TP53 mutations represent one of the most frequent genetic alterations in gliomas and are crucial in the progression of these tumors. (54) The TP53 gene, located on chromosome 17p, encodes the tumor suppressor protein p53, which is pivotal in regulating cell cycle arrest, mediating DNA repair, and initiating apoptosis in response to cellular stress. (55) In gliomas, inactivating mutations in TP53 lead to the loss of p53 function, resulting in tumorigenic features such as resistance to apoptosis, uncontrolled cell proliferation, and genomic instability. (56) These mutations are associated with poor prognosis, as glioma cells lacking functional p53 exhibit enhanced proliferative capacities and increased resistance to therapies like radiation and chemotherapy. The loss of p53 not only contributes to tumor initiation but also facilitates glioma recurrence, highlighting the importance of TP53 mutations in glioma pathophysiology.

Neurofibromin 1 (NF1), a well-known tumor suppressor gene, is frequently mutated in gliomas, particularly in the mesenchymal subtype. (57) NF1 regulates the RTK/Ras/PI3K signaling pathway, and mutations in this gene lead to loss of function, which promotes cellular transformation and drives the transition of glioma cells towards a more aggressive, mesenchymal phenotype. (58) These mutations increase the motility, invasiveness, and resistance of glioma cells, contributing to a more aggressive tumor progression. Furthermore, NF1 mutations enhance the proliferation of neural progenitor cells, providing a source of stem-like cells that fuel glioma growth. (59) This shift toward a more invasive and stem-like state underlines the importance of NF1 mutations in glioma aggressiveness and therapy resistance.

The AKT signaling pathway, a downstream component of the PI3K signaling cascade, plays a significant role in supporting glioma cell survival, proliferation, and migration. (60) AKT is a serine/threonine kinase that is activated by various growth factors and regulates several downstream targets involved in cellular processes such as survival, growth, metabolism, and angiogenesis. In glioma, aberrant activation of the AKT pathway is commonly observed and is associated with enhanced tumor growth, increased cell migration, and resistance to apoptotic. (61) Hyperactivation of AKT contributes to glioma aggressiveness by supporting tumor cell survival under stressful conditions and facilitating metastasis. Given its pivotal role in glioma biology, AKT remains a promising therapeutic target, with inhibitors currently under investigation in clinical trials to block glioma progression and improve treatment outcomes. (62).

The Notch signaling pathway is integral to glioma development, particularly in regulating GSCs, which contribute to tumor self-renewal, resistance to therapies, and recurrence. (63) Notch signaling is involved in a variety of cellular processes, including differentiation, proliferation, and apoptosis. Dysregulation of Notch signaling, whether through hyperactivation or suppression, is associated with the development and progression of gliomas. (64) Hyperactivation of Notch signaling enhances glioma cell proliferation and invasiveness, while also maintaining the stem-like properties of GSCs. The interaction between Notch1 and the CXCL12/CXCR4 axis has been shown to promote tumor cell invasion and sustain glioma stemness, making this axis a potential therapeutic target. (65) Given its crucial role in maintaining tumor-initiating populations, Notch signaling remains a promising target for glioma therapy.

The Wingless/Int1 (Wnt) signaling pathway plays a critical role at different stages of central nervous system (CNS) development and is directly required to regulate self-renewal, proliferation, and differentiation of NPCs in the developing brain. (66) Aberrant activation of the Wnt pathway has been implicated in driving the development and progression of various human cancers.

Another signaling pathway that contributes to GSC invasiveness is the TGF-β pathway. TGF-β signaling plays a critical role in regulating many cellular processes during embryogenesis, cell proliferation, migration, and tissue homeostasis. (67) Although the TGF-β pathway is best known for its tumor suppressor function in epithelial tissues, it also serves as a promoter of tumorigenesis in various solid cancers, including GBM, due to its ability to enhance cell migration and thus cell invasion agent.

