The history of malignant glioma treatment has taught us that the existing combined clinical regimen of surgery and radiotherapy alone cannot yet achieve a cure, although it has improved the quality and/or prolonged the survival of patients to some extent. Biological therapies, including gene therapy, immunotherapy and molecular targeted therapies, have been highly anticipated in the treatment of malignant gliomas, and some have been clearly effective in preclinical studies and even proven safe in phase I clinical trials, but have rarely made it through phase II and phase III clinical trials. This contrast prompted us to analyze the possible reasons for this, with the aim of providing clues to improve the biotherapeutic strategy for malignant glioma.
I. Analysis of gene therapy strategies for malignant glioma
Suicide gene therapy, tumor lysing virus therapy and immunomodulatory therapy are the most common gene therapy strategies for malignant glioma, and we will first analyze their advantages and remaining limitations.
Suicide gene therapy is a drug-activated gene expression to prevent DNA replication in glioma cells. Herpes simplex virus thymidine kinase gene combined with propoxyphene (herpessimplexvirusthymidinekinase/gancyclovir,HSVtk/GCV) and cytosine deaminase/5-fluorocytosine ( cytosinedeaminase/5-fluorocytosine,CD/5-FC) are the most frequently used suicide gene therapy systems [1]. The advantages of suicide gene therapy are that it not only kills tumor cells transduced with the suicide gene, but also the surrounding tumor cells that are not transduced, i.e., the “bystander effect”; only short-term expression of the suicide gene is required; these therapeutic systems preferentially affect proliferating cells and are thus targeted to tumor cells; suicide gene therapy also has the advantage of being a very effective treatment for tumor cells. Gene therapy can also synergize with conventional radiotherapy and enhance sensitivity to conventional therapy. Limitations include the lack of satisfactory gene transfer vectors that limit the efficiency of transfection of tumor cells in vivo, the limited spatial distribution of tumor cells positive for suicide gene transfer, and the inability to track tumor cells that have migrated and dispersed distantly.
The basic idea of tumorolytic virus therapy is to genetically modify the viral genome so that the virus replicates only selectively in glioma cells to achieve a killing effect on tumor cells, and tumorolytic herpes simplex viruses (OncolyticHSVs, OHSVs), tumor-selective replication adenoviruses ( Conditionallyreplicationadenovirus (CRAds), measles virus, and eutherian virus are representative tumorolytic viruses. The advantages of tumor lysing virus therapy are high viral titer, high transfection efficiency to tumor cells, and good distribution within the tumor; additional therapeutic effects can be produced by inserting other therapeutic genes into the tumor lysing virus genome. Limitations include the possibility of immune rejection of the virus by the host; the replicative ability of tumor lysing viruses and potential safety concerns; and the need for local administration of tumor lysing viruses during surgery.
The fundamental aim of immunomodulatory gene therapy is to induce an anti-tumor immune response to kill malignant glioma cells. Cytokine mediation, immune cell recruitment and application of antibody-directed cell carriers are the most basic strategies. The advantage of immunomodulatory gene therapy is that it kills residual tumor cells after surgery through passive or active anti-tumor immunity and also regulates the tumor microenvironment. Limitations include: tumor may induce immunosuppression; lack of dendritic cells with antigen-presenting effects in brain tissue; and secretion of cytokines with immunosuppressive effects by immunosuppressive regulatory T cells.
The following strategies may help to overcome and improve the limitations of gene therapy for malignant glioma.
(i) selection of stem cells as cell carriers for gene therapy: stem cells are tropic for tumors, and using stem cells to carry therapeutic genes may allow better spatial distribution of therapeutic genes and may even track to distantly disseminated glioma lesions [2,3], and stem cell carriers may be immune from clearance by the host immune system and may also act as shields for the therapeutic genes they carry.
