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International audience; A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers opportunity to decipher the kinetics of miRNA activity in vitro, in relation with their intracellular trafficking in glioblastoma cells. In this study we describe in detail the method for production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy. We show that LentiRILES is a robust, highly specific and sensitive miRNA sensor system that can be used in vitro as a single-cell miRNA monitoring method, cell-based screening platform for miRNA therapeutics and as a tool to analyze the structurefunction relationship of the miRNA duplex. Furthermore, we report the kinetic of miRNA activity upon the intracranial delivery of miRNA mimics in an orthotopic animal model of glioblastoma. This information is exploited to evaluate the tumor suppressive function of miRNA-200c as locoregional therapeutic modality to treat glioblastoma. Our data provide evidence that LentiRILES is a robust system, well-suited to resolve the activity of endogenous and exogenously expressed miRNAs for basic research to gene and cell therapy.

INTRODUCTION Adult-type diffuse gliomas comprise IDH-mutant astrocytomas, IDH-mutant 1p/19q codeleted oligodendrogliomas, and IDH-wildtype glioblastomas (GBM). GBM display genomic instability, which is favored by two genetic events leading to massive chromosomal alterations: chromothripsis (CT) and whole-genome duplication (WGD). Those events are scarcely described in IDH-mutant gliomas. The better prognosis of IDH-mutant gliomas may be in part related to their greater genomic stability compared to GBM. METHODS Pangenomic profiles of 301 adult diffuse gliomas were analyzed at initial diagnosis by SNP arrays, including 196 GBM and 105 IDH-mutant gliomas (61 astrocytomas and 44 oligodendrogliomas). We further analyzed 16 IDH-mutant recurrent gliomas (11 astrocytomas and 5 oligodendrogliomas). Tumor ploidy was assessed with Genome Alteration Print and CT events with CTLPScanner and through manual screening. Survival data were compared using Kaplan-Meier method and categorical variables using Chi2 test. RESULTS At initial diagnosis, 37 GBM (18.8%) displayed CT (mainly affecting chromosomes 12, 9 and 7) versus five IDH-mutant gliomas (4.8%) (p = 0.0007). The latter were all high-grade astrocytomas. No oligodendroglioma or grade 2 astrocytoma displayed CT. There was a trend towards higher occurrence of CT in GBM versus high-grade IDH-mutant astrocytomas (p = 0.0589). WGD was detected at initial diagnosis in 18 GBM (9.2%) and 9 IDH-mutant gliomas (of any subtype and grade; 8.6%). Over 75% of chromosomal losses occurred after WGD. Neither CT nor WGD was associated with overall survival in GBM. At recurrence, one grade 4 IDH-mutant astrocytoma displayed CT and another one, WGD with CT. CONCLUSION CT is rare in IDH-mutant gliomas compared to GBM. The absence of CT in oligodendrogliomas and grade 2 astrocytomas may contribute to their greater genomic stability and better prognosis. CT occurrence in high-grade IDH-mutant astrocytomas may underlie aggressive biological behavior. WGD is a rare early event occurring equally in IDH-mutant gliomas and GBM.

International audience; Although old molecules, alkylating agents and platinum derivatives are still widely used in the treatment of various solid tumors. However, systemic toxicity and cellular resistance mechanisms impede their efficacy. Innovative strategies, including local administration, optimization of treatment schedule/dosage, synergistic combinations, and the encapsulation of bioactive molecules in smart, multifunctional drug delivery systems, have shown promising results in potentiating anticancer activity while circumventing such hurdles. Furthermore, questioning of the old paradigm according to which nuclear DNA is the critical target of their anticancer activity has shed light on subcellular alternative and neglected targets that obviously participate in the mediation of cytotoxicity or resistance. Thus, rethinking of the use of these pivotal antineoplastic agents appears critical to improve clinical outcomes in the management of solid tumors.

International audience; Chemokines such as stromal cell-derived factor-1α (SDF-1α) regulate the migration of cancer cells that can spread from their primary tumor site by migrating up an SDF-1α concentration gradient, facilitating their local invasion and metastasis. Therefore, the implantation of SDF-1α-releasing scaffolds can be a useful strategy to trap cancer cells expressing the CXCR4 receptor. In this work, SDF-1α was encapsulated into poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles and subsequently electrospun with chitosan to produce nanofibrous scaffolds of average fiber diameter of 261 ± 45 nm, intended for trapping glioblastoma (GBM) cells. The encapsulated SDF-1α maintained its biological activity after the electrospinning process as assessed by its capacity to induce the migration of cancer cells. The scaffolds could also provide sustained release of SDF-1α for at least 5 weeks. Using NIH3T3 mouse fibroblasts, human Thp-1 macrophages, and rat primary astrocytes we showed that the scaffolds possessed high cytocompatibility in vitro. Furthermore, a 7-day follow-up of Fischer rats bearing implanted scaffolds demonstrated the absence of adverse effects in vivo. In addition, the nanofibrous structure of the scaffolds provided excellent anchoring sites to support the adhesion of human GBM cells by extension of their pseudopodia. The scaffolds also demonstrated slow degradation kinetics, which may be useful in maximizing the time window for trapping GBM cells. As surgical resection does not permit a complete removal of GBM tumors, our results support the future implantation of these scaffolds into the walls of the resection cavity to evaluate their capacity to attract and trap the residual GBM cells in the brain.

