Fig. 2: Radiotherapy is sufficient for epigenetic reprogramming of neural crest to immune-enriched schwannoma.

a Pairwise Pearson correlation coefficients grouped by hierarchical clustering of DNA methylation profiles from the patient (Pt) matched primary and recurrent schwannomas (n = 13). Arrows represent the reprogramming of primary NCS to IES at recurrence. b H&E-stained sections of a patient-matched primary NCS and recurrent IES, showing Verocay bodies (top arrows) and abundant lymphocytes (bottom arrows) with foamy and hemosiderin-filled macrophages (asterisks). Scale bar, 100 μm. Similar findings were observed in two additional matched pairs. c UMAP of 38,754 transcriptomes from single-cell RNA sequencing of HEI-193 cells after treatment with 0Gy, 1.8Gy × 5, or 12.5Gy × 1 (n = 3 independent replicates per condition) of radiotherapy revealing 15 distinct schwannoma cell states. d UMAPs from c with individual transcriptomes split according to triplicate treatment conditions. e Relative composition of cell states from c according to treatment conditions. f Perturb-seq gene-set expression heatmap of 3546 pseudobulked transcriptomes from HEI-193 cells following sgRNA perturbations (columns) across treatment conditions. Expression values are normalized to cells harboring non-targeting control sgRNAs (sgNTC) with 0 Gy. g Number of differentially expressed genes (DEG) from schwannoma cell Perturb-seq with radiotherapy (y-axis) versus 0Gy (x-axis). sgRNA perturbations with ≥40 DEG after radiotherapy compared to control are orange. sgRNA not meeting this threshold are dark gray. sgNTCs are light gray. h Differential gene expression analysis from PTPRG perturbation compared to sgNTC in 0 Gy (left) versus 1.8 Gy × 5 conditions (right). Significant positive (red) or negative (blue) gene expression changes are colored (p < 0.05, |log₂(fold change)| > 1), corresponding to gene ontology (two-sided Fisher’s exact test without adjustments for multiple comparisons). i Feature plot of integrated UMAP from harmonized schwannoma single transcriptomes (Fig. 1b) showing PTPRG expression in schwannoma cells. j TUNEL staining for apoptosis in HEI-193 cells following CRISPRi suppression of PTPRG versus sgNTC after treatment with 0 Gy (n = 3 independent cultures), 1.8 Gy × 5 (n = 3 independent cultures across 2 sgRNAs each), or 12.5 Gy × 1 (n = 3 independent cultures across 2 sgRNAs each) of radiotherapy. Fold changes are normalized to sgNTC in each treatment condition. Lines represent means, and error bars represent the standard error of means (two-sided Student’s t-tests, *p = 0.030, **p = 0.0044). Source data are provided as a Source Data file.