Extended Data Fig. 3: Axonal injury increases with tumour cell density.
From: Axonal injury is a targetable driver of glioblastoma progression
a, YFP mean fluorescence intensity (MFI) in the tumour-involved striatum normalized to intensity in contralateral striatum and plotted as a function of number of tumour cells in the white matter. Each spot represents a mouse at early (light green), intermediate (turquoise), late (dark blue) and terminal (black) stages. Two-tailed Pearson R correlation. p < 0.0001. For early, intermediate and late n = 4, for terminal n = 3 mice. b, Representative images of white matter bundles in the tumour-involved (Tumour) and contralateral (tumour-free; Contra) striatum of Thy1YFP mice bearing intermediate npp tumours. Arrowheads exemplify a heavily- (yellow), moderately- (white) and a lowly- (blue) bundle infiltrated by tdTomato+ tumour cells (red). Scale bar=100 μm. c, Quantification of YFP mean fluorescence intensity (MFI) as a function of tumour cell density in tumour-involved striatal white matter of npp tumour-bearing mice from b. Turquoise line and blue band indicate mean contralateral fluorescence intensity and SD, respectively. Two-tailed Pearson R correlation. n = 6 mice. Each spot represents an individual bundle. d, Representative images of neurofilament staining (NF, yellow) of tumour-involved (tumour) or contralateral (tumour-free, Contra) striatal white matter bundles in WT and Sarm1-/- mice bearing intermediate npp tumours. Moderately- (white arrowhead) and heavily- (yellow arrowhead) infiltrated white matter bundles are highlighted. Scale bar=50 μm. e, Quantification of neurofilament (NF) mean fluorescence intensity (MFI) in tumours depicted in d. Individual white matter bundles are shown. Two-way ANOVA with Tukey’s multiple comparisons. Mean ± SD, WT n = 3, Sarm1-/- n = 3 mice. p < 0.0001 (WT contralateral vs WT tumour); p < 0.0001 (WT tumour vs Sarm1-/- tumour). f, Representative images correlating confocal microscopy images and electron micrographs of intermediate tumours (10.5 weeks post-electroporation) induced in WT (i-iv) and Sarm1-/- mice (v-viii); i, overview image of npp tumours in WT and v, Sarm1-/- mice (bottom). Scale bar=200 μm. Representative of WT n = 4, Sarm1-/- n = 4 mice. ii and vi, Corresponding representative electron micrographs of fluorescence images from i and v. Scale bar=200 μm. Dashed boxes indicate the striatal white matter bundle depicted at higher magnification on the right (iii, iv, vii and viii). Dashed yellow boxes indicates region shown in Fig. 3c. Scale bar=50 μm. g, G-ratios of pathological or intact axons in the tumour-involved striatal white matter of WT mice bearing intermediate npp tumours. Each dot represents an axon. Mean ± SD. Two-sided unpaired t test. n = 4 mice. h-j, Representative immunofluorescence images of intermediate npp tumours generated in Thy1YFP mice and stained for indicated markers of proteinopathies. Bottom panels in h and I indicate successful antibody staining in positive control tissue. Scale bar=200 μm for h and I, Scale bar=50 μm for j. k, Representative immunofluorescence images of intermediate npp tumours generated in WT mice (n = 3 mice) and stained for Hypoxyprobe following pimonizadole administration. Terminal tumours (bottom panels) served as positive control (n = 3 mice). Scale bar=200 μm.l, Representative images of tdTomato+ (red) intermediate npp Thy1YFP tumours stained for phospho-myosin light chain 2 (pMLC2) (grey). Shown are tumour-involved (Tumour) and contralateral (Contra) white matter. Scale bar=100 μm. Images are representative of n = 3 mice.
