γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the adult brain that induces GABAergic signaling mediated via Cl⁻ influx through activating GABA receptors (GABARs), predominantly GABA_ARs (14). A previous study reported on the aberrant expression of chloride extruder K⁺-Cl⁻ cotransporter 2 (KCC2) and Na⁺-K⁺-Cl⁻ cotransporter 1 (NKCC1), which was discovered in the peritumoral area following GABAergic signaling (15). The dysregulation of KCC2 and NKCC1 in the adjacent neurons led to an increasing intracellular Cl⁻ concentration, which resulted in Cl⁻ efflux. However, it was indicated that a high concentration of glutamate in the TME was able to counteract the inhibitory role of GABA via downregulating KCC2 levels (16). Another study reported that glioma cells expressed GABA_ARs when co-cultured with neurons in vitro, and activation of GABA_ARs resulted in inhibition of glioma-cell proliferation (11). Activation of GABA_AR with the exogenous agonist muscimol, however, failed to elicit further inhibition of glioma-cell proliferation. That study attributed the phenomenon to the fact that GABA seemingly acts on glioma stem cells (GSCs) (17). The overexpression of diazepam-binding inhibitor (DBI) in glioma was reported to inhibit GABA_ARs, thereby promoting gliomagenesis. Despite a dearth of GABA_ARs, overexpressed DBI facilitated GBM growth through a lipid metabolism pathway mediated by GABA (18). Therefore, employing agonists or antagonists to disrupt the Cl⁻ current in glioma cells may offer a strategy to inhibit the progress of tumor development.

Glutamate is the predominant excitatory neurotransmitter, which is synthesized and secreted by neurons (19). The association between glutamate receptors and glutamate-induced Ca²⁺ signaling was demonstrated through the upregulation of the Ca²⁺ permeable-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) in glioma, which increased the rate of Ca²⁺ influx. The elevated Ca²⁺ concentration serves to activate the pro-oncogenic Akt and ERK/MAPK signaling pathways (20). It was reported that glutamatergic synapses were formed between neurons and glioma cells (termed neurogliomal synapses), and AMPARs are expressed on post-synapses. AMPAR-mediated neuronal activity has been indicated to induce tumor invasion and growth (12). AMPARs comprise four different types of subunits: GluA1, GluA2, GluA3 and GluA4 (21). Blockade of GluA1 and GluA2 overexpression reduced the proliferation rate of glioma cells by inducing apoptosis and decreasing cell invasiveness and migration (22). A study has demonstrated the role of auxiliary subunits (of the transmembrane AMPAR regulatory protein, cystine-knot AMPAR modulating protein and cornichon homolog families) of AMPARs in promoting gliomagenesis (23).

Activation of N-methyl-D-aspartate receptors (NMDARs) in GBM by extracellular glutamate may potentially boost tumor expansion in vivo (24). However, the expression of NMDARs on neurogliomal synapses and the further impacts on glioma development remain elusive. Previously published studies, however, have confirmed the inhibitory effects on glioma growth via NMDARs (25,26). Metabotropic glutamate receptors (mGluRs) consist of three groups of G-proteins: Groups I (mGluR1 and -5), II (mGluR2 and -3) and III (mGluR4 and -6-8). The group III receptor antagonists were observed to exert their anti-tumor effects in various types of tumor, including hepatoma, melanoma and non-small-cell lung cancer (26). Riluzole has been reported to promote apoptosis in glioma cells via antagonizing mGlu3, which results in the suppression of ERK activation (27). At present, evidence is lacking; however, in terms of whether mGluRs are expressed on neurogliomal synapses, although mGluRs are closely associated with glioma progression, may suggest the involvement of other neuron-glioma interactions.

Serotonin and dopamine are predominant neurotransmitters in the CNS. Of note, a previous study suggested a correlation between depression and gliomagenesis, probably due to the shared molecular pathways and gene networks (28). Dopamine receptor D4 is a receptor that is involved in autophagy and apoptosis, which interferes with glioma-cell proliferation (29). Serotonin exerts an impact on the glutamatergic system through influencing AMPARs and NMDARs (30). Selective serotonin reuptake inhibitor (SSRI) has been indicated to impair glioma cell growth via inducing autophagy and apoptosis (31), although such effects were also observed in patients with a history of long-term drug therapy using tricyclic antidepressants (32). In those patients, the morbidity rate of glioma was reduced. As serotonin receptors are also expressed in glioma cells, it is tempting to hypothesize that SSRI may also exert an influence through impacting neuron-glioma synapses (33).

