In the last three decades, gene therapy has emerged as a promising tool of genome editing that can potentially aid in the treatment of various diseases such as heart failure, neurodegeneration and cancer (69). Gene therapy implicates the correction of a defective gene by introducing a functional and healthy version thus compensating for the abnormal/missing gene (70). Advancements in this field paved the way for considering gene therapy as an adjuvant treatment to conventional chemo- and RT, allowing for a reduced dose of standard modalities in combinatorial approaches.

Several different strategies are currently employed for targeting tumors using gene therapy. One of the common strategies is the delivery of different gene types expressing tumor suppressor genes to compensate for the defective or missing gene, or expressing suicide genes, tumor antigens and growth factors in order to induce apoptosis or improve tumor sensitivity to conventional therapies. Plenty of tumor suppressor genes have been evaluated for gene therapy such as Rb and PTEN (71), and p53 was introduced to treat non-small cell lung carcinoma (72). TNF-related apoptosis inducing ligand and Interleukin-24 (IL-24) are examples of apoptosis inducers that induce apoptosis in various tumor cells with minimal effect on normal cells (73,74).

Another strategy is the gene interference using RNA interference (RNAi) approach to block the expression of an oncogene and, in numerous cases, sensitize tumor cells to RT (75). In vivo and in vitro studies showed that tumor cells treated with antisense RNA targeting c-myc gene and K-ras suppress tumor growth in melanoma and pancreatic cancer cells respectively (76,77). A platform for the treatment of GBM has also been developed using RNAi technology including small interfering (si)RNA, microRNA (miRNA), short hairpin (sh)RNA and long non-coding (lnc)RNAs. siRNA is used in several studies to silence the target proteins that are overexpressed in the GBM (78). Danhier et al (79) in 2015 targeted galectin-1 (gal-1) which is involved in the development and progression of chemoresistance in GBM. Using anti-gal-1 and anti-EGFR siRNA administrated via nanocapsules with TMZ, researchers reported that the median survival rate of mice bearing glioma cell increases (79). Another study using anti-CD73 siRNA reported tumor volume suppression by 60% in C6 glioma rats (80). A first-in-human early phase I clinical trial (NCT03020017) examined the safety and efficiency of NU-0129 drug, which is an RNAi-based spherical nucleic acids (SNAs), in patients with recurrent glioblastoma. SNAs consist of gold nanoparticle cores covalently conjugated with siRNA oligonucleotides specific for the GBM oncogene Bcl2Like12 (siBcl2L12). Results showed successful enrichment of gold particle in tumor-associated endothelium, macrophages and tumor cells, along with decreased expression of tumor-associated Bcl2L12. The study demonstrated the potential of SNA nanoconjugates as a brain-penetrant precision medicine strategy for the systemic management of GBM (81).

GBM cells display abnormal pattern of miRNAs that may result in increased expression of oncogenes or decreased expression of tumor suppressor genes, thus affecting various downstream signaling pathways (78) [reviewed in (82)]. The glioma angiogenesis, for instance, is significantly influenced by miR-26a, which is found in glioma tissues. Additionally, serum exosomes of patients with GBM showed overexpressed miR-21 (83). According to a previous study conducted by Møller et al (84), the microenvironment of GBMs was found to overexpress 256 miRNAs while underexpressing 95 miRNAs when compared with the normal brain. Numerous miRNAs are thus considered as possible biomarker for early diagnosis and prognosis of GBM, and investigated for gene therapy (83,85). Transfection of miR-34a, which is a miRNA normally downregulated in GBM tumor microenvironment (TME), restores normal physiological conditions by inhibiting cell proliferation and invasion, and suppressing tumoral survival in glioma cell lines and glioma xenograft model (86). miR-21 and miR-10b, however, are usually upregulated in GBM cells and exhibit oncogenic properties. In an in vitro model, the inhibition of these miRNAs with synthetic anti-miRNA counterparts prevents cell cycle progression (87).

shRNAs are artificially generated RNA molecules and contain hair-pin-like structures intended to silence target mRNAs. Viel et al (88) in 2013 used shRNA to target O⁶-Methylguanine-DNA-methyltransferase (MGMT), a DNA repair enzyme. Treatment of GBM xenografts with anti-MGMT shRNA and TMZ results in a significant decrease in tumor size (88). Another study showed that knocking down cyclin D1 by shRNA results in the inhibition of cell proliferation and migration and induces apoptosis in GBM cell lines (89). Moreover, Song et al (90) in 2016 used Sox2-shRNA to downregulate Sox2, a gene associated with stemness of CD133 positive GBM cells. The aforementioned study reported that Sox2-shRNA abrogates tumor initiation and drug resistance of these cells, thus, suggesting that RNAi technology could help decreasing GBM cell resistant to anti-GBM treatments.

