Emerging Nano‐Immunotherapeutic Approaches to Glioma

Glioma is a highly invasive and frequently occurring type of brain malignancy in the central nervous system. The prognosis is often poor for glioma patients, despite the substantial advances in diagnosis and therapeutic approaches. The breakthrough discoveries in oncoimmunology have led to innovative and efficacious immunotherapeutic strategies to treat or even cure cancer patients; however, the efficacy of immunotherapy to glioma is disappointing. The unsatisfied outcomes can be explained by local immune resistance, blood–brain barrier (BBB), spatially heterogenous and immunologically specialized glioma microenvironment, etc. The emergence of nanoengineered immunotherapeutics transforms the modalities in glioma immunotherapy under preclinical and clinical settings. Nanotechnology aids the immunotherapeutics crossing the BBB and flicks the switch of immunity locally and systematically for enhanced tumor‐infiltrating effector immune cells against glioma. Herein, the advancement of knowledge in healthy brain immunology and glioma‐associated local and systemic immunosuppression is summarized and highlighted. The clinical development of immunotherapeutic approaches is discussed, such as therapeutic glioma vaccines, dendritic cell vaccines, immune checkpoint blockade, adoptive cell therapy, and new immunotargets. Herein, nanotechnology‐enabled glioma immunotherapy in preclinical and clinical studies is clarified and perspectives on future possibilities of advancing nanoengineered immunotherapeutics to clinical reality for glioma treatment are provided.

Glioma is a highly invasive and frequently occurring type of brain malignancy in the central nervous system. The prognosis is often poor for glioma patients, despite the substantial advances in diagnosis and therapeutic approaches. The breakthrough discoveries in oncoimmunology have led to innovative and efficacious immunotherapeutic strategies to treat or even cure cancer patients; however, the efficacy of immunotherapy to glioma is disappointing. The unsatisfied outcomes can be explained by local immune resistance, blood-brain barrier (BBB), spatially heterogenous and immunologically specialized glioma microenvironment, etc. The emergence of nanoengineered immunotherapeutics transforms the modalities in glioma immunotherapy under preclinical and clinical settings. Nanotechnology aids the immunotherapeutics crossing the BBB and flicks the switch of immunity locally and systematically for enhanced tumorinfiltrating effector immune cells against glioma. Herein, the advancement of knowledge in healthy brain immunology and glioma-associated local and systemic immunosuppression is summarized and highlighted. The clinical development of immunotherapeutic approaches is discussed, such as therapeutic glioma vaccines, dendritic cell vaccines, immune checkpoint blockade, adoptive cell therapy, and new immunotargets. Herein, nanotechnology-enabled glioma immunotherapy in preclinical and clinical studies is clarified and perspectives on future possibilities of advancing nanoengineered immunotherapeutics to clinical reality for glioma treatment are provided.
immunologically distinct from the malignancies elsewhere in the body. [12] Beyond that, the accessibility of immune cells and stimulatory agents from peripheral immune system is constrained by the protection of BBB, which has altered the clinical outcomes of immunotherapeutics. The advancement in understanding the dynamic interplay between brain and peripheral immune system is crucial in developing effective immunotherapeutic interventions. [13,14] In 1921, Shirai first discovered that rat tumors transplanted in the brains of heterogeneous animals grew well, whereas the tumors inoculated in other parts of the body were rejected by the host immune system. [15] Murphy and Tansley observed similar results that the brain was well tolerated with the transplanted heterotopic tissues, escaping the sensitization by the immune system. [16] In 1948, Medawar suggested that the brain lacks lymphatic drainage, thus the grafted non-self-tissues do not trigger antigen-specific T-cell-immunity-mediated rejection. [17] Medawar first proposed the notion of "immune privilege" in CNS. For a very long time, the brain was considered as a completely separated organ from the immune system. Since the 1980s, [18] mounting evidence has revealed the active communications between the brain and the peripheral immune system. For instance, the antigenic substances [18] or T cells [19] were found flowing out from cerebrospinal fluid to cervical lymph nodes (LNs) by crossing cribriform plate and nasal submucosa. In 2015, Louveau et al. first discovered functional lymphatic vessels along the dural sinus. [20] These vessels enable the drainage of immune cells and fluid components to cervical LNs, governed by meningeal lymphatic vasculature. [20,21] This breakthrough discovery in the basic science of brain immunity has led to a new view that the brain is monitored and protected by the immune cells in the borders. This finding also helps to explain one detail observed in Shirais mouse study 100 years ago that rat tumor implanted close to the ventricles did not survive, in which adequate antigens were flushed out to evoke strong immune response against grafted tumors. [15] Since 2015, researchers have been exploring the detailed regions, functions, and characteristics of immune cells tightly intertwined with the brain. For example, Rustenhoven et al. recently discovered that CNS-derived antigens were recognized and processed by dural antigenpresenting cells (APCs), followed by antigen presentation to T cells near the dural sinus, emphasizing that the brain is always in a state of immune detection. [22,23] The attitude shifted from immunologically "privilege" to "unique" now is prevalent in the neuroimmunology research community. [12] The progressively in-depth understanding of cerebral immunity has consolidated the foundation of immunotherapy for glioma, though it remains incompletely understood how exactly the immune cells around the borders talk with the brain.
In parallel, a large body of evidence demonstrates that nanomaterial with at least 1D in the nanometer scale (1-100 nm) has transformed cancer immunotherapy, with numerous products in clinical trials. [24,25] In the context of glioma, nanomaterial aids immunotherapeutics in crossing the brain barriers, owing to the small size, liposolubility, biogenic features, and surface functionality. [26] Additionally, the engineered nanomaterials allow co-delivery of glioma antigens and vaccine adjuvants, [27] and targeted delivery of ICIs [28,29] or immunostimulators (such as agonists, [30] cytokines, [31] or immunogenic cell death [ICD] inducers [32] ). Some advanced nano-immunotherapeutic candidates (such as ClinicalTrials.gov Identifier: national clinical trial (NCT)04573140) are programmed for clinical investigation, opening new avenues for glioma immunotherapy.
Given the advances in the basic science of neuroimmunology and the use of nanomaterials in medicine, it is a thrilling time in the development of nano-immunotherapeutics to improve glioma treatment. Several excellent review articles provide timely overview of this field, primarily focusing on immunotherapy in glioblastoma treatment, [33] micro-and nano-strategies for immune modulation in glioblastoma, [34] brain immunity, [35] immunotherapy, [12] and nanomedicine. [36] This Review first briefly describes current understanding of immune microenvironment of glioma, then summarizes the clinical progress of immunotherapies and potential new immunotargets for glioma treatment. We highlight the preclinical and clinical development of nanotechnology-mediated glioma immunotherapy (Figure 1), by targeting delivery of therapeutic vaccines to lymphoid organs or immune modulators to glioma sites, or co-therapy. The molecular and cellular mechanisms by which the nanoengineering strategies mediate the immunity for enhanced therapeutic efficacy are discussed in detail. The review ends with our perspectives on accelerating the clinical translation of nanoimmunotherapeutics to glioma patients, such as probing the heterogeneous subsets of resident immune cells in brain and tracking the dynamic interplay between immune cells and the brain in the progression of glioma and therapeutic process with nano-immunotherapeutics. This review provides timely summaries of recent advances in the interdisciplinary field of nanotechnology, neuroimmunology, and oncoimmunology for improved glioma therapy.

Healthy Brain Immunology and Glioma-Associated Immunosuppression
It took decades long to understand that the brain is an immunologically specialized rather than privileged organ. [14] The brain structure and BBB limit the entry of foreign invaders and immune cells circulating in the peripheral system, and also create difficulties in identifying the lymphatic system that regulates the brain's immune response. With the advances in technologies, researchers have identified heterogenous populations of immune cells resident inside the brain or at the edges, which protect the healthy brain against infectious diseases ( Figure 2). [14] The immune cells are derived from the bone marrow niches in the skull and vertebrae. [37] The meninges that contain interconnected lymphatic vessels and venous sinuses lay the foundation for the communication between the immune cells and the brain. When the brain tissue is damaged, the danger signals enter the circulation of cerebrospinal fluid in the meninges, which can flow into the bone marrow compartments of the skull through the ossified channels. [38,39] The stem cells in the skull bone marrow then can sense and respond to the migrated signals and differentiate into specialized immune cells, which will return to the cerebrospinal fluids for brain protection. The signals will also enter the parasagittal dura through the superior sagittal sinus into the dural lymphatic vessels, [40] and gathered in the dural sinus, where the signals can be recognized by the circulating immune cells. [22] In addition to circulating myeloid cells, there are also resident and sinuses-derived immune cells in the brain, including substantial microglia, rich perivascular macrophages, and limited T cells. As the largest number of bone marrow cells resident in the CNS, the microglia play an important role in the development of the neuron, the recognition of the innate immune, and the repair of the brain tissue. Perivascular macrophages and CNS-associated macrophages that are derived from the same progenitor cell as microglia can enhance the function of BBB and prevent excessive inflammation. [41] Sinuses-derived T cells migrate into the meninges to monitor the immune clues, and then drain out via the lymphatic vessels. During the cycle, T cells not only perform the immune surveillance, but also promote the maturation of in situ microglia. [42] The diverse immune cells that are made in the bone marrow and surround the brain's margins are essential in protecting the healthy brain.
Glioma is a typical "cold tumor" that is characterized by a low frequency of tumor-infiltrating lymphocytes (TILs) and other effector immune cells. [43] Vaccination strategy hardly promotes Figure 2. A scheme shows current understanding of brain immunology. The brain has its own immune system with microglia resident in the brain and T cells and macrophages at the borders. The immune cells can traffic from sinus to cervical LNs through the functional lymphatic vessels on the back of brain. Created with BioRender.com. the number of infiltrated T cells in glioma and these effector T cells often display an exhausted status. [44] Understanding the detailed mechanism of glioma immunosuppression at molecular and cellular levels is the key to identify potential therapeutic targets for glioma immunotherapy. The suppressive glioma microenvironment is largely caused by the solid enclosure in which the response to inflammatory stimuli and induction of adaptive immunity are tightly regulated. This regulation involves heterogenous communications between glioma cells and the surroundings through direct contact and cytokines, chemokines, or extracellular vehicles. [45,46] For example, the stromal cells in the brain suppress the response to the inflammatory clues secreted from glioma cells via remarkable production of immunosuppressive cytokines, such as transforming growth factor β (TGF-β) and interleukin (IL)-10. [47,48] As the richest nucleated hematopoietic cells, myeloid cells are the main regulators in glioma immunosuppression. [49] Myeloid cells at an immature status often differentiate into dendritic cells (DCs), macrophages, and granulocytes under normal conditions. However, some regulatory molecules under pathological conditions suppress the maturation of myeloid cells, resulting in the development of myeloid-derived suppressor cells (MDSCs) consisting of granulocytic and monocytic subsets. MDSCs block the activity of tumor-specific T cell and natural killer cells via many complex pathways that are dependent on gender. [50][51][52] For example, exosomal miR-1246 from glioma in human bodies promotes the differentiation and inhibitory role of monocytic MDSCs through the dual specificity phosphatase 3/extracellular signal-regulated kinase pathway, particularly under anoxic conditions. [53] Along with MDSCs, tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and neutrophils are recruited around the tumor. [54] TAMs are abundant immune cells, accounting for 30-50% of stromal cells. [55,56] In the process, the expression of genes involved in sensing tumor cells, host defense, and tumor killing is downregulated in microglia, while the expression of genes promoting tumor proliferation is upregulated. [57] T cells are the key effector cells that mediate tumor cell lysis by secreting cytotoxic interferon (IFN)-γ, tumor necrosis factor-α (TNF-α), granzymes, and perforin, while the frequency of infiltrated effector T cells is limited and their cytotoxic function is often impaired in the glioma environment. Generally, freshly generated T cells expressing sphingosine-1-phosphate receptor 1 (S1PR1) have strong affinities to sphingosine-1-phosphate in LNs, spleen, and blood, which drives the emigration of T cells from the thymus or bone marrow to blood circulation system and lymphoid organs. In glioma and other intracranial tumor models, it shows that naive T cells reduce the expression of S1PR1, and thus being sequestered in the bone marrow rather than traveling to the tumor sites. [58] In addition, glioma cells produce inhibitory molecules (e.g., TGF-β and IL-10), promoting the infiltration of Tregs that profoundly inhibit the activation of tumor-specific T cells. [59] Altogether, the heterogenous population of immune cells interacts with glioma cells and stromal cells, mediating the immune suppressive environment for glioma growth and resistance to the immune system sensing glioma. Diverse immunotherapies that aim to reverse the immunosuppressive pathways have been under intensive exploration for enhanced glioma treatment.

