Preclinical evaluation of CXCR4 peptides for targeted radionuclide therapy in glioblastoma.

BACKGROUND: Glioblastoma (GBM), is the most fatal form of brain cancer, with a high tendency for recurrence despite combined treatments including surgery, radiotherapy, and chemotherapy with temozolomide. The C-X-C chemokine receptor 4 (CXCR4) plays an important role in tumour radioresistance and recurrence, and is considered as an interesting GBM target. TRT holds untapped potential for GBM treatment, with CXCR4-TRT being a promising strategy for recurrent GBM treatment. Our study focuses on the preclinical assessment of different 177Lu-labelled CXCR4-targeting peptides, CTCE-9908, DV1-K-DV3, and POL3026 for GBM treatment and exploring some of the radiobiological mechanisms underlying these therapies. RESULTS: All three DOTA-conjugated peptides could be radiolabelled with 177Lu with > 95% radiochemical yield. Binding studies show high specific binding of [177Lu]Lu-DOTA-POL3026 to U87-CXCR4 + cells, with 42% of the added activity binding to the membrane at 1 nM, and 6.5% internalised into the cells. In the presence of the heterologous CXCR4 blocking agent, AMD11070, membrane binding was reduced by 95%, a result confirmed by quantitative in vitro autoradiography of orthotopic GBM xenograft sections. An activity-dependent decrease in cell viability was observed for [177Lu]Lu-DOTA-DV1-K-DV3 and [177Lu]Lu-DOTA-POL3026, along with a slight increase in the induction of apoptotic markers. Additionally, the expression of γH2AX increased in a time-and activity-dependent manner. Ex vivo biodistribution studies with [177Lu]Lu-DOTA-POL3026 show uptake in the tumour reaching a SUV of 1.9 at 24 h post-injection, with higher uptake in the kidneys, lungs, spleen, and liver. Dosimetry estimations show an absorbed dose of 0.93 Gy/MBq in the tumour. A blocking study with AMD11070 showed a 38% reduction in tumour uptake, with no significant reduction observed in µSPECT imaging. Although no brain uptake was observed in the ex vivo biodistribution study, autoradiography on U87-CXCR4 + tumour inoculated mouse brain slices shows non-specific binding in the brain, next to high specific binding to the tumour. CONCLUSIONS: In conclusion, we compared different 177Lu-radiolabelled CXCR4-targeting peptides for their binding potential in GBM, and demonstrated their varied cytotoxic action against GBM cells in vitro, with POL3026 being the most promising, causing considerable DNA damage. Though the peptide's systemic biodistribution remains to be improved, our data demonstrate the potential of [177Lu]Lu-DOTA-POL3026 for CXCR4-TRT in the context of GBM.

Comprehensive profiling of stem-like features in pediatric glioma cell cultures and their relation to the subventricular zone.

Pediatric high-grade gliomas (pHGG) are brain tumors occurring in children and adolescents associated with a dismal prognosis despite existing treatments. Therapeutic failure in both adult and pHGG has been partially imputed to glioma stem cells (GSC), a subset of cancer cells endowed with stem-like cell potential and malignant, invasive, adaptative, and treatment-resistant capabilities. Whereas GSC have largely been portrayed in adult tumors, less information has been provided in pHGG. The aim of our study was to comprehensively document the stem-like capacities of seven in-use pediatric glioma cell cultures (Res259, UW479, SF188, KNS42, SF8628, HJSD-DIPG-007 and HJSD-DIPG-012) using parallel in vitro assays assessing stem cell-related protein expression, multipotency, self-renewal and proliferation/quiescence, and in vivo investigation of their tumorigenicity and invasiveness. Data obtained from in vitro experiments revealed glioma subtype-dependent expression of stem cell-related markers and varying abilities for differentiation, self-renewal, and proliferation/quiescence. Among tested cultures, DMG H3-K27 altered cultures displayed a particular pattern of stem-like markers expression and a higher fraction of cells with self-renewal potential. Four cultures displaying distinctive stem-like profiles were further tested for their ability to initiate tumors and invade the brain tissue in mouse orthotopic xenografts. The selected cell cultures all showed a great tumor formation capacity, but only DMG H3-K27 altered cells demonstrated a highly infiltrative phenotype. Interestingly, we detected DMG H3-K27 altered cells relocated in the subventricular zone (SVZ), which has been previously described as a neurogenic area, but also a potential niche for brain tumor cells. Finally, we observed an SVZ-induced phenotypic modulation of the glioma cells, as evidenced by their increased proliferation rate. In conclusion, this study recapitulated a systematic stem-like profiling of various pediatric glioma cell cultures and call to a deeper characterization of DMG H3-K27 altered cells nested in the SVZ.

