P75 neurotrophin receptor is sequestered in the Golgi apparatus of the U-87 MG human glioblastoma cell line.

International audienceThe P75 neurotrophin receptor (p75NTR) is a cell surface receptor that can induce apoptosis in many cell types. This receptor plays a major role in the development of the central nervous system and is expressed in some adult brain cells. Its implication in cell apoptosis or survival is probably of major importance in cellular homeostasis and thus p75NTR could be implicated in tumor resistance to death. In this study, we investigated the intracellular expression of p75NTR in a human glioblastoma cell line. Detection of p75NTR receptor in Golgi apparatus by immunofluorescence microscopy, or after Golgi apparatus extraction, could be correlated with a decrease of cell apoptosis leading cells to become tumorous. This hypothesis is supported by a loss of ligand-induced apoptosis in this cell line. Our observations show that p75NTR can be sequestered in the Golgi complex and could then be, in part, responsible for the cell resistance to apoptosis and for brain tumor formation

Decrease in Fas-induced apoptosis by the γ-secretase inhibitor is dependent on p75(NTR) in a glioblastoma cell line.

International audiencep75(NTR), a member of the tumor necrosis factor superfamily, plays a key role in numerous physiological processes, including cell survival or apoptosis. Yet, the associated signaling pathways remain poorly understood. Similar to Notch, γ-secretase cleavage is implicated in the p75(NTR) signaling pathway leading to nuclear translocation of the intracellular domain and cell death. Fas receptor activation was found to promote cell death apoptosis in several cell lines. The goal of this study was to determine the respective role of p75(NTR) and Notch in the resistance to Fas-induced apoptosis in the U-87 MG glioblastoma cell line. Using the γ-secretase inhibitor, we investigated the modulation of Fas-induced apoptosis dependent on p75(NTR)-Fas receptor interaction. Whereas the U-87 MG cells expressed the Fas receptor at the cell membrane, apoptosis induced by Fas activation was decreased by the γ-secretase inhibitor. These data suggest that γ-secretase is implicated in p75(NTR) and Fas interaction leading to cell death signaling

EGFR Soluble Isoforms and Their Transcripts Are Expressed in Meningiomas

The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%.Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types

Targeted Sub-Attomole Cancer Biomarker Detection Based on Phase Singularity 2D Nanomaterial-Enhanced Plasmonic Biosensor

Detection of small cancer biomarkers with low molecular weight and a low concentration range has always been challenging yet urgent in many clinical applications such as diagnosing early-stage cancer, monitoring treatment and detecting relapse. Here, a highly enhanced plasmonic biosensor that can overcome this challenge is developed using atomically thin two-dimensional phase change nanomaterial. By precisely engineering the configuration with atomically thin materials, the phase singularity has been successfully achieved with a significantly enhanced lateral position shift effect. Based on our knowledge, it is the first experimental demonstration of a lateral position signal change > 340 μm at a sensing interface from all optical techniques. With this enhanced plasmonic effect, the detection limit has been experimentally demonstrated to be 10–15 mol L−1 for TNF-α cancer marker, which has been found in various human diseases including inflammatory diseases and different kinds of cancer. The as-reported novel integration of atomically thin Ge2Sb2Te5 with plasmonic substrate, which results in a phase singularity and thus a giant lateral position shift, enables the detection of cancer markers with low molecular weight at femtomolar level. These results will definitely hold promising potential in biomedical application and clinical diagnostics

Clinical Relevance of Tumor Cells with Stem-Like Properties in Pediatric Brain Tumors

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors

Clinical management of molecular alterations identified by high throughput sequencing in patients with advanced solid tumors in treatment failure: Real-world data from a French hospital

BackgroundIn the context of personalized medicine, screening patients to identify targetable molecular alterations is essential for therapeutic decisions such as inclusion in clinical trials, early access to therapies, or compassionate treatment. The objective of this study was to determine the real-world impact of routine incorporation of FoundationOne analysis in cancers with a poor prognosis and limited treatment options, or in those progressing after at least one course of standard therapy.MethodsA FoundationOneCDx panel for solid tumor or liquid biopsy samples was offered to 204 eligible patients.ResultsSamples from 150 patients were processed for genomic testing, with a data acquisition success rate of 93%. The analysis identified 2419 gene alterations, with a median of 11 alterations per tumor (range, 0–86). The most common or likely pathogenic variants were on TP53, TERT, PI3KCA, CDKN2A/B, KRAS, CCDN1, FGF19, FGF3, and SMAD4. The median tumor mutation burden was three mutations/Mb (range, 0–117) in 143 patients with available data. Of 150 patients with known or likely pathogenic actionable alterations, 13 (8.6%) received matched targeted therapy. Sixty-nine patients underwent Molecular Tumor Board, which resulted in recommendations in 60 cases. Treatment with genotype-directed therapy had no impact on overall survival (13 months vs. 14 months; p = 0.95; hazard ratio = 1.04 (95% confidence interval, 0.48–2.26)].ConclusionsThis study highlights that an organized center with a Multidisciplinary Molecular Tumor Board and an NGS screening system can obtain satisfactory results comparable with those of large centers for including patients in clinical trials