International audience; MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy.

International audience; This work aims to design biocompatible aerogel sponges that can host and control the release of stromal cell-derived factor-1α (SDF-1α or CXCL12), a key protein for applications ranging from regenerative medicine to cancer therapy (notably for neural tissues). Miscibility of silk fibroin (SF) and hyaluronic acid (HA) was investigated by means of fluorescence and scanning electron microscopy to identify processing conditions. Series of freeze-dried sponges were prepared by associating and cross-linking within the same 3D structure, HA, SF, poly-L-lysine (PLL) and heparin (hep). Aerogel sponges presented high swelling degree and porosity (∼90 %), adequate mean pore diameter (ca. 60 μm) and connectivity for welcoming cells, and a soft texture close to that of the brain (6-13 kPa Young's Modulus). Addition of SF yielded sponges with slower biodegradation. SF-HA and SF-HA-hep sponges retained 75 % and 93 % of the SDF-1α respectively after 7 days and were found to be cytocompatible in vitro.

Despite the tremendous progress made in the field of cancer therapy in recent years, certain solid tumors still cannot be successfully treated. Alongside classical treatments in the form of chemotherapy and/or radiotherapy, targeted treatments such as immunotherapy that cause fewer side effects emerge as new options in the clinics. However, these alternative treatments may not be useful for treating all types of cancers, especially for killing infiltrative and circulating tumor cells (CTCs). Recent advances pursue the trapping of these cancer cells within a confined area to facilitate their removal for therapeutic and diagnostic purposes. A good understanding of the mechanisms behind tumor cell migration may drive the design of traps that mimic natural tumor niches and guide the movement of the cancer cells. To bring this trapping idea into reality, strong efforts are being made to create structured materials that imitate myelinated fibers, blood vessels, or pre-metastatic niches and incorporate chemical cues such as chemoattractants or adhesive proteins. In this review, the different strategies used (or could be used) to trap tumor cells are described, and relevant examples of their performance are analyzed. This work was supported by the “Institut National de la Santé et de la Recherche Médicale” (INSERM), the University of Angers (Angers, France), the MINECO (SAF2017-83118-R), the Agencia Estatal de Investigacion (AEI, Spain), and the Fondo Europeo de Desarollo Regional (FEDER). It is also related to the LabEx IRON “Innovative Radiopharmaceuticals in Oncology and Neurology” as part of the French government “Investissements d’Avenir” program, to the INCa (Institut National du Cancer) MARENGO consortium “MicroRNA agonist and antagonist Nanomedicines for GliOblastoma treatment: from molecular programmation to preclinical validation” through the PL-BIO 2014-2020 grant and to the MuMoFRaT project “Multi-scale Modeling & simulation of the response to hypo-Fractionated Radiotherapy or repeated molecular radiation Therapies” supported by “La Région Pays-de-la-Loire” and by the Cancéropôle Grand-Ouest (tumor targeting and radiotherapy network). MN was a Ph.D. student involved in the Erasmus Mundus Joint Doctorate program for Nanomedicine and pharmaceutical innovation (EMJD NanoFar) and received a fellowship from “La Région Pays-de-la-Loire.” SI

International audience; Internal radiation strategies hold great promise for glioblastoma (GB) therapy. We previously developed a nanovectorized radiotherapy that consists of lipid nanocapsules loaded with a lipophilic complex of Rhenium-188 (LNC(188)Re-SSS). This approach resulted in an 83 % cure rate in the 9L rat glioma model, showing great promise. The efficacy of LNC(188)Re-SSS treatment was optimized through the induction of a T-cell immune response in this model, as it is highly immunogenic. However, this is not representative of the human situation where T-cell suppression is usually encountered in GB patients. Thus, in this study, we investigated the efficacy of LNC(188)Re-SSS in a human GB model implanted in T-cell deficient nude mice. We also analyzed the distribution and tissue retention of LNC(188)Re-SSS. We observed that intratumoral infusion of LNCs by CED led to their complete distribution throughout the tumor and peritumoral space without leakage into the contralateral hemisphere except when large volumes were used. Seventy percent of the (188)Re-SSS activity was present in the tumor region 24 h after LNC(188)Re-SSS injection and no toxicity was observed in the healthy brain. Double fractionated internal radiotherapy with LNC(188)Re-SSS triggered survival responses in the immunocompromised human GB model with a cure rate of 50 %, which was not observed with external radiotherapy. In conclusion, LNC(188)Re-SSS can induce long-term survival in an immunosuppressive environment, highlighting its potential for GB therapy.

