Carole Colin

A new alternative mechanism in glioblastoma vascularization: tubular vasculogenic mimicry

Glioblastoma is one of the most angiogenic human tumours and endothelial proliferation is a hallmark of the disease. A better understanding of glioblastoma vasculature is needed to optimize anti-angiogenic therapy that has shown a high but transient efficacy. We analysed human glioblastoma tissues and found non-endothelial cell-lined blood vessels that were formed by tumour cells (vasculogenic mimicry of the tubular type). We hypothesized that CD133+ glioblastoma cells presenting stem-cell properties may express pro-vascular molecules allowing them to form blood vessels de novo. We demonstrated in vitro that glioblastoma stem-like cells were capable of vasculogenesis and endothelium-associated genes expression. Moreover, a fraction of these glioblastoma stem-like cells could transdifferentiate into vascular smooth muscle-like cells. We describe here a new mechanism of alternative glioblastoma vascularization and open a new perspective for the antivascular treatment strategy.

A2B5 Cells from Human Glioblastoma have Cancer Stem Cell Properties

Glioblastomas, like other cancers, harbor small cell populations with the capability of sustaining tumor formation. These cells are referred to as cancer stem cells. We isolated cells expressing the surface marker A2B5 from three human glioblastomas (GBM) and showed that after grafting into nude mice, they generated dense and highly infiltrative tumors. Then, we extensively studied A2B5+ cells isolated from 11 human GBM. These cells display neurosphere‐like, self‐renewal, asymmetrical cell division properties and have multipotency capability. Stereotactic xenografts of dissociated A2B5+‐derived secondary spheres revealed that as few as 1000 cells produced a tumor. Moreover, flow cytometry characterization of A2B5+‐derived spheres revealed three distinct populations of cells: A2B5+/CD133+, A2B5+/CD133‐ and A2B5‐/CD133‐, with striking proportion differences among GBM. Both A2B5+/CD133+ and A2B5+/CD133‐ cell fractions displayed a high proliferative index, the potential to generate spheres and produced tumors in nude mice. Finally, we generated two green fluorescent protein‐cell lines that display—after serum induction—distinct proliferative and migratory properties, and differ in their CD133 level of expression. Taken together, our results suggest that transformed A2B5+ cells are crucial for the initiation and maintenance of GBM, although CD133 expression is more involved in determining the tumors behavior.

Dysembryoplastic Neuroepithelial Tumors Share with Pleomorphic Xanthoastrocytomas and Gangliogliomas BRAFV600E Mutation and Expression

Pediatric cortical glioneuronal benign tumors mainly include gangliogliomas (GG) [differential diagnoses pilocytic astrocytomas (PA) and pleomorphic xanthoastrocytomas (PXA)] and dysembryoplastic neuroepithelial tumor (DNT). DNT include the specific form and the controversial non‐specific form that lack the specific glioneuronal element. Our aims were to search for BRAFV600E mutation and CD34 expression in DNT, PXA, GG and PA to correlate BRAFV600E mutation with BRAFV600E expression and to evaluate their diagnostic and prognostic values. Ninety‐six children were included. BRAFV600E mutation was studied by sequencing and immunohistochemistry; CD34 expression was analyzed by immunohistochemistry. BRAFV600E mutation was detected in PXA (60%), GG (38.7%), DNT (30%, including 3/11 specific and 3/9 non‐specific forms) and PA (12.5%). BRAFV600E expression was recorded in PXA (60%), GG (45.2%) and DNT (30%). CD34 expression was recorded in PXA (60%), GG (58.1%), DNT (25%) and PA (12.5%). Neither CD34 expression nor BRAFV600E status was predictive of prognosis, except for PA tumors where CD34 expression was associated with a shorter overall survival. In conclusion, DNT shared with PXA and GG, BRAFV600E mutation and/or CD34 expression, which represent molecular markers for these tumors, and we recommend searching for CD34 expression and BRAFV600E mutation in all DNT, especially the non‐specific forms.