Study of Strawberry Notch homolog 1 and 2 expression in human glioblastoma.

PURPOSE: In this work, we aimed to comprehensively document the expression of Strawberry Notch homolog (SBNO) 1 and 2 in glioblastoma (GBM) tissue and patient-derived GBM stem-like cell (GSC) cultures. METHODS: We investigated SBNO1 and SBNO2 expression at the RNA and protein levels in glioma patient tissue and GSCs, respectively by performing immunostainings and qPCR analyses. We also used publicly-available datasets for assessing SBNO1 and SBNO2 gene expression and related copy number alterations. We used lentiviral transduction of SBNO2 to analyze the effect of its expression in patient-derived GSCs. RESULTS: We observed that SBNO2 expression is increased in GBM tissue samples compared to non tumoral brain, or lower-grade gliomas, whereas SBNO1 expression remains unchanged. We hypothesized that such SBNO2 high expression might be linked to copy-number alterations at the level of the 19p13 chromosome section. We located SBNO1 and SBNO2 in different subcellular compartments. Finally, we observed that SBNO2 overexpression induces different phenotypes in different patient-derived GSCs. CONCLUSION: These results provide the first characterization of SBNO1 and SBNO2 expression in glioma tissue, and indicate SBNO2 as highly expressed in GBM.

Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4+ GBM cells: A proof of principle.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, which remains difficult to cure. The very high recurrence rate has been partly attributed to the presence of GBM stem-like cells (GSCs) within the tumors, which have been associated with elevated chemokine receptor 4 (CXCR4) expression. CXCR4 is frequently overexpressed in cancer tissues, including GBM, and usually correlates with a poor prognosis. We have created a CXCR4-retargeted oncolytic herpesvirus (oHSV) by insertion of an anti-human CXCR4 nanobody in glycoprotein D of an attenuated HSV-1 (ΔICP34.5, ΔICP6, and ΔICP47), thereby describing a proof of principle for the use of nanobodies to target oHSVs toward specific cellular entities. Moreover, this virus has been armed with a transgene expressing a soluble form of TRAIL to trigger apoptosis. In vitro, this oHSV infects U87MG CXCR4+ and patient-derived GSCs in a CXCR4-dependent manner and, when armed, triggers apoptosis. In a U87MG CXCR4+ orthotopic xenograft mouse model, this oHSV slows down tumor growth and significantly improves mice survival. Customizing oHSVs with diverse nanobodies for targeting multiple proteins appears as an interesting approach for tackling the heterogeneity of GBM, especially GSCs. Altogether, our study must be considered as a proof of principle and a first step toward personalized GBM virotherapies to complement current treatments.

Glioma Stem Cells in Pediatric High-Grade Gliomas: From Current Knowledge to Future Perspectives.

In children, high-grade gliomas (HGG) and diffuse midline gliomas (DMG) account for a high proportion of death due to cancer. Glioma stem cells (GSCs) are tumor cells in a specific state defined by a tumor-initiating capacity following serial transplantation, self-renewal, and an ability to recapitulate tumor heterogeneity. Their presence was demonstrated several decades ago in adult glioblastoma (GBM), and more recently in pediatric HGG and DMG. In adults, we and others have previously suggested that GSCs nest into the subventricular zone (SVZ), a neurogenic niche, where, among others, they find shelter from therapy. Both bench and bedside evidence strongly indicate a role for the GSCs and the SVZ in GBM progression, fostering the development of innovative targeting treatments. Such new therapeutic approaches are of particular interest in infants, in whom standard therapies are often limited due to the risk of late effects. The aim of this review is to describe current knowledge about GSCs in pediatric HGG and DMG, i.e., their characterization, the models that apply to their development and maintenance, the specific signaling pathways that may underlie their activity, and their specific interactions with neurogenic niches. Finally, we will discuss the clinical relevance of these observations and the therapeutic advantages of targeting the SVZ and/or the GSCs in infants.

