Human glioblastoma stem cells and inflammatory tumor microenvironment: Involvement of nitric oxide synthase 2 (NOS2) expression and activity

Gliomas are classified by the World Health Organization (WHO) grade criteria (I to IV) into multiple specific histologic subtypes, based on cell type of origin and molecular characteristics [1]. Afterward, an increasing number of molecular biomarkers was discovered in these tumors, also including TERT (Telomerase Reverse Transcriptase) promoter mutation, EGFR (Epidermal Growth Factor Receptor) and P53 [2] Grade IV Glioblastoma (GBM) is one of the most lethal brain cancers worldwide since it is the most highly infiltrating and aggressive tumor of the central nervous system, characterized by a high degree of genetic and cellular heterogeneity. GBM accounts for 70–75% of all diffuse glioma diagnoses and has a median overall survival of 14–17 months [3]. The incidence rates of GBM increases with age, with a median age of 64 years at diagnosis and is ranged between 0.6 to 3.7 per 100,000 persons depending on reporting country/organization [4] and with geographic differences [5]. The aims of the study are:  To evaluate the macrophage infiltrate and M2-polarization as well as SOX-2 expression in human GBM sections.  To assess the ability of GBM primary cultures to generate neurospheres and analyze the concordance with the level of SOX-2 expression and M2-polarization.  To analyze the NOS2 expression and activity in neurospheres derived by both human GBM primary cells and GBM cell lines as well as its possible correlation with SOX-2 expression.  To verify the potential functional role of NOS2 activity in glioma biology, through the use of 1400W, a specific NOS2 inhibitor, in terms of cell proliferation and migration rate, clonogenic potential, and capacity of generating neurospheres of GBM cell lines.  To investigate the ability of 1400W to influence autophagic flux as well as the release of EVs by U87MG-derived GSCs.  To assess the effects of EVs derived by 1400W-treated GSCs on adherent U87MG cells (recipient cells) in terms of proliferation index, migration rate, and autophagic flux. In conclusion, the effect induced by the direct addition of 1400W to GSCs or adherent U87MG cells could be mediated by anti-tumoral molecular messages induced by NOS2 inhibition in the origin cells and then transferred to the surrounding recipient cells through the EVs. On the other hand, NOS2 inhibition could induce GSCs to release EVs with a modified cargo that is able to alter and manipulate the tumor inflammatory microenvironment in order to make it less advantageous to tumor growth and invasiveness.