Oncogene silencing consists of the targeted delivery of a particular nucleic acid into tumor cells, which leads to downregulation of specific genes (silencing)50. The extracellular matrix adhesion of glioblastoma multiforme cells is an important step for their invasion and many proteins have been investigated to play critical roles in accelerating this invasion51. The expression of these proteins has been reported to be extremely high in glioblastoma multiforme patients; therefore, silencing of oncogenes that express these proteins would inhibit the spread of glioblastoma multiforme cells51, 52. RabGEF1 is a gene known for its oncogenic role in glioblastoma multiforme as a guanine-nucleotide exchange factor for RAB-5. It has been reported that RabGEF1 is highly upregulated in human glioblastoma and other brain tumors53. Fan et al.54 evealed that downregulation of RabGEF1 inhibited glioblastoma cell proliferation and metastasis and induced autophagy of the cancer cells. GOLPH3 is an oncoprotein that has been reported to play a significant role in glioblastoma oncogenesis, and compelling evidence has demonstrated its role in the regulation of tumor cells as well as the migration and invasion of tumor cells under normal nutrient conditions55. In a recent study, Luo et al.56 silenced the expression of GOLPH3 and observed marked reduction in adhesion of glioblastoma cells. The authors reported that GOLPH3 significantly contributed to the adhesion of glioblastoma cells by regulating the lysosomal degradation of the protein integrin subunit beta 1 (ITGB1) under serum starvation, and its silencing led to a reduction in the adhesion of glioblastoma cells and the levels of ITGB1 protein. Zinc finger E-box-binding homeobox 2 (ZEB2) is a protein-coding gene that plays a critical role in the transcriptional regulation of various cellular functions and is abnormally expressed in brain tumors such as glioblastoma multiforme57. Safaee et al.58 investigated the effect of silencing ZEB2 on the cell cycle, apoptosis, and cytotoxicity of different glioblastoma cell lines and revealed that the suppression of ZEB2 induced apoptosis in all tested cell lines. Furthermore, the authors observed cytotoxic effects and marked reduction in migration of glioblastoma cell lines, suggesting the promising potential of ZEB2 silencing for the treatment of glioblastoma multiforme. Targeting programmed death ligand-1 (PD-L1) or programmed cell death protein 1 (PD-1) is another revolutionary strategy that has been applied to glioblastoma multiforme treatment. Qiu et al.59 studied the role of PD-L1 in glioblastoma multiforme patients and reported that it bound to the Ras oncoprotein in glioblastoma cells which altered gene expression and led to significant acceleration of cellular growth, migration, and invasion pathways. The same authors silenced PD-L1 and revealed that it played a vital role in glioblastoma multiforme cell proliferation and migration. The overexpression of PD-L1 promoted glioblastoma multiforme cell development and invasion in rodents, while its silencing abolished them. Figure 4 presents a summary of the role of PD-L1 in glioblastoma proliferation, migration, and invasion.

Suicide gene-based therapy consists of inserting genes that encode for cytotoxic proteins into glioblastoma cells either directly by inserting the toxin gene to reduce the viability of the cancer cells or indirectly by introducing a gene encoding an enzyme or protein with cytotoxic effects60, 61. Different strategies for suicidal gene delivery have been evaluated; Li et al.62 developed a novel therapy for glioblastoma based on mesenchymal stem cells to counteract the aggressive spread and dissemination of the cancer. The authors genetically engineered the mesenchymal stem cells and integrated the suicide gene TGF-β into their nuclei. Upregulation of TGF-β was clearly observed in the patients’ glioblastoma cells, in contrast to their non-neoplastic cortex cells, suggesting the high selectivity of this therapeutic approach. The same authors monitored the suicide gene products and demonstrated a significant improvement in the clinical efficacy, suggesting great potential for this approach in complicated brain cancers.