Wnt pathway and TGF-β pathway can lead to epithelial-mesenchymal transition(EMT), ultimately leading to loss of epithelial tissue and acquisition of a mesenchymal phenotype.

In gliomas, especially glioblastoma (GBM), the EGFR/MAPK signaling pathway is widely considered to be one of the key axes driving tumor occurrence and progression. EGFR amplification and constitutively active mutations (such as EGFRvIII) can continuously activate the downstream RAS/RAF/MEK/ERK cascade, thereby promoting cell proliferation, inhibiting apoptosis, and enhancing cell invasiveness. Verhaak et al. divided GBM into four subtypes for the first time through large-scale genomic analysis, and pointed out that the Classical subtype is closely related to EGFR amplification and MAPK pathway activation (68), suggesting that abnormalities in this pathway are one of the core characteristics of specific molecular subtypes. In addition, Suvà et al. found that EGFR/MAPK signaling is essential for maintaining the stemness and proliferation potential of glioma stem cells, and its inhibition can induce cell differentiation and reduce the tumor stem cell phenotype (69). Combined with single-cell sequencing technology, Tirosh and his collaborators further revealed that there are complex developmental hierarchies and cell state variations within gliomas, and that the activity of the EGFR/MAPK pathway is highly heterogeneous in different cell subpopulations (70).

ENU is a well-established alkylating mutagen that primarily induces point mutations through the ethylation of DNA bases. (71) This chemical has been widely used to investigate genetic alterations linked to cancer development, including gliomas. ENU exposure in zebrafish typically occurs in a controlled manner, with optimal concentrations ensuring a balance between mutagenicity and toxicity. Studies suggest that repeated exposure to 3-3.5 mM ENU over 2–4 weeks can effectively induce mutagenesis in zebrafish embryos (72) ( Figure 4 ).

For example, Solnica-Krezel et al. exposed male wild-type zebrafish to 3mM ENU for 1 h/day over a pan of 2–4 weeks. (73) After exposure, these males were crossed with wild-type females. The offspring, examined under confocal microscopy, revealed head enlargement and localized masses in the brain vasculature, which are characteristic features of glioma-like tumors. Similarly, Wienholds et al. administered 3.0 mM ENU to four-month-old male zebrafish, repeating the exposure six times. (74) The offspring were subsequently screened for glioma-related markers and tumor phenotypes. Although ENU successfully induces random mutations, its application in glioma modeling is limited by its relatively low efficiency and the non-specific nature of the tumor phenotypes induced, which may affect multiple organs, including the liver and testis, complicating the identification of glioma-specific markers.

MNNG, is another powerful alkylating agent that induces mutations by adding methyl groups to the DNA, leading to the disruption of normal cellular processes and subsequent tumor formation. MNNG has been shown to cause glial cell hyperplasia and abnormal differentiation, facilitating glioma development by activating cellular signaling pathways involved in proliferation and survival. (75).

Several methods have been employed to expose zebrafish to MNNG, including direct immersion, microinjection, and dietary exposure. (76) The direct immersion method involves placing zebrafish embryos or larvae in MNNG solutions, with concentrations of up to 10 ppm for embryos (at 83 hours post-fertilization) and up to 2 ppm for larvae (at three weeks post-hatching). Short-duration exposures to MNNG in these concentrations have been shown to induce glioma-like characteristics, such as head enlargement and abnormal swimming behaviors ( Figure 4 ).

Microinjection allows for more localized mutagenesis by directly injecting MNNG into the zebrafish embryos. This technique enhances the likelihood of developing glioma tumors specifically in the brain, providing a more focused model for glioma research. Lastly, dietary exposure involves feeding zebrafish MNNG-treated food for extended periods, up to three months. This method has been shown to induce a range of mesenchymal tumors, including gliomas, hemangiomas, and sarcomas, highlighting the broad mutagenic potential of MNNG.

Screening of zebrafish exposed to MNNG is conducted through confocal laser microscopy, identifying glioma-related phenotypes in embryos and larvae, such as head enlargement and abnormal swimming patterns. Despite its effectiveness in generating glioma models, MNNG induction can result in tumors in multiple organs, including the liver and testis, complicating the identification of glioma-specific phenotypes.