Therapeutic Glioma Vaccines
Therapeutic vaccination represents one promising type of cancer immunotherapies while it is still under clinical investigation. Cancer vaccines usually consist of selected tumor antigens and adjuvants that boost the activation and antigen presentation of APCs, mainly DCs, to evoke antigen-specific immune response against existing tumor cells. [60] Tumor antigens are often classified into two categories: 1) tumor-specific antigens (TSAs) that are only expressed on tumor cells and completely absent in healthy cells, including neoantigens from nonsynonymous mutations and viral antigens; 2) tumor-associated antigens (TAAs) that are found at upregulated levels on tumor cells while still expressed at a very low level on normal cells, including developmental-specific antigens and tissue-specific antigens. [61] Virus-derived antigens are detected in more than 80% of glioblastoma, representing an attractive antigen candidate.
Post administration of exogenous cancer vaccines, the adjuvant prompts DC maturation. The antigens are uptaken, processed, and then presented by mature DCs on major histocompatibility complex (MHC) class I and MHC class II molecules ( Figure 3). Antigen-carrying DCs migrate into draining LNs (dLNs) to prime naïve T cells via the recognition of T-cell receptors (TCRs) and co-stimulatory receptors. The migratory DCs can transfer the acquired antigens from the periphery to LN-resident DCs for T-cell activation. Tumor cells process endogenous antigen proteins by ubiquitination and proteasome degradation process, and cleavage by peptidases in the cytoplasm and endoplasmic reticulum. The derived epitopes are restrictedly presented on tumor MHC class I molecules, which can be recognized by vaccination-induced antigen-specific effector T cells via MHC class I TCR engagement. The activated cytotoxic T lymphocytes (CTLs) directly kill the tumor cells, while T-helper 1 (Th1) cells are indispensable for the survival, proliferation, and effector function of CTLs and generation of memory CTLs.
Identification and selection of immunogenetic glioma antigens are crucial determinants for the success of cancer vaccines. Several types of vaccine antigens have been extensively explored under clinical studies for the treatment of glioma. For example, Wilms tumor gene1 (WT1) has been well identified as TAAs for gliomas as well as other hematological and solid malignancies, and the clinical studies suggested that WT1 peptide vaccines were capable of inducing immune response against antigens. [62] In a phase I clinical study, patients with recurrent malignant glioma were immunized with both killer human leukocyte antigen (HLA) class I epitopes and helper HLA class II episodes (UMIN000003506), showing enhanced induction of CTLs. [63,64] Another multipeptide vaccine named IMA950 was formulated with nine HLA class I TAAs, two HLA class II restricted peptides, and an adjuvant of granulocyte macrophage colony-stimulating factor (GM-CSF) (NCT01222221) [65][66][67] or polyinosinicpolycytidylic acid stabilized with polylysine and carboxymethylcellulose (NCT01920191). [68] Glioma patients immunized with IMA950 showed excellent tolerance and CD8 þ T-cell immune response. [67,68] Compared with TAAs, TSAs possess several advantages, such as high immunogenicity, strong specificity, and affinity to T cells, low possibility of causing systemic inflammation. Epidermal growth factor receptor variant III (EGFRvIII) is one specific mutant protein-based TSA at advanced clinical development. EGFRvIII is found in 30% of glioma patients, which can continuously activate the rat sarcoma and the phosphoinositide 3-kinase pathway for abnormal cell growth. Vaccines against EGFRvIII are one prospective approach for glioma treatment. For example, a phase 3 randomized trial (NCT01480479) revealed that peptide vaccine rindopepimut against EGFRvIII combined with chemotherapy drug temozolomide (TMZ) improved the survival of patients with newly diagnosed glioblastoma, though rindopepimut alone did not prolong survival in patients. [69,70] Another phase II study (NCT01498328) with a small sample size demonstrated that co-therapy of rindopepimut vaccine and bevacizumab that is a therapeutic monoclonal antibody (mAb) inhibiting angiogenesis showed prolonged survival, which was associated with vaccination-mediated anti-EGFRvIII antibodies. [71] Isocitrate dehydrogenase (IDH) mutations are involved in tumor metabolism of 70% grade II and III gliomas and 10% secondary glioblastoma, which are defined as promising therapeutic targets. IDH1 (R132H) contains neo-epitopes that can be presented on MHC class II molecules, thus IDH1 is an alternative TSA-based vaccine under clinical evaluation. Recent phase I clinical study of IDH1 vaccine showed immunogenicity and side effects but under grade 1 in R132H-mutated grade III and IV gliomas patients. [72] H3.3K27M recently was defined as another type of TSA that has entered clinical trials in patients with diffuse intrinsic pontine glioma (DIPG). [73,74] Timely identification of tumor-specific mutations in individual patients is critically important for generation of neoantigen vaccines. [75] It is a consensus that DC vaccines are a safe and reliable immunotherapeutic modality for glioma treatment, where the vaccines containing DCs loaded or pulsed with tumor antigens are refused back into the patients to initiate T cell activation. DC vaccines were first approved by Food and Drug Administration (FDA) for treating patients with prostate cancer in 2010. Since then, DC vaccines have entered numerous clinical studies to assess the effectiveness in treating other different tumors. Glioma lysates were often used in DC vaccines for glioma treatment. For example, Audencel that was a DC vaccine pulsed with glioma lysis elicited effective immune response in patients with newly diagnosed glioblastoma in a phase II randomized trial (NCT01213407), [76] though this vaccination did not benefit patients in the overall survival. [77] DC vaccines can be hampered by the glioma immunosuppression, thus the combination strategies of DC vaccines and ICIs may yield improved immunotherapeutic effect. DCVax-L is another autologous DC vaccine under clinical exploration for glioma treatment. Encouraged by the promising results in phase I/II trials, DCVax-L entered a phase III trial (NCT00045968) in 2007. The early results from this clinical trial revealed that DCVax-L was safe, [78] and very recent published report confirmed that the addition of DCVax-L to standard of care significantly prolonged the survival of glioblastoma patients. [79] DCVax-L holds immense potential for implementation in clinic. Beyond tumor cell lysates, DC vaccines can be developed by pulsed with stem cell lysates, which showed effective clinical immune response in glioma patients. [80,81] Cytomegalovirus phosphoprotein 65 (pp65) expressed in over 90% of glioblastoma has been used for the generation of DC vaccines, and a phase II clinical study (NCT02366728) observed that vaccination with cytomegalovirus-specific DC vaccine enabled nearly one-third of glioma patients to survive for an exceptional long time. [82]

ICIs
The effectiveness of therapeutic vaccination with glioma antigens is often compromised by the immunosuppressive immune cells or secreted inhibitory molecules in local microenvironment of glioma. The advances in oncoimmunology have revolutionized cancer immunotherapy, by identifying the inhibitory biomarkers expressed on tumor and/or immune cells during the progression of tumor development. These markers inhibit antitumor immune response, promoting tumor immune escape. Therapeutic mAbs-based ICIs are developed to block these immune inhibitory checkpoints, enabling effector immune cells to attack tumor cells. [83] The first ICI-targeting cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) can compete with co-stimulatory molecules CD28 on T cells to bind B7 ligands, thereby impairing T-cell activation. [84] It was in 1996 that researchers found that the administration of anti-CTLA-4 mAbs led to graft immune rejection in mice. [85] Several years later, an anti-CTLA-4 mAb named ipilimumab was approved by FDA for treating advanced melanoma with clear clinical benefits. [86] Soon after the successful clinical study, ICIs have been widely evaluated in clinical settings to treat patients with other tumors. [87] Programmed death 1 (PD-1) expressed on lymphocytes engages with programmed death-ligand 1 (PD-L1) and PD-L 2 on nonlymphoid tissues, dampening the activation of peripheral T cells mainly through src homology-2 domain-containing tyrosine phosphatase-2. [88] ICIs that obstruct PD-1/PD-L1 inhibitory pathways have been clinically proven to restore functional activity of T cells, thereby mediating antitumor immunity. FDA approved anti-PD-1 or PD-L1 mAbs (e.g., pembrolizumab, nivolumab, and atezolizumab), have demonstrated satisfactory clinical efficacy in renal cell carcinoma, melanoma, and non-small cell lung cancer, while still under clinical studies for glioma. Nivolumab did not show obvious benefits in the phase II and III studies in patients with recurrent and newly diagnosed glioblastoma, [89] which could be attributed to the alternation on the expression of PD-1 in gliomas, [90] and the restrictive effect of BBB. Co-therapy of different ICIs showed improved curative effects, while increased the incidence of immune-related adverse events. [91] To address this issue, a phase I clinical trial was established to study the efficacy and safety profile of intracerebral administration of ipilimumab and nivolumab at a low dose in the tumor resection cavity, [92] the clinical outcomes from which demonstrated that this strategy was well tolerated, and associated with encouraging overall survival and a low incidence of immune-related adverse events. The administration of pembrolizumab upregulated IFN-γ and T-cell-related gene expression, enhancing antitumor immune response locally and systematically, and promoting survival benefit of patients with recurrent glioblastoma. [93] Another phase I trial also proved that pembrolizumab monotherapy resulted in long-lasting antitumor activity, [94] suggesting that pembrolizumab might represent efficacious treatment modality for glioma patients in clinic.
Researchers have been devoted to exploring new immunotherapeutic targets to effectively treat patients with glioma. Some promising candidates are found in glioma environment, such as indoleamine 2,3-dioxygenase (IDO) 1, [95,96] T-cell immunoglobulin mucin receptor-3 and galectin-9. [97] An important consideration in ICI therapy is the glioma's structural and immunological characteristics. Nano-immunotherapeutics offer great potential as new therapeutic modality by circumventing the access restrictions to the brain and potentiating ICIs, which will be discussed in Section 4.

Immunostimulators Reversing Immunosuppression
In addition to blockade of inhibitory molecules in tumor microenvironment, activation of co-stimulatory receptors on the surface of immune cells, such as inducible T-cell co-stimulator, CD28 family, and TNF receptor superfamily, can enhance effector immune response against abnormal cancer cells. [98] Some signal pathways play a vital role in activating different subtypes of innate immune cells and initiating adaptive antitumor immune response, such as IL-2 receptor, toll-like receptor (TLR), nuclear factor kappa B, and cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathways. [99] Agonistic antibodies or small-molecule agonists targeting these pathways are emerging approaches to reverse tumor immune suppression, with some advanced candidates entering clinical trials for glioma immunotherapy.
Pattern recognition receptors family, such as TLRs, can sense and recognize pathogen associated molecular patterns. Cytosine-phosphodiester-guanine (CpG) oligonucleotide is a strong agonist sensed by TLR9 for therapeutic activation in cancer treatment. [100] Stimulation of TLR9 promotes the activation of DCs and B cells, inducing potent Th1 immune response against cancerous cells. A phase II clinical study evaluated the safety and therapeutic effect of locally administrated CpG after tumor removal in glioblastoma patients, [101] which did not prolong the survival of patients, though this immunotherapy was well tolerated. In contrast, a phase I clinical study (NCT02026271) of a product named veledimex (IL-12 encoded by an adenovirus) showed acceptable safety and encouraging overall survival in high-grade glioma patients. [102] The combination of veledimex and nivolumab was assessed in phase I clinical trial (NCT03636477), revealing that co-therapy was well tolerated and increased tumor IFN-γ. [103] These encouraging clinical outcomes led to further evaluation of co-therapy of veledimex and ICIs in a phase II clinical trial (NCT04006119).
cGAS is a sensor of double-stranded DNA, which catalyzes the production of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The formulation of complex of cGAS/STING activates STING expressed on the endoplasmic reticulum, producing type I IFNs (IFN-α/β). STING agonists are promising immunotherapeutic modalities for enhancing antitumor immunity in preclinical studies, [104] which potentially can be moved to clinical studies in the future.

