-Independent Translation-Mediated Mechanism

SummaryVarious tumors metastasize via lymph vessels and lymph nodes to distant organs. Even though tumors are hypoxic, the mechanisms of how hypoxia regulates lymphangiogenesis remain poorly characterized. Here, we show that hypoxia reduced vascular endothelial growth factor C (VEGF-C) transcription and cap-dependent translation via the upregulation of hypophosphorylated 4E-binding protein 1 (4E-BP1). However, initiation of VEGF-C translation was induced by hypoxia through an internal ribosome entry site (IRES)-dependent mechanism. IRES-dependent VEGF-C translation was independent of hypoxia-inducible factor 1α (HIF-1α) signaling. Notably, the VEGF-C IRES activity was higher in metastasizing tumor cells in lymph nodes than in primary tumors, most likely because lymph vessels in these lymph nodes were severely hypoxic. Overall, this transcription-independent but translation-dependent upregulation of VEGF-C in hypoxia stimulates lymphangiogenesis in tumors and lymph nodes and may contribute to lymphatic metastasis

Hypoxia Induces VEGF-C Expression in Metastatic Tumor Cells via a HIF-1 α-Independent Translation-Mediated Mechanism.

Various tumors metastasize via lymph vessels and lymph nodes to distant organs. Even though tumors are hypoxic, the mechanisms of how hypoxia regulates lymphangiogenesis remain poorly characterized. Here, we show that hypoxia reduced vascular endothelial growth factor C (VEGF-C) transcription and cap-dependent translation via the upregulation of hypophosphorylated 4E-binding protein 1 (4E-BP1). However, initiation of VEGF-C translation was induced by hypoxia through an internal ribosome entry site (IRES)-dependent mechanism. IRES-dependent VEGF-C translation was independent of hypoxia-inducible factor 1α (HIF-1α) signaling. Notably, the VEGF-C IRES activity was higher in metastasizing tumor cells in lymph nodes than in primary tumors, most likely because lymph vessels in these lymph nodes were severely hypoxic. Overall, this transcription-independent but translation-dependent upregulation of VEGF-C in hypoxia stimulates lymphangiogenesis in tumors and lymph nodes and may contribute to lymphatic metastasis

Characterization of Macrophages and Osteoclasts in the Osteosarcoma Tumor Microenvironment at Diagnosis: New Perspective for Osteosarcoma Treatment?

Biological and histopathological techniques identified osteoclasts and macrophages as targets of zoledronic acid (ZA), a therapeutic agent that was detrimental for patients in the French OS2006 trial. Conventional and multiplex immunohistochemistry of microenvironmental and OS cells were performed on biopsies of 124 OS2006 patients and 17 surgical (“OSNew”) biopsies respectively. CSF-1R (common osteoclast/macrophage progenitor) and TRAP (osteoclast activity) levels in serum of 108 patients were correlated to response to chemotherapy and to prognosis. TRAP levels at surgery and at the end of the protocol were significantly lower in ZA+ than ZA− patients (padj = 0.0011; 0.0132). For ZA+-patients, an increase in the CSF-1R level between diagnosis and surgery and a high TRAP level in the serum at biopsy were associated with a better response to chemotherapy (p = 0.0091; p = 0.0251). At diagnosis, high CD163+ was associated with good prognosis, while low TRAP activity was associated with better overall survival in ZA− patients only. Multiplex immunohistochemistry demonstrated remarkable bipotent CD68+/CD163+ macrophages, homogeneously distributed throughout OS regions, aside osteoclasts (CD68+/CD163−) mostly residing in osteolytic territories and osteoid-matrix-associated CD68−/CD163+ macrophages. We demonstrate that ZA not only acts on harmful osteoclasts but also on protective macrophages, and hypothesize that the bipotent CD68+/CD163+ macrophages might present novel therapeutic targets

Sphingosine Kinase-1 Is Overexpressed and Correlates with Hypoxia in Osteosarcoma: Relationship with Clinicopathological Parameters

The Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway is overexpressed in various cancers, and is instrumental for the adaptation to hypoxia in a number of solid tumor models, but no data are available in osteosarcoma. Here we report that SphK1 and the S1P1 receptor are involved in HIF-1α accumulation in hypoxic osteosarcoma cells. FTY720 (Fingolimod), which targets SphK1 and S1P1, prevented HIF-1α accumulation, and also inhibited cell proliferation in both normoxia and hypoxia unlike conventional chemotherapy. In human biopsies, a significant increase of SphK1 activity was observed in cancer compared with normal bones. In all sets of TMA samples (130 cases of osteosarcoma), immunohistochemical analysis showed the hypoxic marker GLUT-1, SphK1 and S1P1 were expressed in tumors. SphK1 correlated with the GLUT-1 suggesting that SphK1 is overexpressed and correlates with intratumoral hypoxia. No correlation was found between GLUT-1 or SphK1 and response to chemotherapy, but a statistical difference was found with increased S1P1 expression in patients with poor response in long bone osteosarcomas. Importantly, multivariate analyses showed that GLUT-1 was associated with an increased risk of death in flat bone, whereas SphK1 and S1P1 were associated with an increased risk of death in long bones

Expression des voies de signalisation impliquées dans la résistance aux traitements anticancéreux et la dissémination métastatique dans les ostéosarcomes de haut grade

Malgré les progrès considérables obtenus par la polychimiothérapie dans la prise en charge des ostéosarcomes (OS), un nombre important de patients, le plus souvent adolescents ne répond pas au traitement. A ce jour, seul le grade de Huvos et Rosen constitue le facteur pronostique majeur des OS. Nous avons étudié dans une cohorte de 41 patients l'expression tumorale, avant traitement, de protéines impliquées dans les voies de résistance aux traitements anticancéreux et la survie : intégrine b1, b3, b5, ILK, FAK, RhoB, MMP-9, Ang-2 et GSK-3 b et b-caténine. Notre étude montre qu'une plus forte expression de GSK-3 b était associée à une plus mauvaise réponse au traitement et qu'une plus forte expression de FAK était à l'inverse associée à une meilleure réponse au traitement. Nous avons aussi observé qu'une plus forte expression de ILK était corrélée à une plus mauvaise survie. Ainsi, l'étude de l'expression du phénotype tumoral GSK-3 b /FAK, corrélé à la chimiorésistance et l'étude de l'expression de ILK potentiellement impliquée dans la survie, devrait nous permettre d'adapter les protocoles thérapeutiques des OS.TOULOUSE3-BU Santé-Centrale (315552105) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF