Mimicking glioblastoma multiforme microanatomical architecture via patient-derived 3D spheroids

Glioblastoma multiforme (GBM) represents one of the most malignant forms of central nervous system tumors [1]. Despite advances in treatment modalities, prognosis remains poor, due to the remarkable resistance to therapies attributed to molecular intertumoral and intratumoral heterogeneity within GBM [2]. Consequently, to foster the development of more effective antitumor therapies, it is necessary to develop novel experimental models able to mimic the in vivo spatial and molecular organization of GBM, to better understand its biological complexity. Among in vitro models, 3D spheroids mimic the morphological and molecular complexity and heterogeneity of the tumor, as they recapitulate the cell-cell and cell-extracellular matrix interactions present in vivo. Patient-derived 3D spheroids are increasingly considered more physiologically relevant [3]. In this study, our focus lies on regional intratumoral differences and on the potential use of patient-derived 3D spheroids as model able to recapitulate the GBM niches. In GBM patient specimens, we assessed the architecture of vascular niche characterized by pronounced angiogenesis, accompanied by increased VEGF (vascular endothelial growth factor) expression level. In addition, we assessed the morphology of the hypoxic niche that contributes to tumor growth and resistance, wherein HIF (hypoxia-inducible factor) contributes to the upregulation of VEGF and supports cell proliferation in hyperproliferative and invasive niche, overexpressing PCNA (proliferating cell nuclear antigen) and c-KIT (tyrosine-protein kinase KIT). Subsequently, primary cell lines were isolated from GBM tumor tissues and characterized for morphological, phenotypical and growth features. To investigate the spatial organization and cellular heterogeneity of GBM patient-derived 3D spheroids were generated, particularly focusing on the c-KIT pathway. C-kit, also known as stem cell factor receptor (SCF), is a proto-oncogene implicated in both normal growth and development of neoplastic processes, frequently overexpressed and amplified in gliomas. Uncontrolled activity of the SCF/c-KIT pathway by glioma cells can activate brain microvascular endothelial cells, supporting proliferation, angiogenesis, stemness, and metastasis [4]. In summary, we have developed a 3D culture model that faithfully mimics the in vivo architecture of GBM, providing a valuable tool for future mechanistic studies aimed at elucidating the effects of anti-angiogenic therapies targeting the c-KIT pathway.