Fig. 3

Brain colonization suppresses invasiveness of metastatic lung cancer cells. A Transwell migration assay of A549-M0 and M2 cells co-cultured with astrocytes. GFP-labelled M0 and M2 cells (5 × 10⁴/insert) and mCherry-labelled astrocytes (5 × 10⁴/insert) were cultured in the upper wells for 24 h. Migrated A549 cells and astrocytes were counted under a fluorescence microscope. Mean ± SD (n = 3). P, unpaired two-tailed Student’s t-test. B Three-well migration assay of M0 and M2 cells co-cultured with astrocytes. Lung cancer cells and astrocytes were seeded onto either side of the Culture-Insert 2 Well in a 35-mm dish. Cells were then allowed to migrate toward the center and close the wound for 48 h. Overlap areas between two types of cells were measured using ImageJ. Mean ± SD (n = 4). P, unpaired two-tailed Student’s t-test. C Co-culture of A549 spheroids on astrocyte feeder layers. A549-M0 or M2 spheroids (green) were overlaid on top of confluent astrocyte feeder layers (red). Invasion of A549 spheroids was observed after 12 h. Spheroid invasion area was measured by ImageJ