Nanoluciferase-based methods to monitor activation, modulation and trafficking of atypical chemokine receptors.

Chemokines regulate directed cell migration, proliferation and survival and are key components in various physiological and pathological processes. They exert their functions by interacting with seven-transmembrane domain receptors that signal through G proteins (GPCRs). Atypical chemokine receptors (ACKRs) play important roles in the chemokine-receptor network by regulating chemokine bioavailability for the classical receptors through chemokine sequestration, scavenging or transport. Currently, this subfamily of receptors comprises four members: ACKR1, ACKR2, ACKR3 and ACKR4. They differ notably from the classical chemokine receptors by their inability to elicit G protein-mediated signaling, which precludes the use of classical assays relying on the activation of G proteins and related downstream secondary messengers to investigate ACKRs. There is therefore a need for alternative approaches to monitor ACKR activation, modulation and trafficking. This chapter details sensitive and versatile methods based on Nanoluciferase Binary Technology (NanoBiT) and Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) to monitor ACKR2 and ACKR3 activity through the measurement of ß-arrestin and GRK recruitment, and receptor trafficking, including internalization and delivery to early endosomes.

Eccentric Exercise Causes Specific Adjustment in Pyruvate Oxidation by Mitochondria.

INTRODUCTION: The impact of eccentric exercise on mitochondrial function has only been poorly investigated and remains unclear. This study aimed to identify the changes in skeletal muscle mitochondrial respiration, specifically triggered by a single bout of eccentric treadmill exercise. METHODS: Male adult mice were randomly divided into eccentric (ECC; downhill running), concentric (CON; uphill running), and unexercised control groups ( n = 5/group). Running groups performed 18 bouts of 5 min at 20 cm·s -1 on an inclined treadmill (±15° to 20°). Mice were sacrificed 48 h after exercise for blood and quadriceps muscles collection. Deep proximal (red) and superficial distal (white) muscle portions were used for high-resolution respirometric measurements. RESULTS: Plasma creatine kinase activity was significantly higher in the ECC compared with CON group, reflecting exercise-induced muscle damage ( P < 0.01). The ECC exercise induced a significant decrease in oxidative phosphorylation capacity in both quadriceps femoris parts ( P = 0.032 in proximal portion, P = 0.010 in distal portion) in comparison with the CON group. This observation was only made for the nicotinamide adenine dinucleotide (NADH) pathway using pyruvate + malate as substrates. When expressed as a flux control ratio, indicating a change related to mitochondrial quality rather than quantity, this change seemed more prominent in distal compared with proximal portion of quadriceps muscle. No significant difference between groups was found for the NADH pathway with glutamate or glutamate + malate as substrates, for the succinate pathway or for fatty acid oxidation. CONCLUSIONS: Our data suggest that ECC exercise specifically affects pyruvate mitochondrial transport and/or oxidation 48 h after exercise, and this alteration mainly concerns the distal white muscle portion. This study provides new perspectives to improve our understanding of the mitochondrial adaptation associated with ECC exercise.

Correction: In Vivo Tumorigenesis Was Observed after Injection of In Vitro Expanded Neural Crest Stem Cells Isolated from Adult Bone Marrow.

[This corrects the article DOI: 10.1371/journal.pone.0046425.].

Correction: Human bone marrow harbors cells with neural crest-associated characteristics like human adipose and dermis tissues.

[This corrects the article DOI: 10.1371/journal.pone.0177962.].

The expression of B7-H3 isoforms in newly diagnosed glioblastoma and recurrence and their functional role.

