Abstract
The role of the nervous system in the regulation of cancer is increasingly appreciated. In gliomas, neuronal activity drives tumour progression through paracrine signalling factors such as neuroligin-3 and brain-derived neurotrophic factor1,2,3 (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors4,5. The consequent glioma cell membrane depolarization drives tumour proliferation4,6. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity7,8 and strength9,10,11,12,13,14,15. Here we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signalling through the receptor tropomyosin-related kinase B16 (TrkB) to CAMKII, BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. Linking plasticity of glioma synaptic strength to tumour growth, graded optogenetic control of glioma membrane potential demonstrates that greater depolarizing current amplitude promotes increased glioma proliferation. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity17,18,19,20,21,22 that contributes to memory and learning in the healthy brain23,24,25,26. BDNF–TrkB signalling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of glioma TrkB expression robustly inhibits tumour progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of paediatric glioblastoma and diffuse intrinsic pontine glioma. Together, these findings indicate that BDNF–TrkB signalling promotes malignant synaptic plasticity and augments tumour progression.
Main
Gliomas, including glioblastoma and diffuse midline gliomas (DMG), are the most common and lethal primary brain cancers in children and adults27. Progression of glioma is robustly regulated by interactions with neurons1,2,3,4,5, including tumour initiation3,28, growth1,2,3,4,5,28 and invasion5,29. Neuron–glioma interactions include both paracrine factor signalling1,3,28 and electrochemical signalling through AMPA receptor (AMPAR)-mediated neuron-to-glioma synapses4,5. Synaptic integration of high-grade gliomas into neural circuits is fundamental to cancer progression in preclinical model systems4,5,29 and in human patients30. We hypothesized that gliomas may recruit mechanisms of adaptive neuroplasticity to elaborate and reinforce these powerful growth-promoting neuron–glioma interactions, and that neuronal activity-regulated BDNF signalling to the TrkB receptor in glioma cells may have a crucial role in such malignant plasticity.
BDNF–TrkB signalling drives glioma growth
Paediatric gliomas express high levels of the BDNF receptor TrkB (encoded by NTRK2) in malignant cells (Extended Data Fig. 1a,b). Unlike adult glioblastoma31, paediatric high-grade gliomas such as DMGs of the brainstem, also called diffuse intrinsic pontine glioma (DIPG), do not express BDNF (Extended Data Fig. 1a). This suggests a microenvironmental source of BDNF ligand, consistent with previous evidence1. We therefore tested the role of neuronal activity-regulated BDNF secretion into the tumour microenvironment using a genetically engineered mouse model that is deficient in activity-induced expression of BDNF32 (Bdnf-TMKI). This mouse model expresses baseline levels of BDNF ligand, but does not exhibit activity-regulated increases in BDNF expression and secretion owing to a loss of the CREB-binding site in the Bdnf promoter32. We expressed the excitatory, blue-light-gated opsin channelrhodopsin-2 in deep layer cortical projection neurons (Thy1::ChR2) in the Bdnf-TMKI mouse (Fig. 1a) to enable optogenetic stimulation of cortical projection (glutamatergic) neuronal activity. Patient-derived paediatric glioma (DIPG) cells were xenografted into the frontal cortex and subcortical white matter, and following a 5-week period of engraftment, cortical projection neuronal activity was optogenetically stimulated using our established protocol1 (10-min session per day, 20 Hz blue-light stimulation with 30-s on/90-s off cycles) for 1 week. As expected1, we observed an increase in glioma proliferation following optogenetic stimulation of cortical projection neuronal activity in Bdnf wild-type mice. The effects of cortical projection neuronal activity on glioma proliferation were markedly attenuated in Bdnf-TMKI mice lacking activity-regulated BDNF expression and secretion (Fig. 1b–d and Extended Data Fig. 1c,d).
Given this contribution of activity-regulated BDNF to the proliferative influence of neuronal activity in the short term, we next probed the effect of activity-regulated BDNF on the survival of Bdnf wild-type and Bdnf-TMKI mice bearing patient-derived orthotopic paediatric glioma xenografts. We found that the loss of neuronal activity-regulated BDNF expression and secretion exerts a survival advantage in Bdnf-TMKI mice bearing patient-derived DIPG xenografts in the brainstem (Fig. 1e and Extended Data Fig. 1e), concordant with the hypothesis that activity-regulated BDNF signalling robustly influences glioma progression in the context of the brain microenvironment.