GBM cells also express aberrant expressions of lncRNAs associated with pluripotency and tumorigenesis. Akin to miRNAs, several lncRNAs are downregulated while others are upregulated in GBM cells compared with normal human brain tissue (78). Thus, numerous lncRNAs are suggested as biomarkers for GBM tumorigenesis and chemoresistance. For instance, Zhang et al (91) in 2019 provided evidence that knockdown of lncSBF2-AS1 decreases chemoresistance of GBM cell lines to TMZ. These expression patterns of lncRNA revealed a relationship between tumor histopathological differentiation and malignancy grade that could be effectively used to treat GBM (91).

Achieving an effective delivery to the intra-cerebral tumors is the main difficulty in treating GBM. Due to the degradation of therapeutic active molecules, the development and marketing of various nucleic acid-based therapies into clinical trials have been hampered by the uncertainty of effective delivery, therapeutic efficacy and toxicity profile. As enormous research is conducted to investigate the safety and efficacy of gene therapy, delivery tools are thus acquiring a great research focus for more efficient GBM treatment (discussed in ‘Novel Approaches’ section).

In addition to the highly invasive nature of glioblastomas and the presence of BBB, the presence of cancer stem cells is another factor that limits clinical options and treatments of GBMs (92). Glioblastoma stem cells (GSCs), which are self-autonomous units, play a significant role in tumor initiation and growth as well as therapeutic resistance (93). Their prolonged proliferation and ability to metastasize and suppress anti-inflammatory responses are among the most significant mechanisms by which GSCs contribute to tumor malignancy (94). Enriched understanding of GCS biology paved the way for numerous studies that are now being conducted to target GSC. In the present review, major aspects of GSCs recognized as hot target for improved GBM treatment were discussed.

GSCs are affected by differentiated glioblastoma cells (DGC) due to a reciprocal signaling pathway. DGCs express brain-derived neurotrophic factor (BDNF), whereas GSCs express the BDNF receptor NTRK2 which accelerates GSC tumor growth (94). It is thus important to understand the reciprocal signaling between GSCs and DGCs for improved targeted cancer treatment. BDNF induces nerve growth factor (VGF) expression in GSCs through the NTRK2-PI3K-AKT signaling pathway. VGF promotes GSC growth and self-renewal and further secretion of BDNF by DGC for its survival. Thus, BDNF-NTRK2-VGF paracrine signaling pathway enhances the cooperation between DGC and GSC in promoting tumor growth (94). Several studies have thus proposed NTRK and VGF as potential therapeutic targets for GBM treatment (94,95).

CD133 positive GSCs play a significant role in maintaining and proliferating GBM (96). Eliminating CD133 positive stem cells is a method that has proven to increase the survival rate in patients with GBM. Depleting CD133 can be achieved by numerous methods (97). Knocking down BMI1 gene, an oncogene that is involved in the regulation of self-renewal and differentiation of stem cells, may destroy CD133 stem cells (98). Targeting these stem cells is thus another promising target for GBM therapy. Another GCS target is the mitochondria that plays an important role in tumorigenesis and tumor progression, particularly through oxidative phosphorylation (OXPHOS), which is the major source of ATP in numerous types of cancers (99). Among GBM cells, GSCs depend on OXPHOS which requires mitochondrial translation (100). Blocking mitochondrial translation by a bacterial antibiotic quinupristin/dalfopristin (Q/D) does not only suppress the growth of the GSCs, but also dysregulates cell cycle and promotes apoptosis (100). These findings propose that inhibiting mitochondrial translation may be investigated to therapeutically inhibit GSC growth and that Q/D may be considered for the treatment of GBM.

