Bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.

Glioblastoma multiforme (GBM) is the most prevalent and lethal primary intracranial neoplasm in the adult population, with treatments of limited efficacy. Recently, bufotalin has been shown to have anti-cancer activity in a variety of cancers. This investigation aims to investigate the effect of bufotalin on GBM and elucidate its potential underlying mechanism. Our results show that bufotalin not only inhibits the proliferation and epithelial-mesenchymal transition (EMT) but also triggers apoptosis in GBM cells. The result of RNA-seq indicated that bufotalin could induce mitochondrial dysfunction. Moreover, our observations indicate that bufotalin induces an excessive accumulation of intracellular reactive oxygen species (ROS) in GBM cells, leading to mitochondrial dysfunction and the dephosphorylation of AKT. Moreover, bufotalin improved TMZ sensitivity of GBM cells in vitro and in vivo. In conclusion, bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.

Use of circulating tumor cells and microemboli to predict diagnosis and prognosis in diffuse glioma.

OBJECTIVE: Circulating tumor cell (CTC) detection is a promising noninvasive technique that can be used to diagnose cancer, monitor progression, and predict prognosis. In this study, the authors aimed to investigate the clinical utility of CTCs in the management of diffuse glioma. METHODS: Sixty-three patients with newly diagnosed diffuse glioma were included in this multicenter clinical cohort. The authors used a platform based on isolation by size of epithelial tumor cells (ISET) to detect and analyze CTCs and circulating tumor microemboli (CTMs) in the peripheral blood of patients both before and after surgery. Least absolute shrinkage and selector operation (LASSO) and Cox regression analyses were used to verify whether CTCs and CTMs are independent prognostic factors for diffuse glioma. RESULTS: CTC levels were closely related to the degree of malignancy, WHO grade, and pathological subtypes. Receiver operating characteristic curve analysis revealed that a high CTC level was a predictor for glioblastoma. The results also showed that CTMs originate from the parental tumor rather than from the circulation and are an independent prognostic factor for diffuse glioma. The postoperative CTC level is related to the peripheral immune system and patient survival. Cox regression analysis showed that postoperative CTC levels and CTM status are independent prognostic factors for diffuse glioma, and CTC- and CTM-based survival models had high accuracy in internal validation. CONCLUSIONS: The authors revealed a correlation between CTCs and clinical characteristics and demonstrated that CTCs and CTMs are independent predictors for the diagnosis and prognosis of diffuse glioma. Their CTC- and CTM-based survival models can enable clinicians to evaluate patients' response to surgery as well as their outcomes.

New trends in brain tumor immunity with the opportunities of lymph nodes targeted drug delivery.

Lymph nodes targeted drug delivery is an attractive approach to improve cancer immunotherapy outcomes. Currently, the depth of understanding of afferent and efferent arms in brain immunity reveals the potential clinical applications of lymph node targeted drug delivery in brain tumors, e.g., glioblastoma. In this work, we systematically reviewed the microenvironment of glioblastoma and its structure as a basis for potential immunotherapy, including the glial-lymphatic pathway for substance exchange, the lymphatic drainage pathway from meningeal lymphatic vessels to deep cervical lymph nodes that communicate intra- and extracranial immunity, and the interaction between the blood-brain barrier and effector T cells. Furthermore, the carriers designed for lymph nodes targeted drug delivery were comprehensively summarized. The challenges and opportunities in developing a lymph nodes targeted delivery strategy for glioblastoma using nanotechnology are included at the end.

Combination of multi-modal MRI radiomics and liquid biopsy technique for preoperatively non-invasive diagnosis of glioma based on deep learning: protocol for a double-center, ambispective, diagnostical observational study.

Background: 2021 World Health Organization (WHO) Central Nervous System (CNS) tumor classification increasingly emphasizes the important role of molecular markers in glioma diagnoses. Preoperatively non-invasive "integrated diagnosis" will bring great benefits to the treatment and prognosis of these patients with special tumor locations that cannot receive craniotomy or needle biopsy. Magnetic resonance imaging (MRI) radiomics and liquid biopsy (LB) have great potential for non-invasive diagnosis of molecular markers and grading since they are both easy to perform. This study aims to build a novel multi-task deep learning (DL) radiomic model to achieve preoperative non-invasive "integrated diagnosis" of glioma based on the 2021 WHO-CNS classification and explore whether the DL model with LB parameters can improve the performance of glioma diagnosis. Methods: This is a double-center, ambispective, diagnostical observational study. One public database named the 2019 Brain Tumor Segmentation challenge dataset (BraTS) and two original datasets, including the Second Affiliated Hospital of Nanchang University, and Renmin Hospital of Wuhan University, will be used to develop the multi-task DL radiomic model. As one of the LB techniques, circulating tumor cell (CTC) parameters will be additionally applied in the DL radiomic model for assisting the "integrated diagnosis" of glioma. The segmentation model will be evaluated with the Dice index, and the performance of the DL model for WHO grading and all molecular subtype will be evaluated with the indicators of accuracy, precision, and recall. Discussion: Simply relying on radiomics features to find the correlation with the molecular subtypes of gliomas can no longer meet the need for "precisely integrated prediction." CTC features are a promising biomarker that may provide new directions in the exploration of "precision integrated prediction" based on the radiomics, and this is the first original study that combination of radiomics and LB technology for glioma diagnosis. We firmly believe that this innovative work will surely lay a good foundation for the "precisely integrated prediction" of glioma and point out further directions for future research. Clinical trail registration: This study was registered on ClinicalTrails.gov on 09/10/2022 with Identifier NCT05536024.