②In terms of the development of novel tumorolytic viruses, one is to improve the transmission and transduction effect of tumorolytic viral vectors, and the other is to achieve new tumorolytic therapeutic strategies through genetic modifications
In the development of new tumor lysis viruses, we should improve the transmission and transduction of tumor lysis viral vectors and realize new tumor lysis therapeutic strategies through genetic modification, such as expression of tumor necrosis factor α (TNFα), vascular endothelial growth factor (VEGF) specific short hairpin RNA (shorthairpin RNA, shRNA), interleukin 4 (interleukin4, IL), etc. interleukin4,IL-4) [4], and thirdly, the development of novel tumorolytic viruses that specifically target glioma cells, especially glioma stem cells.
(iii) Modulation of the tumor microenvironment and suppression of the host anti-tumor immune response through vaccines for immune gene therapy.
(iv) Synthetic vectors such as liposomes and nanoparticles combined with convection-enhanced delivery to optimize the delivery of therapeutic gene vectors [5].
II. Analysis of immunotherapeutic strategies for malignant glioma
There are three main challenges in immunotherapy of malignant glioma, including immune-editing, reduced antigen delivery and reduced immune cell activation [6]. Immune editing consists of three phases, namely “clearance”, “homeostasis” and “escape”. “Clearance” includes both acquired and natural immune functions against tumors. If the clearance process is complete, the tumor cells are completely eliminated and the immune editing process is finished. If some tumor cells escape “clearance” due to mutation, their relationship with the immune system enters a state of “equilibrium”. At this point, tumor cells are not easily recognized and removed by the immune system because their antigenicity is diminished, but they are under constant pressure to remove and not overgrow. Acquired immunity is the main mechanism to maintain this equilibrium state, and it is generally believed that natural immune mechanisms are not involved in this process. “Escape” is when tumor cells escape immune surveillance and become resistant to antitumor immunity, usually due to genomic instability or downregulation of expression of key antigens. Immune editing has been demonstrated in the treatment of high-grade gliomas, for example, in dendritic cell vaccine therapy against the epidermalgrowthfactorreceptorvarianttypeIII (EGFRvIII) antigen, 82% of patients with recurrent tumors showed deletion of EGFRvIII expression [7 ]. Immunosuppression of the glioma microenvironment leads to reduced antigen recognition and depressed immune cell activation; macrophages and microglia also have a reduced potential for antigen delivery. In vitro experiments revealed that monocytes lost phagocytic activity after exposure to glioma cells; data from in vitro studies also showed that the activity of MHCII-like molecules was significantly reduced in microglia and phagocytes isolated from glioma tissue compared to normal brain tissue [8]. Reduced immune cell activity also contributes to glioma-associated immunosuppression. CD4+ cells isolated from both glioma tissue and peripheral blood of patients exhibited suppression of cellular function, proliferative activity, and interleukin 2 (IL-2) synthesis capacity. Although some studies have found that an increase in CD8+ infiltrating lymphocytes is associated with prolonged patient survival, some studies have reported that most tumor-infiltrating CD8+ cells are not active [9]. The expression of immunosuppressive molecules and the release of immunosuppressive cytokines are similarly associated with reduced immune cell activity.
The way to address the challenges of immunotherapy in malignant glioma is also to enhance antigen delivery, effectively break tumor-induced immune tolerance, and increase the activation of tumor-specific killer cells. While enhancing immune response is an effective tool, it must take into account the range of serious adverse events leading to secondary brain edema and autoimmune disease. Dendritic cell bioimmunotherapy, by enhancing the ability of antigen-presenting cells to develop an effective and durable anti-tumor T-cell response, has clinical applications. However, further methodological maturation is still needed regarding how to culture and obtain potent and activated dendritic cells. Although animal experiments and clinical studies have shown that intrajunctional injection is the most effective route for dendritic cell biotherapy [10], the best way to use dendritic cells for the treatment of brain tumors has yet to be established. However, for the immune status of glioma patients, vaccine therapy appears to be a passive “brake” that limits the response of the immune system to the vaccine. Depletion of regulatory T cells may be one strategy to avoid this “braking” [11].
