Domenico D'Avella

Intratumoral Hypoxic Gradient Drives Stem Cells Distribution and MGMT Expression in Glioblastoma

Glioblastoma multiforme (GBM) are highly proliferative tumors currently treated by surgical removal, followed by radiotherapy and chemotherapy, which are counteracted by intratumoral hypoxia. Here we exploited image guided surgery to sample multiple intratumoral areas to define potential cellular heterogeneity in correlation to the oxygen tension gradient within the GBM mass. Our results indicate that more immature cells are localized in the inner core and in the intermediate layer of the tumor mass, whereas more committed cells, expressing glial fibrillary acidic protein and β‐III‐tubulin, are distributed along the peripheral and neo‐vascularized area, where Smad1/5/8 and Stat3 result to be activated. Moreover, GBM stem cells, identified with the stem cell marker CD133, express high level of DNA repair protein O6‐methylguanine‐DNA‐methyltransferase (MGMT) known to be involved in chemotherapy resistance and highly expressed in the inner core of the tumor mass. Importantly, these cells and, particularly, CD133+ cells result to be resistant to temozolomide (TMZ), the most used oral alkylating agent for the treatment of GBM, which specifically causes apoptosis only in GBM cells derived from the peripheral layer of the tumor mass. These results indicate a correlation between the intratumoral hypoxic gradient, the tumor cell phenotype, and the tumor resistance to chemotherapy leading to a novel concentric model of tumor stem cell niche, which may be useful to define the real localization of the chemoresistant GBM tumor cells in order to design more effective treatment strategies. STEM CELLS 2010;28:851–862

Interaction of Hypoxia‐Inducible Factor‐1α and Notch Signaling Regulates Medulloblastoma Precursor Proliferation and Fate

Medulloblastoma (MDB) is the most common brain malignancy of childhood. It is currently thought that MDB arises from aberrantly functioning stem cells in the cerebellum that fail to maintain proper control of self‐renewal. Additionally, it has been reported that MDB cells display higher endogenous Notch signaling activation, known to promote the survival and proliferation of neoplastic neural stem cells and to inhibit their differentiation. Although interaction between hypoxia‐inducible factor‐1α (HIF‐1α) and Notch signaling is required to maintain normal neural precursors in an undifferentiated state, an interaction has not been identified in MDB. Here, we investigate whether hypoxia, through HIF‐1α stabilization, modulates Notch1 signaling in primary MDB‐derived cells. Our results indicate that MDB‐derived precursor cells require hypoxic conditions for in vitro expansion, whereas acute exposure to 20% oxygen induces tumor cell differentiation and death through inhibition of Notch signaling. Importantly, stimulating Notch1 activation with its ligand Dll4 under hypoxic conditions leads to expansion of MDB‐derived CD133+ and nestin+ precursors, suggesting a regulatory effect on stem cells. In contrast, MDB cells undergo neuronal differentiation when treated with γ‐secretase inhibitor, which prevents Notch activation. These results suggest that hypoxia, by maintaining Notch1 in its active form, preserves MDB stem cell viability and expansion. STEM CELLS 2010;28:1918–1929

Hypoxia and HIF1α Repress the Differentiative Effects of BMPs in High-Grade Glioma†‡§

Hypoxia commonly occurs in solid tumors of the central nervous system (CNS) and often interferes with therapies designed to stop their growth. We found that pediatric high‐grade glioma (HGG)‐derived precursors showed greater expansion under lower oxygen tension, typical of solid tumors, than normal CNS precursors. Hypoxia inhibited p53 activation and subsequent astroglial differentiation of HGG precursors. Surprisingly, although HGG precursors generated endogenous bone morphogenetic protein (BMP) signaling that promoted mitotic arrest under high oxygen tension, this signaling was actively repressed by hypoxia. An acute increase in oxygen tension led to Smad activation within 30 minutes, even in the absence of exogenous BMP treatment. Treatment with BMPs further promoted astroglial differentiation or death of HGG precursors under high oxygen tension, but this effect was inhibited under hypoxic conditions. Silencing of hypoxia‐inducible factor 1α (HIF1α) led to Smad activation even under hypoxic conditions, indicating that HIF1α is required for BMP repression. Conversely, BMP activation at high oxygen tension led to reciprocal degradation of HIF1α; this BMP‐induced degradation was inhibited in low oxygen. These results show a novel, mutually antagonistic interaction of hypoxia‐response and neural differentiation signals in HGG proliferation, and suggest differences between normal and HGG precursors that may be exploited for pediatric brain cancer therapy. STEM CELLS 2009;27:7–17

