Early effects of different brain radiotherapy modalities on circulating leucocyte subpopulations in rodents.

PURPOSES: Lymphopenia is extensively studied, but not circulating leucocyte subpopulations, which however have distinct roles in tumor tolerance. Proton therapy has been shown to have a lesser impact on the immune system than conventional X-ray radiotherapy through lower dose exposure to healthy tissues. We explored the differential effects of brain X-ray and proton irradiation on circulating leucocyte subpopulations. MATERIALS AND METHODS: Leucocyte subpopulation counts from tumor-free mice were obtained 12 hours after 4 fractions of 2.5 Gy. The relationships between irradiation type (X-rays or protons), irradiated volume (whole-brain/hemi-brain) and dose rate (1 or 2 Gy/min) with circulating leucocyte subpopulations (T-CD4+, T-CD8+, B, and NK-cells, neutrophils, and monocytes) were investigated using linear regression and tree-based modeling approaches. Relationships between dose maps (brain, vessels, lymph nodes (LNs)) and leucocyte subpopulations were analyzed and applied to construct the blood dose model, assessing the hypothesis of a direct lymphocyte-killing effect in radiation-induced lymphopenia. RESULTS: Radiation-induced lymphopenia occurred after X-ray but not proton brain irradiation in lymphoid subpopulations (T-CD4+, T-CD8+, B, and NK-cells). There was an increase in neutrophil counts following protons but not X-rays. Monocytes remained unchanged under both X-rays and protons. Besides irradiation type, irradiated volume and dose rate had a significant impact on NK-cell, neutrophil and monocyte levels but not T-CD4+, T-CD8+, and B-cells. The dose to the blood had a heterogeneous impact on leucocyte subpopulations: neutrophil counts remained stable with increasing dose to the blood, while lymphocyte counts decreased with increasing dose (T-CD8+-cells > T-CD4+-cells > B-cells > NK-cells). Direct cell-killing effect of the dose to the blood mildly contributed to radiation-induced lymphopenia. LN exposure significantly contributed to lymphopenia and partially explained the distinct impact of irradiation type on circulating lymphocytes. CONCLUSIONS: Leucocyte subpopulations reacted differently to X-ray or proton brain irradiation. This difference could be partly explained by LN exposure to radiation dose. Further researches and analyses on other biological processes and interactions between leucocyte subpopulations are ongoing. The various mechanisms underlying leucocyte subpopulation changes under different irradiation modalities may have implications for the choice of radiotherapy modalities and their combination with immunotherapy in brain cancer treatment.

Physical Activity Attenuates Brain Irradiation-Associated Skeletal Muscle Damage in the Rat.

PURPOSE: Radiation therapy for brain tumors increases patient survival. Nonetheless, side effects are increasingly reported such as cognitive deficits and fatigue. The etiology of fatigue remains poorly described. Our hypothesis is that the abscopal effects of radiation therapy on skeletal muscle may be involved in fatigue. The present study aims to assess the effect of brain irradiation on skeletal muscles and its relationship with fatigue and to analyze whether physical activity could counteract brain radiation-induced side effects. METHODS AND MATERIALS: Adult Wistar rats were randomly distributed between 4 groups: control (CTL), irradiated (IR), nonirradiated with physical activity (PA), and irradiated with physical activity (IR+PA). IR rats were exposed to a whole-brain irradiation (WBI) of 30 Gy (3 × 10 Gy). Rats subjected to PA underwent sessions of running on a treadmill, 3 times/week for 6 months. The effects of WBI on muscles were evaluated by complementary approaches: behavioral tests (fatigue, locomotion activity), magnetic resonance imaging, and histologic analyses. RESULTS: IR rats displayed a significant fatigue and a reduced locomotor activity at short term compared with the CTL group, which were attenuated with PA at 6 months after WBI. The IR rat's gastrocnemius mass decreased compared with CTL rats, which was reversed by physical activity at 14 days after WBI. Multiparametric magnetic resonance imaging of the skeletal muscle highlighted an alteration of the fiber organization in IR rats as demonstrated by a significant decrease of the mean diffusivity in the gastrocnemius at short term. Alteration of fibers was confirmed by histologic analyses: the number of type I fibers was decreased, whereas that of type IIa fibers was increased in IR animals but not in the IR+PA group. CONCLUSIONS: The data show that WBI induces skeletal muscle damage, which is attenuated by PA. This muscle damage may explain, at least in part, the fatigue of patients treated with radiation therapy.

