(A) Schema of FasL and its conversion by plasmin into sFasL, a diffusible trigger of apoptosis through Fas-FADD signaling. TMD, transmembrane domain; SA, trimeric self-assembly domain; THD, tumor necrosis factor-homology domain. Red crosses, apoptotic cells. a, astrocyte. c, cancer cell. (B) IF with antibodies against GFP (cancer cells), GFAP (reactive astrocytes) and FasL in a mouse brain harboring metastatic cells 21 days after arterial inoculation of H2030-BrM3. (C) Images of astrocyte cultures incubated with exogenous plasminogen (1µM) or no additions. Staining was performed with antibodies against the extracellular domain (ECD) or the intracellular domain of FasL (ICD). (D) Western immunoblotting of supernatants from cultures shown in panel C, using anti-FasL ECD antibodies. (E) Mouse brain slices were incubated with α2-antiplasmin, NS and serpin B2, or no additions. sFasL in tissue lysates was detected by western immunoblotting with anti-FasL ECD. Quantification of band density relative to tubulin (left to right) yielded sFasL:FasL ratios of 1, 0.51, 0.28. (F) GFP+ H2030-BrM3 cells (green) were allowed to infiltrate brain slices in media containing added sFasL or no additions and scored for cleaved caspase-3 (red, in inset). (G, H) Quantification of total GFP+ cells (G), and apoptotic GFP+ cells (H) in the experiments of panels F (orange bars) and I (green bars). Data are averages ± SEM. n=6–10 slices, scoring at least two fields per slice, from at least 2 independent experiments. (I) GFP+ H2030 cells (green) were allowed to infiltrate brain slices in media containing anti-FasL blocking antibody or no additions. Anti-FasL prevented endogenous signals from triggering caspase-3 activation (red, in inset). (J) Depiction of FADD-DD overexpression (yellow shape) to suppress pro-apoptotic Fas signaling in cancer cells. (K) FADD expression in H2030-BrM3 transduced with a FADD-DD vector. (L) Quantification of apoptotic cells following sFasL addition to H2030-BrM3 cells transduced with the indicated vectors. (M, N) Quantification of total GFP+ cells (M), and apoptotic GFP+ cells (N) in brain slices harboring the indicated GFP+ H2030-BrM3 transfectants and/or additions. Data are averages ± SEM, and quantitated as panels G,H. (O) Brain metastatic activity of H2030-BrM3 cells transduced with the indicated vectors and inoculated into the arterial circulation of mice. BLI photon flux was quantitated in cells transduced with control shRNA (n=11), FADD-DD (n=4), NS shRNA (n=14), or this shRNA and FADD-DD (n=12). All P values were determined by Student’s t-test. Scale bars: 25µm (B), 200µm (C), 100µm (F,I), 5µm (insets in F,I).
See also .