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The search for efficient gas carriers for biomedical applications presents a challenging task due to the kinetics of gas adsorption/desorption. This article presents a novel approach utilizing Gd-LTL zeolite crystals for oxygen delivery combined with an MRI study.

CERVOXY COLL; International audience; A growing interest is currently directed to find an efficient NO and CO2 gas carriers for biomedical applications. The gas adsorption properties of sodium- (Na-X) and copper- (Cu-X) containing FAU nanozeolites toward nitric oxide and carbon dioxide were studied. The materials were fully characterised by XRD, TEM, ICP, DLS and in-situ FTIR. The as-prepared Na-X showed higher gas adsorption ability towards carbon dioxide, whereas the ion-exchanged Cu-X was more efficient adsorbent with regards to nitric oxide. In addition, the cytotoxicity tests disclosed that both nanozeolites have no toxicity making them suitable for further tests in biomedical field as gas transporters.

Simple Summary Glioblastoma (GB) is a highly aggressive brain tumor characterized by poor prognosis and high rate of recurrence in response to conventional treatments consisting of tumor resection and radiochemotherapy (RCT). The reasons for this therapeutic failure are mainly due to the complexity of GB biology and its environment. GB progression is highly dependent on its vascularization and inflammatory status. Besides, evidence showed that RCT also induces vascular change and inflammation. In GB patients, Angiopoietin-2 (Ang2), biomarker of poor prognosis is a crucial angiogenic factor also involved in inflammation. Our aim was to clarify the role of Ang2 in RCT-induced changes in the GB environment. To this end, we generated Ang2-overexpressing GL261 cells and characterized tumor progression, as well as inflammation and vascularization, in response to RCT. We showed that Ang2 delays tumor recurrence and makes a lasting improvement in animal survival when combined with conventional RCT. Abstract (1) We wanted to assess the impact of Ang2 in RCT-induced changes in the environment of glioblastoma. (2) The effect of Ang2 overexpression in tumor cells was studied in the GL261 syngeneic immunocompetent model of GB in response to fractionated RCT. (3) We showed that RCT combined with Ang2 led to tumor clearance for the GL261-Ang2 group by acting on the tumor cells as well as on both vascular and immune compartments. (4) In vitro, Ang2 overexpression in GL261 cells exposed to RCT promoted senescence and induced robust genomic instability, leading to mitotic death. (5) Coculture experiments of GL261-Ang2 cells with RAW 264.7 cells resulted in a significant increase in macrophage migration, which was abrogated by the addition of soluble Tie2 receptor. (6) Together, these preclinical results showed that, combined with RCT, Ang2 acted in an autocrine manner by increasing GB cell senescence and in a paracrine manner by acting on the innate immune system while modulating the vascular tumor compartment. On this preclinical model, we found that an ectopic expression of Ang2 combined with RCT impedes tumor recurrence.

CERVOXY; International audience; Approaches able to counteract, at least temporarily, hypoxia, a well-known factor of resistance to treatment in solid tumors are highly desirable. Herein, we report the use of nanosized zeolite crystals as hyperoxic/hypercapnic gas carriers for glioblastoma. First, the non-toxic profile of nanosized zeolite crystals in living animals (mice, rats and non-human primates) and in various cell types is presented. Second, the ability of the nanosized zeolites to act as a vasoactive agent for a targeted re-oxygenation of the tumor after intravenous injection is shown. As attested by an MRI protocol, the zeolites were able to increase oxygenation and blood volume specifically within the brain tumor whilst no changes in the healthy-non tumoral brain-were observed. The first proof of concept for the use of metal-containing nanosized zeolites as a tool for vectorization of hyperoxic/hypercapnic gases in glioblastoma is revealed.

Background Glioblastoma (GB), a highly hypoxic brain tumor (Bekaert et al. 2017), is characterized by a massive macrophage (MΦ) infiltration (Lapa et al. 2015). Hypoxia triggers a shift to a pro-tumoral M2 phenotype in GB (Leblond et al. 2016). Thus, strategies aiming to reduce hypoxia could promote an anti-tumoral M1 phenotype. Among these reoxygenation strategies, we recently developed a new approach with zeolites nanoparticles. These zeolites are able to carry hyperoxic/hypercapnic gases and release them according to a hypoxic gradient. We have demonstrated that the charge balancing cation changes affinity to the gases but also the ability to track zeolite with MRI. Our objective is to study the reoxygenation efficacy of zeolites specifically in the GB and to evaluate their impact on tumor associated MΦ with in vitro and in vivo studies. Methods Faujasite zeolites (FAU, ∼20nm of diameter) were used and modified by ion exchange with various cations (Fe, Gd, Cu, Ag). GB model was obtained by orthotopic glioblastoma cells implantation (U251) in nude rats (ONCOModels/Unicaen). 7T MRI (Bruker/Cyceron) was used to follow zeolites after intravenous injection and for oxygen measurement. Murine bone marrow derived MΦ were prepared and polarized to M1 and M2 using LPS/IFNg or IL4 as previously described (Leblond et al. 2016). Zeolites were added in MΦ medium and their impact on MΦ were evaluated by crystal violet dye assay, flow cytometry (Plateau ICORE/Unicaen) and polarization assays. Results Our results show that zeolites are able to accumulate and release the carried gases specifically in the brain tumor leading to tumor reoxygenation. Regarding the effect on MΦ, our preliminary results show, in vitro, the safety of as-prepared zeolites or Fe, Gd or Cu dopped zeolites on M0, M1 and M2 MΦ cultures. Similarly, no alteration of the cell cycle was observed. As a positive control of cell death, the presence of Ag-dopped zeolites dramatically decreased M0 and M1 MΦ viability. Conclusions Zeolites can deliver oxygen to the brain tumor and may improve the effectiveness of conventional treatments. Zeolites do not exhibit toxicity on primary cultures of MΦ. Additional studies are underway to evaluate the effect of zeolites on the polarization of MΦ, both in vitro and in vivo. Legal entity responsible for the study The authors. Funding Region Normandie, CNRS, Universite de Caen Normandie, Ministere de l’Enseignement Superieur et de la Recherche, European Union-Fonds Europeen de Developpement Regional (FEDER), HABIONOR European project, co-funded by the Normandy County Council, the French State in the framework of the interregional development Contract “Vallee de la Seine” 2015-2020, ARCHADE, Federation pour la Recherche sur le Cerveau (FRC) et INCa (INCA-11699). Disclosure All authors have declared no conflicts of interest.

