Immunomodulation and Cellular Interactions in the Tumor Microenvironment

Glioblastoma (GBM) represents one of the most aggressive and lethal cancers of the central nervous system (CNS), with a poor prognosis and limited therapeutic options. A major obstacle in the development of effective therapies is the presence of the blood–brain barrier (BBB), which tightly regulates molecular transport and hinders drug delivery to the brain parenchyma. Furthermore, current preclinical models, although indispensable, often fail to accurately reproduce the complexity of the human tumor microenvironment and the neurovascular unit, limiting their translational value. In this framework, the work presented in this thesis addresses both the therapeutic modulation of microglia and the development of organ-on-chip models of the BBB and GBM, with the goal of advancing nanomedicine-based strategies and microphysiological systems. Chapters 2 and 3 focus on microglia, the resident immune cells of the CNS and key modulators in brain disorders. In particular, Chapter 2 is focused on the role of microglia in the GBM tumor microenvironment. Lipid-based Magnetic Nanovectors (LMNVs), consisting of a lipid matrix embedding Iron Oxide Nanoparticles (NPs), were developed and tested as magnetothermal transducers capable of inducing microglial polarization. Under alternating magnetic field (AMF) stimulation, LMNVs triggered intracellular calcium signaling, promoting a shift toward a pro-inflammatory M1-like phenotype. This activation was confirmed by upregulation of inflammatory markers (CD40, CD86), increased cytokine secretion (IL-6, IL-8, TNF-α), and a marked anti-tumor effect of microglia-conditioned media on both immortalized and patient-derived GBM cells. These results demonstrate the potential of LMNVs as immunomodulatory nanoplatforms for remote microglial activation against GBM. On the other hand, Chapter 3 explored Polydopamine Nanoparticles (PDNPs) as inhibitors of M1 activation, a key contributor to neuroinflammation in neurodegenerative diseases. PDNPs demonstrated excellent biocompatibility, efficient uptake by human microglia, and significant suppression of IFN-γ-induced oxidative stress, M1 marker expression, and cytokine release. Together, these findings highlight how nanomaterials can be designed to either promote or attenuate microglial activation, depending on the pathological context. Chapter 4 presents the development of a sensorized BBB-on-chip platform. In Part I, the BBB-on-chip model integrates human endothelial cells, astrocytes, and microglia in a microfluidic configuration that mimics the architecture and physiology of the human neurovascular unit. The system incorporates thin-film microelectrodes for real-time monitoring of BBB formation and integrity through electrochemical impedance spectroscopy (EIS). Equivalent circuit modeling allowed the extraction of trans-endothelial electrical resistance (TEER). The device faithfully reproduced shear stress conditions, as confirmed by computational fluid dynamics (CFD) simulations, and successfully discriminated between two chemotherapeutic drugs with different BBB permeability profiles, such as temozolomide and doxorubicin. This work underscores the potential of sensorized BBB-on-chip platforms as physiologically relevant alternatives to animal models for CNS drug discovery and toxicology. In Part II, the model was adapted to a simplified single-chamber configuration with increased cell density, enabling accelerated barrier maturation, suitable for exposure to simulated microgravity using a random positioning machine (RPM) for long-term. Functional and molecular analyses revealed that endothelial cells exhibited increased permeability, tight junction (TJ) gene upregulation (CLDN-5, OCLN, ZO-1), and enhanced oxidative and inflammatory signaling (IL-6, ROS, nitrites, singlet oxygen), indicating barrier weakening under microgravity. Conversely, astrocytes activated protective responses, including IL-6, HSP27, and Annexin V upregulation, without significant extracellular ROS increase, highlighting their ability to counteract the inflammatory effects. These results demonstrate that endothelial cells are the most vulnerable BBB component under altered mechanical loading, while astrocytes provide compensatory feedback that preserves system-level homeostasis. Chapter 5 introduces an impedance-based GBM-on-chip device designed to replicate the three-dimensional microenvironment of tumor foci responsible for recurrence after surgical resection. The platform integrates platinum (Pt) microelectrodes with 3D scaffolds fabricated by two-photon polymerization, which support stable colonization by GBM cells and promote spheroid formation directly in contact with the sensing area. The system enables high-throughput, multiplexed, and real-time impedance monitoring of tumor growth and treatment responses, while its optical transparency allows complementary imaging analysis. Validation studies demonstrated its capacity to detect subtle cytotoxic effects not revealed by conventional end-point immunofluorescence, highlighting its value for drug efficacy screening in physiologically relevant 3D models. Collectively, this thesis combines nanotechnology-based therapeutic strategies with advanced organ-on-chip modeling of the BBB and GBM, providing innovative tools for the study of CNS tumors. The integration of NPs-based immunomodulation with predictive microfluidic and impedance-based tumor models sets the stage for translational approaches in drug discovery and personalized medicine for GBM.