ASPM-associated stem cell proliferation is involved in malignant progression of gliomas and constitutes an attractive therapeutic target

BackgroundASPM (Abnormal Spindle-like Microcephaly associated) over-expression was recently implicated in the development of malignant gliomas.ResultsTo better characterize the involvement of ASPM in gliomas, we investigated the mRNA expression in 175 samples, including 8 WHO Grade II, 75 WHO Grade III and 92 WHO Grade IV tumors. Aspm expression was strongly correlated with tumor grade and increased at recurrence when compared to the initial lesion, whatever the initial grade of the primary tumor. ASPM expression also increased over serial passages in gliomaspheres in vitro and in mouse xenografts in vivo. Lentivirus-mediated shRNA silencing of ASPM resulted in dramatic proliferation arrest and cell death in two different gliomasphere models.ConclusionThese data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and is an attractive therapeutic target in glioblastoma multiforme.

Pilocytic astrocytoma of the optic pathway: a tumour deriving from radial glia cells with a specific gene signature

Pilocytic astrocytomas are WHO grade I gliomas that occur predominantly in childhood. They share features of both astroglial and oligodendroglial lineages. These tumours affect preferentially the cerebellum (benign clinical course) and the optic pathway, especially the hypothalamo-chiasmatic region (poor prognosis). Understanding the molecular basis responsible for the aggressive behaviour of hypothalamo-chiasmatic pilocytic astrocytomas is a prerequisite to setting up new molecular targeted therapies. We used the microarray technique to compare the transcriptional profiles of five hypothalamo-chiasmatic and six cerebellar pilocytic astrocytomas. Validation of the microarray results and comparison of the tumours with normal developing tissue was done by quantitative real-time PCR and immunohistochemistry. Results demonstrate that cerebellar and hypothalamo-chiasmatic pilocytic astrocytomas are two genetically distinct and topography-dependent entities. Numerous genes upregulated in hypothalamo-chiasmatic pilocytic astrocytomas also increased in the developing chiasm, suggesting that developmental genes mirror the cell of origin whereas migrative, adhesive and proliferative genes reflect infiltrative properties of these tumours. Of particular interest, NOTCH2, a gene expressed in radial glia and involved in gliomagenesis, was upregulated in hypothalamo-chiasmatic pilocytic astrocytomas. In order to find progenitor cells that could give rise to hypothalamo-chiasmatic pilocytic astrocytomas, we performed a morphological study of the hypothalamo-chiasmatic region and identified, in the floor of the third ventricle, a unique population of vimentin- and glial fibrillary acidic protein-positive cells highly suggestive of radial glia cells. Therefore, pilocytic astrocytomas of the hypothalamo-chiasmatic region should be considered as a distinct entity which probably originates from a unique population of cells with radial glia phenotype.

In vitro identification and functional characterization of glial precursor cells in human gliomas

Human gliomas including astrocytomas and oligodendrogliomas are defined as being composed of neoplastic astrocytes and oligodendrocytes respectively. Here, on the basis of in vitro functional assays, we show that gliomas contain a mixture of glial progenitor cells and their progeny. We have set up explant cultures from pilocytic astrocytomas, glioblastomas and oligodendrogliomas and studied antigens that characterize glial lineage, from the precursor cells (glial restricted precursors and oligodendrocyte‐type2‐astrocyte/oligodendrocyte precursor cells expressing the A2B5 ganglioside) to the differentiated cells (oligodendrocyte and type‐1 and type‐2 astrocytes). All tumoral explants contain A2B5+ cells and can generate migrating cells with distinctive functional properties according to glioma subtypes. In pilocytic astrocytomas, very few migrating cells are dividing and can differentiate in type‐2 astrocytes or towards the oligodendrocyte lineage. In glioblastomas, most migrating cells are dividing, express A2B5 or glial fibrillary acid protein (GFAP) and can generate oligodendrocytes and type‐1 and type‐2 astrocytes in appropriate medium. Oligodendroglioma explants are made by actively dividing glial precursor cells expressing A2B5 or PSA‐NCAM. Only few cells can migrate and differentiation towards oligodendrocyte lineage does not occur. Isolated A2B5+ cells from both glioblastomas and oligodendrogliomas showed similar genetic alterations as the whole tumour. Therefore, pilocytic astrocytomas contain slowly dividing oligodendrocyte‐type2‐astrocyte/oligodendrocyte precursor cells in keeping with their benign behaviour whereas both glioblastomas and oligodendrogliomas contain neoplastic glial restricted precursor cells. In oligodendrogliomas, these cells are trapped in undifferentiated and proliferating state. The precursor cells properties present in gliomas give new insight into their histogenesis and open up new avenues for research in the field of gliomagenesis.