Notch1, développement normal et cancers

Le récepteur Notch1 est mis en cause dans de nombreux processus biologiques tel que la différenciation, le maintien du potentiel de différenciation, la mort cellulaire et même la cancérisation. Récemment le récepteur Notch1 a été co-immunoprécipité avec la protéine Nov qui appartient à la famille des gènes CCN. Dans un premier temps, nous avons étudié l expression de deux gènes de la famille CCN : CCN1 et CCN3 au cours du développement aviaire. Ce modèle présente de nombreux avantages pour l étude du développement notamment l accessibilité des embryons. Dans ce modèle, nous nous sommes particulièrement intéressés à l épithélium olfactif pour l étude du récepteur Notch1. Cet organe présente la particularité de comporter des cellules nerveuses et d être capable de se renouveler tout au long de la vie suggérant la présence de cellules souches dans cet organe. Des cultures primaires d épithéliums olfactifs d embryons de poulets ainsi que la caractérisation de ces cellules ont été réalisées. Les cellules souches de cet organe ont ensuite été isolées par la méthode de fractionnement par couplage flux / force (SdFFF). Le récepteur Notch1 étant impliqué dans les processus de différenciation neuronale au cours du développement, nous avons étudié le niveau d expression de ces transcrits au cours de la mise en place de l épithélium olfactif aviaire. Nous avons pu mettre en évidence l implication du récepteur Notch1 au cours de l ontogenèse de cet organe. Puis le récepteur a été étudié dans un modèle tumoral glial : U-87 MG. L étude in vitro a permis de montrer la présence du récepteur et de quatre de ses ligands. Les cellules U-87 MG on ensuite été traitées par différentes molécules (FasL, étoposide, déxaméthasone) afin d induire l apoptose. Les variations d expression du récepteur Notch1 en fonction des traitements ont été observées. L utilisation d un inhibiteur des gamma-sécrétases inhibant le clivage S3 du récepteur Notch permet une diminution de l apoptose induite pas FasL dans les cellules U-87 MG. Dans un second temps, un modèle in vivo de glioblastome humain a été mis au point dans le laboratoire. Les molécules utilisées in vitro ont été testées sur notre modèle de xénogreffes. Les traitements par FasL / étoposide / déxaméthasone ont permis une diminution importante de la taille des tumeurs et une amélioration des symptômes cliniques. Il semblait pertinent d étudier la mise en cause du récepteur Notch1 au cours de ce phénomène de cancérisation. Il semble qu une diminution du niveau d expression du récepteur Notch1 soit observée après exposition FasL / étoposide et FasL / étoposide / dexaméthasone.Notch1 receptor participates in several biological processes as cellular differentiation, self renewal potential, apoptosis and carcinogenesis. This protein was shown to have physical interaction with Nov, a member of the CCN gene family. Avian olfactory epithelium is known to have neuronal cell renewal. At first, this potential function was studied using avian olfactory primary cell culture and stem cells were purified using SdFFF method. Notch1 receptor was then studied by in situ hybridization during avian olfactory development in order to know Notch1 expression during neuronal cell production. We determined that Notch1 receptor was implicated during avian olfactory ontogenesis. In a second time, Notch1 receptor was studied in a glioblastoma cell line: U-87 MG cells. In order to induce apoptosis in theses cells different treatments were tested: FasL, etoposide and dexamethazone. Notch1 expression was observed after these different treatments to understand its role during U-87 MG cells apoptosis. We determine that Notch1 expression was modified after treatments and that FasL- induced apoptosis was decreased when Notch1 was inactivated by a gamma-secretase inhibitor. Apoptotic molecules were then tested after U-87 MG cell xenografts. An important decrease of tumour size was observed after treatments. At the end, Notch1 expression was observed in our model. Its expression seemed to be reduced after our treatments in vivo.LIMOGES-BU Sciences (870852109) / SudocSudocFranceF

Oncogramme, a new promising method for individualized breast tumour response testing for cancer treatment.

International audienceBACKGROUND:Breast cancer is the most widely spread cancer in the world, attracting much research and individualized tumour response testing (ITRT) methods are now used to individualize patient chemotherapeutic administrations. A new ITRT method was developed with optimized processing.MATERIALS AND METHODS:Breast tumour fragments were separated and the cells seeded in a foetal calf serum-free defined medium. After various chemotherapeutic treatments, cytotoxicity was determined by cell death detection with calcein acetoxymethyl and ethidium homodimer labelling.RESULTS:The culture medium allowed breast tumour cell proliferation in culture, while preventing fibroblastic cell survival. Moreover, the cell death analysis gave rise to a chemoresistance profile called an Oncogramme, with statistically significant values.CONCLUSION:The Oncogramme is a new ITRT method which can predict patient cell sensitivities to chemotherapeutics and should be validated by a new phase I clinical trial