[Corrigendum] HHLA2 is a novel prognostic predictor and potential therapeutic target in malignant glioma.

Following the publication of this article, an interested reader drew to the authors' attention that, in Fig. 1F on p. 2311 showing a representative high­grade glioma specimen, the data were either duplicated or overlapping with the data featured in Fig. 1D, which showed a low­grade glioma specimen. After having consulted their original data, the authors have realized that the data for Fig. 1D were inadvertently selected incorrectly. The corrected version of Fig. 1, now showing the correct data for the high­magnification high­grade glioma specimen in Fig. 1F, is shown on the next page. The authors sincerely apologize for the error that was introduced during the preparation of this figure, thank the Editor of Oncology Reports for granting them the opportunity to publish a Corrigendum, and are grateful to the reader for alerting them to this issue. The authors also regret any inconvenience that this mistake may have caused. [Oncology Reports 42: 2309-2322, 2019; DOI: 10.3892/or.2019.7343].

The RNA-binding protein CSTF2 regulates BAD to inhibit apoptosis in glioblastoma.

RNA-binding proteins (RBP) regulate several aspects of co- and post-transcriptional gene expression in cancer cells. CSTF2 is involved in the expression of many cellular mRNAs and involved in the 3'-end cleavage and polyadenylation of pre-mRNAs to terminate transcription. However, the role of CSTF2 in human glioblastoma (GBM) and the underlying mechanisms remain unclear. In the present study, CSTF2 was found to be upregulated in GBM, and its high expression predicted poor prognosis. Knockdown CSTF2 induced GBM cell apoptosis both in vitro and in vivo. Specific mechanism studies showed that CSTF2 unstabilized the mRNA of the BAD protein by shortening its 3' UTR. Additionally, an increase in the expression level of CSTF2 decreased the expression level of BAD. In conclusion, CSTF2 binds to the mRNA of the BAD protein to shorten its 3'UTR, which negatively affects the BAD mediated apoptosis and promotes GBM cell survival.

Esterase-responsive and size-optimized prodrug nanoparticles for effective intracranial drug delivery and glioblastoma treatment.

Glioblastoma multiforme (GBM) is the intracranial malignancy with the highest rates of morbidity and mortality. Chemotherapy is often ineffective against GBM due to the presence of the blood-brain barrier (BBB); however, the application of nanotechnology is expected to overcome this limitation. Poly(lactic-co-glycolic acid) (PLGA) is a degradable and nontoxic functional polymer with good biocompatibility that is widely used in the pharmaceutical industry. Previous studies have shown that the ability of PLGA nanoparticles (NPs) to penetrate the BBB is largely determined by their size; however, determination of the optimal PLGA NP size requires further research. Here, we report a tandutinib-based prodrug (proTan), which responds to the GBM microenvironment, that was combined with NPs to overcome the BBB. AMD3100-PLGA NPs loaded with proTan inhibited tumor growth and effectively prolonged the survival of tumor-bearing mice.

A Novel Karyoplasmic Ratio-Based Automatic Recognition Method for Identifying Glioma Circulating Tumor Cells.

Background: Detection of circulating tumor cells (CTCs) is a promising technology in tumor management; however, the slow development of CTC identification methods hinders their clinical utility. Moreover, CTC detection is currently challenging owing to major issues such as isolation and correct identification. To improve the identification efficiency of glioma CTCs, we developed a karyoplasmic ratio (KR)-based identification method and constructed an automatic recognition algorithm. We also intended to determine the correlation between high-KR CTC and patients' clinical characteristics. Methods: CTCs were isolated from the peripheral blood samples of 68 glioma patients and analyzed using DNA-seq and immunofluorescence staining. Subsequently, the clinical information of both glioma patients and matched individuals was collected for analyses. ROC curve was performed to evaluate the efficiency of the KR-based identification method. Finally, CTC images were captured and used for developing a CTC recognition algorithm. Results: KR was a better parameter than cell size for identifying glioma CTCs. We demonstrated that low CTC counts were independently associated with isocitrate dehydrogenase (IDH) mutations (p = 0.024) and 1p19q co-deletion status (p = 0.05), highlighting its utility in predicting oligodendroglioma (area under the curve = 0.770). The accuracy, sensitivity, and specificity of our algorithm were 93.4%, 81.0%, and 97.4%, respectively, whereas the precision and F1 score were 90.9% and 85.7%, respectively. Conclusion: Our findings remarkably increased the efficiency of detecting glioma CTCs and revealed a correlation between CTC counts and patients' clinical characteristics. This will allow researchers to further investigate the clinical utility of CTCs. Moreover, our automatic recognition algorithm can maintain high precision in the CTC identification process, shorten the time and cost, and significantly reduce the burden on clinicians.