As mentioned above, ion currents are involved in neuron-glioma crosstalk. Ion channels exist in the CNS, primarily Na⁺, K⁺, Ca²⁺ and Cl⁻ channels, which regulate cell migration, invasion, proliferation and apoptosis (34). Specifically, the K⁺ channel members include voltage-gated K⁺ channels (VGKC or Kv), calcium-activated K⁺ channels (KCa) and ATP-sensitive K⁺ channels (KATP). Voltage-gated calcium channels (VGCCs) are the most important members of the Ca²⁺ channel group, consisting of the L-, T-, N- and P/Q-types.

The migration of neural progenitor cells and stem cells is influenced by the electric current generated by the local field potential (35). It was recently indicated that the electric current directs the migration and invasion of glioma cells (36). The electrotaxis processes of GBM cell lines are regulated by voltage-gated channels. For instance, in T98G cells, electrotaxis was demonstrated to be mediated via R-type VGCCs, whereas in U251 cells, it was mediated via P/Q-type VGCCs. By contrast, in both T98G and U251 cells, electrotaxis was regulated via A-type VGKCs and acid-sensing ion channels (ASICs) (37). Inhibition of ASICs by benzamil significantly impaired the directedness of the electrotaxis of U251 and T98G cells (37). Of note, P/Q- and N-type VGCC inhibitors derived from spider venom were indicated to markedly inhibit tumor growth in vivo (38). Collectively, these data suggest that electric fields exert important effects on glioma-cell invasion and migration. Therefore, targeting ion channels may be a potential therapeutic strategy for glioma.

Ion channels are employed by cells to regulate their volumes for the purpose of cell migration. Glioma cells accumulate Cl⁻ intracellularly via NKCC1, whereas Cl⁻ channel protein 3 (CLC3) serves to regulate the Cl⁻ efflux (39). To balance the Cl⁻ efflux, glioma cells express KCa1.1 and KCa3.1 channels, which regulate K⁺ influx via Ca²⁺ activation (39). Chlorotoxin, a small neurotoxin of 36 amino acids, causes the internalization of CLC family members, thereby impeding glioma-cell invasion (40). A previous study suggested that specific inhibition of KCa3.1 by TRAM-34 leads to a reduction in the migration and infiltration rates of U87, GL261 and U251 GBM cells (41). Furthermore, blockade of KCa1.1 inhibited the radiation- and hypoxia-induced migration of GBM cells (42).

Apart from cell migration and invasion, ion channels are also involved in regulating the cell cycle, proliferation and apoptosis (39). A previous study revealed that glioma-cell proliferation was inhibited following the blockade of Kv channels by 4-aminopyridine (43). Inhibition of Kv1.1 by KAaH2, a homologous Kv1 blocker from scorpion venom, impaired U87-cell proliferation (44). Ca²⁺-activated K⁺ channels (BK channels) have also been indicated to be involved in regulating proliferation (45). However, the association between neuronal activity and ion channels in terms of how they influence glioma proliferation remains inadequately understood. Hypothetically speaking, neuronal hyperexcitability may promote glioma proliferation, since the activation of ion channels by electric signals is essential for downstream pathway signaling. Considering all of this evidence, ion channels have been indicated to act as an important bridge between neuronal activity and glioma progression, although their role still requires to be confirmed and fully elucidated in further studies.

TMs are a type of tumor protrusion consisting of F-actin and microtubules formed by gliomas, which permit cell-to-cell material transportation (46). Targeted patch-clamp recordings have suggested that a spontaneous excitatory post-synaptic potential arises in glioma cells cocultured with neurons, which induces further Ca²⁺ influx (12). Importantly, TM connectivity is responsible for the distribution of Ca²⁺ ions, which serve as crucial messengers of glioma activity throughout the glioma syncytium (12). Furthermore, NGSs significantly promote glioma invasion through Ca²⁺ signaling, and potentiate glioma proliferation through AMPAR activation (12). In a Drosophila glioma model, Frizzled 1 receptors were indicated to be highly expressed within TMs, where glioma cells were able to vampirize Wingless-related integration site (WNT) ligands from neurons (47) (the process 'vampirization' is defined in that article). The depletion of WNT from neurons led to glioma proliferation and expansion through the JNK/matrix metalloproteinase (MMP) pathway, conversely resulting in a reduction of neuronal synapse activity (47). TM-associated gap junctions have also been indicated to amplify the extracellular K⁺ current and to promote tumor proliferation (48), suggesting that the TM network potentiates the pro-tumoral effects of NGSs.

