Several promising treatments against high-grade gliomas (HGGs) failed to provide significant benefit when translated from the preclinical setting to patients. Improving animal models is fundamental to overcome this translational gap. We have developed and comprehensively characterized in-vivo model based on the orthotopic implantation of CT-2A cells cultured in neurospheres (NS). Anatomical, metabolic (MRS) and perfusion MRI indicated that CT-2A NS-derived tumors showed a more HGG-like behavior, which was supported by survival data, increased glioma stem cell population and enhanced neoangiogenesis. Because of these specific features, the CT-2A NS-derived model represents a high-translational platform for the search of new HGG treatments.