PATIENTS WITH MODERATE HEAD INJURY: A PROSPECTIVE MULTICENTER STUDY OF 315 PATIENTS

OBJECTIVETo analyze the risk factors of worst outcome associated with moderate head injury. METHODSData on patients with moderate head injury were collected prospectively in 11 Italian neurosurgical units over a period of 18 months. Patients older than 18 years with blunt head injury and at least one Glasgow Coma Scale (GCS) score between 9 and 13 were enrolled. The outcome was determined at 6 months using the Glasgow Outcome Scale. RESULTSWe analyzed 315 patients. Initial computed tomographic scans showed a diffuse injury type I or II in 63%, a mass lesion in 35%, and traumatic subarachnoid hemorrhage in 42% of the patients. The risk of progression toward a mass lesion was 23% when the admission computed tomographic scan showed diffuse injury type I or II. An emergency craniotomy was performed in 22% of the patients, delayed surgery was performed in 14%, and both were performed in 25%. A favorable outcome was obtained in 74% of the patients. When the GCS score was 9 or 10, the predictor of worst outcome was a motor GCS score of 4 or lower (odds ratio [OR], 8.08; 95% confidence interval [CI], 1.22–67.35; P = 0.008), but when the GCS score was 11 to 13, the factors associated with worst outcome were neuroworsening (OR, 3.43; 95% CI, 1.45–8.17; P = 0.002), seizures (OR, 7.94; 95% CI, 1.18–64.48; P = 0.02), and medical complications (OR, 4.24; 95% CI, 1.74–10.33; P = 0.0006). CONCLUSIONThere is a high percentage of surgery and worsening on computed tomographic scans in patients with moderate head injury. Neuroworsening, seizures, and medical complications as outcome predictors were more strongly associated with a GCS score of 11 to 13, whereas a low motor GCS score was more outcome-related in patients with GCS scores of 9 and 10.

Neural potential of a stem cell population in the adipose and cutaneous tissues

Abstract Objective: A significant amount of recent interest has been focused on the possibility that adult human stem cells are a realistic therapeutic alternative to embryonic stem cells. Multipotent stem cells that have characteristics reminiscent of embryonic neural crest stem cells have been isolated from several postnatal tissues, including skin, gut, dental pulp and the heart, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. In the present work, we investigated this issue with regard to skin-derived precursors (SKPs) and adipose-derived stem cells (ADSc) Methods: Adult stem cells isolated from skin and from adipose tissue derived from the same adult donor were treated with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Neurospheres obtained were first expanded and evaluated in term of proliferative ability, and then their neuronal differentiation potential was analysed. Results: Adipose- and skin-derived neurospheres grew in suspension as spheres in the presence of the mitogens FGF2 and EGF. With this protocols, the spheres have been able to proliferate and to originate Schwann and glial-like cells. Discussion: In summary, we have demonstrated in this work that multipotent adult precursor cell can be isolated and expanded from two accessible adult tissue sources: skin and adipose tissue. The work described in this paper provides the framework for our attempts to use SKPs or ADSc as autologous adult stem cell population for cell replacement and discovery research.