Short survival of glioblastoma (GBM) patients is due to systematic tumor recurrence. Our laboratory identified a GBM cell subpopulation able to leave the tumor mass (TM) and invade the subventricular zone (SVZ-GBM cells). SVZ-GBM cells escape treatment and appear to contribute to GBM recurrence. This study aims to identify proteins specifically expressed by SVZ-GBM cells and to define their role(s) in GBM aggressiveness and recurrence. The proteome was compared between GBM cells located in the initial TM and SVZ-GBM cells using mass spectrometry. Among differentially expressed proteins, we confirmed B7-H3 by western blot (WB) and quantitative RT-PCR. B7-H3 expression was compared by immunohistochemistry and WB (including expression of its isoforms) between human GBM (N = 14) and non-cancerous brain tissue (N = 8), as well as newly diagnosed GBM and patient-matched recurrences (N = 11). Finally, the expression of B7-H3 was modulated with short hairpin RNA and/or over-expression vectors to determine its functional role in GBM using in vitro assays and a xenograft mouse model of GBM. B7-H3 was a marker for SVZ-GBM cells. It was also increased in human GBM pericytes, myeloid cells and neoplastic cells. B7-H3 inhibition in GBM cells reduced their tumorigenicity. Out of the two B7-H3 isoforms, only 2IgB7-H3 was detected in non-cancerous brain tissue, whereas 4IgB7-H3 was specific for GBM. 2IgB7-H3 expression was higher in GBM recurrences and increased resistance to temozolomide-mediated apoptosis. To conclude, 4IgB7-H3 is an interesting candidate for GBM targeted therapies, while 2IgB7-H3 could be involved in recurrence through resistance to chemotherapy.

Patient-Oriented Perspective on Chemokine Receptor Expression and Function in Glioma.

Gliomas are severe brain malignancies, with glioblastoma (GBM) being the most aggressive one. Despite continuous efforts for improvement of existing therapies, overall survival remains poor. Over the last years, the implication of chemokines and their receptors in GBM development and progression has become more evident. Recently, large amounts of clinical data have been made available, prompting us to investigate chemokine receptors in GBM from a still-unexplored patient-oriented perspective. This study aims to highlight and discuss the involvement of chemokine receptors-CCR1, CCR5, CCR6, CCR10, CX3CR1, CXCR2, CXCR4, ACKR1, ACKR2, and ACKR3-most abundantly expressed in glioma patients based on the analysis of publicly available clinical datasets. Given the strong intratumoral heterogeneity characterizing gliomas and especially GBM, receptor expression was investigated by glioma molecular groups, by brain region distribution, emphasizing tissue-specific receptor functions, and by cell type enrichment. Our study constitutes a clinically relevant and patient-oriented guide that recapitulates the expression profile and the complex roles of chemokine receptors within the highly diversified glioma landscape. Additionally, it strengthens the importance of patient-derived material for development and precise amelioration of chemokine receptor-targeting therapies.

Exploring with [18F]UCB-H the in vivo Variations in SV2A Expression through the Kainic Acid Rat Model of Temporal Lobe Epilepsy.

PURPOSE: The main purpose of this study was to understand how the positron emission tomography (PET) measure of the synaptic vesicle 2A (SV2A) protein varies in vivo during the development of temporal lobe epilepsy (TLE) in the kainic acid rat model. PROCEDURES: Twenty Sprague Dawley male rats were administered with multiple systemic doses of saline (control group, n = 5) or kainic acid (5 mg/kg/injection, epileptic group, n = 15). Both groups were scanned at the four phases of TLE (early, latent, transition, and chronic phase) with the [18F]UCB-H PET radiotracer and T2-structural magnetic resonance imaging. At the end of the scans (3 months post-status epilepticus), rats were monitored for 7 days with electroencephalography for the detection of spontaneous electrographic seizures. Finally, the immunofluorescence staining for SV2A expression was performed. RESULTS: Control rats presented a significant increase in [18F]UCB-H binding at the last two scans, compared with the first ones (p < 0.001). This increase existed but was lower in epileptic animals, producing significant group differences in all the phases of the disease (p < 0.028). Furthermore, the quantification of the SV2A expression in vivo with the [18F]UCB-H radiotracer or ex vivo with immunofluorescence led to equivalent results, with a positive correlation between both. CONCLUSIONS: Even if further studies in humans are required, the ability to detect a progressive decrease in SV2A expression during the development of temporal lobe epilepsy supports the use of [18F]UCB-H as a useful tool to differentiate, in vivo, between healthy and epileptic animals along with the development of the epileptic disease.

