Modello tridimensionale di glioblastoma multiforme per test in vitro di nanomedicine = Three-dimensional glioblastoma multiforme model for in vitro nanomedicine testing

Glioblastoma Multiforme (GBM) is the most malignant primary brain tumor in adults. The current medical treatment is a combination of resection, followed by radiotherapy and chemotherapy, which results in a dismal survival of 14.6 months. Additionally, this treatment regimen is aggressive and poorly selective, resulting in intracranial metastases and systemic side effects. The main features of GBM involve nuclear atypia, highly infiltrative and invasive phenotype and necrosis. Moreover, the heterogeneous composition, characterized by the presence of distinct cell types with different morphologies, genetic mutation and differentiation stage, poses a challenge in the identification of the proper treatment strategy. The presence of a Cancer Stem Cells (CSCs) niche, combined with the heterogeneity of the tumor microenvironment, composed of immune cells, endothelial cells and other infiltrating cells, facilitates invasion, proliferation and angiogenesis. Thus, the tumor microenvironment plays a fundamental role in determining the tumor response to treatment. The complex GBM landscape requires new and more advanced therapies able to concomitantly address the multiple players involved. Current 2D and 3D in vitro models fail to replicate the GBM microenvironment, while in vivo models often lack proper immune response, besides being costly and ethically debated. ??The aim of this work is to design a more reliable in vitro model of GBM comprising CSCs, differentiated GBM cells, resident immune cells, as well as a vascular network. To achieve this, we will use a microfluidic device (OrganoPlate® Graft, MIMETAS) designed to feature i) a vascular channel that can be fully covered by endothelial cells; ii) a graft chamber that hosts the tumor model which can contain multiple cell types; and iii) an intermediate 3D structure that acts as a mimic of the tumor microenvironment, hosting additional cell types as well as a network of micro-vessels. By introducing the angiogenic signals, such vessels can be induced to vascularize the tumor model, allowing perfusion and drug delivery/release experiments. Here, a 3D tumor spheroid model composed of primary human GBM cells (U87), GBM-associated stem cells (GBM8) and human microglia (HMC-3) in different ratios has been developed. Tumor response to the chemotherapeutic agent Bortezomib (BTZ) has been assessed both in 2D and in 3D spheroids at different timepoints and at varying drug concentrations. In parallel, vascularization of the microfluidic channel has been optimized, by testing different conditions using human cerebral microvascular endothelial cells (HBEC-5i). Since BTZ has been shown to possess strong antitumoral activity in GBM models but is not able to cross the blood brain barrier, polymer nanoparticles (NPs) for BTZ delivery have been developed and characterized. Such particles can be exploited to enhance brain-accumulation of the drug while minimizing its adverse effects. The final device will contain the vascularized tumor model and will be used to evaluate drug efficacy, comparing the achieved results with the previous in vivo data with BTZ. NPs-mediated delivery experiments are in progress to determine the ability of NPs to extravasate through the vessel model. If successful, the developed 3D vascularized GBM model can be further implemented by adding additional components, such as astrocytes and neurons and to test drug delivery strategies to brain tumors.