Surgical resection is the first stage of treatment for patients diagnosed with resectable glioblastoma and is followed by a combination of adjuvant radiotherapy and systemic single-agent chemotherapy, which is typically commenced 4-6 weeks after surgery. This delay creates an interval during which residual tumour cells residing in the resection margin can undergo uninhibited proliferation and further invasion, even immediately after surgery, thus limiting the effectiveness of adjuvant therapies. Recognition of the postsurgical resection margin and peri-marginal zones as important anatomical clinical targets and the need to rethink current strategies can galvanize opportunities for local, intraoperative approaches, while also generating a new landscape of innovative treatment modalities. In this Perspective, we discuss opportunities and challenges for developing locoregional therapeutic strategies to target the glioblastoma resection margin as well as emerging opportunities offered by nanotechnology in this clinically transformative setting. We also discuss how persistent barriers to clinical translation can be overcome to offer a potential path forward towards broader acceptability of such advanced technologies.

T cells and their T cell receptors (TCRs) play crucial roles in the adaptive immune system's response against pathogens and tumors. However, immunosenescence, characterized by declining T cell function and quantity with age, significantly impairs antitumor immunity. Recent years have witnessed remarkable progress in T cell-based cancer treatments, driven by a deeper understanding of T cell biology and innovative screening technologies. This review comprehensively examines T cell maturation mechanisms, T cell-mediated antitumor responses, and the implications of thymic involution on T cell diversity and cancer prognosis. We discuss recent advances in adoptive T cell therapies, including tumor-infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR-T) therapy, and chimeric antigen receptor T cell (CAR-T) therapy. Notably, we highlight emerging DNA-encoded library technologies in mammalian cells for high-throughput screening of TCR-antigen interactions, which are revolutionizing the discovery of novel tumor antigens and optimization of TCR affinity. The review also explores strategies to overcome challenges in the solid tumor microenvironment and emerging approaches to enhance the efficacy of T cell therapy. As our understanding of T cell biology deepens and screening technologies advances, T cell-based immunotherapies show increasing promise for delivering durable clinical benefits to a broader patient population.

Glioblastoma is invariably lethal and responds poorly to immune checkpoint blockade. Here, we examined the impact of regulatory T (Treg) cell depletion on glioblastoma progression and immunotherapy responsiveness. In human glioblastoma, elevated Treg cell signatures correlated with poorer survival outcomes, with these cells expressing high levels of CD25. In Nf1-/-Pten-/-EGFRvIII+ glioblastoma-bearing mice, a single dose of non-interleukin-2 (IL-2) blocking (NIB) anti-CD25 (anti-CD25NIB) antibody depleted Treg cells and promoted CD8+ T cell clonal expansion and partial tumor control, further enhanced by programmed cell death-1 (PD1)-blockade. Treg cell depletion induced interferon-γ (IFN-γ)-dependent tumor microenvironment remodeling, increasing Fcγ receptor (FcγR) expression on intratumoral myeloid cells and enhancing phagocytosis. Combination of anti-CD25NIB with anti-EGFRvIII tumor-targeting antibodies resulted in complete tumor control. Anti-human CD25NIB treatment of glioblastoma patient-derived tumor fragments effectively depleted Treg cells and activated CD8+ T cells. These findings underscore the therapeutic relevance of Treg targeting in glioblastoma and unveil potent combination strategies for anti-CD25NIB based on innate cell activation.

Glioblastoma (GBM) is an aggressive primary adult brain tumor that rapidly recurs after standard-of-care treatments, including surgery, chemotherapy and radiotherapy. While immune checkpoint inhibitor therapies have transformed outcomes in many tumor types, particularly when used neoadjuvantly or as a first-line treatment, including in melanoma brain metastases, they have shown limited efficacy in patients with resected or recurrent GBM. The lack of efficacy has been attributed to the scarcity of tumor-infiltrating lymphocytes (TILs), an immunosuppressive tumor microenvironment and low tumor mutation burden typical of GBM tumors, plus exclusion of large molecules from the brain parenchyma. We hypothesized that upfront neoadjuvant combination immunotherapy, administered with disease in situ, could induce a stronger immune response than treatment given after resection or after recurrence. Here, we present a case of newly diagnosed IDH-wild-type, MGMT promoter unmethylated GBM, treated with a single dose of neoadjuvant triplet immunotherapy (anti-programmed cell death protein 1 plus anti-cytotoxic T-lymphocyte protein 4 plus anti-lymphocyte-activation gene 3) followed by maximal safe resection 12 days later. The anti-programmed cell death protein 1 drug was bound to TILs in the resected GBM and there was marked TIL infiltration and activation compared with the baseline biopsy. After 17 months, there is no definitive sign of recurrence. If used first line, before safe maximal resection, checkpoint inhibitors are capable of immune activation in GBM and may induce a response. A clinical trial of first-line neoadjuvant combination checkpoint inhibitor therapy in newly diagnosed GBM is planned (GIANT; trial registration no. NCT06816927 ).

Immune checkpoint inhibitors (ICIs) present clinical benefits in many cancer patients but invariably fail in glioblastoma (GBM), the most common and deadly primary brain tumor. The lack of ICIs efficacy in GBM is attributed to the accumulation of tumor-reprogrammed glioma-associated myeloid cells (GAMs) that create a "cold" immunosuppressive tumor microenvironment (TME), impeding the infiltration and activation of effector T cells. GBM-derived αvβ3/αvβ5-integrin ligands, including SPP1, were shown to mediate the emergence of GAMs. We hypothesized that a combination strategy aiming to block the reprogramming of GAMs using a synthetic 7aaRGD peptide that targets SPP1/integrin signaling might overcome resistance to ICIs and reinvigorate anti-tumor immunity.

Matrigel invasion assay was used to test the efficacy of 7aaRGD in glioma-microglia co-cultures. We determined the impact of 7aaRGD, administered as a monotherapy or combined with PD-1 blockade, on tumor growth, GAMs accumulation and phenotypes, arginase-1 levels and neovasculature in experimental gliomas. The effects of treatments on the tumor immune landscape were dissected using multiparameter flow cytometry, immunocytochemistry, cytokine profiling and RNA-seq analysis of sorted GAMs followed by CITE-seq based data deconvolution.