The aggressive growth of high-grade gliomas leads to a high risk of tumor recurrence, and promising therapeutic measures must have the ability to hunt down and kill tumor cells that remain after surgery and adjuvant therapy; T cell-mediated antitumor immunity specifically tracks and kills disseminated tumor cells without accidentally injuring normal tissue [12]. However, the cells that generate effective anti-tumor T-cell immune responses in the CNS are different from elsewhere, and the exact class of cells responsible for antigen presentation and its mechanisms in gliomas still needs to be investigated, and specific tumor antigens have not yet been identified. Future research should focus on identifying specific antigens and specific immune mechanisms in different types of gliomas, establishing reliable immunotherapeutic pathways and methods for assessing immune responses, and improving and refining the design of clinical trials for immunotherapy.
III. Analysis of molecularly targeted therapeutic strategies for malignant glioma
Research on signal transduction pathways such as tumorigenesis and development, proliferation and apoptosis, angiogenesis, and invasion and migration has promoted the development of targeted drugs. Broadly speaking, tumor-targeted drugs are divided into monoclonal antibody (monoclonal antibodies) class drugs and small molecule drugs. Therapeutic monoclonal antibodies target transmembrane receptors or extracellular growth factors on the cell surface, and can also bind to radionuclides or toxins to exert specific guidance. Small molecule drugs can enter the cell to interact with the target molecule and interfere with the activity of the target protease. Unfortunately, the vast majority of molecularly targeted drugs have little efficacy in malignant gliomas. To date, only bevacizumab (bevacizumab) and cilengitide (cilengitide) have entered phase III clinical trials in malignant glioma [13]. Bevacizumab, a targeted anti-angiogenic agent, has been shown to prolong progression-free survival but not overall survival in patients with glioblastoma, and has a tendency to promote invasive tumor cell migration [14]. The integrin inhibitor cilengitide, an anti-invasive targeted agent, did not provide additional survival benefit in patients with malignant glioma receiving combined radiotherapy [15].
Important constraints on the efficacy of targeted drugs in malignant glioma include the difficulty in determining the dose of targeted drug to be used, the ability of the drug to reach the central nervous system and the biological activity of the drug within the tumor. For conventional cytotoxic antitumor drugs, the optimal dose is determined as the maximum tolerated by the patient at which no significant side effects are induced in the patient. This criterion is clearly not appropriate to simply extend to targeted drugs that act on cellular signaling pathways. Data on targeted drug entry into brain tissue are generally derived from indirect evaluation methods, and direct determination of targeted drug concentrations in fresh glioma tissue has been done sparingly. In future clinical trials, it is possible to design a group of patients who will have their tumors resected, take targeted drugs preoperatively, and directly analyze the distribution and pharmacokinetics of drugs in the resected tumor tissue to determine the level of signaling pathway inhibition and the concentration of targeted drugs to be achieved.
It is also an important challenge that targeted therapy for malignant glioma cannot be “targeted” by identifying potential beneficiary patients in advance. In the future, emphasis should be placed on the collection and preservation of surgical or biopsy tumor samples to test for tumor markers and signaling pathway activity. When these patients are enrolled in phase I clinical trials of new targeted therapeutics, molecular profiles associated with individual responses to therapy can be analyzed in these patients’ tissue specimens; if molecular markers and experimental therapeutic responses are established in phase I clinical trials, the tissue sample pool can be reviewed to provide a more adequate subject population for subsequent phase II clinical trials.
The genetic instability and heterogeneity of malignant gliomas are prominent, and the role of relevant signal transduction pathways and their mutual regulatory mechanisms are not fully understood, which determine the selection of therapeutic targets and the process of drug development [16]. Combined multi-target inhibition is also an important idea for targeted drug development, and in addition to single drugs with multi-target inhibition, the combined application of multiple targeted drugs is also an important attempt. However, even if the developed targeted drugs are tested in two-by-two combinations, the number of trials is very large, so preclinical pre-screening of drugs is very important. Whether targeted drugs can act at the expected target sites, whether they can effectively inhibit downstream signaling pathways, and what are the potential toxic effects are all related to the safety and efficacy of targeted therapy, and these issues also need to be addressed in depth.