Glioblastoma is the most frequent and aggressive primary malignant tumor of the central nervous system with a gloomy prognosis. Platinum derivatives and one among them, cisplatin, exhibited promising results when locally administered into the brain of glioblastoma bearing rats. Nanovectorization of anticancer agents through polymeric nanoparticles may even promote drug accumulation within cells, thus concentrating the drug efficiently at its target. Anchorage of gadolinium complexes on the corona of such smart drug delivery systems could further allow magnetic resonance imaging (MRI) monitoring of the nanoplatform biodistribution in the damaged parenchyma and its therapeutic benefit. For this purpose, a biocompatible amphiphilic triblock copolymer, made of degradable polyester and polycarbonate and bioeliminable polyethylene oxide (PEO), was synthesized by successive ring-opening polymerizations. After micellization in water, gadolinium complexes were grafted onto the PEO micelle corona and the carboxylate functions, located at the surface of the micelle's core, were able to cross-link with Pt(ii) complexes. A macromolecular prodrug was therefore recovered in which more than one third of the carboxylate functions were linked to a platinum atom. By this strategy, stable cisplatin cross-linked nanoparticles were formulated with a mean size in the range of 100.63 ± 12.04 nm consistent with biological investigations. Relaxometry measurements both in water and in plasma at 7 T, 25 °C, confirmed the intrinsic potential of these hybrid nanoparticles as alternative MRI contrast agents with a substantial increase in the r2/r1 ratio by a factor of 3.3 and 2.7, respectively, compared to the conventional low molar mass Gd-DTPA. As a result, their infusion within the striatum of glioblastoma-bearing mice resulted in a hypersignal on T2-weighted MR images that persisted over time. Ultimately, the formulated prodrug exhibited up to 50-fold increased accumulation in human glioblastoma cell lines and up to 32-fold enhanced subsequent Pt-DNA adduct formation in comparison with free cisplatin, thus supporting the potential of this innovative bimodal tool for further applications.

Glioblastoma, the most frequent and lethal form of glioma, displays chromosome instability and recurrent somatic copy number alterations (SCNA). Chromothripsis and whole genome duplication (WGD) have been recently identified in cancer. In the present study, we analyzed SCNA and determine the ploidy pattern in 123 IDH-wild-type glioblastomas, using SNP array data. WGD and chromothripsis events were validated using, respectively, FISH and CTLPScanner. WGD was detected in 11.4% glioblastomas (14/123) and was associated with TP53 mutation (p = 0.0068). It was an early event occurring after the recurrent SCNA observed in diffuse high-grade gliomas. Glioblastomas with WGD were more aneuploid compared to glioblastomas without WGD (p < 0.0001). Chromothripsis occurred in 29.3% glioblastomas (36/123) and mostly affected chromosomes 7, 9 and 12, with amplification of oncogenes (EGFR, MDM2/CDK4), and homozygous deletion of tumor suppressor genes (CDKN2A). There was a significant association between chromothripsis and gene rearrangement at a given locus. WGD is an early genetic event significantly associated to TP53 mutation and leading to chromosome instability and aneuploidy in IDH-wild-type glioblastoma. Chromothripsis recurrently targets oncogenes and tumor suppressor genes that are key players in gliomagenesis and tumor progression. The occurrence of chromothripsis points to underlying gene rearrangements (including gene fusions), potential therapeutic targets in glioblastoma.

International audience; Glioblastoma, the most frequent and lethal form of glioma, displays chromosome instability and recurrent somatic copy number alterations (SCNA). Chromothripsis and whole genome duplication (WGD) have been recently identified in cancer. In the present study, we analyzed SCNA and determine the ploidy pattern in 123 IDH-wild-type glioblastomas, using SNP array data. WGD and chromothripsis events were validated using, respectively, FISH and CTLPScanner. WGD was detected in 11.4% glioblastomas (14/123) and was associated with TP53 mutation (p = 0.0068). It was an early event occurring after the recurrent SCNA observed in diffuse high-grade gliomas. Glioblastomas with WGD were more aneuploid compared to glioblastomas without WGD (p < 0.0001). Chromothripsis occurred in 29.3% glioblastomas (36/123) and mostly affected chromosomes 7, 9 and 12, with amplification of oncogenes (EGFR, MDM2/CDK4), and homozygous deletion of tumor suppressor genes (CDKN2A). There was a significant association between chromothripsis and gene rearrangement at a given locus. WGD is an early genetic event significantly associated to TP53 mutation and leading to chromosome instability and aneuploidy in IDH-wild-type glioblastoma. Chromothripsis recurrently targets oncogenes and tumor suppressor genes that are key players in gliomagenesis and tumor progression. The occurrence of chromothripsis points to underlying gene rearrangements (including gene fusions), potential therapeutic targets in glioblastoma.