In cell-cell communication, gap junctions, which consist of connexin (Cx) proteins, form conductive pores in the plasma membranes between adjacent cells that allow the transportation of cellular material (49). Cx43-based gap junctions and neuronal growth-associated protein-43 (GAP-43) have been indicated to be essential for TM formation (50). Depletion of GAP-43 and Cx43 led to both an impairment of the ability of TMs to form connections and tumor-cell volume reduction in vivo (51). In a subsequent study, suppressing Cx43 with a peptide, TAT-Cx43266-283, inhibited glioma-cell invasiveness, reduced the stemness properties of GSCs and prolonged the survival rate of mice bearing GSC-derived gliomas (52). IMM, a small molecule that is able to induce F-actin polymerization, was demonstrated to hinder TM formation and to prevent glioma invasiveness (53). It is noteworthy that the role of Cx43, a tumor suppressor, appears to be somewhat paradoxical in terms of the overall picture (54). According to a previously published meta-analysis, Cx43 expression was reported to improve the overall survival rate in patients with glioma (55). Hence, the function of Cx43 in glioma should be interpreted with caution in further studies. TM formation was also indicated to be dependent on the EGFR/PI3K signaling pathway, which induces actin cytoskeleton remodeling and initiates TM expansion. The microtubule-targeted agent BAL101553 has entered into clinical trials (Table I). Therefore, targeting TM-associated gap junctions or other pathways may prove to be useful in terms of treating glioma.

The activity of cortical projection neurons promotes glioma growth and progression through neuronal secretion. Neurotrophins (NTs), neuroligins (NLGNs), neurotransmitters and mitogens have all been demonstrated to have participatory roles in neuron-glioma communication (56). In addition to direct (or synaptic) neuronal secretion, glioma cells have also been indicated to be regulated by indirect (or non-synaptic) paracrine and autocrine signaling pathways (57).

NTs are growth factors that are expressed in the nervous system and have an impact on neural growth, survival and biological function. The four types of NTs comprise nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5). NGF has a preferential affinity for tyrosine kinase receptor (Trk)A, whereas BDNF and NT4 have a preference towards TrkB and NT3 has an affinity for TrkC (58).

In the CNS, NGF is secreted predominantly in the cortex, hippocampus and pituitary gland (59). The specific NGF receptors TrkA and p75^NTR have been indicated to be expressed in gliomas (60). The effects of NGF on glioma cells, however, appear to be somewhat paradoxical. Ectopic treatment of the pediatric low-grade glioma cell (PLGG) line Res259 with NGF led to inhibition of cell growth, whereas NGF treatment promoted the growth of another PLGG cell line, Res186 (61). A different study suggested that NGF stimulated U87-cell proliferation through the NOTCH1 receptor signaling pathway (62). The underlying impact of NGF on glioma development, however, requires to be further clarified.

Barreda Tomás et al (63) observed that both progenitor BDNF (pro-BDNF) and BDNF were expressed in glutamatergic and GABAergic neurons of the mouse cortex. Another study demonstrated through an immunocytochemical analysis that BDNF and its receptor, TrkB, are extrasynaptic, whereas BDNF is preferentially located at the glutamatergic synapse (64). It was suggested that BDNF may exert its effects on glioma through NGSs and the paracrine signaling pathway. Targeting BDNF by specific microRNAs inhibited glioma invasion, migration and proliferation (65), indicating the promoting role of BDNF in glioma malignancy. BDNF also induced the synthesis of GluA1 subunits and the synaptic incorporation of CP-AMPARs in primary hippocampal neurons (66). As mentioned above, a high concentration of glutamate in the glioma microenvironment confers neuron hyperexcitability, leading to epilepsy (67). Although AMPARs have been indicated to participate in glutamate-induced epilepsy, BDNF may promote glioma-associated epilepsy due to its capability of regulating the synthesis and incorporation of AMPARs. In addition, glutamate was indicated to stimulate the production of BDNF in neurons (68), which may, in turn, facilitate glioma progression. Of note, pro-BDNF has been observed to exert an inhibitory effect on glioma. A previous study reported that the ratio of pro-BDNF to BDNF was decreased in high-grade glioma, whereas the expression of pro-BDNF was increased. Pro-BDNF was also found to inhibit the growth and invasion of glioma cells via p75^NTR (69).

