Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. Researchers at the International School for Advanced Studies (SISSA) used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7-12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, the researchers show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7-12 when the threshold for spike initiation is significantly reduced. At DIV 7-12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.
Glioma cells-derived exosomes disrupt network synchrony
in mature (DIV 7–12) primary neuron cultures
A–C Representative images of a Fluo-4 loaded DIV 10 control cortical neuron culture (A), of neurons co-cultured for 1 day with U87, mCherry expressing, glioma cells (B), and of neurons exposed for 1 day to U87-derived exosomes (C). ROIs indicate cells from which the representative Ca2+ traces are extracted. Scale bars, 50 µm. D Cross-correlation values of the various experimental groups. Data are shown as average ± standard error, significant differences were assessed with Wilcoxon–Mann–Whitney U test (to compare two groups) or Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test (to compare more than two groups); *p < 0.05; ***p < 0.001; ****p < 0.0001; n = 3 independent cell preparations, four coverslips for each preparation were analyzed and reported as superimposed individual points. E Representative raster plots of spike times, simultaneously detected at 120 spatial channels by MEAs: channels’ numbers are indicated on the y-axis. The spiking activity is displayed over a representative time window of 5 min, preceding the exposure to U87 exosomes (Ctr). At this stage of development (DIV 7–12), network bursts are normally observed. F Representative recording of the same neuronal culture in E after 24 h exosome exposure (Exo), showing a lower occurrence of network bursts. G Pearson correlation coefficients among spike times in different channels over 15 min recordings. The average correlation values across 10 channels were analyzed and their distributions were compared under control conditions and after exosomes, exposure using the Wilcoxon–Mann–Whitney U test; ***p < 0.001; n = 12, an average of 10 channels.