International audience; Glioblastoma is the most frequent and aggressive primary malignant tumor of the central nervous system with a gloomy prognosis. Platinum derivatives and one among them, cisplatin, exhibited promising results when locally administered into the brain of glioblastoma bearing rats. Nanovectorization of anticancer agents through polymeric nanoparticles may even promote drug accumulation within cells, thus concentrating the drug efficiently at its target. Anchorage of gadolinium complexes on the corona of such smart drug delivery systems could further allow magnetic resonance imaging (MRI) monitoring of the nanoplat-form biodistribution in the damaged parenchyma and its therapeutic benefit. For this purpose, a biocom-patible amphiphilic triblock copolymer, made of degradable polyester and polycarbonate and bioelimin-able polyethylene oxide (PEO), was synthesized by successive ring-opening polymerizations. After micelli-zation in water, gadolinium complexes were grafted onto the PEO micelle corona and the carboxylate functions, located at the surface of the micelle's core, were able to cross-link with Pt(II) complexes. A macromolecular prodrug was therefore recovered in which more than one third of the carboxylate functions were linked to a platinum atom. By this strategy, stable cisplatin cross-linked nanoparticles were formulated with a mean size in the range of 100.63 ± 12.04 nm consistent with biological investigations. Relaxometry measurements both in water and in plasma at 7 T, 25 °C, confirmed the intrinsic potential of these hybrid nanoparticles as alternative MRI contrast agents with a substantial increase in the r 2 /r 1 ratio by a factor of 3.3 and 2.7, respectively, compared to the conventional low molar mass Gd-DTPA. As a result, their infusion within the striatum of glioblastoma-bearing mice resulted in a hypersignal on T 2-weighted MR images that persisted over time. Ultimately, the formulated prodrug exhibited up to 50-fold increased accumulation in human glioblastoma cell lines and up to 32-fold enhanced subsequent Pt-DNA adduct formation in comparison with free cisplatin, thus supporting the potential of this innovative bimodal tool for further applications.

Shubaash ANTHIYA and Audrey GRIVEAU contributed equally to this work.; International audience; MicroRNAs (miRNAs) are key regulatory elements coded by the genome. A single miRNA can downregulate expression of multiple genes involved in diverse functions. As cancer is a disease with multiple gene aberrations, developing novel approaches to identify and modulate miRNA-pathways may result in a breakthrough for cancer treatment. With a special focus on glioblastoma, this review provides an up-to-date understanding of miRNA biogenesis, role of miRNA in cancer resistance, essential tools for modulating miRNA expression, emerging list of clinically promising RNAi delivery systems and how they can be adapted for therapy.

Chemokines are known to stimulate directed migration of cancer cells. Therefore, the strategy involving gradual chemokine release from polymeric vehicles for trapping cancer cells is of interest. In this work, the chemokine stromal cell-derived factor-1α (SDF-1α) was encapsulated into nanoparticles composed of poly-(lactic-co-glycolic acid) (PLGA) and a polyethylene glycol (PEG)-PLGA co-polymer to achieve sustained release. SDF-1α, and lysozyme as a model protein, were firstly precipitated to promote their stability upon encapsulation. A novel phase separation method utilising a non-toxic solvent in the form of isosorbide dimethyl ether was developed for the individual encapsulation of SDF-1α and lysozyme precipitates. Uniform nanoparticles of 200–250 nm in size with spherical morphologies were successfully synthesised under mild formulation conditions and conveniently freeze-dried in the presence of hydroxypropyl-β-cyclodextrin as a stabiliser. The effect of PLGA carboxylic acid terminal capping on protein encapsulation efficiency and release rate was also explored. Following optimisation, sustained release of SDF-1α was achieved over a period of 72 h. Importantly, the novel encapsulation process was found to induce negligible protein denaturation. The obtained SDF-1α nanocarriers may be subsequently incorporated within a hydrogel or other scaffolds to establish a chemokine concentration gradient for the trapping of glioblastoma cells.

International audience; Glioblastoma (GBM), the deadliest primary tumor of the central nervous system (CNS), is a clear illustration of the resistance of cancer cells to conventional therapies. Application of combinatorial strategies able to overcome pivotal factors of GBM resistance, particularly within the resection margins, represents an essential issue. This review focuses on the role of iron metabolism in GBM progression and resistance to therapy, and the impact of its pharmaceutical modulation on the disease. Iron, through its involvement in many biological processes, is a key factor in the control of cell behavior and cancer biology. Therefore, targeting cellular iron signaling or taking advantage of its dysregulation in cancer cells may lead to new opportunities for improving treatments and drug delivery in GBM.

