Interfering with the tumour microenvironment of glioblastoma: an in vitro study

Glioblastoma (GBM) is the most aggressive primary malignant tumour of the central nervous system (CNS), characterized by high invasiveness, resistance to therapy and frequent recurrence. Despite the current standard treatment, including surgical resection followed by radiotherapy and chemoradiotherapy, GBM patients face poor prognosis due to therapeutic resistance and tumour recurrence. A major contributor to GBM aggressiveness is the tumour microenvironment (TME), which promotes malignancy through interactions involving soluble factors, direct cell contacts, and extracellular vesicles (EVs). Among these stromal elements, glioma-associated stem cells (GASCs), a non-tumorigenic stromal component of the TME, have been shown to support tumour progression by secreting exosomes. These exosomes enhance glioma cell proliferation, migration, and resistance, particularly affecting glioma stem cells (GSCs), the tumour-initiating population and responsible of resistance to drug treatments. This study explores a novel therapeutic strategy based on reprogramming the tumour-supportive properties of the TME through drug modulation of GASCs and GASC-derived exosomes (GDEs). Two drugs targeting integrin-dependent pathways, anti-integrin α5β1 antibody and bacitracin, were employed to interfere with mechanism known to sustain GBM invasiveness. In vitro, these compounds have shown efficacy in reversing the tumour-supporting function of the TME, reducing GASC activated phenotype such as proliferation, motility and anchorage-independent growth. This supportive function is partly performed by exosomes: in fact, exosomes isolated from untreated GASCs enhanced tumour aggressiveness, whereas those from healthy astrocytes did not exert such effects. Remarkably, exosomes isolated from drug-treated GASCs no longer supported tumour aggressiveness: they impaired tumour cell proliferation, motility, migration/invasion and anchorage-independent growth. These findings indicate that drug treatment can effectively re-educate the TME, converting exosomes from tumour-promoting to tumour-suppressive mediators. Molecular characterization revealed that exosomes from drug-treated GASCs displayed a distinct microRNA profile, characterized by the upregulation of miR-21-5p, miR-34a-5p and miR-100-5p. This modified miRNA cargo suggests that drug-induced changes in the exosomal cargo may underlie their reduced tumour-supporting capacity. Overall, this work provides new insights into the role of GASC-derived exosomes in GBM progression and demonstrate that targeting the TME represents a promising adjuvant strategy to counteract tumour aggressiveness. By modulating exosome-mediated intercellular communication, it may be possible to overcome therapy resistance and improve the clinical management of GBM patients.