Exosomal non-coding RNAs mediate the cross-talk of cancer stem cells from lung-tumor derived brain metastases and microglia cells

Brain metastases are the most common secondary brain tumors in adults. Despite their high frequency and poor prognosis, little research has been performed on lung tumor-derived brain metastases, mainly due to the lack of appropriate experimental models. In this study, we isolated cancer stem cells (CSCs) from fresh specimens of lung-tumor derived brain metastases. The CSCs were analyzed for sphere formation and limiting dilution analyses, stemness markers, ability to generate xenografts and for their interaction with microglia cells. We found that CSCs derived from brain metastases had a high sphere-forming capacity and self-renewal ability comparable to that of glioma stem cells. The CSCs expressed the stemness markers, CD133, Sox2, Klf4, Aldh2a, and CD44 and the lung tumor markers, cytokeratin 7 and CD166. Transplantation of the CSCs or organoids generated from the brain metastases formed xenografts that recapitulated the parental tumors. These xenografts were infiltrated by a large number of amoeboid microglia that expressed high levels of M2 markers. Using co-culture experiments, we further found that CSCs derived from brain metastases induced the polarization of microglia to the M2 phenotype via secreted exosomes. Similarly, M2 microglia cells increased the self-renewal and stemness of the CSCs. RNA sequence analysis identified specific miRNAs and lncRNAs that were associated with the CSC-microglia interactions. Using specific reporters, antagomiRs and CRISPR/Cas9, we demonstrated that miR-21, miR-1246 and the lncRNA TALNEC2 played a major role in the M2 polarization of microglia cells both in vitro and in vivo. These non-coding RNAs were also detected in high levels in circulating serum exosomes of xenografted mice. Finally, we demonstrated that knockdown of miR-21 and TALNEC2 inhibited xenograft growth and prolonged the survival of mice harboring CSC-derived xenografts. In conclusion, we generated CSCs and organoids from lung tumor-derived brain metastases and identified potential therapeutic targets for these tumors. Silencing of miR-21, miR-1246 and TALNEC2 exerted anti-tumor effects in CSCs and modulated the cross-talk of these cells with microglia and possibly with additional immune cells via exosomal delivery.