One promising investigational drug is salinomycin that preferentially eliminates GSCs and other varieties of CSCs. Salinomycin and its derivatives are currently extensively investigated as an anti-CSCs treatment for numerous types of malignancies, however, clinical trials evaluating the potential efficacy of salinomycin in glioblastoma have not yet been published (101). Collectively, these findings suggested that targeting GSCs represents a promising strategy for improved treatment of GBM.

To ensure targeted therapy success, the following key steps should be taken into consideration: identification of targets, identification and development of target-specific small molecules and antibodies followed by pre-clinical studies and clinical trials. Current major challenges in this field include the development of drug resistance and low efficiency to drugs. To date, except for BEV, targeted therapies in GBM patients have not provided significant survival benefits (59,102).

To prevent tumor growth and eradicate tumor cells in patients with cancer, cancer vaccines are intensively studied as a promising therapeutic approach. This method activates the humoral and cellular immunity of patients with cancer (106). To achieve a favorable clinical efficacy, a critical step of cancer vaccine design is antigen selection. An ideal antigen for a cancer vaccine should be specifically expressed and present on all cancer cells-but not in normal cells-, highly immunogenic and essential for cancer cell survival (107). Consequently, the two major classes of antigens employed in cancer vaccine design include tumor-associated antigens (TAA) and tumor-specific antigens (TSA). TAAs are abnormally overexpressed self-antigens derived from tumor cells. However, they may be expressed as well in a subset of normal cells at certain level (108). Differentiation antigens, such as Melan A and CD19, and overexpressed antigens such as HER2 and TROP2 are examples of TAAs (109). Utilizing these antigens may potentially induce autoimmunity against normal tissues and possibly skip T cell recognition, since these antigens are typically deleted from the immune repertoire by central and peripheral tolerance mechanisms (107). TSAs, on the other hand, are exclusively expressed by cancer cells and are not present in normal host cells. This reduces the risk of autoimmune destruction and strongly activates high-affinity antibodies and T cells against the non-self-antigens (110). TSAs include onco-viral antigens, such as E6 and E7 of the human papillomavirus, private neoantigens, and shared neoantigens such as KRAS and p53 (109). To destroy tumor cells, cancer vaccines employ the following mechanism of action. After antigen delivery, tumor antigens are taken up and processed by antigen-presenting cells (APCs) such as dendritic cells (DC). DCs present the antigens to major histocompatibility complex (MHC) class I and II molecules (104). Next, these cells migrate to the vaccine draining lymph nodes (primary site for T cell priming), to recruit and activate immune cells by presenting relevant antigens on MHC I and MHC II to CD8⁺ and CD4⁺ T cells, respectively (106). Activated T lymphocytes differentiate into memory T cells and effector T cells. Effector T cells migrate to the TME, recognize tumor cells, and destroy them through exploiting multiple mechanisms. Cytotoxic T cells can eliminate cancer cells by releasing cytotoxic particles such as perforin and granzymes. Furthermore, they can induce cancer cell apoptosis through direct cell to cell-mediated interactions. Additionally, mediators such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) induce cytotoxicity (111). Besides T cells, B lymphocytes and cells of the innate immune system such as natural killer (NK) cells and macrophages promote tumor eradication (111). Peptide-based, DC-based and personalized vaccines are being explored as potential vaccine therapies in GBM (112,113).

Multiple DC based vaccines are under investigation for the treatment of GBM. One notable DC vaccine is the DCVax^®-L developed by Northwest Biotherapeutics. Newly diagnosed patients with GBM in phase I and II clinical trials who received the vaccine and the standard of care treatment had an overall survival of 36 months and almost 24 months of progression free survival with no major side effects (114). An ongoing randomized, placebo-controlled phase III clinical trial (NCT00045968) involves 348 eligible newly diagnosed GBM participants. The primary objective of the study is to compare the overall survival between patients receiving the vaccine and the control group receiving the standard of care treatment. The treatment cohort will receive two intradermal injections per treatment of DCVax^®-L vaccine at days 0, 10, 20 and at weeks 8, 16, 32, 48, 72, 96 and 120.

