Electrophysiology of glioma: a Rho GTPase-activating protein reduces tumor growth and spares neuron structure and function

Neuro-Oncology Neuro-Oncology 18(12), 1634–1643, 2016 doi:10.1093/neuonc/now114 Neuro-Oncology 2016; 0, 1 – 10, doi:10.1093/neuonc/now114 Advance Access date 13 June 2016 Electrophysiology of glioma: a Rho GTPase-activating protein reduces tumor growth and spares neuron structure and function Eleonora Vannini, Francesco Olimpico, Silvia Middei, Martine Ammassari-Teule, Erik L. de Graaf, Liam McDonnell, Gudula Schmidt, Alessia Fabbri, Carla Fiorentini, Laura Baroncelli, Mario Costa, and Matteo Caleo Corresponding Author: Matteo Caleo, PhD, CNR Neuroscience Institute, via G. Moruzzi 1, Pisa 56124, Italy (). Background. Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. Methods. We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. Results. Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. Conclusions. Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies. Keywords: cytotoxic necrotizing factor 1, dendritic structure, evoked potentials, senescence, visual cortex. Glioblastoma (GBM) is an aggressive form of brain tumor typically associated with a poor prognosis.1 Survival rates and quality of life of the patients have scarcely improved in the last years. Thus, it is urgent to find innovative approaches for GBM treatment. The development of novel therapies requires a better understanding of the biology of glioma cells and their interactions with resident brain cells. In particular, glioma cells are known to release high amounts of glutamate, leading to overexcitation of peritumoral neurons and epileptic seizures,2 with consequent neuronal death by excitotoxicity. This neuronal loss may facilitate glioma invasion3 and underlie cognitive impairments in patients.4,5 A successful brain tumor treatment should therefore aim at protecting neuronal cells to prevent functional deficits and cognitive deterioration, which have a strong impact on patients’ quality of life. Currently, very little information is available about the functional status of peritumoral tissue at the border of the glioma mass. Previous studies have reported accumulation of glutamate6 as well as excitatory actions of gamma-aminobutyric acid (GABA),7 which may explain the occurrence of epileptic seizures. However, how tumor growth reverberates on the activity of single cells and the function of neuronal networks remain completely uncharted. Gathering this information is critical from at least 2 points of view. First, neuronal activity in peritumoral areas can potently influence brain tumor growth via the release of glioma mitogens.8 Second, novel antiglioma Received 3 December 2015; accepted 22 April 2016 # The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: . 1634 1 of 10 CNR Neuroscience Institute, Pisa, Italy (E.V., F.O., L.B., M.C., Mat.C.); CNR Cellular Biology and Neurobiology Institute, Rome, Italy (S.M., M.A.-T.); Fondazione Pisana per la Scienza, Mass Spectrometry and Proteomics, Pisa, Italy (E.L.d.G., L.M.); Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Freiburg, Germany (G.S.); Istituto Superiore di Sanità, Rome, Italy (A.F., C.F.); Scuola Normale Superiore, Pisa, Italy (M.C., Mat.C.) Vannini et al.: Sparing of neuronal function in glioma Materials and Methods For detailed experimental procedures, see the Supplementary Material. Cell Cultures GL261 cells were grown according to American Type Culture Collection protocols. Primary human glioblastoma cells were collected from 2 subjects as described in our previous publication.10 The study was approved by the Human Ethics Committee of the University of Pisa and Pisa Hospital. Cells were treated with CNF1 (3 nM) and incubated for 48 h before b-galactosidase measurements. The percentage of positive cells was determined after counting 3 random fields. performed with Tandem Mass Tag 6-plex labeling, and fractionation was performed using high pH reverse phase fractionation on a BRAVO AssayMAP (Agilent Technologies). Nano-scale liquid chromatography – tandem mass spectrometry analysis was performed on a 50 cm Easyspray column and an Orbitrap Fusion for MS3 analysis (Thermo Fisher Scientific). Data were analyzed using MaxQuant16 and Database for Annotation, Visualization and Integrated Discovery (DAVID)17 software. Animals and Tumor Induction Adult (age .postnatal day 60) C57BL/6J and Thy1-GFP mice were used. All experimental procedures conformed to the European Communities Council Directive #86/609/EEC and were approved by the Italian Ministry of Health. To induce glioma formation, C57BL/6 and Thy1-GFP mice received a stereotaxically guided injection of 40 000 GL261 cells (20 000 cells/mL phosphate buffered saline solution) into the visual cortex (2 mm lateral to the midline and in correspondence with lambda). Five days after injection of GL261 cells (tumor induction), mice were divided into 2 groups. The first group received CNF1 injection, the second, named “vehicle,” Tris – HCl buffer injection (glioma control condition). Immunohistochemistry Glioma volumes were measured in serial cortical sections using Stereo Investigator software (MicroBrightField). To quantify density of glial cells in the peritumoral areas, we used primary antibodies directed against glial fibrillary acidic protein (GFAP) (1:500; Dako) and Iba-1 (1:500; Wako).12 Positive cells were then counted in regions adjacent to the tumor. Assessment of Senescence In vivo Coronal sections were stained (...truncated)