7aaRGD efficiently blocked microglia-dependent invasion of human and mouse glioma cells in vitro. Intratumorally delivered 7aaRGD alone did not reduce tumor growth in orthotopic gliomas but prevented the emergence of immunosuppressive GAMs and led to normalization of peritumoral blood vessels. Combining 7aaRGD with anti-PD-1 antibody resulted in reduced tumor growth, with an increase in the number of proliferating, interferon-ɣ producing CD8+T cells and depletion of regulatory T cells. Transcriptomic profiles of myeloid cells were altered by the combined treatment, reflecting the restored "hot" inflammatory TME and boosted immunotherapy responses. Intratumoral administration of 7aaRGD similarly modified the phenotypes of GAMs in human U87-MG gliomas in immunocompromised mice. Exploration of transcriptomic datasets revealed that high expression of integrin receptor coding genes in pre-treatment biopsies was associated with a poorer response to immune check-point blockade in patients with several types of cancers.

We demonstrate that combining the blockade of SPP1/integrin signaling with ICIs modifies innate immunity and reinvigorates adaptive antitumor responses, which paves the way to improve immunotherapy outcomes in GBM.

Today, treating cancer patients with monoclonal antibodies (mAbs), by targeting immune checkpoints, is one of the most outstanding immunotherapeutic methods. Immune checkpoints are special molecules having regulatory role in immune system responses. Once these molecules are presented on cancer cells, these cells will be capable of evading the immune system through their own specific pathways. This Evasion can be prevented by counterbalancing immune system responses with immune checkpoints related antibodies.

The current study aimed to highlight immunotherapy and its methods, describe the immune checkpoints pathways, outline the immune checkpoint inhibitors (ICIs), and recent advances in this field, and sketch an outlook on the best treatment options for the most prevalent cancers.

This research implemented a narrative review method. A comprehensive literature review on the history, molecular and cellular biology, and the clinical aspects of immune checkpoint molecules was performed to illustrate the pathways involved in various cancers. Also, currently-available and future potential immunotherapies targeting these pathways were extracted from the searched studies.

The immune checkpoint family consists of many molecules, including CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and TIGIT. Attempts to modify these molecules in cancer treatment led to the development of therapeutic monoclonal antibodies. Most of these antibodies have entered clinical studies and some of them have been approved by the Food and Drug Administration (FDA) to be used in cancer patients' treatment plans.

With these novel treatments and the combination therapies they offer, there is also hope for better treatment outcomes for the previously untreatable metastatic cancers. In spite of the beneficial aspects of immune checkpoint therapy, similar to other treatments, they may cause side effects in some patients. Therefore, more studies are needed to reduce the probable side effects and uncover their underlying mechanism.

Based on the data shown in this review, there is still a lack of knowledge about the complete properties of ICIs and the possible combination therapies that we may be able to implement to achieve a better treatment response in cancer patients.

Although with aggressive standards of care like surgical resection, chemotherapy, and radiation, high-grade gliomas (HGGs) and brain metastases (BM) treatment has remained challenging for more than two decades. However, technological advances in this field and immunotherapeutic strategies have revolutionized the treatment of HGGs and BM. Immunotherapies like immune checkpoint inhibitors, CAR-T targeting, oncolytic virus-based therapy, bispecific antibody treatment, and vaccination approaches, etc., are emerging as promising avenues offering new hope in refining patient's survival benefits. However, selective trafficking across the blood-brain barrier (BBB), immunosuppressive tumor microenvironment (TME), metabolic alteration, and tumor heterogeneity limit the therapeutic efficacy of immunotherapy for HGGs and BM. Furthermore, to address this concern, the NanoBioTechnology-based bioinspired delivery system has been gaining tremendous attention in recent years. With technological advances such as Trojan horse targeting and infusing/camouflaging nanoparticles surface with biological molecules/cells like immunocytes, erythrocytes, platelets, glioma cell lysate and/or integrating these strategies to get hybrid membrane for homotypic recognition. These biomimetic nanotherapy offers advantages over conventional nanoparticles, focusing on greater target specificity, increased circulation stability, higher active loading capacity, BBB permeability (inherent inflammatory chemotaxis of neutrophils), decreased immunogenicity, efficient metabolism-based combinatorial effects, and prevention of tumor recurrence by induction of immunological memory, etc. provide new age of improved immunotherapies outcomes against HGGs and BM. In this review, we emphasize on neuro-immunotherapy and the versatility of these biomimetic nano-delivery strategies for precise targeting of hard-to-treat and most lethal HGGs and BM. Moreover, the challenges impeding the clinical translatability of these approaches were addressed to unmet medical needs of brain cancers.

Glioblastoma (GBM) is the most common primary brain malignancy. Few neoantigens have been tested in trials as the cancer vaccine against GBM.

To better understand the neoantigen landscape and its associated tumor microenvironment (TME) for the optimized vaccine design of our initiated GBM trial, we apply the integrative multi-omics approach to comprehensively profile the mutation, HLA typing, TCR/BCR repertoire, immune cell components on the tumor tissue and peripheral blood mononuclear cell (PMBC) specimen of 24 GBM patients.

On average, 148 mutated genes and 200 mutated sites per patient were identified, with no predominant mutated sites and genes in this cohort. Diversified HLA genotypes and expression rate across A, B, and C alleles, with A30:01&A11:01, B13:02, and C06:02, as the most frequent genotypes at respective alleles. Clustered CDR3 of TCR/BCR existed in tumor tissue with decreased richness compared with PMBC. NK and Th1 cells were revealed as the predominant immune cells within the tumor microenvironment (TME). Neoantigens were feasible predicted and designed for each patient, with an average number of 107. Very few neoantigens were shared by more than two patients and no dominant neoantigen could be identified. A minimum of 11-peptide bulk was required to cover this 24-patient cohort, guaranteeing each patient could have at least one neoantigen.

In summary, our data reveals a heterogeneous landscape of the neoantigen and its associated immune TME of GBM, based on which a peptide bulk is feasibly developed to cover these patients as a cohort.

