Extended Data Fig. 5: Analysis of T-cell apoptosis in GBM and TME characterization after Tnfsf10 knockdown in astrocytes.

(a) Reanalysis of spatial transcriptomics data in GBM⁸⁶. Spatially-weighted correlations of TRAIL imputation score from Fig. 2f and non-malignant reactive astrocyte signature³². (b) Spatially annotated bulk RNA-seq from Ivy Glioblastoma Atlas showing signature score of death receptor signalling (R-HSA-73887) split by anatomical niche (left). Pearson correlation analysis of TNFSF10 and TNFRSF10B expression split by anatomical niche. (c) Validation by flow cytometry of Tnfsf10-targeting lentivirus-driven genetic perturbation in TdTomato⁺ Aldh1l1^{Cre-ERT2/TdTomato} reporter cells isolated from naïve mice (n = 8 NTsgRNA, n = 10 sgTnfsf10). (d) Control measurement of TRAIL in a GFP⁺ GL261 line by flow cytometry 15 days after implantation following the lentivirus-driven genetic perturbation in astrocytes (n = 3 NTsgRNA, n = 2 sgTnfsf10). (e) Analysis by qPCR of off-target Tnfsf10 deletion effects in sorted T cells and myeloid cells effects following lentivirus administration as shown in Fig. 3f. (f) Survival analysis following implantation of CRISPR control-edited or Tnfsf10-deleted GL261 lines (n = 4 GL261-NTsgRNA, n = 6 GL261-sgTnfsf10), (g) Survival analysis of mice implanted with genetically engineered glioma model MK007 following TRAIL inactivation in astrocytes as shown in Fig. 3f (n = 10 each). (h) Survival analysis of GL261-implanted Rag2^−/− mice following TRAIL inactivation in astrocytes (n = 5 each). (i,j) Flow cytometry quantification related to Fig. 3h of cleaved caspase-3/7⁺ single-positive (i) and caspase-3/7⁺ SYTOX⁺ double-positive (j) CD4⁺ or CD8⁺ T cell populations. Data shown as mean ± SEM. Unpaired two-tailed t-test used for statistical analysis. Survival analysis by Log-rank (Mantel-Cox) test for (f-h). n indicates biologically independent samples.

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