Molecular Mechanisms of HIF-1α Modulation Induced by Oxygen Tension and BMP2 in Glioblastoma Derived Cells

Background Glioblastoma multiforme (GBM) is one of most common and still poorly treated primary brain tumors. In search for new therapeutic approaches, Bone Morphogenetic Proteins (BMPs) induce astroglial commitment in GBM-derived cells in vitro. However, we recently suggested that hypoxia, which is characteristic of the brain niche where GBM reside, strongly counter-acts BMP effects. It seems apparent that a more complete understanding of the biology of GBM cells is needed, in particular considering the role played by hypoxia as a signaling pathways regulator. HIF-1α is controlled at the transcriptional and translational level by mTOR and, alike BMP, also mTOR pathway modulates glial differentiation in central nervous system (CNS) stem cells. Methodology/Principal Findings Here, we investigate the role of mTOR signaling in the regulation of HIF-1α stability in primary GBM-derived cells maintained under hypoxia (2% oxygen). We found that GBM cells, when acutely exposed to high oxygen tension, undergo Akt/mTOR pathway activation and that BMP2 acts in an analogous way. Importantly, repression of Akt/mTOR signaling is maintained by HIF-1α through REDD1 upregulation. On the other hand, BMP2 counter-acts HIF-1α stability by modulating intracellular succinate and by controlling proline hydroxylase 2 (PHD2) protein through inhibition of FKBP38, a PHD2 protein regulator. Conclusions/Significance In this study we elucidate the molecular mechanisms by which two pro-differentiating stimuli, BMP2 and acute high oxygen exposure, control HIF-1α stability. We previously reported that both these stimuli, by inducing astroglial differentiation, affect GBM cells growth. We also found differences in high oxygen and BMP2 sensitivity between GBM cells and normal cells that should be further investigated to better define tumor cell biology.

Circulating microparticles of glial origin and tissue factor bearing in high-grade glioma: a potential prothrombotic role

Venous thromboembolism (VTE) may complicate the clinical course of glioblastoma multiforme (GBM). Circulating microparticles (MPs) have been associated with cancer-related VTE. Sixty-one consecutive patients with GBM undergoing gross-total (41) or subtotal (20) surgical resection followed by radio-chemotherapy were prospectively evaluated. MPs numbers according to cellular origin and the procoagulant activity of annexin V positive (AV+) MPs (MP-activity) were measured before surgery and then 1 week and 1, 4, and 7 months after surgery. Glial (GFAP+) and endothelial (CD62E+) derived MPs, AV+ and tissue factor-bearing (TF+) MPs were measured using flow cytometry. Baseline levels of GFAP+/TF-, TF+/GFAP-, and GFAP+/TF+ MPs were significantly higher in GBM patients than in healthy controls, and significantly increased at each time point after surgery; at 7 months, a further significant increase over the level found a week after surgery was only seen in the subtotally resected patients. The number AV+/CD62E- MPs increased in GBM patients and correlated with MP activity. TF+/GFAP- MPs numbers were significantly higher in 11 GBM patients who developed VTE than in those who did not (p 0.04). TF+/GFAP- MPs levels above the 90th percentile (calculated in GBM patients without VTE) were associated with a higher risk of VTE (RR 4.17, 95% CI 1.57-11.03). In conclusion, the numbers of glial-derived and/or TF-bearing MPs were high in GBM patients both before and even more after the neoplasm was treated, especially in patients with subtotal resection likely according to disease progression. A contribution of TF+/GFAP- MPs to the risk of VTE is suggested.

Domain-independent neural underpinning of task-switching: An fMRI investigation

The ability to shift between different tasks according to internal or external demands, which is at the core of our behavioral flexibility, has been generally linked to the functionality of left fronto-parietal regions. Traditionally, the left and right hemispheres have also been associated with verbal and spatial processing, respectively. We therefore investigated with functional MRI whether the processes engaged during task-switching interact in the brain with the domain of the tasks to be switched, that is, verbal or spatial. Importantly, physical stimuli were exactly the same and participants performance was matched between the two domains. The fMRI results showed a clearly left-lateralized involvement of fronto-parietal regions when contrasting task-switching versus single task blocks in the context of verbal rules. A more bilateral pattern, especially in the prefrontal cortex, was instead observed for switching between spatial tasks. Moreover, while a conjunction analysis showed that the core regions involved in task-switching, independently of the switching context, were localized both in left inferior prefrontal and parietal cortices and in bilateral supplementary motor area, a direct analysis of functional lateralization revealed that hemispheric asymmetries in the frontal lobes were more biased toward the left side for the verbal domain than for the spatial one and vice versa. Overall, these findings highlight the role of left fronto-parietal regions in task-switching, above and beyond the specific task requirements, but also show that hemispheric asymmetries may be modulated by the more specific nature of the tasks to be performed during task-switching.

