The STEMRI trial: Magnetic resonance spectroscopy imaging can define tumor areas enriched in glioblastoma stem-like cells.

Despite maximally safe resection of the magnetic resonance imaging (MRI)-defined contrast-enhanced (CE) central tumor area and chemoradiotherapy, most patients with glioblastoma (GBM) relapse within a year in peritumoral FLAIR regions. Magnetic resonance spectroscopy imaging (MRSI) can discriminate metabolic tumor areas with higher recurrence potential as CNI+ regions (choline/N-acetyl-aspartate index >2) can predict relapse sites. As relapses are mainly imputed to glioblastoma stem-like cells (GSCs), CNI+ areas might be GSC enriched. In this prospective trial, 16 patients with GBM underwent MRSI/MRI before surgery/chemoradiotherapy to investigate GSC content in CNI-/+ biopsies from CE/FLAIR. Biopsy and derived-GSC characterization revealed a FLAIR/CNI+ sample enrichment in GSC and in gene signatures related to stemness, DNA repair, adhesion/migration, and mitochondrial bioenergetics. FLAIR/CNI+ samples generate GSC-enriched neurospheres faster than FLAIR/CNI-. Parameters assessing biopsy GSC content and time-to-neurosphere formation in FLAIR/CNI+ were associated with worse patient outcome. Preoperative MRI/MRSI would certainly allow better resection and targeting of FLAIR/CNI+ areas, as their GSC enrichment can predict worse outcomes.

Impact of Regorafenib on Endothelial Transdifferentiation of Glioblastoma Stem-like Cells.

Glioblastomas (GBM) are aggressive brain tumours with a poor prognosis despite heavy therapy that combines surgical resection and radio-chemotherapy. The presence of a subpopulation of GBM stem cells (GSC) contributes to tumour aggressiveness, resistance and recurrence. Moreover, GBM are characterised by abnormal, abundant vascularisation. Previous studies have shown that GSC are directly involved in new vessel formation via their transdifferentiation into tumour-derived endothelial cells (TDEC) and that irradiation (IR) potentiates the pro-angiogenic capacity of TDEC via the Tie2 signalling pathway. We therefore investigated the impact of regorafenib, a multikinase inhibitor with anti-angiogenic and anti-tumourigenic activity, on GSC and TDEC obtained from irradiated GSC (TDEC IR+) or non-irradiated GSC (TDEC). Regorafenib significantly decreases GSC neurosphere formation in vitro and inhibits tumour formation in the orthotopic xenograft model. Regorafenib also inhibits transdifferentiation by decreasing CD31 expression, CD31+ cell count, pseudotube formation in vitro and the formation of functional blood vessels in vivo of TDEC and TDEC IR+. All of these results confirm that regorafenib clearly impacts GSC tumour formation and transdifferentiation and may therefore be a promising therapeutic option in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumours.

The m6A RNA Demethylase ALKBH5 Promotes Radioresistance and Invasion Capability of Glioma Stem Cells.

Recurrence of GBM is thought to be due to GBMSCs, which are particularly chemo-radioresistant and characterized by a high capacity to invade normal brain. Evidence is emerging that modulation of m6A RNA methylation plays an important role in tumor progression. However, the impact of this mRNA modification in GBM is poorly studied. We used patient-derived GBMSCs to demonstrate that high expression of the RNA demethylase, ALKBH5, increases radioresistance by regulating homologous recombination (HR). In cells downregulated for ALKBH5, we observed a decrease in GBMSC survival after irradiation likely due to a defect in DNA-damage repair. Indeed, we observed a decrease in the expression of several genes involved in the HR, including CHK1 and RAD51, as well as a persistence of γ-H2AX staining after IR. We also demonstrated in this study that ALKBH5 contributes to the aggressiveness of GBM by favoring the invasion of GBMSCs. Indeed, GBMSCs deficient for ALKBH5 exhibited a significant reduced invasion capability relative to control cells. Our data suggest that ALKBH5 is an attractive therapeutic target to overcome radioresistance and invasiveness of GBMSCs.

Ionizing radiation induces endothelial transdifferentiation of glioblastoma stem-like cells through the Tie2 signaling pathway.

Glioblastomas (GBM) are brain tumors with a poor prognosis despite treatment that combines surgical resection and radio-chemotherapy. These tumors are characterized by abundant vascularization and significant cellular heterogeneity including GBM stem-like cells (GSC) which contribute to tumor aggressiveness, resistance, and recurrence. Recent data has demonstrated that GSC are directly involved in the formation of new vessels via their transdifferentiation into Tumor Derived Endothelial Cells (TDEC). We postulate that cellular stress such as ionizing radiation (IR) could enhance the transdifferentiation of GSC into TDEC. GSC neurospheres isolated from 3 different patients were irradiated or not and were then transdifferentiated into TDEC. In fact, TDEC obtained from irradiated GSC (TDEC IR+) migrate more towards VEGF, form more pseudotubes in MatrigelTM in vitro and develop more functional blood vessels in MatrigelTM plugs implanted in Nude mice than TDEC obtained from non-irradiated GSC. Transcriptomic analysis allows us to highlight an overexpression of Tie2 in TDEC IR+. All IR-induced effects on TDEC were abolished by using a Tie2 kinase inhibitor, which confirms the role of the Tie2 signaling pathway in this process. Finally, by analyzing Tie2 expression in patient GBMs by immunohistochemistry, we demonstrated that the number of Tie2+ vessels increases in recurrent GBM compared with matched untreated tumors. In conclusion, we demonstrate that IR potentiates proangiogenic features of TDEC through the Tie2 signaling pathway, which indicates a new pathway of treatment-induced tumor adaptation. New therapeutic strategies that associate standard treatment and a Tie2 signaling pathway inhibitor should be considered for future trials.

