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.

fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

Insulin promotes cell migration by regulating PSA-NCAM.

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration.

Interferon-γ blocks signalling through PDGFRß in human brain pericytes.

BACKGROUND: Neuroinflammation and blood-brain barrier (BBB) disruption are common features of many brain disorders, including Alzheimer's disease, epilepsy, and motor neuron disease. Inflammation is thought to be a driver of BBB breakdown, but the underlying mechanisms for this are unclear. Brain pericytes are critical cells for maintaining the BBB and are immunologically active. We sought to test the hypothesis that inflammation regulates the BBB by altering pericyte biology. METHODS: We exposed primary adult human brain pericytes to chronic interferon-gamma (IFNγ) for 4 days and measured associated functional aspects of pericyte biology. Specifically, we examined the influence of inflammation on platelet-derived growth factor receptor-beta (PDGFRß) expression and signalling, as well as pericyte proliferation and migration by qRT-PCR, immunocytochemistry, flow cytometry, and western blotting. RESULTS: Chronic IFNγ treatment had marked effects on pericyte biology most notably through the PDGFRß, by enhancing agonist (PDGF-BB)-induced receptor phosphorylation, internalization, and subsequent degradation. Functionally, chronic IFNγ prevented PDGF-BB-mediated pericyte proliferation and migration. CONCLUSIONS: Because PDGFRß is critical for pericyte function and its removal leads to BBB leakage, our results pinpoint a mechanism linking chronic brain inflammation to BBB dysfunction.

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.

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.

Characterisation of PDGF-BB:PDGFRß signalling pathways in human brain pericytes: evidence of disruption in Alzheimer's disease.

Platelet-derived growth factor-BB (PDGF-BB):PDGF receptor-ß (PDGFRß) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer's disease (AD) is well documented. We found that PDGF-BB:PDGFRß signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB. We hypothesised that reduced PDGF-BB:PDGFRß signalling in pericytes may impact on the BBB. We therefore tested the effects of PDGF-BB on primary human brain pericytes in vitro to define pathways related to BBB function. Using pharmacological inhibitors, we dissected distinct aspects of the PDGF-BB response that are controlled by extracellular signal-regulated kinase (ERK) and Akt pathways. PDGF-BB promotes the proliferation of pericytes and protection from apoptosis through ERK signalling. In contrast, PDGF-BB:PDGFRß signalling through Akt augments pericyte-derived inflammatory secretions. It may therefore be possible to supplement PDGF-BB signalling to stabilise the cerebrovasculature in AD.

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.

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.

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.