Hypoxia-induced activation of NDR2 underlies brain metastases from Non-Small Cell Lung Cancer.

The molecular mechanisms induced by hypoxia are misunderstood in non-small cell lung cancer (NSCLC), and above all the hypoxia and RASSF1A/Hippo signaling relationship. We confirmed that human NSCLC (n = 45) as their brain metastases (BM) counterpart are hypoxic since positive with CAIX-antibody (target gene of Hypoxia-inducible factor (HIF)). A severe and prolonged hypoxia (0.2% O2, 48 h) activated YAP (but not TAZ) in Human Bronchial Epithelial Cells (HBEC) lines by downregulating RASSF1A/kinases Hippo (except for NDR2) regardless their promoter methylation status. Subsequently, the NDR2-overactived HBEC cells exacerbated a HIF-1A, YAP and C-Jun-dependent-amoeboid migration, and mainly, support BM formation. Indeed, NDR2 is more expressed in human tumor of metastatic NSCLC than in human localized NSCLC while NDR2 silencing in HBEC lines (by shRNA) prevented the xenograft formation and growth in a lung cancer-derived BM model in mice. Collectively, our results indicated that NDR2 kinase is over-active in NSCLC by hypoxia and supports BM formation. NDR2 expression is thus a useful biomarker to predict the metastases risk in patients with NSCLC, easily measurable routinely by immunohistochemistry on tumor specimens.

Assessment of hypoxia and oxidative-related changes in a lung-derived brain metastasis model by [64Cu][Cu(ATSM)] PET and proteomic studies.

BACKGROUND: Brain metastases (BM) are the most frequent malignant brain tumors. The aim of this study was to characterize the tumor microenvironment (TME) of BM and particularly hypoxia and redox state, known to play a role in tumor growth and treatment resistance with multimodal PET and MRI imaging, immunohistochemical and proteomic approaches in a human lung cancer (H2030-BrM3)-derived BM model in rats. RESULTS: First, in vitro studies confirmed that H2030-BrM3 cells respond to hypoxia with increasing expression of HIF-1, HIF-2 and their target genes. Proteomic analyses revealed, among expression changes, proteins associated with metabolism, oxidative stress, metal response and hypoxia signaling in particular in cortical BM. [64Cu][Cu(ATSM)] PET revealed a significant uptake by cortical BM (p < 0.01), while no uptake is observed in striatal BM 23 days after tumor implantation. Pimonidazole, HIF-1α, HIF-2α, CA-IX as well as GFAP, CTR1 and DMT1 immunostainings are positive in both BM. CONCLUSION: Overall, [64Cu][Cu(ATSM)] imaging and proteomic results showed the presence of hypoxia and protein expression changes linked to hypoxia and oxidative stress in BM, which are more pronounced in cortical BM compared to striatal BM. Moreover, it emphasized the interest of [64Cu][Cu(ATSM)] PET to characterize TME of BM and depict inter-metastasis heterogeneity that could be useful to guide treatments.

Application of a new spectral deconvolution method for in vitro dosimetry in assessment of targeted alpha therapy.