NT-3/TrkC signaling has also been indicated to be necessary for the induction of glioma-cell death through the inhibition of autophagy under hypoxic conditions (70), suggesting a life-supporting role of NT-3/TrkC signaling in GBM in the state of hypoxia. The effect of NT-4/5 on glioma cells, however, remains elusive, and this requires further investigation.

NLGN3 exerts an important role in synaptic function and maturation by binding presynaptic neurexin (71). Venkatesh et al (71) suggested that spontaneous neuronal activity in the cortex induces NLGN3 secretion and promotes glioma-cell proliferation (71). Potentiated neuronal activity also led to an increase in NLGN3 cleavage mediated by MMPs, which may contribute towards mGluR activation (72). In the clinic, NLGN3 expression was observed to be positively correlated with oscillatory brain activity and this was negatively associated with progression-free survival of patients with glioma (73). NLGN3 not only caused an increase in its own expression, but it also led to an upregulation of the sheddase A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in glioma cells (74). Abundant evidence has indicated the pivotal role of NLGN3 in supporting glioma malignancy. In an NLGN3-deficient brain model, human glioma xenografts were indicated to both survive longer and not exhibit any clear signs of glioma-cell infiltration (75), indicating that glioma malignancy may depend on the presence of NLGN3.

NLGN3 activates several oncogenic signaling pathways, including the PI3K/mTOR pathway, and induces transcriptional changes, including the upregulation of numerous synapse-associated genes (76). NLGN3 has also been indicated to induce the expression of Tweety homologue-1, which has a role in the construction of the glioma microtube network in high-grade glioma (77). In addition, protein kinase C (PKC)-induced NLGN3 cleavage is dependent on MMPs, particularly MMP3 and MMP9. MMP3/9 blockade led to inhibition of PKC-induced NLGN3 (78). Collectively, these results suggested that MMP3/9 inhibition may be effective in terms of glioma suppression. Patients with GBM were also indicated to harbor high levels of NLGN3 in the deep regions of the brain, which may partly explain the high recurrence rate of GBM (73).

NLGN3 is cleaved from both cortical neurons and oligodendrocyte precursor cells via ADAM10, the release of which from neurons is dependent on neuronal activity (75). ADAM10 inhibitors have been reported to prevent the release of NLGN3 and to block glioma growth in vivo (75). These data indicate that targeting NLGN3 for gliomas may be a putative therapeutic strategy. Generally speaking, preventing the release of NLGN3 into the glioma microenvironment through the use of ADAM10 inhibitors, blocking certain targets in oncogenic signaling pathways and silencing NLGN3-associated genes are three transformative methods for the treatment of glioma (Fig. 2).

EVs have an indispensable role in cell-cell interaction in the brain microenvironment. A previous study observed how the development of rat cortical neurons led to the secretion of exosomes containing GluA2/3 subunits of AMPARs (79). Similar patterns of exosome secretion were also observed in the differentiated neurons (80). A previous study indicated that exosomes derived from cortical neurons only bind to neurons and not to glial cells (81), suggesting that neuron-derived EVs (NEVs) mediate neuron-to-glioma communication indirectly. This indicated that the secretion of exosomes is activity-dependent and is specifically mediated by glutamatergic activity involving AMPAR and NMDAR (79,80). The glioma microenvironment renders neurons hyperexcitable due to the high glutamate concentration (67). The hyperexcitable neurons are able to promote the secretion of NEVs, which is chiefly regulated by glutamatergic activity. In a previously published study on human neural cultures where MECP2 was knocked down (termed MECP2LOF neural cultures), NEVs were observed to increase neuron proliferation and cell numbers (82). These results suggested that NEVs contain neuroprotective proteins that fulfill an important role in regulating neural circuits and neurogenesis. The impact of NEVs on gliomagenesis, however, remains elusive. It is possible that neurogenesis may increase the formation of NGSs and potentiate neuronal activity to promote glioma growth.

In addition to NEVs, glioma also influences neuronal functions via glioma-derived EVs (GEVs). A previous study suggested that GEVs enhanced the frequency of neuronal spontaneous synaptic responses (83). This indicated that GEV-induced neuron hyperexcitability may promote NLGN3 levels, leading to glioma progression.