Another cancer vaccine that represents a silver lining for GBM immunotherapy is ICT-107, an autologous DC vaccine. Immune cells are pulsed with six synthetic peptide epitopes that recognize and target glioma TAA and stem cell-associated antigens such as HER-2, MAGE-1, AIM-2, TRP2, gp100, and IL13Rα2 expressed in 83% of tumors (115). In an open-label, single-institution, single-arm phase I clinical trial, newly diagnosed patients with GBM who previously received a conventional treatment prior to vaccination had promising responses to ICT-107 (116). The trial achieved its primary endpoint of immunogenicity. The vaccine was well tolerated and efficacious with a median overall survival of 38.4 months in newly diagnosed patients with GBM (116). The aforementioned study was followed by a randomized, double blinded, placebo-controlled phase II trial (NCT01280552). Similarly, the vaccine was well tolerated, however, there was no significant difference in the overall survival when compared with the control group (115). DC vaccines thus offer a favorable immunotherapeutic strategy to treat patients with GBM.

The concept of personalized vaccination in glioblastoma gained interest after two phase I clinical trials that used multi-epitope-based personalized vaccine to target neoantigens only (NCT02287428) (117) or both neoantigens and unmutated tumor-specific antigens (NCT02149225) (118). Keskin et al (117) in 2019 compared whole-exome sequencing data from the surgically removed glioblastoma and matched normal cells in order to discover neoantigens. By identifying the coding mutations for each patient, they chose a pool of 7–20 peptides comprising actionable neoepitopes anticipated to bind to the HLA class I molecules with high affinity. Results reported neoantigen-specific CD4⁺ and CD8⁺ T cell responses migrating into an intracranial glioblastoma tumor with increased number of infiltrating T cells (117). On the other hand, and in order to maximize the amount of actionable epitopes, Hilf et al (118) in 2019 targeted unmutated tumor-specific antigens in addition to neoantigens. For each patient, unmutated antigens were chosen from a common pool of HLA-bound peptides that were specific to glioblastoma and based on unique HLA immunopeptidome data and pre-vaccination T cell reactivity of patients. Vaccination plan involved an actively personalized vaccine of 9 unmutated peptides (APVAC1) followed by a 20-peptide pool that targeted neoantigens (APVAC2). Researchers reported results similar to those aforementioned by Keskin et al, with 29 month median OS (118). Another phase II clinical trial is testing personalized cancer vaccine AV-GBM-1 (developed by AIVITA Biomedical Inc.) based on autologous DCs full of autologous tumor neoantigens (NCT03400917). These neoantigens are derived from tumor-initiating cells following standard surgical resection. The aforementioned study reported significantly improved progression-free survival (PFS) in 57 participants (119). These studies along with others demonstrated the positive effect of immunization using vaccines in patients with GBM.

Cytokine therapy is considered an important immunotherapeutic approach to activate the immune system of cancer patients, recognize cancer cells and destroy them (120). As molecular messengers of the innate and adaptive immunity, these regulators enable cells of the immune system to communicate over short distances in paracrine and autocrine manners (120). To date, only two cytokines are approved by the FDA to treat selected malignancies (121). Interleukin 2 (IL-2) is approved for metastatic renal cell carcinoma and metastatic melanoma treatment, whereas Interferon-α (IFN-α) is approved to treat hairy cell leukemia, follicular melanoma, non-Hodgkin lymphoma and AIDS-related Kaposi's sarcoma (122). Cytokine therapies stimulate the function, survival and proliferation of NK and T cells that mediate immune responses against tumors. Despite their antitumor activity in murine models and clinical treatment of selected human cancers, several issues are associated with this therapeutic approach (120,123). These limitations include the short half-life of most cytokines in vivo, narrow therapeutic windows, severe toxicity at therapeutic doses and lack of efficacy (122,123). While certain cytokines support the growth and invasiveness of glioma cells, others may decrease or inhibit their growth (124). Among the cytokines studied, IL-2, 12, 15 and 21, as well as type 1 interferons, IFN-γ and granulocyte macrophage colony stimulating factor affect various populations of the immune cells with antitumor properties (125). In GBM, cytokines such as transforming growth factor-β (TGF-β), IFN-α, IFN-γ and IL-12 have an antitumor activity decreasing the size of the tumor, inhibiting GBM cell growth and tumorigenesis at the early stages (124).

