Figure 4. Senescent astrocytes promote migration of GBM cells in vitro.

A, GL261 tumors growing in mock-irradiated or pre-irradiated (10 Gy, 30 days) brains were sectioned and stained for phospho-Met (Y1234/1235; red) and DAPI (blue). Representative images are shown (n=3 per cohort). Note robust Met activation in tumor growing in pre-irradiated brain. Scale bar=50 μm. B, Schematic of Boyden Chamber Assay with GL261 cells in the top chamber and mock-irradiated or irradiated (10 Gy, 10 days) primary mouse astrocytes in the bottom chamber. Representative images of GL261 cells on the bottom surface of the trans-well membrane (indicated by *) stained with Alexa Fluor 488 Phalloidin (green). Scale bar=100 μm. C, Plot shows percentage of cells per 40X microscopic field migrating towards the bottom chamber relative to migration towards media alone (n=3, P<0.0001, error bars S.D). Control represents assay with only cell culture media in the bottom chamber (no astrocytes). D, Representative fluorescence images of GL261 cells treated with either IgG or α-HGF antibody in a Boyden Chamber migration assay with mock-irradiated or irradiated primary astrocytes in the bottom chamber. Scale bar=100 μM. E, Plot shows percentage of cells per 40X microscopic field migrating towards the bottom chamber relative to migration towards IgG-treated mock-irradiated astrocytes (n=3, IR/IgG vs IR/αHGF P<0.0001, error bars S.D). F, Representative fluorescence images of GL261 cells treated with either DMSO or Crizotnib in a Boyden Chamber migration assay with mock-irradiated or irradiated primary astrocytes in the bottom chamber. Scale bar=100 μm. G, Plot shows percentage of cells per 40X microscopic field migrating towards the bottom chamber relative to migration towards DMSO-treated mock-irradiated astrocytes (n=3, IR/DMSO vs IR/Crizotinib P=0.0047, error bars S.D).