Glioblastoma (GB) treatment, despite consisting of surgical resection paired with radiation, temozolomide chemotherapy and tumor-treating fields, yields a median survival of 15-20 months. One of the more recently appreciated hallmarks of GB aggressiveness is the co-opting of neurotransmitter signaling mechanisms that normally sustain excitatory synaptic communication in the CNS. AMPA-glutamate receptor (AMPAR) signaling governs the majority of excitatory synaptic activity in the mammalian brain. AMPAR activation in glioma cells activates cellular pathways that enhance proliferation and invasion and confer resistance to approved GB therapeutics. In addition, this review places a specific emphasis on discussing the redefined GB cytoarchitecture that consists of neuron-to-glioma cell synapses, whose oncogenic activity is driven by AMPAR activation on glioma cells, and the discovery of tumor microtubes, which propagate calcium signals throughout the tumor network in order to enhance resistance to complete surgical resection and radiotherapy. These new discoveries notwithstanding, some evidence suggests that AMPAR activation can produce excitotoxicity in tumor cells. This disparity warrants a closer examination at how AMPAR modulation can be leveraged to produce more durable outcomes in the treatment of GB and tumors in peripheral organs that express AMPAR.

Immunotherapy holds significant promise for treating head and neck squamous cell carcinoma (HNSCC), yet responses are limited to a subset of patients. This research investigates whether analyzing the peripheral T-cell receptor (TCR) repertoire could help identify patients who are more likely to benefit from a combination treatment of cetuximab and nivolumab. We report here updated correlative analysis using all samples profiled with deep immunoSEQ assay to study the peripheral TCR repertoires in peripheral blood mononuclear cells from patients enrolled in a phase I/II trial (NCT03370276). TCR repertoires were analyzed in 67 patients. Of these, 64 had available baseline data. Overall, our findings confirm that a more polyclonal peripheral TCR repertoire is associated with improved response to concurrent cetuximab and nivolumab in HNSCC. While the baseline productive Simpson clonality did not reach statistically significant differences in response groups, significant differences were observed within the HPV-negative subgroup and among patients who had received previous ICI therapy. Additionally, the TCR diversity at baseline and early follow-up was associated with overall survival. TRBV/TRBJ gene usage analysis also identified specific gene pairs associated with patient outcomes. Furthermore, our analysis indicates that the TCR clonality patterns are modulated by prior treatment exposures and tumor HPV status, suggesting a cohort expansion within these subgroups for further validation. Together, this study demonstrates the feasibility of leveraging the peripheral T-cell repertoire profiling and clonality dynamics as predictive biomarkers for immunotherapy efficacy in HNSCC.

Glioblastoma (GBM) is the most frequent malignant brain tumor. We recently discovered that oncolytic herpes simplex virus engineered to disable tumor-intrinsic protein kinase R (PKR) signaling (oHSV-shPKR) could increase oHSV oncolysis and antitumor immune response. However, in this study, we show that disabling tumor-intrinsic PKR signaling can also induce the activation of the indoleamine 2,3-dioxygenase (IDO) signaling pathway. Both GBM tumor progression and oHSV intratumoral therapy increased infiltration of IDO+CD11c+ dendritic cells (DC) into the tumor. The coculture of oHSV-infected human GBM neurospheres with monocyte-derived DCs (MoDC) dramatically increased IDO signaling activation in MoDCs through type-I IFN signaling. Addition of IDO inhibitor (indoximod) in the coculture significantly increased MoDC activation and reduced the consumption of tryptophan. Combining indoximod and oHSV significantly inhibited tumor growth and induced antigen-specific CD8+ T-cell activation. These results suggest that inhibition of the IDO pathway could significantly block feedback immunosuppression during oncolytic virotherapy of GBM.

Immune-checkpoint inhibitors (ICIs) have improved clinical outcomes across several solid tumour types. Prominent efforts have focused on understanding the anticancer mechanisms of these agents, identifying biomarkers of response and uncovering resistance mechanisms to develop new immunotherapeutic approaches. This research has underscored the crucial roles of the tumour microenvironment and, particularly, tumour-infiltrating lymphocytes (TILs) in immune-mediated tumour elimination. Numerous studies have evaluated the prognostic and predictive value of TILs and the mechanisms that govern T cell dysfunction, fuelled by technical developments in single-cell transcriptomics, proteomics, high-dimensional spatial platforms and advanced computational models. However, questions remain regarding the definition of TILs, optimal strategies to study them, specific roles of different TIL subpopulations and their clinical implications in different treatment contexts. Additionally, most studies have focused on the abundance of major TIL subpopulations but have not developed standardized quantification strategies or analysed other crucial aspects such as their functional profile, spatial distribution and/or arrangement, tumour antigen-reactivity, clonal diversity and heterogeneity. In this Review, we discuss a conceptual framework for the systematic study of TILs and summarize the evidence regarding their biological properties and biomarker potential for ICI therapy. We also highlight opportunities, challenges and strategies to support future developments in this field.

Gliomas, particularly glioblastoma (GBM), are among the most challenging brain tumors due to their complex and dynamic tumor microenvironment (TME). The TME plays a pivotal role in tumor progression, immune evasion, and resistance to therapy through intricate interactions among glioma cells, immune components, neurons, astrocytes, the extracellular matrix, and the blood-brain barrier. Targeting the TME has demonstrated potential, with immunotherapies such as checkpoint inhibitors and neoadjuvant therapies enhancing immune responses. Nonetheless, overcoming the immunosuppressive landscape and metabolic adaptations continues to pose significant challenges. This review explores the diverse cellular and molecular mechanisms that shape the glioma TME. A deeper understanding of these mechanisms holds promise for providing novel therapeutic opportunities to improve glioma treatment outcomes.