Adoptive Cell Therapy
Adoptive cell therapy (ACT) is a form of immunotherapy by using autologous T lymphocytes from patients to eliminate cancerous cells. TILs are the first and most studied ACT, where TILs with antitumor activity were harvested from a patient's tumor biopsy, followed by proliferative expansion mediated by IL-2, and refusion back to patients. As early as the 1980s, autologous TILs showed the capability of regressing metastatic melanoma in patients. [105] A phase II study (NCT01585428) of TILs-based immunotherapy demonstrated impressive therapeutic effects (complete regression) for treating patients with metastatic cervical cancer. [106] Currently, immunotherapy using TILs showed clinical efficacy in a variety of tumors but not in glioma. [107] To broaden the implementation of ACT immunotherapy, genetically engineered T cells with antigen-specific receptors, including CARs and TCRs, have been developed to improve the identification and elimination of tumor cells. TCR-T cells recognize the complexes of tumor antigen peptides and MHC molecules for tumor killing. [108] At the beginning of 2022, FDA approved TCR therapeutic, namely Kimmtrak, containing tumor antigen gp100-targeted TCR and CD3-specific singlechain variable fragment for the treatment of melanoma. [109] Recently, a TCR therapeutic showed promising results in MHC-humanized glioma models, [110] which might pave the path to clinical trials. Different from TCR-T cells, CAR-T cells mediate antigen-targeting process independent of MHC molecules and co-stimulatory domains are inosculated for enhanced granzymes and cytokines release. [111] FDA has approved several CAR-T cell therapies for the treatment of hematological malignancies with curative effects, while current CAR-T-cell therapies showed limited efficacy in solid tumors including glioma. Early clinical results have confirmed the feasibility, safety, and certain effectiveness of CAR-T therapy in glioblastoma patients. [112] Recently, CAR-T cells modified with disialoganglioside binding domain exhibited clinical safety and benefits in three of four glioma patients. [113] However, the number of participants is small, and further clinical studies on a large population of patients are required to determine the therapeutic efficacy.

Oncolytic Virus Therapy
Despite the remarkable success of ICIs in clinic, small proportions of patients respond to ICIs at a rate of 10-30%. Oncolytic virus therapy represents a promising strategy to potentiate the patient response to ICIs. In oncolytic virus therapy, the genetically modified viruses with low toxicity can amplify immune response against cancers through multiple mechanisms, such as lysing tumor cells to release immunogenetic antigens, and altering tumor-associated ECs, inflaming tumor microenvironment by antiviral-machinery-mediated type I IFNs. [114] Oncolytic virus agents that were derived from human herpes simplex virus 1 (HSV-1) and genetically modified with GM-CSF cytokine were the first and only approved by FDA for treating melanoma patients in 2015. [115] These encouraging results fostered the clinical applications of HSV for the treatment of other tumors. For example, G207 and HSV-1716, HSV-1 variants, were clinically approved safe in glioblastoma patients under phase I and II studies. [116][117][118][119] Recently, a phase I clinical trial (NCT02457845) showed that intratumoral G207 alone or combined with radiation therapy reversed tumor immunosuppressive environment with an acceptable adverse-event profile in patients with pediatric high-grade glioma. [120] G47Δ (Delytact/Teserpaturev) is constructed by depletion of the a47 gene in G207, strengthening the oncolytic activity and immunogenicity of cancer cells. [121] The phase I clinical trial of G47Δ in patients with malignant and recurrent glioblastoma demonstrated that 12 of 13 patients survived for over 1 year, leading to the approval of oncolytic therapy for glioma treatment by the Ministry of Health, Labour and Welfare in 2021. The remarkable success represents a milestone in glioma immunotherapy (UMIN000002661 and UMIN000015995). [122,123] Along with HSV-1, oncolytic therapy using other types of viruses confirmed promising and safe clinical outcomes for glioma therapy. [124] DNX-2401 is an oncolytic adenovirus selectively replicating in tumors. The latest clinical trial (NCT03178032) results indicated that intratumoral administration of DNX-2401 combined with radiation therapy enhanced the effector function of T cells, leading to tumor shrinkage in DIPG patients. [125] Oncolytic virus therapy is an emerging option for treating malignant glioma. Oncolytic viruses preferentially replicate in tumor, remodeling the tumor microenvironment to trigger potent antitumor immune response. Most oncolytic viruses are administrated in patients by stereotactic injection, though some viruses perform well in BBB penetration. Potential systemic toxicity is regarded as one of the most important considerations.
The newly discovered immunotargets that are under ongoing clinical studies for glioma treatment are listed in Table 1.

Emerging Nanotechnology-Enhanced Glioma Immunotherapy
Current immunotherapeutic strategies introduced earlier often show disappointing potency in patients with glioma and unwanted side effects. Recent advances in nanotechnology-based platforms enable encapsulation, protection, and modulation of the action of immunotherapeutics, thereby overcoming physical obstacles of BBB and glioma resistance to immunotherapy. Precise tuning the chemophysical properties of nanoplatforms allows targeting to the specific tissues, subsets of cells, and intracellular compartments, avoiding systematic toxicities associated with administrated free immunodrugs. [34]

Nanoadjuvanted Vaccines for Glioma Treatment
Despite the great progress in therapeutic vaccines for cancer treatment, it remains a pressing challenge to selectively deliver vaccines to lymphoid organs, initiating profound antitumor immunity. For instance, Hailemichael et al. immunized mice with melanoma neoantigen peptides (gp100) delivered by incomplete Freund's adjuvant (IFA) that is often used in cancer vaccines under clinical studies. [126] The results revealed that the water-in-oil emulsion of IFA formed a depot at the skin, thus most tumor-antigen-specific T cells stayed in a dysfunctional state at vaccine injection sites, leading to poor tumor eradication. To avoid persistent vaccine-deposition-mediated T-cell accumulation and apoptosis at immunization sites, the authors attempted to vaccinate mice with aqueous peptide vaccines in the absence of IFA but failed to prime T cells in dLNs. Subcutaneous injection of free small-size peptide vaccines may cause rapid diffusion to peripheral blood vessels or immune tolerance in the absence of adjuvants. [127] Hence, researchers endeavored to develop vaccine delivery systems that can co-transport neoantigen peptides and adjuvants to dLNs. Given the small size, nanomaterial enables efficient drainage to dLNs by passively penetrating EC layers. [128] For example, synthetic high-density lipoprotein (sHDL) nanodiscs that closely mimic the features of natural lipoprotein are promising and representative candidates for vaccine delivery. [129] Kuai et al. reported that sHDLs (around 10.5 nm in diameter) enabled co-delivery of tumor antigen peptides and small-molecule adjuvant CpG in skin dLNs, thereby markedly enhancing CD8 þ T responses against mouse colon and melanoma cancer. [130] Recently, the research team demonstrated the potential of sHDL in delivering therapeutic peptide vaccines for glioma treatment. [27] sHDLs were constructed with 1,2-dimyristoyl-sn-glycero-3hosphocholine and 22-amino-acid ApoA1 mimetic peptide, where pyridyl disulfide-modified phospholipid was conjugated with sHDL for reduction-sensitive release of screened neoantigens (AALLNKYLA, MSLQFMTL, and GAIFNGFTL) covalently linked with cholesterol-modified CpG1826. [131] In a therapeutic regimen, GL261 glioma tumor-bearing mice immunized with the engineered nanovaccine showed significant induction of neoantigenspecific T cells in blood, and markedly regressed established tumor, particularly when combined anti-PD-L1 antibodies. Tumor microenvironment analysis displayed a substantial increase in tumor-infiltrating CD8 þ T and M1-like macrophages, which explained the potent vaccine-mediated antitumor immunity. Mice remained tumor free when rechallenged with GL261 glioma cells in the contralateral brain, indicating the successful establishment of long-lasting memory immune response. Nanomaterials represent a powerful delivery platform for engineering fascinating therapeutic vaccines against glioma.
Alternatively, tumor-derived exosomes that are 30-100 nm vesicles containing donor tumor-related proteins and nucleic acids were proved to be able to initiate CTLs superior to tumor lysates in DC vaccines. [132] For example, Liu et al. loaded DCs with C6 glioma cells-derived exosomes and α-galactosylceramide that is an adjuvant activating invariant natural killer T cells. [133] The developed DC vaccines induced augmented tumor-specific CTLs, reduced immunosuppressive cytokines and significantly improved mouse survival rate, compared to DC vaccines pulsed with tumor lysates. In addition, exosomes are easy to store and extracted, thus being an appropriate and potential DC vaccine platform for future clinical translation.

Nanotechnology-Delivered ICIs for Glioma Treatment
Along with therapeutic vaccination, ICIs have been extensively used in clinical trials for treating glioma by restoring T-cell cytotoxic function, such as mAbs against inhibitory receptors of CTLA-4, IDO, and PD-1/PD-L1. [10] Nevertheless, glioma patients are often resistant to ICI immunotherapy. [12] One of the key reasons is the existence of BBB limiting the penetration of immunotherapeutic mAbs into glioma sites, [134] thus diverse strategies have been explored to improve the intracerebral delivery efficiency. [135] One predominant approach is to conjugate the drugs with ligands that bind to the receptors expressed on ECs or glioma cells, triggering receptor-mediated transcytosis to cross the BBB. For instance, acetylcholine and choline analogues can interact with choline transporter that is over distributed on both ECs and glioma cells. [136,137] Recently, Wang et al. utilized 3-(bromomethyl)-4-methyl-2,5-furandione (MMfu) acting as a pH-sensitive linker to generate a polymer nanocapsule for delivery of anti-PD-L1 mAbs to glioma (Figure 4a). [28] The engineered nanocapsule displayed a diameter of around 45 nm (Figure 4b In addition to choline-transporter-mediated transcytosis, lowdensity lipoprotein receptor and transferrin receptor (TfR) are alternative mediators for enhanced BBB penetration. [29,138]  For instance, Galstyan et al. functionalized poly(β-L-malic acid) nanoparticles with anti-mouse TfR antibody, which enabled trans-BBB delivery of anti-PD-1 or anti-CTLA-4 mAbs for glioma immunotherapy. [29] The immune nanotherapeutics efficiently modulated glioblastoma environment with increased tumorinfiltrating effector CD8 þ T cells and natural killer cells while decreased immunosuppressive Tregs. Glioblastoma-bearing mice received nanotherapeutics demonstrated enhanced survival rate compared to those received free anti-PD-1 or anti-CTLA-4 mAbs. These preclinical studies have demonstrated that ICIs formulated in nanoplatforms that facilitate BBB cross are a talented strategy to potentially overcome the resistance of glioma to ICI immunotherapy.

Nano-Stimulators for Glioma Treatment
Relative to other malignancies, glioma microenvironment contains a low frequency of tumor-infiltrating T cells but abundant immunosuppressive cells, such as MDSCs, TAMs, and Tregs. [54] To reverse local immunosuppression and promote tumor infiltration of effector T cells, therapeutic immunostimulators that act on distinct complex signaling pathways have been assessed in preclinical animal models and clinical studies for enhanced glioma immunotherapy. However, these stimulators post administration tend to induce systemic inflammatory response, rapid blood clearance, and inefficient delivery to glioma sites, seriously hindering their clinical progress. The advanced nanomaterials Reproduced with permission. [28] Copyright 2022, American Chemical Society.
that can be engineered to precisely deliver the immunostimulators to the sites of interest hold immense potential for safe and efficacious glioma treatment.