In a multicenter phase I dose escalation clinical trial (NCT02026271), 31 patients with recurrent high grade glioma were treated with Ad-RTS-hIL-12, an inducible adenoviral vector that encodes the hIL-12 p70 transgene put under the control of the RTS gene switch (126). The oral administration of 20 mg of the hIL-12 activator veledimex (VDX) showed an improved safety and tolerability profile, less severe cytokine severe syndrome, increased but tolerable IFN-γ and tumor infiltrating lymphocyte generation and improved median overall survival of 12.7 months (126). The efficacy and safety of a single tumoral injection of Ad-RTS-hIL-12 with VDX in combination with the FDA-approved antibody cemiplimab completed its phase II trial targeting patients with recurrent or progressive GBM (NCT04006119). Preliminary data revealed a tolerated outcome of the combined treatment with elevated levels of serum cytokine and a significant increase in circulating cytotoxic T cells (127).

The immunomodulating effects of interferons offer another possible therapeutic potential in treating malignant gliomas. IFN-γ, for instance, has an array of antitumor properties such as inducing programmed cell death, inhibiting glioma proliferation and angiogenesis, and enhancing tumor immunogenicity (128). Thus, researchers consider it as an immunotherapeutic option. Furthermore, a phase I open-label trial evaluated the combination of INF-β and conventional therapy with TMZ. Out of the 23 enrolled patients, 16 were newly diagnosed with high-grade glioma and 7 with recurrent high-grade glioma. Newly diagnosed patients received RT, intravenous INF-β and TMZ, and had a median overall survival time of 17.1 months. The study outcome emphasized that the combination therapy caused minimal toxicity (129).

The role of multiple cytokines remians under study in regards to their clinical effectiveness to treat gliomas. The safest and most effective dosages of the cytokines are being delineated with some considered as a promising adjunct to other therapies (130).

Adoptive cell therapy (ACT). ACT is an immunotherapeutic approach that developed rapidly in the recent years and is considered a promising and effective anticancer treatment. This treatment method involves the isolation of autologous T lymphocytes with antitumor activity of a cancer patient, ex vivo expansion, and the adoptive transfer of the amplified tumor-resident or engineered T cells back to patients (131,132). There are three categories of ACT used to modulate the immune system and target cancer cells: i) ACT with tumor infiltrating lymphocytes (TIL), ii) ACT with genetically modified peripheral blood T cells such as T cell receptor (TCR), and iii) chimeric antigen receptor (CAR) gene therapies (133). TIL are genetically unmodified killer lymphocytes that infiltrate the TME (134). These cells, however, can lose their tumor eliminating ability due to immunosuppressive factors of the TME. Accordingly, the number of specific TIL should be abundant to enhance the efficacy of TIL therapy before intravenous adoptive transfer into the patient (134). T cells equipped with a TCR can recognize peptides presented on MHC molecules and target cancer cells. Nevertheless, and to overcome the limitation of antigen presentation, CAR-modified T cells recognize various types of antigens regardless of their presentation on MHC molecules (135). The aforementioned categories have different mechanisms of action to eliminate cancer cells.

Among the three aforementioned ACT approaches, CAR-T cell gene therapy gained ample interest in eradicating cancer cells. These modified T cells can recognize and bind surface expressed antigens through the single-chain variable fragment recognition domain. After recognition, T cells mediate tumor elimination via three different mechanisms: the perforin and granzyme axis, cytokine secretion and Fas and Fas ligand axis (136). Currently, 24 clinical trials are studying the effectiveness of CAR-T cell therapy on GBM (Accessed through Clinicaltrials.gov on July 1, 2022). Amongst these, one is in the early phase I study, 21 in phase I study and 2 studies in phase II clinical trial. Since its discovery, multiple CARs were developed to target GBM antigens including, but not limited to, IL13Rα2, EGFRvIII, HER2 and CD70 (137).