Despite multimodal therapies, the treatment of glioblastoma remains challenging. In addition to the very complex mechanisms of cancer cells, including specialized phenotypes that enable them to proliferate, invade tissues, and evade immunosurveillance, they exhibit a pronounced resistance to chemo- and radiotherapy. More advanced tumors create a hypoxic environment that supports their proliferation and survival, while robust angiogenesis ensures a constant supply of nutrients. In GBM, these structures are very pronounced and contribute to the creation and maintenance of a highly immunosuppressive microenvironment that promotes tumor growth and immune escape. In addition, the high accumulation of immunosuppressive tumor-infiltrating leukocytes and other cells, the pronounced expression of immune checkpoint molecules, and the low mutational burden, i.e., the low number of neoantigens, are hallmarks of GBM and contribute to the challenge of therapeutic approaches. Here, we review a number of mechanisms that GBM exploits to support tumor growth and potential treatments. These include new chemotherapeutics, tumor treating fields, and small molecules, including compounds targeting angiogenesis or blockers of tyrosine kinases that inhibit tumor cell proliferation and survival. In addition, we focus on immunotherapies such as immune checkpoint blockade or cell therapies, in particular vaccination with dendritic cells and CAR-T cells, which can either kill GBM cells directly or bypass immunosuppression by modulating the tumor microenvironment or boosting the patient's own immune response.

Patients with unmethylated O6-methylguanine-DNA methyltransferase promoter (uMGMT) glioblastoma (GBM) have a poor prognosis. Inflammatory response can affect the prognosis, for it may have a significant impact on the tumor microenvironment (TME). This study aims to identify a prognostic signature of inflammation-related genes, which can predict the prognosis of uMGMT GBM patients.

We examined the gene expression, somatic mutations, and overall survival of 159 GBM patients with uMGMT using the TCGA and CGGA databases. We identified molecular subtypes of uMGMT GBM patients based on the expression of inflammation-related genes. Furthermore, we determined principal component analysis (PCA), gene ontology (GO) analysis, pathway analysis and immune infiltration analysis between high and low-inflammation subtypes. We also examined the spatial and longitudinal heterogeneity of these two subtypes. The LASSO-Cox analyses were used to develop an inflammation-related prognostic model.

Our findings indicate that patients with uMGMT GBM can be divided into high-inflammation and low-inflammation subtypes. Patients with high levels of inflammation are more likely to develop an immunosuppressive microenvironment, which stimulates the production of immunosuppressive cytokines, immune checkpoints, and immunosuppressive cells. Nine inflammation-related genes (EREG, BDKRB1, DCBLD2, CD14, AHR, CLEC5A, LTA, SLC4A4, and LY6E) were found to have excellent predictive potential for patient survival in the prognostic model.

In conclusion, we created a new prognostic model including 9 inflammation-related genes. This model has produced meaningful results in evaluating patient prognosis, which may help with future therapeutic strategies for patients with uMGMT GBM.

The standard of care in high-grade gliomas has remained unchanged in the past 20 years. Efforts to replicate effective immunotherapies in non-cranial tumors have led to only modest therapeutical improvements in glioblastoma (GB). Here, we demonstrate that intratumoral administration of recombinant interleukin-12 (rIL-12) promotes local cytotoxic CD8 POS T cell accumulation and conversion into an effector-like state, resulting in a dose-dependent survival benefit in preclinical GB mouse models. This tumor-reactive CD8 T cell response is further supported by intratumoral rIL-12-sensing dendritic cells (DCs) and is accompanied by the co-stimulatory receptor 4-1BB expression on both cell types. Given that DCs and CD8 POS T cells are functionally suppressed in the tumor microenvironments of de novo and recurrent glioma patients, we tested whether anti-tumor response at the rIL-12-inflamed tumor site could be enhanced with 4-1BBL, the ligand of 4-1BB. 4-1BBL was delivered using an adeno-associated virus (AAV) vector targeting GFAP-expressing cells and resulted in prolonged survival of rIL-12 treated GB-bearing mice. This study establishes that tumor antigen-specific CD8 T cell activity can be directed using an AAV-vector-mediated gene therapy approach, effectively enhancing anti-GB immunity.

The synergistic approach of combining photodynamic immunotherapy with endogenous clearance of PD-L1 immune checkpoint blockade therapy holds promise for enhancing survival outcomes in glioblastoma (GBM) patients. The observed upregulation of O-GlcNAc glycolysis in tumors may contribute to the stabilization of endogenous PD-L1 protein, facilitating tumor immune evasion. This study presents a pH-adapted excited state intramolecular proton transfer (ESIPT)-isomerized β-ketoamide-based covalent organic framework (COF) nanoplatform (denoted as OT@COF-RVG). Temozolomide (TMZ) and OSMI-4 (O-GlcNAc transferase inhibitor) were integrated into COF cavities, then modified on the surface with polyethylene glycol and the rabies virus peptide RVG-29, showing potential for sensitizing TMZ chemotherapy and initiating photodynamic therapy (PDT). By inhibiting O-GlcNAc and promoting lysosomal degradation of PD-L1, OT@COF-RVG enhanced the effectiveness of immune checkpoint blockade (ICB) therapy. Additionally, treatment with OT@COF-RVG led to a notable elevation in reactive oxygen species (ROS) levels, thereby re-establishing an immunostimulatory state, inducing immunogenic cell death (ICD). In summary, our research unveiled a correlation between O-GlcNAc in GBM and the evasion of immune responses by tumors, while showcasing the potential of OT@COF-RVG in reshaping the immunosuppressive microenvironment of GBM and offering a more effective approach to immunotherapy in clinical settings.

High-grade gliomas are devastating cancers that remain incurable with standard surgical resection and radiochemotherapy. Although beneficial against neoplasms, radiation lowers lymphocyte counts, weakens immune activation, and recruits suppressive myeloid cells impairing immune responses. Tumor environments treated with radiation experience long-term immunosuppression, reducing immunotherapy effectiveness and contributing to recurrence. Investigating pre-radiation treatments in newly diagnosed patients could identify active agents, assess immunotherapy impact, and enable multiomic analyses without radiation-induced confounding factors. This literature review was conducted to describe the feasibility, safety, and outcomes of postsurgical, pre-radiation clinical trials for adults with newly diagnosed high-grade glioma.

A systematic review was performed of the English-language literature reporting results of clinical trials for adults with newly diagnosed high-grade glioma administered postsurgical treatment prior to radiation therapy. A search was conducted in PubMed and references cited in research and review articles were also considered.