BACKGROUND: The improvement of in vitro assessment of targeted alpha therapy (reproducibility, comparability of experiments…) requires precise evaluation of the dose delivered to the cells. To answer this need, a previous study proposed an innovative dosimetry method based on α-spectroscopy and a specific deconvolution process to recover the spatial distribution of 212 Pb isotopes inside in vitro culture wells. Nevertheless, although promising, the deconvolution method was time consuming and only tested for a simple isotope decay chain. PURPOSE: The purpose of this work is to propose a new matrix deconvolution method of α spectra based on a constrained-non-negative-maximum-likelihood decomposition, both faster and offering a greater modelling flexibility, allowing to study independently the kinetics of each of the daughter nuclides of complex decay chains (illustrated here with 223 Ra) in in vitro culture wells. METHODS: Firstly, the performance of the new method was fully evaluated through Monte Carlo simulations of in vitro irradiations. Different spatial distributions of 212 Pb and 223 Ra, the corresponding α spectra measured by a silicon detector and the doses delivered to the cells were simulated with Geant4. The deconvolution results were then compared to the simulation results. Secondly, measurements were carried out in culture wells without cells containing 15 kBq of 212 Pb or 9.3 kBq of 223 Ra, placed above silicon detectors recording α spectra in real time. The matrix deconvolution was then applied to determine the spatial and temporal distribution of all α-emitting daughters of studied isotopes. RESULTS: The matrix deconvolution was proved to recover the simulated distribution gradients, ensuring simulated doses within 3 % for both tested radionuclides, with errors on dose normally distributed around the reference value (consequently not exhibiting any bias), even in the case of complex decay chains as 223 Ra. The experimental study of 212 Pb and 223 Ra showed highly inhomogeneous distributions and time evolution of the concentration gradients, consistent with the previous study. Furthermore, it highlighted the complex kinetics of 223 Ra with different distributions of its α-emitting daughters (219 Rn, 215 Po, 215 At, 211 Bi, 211 Po). CONCLUSIONS: This study validates a new deconvolution method, fast and flexible, that proved to be accurate and reliable. This method allowed to reveal the complexity of isotopes kinetics in in vitro experiments, especially with complex decay chains. Experimental dosimetry, necessary to improve reliability of in vitro studies in targeted alpha therapy, is demonstrated to be feasible with the proposed method.

Beyond lymphopenia, unraveling radiation-induced leucocyte subpopulation kinetics and mechanisms through modeling approaches.

Leucocyte subpopulations in both lymphoid and myeloid lineages have a significant impact on antitumor immune response. While radiation-induced lymphopenia is being studied extensively, radiation effects on lymphoid and myeloid subtypes have been relatively less addressed. Interactions between leucocyte subpopulations, their specific radiation sensitivity and the specific kinetics of each subpopulation can be modeled based on both experimental data and knowledge of physiological leucocyte depletion, production, proliferation, maturation and homeostasis. Modeling approaches of the leucocyte kinetics that may be used to unravel mechanisms underlying radiation induced-leucopenia and prediction of changes in cell counts and compositions after irradiation are presented in this review. The approaches described open up new possibilities for determining the influence of irradiation parameters both on a single-time point of acute effects and the subsequent recovery of leukocyte subpopulations. Utilization of these approaches to model kinetic data in post-radiotherapy states may be a useful tool for further development of new treatment strategies or for the combination of radiotherapy and immunotherapy.

Magnetic resonance imaging of hypoxia in acute stroke compared with fluorine-18 fluoromisonidazole-positron emission tomography: A cross-validation study?

Acute ischemic stroke results in an ischemic core surrounded by a tissue at risk, named the penumbra, which is potentially salvageable. One way to differentiate the tissues is to measure the hypoxia status. The purpose of the current study is to correlate the abnormal brain tissue volume derived from magnetic resonance-based imaging of brain oxygen saturation (St O2 -MRI) to the fluorine-18 fluoromisonidazole ([18 F]FMISO) positron emission tomography (PET) volume for hypoxia imaging validation, and to analyze the ability of St O2 -MRI to depict the different hypoxic tissue types in the acute phase of stroke. In a pertinent model of stroke in the rat, the volume of tissue with decreased St O2 -MRI signal and that with increased uptake of [18 F]FMISO were equivalent and correlated (r = 0.706; p = 0.015). The values of St O2 in the tissue at risk were significantly greater than those quantified in the core of the lesion, and were less than those for healthy tissue (52.3% ± 2.0%; 43.3% ± 1.9%, and 67.9 ± 1.4%, respectively). A threshold value for St O2 of ≈60% as the cut-off for the identification of the tissue at risk was calculated. Tissue volumes with reduced St O2 -MRI correlated with the final lesion (r = 0.964, p < 0.0001). The findings show that the St O2 -MRI approach is sensitive for the detection of hypoxia and for the prediction of the final lesion after stroke. Once validated in acute clinical settings, this approach might be used to enhance the stratification of patients for potential therapeutic interventions.

Internalization study of nanosized zeolite crystals in human glioblastoma cells.