Inhibiting Integrin ß8 to Differentiate and Radiosensitize Glioblastoma-Initiating Cells.

Glioblastomas (GB) are malignant brain tumors with poor prognosis despite treatment with surgery and radio/chemotherapy. These tumors are defined by an important cellular heterogeneity and notably contain a subpopulation of GB-initiating cells (GIC), which contribute to tumor aggressiveness, resistance, and recurrence. Some integrins are specifically expressed by GICs and could be actionable targets to improve GB treatment. Here, integrin ß8 (ITGB8) was identified as a potential selective target in this highly tumorigenic GIC subpopulation. Using several patient-derived primocultures, it was demonstrated that ITGB8 is overexpressed in GICs compared with their differentiated progeny. Furthermore, ITGB8 is also overexpressed in GB, and its overexpression is correlated with poor prognosis and with the expression of several other classic stem cell markers. Moreover, inhibiting ITGB8 diminished several main GIC characteristics and features, including self-renewal ability, stemness, migration potential, and tumor formation capacity. Blockade of ITGB8 significantly impaired GIC cell viability via apoptosis induction. Finally, the combination of radiotherapy and ITGB8 targeting radiosensitized GICs through postmitotic cell death. IMPLICATIONS: This study identifies ITGB8 as a new selective marker for GICs and as a promising therapeutic target in combination with chemo/radiotherapy for the treatment of highly aggressive brain tumors.

FGFR1/FOXM1 pathway: a key regulator of glioblastoma stem cells radioresistance and a prognosis biomarker.

Glioblastoma are known to be aggressive and therapy-resistant tumors, due to the presence of glioblastoma stem cells inside this heterogeneous tumor. We investigate here the involvement of FGFR1 in glioblastoma stem-like cells (GSLC) radioresistance mechanisms. We first demonstrated that the survival after irradiation was significantly diminished in FGFR1-silenced (FGFR1-) GSLC compared to control GSLC. The transcriptome analysis of GSLCs FGFR1(-) showed that FOX family members are differentially regulated by FGFR1 inhibition, particularly with an upregulation of FOXN3 and a downregulation of FOXM1. GSLC survival after irradiation was significantly increased after FOXN3 silencing and decreased after FOXM1 inhibition, showing opposite effects of FGFR1/FOX family members on cell response to ionizing radiation. Silencing FGFR1 or FOXM1 downregulated genes involved in mesenchymal transition such as GLI2, TWIST1, and ZEB1 in glioblastoma stem-like cells. It also dramatically reduced GSLC migration. Databases analysis confirmed that the combined expression of FGFR1/FOXM1/MELK/GLI2/ZEB1/TWIST1 is significantly associated with patients overall survival after chemo-radiotherapy treatment. All these results, associated with our previous conduced ones with differentiated cells, clearly established that FGFR1-FOXM1 dependent glioblastoma stem-like cells radioresistance pathway is a central actor of GBM treatment resistance and a key target to inhibit in the aim to increase the sensitivity of GBM to the radiotherapy.

Engineering of adult human neural stem cells differentiation through surface micropatterning.

Interaction between differentiating neural stem cells and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. To restore damage brain area by tissue engineering, the biomaterial scaffold has to mimic this microenvironment to allow organized tissue regeneration. To establish the validity of using microgrooved surfaces in order to simultaneously provide to primary adult human neural stem cells a permissive growth environment and a guide for neurite outgrowth in a pre-established direction, we have studied the long-term culture of adult human neural stem cells from patient biopsies on microgrooved polymers. By exploiting polymer moulding techniques, we engineered non-cytotoxic deep microstructured surfaces of polydimethylsiloxane (PDMS) exhibiting microchannels of various widths. Our results demonstrate that precoated micropatterned PDMS surfaces can serve as effective neurite guidance surfaces for human neural stem cells. Immunocytochemistry analysis show that channel width can impact strongly development and differentiation. In particular we found an optimal microchannel width, that conciliates a high differentiation rate with a pronounced alignment of neurites along the edges of the microchannels. The impact of the microstructures on neurite orientation turned out to be strongly influenced by cell density, attesting that cell/surface interactions at the origin of the alignment effect, are in competition with cell/cell interactions tending to promote interconnected networks of cells. Considering all these effects, we have been able to design appropriate structures allowing to obtain neuron development and differentiation rate comparable to a plane unpatterned surface, with an efficient neurite guidance and a long-term cell viability.