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Inhibition of tumor progression and M2 microglial polarization by extracellular vesicle-mediated microRNA-124 in a 3D microfluidic glioblastoma microenvironment.

ABSTRACT: Background: Glioblastoma (GBM) is one of the most aggressive types of brain cancer. GBM progression is closely associated with microglia activation; therefore, understanding the regulation of the crosstalk between human GBM and microglia may help develop effective therapeutic strategies. Elucidation of efficient delivery of microRNA (miRNA) via extracellular vesicles (EVs) and their intracellular communications is required for therapeutic applications in GBM treatment. Methods: We used human GBM cells (U373MG) and human microglia. MiRNA-124 was loaded into HEK293T-derived EVs (miR-124 EVs). Various anti-tumor effects (proliferation, metastasis, chemosensitivity, M1/M2 microglial polarization, and cytokine profile) were investigated in U373MG and microglia. Anti-tumor effect of miR-124 EVs was also investigated in five different patient-derived GBM cell lines (SNU-201, SNU-466, SNU-489, SNU-626, and SNU-1105). A three-dimensional (3D) microfluidic device was used to investigate the interactive microenvironment of the tumor and microglia. Results: MiR-124 EVs showed highly efficient anti-tumor effects both in GBM cells and microglia. The mRNA expression levels of tumor progression and M2 microglial polarization markers were decreased in response to miR-124 EVs. The events were closely related to signal transducer and activator of transcription (STAT) 3 signaling in both GBM and microglia. In 3D microfluidic experiments, both U373MG and microglia migrated to a lesser extent and showed less-elongated morphology in the presence of miR-124 EVs compared to the control. Analyses of changes in cytokine levels in the microfluidic GBM-microglia environment showed that the treatment with miR-124 EVs led to tumor suppression and anti-cancer immunity, thereby recruiting natural killer (NK) cells into the tumor. Conclusions: In this study, we demonstrated that EV-mediated miR-124 delivery exerted synergistic anti-tumor effects by suppressing the growth of human GBM cells and inhibiting M2 microglial polarization. These findings provide new insights toward a better understanding of the GBM microenvironment and provide substantial evidence for the development of potential therapeutic strategies using miRNA-loaded EVs.

SUBMITTER: Hong S

PROVIDER: S-EPMC8490520 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. As miR-124 expression is repressed in various cancer types (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared with regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 reintroduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression.

Project description:Abstract Background Melatonin (MEL), an endogenous hormone, has been widely investigated in neurological diseases. Microglia (MG), a resident immunocyte localizing in central nervous system is reported to play important functions in the animal model of temporal lobe epilepsy (TLE). Some evidence showed that MEL influenced activation of MG, but the detailed model of action that MEL plays in remains uncertain. Methods In this study, we established a model of TLE in mice by stereotactic injection of kainic acid (KA). We treated the mice with MEL. Lipopolysaccharide, ROCK2‐knockdown (ROCK‐KD) and ‐overexpression (ROCK‐OE) of lentivirus‐treated cells were used in cell experiments to simulate an in vitro inflammatory model. Results The results of electrophysiological tests showed that MEL reduced frequency and severity of seizure. The results of behavioral tests indicated MEL improved cognition, learning, and memory ability. Histological evidences demonstrated a significant reduction of neuronal death in the hippocampus. In vivo study showed that MEL changed the polarization status of MG from a proinflammatory M1 phenotype to an anti‐inflammatory M2 phenotype by inversely regulating the RhoA/ROCK signaling pathway. In cytological study, we found that MEL had a significant protective effect in LPS‐treated BV‐2 cells and ROCK‐KD cells, while the protective effect of MEL was significantly attenuated in ROCK‐OE cells. Conclusion MEL played an antiepileptic role in the KA‐induced TLE modeling mice both in behavioral and histological levels, and changed MG polarization status by regulating the RhoA/ROCK signaling pathway. 1. Melatonin (MEL) reduces frequency and severity of seizure. 2. MEL regulates the ROCK2 signaling pathway rather than ROCK1 to promote microglial polarization toward an anti‐inflammatory direction. 3. RhoA/ROCK2 signaling pathway might be a potential mechanism in the status epilepticus modeling mice. Highlights • Melatonin (MEL) reduces frequency and severity of seizure.• MEL regulates the ROCK2 signaling pathway rather than ROCK1 to promote microglial polarization toward an anti‐inflammatory direction.• RhoA/ROCK2 signaling pathway may be important in the temporal lobe epilepsy modeling mice.

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Inhibition of tumor progression and M2 microglial polarization by extracellular vesicle-mediated microRNA-124 in a 3D microfluidic glioblastoma microenvironment.

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