Gene therapy of glioblastoma with suicide genes using viral vectors has been also studied. The herpes simplex virus thymidine kinase (HSVtk) gene was inserted into glioblastoma cells using a viral vector; the expression of the gene led to the production of HSVtk protein, which phosphorylated and transformed the non-toxic prodrug ganciclovir into toxic ganciclovir-triphosphate63. Toxic ganciclovir-triphosphate produced from transfected cells was transported into neighboring cancer cells through gap junctions and led to a widespread and strong antitumor effect, even in cells that had not been genetically modified. Park et al.64 used the same method and combined the thymidine kinase gene with curcumin; the combined delivery of both the thymidine kinase gene and curcumin had higher therapeutic effects on glioblastoma than either treatment alone.

Uncontrolled and rapid growth of tumor cells leads to hypoxia and subsequent secretion of cellular angiogenesis signals such as angiopoietins, IL-8, fibroblast growth factor-2, or vascular endothelial growth factor to secure the oxygen and nutrient supply to the tumor cell, a vital process for tumor progression65, 66. Two major strategies have been pursued to target glioblastoma cell angiogenesis: upregulating the expression of anti-angiogenic factors and downregulating the expression of pro-angiogenic factors60. Sousa et al.67 developed an anti-angiogenic monoclonal antibody for glioblastoma multiforme treatment in animal models using bevacizumab-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles to decrease off-target organ toxicity and to circumvent the blood–brain barrier. The monoclonal antibodies were administrated intranasally and showed high brain bioavailability 7 days after administration. The authors reported significant reduction in tumor growth 14 days after administration which resulted from the high anti-angiogenic effect. Some studies have suggested that resistance to anti-angiogenic therapy may develop as a result of immune activation68, 69, specifically as a result of the action of pro-angiogenic M2-polarized macrophages. Some researchers have aimed to reprogram or inhibit the M2 phenotype to prevent the development of resistance to anti-angiogenic therapy and have revealed the phenotype to augment strong anti-angiogenic activity in animal models70, 71, 72. Glioblastomas are characterized by the release of vascular endothelial growth factor, which acts as a regulator and promoter of angiogenesis. Some anti-angiogenic therapies target this regulator or its receptor to inhibit the growth of glioblastoma cells, using monoclonal antibodies against vascular endothelial growth factor in addition to tyrosine kinase inhibitors that target the receptors of vascular endothelial growth factor73, 74.

Exosomes are a type of extracellular nanovesicle that have an important role in intercellular communications and consist of biological macromolecules such as proteins, DNA, and/or RNA75. The exact content of exosomes is dependent of the type of cell from which they are derived and its physiological condition76, 77. Importantly, many studies have revealed that after internalization by a secondary cell, the released exosomes induced significant phenotypic alterations depending on their content78, 79. Tumor cells generally secrete higher quantities of exosomes compared with normal cells; these secretions have been reported to play a vital role in promoting tumor progression by inducing malignant transformation of normal cells, cancer-associated fibroblast transformation, tumor escape from the immune system, angiogenesis, and metastasis80. Glioblastoma stem cells have been used to release exosomes containing microRNAs to mediate cellular communication; Tian et al.81 investigated whether glioblastoma stem cell-derived exosomes that contained the microRNA miR-26a could influence angiogenesis in microvessel endothelial cells in glioblastoma. The authors reported that, in glioblastoma, the tumor suppressor gene PTEN was downregulated while miR-26a was upregulated; this finding was explained by the activation of the PI3K/Akt signaling pathway by miR-26a, which led to PTEN downregulation. Furthermore, the authors reported that glioblastoma stem cell-derived exosomes containing miR-26a promoted the proliferation, migration, tube formation, and angiogenesis of tumor cells, suggesting that targeting of these exosomes could represent a potential therapeutic approach. Domenis et al.82 investigated the properties of immune-mediated tumor-derived exosome release upon activation of toll-like receptor 4 and found that treatment of the tumor influenced tumor-derived exosome composition and boosted its immunosuppressive ability, suggesting that the activation of toll-like receptor 4 supports tumor progression by stimulating the excretion of more effective immunosuppressive exosomes from the cells. This excretion of exosomes allows cancer cells to escape immune surveillance. Hypoxia-mediated stress in glioblastoma multiforme produced qualitative and quantitative changes in exosome content, with significant elevation of protein-lysine 6-oxidase, vascular-derived endothelial factor, and thrombospondin-1, which were all associated with tumor progression, metastasis, and angiogenesis83. Kore et al.83 demonstrated that hypoxia-related exosomes induced significant differential gene expression in recipient glioblastoma cells, showing a marked upregulation of small nucleolar RNA, C/D box 116–21 transcript, among others, and significant downregulation of voltage-gated potassium channels. Glioblastoma cell-derived exosomes are potential novel therapeutic targets. A significant number of publications address the qualitative and quantitative changes in these nanovesicles and their role in tumor development, and promote the development of preclinical and clinical trials involving these potential new targets84, 85, 86.