Therapeutic targeting of TrkB
Genetic expression patterns of the neurotrophin receptors in DMG tumours (Extended Data Fig. 1a), suggests that BDNF acts on glioma cells through the TrkB (encoded by NTRK2) receptor and that BDNF is a key neurotrophin to which paediatric glioma cells respond. Concordantly, the neurotrophins NGF and NT-3, which signal through TrkA and TrkC receptors, respectively, did not affect glioma cell proliferation in vitro. NT-4, a neurotrophin that also signals through TrkB, promotes glioma proliferation similarly to BDNF (Extended Data Fig. 1f). We therefore tested the effects of genetic or pharmacological TrkB blockade on growth of paediatric gliomas. We used CRISPR technology to delete NTRK2 from human, patient-derived glioma cells (referred to as NTRK2-knockout (KO)). The knockout used a direct deletion in exon 1 of NTRK2, resulting in an approximately 80% decrease in TrkB protein levels (Extended Data Fig. 1g,h). Mice were xenografted orthotopically with patient-derived cells in which NTRK2 was wild type (Cas9 control) or had been CRISPR-deleted (NTRK2-KO). Mice bearing orthotopic xenografts of NTRK2-KO DIPG in the brainstem or NTRK2-KO paediatric cortical glioblastoma in the frontal cortex exhibited a marked increase in overall survival compared with littermate controls xenografted with NTRK2 wild-type cells (Fig. 1f and Extended Data Fig. 1i,j). Proliferation of NTRK2-KO glioma cells was similar in wild-type mice and in mice lacking activity-regulated BDNF (Bdnf-TMKI) following optogenetic stimulation of cortical projection neuronal activity, indicating that the loss of activity-regulated BDNF does not exert effects that are independent of glioma TrkB signalling (Extended Data Fig. 1k).
We next performed preclinical efficacy studies of pan-Trk inhibitors. Trk inhibitors have recently been developed for treatment of NTRK-fusion malignancies, including for NTRK-fusion infant gliomas33,34,35. Here, we tested the preclinical efficacy of these inhibitors in NTRK non-fusion gliomas such as DIPG. We first assessed the ability of entrectinib to cross the blood–brain barrier and found that systemic entrectinib (120 mg kg−1, oral administration) reduced pharmacodynamic markers of TrkB signalling, including TrkB phosphorylation and downstream ERK phosphorylation in brain tissue (Extended Data Fig. 2a–c). Treatment of an aggressive patient-derived paediatric glioma (DIPG) orthotopic xenograft model with entrectinib increased overall survival compared to vehicle-treated controls (Fig. 1g and Extended Data Fig. 2d). Although entrectinib decreased the proliferation rate of xenografted NTRK2 wild-type DIPG cells in vivo, it did not further decrease the proliferation rate of NTRK2-KO glioma xenografts (Extended Data Fig. 2e,f), demonstrating that the mechanism of action of entrectinib in DIPG is mediated through TrkB.
BDNF regulates neuron–glioma interactions
We previously found that BDNF is one of multiple paracrine factors that can increase glioma proliferation in response to neuronal activity1,3, albeit not as robustly as other neuron–glioma signalling mechanisms1. To confirm the relative contribution of activity-regulated BDNF ligand to the mitogenic effect of activity-regulated secreted factors, we optogenetically stimulated cortical explants from Bdnf-TMKI or Bdnf wild-type mice, collected conditioned medium, and tested the effects of conditioned medium on glioma cell proliferation in vitro using our well-validated experimental paradigm1. Exposure of patient-derived glioma cultures to conditioned medium from optogenetically stimulated Bdnf wild-type cortical explants increased tumour cell proliferation rate, as we have previously shown1 (Extended Data Fig. 3a,b). Conditioned medium collected from optogenetically stimulated Bdnf-TMKI cortical explants elicited a mildly reduced proliferative response of glioma cells in monoculture compared with conditioned medium from wild-type cortical explants, indicating a small direct mitogenic effect of activity-regulated BDNF ligand secretion (Extended Data Fig. 3b), as expected1.
Testing the effects of BDNF alone on glioma proliferation in vitro, we found that the addition of recombinant BDNF (100 nM) increases paediatric glioma (DIPG) cell proliferation from a rate of around 20% to around 30%. This effect is completely abrogated—as expected—with CRISPR knockout of NTRK2 and by pharmacological inhibition with entrectinib or larotrectinib (Fig. 1h and Extended Data Fig. 3c). A similarly modest increase in proliferation was observed in a range of patient-derived glioma monocultures exposed to BDNF, including thalamic DMG and paediatric cortical glioblastoma (Extended Data Fig. 3d).
Co-culture with neurons elicits a robust increase in glioma cell proliferation rate from around 20% to around 60%, underscoring the powerful effects of neurons on glioma proliferation that include neuroligin-3 (NLGN3) signalling and neuron-to-glioma synaptic mechanisms1,3,4,5. We sought to investigate the relative contribution of BDNF–TrkB signalling in neuron–glioma interactions using neuronal co-culture with NTRK2 wild-type or NTRK2-KO glioma cells. In the absence of neurons, TrkB loss alone does not reduce paediatric glioma cell proliferation (Fig. 1i), consistent with the lack of BDNF ligand expression in paediatric glioma cells (Extended Data Fig. 1a). However, TrkB loss in glioma cells co-cultured with neurons resulted in a marked reduction in neuron-induced proliferation, decreasing the glioma cell proliferation rate from around 60% to around 30%. This reduction is disproportionate to the loss accounted for by BDNF mitogenic signalling alone, as described above (Fig. 1h). The magnitude of the change in glioma proliferation elicited by TrkB loss in response to BDNF ligand alone compared with that in the context of neuron co-culture (Fig. 1h,i) suggests that BDNF may have a more complex role in neuron–glioma interactions than simply as an activity-regulated growth factor.