International audience; The controlled delivery of proteins within calcium carbonate (CaCO 3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated proteins and the particles. This poses a great limitation on the successful loading of proteins that have no ionic affinity to CaCO 3. In this study, we explored the use of polysaccharide–protein interactions to strongly enhance the encapsulation of proteins in CaCO 3 microparticles. Previously, Vandevenne and colleagues inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into a b-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study we showed that this hybrid protein can associate with HA and be successfully loaded into vaterite CaCO 3 microparticles using supercritical CO 2 (ScCO 2) technology aided by the templating effect of HA on CaCO 3. The presence of ChBD inserted into BlaP increased the encapsulation of the protein by 6-fold when complexed with HA. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP to achieve release of the protein from the micro-particles by protease cleavage. Our results showed that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from o20% to 87%. In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 82% of its activity and efficient release of the protein from the microparticles was achieved by protease cleavage.

BACKGROUND Genomic rearrangements are thought to occur in a stepwise manner during cancer development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome (chr) rearrangements in a one-step catastrophic event termed chromothripsis (CT). In this scenario, the chr shatters into tens to hundreds of pieces, some of which are then stitched together in a mosaic patchwork of genomic fragments. Such random stitching may give rise to fusion genes with oncogenic potential. CT has not been thoroughly studied in diffuse gliomas, such as glioblastomas (GB). The aim of the project was to detect CT in GB and identify novel fusion genes in CT regions. Those genes are potential therapeutic targets. MATERIAL AND METHODS 170 de novo IDH wild-type GB were screened for CT patterns using whole genome SNP arrays. RNA sequencing was performed in 52 cases with CT features to identify novel fusion genes within the rearranged genomic regions. Expression levels of transcripts were measured using FPKM (fragments per kilobase of exon per million fragments mapped) normalization method. Candidate fusion genes were validated by RT-PCR followed by Sanger sequencing. RESULTS Out of 66 GB with CT patterns (66/170, 38.8%), 40 cases (out of 52 RNA-sequenced cases, 77%) harbored at least one fusion gene within CT regions, which were mostly detected on chr 7, 9, and 12. Overall, 137 candidate fusion genes were identified, 26 of which with a predicted open reading frame. Twelve out of 26 fusions were validated by RT-PCR followed by Sanger sequencing (validation of the remaining fusions is ongoing). The fusions involved either well-known key oncogenes such as EGFR, MDM2, and MET or potential oncogenes (CPM, PPM1H, SEPT14, SEC61G) with a yet-to-discover role in GB pathogenesis. GB with CT on chr 12 (11/66, 16.7%) showed a distinct transcript expression profile at the locus 12q13-15, particularly in the vicinity of MDM2 and CDK4 genes. In our cohort, this so-called “multi-aberration region” recurrently harbored gene fusions, involving, among others, CPM (12q15). CPM, encoding an extracellular membrane-bound carboxypeptidase, is recurrently amplified in liposarcoma and is a potential oncogene involved in the EGFR signaling pathway. Three cases (3/26, 11.5%) harboring an in-frame gene fusion involving CPM showed higher levels of CPM transcripts and did not exhibit any EGFR amplification. Gene fusions involving CPM might constitute an alternative mechanism of EGFR pathway activation in de novo IDH wild-type GB. CONCLUSION The occurrence of CT points to underlying gene fusions in IDH wild-type GB. Such fusions may drive tumorigenesis and represent promising therapeutic targets. We identified CPM as a recurrent fusion partner and a potential alternative mechanism of EGFR signaling pathway activation in de novo IDH wild-type GB.

Internal radiation strategies hold great promise for glioblastoma (GB) therapy. We previously developed a nanovectorized radiotherapy that consists of lipid nanocapsules loaded with a lipophilic complex of Rhenium-188 (LNC

Objective: microRNA assessments in biological samples can be performed by different methods that mainly rely on hybridization process, qPCR or RNA sequencing. With the aim to detect and validate microRNA biomarkers in tumor samples, we challenged the consistency of the quantitative results obtained with the different methods. Methods: We measured microRNA concentrations in several biological samples such as cultured tumor cells or tumor tissues (frozen tissues or FFPE samples) using different microRNA assay methods, in particular hybridization to Affymetrix TM arrays, qPCR and digital droplet qPCR (Biorad TM ) based on Taqman microRNA assays (Life Technologies TM ). We also compared our results to other data that have been obtained with different technical approaches and available in the literature. Results: We found poor consistency for the microRNA amounts measured in the samples assayed by the different methods. Both technical platforms and microRNA assays protocols may be responsible for the observed inconsistencies. Conclusion: When assaying microRNAs for clinical purpose or fundamental researches it seems necessary to keep in mind the specific pitfalls of all the microRNA detection methods such as those we disclose here. Obviously, valid inter sample comparisons and meaningful multicenter studies can only be obtained when microRNA assessments are strictly performed with identical technical approaches and reagents.