While the use of nanozeolites for cancer treatment holds a great promise, it also requires a better understanding of the interaction between the zeolite nanoparticles and cancer cells and notably their internalization and biodistribution. It is particularly important in situation of hypoxia, a very common situations in aggressive cancers, which may change the energetic processes required for cellular uptake. Herein, we studied, in vitro, the kinetics of the internalization process and the intracellular localization of nanosized zeolite X (FAU-X) into glioblastoma cells. In normoxic conditions, scanning electron microscopy (SEM) showed a rapid cell membrane adhesion of zeolite nanoparticles (< 5 min following application in the cell medium), occurring before an energy-dependent uptake which appeared between 1 h and 4 h. Additionally, transmission electron microscopy (TEM) and flow cytometry analyzes, confirmed that the zeolite nanoparticles accumulate over time into the cytoplasm and were mostly located into vesicles visible at least up to 6 days. Interestingly, the uptake of zeolite nanoparticles was found to be dependent on oxygen concentration, i.e. an increase in internalization in severe hypoxia (0.2 % of O2) was observed. No toxicity of zeolite FAU-X nanoparticles was detected after 24 h and 72 h. The results clearly showed that the nanosized zeolites crystals were rapidly internalized via energy-requiring mechanism by cancer cells and even more in the hypoxic conditions. Once the zeolite nanoparticles were internalized into cells, they appeared to be safe and stable and therefore, they are envisioned to be used as carrier of various compounds to target cancer cells.

First-in-Man Noninvasive Initial Diagnostic Approach of Primary CNS Lymphoma Versus Glioblastoma Using PET With 18 F-Fludarabine and l -[methyl- 11 C]Methionine.

OBJECTIVES: This study sought to assess 18 F-fludarabine ( 18 F-FLUDA) PET/CT's ability in differentiating primary central nervous system lymphomas (PCNSLs) from glioblastoma multiformes (GBMs). PATIENTS AND METHODS: Patients harboring either PCNSL (n = 8) before any treatment, PCNSL treated using corticosteroids (PCNSLh; n = 10), or GBM (n = 13) were investigated with conventional MRI and PET/CT, using 11 C-MET and 18 F-FLUDA. The main parameters measured with each tracer were SUV T and T/N ratios for the first 30 minutes of 11 C-MET acquisition, as well as at 3 different times after 18 F-FLUDA injection. The early 18 F-FLUDA uptake within the first minute of injection was equally considered, whereas this parameter was combined with the later uptakes to obtain R FLUDA 2 and R FLUDA 3 ratios. RESULTS: No significant differences in 11 C-MET uptakes were observed among PCNSL, PCNSLh, and GBM. With 18 F-FLUDA, a clear difference in dynamic GBM uptake was observed, which decreased over time after an early maximum, as compared with that of PCNSL, which steadily increased over time, PCNSLh exhibiting intermediate values. The most discriminative parameters consisting of R FLUDA 2 and R FLUDA 3 integrated the early tracer uptake (first 60 seconds), thereby provided 100% specificity and sensitivity. CONCLUSIONS: 18 F-FLUDA was shown to likely be a promising radiopharmaceutical for differentiating PCNSL from other malignancies, although a pretreatment with corticosteroids might compromise this differential diagnostic ability. The diagnostic role of 18 F-FLUDA should be further investigating, along with its potential of defining therapeutic strategies in patients with PCNSL, while assessing the treatments' effectiveness.

The Reactive Species Interactome in the Brain.

Significance: Redox pioneer Helmut Sies attempted to explain reactive species' challenges faced by organelles, cells, tissues, and organs via three complementary definitions: (i) oxidative stress, that is, the disturbance in the prooxidant-antioxidant defense balance in favor of the prooxidants; (ii) oxidative eustress, the low physiological exposure to prooxidants; and (iii) oxidative distress, the supraphysiological exposure to prooxidants. Recent Advances: Identification, concentration, and interactions are the most important elements to improve our understanding of reactive species in physiology and pathology. In this context, the reactive species interactome (RSI) is a new multilevel redox regulatory system that identifies reactive species families, reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species, and it integrates their interactions with their downstream biological targets. Critical Issues: We propose a united view to fully combine reactive species identification, oxidative eustress and distress, and the RSI system. In this view, we also propose including the forgotten reactive carbonyl species, an increasingly rediscovered reactive species family related to the other reactive families, and key enzymes within the RSI. We focus on brain physiology and pathology to demonstrate why this united view should be considered. Future Directions: More studies are needed for an improved understanding of the contributions of reactive species through their identification, concentration, and interactions, including in the brain. Appreciating the RSI in its entirety should unveil new molecular players and mechanisms in physiology and pathology in the brain and elsewhere.