Background Glioblastoma (GB) are brain tumors with a poor prognosis despite multimodal treatment combining resection, chemotherapy (CT) and radiotherapy (RT). The rich vascularization of these tumors led to the introduction of anti-angiogenic therapy with most efforts focused on the vascular endothelial growth factor (VEGF). However, the angiopoietins (Ang) have emerged as alternative regulators of angiogenesis. In particular, in GB, Ang2 is up-regulated and stimulates tumor angiogenesis in concert with VEGF but also activates pro-angiogenic functions of macrophages. However, Ang2 functions are context-dependent. Therefore, we sought to elucidate the involvement of Ang2 in the interaction of glioma response to CT and RT, both therapeutic modalities known to alter tumor angiogenesis and inflammation. Methods To recapitulate high levels of Ang2 in GB patients, Ang2 was overexpressed in murine glioma cells (GL261-Ang2). Effects of Ang2 were studied on an orthotopic syngenic model of GB (GL261 cells) in response to combined CT/RT. C57bl/6 mice were co-treated with temozolomide (TMZ 10 mg/kg; i.p.) and brain tumors were irradiated with X-rays (4 Gy) at 7, 9 and 11 days post-cell injection. The tumor growth and its microenvironment were followed by MRI and immunohistology analyses. Results We showed that, in this model, the chronic overexpression of Ang2 does not modify tumor progression, but leads to a decrease in vessel density (-39±10%, p 3 months) compared with treated GL261 tumors (18±3 days). In vitro, no difference in the chemo-radiosensitivity of GL261 and GL261-Ang2 cells was noticed, suggesting a paracrine effect of Ang2 on the tumor microenvironment. Accordingly, we showed that Ang2 sensitizes the tumor vasculature to CT/RT and sustains inflammatory cells in the tumor microenvironment until 3 months post-treatment. Conclusions These results suggest that Ang2 might influence the therapeutic response of GB by acting on angiogenesis and inflammation. Legal entity responsible for the study E. Petit. Funding This study was funded by the Region Normandie, the Centre National de la Recherche Scientifique (CNRS), the Universite de Caen Normandie (UNICAEN), the European Union-Fonds Europeen de Developpement Regional (FEDER), ARCHADE, HABIONOR European project, la Federation pour la Recherche sur le Cerveau par l’operation Rotary «Espoir en tete » (FRC), EdNBise 497 - Normandie Universite. Disclosure All authors have declared no conflicts of interest.

Introduction Glioblastoma (GB) are known to be highly hypoxic and both hypoxia inducible factors, HIF-1 and HIF-2, have been implicated in their growth and resistance to treatments. Recently, it was proposed that a severe chronic hypoxia would enhance HIF-2 expression at the expense of HIF-1 through HAF (hypoxia-associated factor). HAF is expressed in many cancers including high and low grade glioma. However, its implication in GB growth and its treatments is poorly documented (Koh et al., 2011). In this context, we conducted in vivo and in vitro studies to identify whether HAF expressed by glioma cells modulates tumour growth and efficacy of GB conventional treatments, i.e. chemotherapy (temozolomide, TMZ) and radiotherapy (X-rays). Material and methods Stable inhibition of HAF expression was established in human glioblastoma cells by RNA interference (U251shHAF). Orthotopic GB models were developed in mice (8/group) for U251shHAF and U251Sc cells (scrambled-shRNA infected cells) as control. Tumour development was assessed with 7T MRI (T2w sequence). At the end of the experiments, an immunohistology study was performed to characterise the vascularisation (PECAM), glial (GFAP) and inflammatory (CD68) reactions. In vitro, the radio- and chemosensitivity of U251shHAF were studied by clonogenic assay and cell cycle analysis following X-rays irradiation (X-RAD 225Cx) or TMZ exposition. Annexin-V binding and propidium iodure uptake followed by flow cytometry was used to quantify apoptotic and necrotic cells. Results and discussions The stable inhibition of HAF expression in U251 cells leads to around 70% of its extinction in either normoxia or hypoxia (1% O2). Accordingly, the expression of VEGFA and CAIX, both known as HIF-1 and HIF-2 dependent genes, was decreased in U251shHAF cultured in hypoxia (1% O2) compared to U251Sc cells. Loss of function of HAF leads to a significant growth delay of U251shHAF tumours of 3 weeks compared to U251Sc tumours, although both tumours display similar vascularisation, glial and inflammation reactions. In other hand, HAF silencing in glioma cells does not modified their sensitivity to X-rays or TMZ as suggested by the similar results obtained for both U251shHAF and U251Sc cells, through clonogenic assay, cell cycle and apoptosis analyses. Conclusion Our results suggest that HAF might be of poor prognosis for GB since its inhibition in glioma cells reduces tumour growth without alleviating glioma cell chemo- and radioresistance.