Background Overcoming resistance to treatment is an essential issue in many cancers including glioblastoma (GBM), the deadliest primary tumor of the central nervous system. As dependence on iron is a key feature of tumor cells, using chelators to reduce iron represents an opportunity to improve conventional GBM therapies. The aim of the present study was, therefore, to investigate the cytostatic and cytotoxic impact of the new iron chelator deferasirox (DFX) on human GBM cells in well-defined clinical situations represented by radiation therapy and mild-hypoxia. Results Under experimental normoxic condition (21 % O2), deferasirox (DFX) used at 10 μM for 3 days reduced proliferation, led cell cycle arrest in S and G2-M phases and induced cytotoxicity and apoptosis in U251 and U87 GBM cells. The abolition of the antineoplastic DFX effects when cells were co-treated with ferric ammonium sulfate supports the hypothesis that its effects result from its ability to chelate iron. As radiotherapy is the main treatment for GBM, the combination of DFX and X-ray beam irradiation was also investigated. Irradiation at a dose of 16 Gy repressed proliferation, cytotoxicity and apoptosis, but only in U251 cells, while no synergy with DFX was observed in either cell line. Importantly, when the same experiment was conducted in mild-hypoxic conditions (3 % O2), the antiproliferative and cytotoxic effects of DFX were abolished, and its ability to deplete iron was also impaired. Conclusions Taken together, these in vitro results could raise the question of the benefit of using iron chelators in their native forms under the hypoxic conditions often encountered in solid tumors such as GBM. Developing new chemistry or a new drug delivery system that would keep DFX active in hypoxic cells may be the next step toward their application. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2074-y) contains supplementary material, which is available to authorized users.

International audience; The anti-tumour effect of ferrociphenol (FcdiOH)-loaded lipid nanocapsules (LNCs), with or without a DSPE-mPEG2000 coating, was evaluated on an orthotopic gliosarcoma model after administration by convection-enhanced delivery (CED) technique or by intra-carotid injection. No toxicity was observed by MRI nor by MRS in healthy rats receiving a CED injection of FcdiOH-LNCs (60 μL, 0.36 mg of FcdiOH/rat) when the pH and osmolarity had been adjusted to physiological values prior to injection. At this dose, the treatment by CED with FcdiOH-LNCs significantly increased the survival time of tumour-bearing rats in comparison with an untreated group (28.5 days vs 25 days, P = 0.0009) whereas DSPE-mPEG2000–FcdiOH-LNCs did not exhibit any efficacy with a median survival time of 24 days. After intra-carotid injection (400 μL, 2.4 mg of FcdiOH/rat), hyperosmolar DSPE-mPEG2000–FcdiOH-LNCs markedly increased the median survival time (up to 30 days, P = 0.0008) as compared to the control (20%). This was strengthened by their evidenced accumulation in the tumour zone and by the measure of the fluorescent brain surface obtained on brain slides for these DiI-labelled LNCs, being 3-fold higher than for the control. These results demonstrated that, depending upon the administration route used, the characteristics of LNC suspensions had to be carefully adapted.

Introduction L’utilisation de nanocapsules lipidiques chargees en rhenium-188 (188Re-SSS-LNC) pour la radiotherapie interne vectorisee du glioblastome constitue une alternative innovante qui a presente des resultats prometteurs. Le systeme nanocapsulaire lipidique utilise a montre une capacite a encapsuler le rhenium-188 sous forme d’un complexe, le bis(trithioperoxybenzoate)(dithiobenzoate) de rhenium. Deux programmes de recherche ont ete soutenus dans l’objectif du transfert a la clinique de cette nouvelle technologie. Dans cet objectif l’automatisation de la production et sa caracterisation constituent des etapes clefs. Les donnees presentees concernent l’automatisation de la production de nanocapsules lipidiques chargees en technetium-99 m (99mTc-SSS-LNC), utilisee comme modele du rhenium-188. Materiels et methodes Apres une etape de developpement et d’optimisation des conditions, 6 production (3 avec 0,5 GBq et 3 avec 5 GBq) ont ete realisees a l’aide d’un automate Synchrom R&D, Raytest® pilote par le logiciel Gynastar®, Raytest®. Chacune de ces productions a ete decrite par des indicateurs de procede et une caracterisation du produit final (activite et activite volumique, distribution en taille et potentiel zeta des particules et purete radiochimique PRC). En parallele, une methode de caracterisation de la PRC par chromatographie d’exclusion de taille a ete developpee (colonne ultrahydrogel, Waters®, injecteur pompe HPLC et detecteur UV Series 200, Perkin Elmer®, detecteur de radioactivite Radiomatic 150 TR, Perkin Elmer®). Resultats Un temps de production de 90 min ± (10) a ete observe. Les rendements de production ont ete de 44 % ± (1,7) pour les productions basse activite (0,5 GBq) et de 37 % ± (1,3) pour les productions haute activite (5 GBq). En fin de production, l’activite moyenne est comprise entre 291 MBq et 1501 MBq (activite volumique comprise entre 70 MBq/mL – 395 MBq/mL). Les nanoparticules obtenues presentent une taille de 64 nm ± (2), un PdI de 0,089 ± (0,019) et un potentiel zeta de -10 mV ± (2). Pour chaque production, une valeur de PRC superieure a 98 % a ete retrouvee. Conclusion La production automatisee en 99mTc-SSS-LNC developpee a presente un rendement, une distribution en taille et une PRC compatibles avec une utilisation en clinique. Les productions de nanocapsules chargees en rhenium-188 sont en cours d’optimisation et de caracterisation.