Radioimmunotherapy for Brain Metastases: The Potential for Inflammation as a Target of Choice.

Brain metastases (BM) are frequently detected during the follow-up of patients with malignant tumors, particularly in those with advanced disease. Despite a major progress in systemic anti-cancer treatments, the average overall survival of these patients remains limited (6 months from diagnosis). Also, cognitive decline is regularly reported especially in patients treated with whole brain external beam radiotherapy (WBRT), due to the absorbed radiation dose in healthy brain tissue. New targeted therapies, for an earlier and/or more specific treatment of the tumor and its microenvironment, are needed. Radioimmunotherapy (RIT), a combination of a radionuclide to a specific antibody, appears to be a promising tool. Inflammation, which is involved in multiple steps, including the early phase, of BM development is attractive as a relevant target for RIT. This review will focus on the (1) early biomarkers of inflammation in BM pertinent for RIT, (2) state of the art studies on RIT for BM, and (3) the importance of dosimetry to RIT in BM. These two last points will be addressed in comparison to the conventional EBRT treatment, particularly with respect to the balance between tumor control and healthy tissue complications. Finally, because new diagnostic imaging techniques show a potential for the detection of BM at an early stage of the disease, we focus particularly on this therapeutic window.

Connexin 43 and Sonic Hedgehog Pathway Interplay in Glioblastoma Cell Proliferation and Migration.

Glioblastoma (GBM) represents the most common primary brain tumor within the adult population. Current therapeutic options are still limited by high rate of recurrences and signalling axes that promote GBM aggressiveness. The contribution of gap junctions (GJs) to tumor growth and progression has been proven by experimental evidence. Concomitantly, tumor microenvironment has received increasing interest as a critical process in dysregulation and homeostatic escape, finding a close link between molecular mechanisms involved in connexin 43 (CX43)-based intercellular communication and tumorigenesis. Moreover, evidence has come to suggest a crucial role of sonic hedgehog (SHH) signalling pathway in GBM proliferation, cell fate and differentiation. Herein, we used two human GBM cell lines, modulating SHH signalling and CX43-based intercellular communication in in vitro models using proliferation and migration assays. Our evidence suggests that modulation of the SHH effector smoothened (SMO), by using a known agonist (i.e., purmorphamine) and a known antagonist (i.e., cyclopamine), affects the CX43 expression levels and therefore the related functions. Moreover, SMO activation also increased cell proliferation and migration. Importantly, inhibition of CX43 channels was able to prevent SMO-induced effects. SHH pathway and CX43 interplay acts inducing tumorigenic program and supporting cell migration, likely representing druggable targets to develop new therapeutic strategies for GBM.

Multimodal evaluation of hypoxia in brain metastases of lung cancer and interest of hypoxia image-guided radiotherapy.

Lung cancer patients frequently develop brain metastases (BM). Despite aggressive treatment including neurosurgery and external-radiotherapy, overall survival remains poor. There is a pressing need to further characterize factors in the microenvironment of BM that may confer resistance to radiotherapy (RT), such as hypoxia. Here, hypoxia was first evaluated in 28 biopsies from patients with non­small cell lung cancer (NSCLC) BM, using CA-IX immunostaining. Hypoxia characterization (pimonidazole, CA-IX and HIF-1α) was also performed in different preclinical NSCLC BM models induced either by intracerebral injection of tumor cells (H2030-Br3M, H1915) into the cortex and striatum, or intracardial injection of tumor cells (H2030-Br3M). Additionally, [18F]-FMISO-PET and oxygen-saturation-mapping-MRI (SatO2-MRI) were carried out in the intracerebral BM models to further characterize tumor hypoxia and evaluate the potential of Hypoxia-image-guided-RT (HIGRT). The effect of RT on proliferation of BM ([18F]-FLT-PET), tumor volume and overall survival was determined. We showed that hypoxia is a major yet heterogeneous feature of BM from lung cancer both preclinically and clinically. HIGRT, based on hypoxia heterogeneity observed between cortical and striatal metastases in the intracerebrally induced models, showed significant potential for tumor control and animal survival. These results collectively highlight hypoxia as a hallmark of BM from lung cancer and the value of HIGRT in better controlling tumor growth.

