Figure 7: Primary and micronuclear ruptures are associated with cGAS-STING and interferon signaling.

A, Schematic of t-CyCIF imaging to analyze inflammatory and DNA damage states in the glioma TMA. B, Representative CyCIF images of primary IDH-WT GBM showing numerous BAF foci and heterogeneous expression of inflammatory markers in tumor cells. C,D, Representative t-CyCIF images showing a large PN bleb (C) and MN (D) with NE rupture (BAF+) and co-accumulation of TREX1, and pH2AX. E, Representative t-CyCIF images of primary IDH-WT GBM cells showing co-localization of BAF and cGAS at ruptured PN (top row) and MN (bottom row), nuclear translocation of IRF3, NF-kB, and pSTAT3, and expression of IFITM1/2/3 (top row). STING showed substantial background staining (bottom row). F, Probability density analysis of pH2AX, cGAS, and TREX1 signal in PN and MN BAF foci normalized to Hoechst signal, showing significantly greater signal in MN rupture relative to PN ruptures for each marker (p<1x10⁻⁴⁰). G,H, Quantification of interferon signaling-related markers in 82 primary IDH-WT GBM comparing cells with ruptured and unruptured PN (G) and the nearest PN associated with ruptured MN (H). p<0.05, **p<0.01; t-test, two-sided, unpaired. I, Schematic of super-resolution 3D CyCIF markers and image processing. J, Overview of 3D super-resolution CyCIF of 20µm FFPE GBM tissue. K,L, Representative 3D surface rendering of primary IDH-WT GBM tumor cells with a BAF-positive ruptured primary nuclear bleb (K) and BAF-positive ruptured micronucleus (L). BAF forms a ‘shell’ around each ruptured structure. M-P, Representative 3D surface rendering of primary IDH-WT GBM tumor cells with PN rupture, showing frequent co-localization of BAF and CHMP4B consistent with ESCRT-III recruitment and repair (M). PN ruptures, indicated by BAF, occur at sites of reduced lamin B1 expression (N). Super-resolution CyCIF shows frequent co-localization of cGAS (O), and Emerin (P) at BAF-positive rupture sites. Q, Representative 3D surface rendering of primary IDH-WT tumor cells with a micronuclear rupture, showing co-localization of CHMP4B, cGAS, and STING. R, Super-resolution CyCIF imaging reveals complex nuclear bridges connecting multiple tumor cell nuclei (arrows; yellow coloration indicates contiguous nuclear structures).