Extended Data Fig. 1: TrkB is the key receptor mediating neuronal BDNF signaling in glioma.

a, Left, Primary human biopsy single cell transcriptomic data³⁶ illustrating the expression of the neurotrophin family genes in H3K27M⁺ DMG (red; n = 2,259 cells, 6 study participants), tumor associated, non-malignant immune cells (blue; n = 96 cells, 5 participants) and oligodendrocytes (green; n = 232 cells). Right, NTRK2 and BDNF expression in H3K27M⁺ DMG malignant single cells primary human biopsy single-cell transcriptomic data from each of 6 study participants (case numbers denoted on x axis). For each individual violin plot, the y axis represents expression log₂ (transcripts per million) and the x axis represents number of individual cells with indicated expression value. b, Expression levels of neurotrophin receptors analysed from previously published^59,66,67 and newly reported (GEO# GSE222560) bulk RNA sequencing of human autopsy pediatric DMG (n = six patient-derived glioma samples SU-DIPG-IV, SU-DIPG-VI, SU-DIPG-XIII-P, SU-DIPG-XIII-FL, SU-DIPG-21 and SU-DIPG-25; means 2.36 NTRK1, 22.73 NTRK2, 8.688 NTRK3, 5.439 NGFR FPKM; NTRK2 minimum 0.03273, 25% percentile 1.537, median 6.873, 75% percentile 11.10, maximum 12.34; BDNF minimum 0.01429, 25% percentile 0.01499, median 0.03367, 75% percentile 0.04565, maximum 0.1951). c Model for optogenetic stimulation of ChR2-expressing neurons (blue) in microenvironment of glioma xenograft (green); light blue rectangle denotes region of analysis. P, postnatal day. d, Proliferation index of SU-DIPG-XIII-FL glioma xenografted to mice with neurons expressing Channelrhodopsin (ChR2 + ) in a wild-type or Bdnf-TMKI genetic background (Fig. 1a) after neuronal optogenetic stimulation (quantified by confocal microscopy of EdU + /HNA cells, as in representative Fig. 1c, n = 7 wild-type ChR2+ mice, 8 Bdnf-TMKI ChR2+ mice, P = 0.0007). e, Representative image of tumor burden in a mouse brain (sagittal section) bearing orthotopic xenograft of SU-DIPG-XIII-P* xenografted to the pons at endpoint. Survival analysis presented in Fig. 1e. White denotes HNA (tumor cells); DAPI nuclei are shown in blue (Scale bar = 2000 µm). f, Proliferation rate of SU-DIPG-XIII-FL cultures treated with recombinant proteins NGF, BDNF, NT3, NT4 (100 μM each), compared to vehicle control (quantified by confocal microscopy of EdU + /DAPI cells, as in representative Fig. 1h, n = 4 coverslips/group, Control vs BDNF P = 0.016, Control vs NT4 P = 0.0074). g, Representative western blot analysis of TrkB protein levels in wild-type, Cas9-control and NTRK2 KO cultures (SU-DIPG-VI, SU-pcGBM2, SU-DIPG-XIII-FL), using indicated antibodies. h, Quantification of g, with levels of TrkB normalized to total protein loading using ß-actin levels and compared to wild-type, Cas9-scramble control, cultures (y axis is in arbitrary units, n = 3 technical replicates, DIPGVI WT vs NTRK2 KO P = 0.0019, DIPGXIII WT vs NTRK2 KO P = 0.0002, pcGBM2 WT vs NTRK2 KO P = 0.0013). i-j, Representative images of tumors at survival endpoint for Fig. 1f. i, Orthotopic xenograft of SU-DIPG-VI into pons (sagittal section of mouse brain; scale bar = 2000 µm), and in j, cortical orthotopic xenograft of SU-pcGBM2 (coronal section of mouse brain). White denotes HNA (tumor cells); Green denotes GFP (tumor cells); DAPI nuclei are shown in blue (scale bar = 2000 µm). k, Proliferation index of NTRK2 KO SU-DIPG-VI glioma xenografted to mice with neurons expressing Channelrhodopsin (ChR2 + ) in a wild-type or Bdnf-TMKI genetic background after neuronal optogenetic stimulation (quantified by confocal microscopy of EdU + /HNA cells, as in representative Fig. 1c, n = 5 wild-type ChR2+ mice, n = 4 BDNF-TMKI ChR2+ mice). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. Two-tailed unpaired Student’s t-test for d and k, one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis for f and two-tailed one sample t-test for h.