Nano-Stimulators-Targeting TLR-Signaling Pathway
Free CpG molecules that target TLR9 are serum unstable caused by endonucleases, markedly decreasing CpG immunopotency.
In glioma, activation of CpG-TLR9 pathway was found to increase tumor invasiveness and maintain cancer stem cells. [139] To reduce pro-tumor effect while retaining immune stimulation capability, CpG molecules were noncovalently conjugated with single-walled carbon nanotubes (SWNT), which enabled eradication of established gliomas with a long-term tumor free in murine models. [140,141] Mechanistically, CpG/SWNT potentiated the production of proinflammatory cytokines by monocytes for enhanced antitumor immunity. Neural stem cells (NSCs) can traffic to hypoxic areas of glioma and other tumors, being explored for delivery of antitumor therapeutics in clinical trials. [142,143] For this reason, Adamus et al. loaded NSCs with CpG conjugated with antisense oligonucleotides that target signal transducer and activator of transcription (STAT)3, a key promoter of resistance to cancer treatment. [144] Following cellular uptake, NSCs secreted exosomes with CpG-STAT3 antisense oligonucleotide conjugates, which strongly activated glioma-associated microglia and showed potent therapeutic effect against orthotopic GL261 tumors. Recently, Wei et al. engineered apolipoprotein E (ApoE) peptide-functionalized polymersomes to co-deliver CpG and granzyme B, [145] in which ApoE promoted the penetration of BBB to glioma sites, granzyme B induced ICD, and CpG-activated DCs and triggered tumor-antigen-specific T-cell immunity. Glioma-bearing mice received the engineered nanoimmunotherapeutics evidently delayed tumor progression.
Beyond delivery function, some nanoparticles are natural stimulators activating TLRs, such as virus-like particles (VLPs, 20-500 nm) that present a 3D structure composed of viral capsid proteins in the absence of virus nucleic acids. [146] Kerstetter-Fogle et al. investigated the potency of plant VLPs from cowpea mosaic virus for glioma immunotherapy. [147] Following intracerebral administration of plant VLPs, tumor-infiltrating neutrophils, natural killer cells, and adaptive immune cells were profoundly recruited in glioma sites of mice, leading to significant tumor regression. Nano-immunotherapeutics that target TLR-signaling pathways offer a potent and safe strategy to enhance the potency of glioma immunotherapy.

Nano-Stimulators-Targeting STING-Signaling Pathway
As a cGAMP mimetic, SR-717 is a non-nucleotide STING agonist, displaying antitumor activity. [148] To explore the therapeutic potential of SR-717 in glioma treatment, Wang et al. genetically engineered human heavy-chain ferritin (HFn) and Arginine-glycine-aspartate (RGE) fusion proteins that can self-assemble into nanoparticles to deliver SR-717 to brain tumor (Figure 5a). [30] HFn can bind to TfR1 overexpressed on ECs of BBB, thereby efficiently crossing BBB via receptor-mediated transcytosis. RGE, a tumor-targeting peptide, enabled a superb glioma-homing ability of encapsulated SR-717 (Figure 5a). Following intravenous injection of the designed nanoparticles, this dual-targeting nanoparticle increased the accumulation of SR717 in tumors, promoted GL261 tumor remission, and prolonged mice survival (Figure 5b). Mechanistically, the engineered nanoparticles enhanced the activation of STING (Figure 5c) and infiltration of effector CD8 þ T cells in tumor (Figure 5d). Cyclic diguanylate monophosphate (cdGMP) is another STING agonist, which can activate APCs via boosting pro-inflammatory type I IFN secretion. [149] To reduce off-target-associated adverse effects of cdGMP, Bielecki et al. loaded cdGMP into inorganic mesoporous silica nanoparticles (60 nm). [150] The abundance of protonatable amine groups in mesoporous silica nanoparticles enabled endosomal escape of cdGMP and subsequent STING activation in the cytoplasm, promoting activation APCs and adaptive immunity against glioma. Relative to other types of cancers, the immunotherapeutic potency of STING agonists formed in nanoplatforms is less investigated in glioma treatment.

Nano-Stimulators-Targeting Other Signaling Pathways Associated with TAMs
Along with TLR and STING stimulants, alternative strategies are constructed to target-specific immunosuppressive factors or signaling pathways to reduce or reprogram immunosuppressive cells in glioma. Microglia and macrophages are the main immunosuppressive components in glioma (up to 30% of glioma mass). [151] Most TAMs are polarized from antineoplastic M1 type to tumor-promoting M2 type, [152] leading to rapid cancer cell proliferation. Therefore, diverse small molecules have been explored to target some crucial factors that regulate the polarization of TAMs for glioma immunotherapy. [153] For example, plantderived chlorogenic acid (CHA) is capable of reversing the polarization of TAMs by activating STAT1-signaling pathway while inhibiting STAT6 pathway. [154] Phase I clinical studies revealed that CHA was a safe and promising therapeutic molecule in cancer therapy. [155] However, its rapid clearance in the body required repeated intramuscular administration to maintain therapeutic effect, leading to poor patient compliance. In an attempt to address these issues, Ye et al. encapsulated CHA in mannosylated liposomes for glioma therapy (Figure 6a). [156] Mannosylated liposomes were inherently capable of targeting mannose receptors (also known as CD206) expressed on the surface of TAMs and shifting M2 phenotype to M1 phenotype against tumor (Figure 6a). [157][158][159] The mannosylated polyethylene glycol (PEG)-liposome substantially promoted its accumulation at tumor sites (Figure 6b), thereby potently inhibiting glioma growth (Figure 6c). The "depot effect" of PEGylated liposomes contributed to the infiltration of memory T cells which mediated long-term immunotherapeutic effect against glioma cells. [160] Nucleic-acid-based therapeutics are complementary approaches to edit, inhibit, or replace the transcription factors that regulate the development of TAMs. [161] To exert their functions in vivo, nucleic acids often require delivery platforms to protect them from degradation by nucleases and promote the expression of encoded protein molecules. For instance, Stephan and colleagues engineered poly(β-amino ester) polymers decorated with di-mannose-targeting moieties to deliver in vitro transcribed messenger RNA (mRNA) encoding IFN regulatory factor 5 and its upstream signal IKKβ (a phosphorylated kinase) that are transcription factors reprograming TAMs to M1 phenotype for glioma treatment. [162] This nano-mRNA platform exhibited therapeutic potency in multiple other mouse tumor models (ovarian cancer and melanoma) and excellent safety profile with repeated doses. In addition to mRNA immunotherapeutics, miRNAs, such as miR-155, are key regulatory molecules skew TAMs polarization into M1 phenotype. For this reason, Zhang and co-workers designed a virus-like nanogel to deliver miR-155 for glioma treatment (Figure 6d). [163] miR-155 molecules were encapsulated in the nanogel cross-linked by DNA-conjugated polymers, followed by coating with erythrocyte membranes that promoted cell penetration, and modification with M2pep and A2 peptides that targeted microglia and macrophages (denoted as Vir-Gel, Figure 6d). Post administration, the delivered miRNA downregulated the inflammatory suppressor of cytokine signaling 1 (SOCS1), thereby promoting the production of immunogenic cytokines. The secreted cytokines in turn reprogramed microglia and macrophages polarizing to M1-type macrophages against glioma cells. Vir-Gel therapy profoundly suppressed the growth of glioma and prolonged the survival time of mice (Figure 6e). The frequency of M2-type microglia and macrophages remarkedly decreased in glioma (Figure 6f ), which explained the improved antitumor activity. In another study, Figure 5. Genetically engineered human heavy-chain ferritin nanoparticles (HFn NPs) decorated with RGE and encapsulated with a stimulator of interferon genes (STING) agonist SR717 enable efficacious treatment of glioma. a) Schematic illustration of the preparation procedures and immunotherapy mechanism of SR717@RGE-HFn NPs. b) An experimental timeline, average tumor weight, and survival curves of mice intracranially inoculated with GL261 cells and received different treatments. c) The expression of p-STING, p-TBK1, and p-IRF3 was measured by western blot analysis. d) The percentages of infiltrating CD8 þ T cells in glioma tissues posttreatments as indicated. Reproduced with permission. [30] Copyright 2022, KeAi Publishing Ltd.
www.advancedsciencenews.com www.small-structures.com recurrence after surgical resection. To reverse the local immunosuppression of glioma, Zhang et al. designed a hydrogel to postoperatively co-deliver CXCL12/CXCR4 axis inhibitor (AMD3100) chelated with metal Zn (II) and CpG/poly-L-lysine nanoparticles. [165] This paradigm effectively blocked tumor recruitment of microglia and macrophages, and activated effector T cells, displaying potent immunotherapeutic effects against glioma. Immunotherapeutic molecules formulated in nanoparticles are a promising strategy for improved anti-glioma effect. In view of that the chemophysical features (such as surface charge and sizes) are determinants for organ selectivity of nanoparticles, [166] exploration of activity-structure correlation will advance the development of nano-immunotherapeutics for glioma treatment.

Nanotechnology-Mediated Combination Therapy for Glioma Treatment
Cancer patients often can benefit from co-therapy, such as the combination of immunotherapy with chemotherapy, radiotherapy, or surgery. The immunotherapeutic effect can be improved by combination with other treatments that induce immunogenic factors and promote immune response against tumors, providing survival benefits in cancer patients. In preclinical and clinical studies, combination therapy regimens have been extensively explored for efficacious glioma treatment.

Immunotherapy Combined with Therapeutics Targeting Glioma Cells
Despite the promising prospects of cancer immunotherapy, the results from clinical trials have shown that mono-immunotherapy is still suboptimal to fight against cancerous cells. [167] For example, ICIs have become the frontier of tumor immunotherapy, but the response rate is relatively low (around 10-25%) in clinic. In parallel, nanotherapeutics that target glioma growth pathway have exhibited a great potential in preclinical studies, such as kinase inhibitors of sorafenib [168] and volasertib. [169] The combined treatment modalities can potentially alter the immunosuppressive environment, thereby enhancing tumor sensitivity to ICIs, such as therapeutic agents targeting a suppressor gene p53 that is often found lost in human cancer and associated with tumor growth. [170] A nanotherapeutic agent SGT-53 contained a cationic liposome decorated with a single-chain fragment variable for recognition of TfR overexpressed on tumor cells, and a plasmid encoding human wild-type p53. [170] The clinical studies of SGT-53 showed that the restoration of p53-expression-mediated therapeutic activity in tumor patients. [171,172] Kim et al. for the first time evaluated the therapeutic effectiveness of SGT-53 in the combination with anti-PD-1 mAbs against glioma. [170] The results revealed that SGT-53 improved the immunogenicity of apoptotic glioma cells, leading to promoted infiltration of T cells against glioma. The combination therapy of SGT-53 and anti-PD-1 mAbs significantly inhibited tumor growth, while anti-PD-1 mAbs alone demonstrated limited therapeutic effect.
Nicotinamide phosphoribosyltransferase is an enzyme that can transform nicotinamide to nicotinamide mononucleotide, maintaining cellular energetics and providing essential substrates for cancer cell proliferation. Therefore, nicotinamide phosphoribosyltransferase inhibitors are an attractive strategy for glioma therapy, while these inhibitor molecules tend to cause adverse reactions on normal cells, such as GMX1778. To reduce system toxicity of GMX1778, Li et al. encapsulated GMX1778 into polymer particles. [173] Intratumor injection of nanoparticles loaded with GMX1778 led to upregulated expression of PD-L1 on glioma cells. The combination therapy of GMX1778 and anti-PD-1 mAbs substantially enhanced the recruitment of effector T cells and reduced the infiltration of tumor-promoting M2-type macrophages, thereby enhancing the therapeutic effect against glioma.
Different from the inhibitory function of PD-1/PD-L1 on effector T cells, CD47 expressed on cancer cells can bind signalregulatory protein α (SIRPα) on macrophages via ligand-receptor interaction, [174] inhibiting the phagocytosis capability of macrophages. [175] Thus, therapeutic strategies have been explored to blockade CD47-SIRPα inhibitory signaling pathway to restore macrophage-phagocytosis-mediated antitumor function. [176] To augment the therapeutic efficacy, Liu et al. recently designed ionizable cationic lipid nanoparticles (LNPs) with optimized amine headgroups to deliver small interfering RNA (siRNA) molecules to silence both PD-L1 and CD47 molecules expressed on glioma cells. [177] The optimized cationic nanocomplexes demonstrated effective BBB penetration via interacting with anionic cell membranes, and targeted delivery of siRNA molecules to the brain, decreasing expression levels of PD-L1 and CD47 proteins for enhanced glioma cancer immunotherapy.
The heterogenous interactions between immune cells and structural cells (such as cancer cells and stromal cells) support the immunosuppressive environment for glioma growth. Mono-immunotherapy with blockade therapeutics on one inhibitory receptor pathway is not potent enough to effectively eliminate cancer cells. The accumulated evidence has demonstrated that the combined strategies targeting cancer or immune cells modulate immunosuppression environment, leading to synergetic effect on glioma treatment.