From 1991 to 2024, 52 clinical trials were identified: 3 phase I, 38 phase II, 4 phase III, and 7 of unknown phase. Nine trials were randomized, 24 were multicenter trials, 21 investigated temozolomide-containing regimens, and 12 focused on inoperable tumors, involving a total of 2737 patients.

Pre-radiation neoadjuvant studies are feasible and may identify active drugs. This is particularly relevant in the era of personalized medicine with brain-penetrant drugs, targeted therapy, and immuno-oncology advancements. Investigating pre-radiation treatments in newly diagnosed high-grade glioma is a viable approach to rapidly identify active and inactive regimens while the immune system and tumor microenvironment remain intact.

Glioblastoma (GBM), the most common and aggressive primary central nervous system (CNS) tumor in adults, continues to have a dismal prognosis. Across hundreds of clinical trials, few novel approaches have translated to clinical practice while survival has improved by only a few months over the past three decades. Randomized controlled trials of immune checkpoint inhibitors (ICIs), which have seen impressive success for advanced or metastatic extracranial solid tumors, have so far failed to demonstrate a clinical benefit for patients with GBM. This has been secondary to GBM heterogeneity, the unique immunosuppressive CNS microenvironment, immune-evasive strategies by cancer cells, and the rapid evolution of tumor on therapy. This review aims to summarize findings from major clinical trials of ICIs for GBM, review historic failures, and describe currently promising avenues of investigation. We explore the biological mechanisms driving ICI responses, focusing on the role of the tumor microenvironment, immune evasion, and molecular biomarkers. Beyond conventional monotherapy approaches targeting PD-1, PD-L1, CTLA-4, we describe emerging approaches for GBM, such as dual-agent ICIs, and combination of ICIs with oncolytic virotherapy, antigenic peptide vaccines, chimeric antigenic receptor (CAR) T-cell therapy, along with nanoparticle-based delivery systems to enhance ICI efficacy. We highlight potential strategies for improving patient selection and treatment personalization, along with real-time, longitudinal monitoring of therapeutic responses through advanced imaging and liquid biopsy techniques. Integrated radiomics, tissue, and plasma-based analyses, may potentially uncover immunotherapeutic response signatures, enabling early, adaptive therapeutic adjustments. By specifically targeting current therapeutic challenges, outcomes for GBM patients may potentially be improved.

Glioblastoma (GBM) is a highly aggressive primary brain tumor that carries a grim prognosis. Because of the dearth of treatment options available for treatment of GBM, Chimeric Antigen Receptor (CAR)-engineered T cell and Natural Killer (NK) therapy could provide alternative strategies to address the challenges in GBM treatment. In these approaches, CAR T and NK cells are engineered for cancer-specific immunotherapy by recognizing surface antigens independently of major histocompatibility complex (MHC) molecules. However, the efficacy of CAR T cells is hindered by GBM's downregulation of its targeted antigens. CAR NK cells face similar challenges, but, in contrast, they offer advantages as off-the-shelf allogeneic products, devoid of graft-versus-host disease (GVHD) risk as well as anti-cancer activity beyond CAR specificity, potentially reducing the risk of relapse or resistance. Despite CAR T cell therapies being extensively studied in clinical settings, the use of CAR-modified NK cells in GBM treatment remains largely in the preclinical stage. This review aims to discuss recent advancements in NK cell and CAR T cell therapies for GBM, including methods for introducing CARs into both NK cells and T cells, addressing manufacturing challenges, and providing evidence supporting the efficacy of these approaches from preclinical and early-phase clinical studies. The comprehensive evaluation of CAR-engineered NK cells and CAR T cells seeks to identify the optimal therapeutic approach for GBM, contributing to the development of effective immunotherapies for this devastating disease.

Glioblastoma (GBM) is the most common primary malignant brain tumor, with fewer than 5% of patients surviving five years after diagnosis. The introduction of immune checkpoint inhibitors (ICIs), followed by chimeric antigen receptor (CAR) T-cell therapy, marked major advancements in oncology. Despite demonstrating efficacy in other blood and solid cancers, these therapies have yielded limited success in clinical trials for both newly diagnosed and recurrent GBM. A deeper understanding of GBM's resistance to immunotherapy is essential for enhancing treatment responses and translating results seen in other cancer models.

In this review, we examine clinical trial outcomes involving ICIs and CAR-T for GBM patients and explore the evasive mechanisms of GBM and the tumor microenvironment.

Multiple clinical trials investigating ICIs in GBM have shown poor outcomes, with no significant improvement in progression-free survival (PFS) or overall survival (OS). Results from smaller case studies with CAR-T therapy have warranted further investigation. However, no large-scale trials or robust studies have yet established these immunotherapeutic approaches as definitive treatment strategies. Future research should shift focus from addressing the scarcity of functional T cells to exploiting the abundant myeloid-derived cells within the tumor microenvironment.

Translating these therapies into effective treatments for glioblastoma in humans remains a significant challenge. The highly immunosuppressive nature of GBM and its tumor microenvironment continue to hinder the success of these innovative immunotherapeutic approaches. Targeting the myeloid-derived compartment may lead to more robust and sustained immune responses.

Glioblastoma (GBM) cells leverage complex endogenous and environmental regulatory mechanisms to drive proliferation, invasion, and metastasis. Tumor immune evasion, facilitated by a multifactorial network, poses a significant challenge to effective therapy, as evidenced by the limited clinical benefits of monotherapies, highlighting the adaptive nature of immune evasion. This review explores glioblastoma's immune evasion mechanisms, the role of ICIs in the tumor microenvironment, and recent clinical advancements, offering theoretical insights and directions for monotherapy and combination therapy in glioblastoma management.

In recent years, significant breakthroughs have been made in cancer therapy, particularly with the development of molecular targeted therapies and immunotherapies, owing to advances in tumor molecular biology and molecular immunology. High-grade gliomas (HGGs), characterized by their high malignancy, remain challenging to treat despite standard treatment regimens, including surgery, radiotherapy, chemotherapy, and tumor treating fields (TTF). These therapies provide limited efficacy, highlighting the need for novel treatment strategies. Molecular targeted therapies and immunotherapy have emerged as promising avenues for improving treatment outcomes in high-grade gliomas. This review explores the current status and recent advancements in targeted and immunotherapeutic approaches for high-grade gliomas.

