Irradiation-induced M1 Microglia Affect Brain Metastatic Colonization of A549 Cell Lines via miR-9/CDH1 Axis

Abstract Background and purpose: Brain metastasis is among the leading causes of death in patients with non-small-cell lung cancer (NSCLC). Through yet unknown mechanisms, prophylactic cranial irradiation (PCI) can significantly decrease the incidence of brain metastases. We propose that PCI probably exerts indirect anti-tumoral effects by turning cerebral “soil” unfavorable for the colonization of metastatic tumor “seeds”. This study aims to reveal how PCI regulates the brain microenvironment conducing to a reduction in brain metastases. Materials and methods: Key marks of M1/M2 microglia types and mesenchymal-to-epithelial transition (MET) were analyzed by qRT-PCR and Western Blot in vitro. The target miR-9-5p was obtained by miRNA array analysis and confirmed by qRT-PCR in microglia. miRTarBase and TargetScan were used to predict the target genes of miR-9-5p, which were assessed by luciferase activity assay. Anti-metastatic effects of irradiation on the brain were evaluated by intravital imaging using a brain metastatic NSCLC A549-f3 cell line in a nude mouse model. Results: We found the microglia can polarize into M1 type after irradiation and the irradiated microglia inhibited mesenchymal-epithelial transition of metastatic tumor cells, which decreased their adhesion and colonization capabilities in brain. We discovered up-regulated miR-9 was related with inhibition of MET process. Further overexpression/silencing experiments indicated that irradiated M1-type microglia inhibited A549 mesenchymal-epithelial transition, mediated by miR-9 up-regulation and secretion. Additionally, these results were confirmed in mice model, as low dose irradiation increased miR-9 level in the brain microenvironment and reduced brain metastases of metastatic tumor cells. Conclusions: We demonstrated that miR-9 secreted by irradiated M1-type microglia played important role in inducing A549 cell lines into mesenchymal phenotype, and further decreased their localization capabilities in brain. Our findings emphasized the modulating effect of irradiation on metastatic soil, and the cross-talk between tumor cells and the metastatic microenvironment. More importantly, our findings provided new perspectives for effective anti-metastasis therapies, especially for NSCLC patients with high risk of brain metastasis.