To establish the therapeutic relevance of new nanocarriers, rationalization of knowledge on their interactions with biological structures is essential. In the present study, we have investigated endocytosis and intracellular trafficking of lipid nanocapsules (LNCs) in rat glioma cells. Radiolabelled and fluorescent LNCs were synthesized by using a phase inversion process that follows the formation of an oil/water microemulsion containing triglycerides, lecithins and a non-ionic surfactant, the hydroxystearate of poly (ethylene glycol) (HS-PEG). Our data revealed that LNCs were rapidly accumulated within cells (from 2 min exposure) through active and saturating mechanisms involving endogenous cholesterol with a major contribution of clathrin/caveolae-independent pathways. Although initially present in endosomes, LNCs can bypass the endo-lysosomal compartment with only 10% of the cell-internalized fraction found in isolated lysosomes after 2 h exposure. As demonstrated by use of lysosomal probes, LNCs reverted lysosome integrity similarly to V-ATPase inhibitors and in a size-dependent fashion with best efficiency for small nanoparticles. When loaded with paclitaxel, smallest LNCs also triggered the best cell death activity. Those LNC properties are ascribed to the proportion of HS-PEG they provided to the cell. They are important to consider toward the development of nanomedicines that use drugs sensitive to lysosomal degradation or that need to reach extra endo-lysosomal targets.

International audience; Background: Glioma, notably glioblastoma multiforme, is characterized by extensive inter-and intra-tumoral heterogeneity. Surprisingly, the potential for differentiation of glioma cells has not been systematically analyzed and included in patient stratification methods. In the current study, retinoic acid (RA), a neuronal differentiation agent, was assessed for the pro-differentiative and anti-proliferative effects on glioma cells. Methods: Using RA-responsive glioma culture as an experimental paradigm, we analyzed the differentiation process both by videomicroscopy and at the mRNA (RNA-seq and reverse transcription-quantitative-polymerase chain reaction) and proteomic levels. Results: Glioma cells can differentiate into neurons in response to RA by (i) extending ultra-long cytoplasmic extensions, (ii) using these extensions to move from cell to cell either by perikaryal translocation or in a "spider-flight" like process, (iii) slowing their cell cycling, (iv) acquiring several neuronal differentiation markers such as MAPT, GAP43, DCX, NRCAM, NeuroD2, NeuroG2, and NeuN, and (v) decreasing the expression of several genes associated with glioma aggressiveness. Conclusion: These results indicate the existence of a subgroup of patients harboring RA-responsive glioma cells amenable to differentiation therapy, and stratifying such patients with a functional test is easily achievable. This provides the first step to reassess the potential of RA in the context of personalized medicine.

Many arguments support the development of local therapies for malignant gliomas. Simple injections of antimitotic agents into the surgical cavity has been replaced by more sophisticated systems. Tissues can be infused with complex prolonged-release polymeric or lipidic systems with macroscopic, microscopic and now even nanometric particles. But, as for any drug, the developments of these new agents has been long and only very few reach the stage of the clinic trials.

International audience; Hypoxia is a characteristic feature of solid tumors leading to the over expression of hypoxia-inducible factor (HIF)-1α protein and therefore to a specific cellular behavior. However, even though the oxygen tension in tumors is low (However, under both hypoxic and normoxic conditions, glucose is used for de novo synthesis of pyrimidine since the 13C label is found both in the uracil and ribose moieties. Labeling of the ribose ring demonstrates that U87-MG glioma cells use the reversible branch of the non-oxidative pentosephosphate pathway. Interestingly, stereotactic implantation of U87-MG cells grown under normoxia or mild hypoxia within the striatum of nude mice led to differential growth; the cells grown under hypoxia retaining an imprint of the oxygen adaptation as their development is then slowed down.