The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitro model and the choice of cellular response assay are critical for reliable predictions. Fluorescence lifetime imaging microscopy (FLIM) is a powerful, non-destructive tool that enables direct monitoring of cellular metabolism on a label-free basis with a potential to resolve metabolic rearrangements in immune cells associated with their reactivity.

The aim of the study was to develop a patient-derived glioma explant model enriched by autologous peripheral lymphocytes and explore FLIM of the redox-cofactor NAD(P)H in living lymphocytes to measure the responses of the model to immune checkpoint inhibitors.

The light microscopy, FLIM of NAD(P)H and flow cytometry were used.

The results demonstrate that the responsive models displayed a significant increase in the free NAD(P)H fraction α1 after treatment, associated with a shift towards glycolysis due to lymphocyte activation. The non-responsive models exhibited no alterations or a decrease in the NAD(P)H α1 after treatment. The FLIM data correlated well with the standard assays of immunotherapy drug response in vitro, including morphological changes, the T-cells activation marker CD69, and the tumor cell proliferation index Ki67.

The proposed platform that includes tumor explants co-cultured with lymphocytes and the NAD(P)H FLIM assay represents a promising solution for the patient-specific immunotherapeutic drug screening.

Glioblastoma multiforme (GBM) is the most common and aggressive adult glioma (16-month median survival). Its immunosuppressive microenvironment limits the efficacy of immune checkpoint inhibitors (ICIs).

To assess the effects of the ICIs antibodies anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (anti-PD-L1) in treating adults with diffuse glioma.

We searched CENTRAL, MEDLINE, Embase, and clinical trials registers on 8 March 2024.

We included randomised controlled trials (RCTs) evaluating adults with diffuse glioma treated with anti-PD-1/PD-L1 compared to placebo or other therapies used alone or with other ICIs. Primary outcomes were overall survival (OS), progression-free survival (PFS), and serious adverse events (SAE). Secondary outcomes were overall response rate (ORR), quality of life (QoL), and less serious AEs.

We followed standard Cochrane methods.

We included seven RCTs evaluating anti-PD-1 treatment in recurrent (N = 4) and newly diagnosed (N = 3) grade 4 glioma participants. The analysis encompassed 1953 participants; sample sizes ranged from 35 to 716. Meta-analyses were not possible due to heterogeneity and the small number of studies. Most trials had high risk of bias. Nivolumab versus bevacizumab in people with recurrent GBM (1 trial, 369 participants) Nivolumab probably does not increase OS (hazard ratio (HR) 1.04, 95% confidence interval (CI) 0.83 to 1.30; 1.3% more, 95% CI 6.30 fewer to 7.80 more; 369 participants; moderate-certainty evidence) or PFS (HR 1.97, 95% CI 1.57 to 2.48; 16.40% more, 95% CI 12.40 more to 19.00 more; 369 participants; moderate-certainty evidence). The evidence for SAE is very uncertain (risk ratio (RR) 1.20, 95% CI 0.74 to 1.92; 347 participants). Nivolumab probably does not increase ORR (RR 0.34, 95% CI 0.18 to 0.63; 309 participants; moderate-certainty evidence), but may not increase less serious AEs (RR 1.03, 95% CI 0.96 to 1.10; 347 participants; low-certainty evidence). Nivolumab plus bevacizumab 10 mg/kg versus nivolumab plus bevacizumab 3 mg/kg in people with recurrent GBM (1 trial, 90 participants) Nivolumab plus bevacizumab 10 mg/kg may not increase OS (HR 1.39, 95% CI 0.86 to 2.25; 9.90% more, 95% CI 5.20 fewer to 18.80 more; 90 participants; low-certainty evidence). The evidence for PFS (HR 1.23, 95% CI 0.78 to 1.93; 5.80% more, 95% CI 8.20 fewer to 14.20 more; 90 participants) and SAE (RR 1.19, 95% CI 0.79 to 1.79; 90 participants) is very uncertain. Nivolumab may not increase less serious AEs (RR 1.02, 95% CI 0.96 to 1.09; low-certainty evidence; 90 participants). Pembrolizumab plus bevacizumab versus pembrolizumab in people with recurrent GBM (1 trial, 80 participants) The evidence for OS (HR 1.03, 95% CI 0.65 to 1.63; 0.30% more, 95% CI 7.60 fewer to 2.90 more; 80 participants), PFS (HR 0.97, 95% CI 0.61 to 1.54: 0.40% fewer, 95% CI 9.20 fewer to 2.80 more; 80 participants), SAE (RR 1.32, 95% CI 0.75 to 2.42; 80 participants), and ORR (RR 12.76, 95% CI 0.77 to 210.27; 80 participants) is very uncertain. Pembrolizumab plus bevacizumab may not increase less serious AEs (RR 1.04, 95% CI 0.96 to 1.13; 80 participants; low-certainty evidence). Neoadjuvant (before surgical resection) and adjuvant (after surgical resection) pembrolizumab versus adjuvant-only pembrolizumab in people with recurrent GBM (1 trial, 35 participants) The evidence for OS (HR 0.39, 95% CI 0.17 to 0.92; 25.20% fewer, 95% CI 37.10 fewer to 2.10 fewer; 35 participants), PFS (HR 0.43, 95% CI 0.20 to 0.91; 30.10% fewer, 95% CI 52.20 fewer to 3.60 fewer; 35 participants), and SAE (RR 1.00, 95% CI 0.31 to 3.28; 32 participants) is very uncertain. Nivolumab plus radiotherapy versus temozolomide plus radiotherapy in people with newly diagnosed unmethylated GBM (1 trial, 560 participants) Nivolumab plus radiotherapy probably does not increase OS (HR 1.31, 95% CI 1.09 to 1.58 months; 8.30% more, 95% CI 2.80 more to 12.90 more; 560 participants) and PFS (HR 1.38, 95% CI 1.15 to 1.65 months; 7.50% more, 95% CI 3.60 more to 10.30 more; 560 participants; moderate-certainty evidence). The evidence for SAE is very uncertain (RR 0.87, 95% CI 0.65 to 1.18; 553 participants). It may not increase ORR (RR 1.08, 95% CI 0.43 to 2.69; 560 participants; low-certainty evidence) and probably does not increase less serious AEs (RR 1.00, 95% CI 0.96 to 1.04; 560 participants; moderate-certainty evidence). The evidence for time to deterioration of QoL is very uncertain (HR 0.76, 95% CI 0.59 to 0.99; 560 participants). Nivolumab plus temozolomide plus radiotherapy versus placebo plus temozolomide plus radiotherapy in people with newly diagnosed methylated GBM (1 trial, 716 participants) Nivolumab plus temozolomide plus radiotherapy probably does not increase OS (HR 1.10, 95% CI 0.92 to 1.32; 3.50 more, 95% CI 3.80 fewer to 9.60 more; 716 participants) and PFS (HR 1.10, 95% CI 0.92 to 1.32; 3.00 more, 95% CI 3.50 fewer to 7.90 more; 716 participants), and probably increases SAE (RR 2.91, 95% CI 2.05 to 4.12; 709 participants; moderate-certainty evidence). It does not increase less serious AEs (RR 1.02, 95% CI 1.00 to 1.04; 709 participants; high-certainty evidence). Adjuvant nivolumab plus temozolomide versus temozolomide in older people with GBM (1 trial, 103 participants) Nivolumab plus temozolomide probably does not increase OS (HR 0.85, 95% CI 0.54 to 1.33; 3.10 fewer, 95% CI 15.80 fewer to 3.60 more; 103 participants; moderate-certainty evidence) and PFS (HR 0.77, 95% CI 0.49 to 1.19; 5.40 fewer, 95% CI 19.10 fewer to 2.40 more; 103 participants; moderate-certainty evidence). The evidence for SAE is very uncertain (RR 1.58, 95% CI 0.88 to 2.81; 103 participants). The evidence for QoL is very uncertain (results only reported graphically; 103 participants).