To assess the feasibility, safety, anti-glioblastoma activity and the persistence of T cells in patients, an open-label phase I dose-escalation study tested the activity of HER2-specific CAR-modified virus-specific T cells (VSTs) (NCT01109095). 17 HER2 positive patients with progressive recurrent GBM received the treatment. The infusion of VSTs was safe and well tolerated with no dose-limiting effects and the median overall survival was 24.5 months from diagnosis. It is worth mentioning that although T cells were present in the peripheral blood, HER2-CAR VSTs did not expand (138). Another clinical study utilized a retroviral vector containing the CAR that recognizes EGFRvIII tumor antigen (NCT01454596). A total of 18 patients with malignant gliomas expressing EGFRvIII received immunotherapy to evaluate the safety and feasibility of the T cells expressing anti-EGFRvIII CAR. One more study evaluated the clinical benefit of CAR-engineered autologous primary human CD8⁺ T lymphocytes against IL13 receptor α2 (IL-13Rα2) in 3 patients with recurrent GBM (NCT00730613). IL-13Rα2 is reported to be overexpressed in more than 50% of patients with GBM. In this first human pilot study, the intracranial administration of IL-13Rα2 specific CAR-T cells exhibited anti-glioma activity which was promising for CAR-T cell immunotherapy (139).

The outcomes of CAR-T cell immunotherapy is promising as it displays efficacy with minimal toxicity. Nonetheless, limitations such as tumor heterogeneity and the heterogonous expression of antigens as well as the function of T lymphocytes at the sites of the tumor render it difficult to eradicate the tumor (137).

Using monoclonal antibodies, researchers are able to target cancer cells by utilizing immune checkpoint inhibitors. These monoclonal antibodies are involved mainly to free T lymphocytes from the negative regulation of several immune checkpoint proteins, which are in turn involved in helping cancer cells evade the immune system (140,141). Cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1) are highly studied targets for inhibition (104). CTLA-4 and PD-1 are proteins expressed on cytotoxic T cells. The former interacts with cluster differential 80 (CD80) and induces T cell immunosuppression. Cluster differential 28 (CD28) protein, however, can also interact with CD80 and induce T cell activation. Similar to CTLA-4, once PD-1 binds to PD-L1, a protein expressed on tumor cells and APCs, it negatively regulates the T cell activation (142). Accordingly, and to surpass the negative regulation, immune checkpoint inhibitors are developed. The FDA approved several immune checkpoint inhibitor antibodies for several cancer types including, but not limited to, melanoma, renal cell carcinoma, NSCLC, small cell lung cancer and head and neck squamous cell cancer (143). Ipilimumab is used to target CTLA-4, pembrolizumab, nivolumab and cemiplimab for PD-1, and atezolizumab, avelumab and durvalumab for PD-L1 (143).

In patients with GBM, immune checkpoint inhibitors have so far demonstrated limited activity in regard to efficacy, safety and toxicity. In a randomized, open-label multi-institution pilot study, the immunotherapeutic activity of pembrolizumab, an anti-PD-1 monoclonal antibody, was examined on 35 patients with recurrent surgically respectable GBM (NCT02337686). The aforementioned study included 16 patients for neoadjuvant therapy and 19 in the adjuvant group to receive only the monoclonal antibody. Overall, pembrolizumab was well tolerated in the neoadjuvant group with improved overall and progression-free survival. The neoadjuvant arm had a median overall survival of 13.7 months, whereas the adjuvant arm had 7.5 months of median overall survival. Notably, PD-1 blockade alters the gene expression profile of the patients with a transcriptional increase in INF-γ genes, increased T cell expression and enhanced T cell clonal expansion (144).

Following the evaluation of nivolumab safety and tolerability in phase I cohorts of CheckMate 143 with recurrent GBM (145), a phase III clinical trial was conducted. In this open-label, multicenter, randomized phase III trial, the PD-1 blocking monoclonal antibody, nivolumab, was compared with bevacizumab to determine its survival benefit. A total of 369 patients were randomized to receive nivolumab or bevacizumab after standard radiation and TMZ therapy. The CheckMate 143 phase III clinical trial (NCT02017717) did not meet its primary endpoint. The median overall survival was almost similar with nivolumab and bevacizumab, 9.8 months vs. 10.0 months respectively. Likewise, the median progression free survival and treatment-related grade 3/4 adverse events were similar among both groups (146).