Chemotherapy Combined with Immunotherapy
Despite that therapeutic glioma vaccines evoke profound tumorspecific T cells, it remains a challenge to recruit a substantial frequency of effector immune cells infiltrating at glioma sites caused by the immunosuppressive factors. Nanotechnologymediated chemotherapy offers a targeted delivery strategy of systemically administrated chemotherapeutics to glioma, in which tumor-infiltrating DCs can source tumor antigens from immunogenic dying cells. The activated DCs then migrate tumor dLNs to present antigens to naïve T cells, evoking tumor-antigenspecific T-cell immunity. The effector T cells will travel to tumor sites to produce cytokines to kill glioma cells and reverse immunosuppressive environment. Thus, co-administration of chemo-and immunotherapeutics in nanoplatforms is a prevalent regimen in glioma treatment.
Among chemotherapeutics, TMZ is the first-choice alkylating agent for glioma treatment. TMZ administration causes nucleotide mismatch and cell cycle arrest by alkylating the genomic DNA, thereby leading to tumor cells. [178] The favorable lipophilicity of TMZ endows its BBB penetration capability for glioma treatment, while TMZ resistance often occurs via multiple mechanisms, such as DNA repair pathways, survival autophagy, and glioma stem cell proliferation. OTX015 (abbreviated as OTX) is an inhibitor of bromodomain containing protein 4 that can increase the response of tumor cells to TMZ, by reducing the DNA damage repair response. [179] However, OTX performs poorly in BBB penetrability and glioma targeting. To achieve targeted co-delivery of OTX and TMZ to glioma, red blood cell membrane-coated acetal-dextran nanoparticles were engineered by encapsulating TMZ and OTX inside the core via a sonication method (Figure 7a). [180] The designed nanoparticle displayed a diameter of around 150 nm (Figure 7b), in which the embedded acetal-dextran enabled pH-responsive drug release under acidic tumor environment by cleaving acetal groups, the erythrocyte membrane modified with ApoE-promoted BBB crossing by targeting LPR. The combination of TMZ-and OTX-promoted ICD of glioma cells (Figure 7c), DC activation (Figure 7d), and T-cell priming increased the infiltration of CTLs at tumor sites. In addition, the OTX effectively inhibited the expression of PD-L1 on glioma cells. The combined ICD effect and PD-L1 inhibition effectively reversed the immunosuppressive microenvironment, thus profoundly suppressing glioma growth (Figure 7e).
TGF-β inhibits the proliferation of T and B cells for the immune escape of tumor cells, which also decreased TMZ sensitivity. Therefore, Qiao et al. designed dual-targeting nanotheranostic system (angiopep-2-modified phosphoethanolaminepolycarboxybetaine [DSPE-PCB]) to deliver siRNA against TGF-β, promoting the sensitivity of glioma cells to co-delivered TMZ. [181] The zwitterionic lipid DSPE-PCB aided the lysosomal escape, which was essential for cytosolic delivery of siRNA molecules and subsequent silencing function. Reactive oxygen species (ROS)-responsive polymer decorated on the surface of nanoparticles enabled controlled release of therapeutics at tumor sites while encapsulated iron oxide nanoparticles allowed magnetic resonance imaging track. The imaging guided nanoplatform showed potent therapeutic effect on glioma.
Different from alkylating agents, taxanes, such as paclitaxel (PTX), docetaxel (DTX), and cabazitaxel, resist tumor proliferation by stabilizing microtubules and inhibiting the mitosis of cancer cells. [182] To examine the efficacy of PTX-induced ICD in increasing glioma response to ICIs, Sun et al. linked PTXloaded PEG-poly(ε-caprolactone) copolymers with PD-L1 mAbs, showing significantly enhanced glioma treatment. [183] Nano-micelles co-delivered PTX and imiquimod that is a TLR7 agonist for TAM polarization showed cooperative therapeutic effects against glioma. [184] To enhance the combination therapy effect, Wang et al. constructed a 3D-network hydrogel (PLGA 1750 -PEG 1500 -gel 1750 ) for in situ sustained release of PTX-CpG nanoparticles postoperative intracavitary administration. [185] In this design, glioma-homing peptide (Pep-1) and APC-targeting molecule (mannitol) were modified on PTX and CpG nanoparticles embedded in the hydrogel responsive to body temperature. The sustained release of chemo-and immunonanotherapeutics was able to regress the residual glioma postsurgery and successfully prevent the recurrence of glioma.
Cell-membrane-coated nanoparticles represent an attractive platform, [186] which assists the encapsulated therapeutics in overcoming biological barriers to effectively combat cancerous cells.
Hao et al. utilized cell membranes to coat hydrophobic chemodrug nanosuspensions with a high loading capacity for glioma treatment. [187] Specifically, the authors fused cell membranes derived from glioma C6 cells and DCs (Figure 7f ). Glioma cell membrane provided multiple antigens and domains that target glioma, while DC membrane could activate T cells under antigen stimulation. Compared with traditional DC vaccines, C6 and DC hybrid membranes ([C6&DC]m) displayed enhanced biosafety and storage of DTX nanosuspensions (DNS) that was prepared via an ultrasonic precipitation method. Driven by C6-cell-membrane-mediated homotypic targeting, the constructed DNS-[C6 & DC]m showed significantly increased accumulation in glioma. DC membrane together with tumor antigens facilitated antigen presentation to activate T cells. The multifunctional biomimetic nanoparticles demonstrated combined effect of chemotherapy and immunotherapy, thereby prolonging the survival of mice with glioma (Figure 7g). Cell-membrane-coated nanoplatforms harness the potential of co-therapy of chemo-and immunotherapeutics in the treatment of glioma.
Some anticancer chemical drugs are derived from a mutated strain of Streptomyces peucetius, anthracyclines, such as daunorubicin, doxorubicin (DOX), and mitoxantrone. Mechanistically, these drugs bind with the basic groups, inhibiting DNA replication and RNA synthesis. Additionally, this type of anticancer drugs blocks topoisomerase II, obstructing cancer cell proliferation. Several other molecular mechanisms are also involved, such as the production of oxygen-free radicals and mitochondrial dysfunction. However, side effects on normal tissues, especially myocardial tissue, are the major concern of these potent anticancer drugs. [188] DOX was a water-soluble chemodrug approved by FDA in 1974, which is the first-line anticancer drug for a wide spectrum of tumors. To avoid systemic toxicity, DOX molecules are formulated in LNPs, named Doxil that has been proven for clinical use in humans since 1995. Preclinical experiments have shown that DOX nanoformulations were a powerful inducer of damageassociated molecular patterns, which potentially modulate glioma-infiltrating immune cells. [189,190] To increase potency of DC vaccines, Chen and colleagues utilized DCs to deliver nano-DOX that was formed by polyglycerol nanodiamonds and DOX molecules. [191] Results displayed that DCs successfully released DOX to glioma cells, promoting the production of damage-associated molecular patterns and tumor antigens and subsequent T-cell activation against glioma. [192] Mitoxantrone is another anthracycline without amino sugar structure, thus not producing free radicals that are toxic to the heart. To prevent glioblastoma recurrence, Zhang et al. constructed zinc 2-methylimidazole (ZIF-8) for co-delivery of mitoxantrone and immunomodulators, to evoke tumoricidal immunity post intracranial injection. [193] The metal-organic framework of ZIF-8 enabled efficient mitoxantrone loading capacity and acidic degradation in tumor microenvironment. The encapsulated siRNA-targeting IDO1 was to revert IDO1mediated immunosuppression. Glioma-associated macrophage membrane was used to camouflage the nanoparticles (denoted as tumour-homing immune nanoregulator (THINR)), which allowed glioma-homing capability. THINR and CXCL10 were encapsulated in an oligopeptide hydrogel (denoted as THIN-CXCL10@Gel) for sustained release. Post intratumor injection, the designed nanosystem with multiple functions turned the glioma immunosuppressive microenvironment from "cold" to "hot", inhibited glioma growth, produced ICD to active DCs and prime T cells, and silenced IDO1-Tregs-mediated immune  suppression. THINR-CXCL10@Gel substantially improved the infiltration of CTLs in glioma, and prevented tumor recurrence. Disulfiram was originally approved as a treatment for alcoholism via inhibiting aldehyde dehydrogenase in 1951, [194] which was found potent against cancer in 1977, particularly combined with copper. [195] Disulfiram and copper have synergetic effects on cancer treatment in diverse mechanisms, including upregulation of ROS, promoting apoptosis of tumor cells, and triggering ICD-mediated antitumor immunity. In terms of modulation of the suppressive immune environment, honokiol is regarded as a powerful immunomodulator, which regulates the glycolysis and autophagy of tumor cells and TAMs polarization toward M1 type via blocking the mammalian target of rapamycin-signalingpathway-mediated immune suppression. [196] To achieve combined anticancer medicaments for glioma treatment, Zheng et al. constructed a liposome system encapsulated with disulfiram/copper and honokiol, and modified with D CDX peptide (GREIRTGRAERWSEKF)-targeting α7 nicotinic acetylcholine receptors overexpressed on glioma cells, ECs, and TAMs. [197] This combination therapy with a "three-birdsone-stone" strategy displayed promising anti-glioblastoma properties by remodeling glioma metabolism and suppressive immune environment. Co-therapy of camptothecin and curcumin delivered by neurotransmitter-modified liposomes demonstrated as an alternative approach alleviating the immunosuppressive environment. [198] Cell-penetrating peptide TAT (CYGRKKRRQRRR)-modified lactoferrin encapsulated simvastatin and fenretinide, which activated STAT6-pathway-mediated TAMs repolarization and ROS-induced mitochondrial apoptosis in glioma cells. [199] Recent advanced nanotechnology with the ability to concentrate the chemotherapeutics within glioma cells is of significance for improving immunotherapy, by promoting the immunogenicity of dying cancer cells, initiating effector T-cell immunity, and reverting tumor suppression. The intriguing nanoplatforms as discussed earlier in preclinical studies offer promising results and strong motivation to evaluate the proposed concepts in glioma treatment. Further exploration of these treatment modalities in safety and efficacy will advance their investigation in clinical studies.

