Hypoxic glioblastoma-cell-derived extracellular vesicles impair cGAS-STING activity in macrophages.

BACKGROUND: Solid tumors such as glioblastoma (GBM) exhibit hypoxic zones that are associated with poor prognosis and immunosuppression through multiple cell intrinsic mechanisms. However, release of extracellular vesicles (EVs) has the potential to transmit molecular cargos between cells. If hypoxic cancer cells use EVs to suppress functions of macrophages under adequate oxygenation, this could be an important underlying mechanism contributing to the immunosuppressive and immunologically cold tumor microenvironment of tumors such as GBM. METHODS: EVs were isolated by differential ultracentrifugation from GBM cell culture supernatant. EVs were thoroughly characterized by transmission and cryo-electron microscopy, nanoparticle tracking analysis (NTA), and EV marker expression by Western blot and fluorescent NTA. EV uptake by macrophage cells was observed using confocal microscopy. The transfer of miR-25/93 as an EV cargo to macrophages was confirmed by miRNA real-time qPCR. The impact of miR-25/93 on the polarization of recipient macrophages was shown by transcriptional analysis, cytokine secretion and functional assays using co-cultured T cells. RESULTS: We show that indirect effects of hypoxia can have immunosuppressive consequences through an EV and microRNA dependent mechanism active in both murine and human tumor and immune cells. Hypoxia enhanced EV release from GBM cells and upregulated expression of miR-25/93 both in cells and in EV cargos. Hypoxic GBM-derived EVs were taken up by macrophages and the miR-25/93 cargo was transferred, leading to impaired cGAS-STING pathway activation revealed by reduced type I IFN expression and secretion by macrophages. The EV-treated macrophages downregulated expression of M1 polarization-associated genes Cxcl9, Cxcl10 and Il12b, and had reduced capacity to attract activated T cells and to reactivate them to release IFN-γ, key components of an efficacious anti-tumor immune response. CONCLUSIONS: Our findings suggest a mechanism by which immunosuppressive consequences of hypoxia mediated via miRNA-25/93 can be exported from hypoxic GBM cells to normoxic macrophages via EVs, thereby contributing to more widespread T-cell mediated immunosuppression in the tumor microenvironment.

Radio-chemotherapy of glioblastoma cells promotes phagocytosis by macrophages in vitro.

BACKGROUND AND PURPOSE: Immunotherapy is actively explored in glioblastoma (GBM) to improve patient prognosis. Tumor-associated macrophages (TAMs) are abundant in GBM and harnessing their function for anti-tumor immunity is of interest. They are plastic cells that are influenced by the tumor microenvironment, by radio-chemotherapy and by their own phagocytic activity. Indeed, the engulfment of necrotic cells promotes pro-inflammatory (and anti-tumoral) functions while the engulfment of apoptotic cells promotes anti-inflammatory (and pro-tumoral) functions through efferocytosis. MATERIALS AND METHODS: To model the effect of radio-chemotherapy on the GBM microenvironment, we exposed human macrophages to supernatant of treated GBM cells in vitro. Macrophages were derived from human monocytes and GBM cells from patient-resected tumors. GBM cells were exposed to therapeutically relevant doses of irradiation and chemotherapy. Apoptosis and phagocytic activity were assessed by flow cytometry. RESULTS: The phagocytic activity of macrophages was increased, and it was correlated with the proportion of apoptotic GBM cells producing the supernatant. Whether uptake of apoptotic tumor cells could occur would depend upon the expression of efferocytosis-associated receptors. Indeed, we showed that efferocytosis-associated receptors, such as AXL, were upregulated. CONCLUSIONS AND PERSPECTIVES: We showed that macrophage phagocytic activity increased when exposed to supernatant from GBM cells treated by radio-chemotherapy. However, as efferocytosis-associated receptors were up-regulated, this effect could be deleterious for the anti-GBM immune response. We speculate that by inducing GBM cell apoptosis in parallel to an increase in efferocytosis receptor expression, the impact of radio-chemotherapy on phagocytic activity could promote anti-inflammatory and pro-tumoral TAM functions.