In the glioma microenvironment, microglia and tumor-associated macrophages (TAMs) are the dominant immune cells assisting neuronal regulation in glioma cells. Microglia/microphages exert their immune-modulating effects through mediating the neurotransmitter functioning processes, as well as via releasing regulatory EVs. Novel immunotherapies may be devised based on neuron-microglia/macrophage-glioma interactions.

Microglia regulate synapse formation by forming direct contacts with glioma cells and secreting growth factors, such as BDNF and interleukin-10 (IL-10) (92). On the other hand, GSCs were observed to regulate the secretion of IL-10 from microglia, suggesting the possible presence of a bidirectional communicative process. Through this bilateral talk, the secretion of IL-10 by microglia was altered, which may interfere with normal synapse formation (93). It was reported that activated microglia were able to increase glutamatergic synapses on neurons with perineuronal 'nets' (94), possibly via microglial chemokine C-X-X-X-C motif ligand 1 (CX3CL1) signaling (95), promoting glioma growth and invasion. It was indicated that depletion of GluA2 on the microglial membrane resulted in an increase in the expression of tumor necrosis factor-α (TNF-α), which, in turn, may enhance AMPAR levels on the neuronal membrane (96). An increased expression of AMPARs on neurons promoted neuron hyperexcitability and death in an environment containing a high concentration of glutamate. As the overexpression of GluA2 inhibits GBM proliferation (97), this treatment was able to both ameliorate the TME and protect the neurons simultaneously. In addition, IL-β and TNF-α secreted by microglia were indicated to decrease the levels of mGlu5 in astrocytes, which impaired glutamate uptake and led to a further increase in the extra-cellular glutamate concentration (98). GABA was also indicated to exert anti-tumor effects via GABA-GABA_AR in the neuron-microglia-tumor axis (11). Microglia-released IL-1β and TNF-α inhibited GABAergic synaptic activities in neurons, which supported glioma growth (99). Taken together, TAMs act as a bridge in neuron-glioma interactions via pro-inflammatory cytokines acting as mediators. Targeting microglia/macrophages therefore offers a promising strategy for the treatment of glioma.

Colony-stimulating factor 1 (CSF-1) is produced by glioma cells and is highly expressed in TAMs (100). CSF-1 inhibition was observed to significantly reduce both the number of TAMs and glioma progression in vivo (101), whereas administration of PLX3397, a CSF-1 inhibitor, had no efficacy in recurrent GBM according to a phase II clinical trial (101).

EVs fulfill an important role as secondary messengers within microglia-glioma communication networks. GEVs under hypoxic conditions skew macrophages towards the M2-type, which was subsequently indicated to support the proliferation and migration of U87 cells in vitro and in vivo (102). Microglia have a critical role in the 'tripartite synapse', which is formed by an astrocyte and two neurons. The normal functions of the astrocyte-neuron crosstalk are supported by microglia and neurons in turn, which regulates the activation and motility of the microglia (95). Neurons maintain the homeostatic phenotype of microglia via CX3CL1-CX3CR signaling (95,103), which may have an important role in glioma progression by regulating the function of microglia. Reciprocally, microglia-derived EVs (MEVs) also mediate neuronal growth and activity by increasing the miniature excitatory postsynaptic current via the membrane components of MEVs. In addition, MEVs have been demonstrated to exert roles in neuritogenesis and neuroprotection, which may potentiate the neuron-glioma circuit. In addition, inhibition of neutral sphingomyelinase 2 successfully impeded the release of EVs from the brain and aggravated the symptoms in a Parkinson's disease mouse model, suggesting that the crosstalk between neurons, microglia and glioma cells based on EVs may be a possible option for therapeutic intervention (104).

It is theoretically feasible that viro-immunotherapy may be utilized to reverse the immunosuppressive environment by introducing oncolytic viruses (OVs). OVs exert antitumor effects through polarizing TAMs towards the M1 phenotype, which has the effect of upregulating the pro-inflammatory response (105). A pro-inflammatory TME leads to inhibition of glioma growth. It is also possible that engineered MEVs are used to manipulate the neuron-glioma circuit for better tumor suppression and neuroprotection.