Radiotherapy Combined with Immunotherapy
Radiotherapy can directly destroy cancer cells by energetic particles or waves, including X-ray, γ ray, electron, and proton. Recently, accumulated experimental evidence has proved that radiotherapy can promote nanotherapeutics to penetrate the BBB for enhanced glioma therapy. In addition, radiotherapy triggered immunogenic cancer cell death by triggering extensive DNA damage, leading to release of dsDNA, ssDNA, dsRNA, and ssRNA, which promotes the immune system against cancer cells. [200,201] The intrinsic features of radiotherapy lay the foundation for the combination of radiotherapy and immunotherapy.
Radiotherapy kills cells mainly by destroying DNA strands and producing free radicals after reacting with water molecules, especially for tumor cells with a high proliferation rate. To avoid inevitable damage to normal cells, radiosensitizers have been explored to target tumor sites and increase the sensitivity of cancer cells to radiotherapy. [202] For example, gold nanoparticles (AuNPs) can substantially increase the dose of radiation via photoelectric effect and Compton scattering, which promotes ROS production for enhanced radiotherapy therapeutic effect. Given the characteristics of easy preparation, rapid distribution, and discharge, AuNPs are predominant radiosensitizers in radiotherapy. To explore the combined therapeutic effect of radiotherapy and immunotherapy in glioma treatment, Chen et al. coated AuNPs radiosensitizer with immunogenetic bacterial outermembrane vesicles (OMVs). [203] OMVs derived from gram-negative bacteria are nanoscale lipid bilayer vesicles, containing abundant outer-membrane proteins and participating in antitumor response in an IFN-γ-dependent manner. [204] Combined with 2 Gray (Gy) radiotherapy, the stable co-suspension solution (Au-OMV) displayed effective killing effect on glioma cells after intratumoral injection, which was contributed by the production of ROS and TNF-α. The enhanced therapeutic effect was also observed in the combination of CpG and Au-NPs-amplified radiotherapy in glioma treatment. [205] Wei et al. demonstrated that designed ApoE-modified chimeric biodegradable polycarbonate vesicles for BBB permeable delivery of CpG molecules to glioma sites. [206] Nano-CpG showed modest immunotherapy efficacy against glioma while radiotherapy profoundly boosted the immunotherapeutic effect. Kadiyala et al. reported the combined co-therapy of three treatment modalities by the use of sHDL nanoplatform, [207] in which DTX and CpG were encapsulated by sHDL. Post intratumoral injection of DTX-sHDL-CpG and radiotherapy, complete tumor regression was observed in mice bearing glioblastoma with a long-term survival and effective prevention of tumor rechallenge.
In addition to radiosensitizers, boron neutron capture therapy can also reduce damage to normal tissues. Boron neutron capture therapy kills cancerous cells via nuclear capture and fission reactions, which need sufficient 10 B aggregation in tumor cells and a collimated beam of neutrons to arouse 10 B (n, α) 7 Li capture reaction. [208] To increase the targeting efficacy and concentrate boron neutron capture therapy drugs at glioma, Chen et al. utilized DOX to deliver carborane (CB) (Figure 8a), which allowed the nucleus penetration via the nuclear pore complex. [209] DOX-CB was encapsulated in cationic liposomes modified with a homing peptide of iRGD (a cyclic peptide composed of 9 amino acids), which enabled targeting to α v integrins overexpressed on glioma vascular ECs and facilitated accumulation of nanoparticles in glioma. In parallel, a plasmid containing singleguide RNA sequences to knock out CD47 (Figure 8b,c) that binds to SIRPα expressed on macrophages, enhancing the capability of macrophages in phagocytosis of glioma cells. [210] Under irradiation of neutron beam, the engineered nanoparticles with combined therapeutic effect showed significantly prolonged survival of mice bearing GL261 glioma compared with other treatment modalities (Figure 8d).
In the context of glioma treatment, the penetration of BBB is essential for exerting the combined functions. Fortunately, radiotherapy offers additional merits in promoting the transport of nanotherapeutics to brain tumors via TAMs-mediated vascular bursts. [211,212] For example, Erel-Akbaba et al. demonstrated that radiation aided BBB penetration of nanotechnology-based siRNA for enhanced glioma immunotherapy (Figure 9a,b). [213] Specifically, the authors constructed solid lipid nanoparticles (SLNs) via the microemulsion dilution method (Figure 9a), where cationic lipid Esterquat was used to absorb siRNA molecules while 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-dibenzocyclooctyne (DSPE-PEG (2000)-DBCO) was incorporated to functionalize SLNs for grafting glioma-penetrating peptide of iRGD via click chemistry. The engineered monolayer SLNs (denoted as iRGD-SLN) with low molecular fluidity and strong stability enabled controlled release of delivered cargoes. In vivo imaging revealed that radiation significantly promoted the accumulation of iRGD-SLN at brain tumor sites (Figure 9c). The delivered siRNAs were effective against both EGFR and PD-L1 for combined immunotherapy in glioblastoma treatment. In combination with 5 Gy of X-ray radiation, iRGD-SLN-siEGFR/PD-L1 showed remarkable Figure 8. A cationic liposome-based nanoparticle combines nuclear-targeting doxorubicin-carborane (DOX-CB) drug and CD47-targeting clustered regularly interspersed short Palindromic repeats (CRISPR)-Cas9 system to combat glioblastoma. a) Schematic depiction of the fabrication process and co-therapy mechanism of DOX-CB@lipo-pDNA-internalizing RGD (iRGD). b) A diagram of constructed CRISPR-Cas9 system for CD47 gene editing. c) CD47 expression levels in GL261 cells after each treatment. d) Percentages of survival of mice implanted with GL261 tumor and received different nanotherapeutics. Reproduced with permission. [209] Copyright 2022, BioMed Central.   [213] (e-h) [214] Copyright 2019, American Chemical Society. regression of brain glioma (Figure 9d). Similar concept was also validated in extracellular vehicles-based nanoplatforms. [214] Extracellular vehicles are secreted from endogenous cells, with low immunogenicity and excellent stability. [215,216] Tian et al. harvested extracellular vehicles (Figure 9e,f ) from ReNcell VM cells (a neural progenitor cell line) and conjugated with a glioma-targeting ligand c(RGDyK) (denoted as RGD-extracellular vehicles (EV)). The conjugation of Cy5.5 was to assess the biodistribution of RGD-EV. In vivo imaging results indicated that radiotherapy significantly enhanced targeted delivery of RGD-EV to brain. However, radiation elevated the expression of PD-L1 on tumor cells, TILs, and microglia (Figure 9g). RGD-EV delivered small interfering RNA molecules that could silence PD-L1 (siPD-L1) effectively silenced PD-L1 expression, thus the combination of 5 Gy of X-ray radiation and RGD-EV-siPD-L1 dramatically inhibited glioma growth. Accumulating research has revealed that PD-L1 is overexpressed on tumor-associated myeloid cells (TAMCs), which is a key subset of immune cells driving tumor immunosuppression. To target PD-L1 þ TAMCs, Zhang et al. constructed an anti-PD-L1 mAbs-modified LNPs (denoted as αPD-L1-LNP) to deliver dinaciclib (Dina) that inhibits key enzymes associated with PD-L1 production of TAMCs, thereby promoting therapeutic effect of CTLs against glioma, particularly in combination with radiotherapy (Figure 10a). [217] The engineered LNP and αPD-L1-LNP displayed a size of less than 100 nm (Figure 10b). Cocultured experiments showed that TAMCs suppressed the proliferative expansion of CD8 þ T cells, while pretreatment TAMCs with αPD-L1-LNP/Dina blocked inhibition function of TAMCs (Figure 10c). The conjugation of αPD-L1 promoted the targeting efficacy of αPD-L1-LNP to diverse subsets of PD-L1þ immune cells, particularly TAMCs (Figure 10d). Radiation dramatically increased the population of PD-L1positive TAMCs (Figure 10e), thereby significantly enhancing the therapeutic effect of αPD-L1-LNP/Dina against glioma (Figure 10f ). Co-therapy of radiotherapy and αPD-L1-LNP/ Dina remarkedly eliminated the number of TAMCs and TAMs ( Figure 10f ) and profoundly downregulated PD-L1 expression on TAMCs (Figure 10h) in glioma, though radiotherapy alone promoted the infiltration of these immunosuppressive cells. The targeting efficacy of αPD-L1-LNP was also proven in human TAMCs derived from glioblastoma patients. The co-therapy of radiotherapy and nano-immunotherapeutic holds great potential in clinical translation for enhanced glioma treatment.
It has been discovered that radiation induced upregulation of CXCL12/CXCR4, which in turn promoted the growth and metastasis of glioma via multiple mechanisms, such as expanding blood vessels, promoting the migration of tumor stem cells and the infiltration of immunosuppressive MDSCs. [218] CXCL12/CXCR4 antagonist, AMD3100, is a promising therapeutic candidate to revert CXCL12/CXCR4-mediated immunosuppression, while the therapeutic effect of AMD3100 is hampered by its incapability of crossing BBB. Recently, Alghamdi et al. engineered iRGD-modified synthetic protein nanoparticle (SPNP) to facilitate BBB penetration capability of AMD3100 for glioma treatment combined with radiotherapy. [32] SPNP was constructed by human serum albumin (HSA), bovine serum albumin (BSA) and bifunctional macromerm (N-hydroxysuccinimide-oligoethylene glycol-N-hydroxysuccinimide) (Figure 11a). SPNP-AMD3100 showed a size of around 100 nm (Figure 11b). Glioblastoma cells treated with SPNP-AMD3100 and radiation showed significantly promoted release of immunogenic cell-death-associated high mobility group box-1 protein (HMGB1) (Figure 11c), evoking immune response against glioma. With the aid of radiation, CXCR4 blockade SPNP profoundly prolonged the survival of mice with OL61 glioma (Figure 11d,e), and effectively prevented tumor rechallenge (Figure 11f ), which was associated with reduction of glioma-infiltrating CXCR4þ monocytic MDSCs by blocking CXCL12/ CXCR4 signaling, and induction of memory response triggered by ICD. This fascinating combination therapy of radiotherapy and nano-CXCL12/CXCR4 antagonist provides promising preclinical evidence for the implementation in clinic.
Together, accumulating evidence proves that engineered nanotechnology not only enhances radiotherapy-mediated eradication of tumors, but also reshapes the tumor immune environment for enhanced immunotherapy. More importantly, radiation increases the accumulation of nanotherapeutics in brain by enhancing BBB penetration ability. Radiotherapy mediates enhanced immunogenicity or suppression in glioma, depending on the dose of radiation and the characteristics of glioma models. The cooperation of nanotechnology delivered radiosensitizers and immunodrugs is an effective paradigm for cancer treatment, which holds immense potential in clinical translation.

Photodynamic Therapy Combined with Immunotherapy
Photodynamic therapy (PDT) is a relatively less invasive cancer treatment modality, in which photosensitizers are activated by a specific wavelength of light, transfer the energy to molecular oxygen, and subsequently produce cytotoxic ROS to kill cancer cells. [219] Different from radiotherapy and chemotherapy that tend to induce immunosuppressive factors, PDT causes destruction of cancer cells to release immunogenetic antigens, and actuate inflammation to secrete pro-inflammatory cytokines that promote the recruitment of neutrophils and DCs, thereby promoting immune response against cancerous cells. Thus, combination therapy of PDT and ICIs represents an attractive approach for enhanced glioma treatment. For example, Xu et al. engineered nanocomplexes (%30 nm) self-assembled by immunoglobulin G (IgG), a photosensitizer of Chlorine e6 (Ce6) and polyvinylpyrrolidone (Figure 12a,b). [220] IgG promoted the accumulation of Ce6 at glioma. The fluorescent properties of Ce6 enabled an intraoperative fluorescence image-guided surgery for accurate resection (Figure 12c,d). Post-surgery, co-administration of PDT and anti-PD-L1 mAbs significantly improved the survival time of mice (Figure 12c).
The conversion of oxygen into ROS is the key to PDT efficacy, while the inherent hypoxic environment in glioma which is characterized by rich hydrogen peroxide, hampers PDT potency. In addition, hypoxia induces TAMs polarization to pro-tumor M2 phenotype, which accelerates the immunosuppressive environment in glioma. To address hypoxia-associated suppression on therapeutics, Sunil et al. utilized a ROS-responsive thio-ketal linker to conjugate with Nutlin-3a that was capable of producing  oxygen, followed by encapsulation with a photosensitizer of protoporphyrin IX into DSPE-PEG micelles for enhanced glioma treatment. [221] Bacteria-based delivery system can penetrate BBB via multiple pathways, including extracellular and transcellular pathways. [222,223] To increase the transport of nano-photosensitizers, Figure 11. A synthetic protein nanoparticle decorated with iRGD promotes immunotherapy efficacy of CXCL12/CXCR4 antagonist AMD3100, particularly in combination with radiotherapy. a) The addition sequence of different components in the preparation process of AMD3100-SPNP. b) A scanning electron microscope (SEM) image of AMD3100-SPNP. c) Enzyme-linked immunosorbent assay analysis of HMGB1 released from different mouse glioblastoma cells pretreated with each formulation (from left to right: saline; AMD3100; irradiation; AMD3100þ irradiation). d) A diagram of therapeutic treatment regimen. e) Survival curves of mice implanted with OL61 tumor and received different treatments or f ) rechallenged with OL61 tumor. Reproduced with permission. [32] Copyright 2022, American Chemical Society. Sun et al. constructed a Trojan bacteria system to load indocyanine-green-silicon nanoparticles for glioma photothermal immunotherapy (Figure 13a). [224] Under 808 nm irradiation, indocyanine-green-mediated PDT destroyed the glioma cells and bacterial cells, which led to the release of TAAs and PAMPs, and subsequent activation of DCs (Figure 13b). PDTbacterial-mediated in situ vaccination remarkably promoted the infiltration of effector CD8 þ T cells (Figure 13c) and NK cells in tumor sites, which significantly suppressed glioma growth (Figure 13d). Reproduced with permission. [220] Copyright 2019, American Chemical Society. To sum up, nanoplatforms enable BBB penetration of photosensitizers and stimulatory agents for enhanced co-therapy of glioma. This combination strategy can be further improved by incorporating additional immunogenic platforms. Nanotechnology-amplified photodynamic immunotherapy represents an appealing type of glioma treatment, with immense potential for clinical translation.