To explore glioma driven by specific oncogenic pathways, as shown in Figure 3B , Marie Mayrhofer et al. utilized the Gal4-UAS binary expression system, which enables targeted expression of oncogene in zebrafish. (77) In this model, the Ethmz5 driver line expresses a codon-optimized Gal4 transcription factor under the control of the zic4 enhancer, which is specifically active in proliferative regions of the developing CNS. By crossing this driver line with zebrafish carrying various oncogenes under the UAS promoter, the researchers were able to activate the EGFR/RAS/ERK/AKT pathway, which is critical in glioma pathogenesis. (78) This activation led to tumor-like growths in the zebrafish brain.

Brain imaging of zebrafish at juvenile and adult stages (ranging from 1–14 months) revealed vsignificant malformations, particularly in the telencephalon, fourth ventricle, and diencephalon. Tumor incidence was assessed over time, with glioma-like growths appearing in 36.6% of zebrafish by 6 months and 49% by 9 months of age upon activation of the AKT pathway. These findings underscore the efficacy of the EGFR/RAS/ERK/AKT pathway as a driver of glioma in zebrafish and highlight the suitability of zebrafish models for studying glioma progression in an age-dependent manner.

Glioma development in humans is frequently associated with mutations in key tumor suppressor genes, including RTK/Ras/PI3K, RB, and TP53 pathways. (79) To replicate these genetic alterations in zebrafish, Luo et al. employed the CRISPR/Cas9-based gene editing to targeted the Nf1, Tp53, and Rb1 genes. (80) Using a modified U6–3 promoter vector, the researchers inserted specific guide RNA (gRNA) sequences to induce precise gene deletions in these critical tumor suppressor genes. A Cas9-T2A-mCherry construct was used to facilitate gene expression under the control of the gfap promoter, which is active in glial cells, ensuring that the genetic modifications occurred specifically in the brain. Embryos were injected with the CRISPR constructs, and successful modifications were confirmed through histological analysis, immunohistochemistry, and confocal microscopy. Zebrafish embryos exhibiting a “curved body” phenotype were identified as candidates for further study, as this phenotype correlated with the development of cerebellar gliomas and associated motor dysfunctions. Histological analysis of the edited zebrafish revealed significant structural disruption in the brain, with gliomas invading the fourth ventricle, similar to human glioma progression. These findings were confirmed by hematoxylin and eosin staining and confocal imaging, which demonstrated the presence of glioma tissues in these regions. This genetic engineering approach not only mimicked the key genetic alterations found in human gliomas but also provided a powerful tool for dissecting the molecular mechanisms of gliomagenesis. The zebrafish model allowed for detailed study of the roles of Nf1, Tp53, and Rb1 mutations in glioma formation and progression.

The U87 cell line, one of the most widely used glioblastoma models, is known for its aggressive invasion and growth properties in vivo. In a study by Yang et al. (81) U87 cells were transfected with a red fluorescence protein (RFP) plasmid and microinjected into zebrafish embryos at 36 hours post-fertilization. The embryos were cultured at 35°C and monitored for tumor growth using confocal microscopy. The results revealed that U87 cells, once injected, formed secondary tumor nodules, which were typically located around the neurons, blood vessels, and leptomeninges, indicative of glioma’s characteristic pattern of invasion. Furthermore, tumor progression was associated with a significant reduction in embryo survival rates, which decreased to 40% when approximately 1,000 cells were injected. U87 cells were found to occupy nearly 20% of the zebrafish brain, highlighting their invasive nature ( Figure 5 ).

