Anne Gomez-Brouchet

Hypoxia Induces VEGF-C Expression in Metastatic Tumor Cells via a HIF-1α-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 ofxa0hypoxia-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.

Osteoblast-derived sphingosine 1-phosphate to induce proliferation and confer resistance to therapeutics to bone metastasis-derived prostate cancer cells

Sphingosine 1‐phosphate (S1P) plays important roles in cell proliferation, differentiation or survival mainly through its surface G‐protein‐coupled receptors S1P1−5. Bone represents the major site of metastasis for prostate cancer (CaP) cells, which rely on bone‐derived factors to support their proliferation and resistance to therapeutics.

CD163-positive tumor-associated macrophages and CD8-positive cytotoxic lymphocytes are powerful diagnostic markers for the therapeutic stratification of osteosarcoma patients: An immunohistochemical analysis of the biopsies fromthe French OS2006 phase 3 trial

ABSTRACT 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.

Beneficial effects of levobupivacaine regional anaesthesia on postoperative opioid induced hyperalgesia in diabetic mice

BackgroundDiabetic neuropathy is one of the most common complications of diabetes and causes various problems in daily life. The aim of this study was to assess the effect of regional anaesthesia on post surgery opioid induced hyperalgesia in diabetic and non-diabetic mice.MethodsDiabetic and non-diabetic mice underwent plantar surgery. Levobupivacaine and sufentanil were used before surgery, for sciatic nerve block (regional anaesthesia) and analgesia, respectively. Diabetic and non-diabetic groups were each randomly assigned to three subgroups: control, no sufentanil and no levobupivacaine; sufentanil and no levobupivacaine; sufentanil and levobupivacaine. Three tests were used to assess pain behaviour: mechanical nociception; thermal nociception and guarding behaviours using a pain scale.ResultsSufentanil, alone or in combination with levobupivacaine, produced antinociceptive effects shortly after administration. Subsequently, sufentanil induced hyperalgesia in diabetic and non-diabetic mice. Opioid-induced hyperalgesia was enhanced in diabetic mice. Levobupivacaine associated to sufentanil completely prevented hyperalgesia in both groups of mice.ConclusionThe results suggest that regional anaesthesia can decrease opioid-induced hyperalgesia in diabetic as well as in non-diabetic mice. These observations may be clinically relevant for the management of diabetic patients.

Remodelling of vertebral endplate subchondral bone in scoliosis: A micro-CT analysis in a porcine model

BACKGROUNDnDisc degeneration has been correlated with alteration of bone density of adjacent vertebral bodies. Abnormal mechanical loading appears in scoliosis as compared to normal spines. How vertebral endplate was remodelled in scoliosis is not well understood.nnnMETHODSnWe conducted a micro-CT analysis of subchondral bone of the vertebral endplate at the curve apex in a porcine scoliosis model. Two adjacent thoracic T(5)-T(6) and lumbar L(1)-L(2) levels were instrumented in six four-week-old pigs with a custom offset implant connected by a flexible stainless steel wire. Two months after implantation, three cylindrical specimens were harvested into the vertebral endplate of each of the scoliosis levels: centre, convexity and concavity, and from the dorsal T(9)-T(10) vertebral units obtained from nine three-month-old non-instrumented pigs used as controls. Micro-CT analysis was carried out on each specimen.nnnFINDINGSnIn the concavity of the scoliotic spine, bone volume fraction, trabecular thickness, and trabecular separation significantly increased whereas in the convexity, only trabecular separation increased. Connectivity index and trabecular number decreased significantly.nnnINTERPRETATIONnThis was the first micro-CT study of subchondral bone microarchitecture of the scoliotic vertebral end plate. At the curve apex, increased compression in the concavity induced an osteogenic process. In the convexity, diminished compression caused an osteolytic process with a local resorption. Clinically, the unbalanced tissue remodelling could play a role in the convective and diffusive transports into the end plate, which is of prime importance for the segment homeostasis in scoliosis treatment with or without surgery.

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

BACKGROUNDnAbnormal 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.nnnMETHODSnFlexible 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.nnnFINDINGSnA three-dimensional deformity was obtained in all six animals with an average of 42° right thoracic curve, 44° lordosis and 21° 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.nnnINTERPRETATIONnThe 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.

Establishment and characterization of in vivo orthotopic bioluminescent xenograft models from human osteosarcoma cell lines in Swiss nude and NSG mice

