Extended Data Fig. 1: Early tumour cells home to white matter.

a, Schematic of constructs used for in vivo electroporation in this study. From top to bottom: piggyBase. Basal tdTomato control piggyBac construct (tdTom) for integration of tdTomato alone; npp piggyBac construct for tumour induction. Adapted from (Clements et al.²⁰). b, Quantification of the percentage of striatal area occupied by MBP⁺ white matter in mice bearing npp tumours at indicated stages of tumour development. Tumour-involved striatum was quantified. One-way ANOVA with Tukey’s multiple comparisons. Mean ± SD, early, intermediate and late, n = 4 mice; for terminal, n = 3 mice. p = 0.015 (early vs late); p = 0.0333 (intermediate vs late). c, Time course analysis of SVZ/striatal brain regions of mice electroporated with tdTom construct. Representative images confirm efficient targeting and minimal migration in white matter regions adjacent to the SVZ in the absence of mutations. Scale bar=500 μm. Representative of n = 3 mice for each time point. d, Representative image of an olfactory bulb from a mouse collected at 4 weeks post-electroporation shown in c, confirming that tdTom-electroporated NSCs predominantly give rise to new olfactory neurons. Scale bar=500 μm. Representative of n = 3 mice. e, Representative images of tdTomato⁺ (red) npp tumours quantified in Fig. 1c. Tumours were collected at indicated stages and stained for the proliferation marker Ki67⁺ (grey), white matter marker (myelin basic protein, MBP, green) and DAPI (blue). Arrowheads highlight examples of tumour cell proliferation within (white) and outside (yellow) white matter. Scale bar=100 μm. Representative of early n = 4, Intermediate n = 4, Late n = 4, Terminal n = 3 mice. f, Representative images of indicated GFP-labelled PDX tumours collected early or at terminal disease (corresponding to 56 and 190 days for GBM2, and 70 and 143 for GL23, 34 and 56 days for GCGR E43 and 60 and 89 days for GCGR L5, respectively), stained for MBP (red). Dashed boxes denote regions shown at higher magnification in inset. Scale bar=500 μm. Images are representative of the following numbers of mice; GBM2 n = 3 early, n = 4 late; GL23 n = 3 early, n = 4 late; GCGRL5 n = 3 early, n = 3 late; GCGR E43 n = 3 early, n = 5 late. g, Quantification of percentage of GFP⁺ tumour cells located in white or grey matter within the striatum of PDX models shown in f. Comparison between %GFP⁺ cells in white matter and grey matter: two-way ANOVA with Tukey’s multiple comparisons. p < 0.0001 (early WM vs early GM). Comparison of the distribution of GFP⁺ cells (calculated as %GFP⁺ cells in WM - %GFP⁺ cells in GM) between early and terminal stages: Two-sided paired t test.; p = 0.0127 (early vs terminal). Mean ± SD. Early n = 4, Terminal n = 4 mice.

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