Sonodynamic Therapy (SDT) Combined with Immunotherapy
Sonodynamic therapy (SDT) employs low-intensity ultrasound to activate sonosensitizers that are selectively accumulated in tumor, generating ROS to cause cell death. SDT is a promising and noninvasive modality for glioma treatment. [225] In addition, Figure 13. Bacteria loaded with indocyanine green, glucose polymer, and silicon nanoparticles for glioblastoma photothermal immunotherapy. a) Schematic illustration of mechanism for photothermal immunotherapy. Flow cytometry analysis of the percentages of b) mature DCs, c) gliomainfiltrating CD8 þ T cells, and d) bioluminescence imaging in situ glioblastoma sizes in mice after various treatments as indicated. Reproduced with permission. [224] Copyright 2022, Nature Portfolio. endogenous and exogenous stimuli-responsive nanotherapeutics allow site-and time-specific delivery fashion for safe cancer treatment, of which advanced nanoscale platforms that are responsive to external stimuli, such as radiation, light, ultrasound, and electric field, have shown promising results in the combination therapy of immunotherapy and other treatment modalities. For instance, Li et al. utilized neutrophils for targeted delivery of a multiple-function-responsive nanostructure, where ZnGa 2 O 4 : Cr 3þ (ZGO) was coated with a layer of mesoporous TiO 2 , which together with PTX and anti-PD-1 mAbs were loaded in a ROSresponsive liposome (Figure 14a). [226] Glioma inflammatory environment promoted the recruitment of neutrophils, thereby facilitating the accumulation of nanotherapeutics. ZGO possessed persistent luminescence under near-infrared-light irradiation. Post intravenous injection, nanotherapeutics successfully reached the glioma area, detected by luminescence imaging (Figure 14b). Upon sonication, TiO 2 -induced ROS generation triggered the release of PTX and immunotherapeutics (anti-PD-1 mAbs) from the liposomes, which resulted in potent antitumor effect (Figure 14c), prolonged survival of mice bearing glioblastoma and preventing tumor recurrence. The emerging co-therapy of SDT and nano-immunotherapy has profoundly enhanced cancer treatment, representing a highly potent paradigm. [227] However, this strategy has been rarely explored in glioma treatment, requiring further investigation to unveil its potential.

Other Therapies Combined with Immunotherapy
Ferroptosis is an iron-dependent programmed cell death driven by excessive Fe 2þ and phospholipid peroxidation. This biological process can be regulated by depletion of glutathione and decreasing glutathione peroxidase 4 (GPX4), which results in the production of abundant hydroxyl radicals through the Fenton reaction, ultimately killing tumor cells. In contrast to other types of cell death, such as apoptosis, necroptosis, and pyroptosis, which are proven to be engaged with the immune system, the biological role of ferroptosis is largely obscure. Some experimental evidence suggested that the impaired function of GPX4 might induce the secretion of lipid immunostimulators, triggering the sense and response of immune cells. To explore the potential synergic effect of ferroptosis and immunotherapy in nanotherapeutics, Liu et al. engineered microglia BV2 (a type of microglial cell derived from C57/BL6 murine)-membrane-coated Fe 3 O 4 -siPD-L1 nanocomplexes linked by the S-S bond (Figure 15a). [228] Given that microglial cells tend to be recruited by glioblastoma cells through chemokines, [229] BV2 membrane enabled promoted accumulation of nanotherapeutics at tumor site, thereby  enhancing therapeutic effect with prolonged survival of mice bearing drug-resistant glioblastoma (Figure 15b). It was observed that Fe irons released from Fe 3 O 4 nanoparticles downregulated the expression of GPX4 ( (Figure 15c), inducing glioma ferroptosis. The immunogenic tumor antigens released from ferroptosismediated dying cells were able to activate DCs, evoking anti-glioma effector T-cell immunity (Figure 15a). In parallel, the delivered siPD-L1 molecules successfully silenced PD-L1 expression in glioma, which potentiated the cytotoxic function of CTLs. The cooperative effect of Fe 3 O 4 nanoparticles and siPD-L1 explained the significantly enhanced glioma treatment effect. The exemplified nano-immunotherapeutics and combined nanotherapeutics for enhanced glioma treatment are summarized in Table 2. Inorganic nanoplatforms showed advantages in enhanced biological stability and engineering flexibility, such as tunable sizes, shapes, roughness, and chirality. However, most candidates entered clinical studies are organic nanotherapeutics with excellent safety profiles and easy scale-up production (please refer to details in Section 5).

Emerging Nanotechnology-Mediated Glioma Immunotherapy in Clinical Trials
Mono-immunotherapeutics or combined therapeutic agents in nanoplatforms enabled amplified therapeutic outcomes and reduced adverse effects in preclinical animal studies, which advanced their exploration in clinical trials. For example, the heat shock protein glycoprotein 96 kDa (HSP-gp96)-based carriers are the most studied in clinical trials ( Table 3). HSP-gp96 is an Figure 15. BV2 cell membrane co-delivers Fe 3 O 4 and siPD-L1 for enhanced ferroptosis and anti-PD-L1 immunotherapy against drug-resistant glioblastoma. a) Schematic illustration of preparation procedure and co-therapy mechanism of Fe 3 O 4 -siPD-L1@M -BV2 . b) Survival curves of mice treated with different therapeutics. c) Western blot analysis of expression of some key proteins in glioblastoma tissue. Reproduced with permission. [228] Copyright 2022, BioMed Central.
www.advancedsciencenews.com www.small-structures.com Antigens and adjuvants are codelivered to dLNs for the enhancement of the antigen presentation process; anti-PD-L1 antibodies suppress immune checkpoints.
Intravenous injection GL261 cells [29] SWNT CpG TLR9-pathway-mediated immune activation and the inhibition of cell migration.
Intracranial injection GL261 cells [147] HFn modified with RGE SR717 STING agonists are mediated to cross the BBB through TfR1 and RGE.
Intravenous injection G422 cells [157] PEGylated liposomes CHA STAT-signaling-mediated polarization reversal of TAMs with reduced administration frequency and improved memory T-cells infiltration.
In a 3D microfluidic model U373MG cells [164] Zn (II) and dendrigraft poly-Llysine AMD3100 and CpG Continuous drug release in the hydrogel for postoperative inhibition of TAMs recruitment and activation of CTLs.
Intravenous injection U87 MG cells [191] ZIF-8 coated with glioma-associated macrophage membrane and hydrogel Mitoxantrone, CXCL10, and siIDO1 Continuous release of MIT and immunomodulator in the postoperative area of glioma.
Postoperative inoculation GL261 cells [193] Liposomes modified with DCDX Honokiol and disulfiram/copper Targeted regulation of metabolism and immune environment.

Intravenous and intranasal injection
LCPN cells [206] sHDL DTX, CpG, and 2 Gy of X-Ray radiation Radiotherapy enhances the antitumor effect of DTX and CpG.
Intracranial administration GL261 cells [217] HAS modified with OEG and iRGD AMD3100 and 2 Gy of X-Ray radiation CXCR4-pathway-mediated immunomodulation and radiationmediated ICD reinforce each other.

Intravenous injection
OL61, RPA, and ArfÀ/À cells [32] Chlorine globulin Anti-PD-L1 antibody and 660 nm laser irradiation at a dose of 458 mJ The mixture of Ce6 and anti-PD-L1 antibody promotes red-light fluorescence image-guided surgery while enabling stereotactic PDT and ICB.
In vitro spheroid model U87 cells [221] NEs loaded with ZGO core and TiO2 shell PTX-carried liposomes and anti-PD-1 antibody Ultrasound irradiation enhances the antitumor effect of PTX and anti-PD-1 antibody under optical imaging guidance.
Intravenous injection GL261 cells [228]  intracellular chaperone protein that can naturally bind to carcinoembryonic antigen and progenitor cell antigen. HSP-gp96adjuvanted cancer vaccines can be internalized by DCs via CD91 receptors, which assisted the presentation of peptide vaccines via the MHC molecules, thereby enhancing immune response against antigens. An early phase 1/2 clinical trial of HSP-gp96 peptide complexes (denoted as HSPPCs), showed obvious peripheral immune response and immune cell infiltrates in tumor sites in 11 out of 12 patients with recurrent glioblastoma (NCT00293423). [230] The encouraging clinical data warranted a phase 2 trial, where HSPPCs further confirmed its safety and efficacy in 41 patients. [231] Patients with fewer lymphocyte counts showed decreased survival, suggesting that pretreatment lymphopenia might impact the vaccine efficacy. Another phase 1 clinical study (NCT02122822) revealed that HSPPC vaccination together with standard treatments was safe and effective in patients with newly diagnosed gliomas. [232] A phase 2 trial confirmed the effectiveness of combined strategy of HSPPC vaccination and standard treatments in glioblastoma patients, displaying improved survival (NCT00905060). [233] In this clinical study, PD-L1 expression on peripheral myeloid cells was found as a potential factor affecting vaccine efficacy, thus another ongoing clinical study is assessing the curative effect and safety of co-therapy of HSPPC-96, anti-PD-1 mAb pembrolizumab (NCT03018288), and radiation therapy in glioblastoma patients. HSPPC vaccination combined with dual ICIs of anti-PD-1 (balstilimab) and anti-CTLA-4 (zalifrelimab) was also under clinical evaluation (NCT04943848). In another phase 2 clinical trial study (NCT01814813), the co-therapy of HSPPC vaccine and bevacizumab that is an approved humanized mAb-targeting EVGF was evaluated in patients with recurrent glioblastoma, the results of which were not yet reported. To further assess the safety and effectiveness of HSPPCs, a large phase 2 clinical study (NCT03650257) is actively recruiting patients with glioblastoma. In view of the aforementioned extensive clinical research, HSP-based cancer vaccines are promising products for future clinical use in the treatment of glioblastoma patients. Along with HSP nanotechnology, tumor-selective replicating oncolytic adenoviruses that are %70-90 nm in diameter not only can directly dissociate tumor cells, but also potentiate antitumor immunity, being an important branch of immunotherapy for glioma treatment in clinical trials. Nevertheless, oncolytic viruses have poor ability in penetration of biological barriers, which makes them unable to completely spread the tumor tissue post intraluminal injection. NSCs are CNS-derived multipotent progenitor cells with inherent capability to penetrate into the tumor bed. Hence, these NSCs were engineered to deliver an oncolytic adenoviral CRAd-S-pk7, which has been genetically modified to replicate more efficiently and selectively target glioma cells in the brain. [234] NSC-CRAd-S-pk7 was proven well tolerated and safe and feasible in glioblastoma patients under a phase 1 clinical study (NCT03072134), [234] supporting further evaluation in phase 2/3 clinical trials. Another phase 1 clinical study (NCT05139056) is underway to assess the effectiveness of NSC-CRAd-S-pk7 administrated with multiple doses in glioma patients.
Beyond peptide vaccines, mRNA vaccines are revolutionizing cancer immunotherapy, and research results ranging from lab and clinical exploration in treating glioma have been promising.
For example, mRNA immunotherapeutic that encodes glioma viral antigens of cytomegalovirus pp65-loaded DC vaccine was proven highly potent, which significantly suppressed tumor growth in mice when preconditioning immunization sites with immunogenic tetanus toxoid. [235] Patients with glioblastoma received tetanus toxoid and DC vaccines showed significantly enhanced overall survival, which was found associated with pp65-specific immune response. [235] Nevertheless, electroporation was applied to enhance RNA transfection in DC vaccine preparation, which might cause substantial cell death. LNPs that possess advantages of excellent safety, stability, and easy commercial production, are commercially used to deliver mRNA vaccines in clinic against COVID-19. This striking success of LNP-mRNA promoted the investment in clinical investigation of LNP-mRNA therapeutics for cancer immunotherapy. [236] For instance, commercially available cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane was selected in the formulation of LNPs to deliver tumor-derived mRNAs, thereby profoundly activating DCs in vivo post intravenous administration. [237] The engineered RNA-LNPs displayed superior capability in promoting the maturation of immune cells and expanding T-cell immunity compared to Freund adjuvant-assisted peptide vaccines in mice with invasive high-grade glioma. [237] Further animal experiments revealed that RNA-LNPs reprogrammed systemic immunity with increased PD-L1 expression, which enabled mice to respond to ICIs. [238] These promising results supported an ongoing phase 1 clinical trial (NCT04573140) of RNA-LNPs in glioblastoma patients, assessing the tolerated dose and effectiveness.
Despite that only a few nano-immunotherapeutic strategies are currently under clinical investigation, the breakthrough knowledge in brain immunology and nano-engineering science will advance new treatment modalities for glioma with enhanced effectiveness.

