Extended Data Fig. 4: Axonal injury is accompanied by neuroinflammation.

a-c, Representative images of GFAP (magenta) and CD68 (turquoise) staining in brains from age-matched healthy control mice or within early, intermediate, late and terminal npp tumours generated in WT mice. Scale bar=500 μm. Control n = 6, early n = 6, intermediate n = 7, late n = 7 and terminal n = 4 mice. b-c, Quantification of GFAP area (b) and CD68 integrated density (c) within the tumour region of samples from a, plotted as fold change over levels in brain region-matched controls. Mean ± SEM. Multiple two-sided unpaired t tests. Control n = 6, early n = 6, intermediate n = 7, late n = 7 and terminal n = 4 mice. In b, p < 0.0001 for control vs early, control vs intermediate and control vs terminal; p = 0.0004 control vs late; p = 0.0281 early vs late; p = 0.0217 early vs terminal; p = 0.0494 intermediate vs late. In c, p < 0.0001 for control vs terminal and intermediate vs terminal; p = 0.0024 control vs early; p = 0.0150 control vs intermediate; p = 0.0023 control vs late; p = 0.0005 early vs terminal; p = 0.0112 intermediate vs late. d, Representative images of GFAP⁺ astrocytes surrounding tumour-involved striatal white matter bundles in mice bearing intermediate npp tumours. Contralateral tumour-free striatum (Contra) is shown on the right. Scale bar=100 μm. Images are representative of n = 6 mice. e, Quantification of GFAP⁺ astrocyte density as a function of tumour cell density in tumour-involved striatal bundles in mice bearing intermediate npp tumours. Each point represents a bundle, ≥5 bundles in tumour-involved striatum per mouse were quantified. Turquoise line and blue band indicate mean contralateral GFAP⁺ cell density and SD, respectively. Two-tailed Pearson R correlation. p < 0.0001. n = 6 mice. f, Representative images of CD68⁺/Iba1⁺ microglia in tumour-involved striatal white matter bundles in mice bearing intermediate npp tumours. Contralateral tumour-free striatum (Contra) is shown on the right. Scale bar=100 μm. Images are representative of n = 4 mice. g, Quantification of CD68 integrated intensity (IntDen) as a function of tumour cell density in tumour-involved striatal bundles in mice bearing intermediate npp tumours. Each point represents a bundle, ≥5 bundles in tumour-involved striatum per mouse were quantified. Turquoise line and blue band indicate mean contralateral CD68 integrated density and SD, respectively. ±1 SD range. Two-tailed Pearson R correlation. p < 0.0001. n = 4 mice. h-l, Flow cytometry analysis of indicated immune populations in age-matched control healthy brains or early, intermediate and late npp tumour generated in WT mice. Mean ± SD. For control, early, and late n = 4, for intermediate n = 3 mice. Multiple two-sided unpaired t tests. In h; p = 0.0011 (control vs early); p = 0.0062 (control vs intermediate); p < 0.0001 (control vs late); p = 0.0062 (early vs intermediate); p < 0.0001 (early vs late). In i; p = 0.0037 (control vs intermediate); p < 0.0001 (control vs late); p = 0.0041 (early vs intermediate); p < 0.0001 (early vs late). In j; p = 0.0124 (control vs intermediate); p = 0.001 (control vs late); p = 0.0151 (early vs intermediate); p = 0.0012 (early vs late). In k; p < 0.0001 (control vs intermediate); p = 0.0008 (control vs late); p < 0.0001 (early vs intermediate); p = 0.0008 (early vs late); p = 0.0097 (late vs terminal); In l; p = 0.0353 (control vs intermediate); p = 0.0078 (control vs late); p = 0.0434 (early vs intermediate); p = 0.0092 (early vs late). m-n, Quantification of GFAP area (m) CD68 integrated density (n) within the tumour region of indicated early and terminal PDX models plotted as fold change relative to contralateral tumour-free brain tissue. Each point represents a tumour. n = 5 mice. Mean ± SEM. Multiple two-sided paired t tests. In m, p = 0.0023 for contralateral vs early; p = 0.0016 for contralateral vs terminal.

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