Immunization gene therapy is a broad therapeutic approach that involves enhancing the efficacy of the patient’s immune system against glioblastoma multiforme cells using chimeric antigen receptor T (CAR-T) cell therapy, tumor vaccine therapy, or cytokine gene therapy87. In a recent study, Agliardi et al.88 used genetically engineered CAR-T cells targeting receptor variant III of the glioblastoma tumor-specific epidermal growth factor in a mouse model and revealed that the engineered cells alone were unable to fully control the established tumors; however, the authors achieved lasting antitumor response when they combined single and locally delivered doses of IL-12. The cytotoxicity of CAR-T cells was significantly enhanced in the presence of IL-12, which also reshaped the tumor microenvironment, decreased the number of regulatory T cells, and increased the infiltration rate of pro-inflammatory CD4⁺ T cells89. In a similar study, Tang et al.90 transduced a CAR-targeting B7-H3 transmembrane protein into T cells using a lentivirus. The authors assessed the antitumor effects of the transmembrane protein B7-H3-specific CAR-T cells in vitro and in vivo with primary and glioblastoma cell lines and revealed that B7-H3 expression levels were highly correlated with malignancy grade; in addition, most of the clinical glioma samples were positive for B7-H3. The study also revealed the association of B7-H3 with poor survival rates in both low-grade glioma and glioblastoma patients. The results of cytotoxic and ELISA assays confirmed the specific antitumor effects of the engineered CAR-T cells on both cell lines. The authors added that the median survival time of the CAR-T-cell-treated group in the orthotropic glioblastoma models was significantly longer than that of the control group, suggesting promising potential for targeting CAR-T in glioblastoma multiforme treatment.

Tumor vaccination has also been studied in glioblastoma multiforme and involves presenting the tumor-specific antigens to the immune system and triggering an immune response against the antigens91. Dendritic cell-based vaccines are one of the novel strategies being tested in recent clinical trials that mediate anticancer immune reactions92. A number of dendritic cell-based vaccines for glioblastoma multiforme have been developed and are currently undergoing clinical investigation93. Erhart et al.94 found that glioblastoma multiforme patients with pre-existing antitumor characteristics seemed to survive longer under dendritic cell-based immunotherapy (Audencel) than patients who lacked these characteristics. The authors reported the inability of Audencel therapy to induce a significant clinical response, but the treatment had a strong effect on the immune system. The pre-vaccination blood count of CD8⁺ T lymphocytes and the in vitro production capacity of the enzyme-linked immunosorbent spot granzyme B upon exposure to the tumor antigen were significantly correlated with overall patient survival. Dendritic cell-based immunotherapy for glioblastoma multiforme led to significant upregulation of many cytokines promoting Th1 activation, better antitumor response, higher post-vaccination levels of IFNγ; in addition, the levels of CD8⁺ cells in the patients’ blood were indicative of better prognosis compared with unvaccinated patients94. Other studies proposed immune checkpoint inhibitors for the enhancement of glioblastoma multiforme treatment95, 96, 97. Current efforts focus on the combination of immune checkpoint inhibitors with different glioblastoma treatment modalities such as LAG3 and TIM3 checkpoint inhibitors98, 99. Khaddour et al.100 reviewed three novel techniques involving immune checkpoint inhibitors, including the administration of adjuvant immune checkpoint inhibitors following surgical resection of glioblastoma, patient selection for treatment with immune checkpoint inhibitors, and combining immune checkpoint inhibitors with other novel therapies (Figure 5). Recently, a variety of receptors, molecules, and pathways have been investigated and have emerged as potential targets for combined immune checkpoint inhibitors, such as TGF-β inhibitors, CD47 blockade, and colony-stimulating factor-1 ligand inhibitors101. Rationally designed immunization gene therapy and combinatorial approaches offer promising treatments for glioblastoma multiforme.