To explore possible roles for BDNF–TrkB signalling in glioma pathophysiology, we examined gene-expression relationships between TrkB and other gene programmes at the single-cell level using available single-cell transcriptomic data from human H3K27M-mutated DMG primary biopsy tissue36. NTRK2 is expressed in the majority of glioma cells at varying levels across the defined cellular subpopulations that comprise DMGs, including oligodendrocyte precursor cell-like tumour cells (OPC-like), astrocyte-like tumour cells (AC-like) and oligodendrocyte-like tumour cells (OC-like) (Extended Data Fig. 4a,b). As previously demonstrated4, synaptic gene expression is enriched in the oligodendroglial compartments of the tumour (oligodendrocyte-like and oligodendrocyte precursor cell-like cellular subpopulations), whereas tumour microtube-associated gene expression is enriched in the astrocyte-like compartment (Extended Data Fig. 4c). Expression correlation analyses identified different patterns of genes in each cellular compartment that correlate with NTRK2 expression (Extended Data Fig. 4d). Examples of genes that are strongly correlated with NTRK2 in the astrocyte-like compartment include GJA1, TTHY1, GRIK1 and KCNN3; TTHY1 and GJA1 are known to have crucial roles in tumour microtube formation and connectivity in adult high-grade gliomas37,38. In the OC-like compartment, NTRK2 expression correlates with NRXN2, NLGN3, CSPG4, PDGFRA, FGFR1, CNTN1, SLIT2, IGF1R and CACNG5, and in the OPC-like compartment it correlates with NRXN2, NRXN1, NLGN4X, SYT11, CREB5, SRGAP2C, CSPG4, ASCL1, PI3KR3, CDK6, EGFR and EPHB1 (Extended Data Fig. 4d). Gene Ontology analyses of these differentially correlated genes in each cellular sub-compartment revealed correlation of NTRK2 with processes of synaptic communication and neural circuit assembly (Extended Data Fig. 4e–g). In the OPC-like compartment, NTRK2 expression correlated with postsynaptic organization, axon guidance, neuronal projection guidance, neuronal migration, ERK signalling cascades and the AKT signalling cascade, consistent with the hypothesized role of TrkB in neuron-to-glioma synapses, consequent effects of AMPAR-mediated synaptic signalling on tumour migration29 and expected signalling consequences of TrkB activation. In the OC-like compartment, the gene sets correlated with NTRK2 expression involve synaptic organization, modulation of synaptic transmission, synaptic plasticity, and learning and memory. In the astrocyte-like compartment, which tends to engage in extensive tumour microtube connectivity37, NTRK2 expression correlated with genes involved in axon guidance and neuronal projection morphogenesis. Together, these single-cell transcriptomic analyses support potential roles for TrkB signalling in neuron-to-glioma synaptic biology as well as glioma-to-glioma network formation, with TrkB correlated with distinct processes in astrocyte-like and oligodendroglial-like cellular subpopulations.
Relationship between TrkB and AMPAR signalling
Glutamatergic neuron-to-glioma synapses are mediated by calcium-permeable AMPARs in both paediatric and adult gliomas, and robustly regulate glioma progression4,5,29. In the healthy brain, BDNF–TrkB signalling regulates glutamatergic synaptic transmission through several mechanisms9,10,11,12,13,14,15,39. To explore the hypothesis that the growth-promoting effects of activity-regulated BDNF–TrkB signalling in glioma involves modulation of synaptic biology, we explored whether the effects of glioma TrkB signalling are related to or independent of AMPAR signalling. We found that pharmacologically blocking AMPARs or genetically blocking TrkB through NTRK2 knockout decreased tumour cell proliferation in vivo or in neuron–glioma co-culture (Fig. 1j–m and Extended Data Fig. 5a,b). However, we found no additive effect of blocking AMPARs and TrkB, suggesting a relationship between the mechanisms.
Glioma glutamatergic current strength
We hypothesized that BDNF–TrkB signalling may function in glioma to strengthen neuron-to-glioma synapses. In healthy neurons, activity-regulated plasticity23,24,40 of synaptic strength—the evoked amplitude of the postsynaptic current—dynamically modulates neural circuit function41, and these synaptic changes are thought to underlie learning and memory42. One form of plasticity of synaptic strength involves increased AMPAR trafficking to the postsynaptic membrane43,44. Glutamatergic neurotransmission through NMDAR (N-methyl-D-aspartate receptor) and consequent calcium signalling can increase AMPAR trafficking to the postsynaptic membrane17,18,19,20,21,22,45, but glioma cells do not strongly express NMDAR genes4. Another activity-regulated mechanism that can promote AMPAR trafficking to the membrane is BDNF–TrkB signalling and consequent stimulation of the CAMKII calcium signalling pathway9,10,11,12,13,14,15. Concordantly, inhibition of CAMKII reduces the proliferation-inducing effects of neuron–glioma co-culture (Extended Data Fig. 5c,d). We therefore tested the hypothesis that BDNF–TrkB signalling could induce plasticity of the malignant synapse—that is, it could increase the amplitude of glioma excitatory postsynaptic currents (EPSCs)….