Although there are still many challenges and problems in the biologic treatment of malignant glioma, some of which are due to the limitations of current scientific and technological capabilities and the level of knowledge, it does not mean that there is nothing we can do for the biologic treatment of malignant glioma. There are still some basic principles that need to be adhered to when designing biologic therapies, and there is no doubt that preclinical studies need to be strengthened, and clinical trials need to be optimized and innovated in terms of homogeneous patient inclusion, dosing, route of administration, efficacy assessment, and individualized protocol implementation. We attempt to propose the following strategies for the common problems of malignant glioma involving the prospect of biologic therapy.
First, robust and rigorous preclinical biologic therapy studies require satisfactory animal models of glioma to facilitate a more reliable determination of the clinical translational value of a biologic therapy in preclinical experiments, but such animal models of glioma are currently lacking. The establishment of animal models that can mimic the tumor microenvironment, heterogeneity, growth pattern, histology and anti-tumor immune response of human glioma is better to determine the pharmacokinetics and pharmacodynamics of biologic therapies.
Second, biologic therapy for malignant gliomas will remain for a long time as a complement to conventional therapy, and will still need to be combined with surgery and radiotherapy. Since most biologic therapeutic measures only keep the tumor under control, at least at this stage, the combination with conventional therapy offers ethical advantages and better therapeutic outcomes than the application of biologic therapy alone. Gene therapy, immunotherapy and targeted therapy can cross over each other in the application of protocols to bring out their respective advantages and play a synergistic role in treatment, but the synergistic advantages among multiple biologic therapies require a scientific evaluation system
Third, for biologic therapy trials of malignant gliomas, patient enrollment criteria generally require a histologic diagnosis of grade III/IV glioma. In the inclusion of patients in future clinical trials, tissue staging criteria may need to be more detailed to exclude the presence of confounding prognostic factors due to generalized histological staging. Molecular typing of glioblastoma has been documented and includes four types: classic, mesenchymal, proto-neurological, and neurological, with different typing not showing the same response to conventional treatment and survival benefit [17]. This idea of molecular typing of malignant gliomas based on histologic typing is worthy of reference in biologic therapy research, which will not only lead to a more homogeneous and balanced inclusion and grouping of patients in biologic therapy clinical trials, but also help to find important molecular targets in each group in the future, and to develop appropriate therapeutic interventions and optimal targeted treatment options.
Fourth, it has long been found in the clinical management of malignant gliomas that patients with the same histological diagnosis of glioma differ greatly in their clinical course, prognosis, tumor treatment response and treatment tolerance, risk of recurrence, and long-term complications of treatment. The heterogeneity in human tumor biology and individualized genomic variation require more personalized tumor treatment regimens. Biological therapy for malignant gliomas should also focus on individualized biological information and be able to adjust biological treatment regimens accordingly in real time. Each individual tumor has a unique profile of variation in tumor molecular genetics (DNA, mRNA, microRNA) and epigenetics, in addition to the individual tumor pathological histological characteristics. A higher level strategy for the biologic treatment of malignant glioma is to develop targeted gene therapy, immunotherapy, and targeted therapies based on the molecular pathology of the patient to form a “cocktail” of drugs that work in combination. Even if we cannot fundamentally cure glioma in the future, we can still achieve survival with tumor or greatly extend the survival of patients with malignant glioma.
Fifth, if biologic therapeutic strategies are to be effective in malignant glioma, they must first cross the blood-brain barrier and act on the tumor entity at high concentrations, and biologic factors are not highly targeted, so we need to improve the delivery system of biologic drugs. Convection-enhanced delivery and controlled-release delivery systems are important strategies.
In conclusion, breakthroughs in the biologic treatment of malignant gliomas still require advances in basic research, improvements in biologic treatment techniques and protocols, and the development of diagnostic and therapeutic efforts around individualized protocols.