Glioblastoma Multiforme: new insights on its three-dimensional in vitro modeling for a reliable drug and nanomedicine screening

Glioblastoma multiforme (GBM) is the most common malignant, primary brain tumor with a median overall survival of only 15 months. The major challenges in treating GBM are the self-renewal capability of GBM cells and GBM stem cells (GSCs) which drive the development of treatment-resistant tumor variants; the heterogeneous composition, consisting of different cell types and of tumor cells at different stages of mutation; and the presence of the blood brain barrier (BBB), which acts as a protective boundary between the circulatory system and the brain parenchyma, hampering drug access to the central nervous systems (CNS). Therefore, treatment options for GBM are extremely limited, highlighting the need for newer and more efficient therapies able to address the above challenges. In this contest, nanomedicine-based therapies are extremely interesting as they have the potential to bypass the BBB, thereby extending drug accumulation in the CNS, and to efficiently target different cell populations composing the GBM microenvironment (TME). Unfortunately, nanomedicine require extensive testing, which is unfeasible with traditional animal models. Therefore, three-dimensional (3D) models of GBM representing a powerful and versatile alternative to animal testing, are needed for efficient validation of nanomedicine and, more in general, of other therapeutic options. The primary goal of this thesis was to develop and characterize different in vitro 3D GBM models, focusing on the preparation of multicellular tumor spheroids (MTS), and to apply them for the testing of core-shell drug-loaded nanoparticles (NPs). For this purpose, different cell lines were used, namely, GBM cells (U87-MG), GSCs (GBM-8), microglia (HMC3), and astrocytes (HASTR-ci35). MTS with different compositions were successfully obtained and used to test NPs loaded with the proteasome inhibitor, Bortezomib (BTZ), using the unencapsulated drug as control. Results confirmed that NPs treatment was overall less cytotoxic, proving the importance of the encapsulation of drug for a better targeting and for minimal adverse effects. Moreover, the inclusion of different cell lines provides better mimicry of the heterogenous cell composition of GBM TME resulting in chemoresistance. The invasion mechanism of GBM was studied by embedding MTS in two different hydrogels, simulating the extracellular matrix (ECM), to identify the effect of matrix stiffness on tumor invasion. Moreover, since the BBB is one of the main actors of the GBM progression, MTS of the BBB were successfully created, by mixing brain vascular endothelial cells (HBEC-5i), pericytes (HVBPC) and astrocytes. Lastly, the GBM model complexity was increased by adopting a microfluidic platform (OrganoPlate® Graft, MIMETAS), composed by a central chamber housing the MTS within an ECM gel and two lateral perfusion channels. The latter were seeded with HBEC-5i to mimic brain blood vessels. Thus, an in vitro brain capillary network was obtained with dense homogenous vessel and well-branched sprouts. Thanks to this, the infiltration capabilities of HMC3 and NPs towards the tumor mass in the host chamber was assessed. These promising results pave the way to the possibility of increasing the model complexity, for instance, by varying the properties of the hydrogel matrix or introducing 3D-bioprinting approaches. Moreover, the microfluidic model could be used to investigate nanocarrier- and cell-mediated transport through the BBB to ensure targeted and effective drug delivery.