In recurrent GBM, nivolumab alone probably has no benefit. Anti-PD1 plus bevacizumab may also be ineffective based on low- to very low-certainty evidence. Neoadjuvant plus adjuvant pembrolizumab may improve OS and PFS, but this was based on only one small trial and very low-certainty evidence. In newly diagnosed GBM, nivolumab plus radiotherapy in unmethylated and plus radiotherapy plus temozolomide in methylated GBM probably has no benefit. In older participants, adjuvant nivolumab probably offers no benefit.

By simultaneously staining multiple immunomarkers on a single tissue section, multiplexed immunohistochemistry (mIHC) enhances the amount of information that can be observed in a single tissue section and thus can be a powerful tool to visualise cellular interactions directly in the tumour microenvironment. Performing mIHC remains technically and practically challenging, and this technique has many limitations if not properly validated. However, with proper validation, heterogeneity between histopathological images can be avoided.

This review aimed to summarize the currently used methods and to propose a standardised method for effective mIHC.

An extensive literature review was conducted to identify different methods currently in use for mIHC.

Guidelines for antibody selection, panel design, antibody validation and analytical strategies are given. The advantages and disadvantages of each method are discussed.

This review summarizes widely used pathology imaging software and discusses the potential for automation of pathology image analysis so that mIHC technology can be a truly powerful tool for research as well as clinical use.

Gliomas and glioblastomas (GBM) are common primary malignant brain tumors, which are highly malignant and have a poor prognosis. The presence of cancer stem cells with unrestricted proliferative capacity and ability to generate glial neoplastic cells, the diffuse nature of GBM, and other specific factors of GBM contribute to poor results of drug therapy in patients with GBM. Despite the worldwide efforts to improve the treatment, many novel anti-GBM drugs are active just in vitro, in silico, and in preclinical trials, and they sometimes demonstrate poor or no activity in clinical trials. In this paper, we have casually selected and analyzed the most promising evidence-based results related to glioblastoma treatment at FDA and Clinical Trials.gov databases. It was observed that the most prospective trend in the development of anti-GBM drugs is combination therapy vs. monotherapy. Our analysis of clinical trials has allowed us to predict that the most promising combination therapy that has shown the best results in patient's surveillance should include drugs that block different growth-promoting signals in glioblastoma cells and that are activated by the V600E BRAF mutation. One drug should inhibit signals from the BRAF protein, whereas the second drug in combination should inhibit signals from the MEK protein.

The content of this review is based on information obtained from PubMed, ClinicalTrials.- gov, and the U.S. Food and Drug Administration (https://www.fda.gov/). In ClinicalTrials.gov, we retrieved studies published from January 1, 2015. In the data search, "Glioblastoma" was used as the keyword. A study was deleted if it studied remedies for concomitant tumor diseases, as well as if it did not include descriptions of treatment methods and/or if GBM was not mentioned. The analysis of the effectiveness of treatment was carried out according to the increasing overall survival in GBM patients, compared to the gold standard for this cancer.

GBM patients treated with novel immunotherapy agents and drugs acting on epigenetic factors and receptor tyrosine kinase inhibitors have shown encouraging potential for future development in clinic. However, combinations of drugs have led to more significant improvements in the results and an increase in life expectancy of patients. For example, the combination of nivolumab and ipilimumab showed a 72% increase in life expectancy compared to using nivolumab alone (9.8 vs. 16.85).

Combining anti-GBM drugs appears to be a key direction for increasing treatment effectiveness and overall survival. Radiotherapy of GBM can increase the effect of combination drug therapy.