NSCs have the property of tropism towards glioma and these are dependent on interactions between growth factors and receptors (106). This property of NSCs paves the way towards a novel approach of developing NSC-based drug delivery systems. On this basis, murine NSCs transfected with adenoviral vector containing TNF-related apoptosis-inducing ligand (TRAIL) genes were able to successfully migrate towards glioma cells and express TRAIL in vivo, thereby significantly increasing the rate of tumor apoptosis (107). Furthermore, IL-23-expressing NSCs derived from bone marrow stem cells have been utilized to treat glioma in an animal model, where the survival time of mice was significantly prolonged; these effects were attributed to the potentiated activity of CD8+ T cells, CD4+ T cells and natural killer cells (108). Recently, researchers have loaded NSCs with oncolytic viruses to obtain more effective therapeutic effects on gliomas. Batalla-Covello et al (109) observed that the virus-loaded NSCs successfully migrated towards glioma and exerted anti-glioma effects in vivo. These findings demonstrated the feasibility and efficacy of using an NSC-based delivery system. It is noteworthy that researchers have observed a close contact (possibly physical) between NSCs and glioma cells through the use of immunohistochemistry and fluorescence immunohistochemistry (107-111). More importantly, Benmelouka et al (108) observed that the transplanted NSCs were capable of differentiating into neurons, astrocytes and oligodendrocytes in vivo. Therefore, NSCs may incorporate into the neuron-glioma circuit or neuron-glial network and form NGSs between neurons and gliomas. To use NSCs as a vehicle to deliver drugs, including small-molecule inhibitors, nucleotides such as miRNAs and oligonucleotides to target receptors and channels with both precision and efficiency may prove to be beneficial in the treatment of glioma, and this approach warrants further investigation.

The application of emergent optogenetic tools with their great precision and high safety provides the possibility of treating gliomas by neuronal manipulation. Optogenetic tools are able to modulate the activation of receptors on the neuronal membrane, which affects the tumor behavior via neuron-glioma activities (15). In a recent exploratory study, Trks were engineered with a photosensory core module of DrBphP, named Dr-Trk opto-kinase, which was successfully suppressed by far-red (FR) light and reactivated by near-infrared light (112). It is notable that the optical modulation may be limited to a single molecule of Dr-Trk, whereas, by contrast, even selective chemical receptor tyrosine kinase inhibitors usually suppress several targets simultaneously. Furthermore, the inhibitory effects of FR light were similar to pharmacological inhibition (112). In addition to the effects of modulation on membrane receptors, optogenetic technology may also be used to regulate neuronal activity. It was demonstrated that optical stimulation on GABAergic interneurons resulted in impaired glioma proliferation. The sensory stimulation promoted glioma cell growth, whereas this effect was limited to glioma cells in the visual cortex (15). Based on the insight gained on neuron-glioma interaction, optogenetic technology harbors great therapeutic potential for the future due to its non-invasiveness and accuracy.

The neuron organoids, mostly derived from embryonic stem cells or induced pluripotent cells, recapitulate multiple structures and functions similar to those found in the human brain (113). The co-culture system provided a suitable microenvironment for glioma development and interplay with other TME determinants, including astrocytes, oligodendrocytes, microglia and neurons (114). Brain organoids and GSCs constitute an ad hoc three-dimensional system, which may be used to further verify the functions of above-mentioned neuronal interacting factors, including neurotransmitters and transsynaptic proteins (including NLGN3 and its modifying enzyme, ADAM10). The super-resolution imaging technique revealed that GSCs develop an enhanced tropism for mature neurons by forming direct hemi-synapses containing pre-synaptic vesicles, for which a complete explanation of the internal structure awaits further investigation (115). Of note, the co-culture system provides an 'off-the-wall' methodology for advanced studies on glioma invasion and screening anti-invasive compounds. Single-cell transcriptomics combined with neural organoids have provided a novel perspective in terms of studying glioma heterogeneity and invasion, as well as the transcriptomic characterization of surrounding organoid cells upon glioma interaction. Therefore, specific transcriptional changes underlying therapeutic targets were denoted to enable effective drugs to be screened in a patient-specific manner (116). An up-to-date study has accumulated evidence of cognitive and electrophysiological neural responses in patients with glioma to analyze the impacts of functional integration on clinical outcomes. The results obtained suggested that gliomas with increased functional connectivity are correlated with higher aggressiveness through remodeling functional neural circuits in certain cortex areas (117). It is theoretically feasible that the co-culture of neuron organoids and glioma, as a more characteristic and accessible tool, may be applied to study the integration of glioma with neural networks and this technology may give rise to a therapeutic strategy that affords cognitive outcomes and survival.