Fig. 2: Tumour-infiltrated circuits exhibit areas of synaptic remodelling characterized by glioma cells expressing synaptogenic factors.
From: Glioblastoma remodelling of human neural circuits decreases survival
a, Single-cell RNA-seq feature plot analysis of THBS1 in HFC (n = 6,666 cells, 3 participants) tissues; within HFC samples, THBS1 is primarily in glioblastoma cells (circled). b, TSP-1 immunofluorescence analysis of nestin-positive tumour cells in HFC and LFC tissues. n = 13 (HFC) and n = 11 (LFC) sections, 3 per group. P = 0.000073. Scale bar, 50 µm. The box plot shows the median (centre line), interquartile range (box limits) and minimum and maximum values (whiskers). c, The synapsin-1 puncta count in HFC and LFC glioblastoma tissue samples. n = 25 regions, 4 per group. P = 0.000014. Red, synapsin-1 (presynaptic puncta); white, neurofilament heavy and medium (neurons). Scale bar, 10 µm. Inset: magnified view of synapsin-1 puncta on neurons. Scale bar, 3 µm. d, PSD95 puncta count. n = 7 (HFC) and n = 9 (LFC) sections, 3 per group. P = 0.04. Red, PSD95 (postsynaptic puncta); white, neurofilament heavy and medium chains (NFH/M) (neurons). Scale bar, 10 µm. Inset: magnified view of PSD95 puncta on neurons. Scale bar, 3 µm. e, Representative confocal images showing synaptic punctum colocalization (yellow arrows). Red, synapsin-1; green, homer-1 (postsynaptic puncta); white, MAP2 (neurons); blue, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar, 10 µm. Quantification of the number of colocalized pre- and postsynaptic puncta (n = 13 (HFC) and n = 10 (LFC) regions, 2 per group; P = 0.005) and homer-1 puncta size in neuron–glioma co-culture (P = 0.000024). f, TSP-1 rescue of induced neuron (iN) organoids in co-culture with HFC and LFC cells for 6 h. Scale bar, 300 µm. Quantification of glioblastoma (GBM) cell integration measured on the basis of the fluorescence intensity of RFP-positive glioblastoma cells in the organoids. Significant differences between HFC and LFC groups (asterisks) and LFC and LFC + TSP-1 (hash) are indicated. n = 2 (HFC and LFC groups) and n = 1 (LFC + TSP-1 group). Scale bar, 300 µm. g, Representative MEA raster plots showing individual spikes (tick mark), bursts (cluster of spikes in blue) and synchronized network bursts (pink) after 48 h co-culture of cortical neurons (CN) with HFC and LFC cells (outlined in red and blue, respectively). Quantification of network burst frequency (Hz) (n = 2 (CN only), n = 3 (CN + HFC) and n = 4 (CN + LFC); P = 0.05) and network synchrony (area under normalized cross-correlation; n = 2 (CN only), n = 3 (CN + HFC) and n = 4 (CN + LFC); P = 0.0129 (CN versus CN + HFC); P = 0.0308 (CN + HFC versus CN + LFC)). Data are mean ± s.e.m. (b–g). P values were determined using two-tailed Student’s t-tests (b–f) and one-way analysis of variance (ANOVA) with Tukey’s post hoc test (g). *P < 0.05, **P < 0.01, ****P < 0.0001; NS, not significant.
