028 | Modelling glioblastoma multiforme microanatomical complexity with patient-derived 3D spheroids: a promising therapeutic tool for advancing precision medicine

Alessandra Maria Vitale¹, Giuseppa D’Amico¹, Martina Di Marco¹, Giuseppina Poppa², Pierlorenzo Maria Veiceschi³, Alessandro Lo Giudice¹, Francesca Chiara Cecala¹, Paola Limoncelli², Ilaria Giusti², Vincenza Dolo², Claudia Campanella¹, Francesca Rappa¹, Rosario Barone¹, Giovanni Tringali³, Francesco Cappello¹, Celeste Caruso Bavisotto¹ | ¹Institute of Human Anatomy and Histology, Department of Biomedicine, Neuroscience and Advanced Diagnostics, University of Palermo, Italy; ²Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy; ³Department of Neurosurgery, National Relevance and High Specialization Hospital Trust ARNAS Civico, Palermo, Italy.

Abstract

Glioblastoma multiforme (GBM) is one of the most aggressive and malignant primary brain tumors. Despite advances in therapeutic strategies, patients’ prognosis remains extremely poor, largely due to the high rates of tumor recurrence and the development of resistance to standard treatments [1]. In this context, the development of robust preclinical models that faithfully recapitulate the biological complexity and heterogeneity of GBM is critical for the design of more effective, targeted, and personalized therapeutic strategies. Patient-derived three-dimensional (3D) culture systems, including tumor spheroids, are emerging as powerful tools in GBM research, capable of closely mimicking in vivo tumor microarchitecture and functional complexity compared to conventional two-dimensional models [2]. In our study, we established a robust and stable patient-derived 3D GBM spheroid model. Long-term monitoring of spheroids demonstrated a reproducible and dynamic morphological evolution, characterized by a biphasic growth pattern, with preserved viability and structural integrity for up to 14 days in culture. Immunomorphological analyses revealed that the spheroids effectively recapitulated key features of the native tumor, including spatial organization and cellular heterogeneity, as evidenced by the non-uniform distribution of specific molecular markers and the formation of functional niches. Notably, GBM spheroids developed a central necrotic core, characterized by apoptotic cells, upon the establishment of hypoxic conditions, surrounded by an outer proliferative layer, closely resembling the metabolic and functional gradients observed in native GBM tissue. We then focused on the c-KIT pathway. c-KIT is a proto-oncogene implicated in both normal growth and development and in neoplastic processes, frequently overexpressed and amplified in malignant gliomas [3]. Aberrant activation of the SCF/c-KIT pathway in glioma cells promoted the activation and proliferation of brain microvascular endothelial cells, inducing angiogenesis and ultimately contributing to tumor resistance and progression. On the contrary, downregulation of the SCF/c-KIT pathway has been demonstrated to inhibit tumor-mediated angiogenesis and glioma growth in vivo [4]. Overall, our patient-derived 3D GBM model reflects the intrinsic heterogeneity and complexity of GBM and represents a reliable and biologically relevant platform for studying tumor behavior and evaluating novel therapeutic strategies, such as anti-angiogenic therapies targeting the SCF/c-KIT pathway.