The limited success of immune checkpoint inhibitors (ICIs) in the adjuvant setting for glioblastoma highlights the need to explore administering ICIs prior to immunosuppressive radiation. To address the feasibility and safety of this approach, we conducted a phase I study in patients with newly diagnosed Grade 3 and Grade 4 gliomas. Patients received nivolumab 300 mg every 2 weeks and ipilimumab 1 mg/kg every 6 weeks until disease progression or unacceptable toxicity. Fifteen patients were treated, with four patients on dexamethasone at treatment initiation and five tumors having MGMT promoter methylated. Treatment began a median of 38 days post-surgery. The most common treatment-related adverse events (AEs) were rash, pruritus, fatigue, nausea, and anorexia. Grade 3 AEs were lipase increased (n = 2), anorexia (n = 1), pruritus (n = 1), and rash (n = 3), and one Grade 4 cerebral edema occurred. Median progression-free survival (mPFS) was 1.3 months and median overall survival (mOS) was 19.3 months (95% CI, 12.9-NA). Three patients deferred conventional radiochemotherapy for over seven months while ten eventually received it. Progressing tumors tended to exhibit higher LAG-3 levels at baseline compared to shrinking tumors. Analysis of paired pre-treatment and post-progression tissue (n = 5) showed trends of up-regulated TGF-β, ERBB2, ERBB3, and ERBB4 signaling pathways, downregulated PPAR signaling, decreased B cell proportions, and increased monocytes proportions in tumors post-treatment. We show nivolumab plus ipilimumab can be safely administered prior to standard radiotherapy for newly diagnosed gliomas and is operationally feasible. Clinicaltrials.gov NCT03425292 registered February 7, 2018.

Glioblastoma (GBM) is an aggressive and lethal type of brain tumor in human adults. The standard of care offers minimal clinical benefit, and most GBM patients experience tumor recurrence after treatment. In recent years, significant advancements have been made in the development of novel immunotherapies or other therapeutic strategies that can overcome immunotherapy resistance in many advanced cancers. However, the benefit of immune-based treatments in GBM is limited because of the unique brain immune profiles, GBM cell heterogeneity, and immunosuppressive tumor microenvironment. In this review, we present a detailed overview of current immunotherapeutic strategies and discuss the challenges and potential molecular mechanisms underlying immunotherapy resistance in GBM. Furthermore, we provide an in-depth discussion regarding the strategies that can overcome immunotherapy resistance in GBM, which will likely require combination therapies.

Glioblastoma (GBM) is the most common central nervous system malignancy in adults. GBM may be classified as grade IV diffuse astrocytoma according to the 2021 World Health Organization revised classification of central nervous system tumors, which means it is the most aggressive, invasive, undifferentiated type of tumor. Immune checkpoint blockade (ICB), particularly anti‑programmed cell death protein‑1 (PD‑1)/PD‑1 ligand‑1 immunotherapy, has been confirmed to be successful across several tumor types. However, in GBM, this treatment is still uncommon and the efficacy is unpredictable, and <10% of patients show long‑term responses. Recently, numerous studies have been conducted to explore what factors may indicate or affect the ICB response rate in GBM, including molecular alterations, immune expression signatures and immune infiltration. The present review aimed to summarize the current progress to improve the understanding of immunotherapy for GBM.

Glioma is one of the common tumors in the central nervous system, and its treatment methods (surgery, radiotherapy, and chemotherapy) lack specificity and have a poor prognosis. With the development of immunology, cell biology, and genomics, tumor immunotherapy has ushered in a new era of tumor therapy, achieving significant results in other invasive cancers such as advanced melanoma and advanced non-small cell lung cancer. Currently, the clinical trials of immunotherapy in glioma are also progressing rapidly. Here, this review summarizes promising immunotherapy methods in recent years, reviews the current status of clinical trials, and discusses the challenges and prospects of glioma immunotherapy.

Combination immunotherapy holds promise for improving survival in responsive glioblastoma (GBM) patients. Programmed death-ligand 1 (PD-L1) expression in immune microenvironment (IME) is the most important predictive biomarker for immunotherapy. Due to the heterogeneous distribution of PD-L1, post-operative histopathology fails to accurately capture its expression in residual tumors, making intra-operative diagnosis crucial for GBM treatment strategies. However, the current methods for evaluating the expression of PD-L1 are still time-consuming.

To overcome the PD-L1 heterogeneity and enable rapid, accurate, and label-free imaging of PD-L1 expression level in GBM IME at the tissue level.

We proposed a novel intra-operative diagnostic method, Machine Learning Cascade (MLC)-based Raman histopathology, which uses a coordinate localization system (CLS), hierarchical clustering analysis (HCA), support vector machine (SVM), and similarity analysis (SA). This method enables visualization of PD-L1 expression in glioma cells, CD8+ T cells, macrophages, and normal cells in addition to the tumor/normal boundary. The study quantified PD-L1 expression levels using the tumor proportion, combined positive, and cellular composition scores (TPS, CPS, and CCS, respectively) based on Raman data. Furthermore, the association between Raman spectral features and biomolecules was examined biochemically.

The entire process from signal collection to visualization could be completed within 30 min. In an orthotopic glioma mouse model, the MLC-based Raman histopathology demonstrated a high average accuracy (0.990) for identifying different cells and exhibited strong concordance with multiplex immunofluorescence (84.31 %) and traditional pathologists' scoring (R2 ≥ 0.9). Moreover, the peak intensities at 837 and 874 cm-1 showed a positive linear correlation with PD-L1 expression level.

This study introduced a new and extendable diagnostic method to achieve rapid and accurate visualization of PD-L1 expression in GBM IMB at the tissular level, leading to great potential in GBM intraoperative diagnosis for guiding surgery and post-operative immunotherapy.

Postoperative radiotherapy currently stands as the cornerstone of glioblastoma (GBM) treatment. Nevertheless, low-dose radiotherapy has been proven ineffective for GBM, due to hypoxia in the GBM microenvironment, which renders the resistance to radiation-induced cell death. Moreover, the overexpression of the PLK1 gene in glioma cells enhances GBM proliferation, invasion, metastasis, and resistance to radiation. This study introduced a hybrid membrane-camouflaged biomimetic lipid nanosensitizer (CNL@miPA), which efficiently encapsulated gold nanoclusters (PA) and miR-593-5p by a chimeric membrane derived from lipids, cancer cells, and natural killer cells. CNL@miPA exhibited exceptional blood-brain barrier and tumor tissue penetration, effectively ameliorating hypoxia and synergizing with radiotherapy. By enabling prolonged miRNA circulation in the bloodstream and achieving high enrichment at the tumor site, CNL@miPA significantly suppressed tumor growth in combination treatment, thereby significantly extending the survival period of treated mice. Overall, the developed biomimetic nanosensitizer represented an efficient and multifunctional targeted delivery system, offering a novel strategy for gene-radiotherapy of GBM.