Simultaneous Mapping of Vasculature, Hypoxia, and Proliferation Using Dynamic Susceptibility Contrast MRI, 18F-FMISO PET, and 18F-FLT PET in Relation to Contrast Enhancement in Newly Diagnosed Glioblastoma.

Conventional MRI plays a key role in the management of patients with high-grade glioma, but multiparametric MRI and PET tracers could provide further information to better characterize tumor metabolism and heterogeneity by identifying regions having a high risk of recurrence. In this study, we focused on proliferation, hypervascularization, and hypoxia, all factors considered indicative of poor prognosis. They were assessed by measuring uptake of 18F-3'-deoxy-3'-18F-fluorothymidine (18F-FLT), relative cerebral blood volume (rCBV) maps, and uptake of 18F-fluoromisonidazole (18F-FMISO), respectively. For each modality, the volumes and high-uptake subvolumes (hot spots) were semiautomatically segmented and compared with the contrast enhancement (CE) volume on T1-weighted gadolinium-enhanced (T1w-Gd) images, commonly used in the management of patients with glioblastoma. Methods: Dynamic susceptibility contrast-enhanced MRI (31 patients), 18F-FLT PET (20 patients), or 18F-FMISO PET (20 patients), for a total of 31 patients, was performed on preoperative glioblastoma patients. Volumes and hot spots were segmented on SUV maps for 18F-FLT PET (using the fuzzy locally adaptive bayesian algorithm) and 18F-FMISO PET (using a mean contralateral image + 3.3 SDs) and on rCBV maps (using a mean contralateral image + 1.96 SDs) for dynamic susceptibility contrast-enhanced MRI and overlaid on T1w-Gd images. For each modality, the percentages of the peripheral volumes and the peripheral hot spots outside the CE volume were calculated. Results: All tumors showed highly proliferated, hypervascularized, and hypoxic regions. The images also showed pronounced heterogeneity of both tracers regarding their uptake and rCBV maps, within each individual patient. Overlaid volumes on T1w-Gd images showed that some proliferative, hypervascularized, and hypoxic regions extended beyond the CE volume but with marked differences between patients. The ranges of peripheral volume outside the CE volume were 1.6%-155.5%, 1.5%-89.5%, and 3.1%-78.0% for 18F-FLT, rCBV, and 18F-FMISO, respectively. All patients had hyperproliferative hot spots outside the CE volume, whereas hypervascularized and hypoxic hot spots were detected mainly within the enhancing region. Conclusion: Spatial analysis of multiparametric maps with segmented volumes and hot spots provides valuable information to optimize the management and treatment of patients with glioblastoma.

The Impact of Resampling and Denoising Deep Learning Algorithms on Radiomics in Brain Metastases MRI.

BACKGROUND: Magnetic resonance imaging (MRI) is predominant in the therapeutic management of cancer patients, unfortunately, patients have to wait a long time to get an appointment for examination. Therefore, new MRI devices include deep-learning (DL) solutions to save acquisition time. However, the impact of these algorithms on intensity and texture parameters has been poorly studied. The aim of this study was to evaluate the impact of resampling and denoising DL models on radiomics. METHODS: Resampling and denoising DL model was developed on 14,243 T1 brain images from 1.5T-MRI. Radiomics were extracted from 40 brain metastases from 11 patients (2049 images). A total of 104 texture features of DL images were compared to original images with paired t-test, Pearson correlation and concordance-correlation-coefficient (CCC). RESULTS: When two times shorter image acquisition shows strong disparities with the originals concerning the radiomics, with significant differences and loss of correlation of 79.81% and 48.08%, respectively. Interestingly, DL models restore textures with 46.15% of unstable parameters and 25.96% of low CCC and without difference for the first-order intensity parameters. CONCLUSIONS: Resampling and denoising DL models reconstruct low resolution and noised MRI images acquired quickly into high quality images. While fast MRI acquisition loses most of the radiomic features, DL models restore these parameters.