GBM9 cells, derived from patient cortically localized glioblastomas, represent a highly aggressive and clinically relevant glioma model. Welker et al. (82) employed xenotransplantation of both serum-grown adherent GBM9 cells and neurospheres into zebrafish embryos at 36 hours post-fertilization. Confocal imaging over time revealed extensive tumor progression with significant brain infiltration. Notably, zebrafish xenografted with GBM9 cells exhibited abnormal swimming behaviors, including twitching and circling, suggesting impaired brain function. The study showed dose-dependent lethality, with zebrafish injected with over 25 cells exhibiting 100% mortality, while the median survival time decreased significantly with higher cell numbers. These results emphasize the invasiveness and lethal potential of GBM9 cells, making them a valuable tool for modeling aggressive gliomas and evaluating potential therapies.

The U251 cell line, derived from human astrocytoma, is frequently used to study glioma progression, particularly in examining tumor-cell interactions with the vasculature. In research by Gamblea et al. (83) U251MG cells were cultured and labeled with CM-dI fluorescent dye before microinjection into the hindbrain ventricle of zebrafish embryos. Tumor progression was monitored using confocal microscopy at 1 and 4 days post-injection, revealing that U251MG cells adhered to the blood vessels within deep brain regions and formed microtumors. The cells also exhibited invasive behavior, with pseudopodia extending toward surrounding brain structures (84). The interactions between U251MG cells and the vasculature were further elucidated using time-lapse imaging, which revealed that U251MG cells integrated with blood vessels and exhibited significant invasion into the zebrafish brain parenchyma, mimicking key features of human glioma progression ( Figure 6 ).

While human glioma cell lines such as U87 and U251 have historically provided insights into basic tumor biology, their prolonged in vitro culturing has significantly altered key features of glioma, including invasiveness, genomic heterogeneity, and stemness. Given the increasing availability of patient-derived glioma stem-like cells and organoid-based models (88), future studies should prioritize more physiologically relevant systems for both mechanistic research and translational validation.

In general, the chemical mutagenesis zebrafish model is easy to operate and can be screened in large quantities. It is suitable for preliminary exploration of carcinogenic mechanisms. However, its disadvantage is that carcinogenesis is highly random and the tumor type cannot be determined. Gene editing to establish a glioma model can be used through CRISPR/Cas9, TALEN or transgenic strategies to target and regulate key oncogenes or tumor suppressor genes (such as Tp53, IDH1, EGFR, PDGFRA) in zebrafish to induce brain glioma formation. The disadvantage is that the cycle is long and the technical difficulty is high. Xenotransplantation modeling is rapid, and the transparency of zebrafish can be used to observe dynamic processes such as tumor cell migration, invasion, and angiogenesis. In terms of limitations, early embryonic stages are usually used, and there is no mature immune system. In addition, human cells are transplanted, which cannot simulate immune responses. In recent years, the development of TEAZ (Transgene Electroporation in Adult Zebrafish) technology has provided a new approach for the construction of zebrafish tumor models. This method introduces DNA constructs containing specific genes into adult zebrafish through electroporation, achieving spatiotemporal specific induction of tumors. Compared with traditional transgenic methods, TEAZ is easier to operate and is suitable for individuals with intact immunity, providing a powerful tool for studying the occurrence, progression and metastasis of tumors (89).

Angiogenesis, the formation of new blood vessels, is a hallmark of glioma and a crucial factor in tumor growth, ensuring an adequate supply of nutrients and oxygen to rapidly proliferating cancer cells. Notably, TGF-β1 was shown to significantly enhance angiogenesis, while JNK pathway inhibition markedly reduced vessel formation, highlighting JNK as a potential target for limiting tumor vascularization. In contrast, inhibitors of p38 MAPK, ERK, and PI3K pathways did not impact angiogenesis, underlining the specificity of the JNK signaling pathway in glioma vascular responses.

Further extending these findings, Umans et al. (100) utilized advanced imaging techniques in zebrafish to model perivascular GBM invasion. Their study quantified changes in blood vessel volume and glut1 signal intensity during tumor growth. By observing glioma cells’ interaction with tumor-associated and non-tumor-associated blood vessels, they identified critical tumor-vascular interactions that support glioma cell invasion. This research provides valuable insights into how glioma cells manipulate the vascular niche to facilitate tumor progression and invasion, pinpointing novel therapeutic targets aimed at disrupting these interactions.