The activity of a CTLA-4 blocker, ipilimumab, was investigated alone or in combination with other immune checkpoint inhibitors such as nivolumab. In NCT02311920 phase I trial, patients with newly diagnosed GBM received ipilimumab, nivolumab, or the combination after standard chemoradiotherapy with adjuvant TMZ. The aforementioned study reported that the treatments were safe and tolerated, with no grade 5 adverse events (147). In patients with recurrent GBM, a recent study showed that the intratumoral and intracavitary administration of ipilimumab and nivolumab combination is under phase I trial (NCT03233152) with median overall survival of 71 weeks (148).

Glioma cells have often different mechanisms to escape immune surveillance. This is achieved by activating immune checkpoint ligands. Accordingly, ligand inhibition is considered a promising immunotherapeutic approach to treating GBM (137).

The enhanced understanding of the GBM biology discussed, was in part, possible due to an expansion of murine preclinical GBM models. Current models include glioblastoma cell-line xenografts, patient-derived xenografts (PDX) and genetically engineered mouse (GEM) models [reviewed in (160)]. PDX are anticipated to serve as effective preclinical models in translational research, compared with glioblastoma cell-line xenografts, since they retain the genetic and histological characteristics of the parent tumor. However, they do not entirely reflect the antitumor immunity of the host. On the other hand, GBM GEM models allow for the identification of specific genomic changes involved in tumor initiation and development and can provide information about the consequences brought on by certain mutations. GEM models can be used to examine how the microenvironment affects tumor biology and are thus helpful for evaluating therapeutic plans as well (160,161). Although mice have historically been the best organism for simulating the genetic and physiological characteristics of cancer, its translational potential has been constrained by the substantial differences between mice and humans. Therefore, and in order to bridge the translational gap between murine animal studies and human clinical trials, investigations in large-animal models of several cancer types have evolved. It is reasoned that anatomy and physiology of large animals has an improved applicability to human disease and could thus reliably recapitulate human GBM. For instance, GBM can develop spontaneously in canines, however, this event is relatively rare and it is thus difficult to reproduce experiments (162,163). On the other hand, several reproducible models of glioma have been successfully developed in pigs, evolving as the most promising preclinical large-animal models (164–167). Xenograft and genetically engineered porcine models are thus being studied as prospective research tools as a promising transitional step between murine models and human clinical trials (163).

Drug resistance and normal tissue damage are not the only factors limiting GBM cancer treatment. The BBB and the plasticity and heterogeneity of both GBM and TME are challenges hindering successful GBM treatment. In GBM, the integrity and function of the BBB is impaired resulting in a resistant barrier known as Blood Brain Tumor Barrier (BBTB). BBTB is characterized by overexpression of efflux transporters thus restricting drug delivery to the brain (168–171). Additionally, the multiple cellular states and the existence of ‘cancer stem cell likes’ add to the complexity of the heterogeneous cancer cell populations within the same tumor (172,173). To overcome these challenges, the genetic, epigenetic and molecular profile of each patient should be evaluated.

In conclusion, despite its adverse side effects, chemotherapy remains the first line therapy for cancer, solely or in combination with RT and surgery. However, late sequelae of these modalities are of a big concern particularly in regard of young cancer patients who are expected to live longer. Thus, improved survival health and quality of life for cancer survivors are a public health priority. Improved understanding of the molecular aspects of a tumor initiated the emergence of biology-driven therapeutic means and made personalized-cancer therapy possible. Doctors can customize cancer treatment to a tumor of a patient thanks to personalized or precision medicine. With the use of precision medicine, clinicians can select the treatment that is most likely to be effective based on the exact genetics of that patient's particular cancer rather than using a ‘passe-partout’ strategy. Precision medicine is beginning to be utilized more frequently to treat cancer patients, thanks to developments resulting in quicker and less expensive gene sequencing. It is also advised that physicians examine the structure of BBB to evaluate the ability of a drug to cross the BBB before enrolling patients in any clinical trials. Along with advanced technologies in conventional methods, immunotherapy and targeted therapy provide a hope for improved survival and quality of life for the patients. Finally, the effectiveness of the GBM treatment may be improved by using a combination therapy approach that targets both tumor cells and cells in the surrounding TME. While healthcare is a huge market, it is worth to note that changing the routine of cancer therapy is not easy. It is therefore more challenging now for an oncologist to navigate through different therapeutic options (both single and combinatorial) to select the optimal treatment whilst ethics are to be respected.