Ruthenium tris(2,2'-bipyridyl) complex encapsulated in nanosized faujasite zeolite as intracellular localization tracer.

Designing zeolites for medical applications is a challenging task that requires introducing new functionalities without altering the intrinsic properties such as morphology, crystallinity, colloidal stability, surface charge, and porosity. Herein, we present the encapsulation of luminescent ruthenium-tris(2,2'-bipyridyl) complex in faujasite (FAU) zeolite nanocrystals (Ru(bpy)3-FAU) and their use as an intracellular localization tracer. Upon exciting the Ru(bpy)3-FAU zeolite at 450 nm, the sample gives rise to an orange-red emission at 628 nm, thus permitting its use for cellular imaging and localization of the zeolite nanoparticles. The nanosized Ru(bpy)3-FAU zeolite is characterized in terms of size, charge, crystallinity, morphology, porosity, thermal stability, and sorption capacity. The potential toxicity of Ru(bpy)3-FAU on U251-MG glioblastoma cells was evaluated. A safe concentration (50-100 µg/ml) for the Ru(bpy)3-FAU zeolite is identified. The luminescent properties of the ruthenium complex confined in the zeolite nanocrystals allow their localization in the U251-MG cells with a main accumulation in the cytoplasm. The Ru(bpy)3-FAU nanosized zeolite is a potential candidate for biological applications for being stable, safe, capable of loading respiratory gases, and easily probed in the cells owing to its luminescent properties.

Angiopoietin-2 Combined with Radiochemotherapy Impedes Glioblastoma Recurrence by Acting in an Autocrine and Paracrine Manner: A Preclinical Study.

(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.

The Role of Hypoxia and SRC Tyrosine Kinase in Glioblastoma Invasiveness and Radioresistance.

Advances in functional imaging are supporting neurosurgery and radiotherapy for glioblastoma, which still remains the most aggressive brain tumor with poor prognosis. The typical infiltration pattern of glioblastoma, which impedes a complete surgical resection, is coupled with a high rate of invasiveness and radioresistance, thus further limiting efficient therapy, leading to inevitable and fatal recurrences. Hypoxia is of crucial importance in gliomagenesis and, besides reducing radiotherapy efficacy, also induces cellular and molecular mediators that foster proliferation and invasion. In this review, we aimed at analyzing the biological mechanism of glioblastoma invasiveness and radioresistance in hypoxic niches of glioblastoma. We also discussed the link between hypoxia and radiation-induced radioresistance with activation of SRC proto-oncogene non-receptor tyrosine kinase, prospecting potential strategies to overcome the current limitation in glioblastoma treatment.

Nanosized zeolites as a gas delivery platform in a glioblastoma model.

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.

Impact of Hypoxia on Carbon Ion Therapy in Glioblastoma Cells: Modulation by LET and Hypoxia-Dependent Genes.

Tumor hypoxia is known to limit the efficacy of ionizing radiations, a concept called oxygen enhancement ratio (OER). OER depends on physical factors such as pO2 and linear energy transfer (LET). Biological pathways, such as the hypoxia-inducible transcription factors (HIF), might also modulate the influence of LET on OER. Glioblastoma (GB) is resistant to low-LET radiation (X-rays), due in part to the hypoxic environment in this brain tumor. Here, we aim to evaluate in vitro whether high-LET particles, especially carbon ion radiotherapy (CIRT), can overcome the contribution of hypoxia to radioresistance, and whether HIF-dependent genes, such as erythropoietin (EPO), influence GB sensitivity to CIRT. Hypoxia-induced radioresistance was studied in two human GB cells (U251, GL15) exposed to X-rays or to carbon ion beams with various LET (28, 50, 100 keV/µm), and in genetically-modified GB cells with downregulated EPO signaling. Cell survival, radiobiological parameters, cell cycle, and ERK activation were assessed under those conditions. The results demonstrate that, although CIRT is more efficient than X-rays in GB cells, hypoxia can limit CIRT efficacy in a cell-type manner that may involve differences in ERK activation. Using high-LET carbon beams, or targeting hypoxia-dependent genes such as EPO might reduce the effects of hypoxia.