A Composite Sketch of Fast-Spiking Parvalbumin-Positive Neurons.

Parvalbumin-positive neurons are inhibitory neurons that release GABA and are mostly represented by fast-spiking basket or chandelier cells. They constitute a minor neuronal population, yet their peculiar profiles allow them to react quickly to any event in the brain under normal or pathological conditions. In this review, we will summarize the current knowledge about the fundamentals of fast-spiking parvalbumin-positive neurons, focusing on their morphology and specific channel/protein content. Next, we will explore their development, maturation, and migration in the brain. Finally, we will unravel their potential contribution to the physiopathology of epilepsy.

The Subventricular Zone, a Hideout for Adult and Pediatric High-Grade Glioma Stem Cells.

Both in adult and children, high-grade gliomas (WHO grades III and IV) account for a high proportion of death due to cancer. This poor prognosis is a direct consequence of tumor recurrences occurring within few months despite a multimodal therapy consisting of a surgical resection followed by chemotherapy and radiotherapy. There is increasing evidence that glioma stem cells (GSCs) contribute to tumor recurrences. In fact, GSCs can migrate out of the tumor mass and reach the subventricular zone (SVZ), a neurogenic niche persisting after birth. Once nested in the SVZ, GSCs can escape a surgical intervention and resist to treatments. The present review will define GSCs and describe their similarities with neural stem cells, residents of the SVZ. The architectural organization of the SVZ will be described both for humans and rodents. The migratory routes taken by GSCs to reach the SVZ and the signaling pathways involved in their migration will also be described hereafter. In addition, we will debate the advantages of the microenvironment provided by the SVZ for GSCs and how this could contribute to tumor recurrences. Finally, we will discuss the clinical relevance of the SVZ in adult GBM and pediatric HGG and the therapeutic advantages of targeting that neurogenic region in both clinical situations.

MKP1 phosphatase is recruited by CXCL12 in glioblastoma cells and plays a role in DNA strand breaks repair.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor in the central nervous system. Previously, the secretion of CXCL12 in the brain subventricular zones has been shown to attract GBM cells and protect against irradiation. However, the exact molecular mechanism behind this radioprotection is still unknown. Here, we demonstrate that CXCL12 modulates the phosphorylation of MAP kinases and their regulator, the nuclear MAP kinase phosphatase 1 (MKP1). We further show that MKP1 is able to decrease GBM cell death and promote DNA repair after irradiation by regulating major apoptotic players, such as Jun-N-terminal kinase, and by stabilizing the DNA repair protein RAD51. Increases in MKP1 levels caused by different corticoid treatments should be reexamined for GBM patients, particularly during their radiotherapy sessions, in order to prevent or to delay the relapses of this tumor.

Relevance of Translation Initiation in Diffuse Glioma Biology and its Therapeutic Potential.

Cancer cells are continually exposed to environmental stressors forcing them to adapt their protein production to survive. The translational machinery can be recruited by malignant cells to synthesize proteins required to promote their survival, even in times of high physiological and pathological stress. This phenomenon has been described in several cancers including in gliomas. Abnormal regulation of translation has encouraged the development of new therapeutics targeting the protein synthesis pathway. This approach could be meaningful for glioma given the fact that the median survival following diagnosis of the highest grade of glioma remains short despite current therapy. The identification of new targets for the development of novel therapeutics is therefore needed in order to improve this devastating overall survival rate. This review discusses current literature on translation in gliomas with a focus on the initiation step covering both the cap-dependent and cap-independent modes of initiation. The different translation initiation protagonists will be described in normal conditions and then in gliomas. In addition, their gene expression in gliomas will systematically be examined using two freely available datasets. Finally, we will discuss different pathways regulating translation initiation and current drugs targeting the translational machinery and their potential for the treatment of gliomas.