An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients.

BackgroundThe molecular characteristics associated with the response to treatment in glioblastomas (GBMs) remain largely unknown. We performed a retrospective study to assess the genomic characteristics associated with the response of GBMs to either first-line chemotherapy or radiation therapy. The gene expression (n = 56) and genomic profiles (n = 67) of responders and non-responders to first-line chemotherapy or radiation therapy alone were compared on Affymetrix Plus 2 gene expression arrays and BAC CGH arrays.ResultsAccording to Verhaak et al.s classification system, mesenchymal GBMs were more likely to respond to radiotherapy than to first-line chemotherapy, whereas classical GBMs were more likely to respond to first-line chemotherapy than to radiotherapy. In patients treated with radiation therapy alone, the response was associated with differential expression of microenvironment-associated genes; the expression of hypoxia-related genes was associated with short-term progression-free survival (< 5 months), whereas the expression of immune genes was associated with prolonged progression-free survival (> 10 months). Consistently, infiltration of the tumor by both CD3 and CD68 cells was significantly more frequent in responders to radiotherapy than in non-responders. In patients treated with first-line chemotherapy, the expression of stem-cell genes was associated with resistance to chemotherapy, and there was a significant association between response to treatment and p16 locus deletions. Consistently, in an independent data set of patients treated with either radiotherapy alone or with both radiotherapy and adjuvant chemotherapy, we found that patients with the p16 deletion benefited from adjuvant chemotherapy regardless of their MGMT promoter methylation status, whereas in patients without the p16 deletion, this benefit was only observed in patients with a methylated MGMT promoter.ConclusionDifferential expression of microenvironment genes and p16 locus deletion are associated with responses to radiation therapy and to first-line chemotherapy, respectively, in GBM. Recently identified transcriptomic subgroups of GBMs seem to respond differently to radiotherapy and to first-line chemotherapy.

Relevance of combinatorial profiles of intermediate filaments and transcription factors for glioma histogenesis

In order to define specific markers for histogenesis of three well‐characterized subgroups of human gliomas (pilocytic astrocytomas, glioblastoma multiforme and oligodendrogliomas), we studied the expression of relevant markers that characterize gliomagenesis, by immunohistochemistry and in situ hybridization. They include the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin and nestin, the transcription factors Olig2, Nkx2.2 and Sox10, and the proteolipid protein transcripts plp/dm20. We show that the three major categories of human gliomas express a combinatorial profile of markers that gives new insights to their histogenesis and may help diagnosis. Pilocytic astrocytomas strongly express GFAP, vimentin, Olig2, Nkx2.2 and Sox10 but not nestin. In contrast, glioblastomas strongly express GFAP, vimentin and nestin but these tumours are heterogeneous regarding the expression of the transcription factors studied. Finally, in oligodendrogliomas, intermediate filament proteins are generally not observed whereas Olig2 was found in almost all tumour cells nuclei while only a subpopulation of tumour cells expressed Nkx2.2 and Sox10.

Saccharomyces boulardii Improves Intestinal Cell Restitution through Activation of the α2β1 Integrin Collagen Receptor

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohns disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex) is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2β1 integrin activity was assessed using adhesion assays and the analysis of α2β1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2β1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2β1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Outcome analysis of childhood pilocytic astrocytomas: a retrospective study of 148 cases at a single institution.

Pilocytic astrocytomas (PAs) are characterized by an excellent prognosis although several factors of adverse outcome have been reported. The mitogen‐activated protein kinase pathway plays a major role in their tumorigenesis.