Osteosarcoma is one of the most common primary bone tumors in childhood and adolescence. Metastases occurrence at diagnosis or during disease evolution is the main therapeutic challenge. New drug evaluation to improve patient survival requires the development of various preclinical models mimicking at best the complexity of the disease and its metastatic potential. We describe here the development and characteristics of two orthotopic bioluminescent (Luc/mKate2) cell‐derived xenograft (CDX) models, Saos‐2‐B‐Luc/mKate2‐CDX and HOS‐Luc/mKate2‐CDX, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system allowed both longitudinal computed tomography (CT) and bioluminescence real‐time follow‐up of primary tumor growth and metastatic spread, which was confirmed by histology. The murine immune context influenced tumor engraftment, primary tumor growth, and metastatic spread to lungs, bone, and spleen (an unusual localization in humans). Engraftment in NSG mice was found superior to that found in nude mice and intratibial bone environment more favorable to engraftment compared to paratibial injection. The genetic background of the two CDX models also led to distinct primary tumor behavior observed on CT scan. Saos‐2‐B‐Luc/mKate2‐CDX showed osteocondensed, HOS‐Luc/mKate2‐CDX osteolytic morphology. Bioluminescence defined a faster growth of the primary tumor and metastases in Saos‐2‐B‐Luc/mKate2‐CDX than in HOS‐Luc/mKate2‐CDX. The early detection of primary tumor growth and metastatic spread by bioluminescence allows an improved exploration of osteosarcoma disease at tumor progression, and metastatic spread, as well as the evaluations of anticancer treatments. Our orthotopic models with metastatic spread bring complementary information to other types of existing osteosarcoma models.

EWSR1-PATZ1 gene fusion may define a new glioneuronal tumor entity: EWSR1-PATZ1 gene fusion

We investigated the challenging diagnostic case of a ventricular cystic glioneuronal tumor with papillary features, by RNA sequencing using the Illumina TruSight RNA Fusion panel. We did not retrieve the SLC44A1‐PRKCA fusion gene specific for papillary glioneuronal tumor, but an EWSR1‐PATZ1 fusion transcript. RT‐PCR followed by Sanger sequencing confirmed the EWSR1‐PATZ1 fusion. It matched with canonic EWSR1 fusion oncogene, juxtaposing the entire N‐terminal transcriptional activation domain of EWSR1 gene and the C‐terminal DNA binding domain of a transcription factor gene, PATZ1. PATZ1 protein belongs to the BTB‐ZF (broad‐complex, tramtrack and bric‐à‐brac ‐zinc finger) family. It directly regulates Pou5f1 and Nanog and is essential to maintaining stemness by inhibiting neural differentiation. EWSR1‐PATZ1 fusion is a rare event in tumors: it was only reported in six round cell sarcomas and in three gliomas of three exclusively molecular studies. The first reported glioma was a BRAFV600E negative ganglioglioma, the second a BRAFV600E negative glioneuronal tumor, not otherwise specified and the third, very recently reported, a high grade glioma, not otherwise specified. In our study, forty BRAFV600E negative gangliogliomas were screened by FISH using EWSR1 break‐apart probes. We performed methylation profiling for the index case and for seven out of the ten FISH positive cases. The index case clustered apart from other pediatric low grade glioneuronal entities, and specifically from the well‐defined ganglioglioma methylation group. An additional pediatric intraventricular ganglioglioma clustered slightly more closely with ganglioglioma, but showed differences from the main ganglioglioma group and similarities with the index case. Both cases harbored copy number variations at the PATZ1 locus. EWSR1‐PATZ1 gene fusion might define a new type of glioneuronal tumors, distinct from gangliogliomas.

Exostosis osteocartilaginosa solitaria y múltiple

El punto comun de la exostosis osteocartilaginosa solitaria y la exostosis multiple es la expresion tumoral uniforme caracterizada por un doble componente, oseo y cartilaginoso, que evoluciona a lo largo del crecimiento. Ambas pertenecen al ambito de la cirugia ortopedica pediatrica y del adulto. En su forma unica, la exostosis osteocartilaginosa u osteocondroma tiene una semiologia clinica y radiologica tipica que permite formular un diagnostico de certeza sin biopsia previa: tumor de superficie en estricta continuidad radiologica con la cortical metafisaria (de donde proviene) con inclusion de un capuchon cartilaginoso. Rara vez plantea dificultades de diagnostico diferencial con un osteosarcoma paraoseo. El riesgo de degeneracion es excepcional en la forma solitaria, en la cual la exeresis esta indicada en las formas sintomaticas, en especial compresivas y localizadas en el femur proximal, el omoplato y la pelvis. La exostosis multiple es infrecuente, secundaria a tres mutaciones cromosomicas de transmision autosomica dominante. En dos tercios de los casos hay un antecedente familiar que permite sospechar el diagnostico en un recien nacido de padres afectados. La aparicion esporadica es posible y obedece a una mutacion de novo. La exostosis multiple se caracteriza por una gran heterogeneidad clinica en relacion con el numero y el volumen de las unidades tumorales y con su diseminacion. Las localizaciones mas frecuentes, con repercusion sobre el crecimiento, son la muneca, la rodilla y el tobillo. Las indicaciones terapeuticas respecto a estas localizaciones se han precisado mejor para prevenir las deformaciones ortopedicas, cuya repercusion funcional y social no es nada desdenable. El riesgo de degeneracion condrosarcomatosa en el adulto es claramente mas elevado, sobre todo en las formas graves y en las cinturas escapular y pelvica. Los progresos de la genetica permitiran la identificacion precoz de las formas graves en el plano funcional y las de riesgo oncologico.