Endoscopic anatomy of the fourth ventricle: Laboratory investigation

OBJECTnMicrosurgical anatomy of the fourth ventricle has been comprehensively addressed by masterly reports providing classic descriptions of this complex region. Neuroendoscopy could offer a new, somewhat different perspective of the inside view of the fourth ventricle. The purpose of this study was to examine from the anatomical point of view the access to the fourth ventricle achieved by the endoscopic transaqueductal approach, to enumerate and describe the anatomically identifiable landmarks, and to compare them with those described during microsurgery.nnnMETHODSnThe video recordings of 52 of 75 endoscopic explorations of the fourth ventricle performed at the authors institution for different pathological conditions were reviewed and evaluated to identify and describe every anatomical landmark. According to the microsurgical anatomy, at least 23 superficial structures are clearly identifiable in the fourth ventricle, and they represent the comparative basis of parallel endoscopic anatomy of the structures found during the fourth ventricle navigation.nnnRESULTSnThe following anatomical structures were identified in all cases: median sulcus, superior and inferior vela medullare, choroid plexus, inferior fovea, hypoglossal and vagal triangles, area postrema, obex, canalis medullaris, lateral recess, and the foramina of Luschka and Magendie. The median eminence, facial colliculus, striae medullaris, auditory tubercle, and inferior fovea were seen in the majority of cases. The locus caevruleus could never be seen.nnnCONCLUSIONSnOn the whole, 20 anatomical structures could consistently be identified by exploring the fourth ventricle with a fiberscope. Neuroendoscopy offers a quite different outlook on the anatomy of the fourth ventricle, and compared with the microsurgical descriptions it seems to provide a superior and detailed visualization, particularly of the structures located in the inferior triangle.

Carmustine Wafer Implantation When Surgical Cavity Is Communicating with Cerebral Ventricles: Technical Considerations on a Clinical Series

BACKGROUNDnImplantation of carmustine (1,3-bis (2 chloroetyl)-1-nitrosurea [BCNU]) wafers is an approved local treatment after surgical removal of high-grade gliomas. Safety data have been largely reported by phase III studies. The communication between the final surgical cavity and the ventricular cavities is supposed to be a relative contraindication for positioning of the wafers because of the possible development of hydrocephalus. However, at present there are neither data about this topic published with the exception of a few case reports, nor any proposals for selection criteria for wafer implantation in such circumstances. Furthermore, there are no technical suggestions in literature put forward for the surgical repairing of ventricular defects. Our study was particularly focused on addressing these 3 issues.nnnMETHODSnForty-three patients affected by a high-grade glioma underwent surgical removal and BCNU wafer implantation between March 2007 and September 2009 at the Department of Neurosurgery of Padua. Among them, we retrospectively reviewed clinical, surgical, and radiological data of 9 patients who had been treated with carmustine wafers after surgical repair of communication between the surgical cavity and the ventricular cavities. We also focused on the technical details concerning wafers positioning in this particular situation.nnnRESULTSnVentricular defects were present in the atrium in 4, frontal horn in 3, and temporal horn in 2 cases. The maximum diameter of the defect was between 6 and 10 mm. In all cases, the defect was intraoperatively repaired in the same way, and up to 8 wafers were implanted in the surgical cavity. In the series reported, no cases of hydrocephalus were detected.nnnCONCLUSIONSnIn our experience, integrity of wafers, size of ventricular wall defect, and accuracy in repairing the defect were crucial issues. Nevertheless, more experience and prospective studies would be helpful to clarify both in what measure ventricular opening affects safety data and the best reliable way of repairing ventricular defects when BCNU wafers are implanted.