Fig. 3: TSP1-KO reduces glioma-associated neuronal hyperexcitability.

a Scatter plot illustrating differential gene expression among three RNA sequencing datasets: SB28 (GSE127075), GL261 (GSE94239), and normal murine brain (E-MTAB-6081). Thbs1 is highlighted in red. b Representative confocal images of neonatal mouse cortical neurons (mCN) co-cultured with SB28-TSP1-WT or KO cells for 24 h, showing synaptic puncta colocalization (yellow arrows). Green, synapsin-1 (pre-synaptic); red, Homer-1 (post-synaptic); white, MAP2 (neurons); cyan, GFP (SB28 cells); blue, DAPI. Scale bar, 10 µm. The bar plot quantifies colocalized pre- and post-synaptic puncta on neurites from 12 fields (135 µm × 135 µm) per group across 3 experiments (mean: 1.23 [WT] vs. 0.56 [KO] per 10 µm neurite; p = 0.004). c Representative GCaMP calcium imaging traces of neonatal mouse cortical neurons alone (mCN only [top]) or co-cultured with SB28-TSP1-WT (middle) or KO (bottom) cells. d Bar plots comparing calcium imaging data across the three conditions, quantifying synchronized neuronal activity as calcium events per neuron (regions of interest [ROIs]: n = 5 for mCN only, n = 8 for mCN + WT, n = 7 for mCN + KO). e Enrichment plots and volcano plots summarizing GSEA with the gene set Synaptic Transmission Glutamatergic (GO:BP). Positive normalized enrichment scores (NES) indicate upregulation in WT tumors vs. KO. In volcano plots, gene set members are highlighted, leading-edge genes are labeled, and genes exceeding log₂FC or adjusted p value thresholds are shown at the edges. f Representative traces of spontaneous excitatory postsynaptic currents (EPSCs) from pyramidal neurons identified in mouse cortical layers 5 and 6 (L5/6) near GFP-positive TSP1-WT or KO tumor lesions (400 to 800 μm in distance). g Cumulative frequency distributions of inter-event intervals (IEIs) from the same datasets as Supplementary Fig. 15b (WT: n = 2938 events; KO: n = 1315 events). p values were calculated using the two-sided Welch’s unpaired t-test (b), one-way analysis of variance (ANOVA) with Tukey’s post hoc test (d), and the two-sided Kolmogorov-Smirnov (K-S) test g. For details on statistical tests used in (e), see “Methods”. Data are mean ± s.e.m. b, d. FC fold change, NS not significant. Source data are provided as a Source Data file.