Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O₂) and to physioxia (5% O₂). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O₂ to 1% O₂. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.

Anti-tumor responses can be achieved via the stimulation of the immune system, a therapeutic approach called cancer immunotherapy. Many solid tumor types are characterized by the presence of immune-suppressive tumor-associated macrophage (TAMs) cells within the tumor microenvironment (TME). Moreover, TAM infiltration is strongly associated with poor survival in solid cancer patients and hence a low responsiveness to cancer immunotherapy. Therefore, 2′3′ Cyclic GMP-AMP (2′3′ cGAMP) was employed for its ability to shift macrophages from pro-tumoral M2-like macrophages (TAM) to anti-tumoral M1. However, cGAMP transfection within macrophages is limited by the molecule’s negative charge, poor stability and lack of targeting. To circumvent these barriers, we designed nanocarriers based on poly(amidoamine) dendrimers (PAMAM) grafted with D-glucuronic acid (Glu) for M2 mannose-mediated endocytosis. Two carriers were synthesized based on different dendrimers and complexed with cGAMP at different ratios. Orthogonal techniques were employed for synthesis (NMR, ninhydrin, and gravimetry), size (DLS, NTA, and AF4-DLS), charge (DLS and NTA), complexation (HPLC-UV and AF4-UV) and biocompatibility and toxicity (primary cells and hen egg chorioallantoic membrane model) evaluations in order to evaluate the best cGAMP carrier. The best formulation was selected for its low toxicity, biocompatibility, monodispersed distribution, affinity towards CD206 and ability to increase M1 (STAT1 and NOS2) and decrease M2 marker (MRC1) expression in macrophages.

Glioblastoma (GBM) is an aggressive brain cancer with a highly immunosuppressive tumor microenvironment (TME), invariably infiltrated by tumor-associated macrophages (TAMs). These TAMs resemble M2 macrophages, which promote tumor growth and suppress immune responses. GBM cells secrete extracellular vesicles (EVs) containing microRNA-25, which inhibits the cGAS-STING pathway and prevents TAMs from adopting a pro-inflammatory M1 phenotype. This study characterizes antimiR-25/cGAMP nanocomplexes (NCs) for potential therapeutic applications. A particle size analysis revealed a significant reduction upon complexation with antimiR-25, resulting in smaller, more stable nanoparticles. Stability tests across pH levels (4–6) and temperatures (25–37 °C) demonstrated their resilience in various biological environments. Biological assays showed that antimiR-25 NCs interacted strongly with transferrin (Tf), suggesting potential for blood–brain barrier passage. The use of cGAMP NCs activated the cGAS-STING pathway in macrophages, leading to increased type I IFN (IFN-β) production and promoting a shift from the M2 to M1 phenotype. The combined use of cGAMP and antimiR-25 NCs also increased the expression of markers involved in M1 polarization. These findings offer insights into optimizing antimiR-25/cGAMP NCs for enhancing immune responses in GBM.