Figure 4. Gap junctions induce cytosolic dsDNA response in astrocytes.

a, MDA231-BrM2 cells expressing control shRNA (Ctrl sh) or shRNA targeting Cx43, were cultured for 18 h with or without astrocytes, and subjected to immunobloting analysis of phosphorylated TBK1 and IRF3 (n=3 independent experiments). b, Representative images of dual immunofluorescent staining of IRF3 and GFP. DAPI, nuclear staining. In co-cultures: white arrows, nuclear accumulation of IRF3 in astrocytes; green arrows, even distribution of IRF3 in GFP+ MDA231-BrM2 cells. Scale bar, 20 μm. (n=2 independent experiments). c, Mouse astrocytes (Astro) expressing control shRNA (Ctrl sh) or shRNA targeting STING, were cultured for 18 h with or without LLC BrM cells (BrM), and subjected to immunobloting analysis of phosphorylated IRF3 (c); d, LLC BrM growth in syngeneic C57Bl6 mice hosts wild-type (+/+) or knock-out (−/−) for STING. Quantification of BLI signal from brain metastases formed in +/+ and −/− host mice (e). (n=11 mice in each group; 2 independent experiments). e, MDA231-BrM2 alone, astrocytes alone, or 18 h co-cultures, were harvested for sample preparation and cGAMP analysis by LC-MS/MS. Representative chromatograms (e) and quantitation (f) are presented (n= 5 biological replicates in 3 independent experiments). Refer also to Supplementary Material. g, Human astrocytes (Astro), were cultured for 18 h with or without H2030 BrM cells (BrM) expressing control shRNA (Ctrl sh) or shRNA targeting cGAS, and subjected to immunobloting analysis of phosphorylated IRF3, (2 independent experiments). h, Schematic summary of gap junction mediated anti-dsDNA response, production of IFNα and TNFα in astrocytes, and consequent activation of STAT1 and NF-κB pathways in cancer cells to support brain metastasis.