Figure 5

Abnormal expression of different DNA repair genes is frequently associated with tumorigenesis, although the role of these repair genes in the progression and development of glioblastoma remains unclear102. Repairing the damage in tumor genes involves induction of limited, selective alterations to the genetic material of tumor cells, which is consider an advantage of this approach; however, this process contributes to the development of resistance to genotoxic therapies based on tumor-driving cells103. Struve et al.104 observed significantly elevated expression of several DNA mismatch repair proteins in epidermal growth factor receptor variant III cells and samples from glioblastoma patients; this expression was most pronounced for DNA mismatch repair protein 2 and 6. Epidermal growth factor receptor variant III-specific knockdown reduced mismatch repair protein expression, thereby increasing resistance to the alkylating agent temozolomide and demonstrating that the oncoprotein epidermal growth factor receptor variant III sensitizes a fraction of glioblastoma through the upregulation of DNA mismatch repair proteins. In separate study, Lin et al.105 revealed that the inhibition of RNA-binding protein Musashi-1 in glioblastoma multiforme patients radiosensitized tumors, prevented cancer stem cell selection in radiotherapy, and reduced tumor invasion. The authors reported that Musashi-1 enhanced tumor invasion via vascular cell adhesion protein 1 and modulated glioblastoma multiforme radioresistance via hyperactivation of the DNA damage response process through evasion of apoptosis and increased homologous recombination repair. Therefore, knockdown of Musashi-1 induced the accumulation of DNA damage in irradiated glioblastoma multiforme cells and promoted their depletion in vitro. Kun et al.102 performed clustering to screen for potentially abnormal DNA repair genes associated with glioblastoma multiforme prognosis and revealed that five DNA repair genes (CDK7, RFC2 DDB2, RNH1, FAH, and RNH1) were significantly related to glioblastoma multiforme prognosis. The prognosis of glioblastoma multiforme can be predicted from the expression of DNA repair genes, which may also represent future therapeutic targets.

Genome editing-based therapy consists of the modification of whole-body intracellular DNA in a sequence-specific manner via substitution, insertion, deletion, or integration106. Maeder et al.107 reviewed the most common nucleases used for genome editing, including transcription activator-like effector nucleases, zinc finger nucleases, meganucleases, and the CRISPR/Cas9 system. Among these strategies, CRISPR nuclease Cas9 is the most advanced and versatile system of gene editing technology; it is characterized by highly specific recognition of its target chromosomal DNA and eventual gene disruption108, 109. This technology opens multiple avenues for the treatment of cancers such as glioblastoma multiforme. Rosenblum et al.108 utilized CRISPR-encapsulating lipid nanoparticles to promote therapeutic gene editing in vitro through the disruption of key glioblastoma multiforme survival genes in murine and human glioblastoma cell lines and in vivo in an aggressive syngeneic glioblastoma mouse model. The authors revealed that a CRISPR lipid nanoparticle-based platform could potentially be developed and utilized in human clinical trials as a new therapeutic modality for glioblastoma multiforme. To test and confirm the role of the CRISPR/Cas9 system in the induction of significant gene mutations in animal genome atlases of glioblastoma multiforme, Chow et al.110 analyzed a cloned mTSG library into an adeno-associated virus vector encoding sgRNA that targeted Trp53 and astrocyte-specific GFAP-Cre (frequently mutated in glioblastoma multiforme). The authors reported that, after using the adeno-associated virus vector-based CRISPR/Cas9, many genes were significantly mutated, suggesting that the CRISPR/Cas9 system could play a critical role in glioblastoma multiforme treatment. In another recent investigation, MacLeod et al.111 used the CRISPR-Cas9 system in patient-derived glioblastoma multiforme stem cells to elucidate the function of the coding genome. The authors identified actionable pathways responsible for the proliferation of cells and revealed the gene-essential circuitry of glioblastoma stemness and growth. The authors also revealed the mechanisms of temozolomide resistance in glioblastoma cells that could lead to combination strategies.