Safety of Nano-Immunotherapeutic in Preclinical and Clinical Studies
Despite extensive research efforts devoted to the development of advanced nano-immunotherapeutics for the treatment of glioma, the safety of these emerging treatment modalities is always paramount in both preclinical and clinical exploration. Nanoparticles are now available in a variety of sizes, shapes, topologies, solubility, and surface chemistries. Once they exert their functions in vivo, the fate of nanotherapeutics remains elusive, such as whether the nanotherapeutics would be completely degraded under physiological conditions, cleared by phagocytosis in liver, or excreted by the kidneys. The unresolved research questions might be associated with the potential nanotoxicity that is a burning matter for their clinical translation.
Inorganic nanoparticles are relatively inert compared to their organic counterparts. Researchers have applied various technologies, such as noninvasive X-ray imaging to trace gold nanoparticles in vivo, [239] to understand the nano-bio interactions, biodistributions in organs, and biological alternations in nanocell interactions in a healthy or disease model. The elimination or potential accumulation of nanoparticles in the body heavily relies on their physicochemical properties. For example, small-sized nanoparticles (less than 6 nm) tend to be cleared via renal cortex. [240] Surface PEGylation shields the nanotherapeutics from aggregation, protein corona formulation, and phagocytosis by circulating immune cells, which prolongs their circulation time. The fundamental studies into biodegradability, clearance, and nano-bio cellular interactions in different animal models (e.g., nonhuman primates) will expedite the clinical translation of nanotherapeutics.
Biomimetic organic nanoparticles, such as cell-membranebased nanoparticles, cell-derived carriers, and extracellular vehicles systems, are platforms at advanced clinical for glioma immunotherapy with well-verified safety. For example, NSCsbased therapeutics under a first-in-human trial was proven well tolerated in patients with recurrent glioblastoma, where dose-limiting toxicity was not detected. [142] extracellular vehicles are nano-sized vesicles secreted from various cells, participating in cell communication, antigen presentation, tumor invasion, and other biological functions. [241] Given the inherited immunogenicity, tumors-derived extracellular vehicles are a great choice for therapeutic glioma vaccines with excellent safety profiles. [133] Accumulated evidence in clinic has demonstrated the effectiveness and safety of LNP-mRNA technology. The transformative technology has gained increasing attention in cancer immunotherapy, including glioma mRNA vaccines. In addition to the safety concerns of nanoparticles, immunologic toxicities, such as immune-related adverse events, represent a challenge to identify and manage. It is crucial to develop reliable animal models that closely resemble the immune systems of humans, so that the side effects and undeniable benefits of immunotherapeutics in nanoplatforms can be well evaluated, recognized, and managed. Current preclinical experiments revealed that most nanoimmunotherapeutics demonstrated low toxicity in vivo and in vitro. For instance, anti-PD-L1 mAbs-loaded MP-3 copolymer displayed negligible cytotoxicity, without obvious damage in major organs post intravenous administration. [28] To hepatorenal toxicity of nanotherapeutics, the levels of alanine aminotransferase, aspartate transaminase, and other indicators in serum were found within normal range, showing acceptable safety. [30,163] Beyond these characteristics, the pro-inflammatory cytokines are generally measured as markers to assess the possibility of inflammation-associated immunotoxicity in healthy individuals.
In the context of external stimulation-mediated nanoimmunotherapy for glioma, the safety profile of the stimuli (e.g., radiation, light, and ultrasound) should be discussed and evaluated by considering the possibility of non-targeted and dose-dependent injury induced by external treatment. For example, radiation alters the dose-toxicity relationship of nanoparticles. [203] The biocompatibility of AuNPs in cells was found significantly decreased (from 80% to 30%) when exposed to 2 Gy of radiation. In glioma nano-immunotherapy, most studies were conducted under 2-6 Gy radiation in combination with nanotherapeutics, where the biosafety assays, such as body weight change of mice, [206] histological stains of the brain tissue, and hepatorenal toxicity-associated blood marker analysis. [32] A comprehensive evaluation of the safety profile of nanoimmunotherapeutics in both preclinical and clinical studies was essential for their further clinical investigation and implementation in clinic. The complexities in the combined therapeutic strategies increase the assessment aspects, such as in vitro cytotoxicity, protein corona formulation, in vivo biodistribution, degradation and accumulations, clearance pathways, pharmacokinetics of immunotherapeutics, hepatorenal toxic effects, and inflammation-mediated immunotoxicity.

Conclusions and Future Perspectives
Immunotherapy has revolutionized the treatment modalities of cancer patients. Nonetheless, malignant gliomas remain universally fatal with little improvement by immunotherapy. These failures are mainly caused by several challenges that are particularly associated with the brain. Glioma is a challenging tumor lesion located in a complex environment where the brain is physically less connected with the host immune system than those in other organs. The immunotherapeutics hardly access to tumor sites to exert their biological functions. Glioma is extremely heterogenous and rarely has identified mutations, creating difficulties to discover biomarkers for immunotherapeutic targets. The profound immunosuppressive features of glioma thwart the attempts of synergistic combination therapy, such as standard radiation or chemotherapy combined with immunotherapy. Advanced nanotechnology represents an effective and promising strategy to improve the therapeutic outcome of immunotherapeutics, with immense potential to benefit glioma patients. The use of nanosized platforms with desired functionality enhances the capability of embedded immunotherapeutic agents in crossing BBB. By manipulating the chemical compositions and physical properties of nanoparticles, additional immunomodulation of glioma microenvironment can be achieved by the intrinsic immunostimulatory function of nanoparticles. Beyond these characteristics, the combination of different therapeutic agents and stimulatory drugs in one nanoplatform can maximize the synergistic effect against glioma. We herein summarize recent progress of nano-immunotherapeutic approaches in both preclinical and clinical studies for enhanced glioma treatment (Table 2 and 3). The associated mechanisms and local immunomodulation are discussed in these advanced nanotechnology-mediated treatment modalities, which cover nano-immunotherapy alone or combined with chemotherapy, radiotherapy, photodynamic, or sonodynamic therapy. Despite the undenied benefits and rapid advancement, there are still several challenges facing the clinical translation of emerging nanoimmunotherapeutics in malignant glioma treatment ( Figure 16).
Advances in Brain and Glioma Immunology. A predominant limiting factor in exploring efficacious immunotherapeutic strategies for glioma is current inadequate knowledge of cerebral immunity. It remains a challenge to fully understand the molecular mechanisms and immunological function of heterogeneous subsets of immune cells infiltrating in brain and glioma. Conventional molecular and biological methods tend to yield averaged gene or protein expression in homogenized brain tissue cells, while single-cell RNA (scRNA)-sequencing technology was developed in 2014, bringing fine-grained RNA information of each cell. For example, scientists performed scRNA sequencing on cerebrospinal fluid samples from healthy individuals, which revealed that the signal of monocytes to clonal CD8 þ T-cellsmediated immune dysregulation during healthy brain aging. [242] By analyzing scRNA-sequencing data of human glioma tissues, researchers discovered nine distinct subsets of myeloid cells as prognostic markers and identified S100A4 as an immunotherapeutic target by reprogramming suppressive myeloid cells and T cells infiltrating into glioma. [243] In scRNA-sequencing technology, single cells need to be isolated from the tissue to discern the subtypes of cells based on their unique gene expression profile. Spatial transcriptomics technology enables scientists to obtain transcriptomic data in known positional contexts of a small group of cells in brain tissue. The integration of these advanced "omics" technologies delivers high throughput at single-cell and spatial resolutions, offering advanced strategies in immune cell typing and characterizing their roles in brain immune regulation. [244] The advanced knowledge in brain and glioma immunology will lead to novel and effective immunotherapeutic targets.
Precise Nano-Engineering Strategies. The restricted permeability of immunotherapeutics across the BBB accounts for the unsatisfied efficacy in glioma treatment. Nanotechnology attempts to increase the transport of immunotherapeutics across the barrier to glioma sites via several mechanisms. For example, small nanoparticles with lipophilic and cationic properties are advantageous in passive diffuse crossing the EC membranes or paracellular transport via tight junctions. The conjugation of appropriate ligands on the surface of nanoparticles enables receptor-mediated transport. The active targeting routes that are under extensive exploration include insulin receptor, TfR, lactoferrin receptor, opioid receptor, and insulin-like growth factor-1 receptors expressing on BBB. Adsorptive-mediated transcytosis is a nonspecific strategy through the electrostatic interaction of the negatively charged EC membranes and cationic nanoparticles. Disruption of BBB integrity is an alternative strategy for improving the delivery of immunotherapeutic agents to brain tumor sites. The configurations of nanoparticles should be precisely engineered with unique desired properties for increasing their access to glioma, while a multitude of factors should be considered and tailored for this purpose.
Identification of Novel Glioma TAA. The advances in omics technologies enable fine profiling of the immuno-features of healthy brain and glioma tissues, which offers a possibility of discovering novel TAAs for specific immunotherapy. The potential glioma antigen can be screened by exploring the transcripts that display minimal in healthy tissue but are overexpressed in glioma. Researchers are keen to define and identify immunogenic peptides that are naturally presented by appropriate MHC molecules expressed by glioma cells and are effective in activating immune responses against glioma. Despite numerous TAAs identified for developing glioma vaccines with some promising candidates under clinical investigation, the therapeutic outcomes are uneven in various evaluation settings. Innovative approaches are still required to explore potentially powerful candidates by identifying and validating highly immunogenic peptides that are capable of evoking potent antitumor immune responses, particularly in the combination of emerging nano-stimulators.
Close Interprofessional Collaboration. Developing powerful nano-immunotherapeutics needs close and intensive www.advancedsciencenews.com www.small-structures.com cooperation among scientists from multidisciplinary fields that involve materials science, neuroimmunology, oncology, bioinformatics, and medicine. Professionals in each field will bring their unique knowledge, talents, and skills, leading to increased productivity and more innovation. Scientific collaborations are decisive factors in fully understanding that the mechanism of nanotechnology enabled glioma immunotherapy, which will ultimately provide insights and guidelines for developing effective nano-immunotherapeutics. For example, multi-omics technologies will substantially advance the understanding of the interactions between the nanoparticles with high diversity and immune infiltrates with high heterogeneity at glioma, bringing enormous opportunities to discover novel biomarkers and to build up structure-activity relationship. However, large-scale single-cell data poses numerous challenges that require development of appropriate computational strategies. Bioinformaticians with adequate expertise in computational and biological science ensure that the massive data can be analyzed in an unbiased manner and interpreted correctly with accurate quality control on raw data. Thus, the collaborative team will tackle these challenges that require a breadth of knowledge and capabilities, facilitating the clinical translation of safe and effective nano-immunotherapeutics in glioma treatment.
Effective Evidence Exchanges across Preclinical and Clinical Boundaries. The preclinical studies evaluated the safety and efficacy of nanotherapeutic candidates validated in cells or animals, the encouraging outcomes from which will potentially support their first-human clinical trial. The relevance of animal models of immunotherapy in glioma treatment tends to be questioned, as the promising candidates failed to prove their efficacy in clinical studies. It is critically important to bridge the wide gap between preclinical research and clinical medicine. The clinicians should make effective evidence exchanges with the preclinical researchers. The positive or negative feedbacks from the clinical research will foster the development of reliable preclinical animals that closely resemble the features of neuroimmune system and brain oncology in humans. The dialog between researchers in the lab and clinicians in clinic will help the implementation of nano-immunotherapeutics in glioma treatment.
The emerging nano-engineering strategies have advanced the therapeutic efficacy of immuno-drugs by promoting BBB permeability and enhancing immune response against glioma. Despite that advanced nano-immunotherapy alone or in the combination with other therapies showed enormous benefits to glioma treatment in preclinical studies while their clinical investigation currently is at relatively less advanced stage, accumulated knowledge in brain and glioma immunity together with the advances in precise nanotechnology will accelerate the translation of nanotechnology-amplified glioma immunotherapy into clinical practices.