Fibroblast and epidermal growth factors (FGF-2 and EGF) are powerful mitogens for neural precursor cells isolated from the developing striatum and grown as neurospheres. However, questions remain as to the exact role of each of these molecules, and how the proteoglycan heparin may modify their behavior. Here, we show that FGF-2 is remarkably unstable in culture media, but that heparin could completely prevent its degradation, which led to faster cell growth rates. In addition, heparin significantly increased the number of cells within the E14 striatum responding to a brief pulse of FGF-2. In contrast, EGF was unable to stimulate the growth of E14 striatal precursors. However, EGF could induce the division of E18 striatal precursors as neurospheres and acted synergistically with FGF-2. FGF-2/heparin neurospheres generated significantly more neurons than EGF neurospheres. Interestingly, the addition of heparin to EGF neurospheres, which had no effects on EGF stability or growth rates, increased the numbers of neurons generated to that seen for FGF-2/heparin neurospheres. EGF neurospheres were found to produce FGF-2, but addition of heparin did not affect its concentration within cells or in the medium suggesting this released FGF-2 may already be bound to a proteoglycan. In addition, expanding cells with EGF plus heparin in the presence of an FGF-2 blocker did not have a significant effect on the number of neurons generated confirming that the increase in neuronal number is through a mechanism which is independent of FGF-2.

Glial cell line-derived neurotrophic factor (GDNF) is a protein with potent trophic actions on dopaminergic neurons, which is under investigation as a therapeutic agent for the treatment of neurodegenerative disorders, including Parkinson's disease. The aim of this work was to develop GDNF-loaded microspheres, which could be implanted by stereotaxy in the brain and could offer an alternative strategy in the treatment of Parkinson's disease. A w/o/w extraction-evaporation technique was chosen to prepare protein-loaded microspheres. An in vitro release study of the protein was required to assess the retention of integrity and the performance of the microsphere formulation with regard to sustained release. In order to assess the in vitro release profile of the GDNF-loaded microspheres, a preliminary study was performed to select an appropriate buffer for GDNF stabilization, using experimental designs. GDNF was measured by both enzyme-linked immunosorbant assay (ELISA) and radioactivity using (125)I-GDNF. The GDNF-loaded microsphere release profile was assessed in a low continuous flow system, and showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses.

Composee de l’agencement de differents collagenes, proteoglycanes et glycoproteines, la matrice extracellulaire constitue un substrat essentiel pour la morphogenese tissulaire et procure soutien et flexibilite aux tissus matures. De plus, elle agit en tant qu’entite epigenetique d’information dans le sens ou elle transmet et integre des signaux extracellulaires par le biais de divers recepteurs de la membrane plasmique cellulaire. Parmi les molecules de la matrice extracellulaire presentes au cours du developpement du systeme nerveux central (SNC), la tenascine-C (TN-C) possede un profil d’expression extremement particulier non seulement quant a sa distribution spatio-temporelle, mais, egalement, quant aux isoformes synthetisees. De nombreuses etudes realisees in vitro ont suggere des roles specifiques de la TN-C dans le developpement du SNC, notamment dans la migration de precurseurs neuronaux ou dans la croissance et le guidage neuritiques. Aussi, l’invalidation du gene codant pour la TN-C chez la souris, initialement decrite sans phenotype structural apparent, a permis d’explorer plus en detail et in vivo l’implication potentielle de cette glycoproteine dans l’ontogenese du SNC. Les resultats recemment obtenus tendent a confirmer les travaux plus anciens mais aussi a reveler des fonctions nouvelles, en particulier dans la proliferation des precurseurs oligodendrocytaires.

Accumulating data have provided evidence that 1 alpha,25 dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] is involved in brain function. Thus, the nuclear receptor for 1,25-(OH)(2)D(3) has been localized in neurons and glial cells. Genes encoding the enzymes involved in the metabolism of this hormone are also expressed in brain cells. The reported biological effects of 1,25-(OH)(2)D(3) in the nervous system include the biosynthesis of neurotrophic factors and at least one enzyme involved in neurotransmitter synthesis. 1,25-(OH)(2)D(3) can also inhibit the synthesis of inducible nitric oxide synthase and increase glutathione levels, suggesting a role for the hormone in brain detoxification pathways. Neuroprotective and immunomodulatory effects of this hormone have been described in several experimental models, indicating the potential value of 1,25-(OH)(2)D(3) pharmacological analogs in neurodegenerative and neuroimmune diseases. In addition, 1,25-(OH)(2)D(3) induces glioma cell death, making the hormone of potential interest in the management of brain tumors. These results reveal previously unsuspected roles for 1,25-(OH)(2)D(3) in brain function and suggest possible areas of future research.