Unravelling the metastasis-preventing effect of miR-200c in vitro and in vivo

Advanced breast cancer as well as insufficient treatment can lead to the dissemination of malignant cells from the primary tumor to distant organs. Recent research has shown that miR-200c can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear, whether sole miR-200c expression is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs including lung, liver, brain, and spleen has revealed a dramatically reduced metastatic burden of mice with miR-200c expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective and single cell migration in vitro, utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective cell migration has resulted in confluence dependent motility of cells with altered miR-200c expression. Additionally, scratch assays have shown enhanced predisposition of miR-200c negative cells to leave cell clusters. The in-between stage of collective and single cell migration was validated using transwell assays, which have displayed reduced migration of miR-200c positive cells. Finally, to measure migration on single cell level, a novel assay on dumbbell shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that exclusive expression of miR-200c impedes metastasis formation in vivo and migration in vitro and highlight miR-200c as metastatic suppressor in breast cancer.