In recent years, the zebrafish model has not only made progress in the growth and invasion mechanisms of gliomas, but has also been gradually used to analyze the tumor-related immune microenvironment. Mai Nguyen-Chi et al. (101) used the zebrafish in situ transplantation model to observe tumor-induced macrophage aggregation and M1/M2 polarization dynamics, revealing the key role of the innate immune system in the early progression of tumors; Zhang et al. (102) combined transgenic fluorescent labeling technology to track the regulatory mechanism of glioma cells on neutrophil chemotaxis in real time. These results show that zebrafish not only have imaging and intervention advantages, but can also be used to establish a preliminary screening platform for microenvironment intervention targets.

Zebrafish models are increasingly used for high-throughput drug screening, enabling rapid evaluation of therapeutic efficacy and safety in vivo. He et al. (103) demonstrated the potential of zebrafish for testing combination therapies by investigating the effects of ionizing radiation and temozolomide in U251 glioma cell xenografts. Their results showed a significant reduction in tumor size with combined treatment, which was further enhanced by pre-treatment with temozolomide, without notable toxicity to embryonic development. These findings underscores the utility of zebrafish models in optimizing glioma treatment regimens by balancing therapeutic efficacy with safety.

Similarly, Li et al. (104) compared conventional radiotherapy with pulsed low-dose rate radiation using zebrafish glioma models. They found that pulsed radiation more effectively controlled glioma growth while minimizing damage to normal tissues, suggesting it could be a promising alternative to traditional radiotherapy. This highlights the zebrafish model’s capacity to simulate and evaluate treatment responses, facilitating the development of more refined and targeted radiotherapeutic strategies for glioma.

Overcoming the BBB remains one of the most significant challenges in glioma therapy. Zebrafish models have been instrumental in exploring innovative drug delivery systems designed to address this issue. Jia et al. (105) developed a novel approach using neutrophil-derived exosomes loaded with doxorubicin hydrochloride (DOX) to target gliomas. Exosomes, known for their ability to cross the BBB and their excellent biocompatibility, (106) effectively delivered DOX to brain tumors in zebrafish models. This method reduced systemic toxicity and improved therapeutic outcomes, demonstrating the potential of exosome-based drug delivery as a minimally invasive strategy for treating gliomas.

Additionally, the effects of dl-nordihydroguaiaretic acid (Nordy) were explored in zebrafish glioma models by researchers investigating its impact on GSCs. (107) Nordy was found to inhibit GSCs proliferation and promote their differentiation into astrocyte-like cells, reducing tumor invasiveness and angiogenesis that targeting GSCs through differentiation therapies could offer a novel approach for managing aggressive gliomas, complementing existing treatments by addressing the tumor’s stem cell compartment (108).

The latest studies have shown that the intestinal microbiota can indirectly affect the immune microenvironment of brain tumors, especially gliomas, by regulating systemic immune responses. (109) Microbial metabolites and the cytokine network mediated by them have been found to regulate the polarization state of tumor-associated macrophages, the infiltration ability of T cells, and the local immunosuppression state of tumors. Although the research on the gut-brain-immune axis in the field of glioma is still in its early stages, this direction can provide new targets for immunotherapy. Zebrafish have potential in studying host-microbe interactions, and their innate immune system is highly conserved with humans. In the future, introducing microbial manipulation technologies (such as germ-free zebrafish and colonization models) into zebrafish glioma models is expected to further clarify how microbial signals shape the immune landscape of glioma.

Through these studies, zebrafish models have proven to be powerful tools for both understanding the complex TME and for therapeutic screening. They provide unique opportunities to study glioma progression in real-time, evaluate the efficacy of novel treatment regimens, and explore cutting-edge drug delivery methods, all while reducing the need for more complex and costly mammalian models.