Rho family GTPase 1 (RND1), a novel regulator of p53, enhances ferroptosis in glioblastoma.

BACKGROUND: Ferroptosis is an iron dependent cell death closely associated with p53 signaling pathway and is aberrantly regulated in glioblastoma (GBM), yet the underlying mechanism needs more exploration. Identifying new factors which regulate p53 and ferroptosis in GBM is essential for treatment. METHODS: Glioma cell growth was evaluated by cell viability assays and colony formation assays. Lipid reactive oxygen species (ROS) assays, lipid peroxidation assays, glutathione assays, and transmission electron microscopy were used to assess the degree of cellular lipid peroxidation of GBM. The mechanisms of RND1 in regulation of p53 signaling were analyzed by RT-PCR, western blot, immunostaining, co-immunoprecipitation, ubiquitination assays and luciferase reporter assays. The GBM-xenografted animal model was constructed and the tumor was captured by an In Vivo Imaging System (IVIS). RESULTS: From the The Cancer Genome Atlas (TCGA) database, we summarized that Rho family GTPase 1 (RND1) expression was downregulated in GBM and predicted a better prognosis of patients with GBM. We observed that RND1 influenced the glioma cell growth in a ferroptosis-dependent manner when GBM cell lines U87 and A172 were treated with Ferrostatin-1 or Erastin. Mechanistically, we found that RND1 interacted with p53 and led to the de-ubiquitination of p53 protein. Furthermore, the overexpression of RND1 promoted the activity of p53-SLC7A11 signaling pathway, therefore inducing the lipid peroxidation and ferroptosis of GBM. CONCLUSIONS: We found that RND1, a novel controller of p53 protein and a positive regulator of p53 signaling pathway, enhanced the ferroptosis in GBM. This study may shed light on the understanding of ferroptosis in GBM cells and provide new therapeutic ideas for GBM.

SAA1 knockdown promotes the apoptosis of glioblastoma cells via downregulation of AKT signaling.

Serum amyloid A1 (SAA1) is an inflammatory associated high-density lipoprotein. And It is also considered as a predictor and prognostic marker of cancer risk. However, its role and mechanisms in glioblastoma (GBM) still unclear. In this study, we validate that SAA1 is up-regulated in GBM, and its high expression predicts poor prognosis. SAA1 knockdown promotes the apoptosis of GBM cell. Mechanistically, SAA1 knockdown can inhibit serine/threonine protein kinase B (AKT) phosphorylation, thereby regulating the expression of apoptosis-related proteins such as Bcl2 and Bax, leading to GBM cell death. Moreover, Gliomas with low SAA1 expression have increased sensitivity to Temozolomide (TMZ). Low SAA1 expression segregated glioma patients who were treated with Temozolomide (TMZ) or with high MGMT promoter methylation into survival groups in TCGA and CGGA dataset. Our study strongly suggested that SAA1 was a regulator of cells apoptosis and acted not only as a prognostic marker but also a novel biomarker of sensitivity of glioma to TMZ.

Notch intracellular domain regulates glioblastoma proliferation through the Notch1 signaling pathway.

Notch intracellular domain (NICD), also known as the activated form of Notch1 is closely associated with cell differentiation and tumor invasion. However, the role of NICD in glioblastoma (GBM) proliferation and the underlying regulatory mechanism remains unclear. The present study aimed to investigate the expression of NICD and Notch1 downstream gene HES5 in human GBM and normal brain samples and to further detect the effect of NICD on human GBM cell proliferation. For this purpose, western blotting and immunohistochemical staining were performed to analyze the expression of NICD in human GBM tissues, while western blotting and reverse-transcription quantitative PCR experiments were used to analyze the expression of Hes5 in human GBM tissues. A Flag-NICD vector was used to overexpress NICD in U87 cells and compound E and small interfering (si) Notch1 were used to downregulate NICD. Cellular proliferation curves were generated and BrdU assays performed to evaluate the proliferation of U87 cells. The results demonstrated that compared with normal brain tissues, the level of NICD protein in human GBM tissues was upregulated and the protein and mRNA levels of Hes5 were also upregulated in GBM tissues indicating that the Notch1 signaling pathway is activated in GBM. Overexpression of NICD promoted the proliferation of U87 cells in vitro while downregulation of NICD by treatment with compound E or siNotch1 suppressed the proliferation of U87 cells in vitro. In conclusion, NICD was upregulated in human GBM and NICD promoted GBM proliferation via the Notch1 signaling pathway. NICD may be a potential diagnostic marker and therapeutic target for GBM treatment.

Circulating Tumor Cells for Glioma.

Liquid biopsy has entered clinical applications for several cancers, including metastatic breast, prostate, and colorectal cancer for CTC enumeration and NSCLC for EGFR mutations in ctDNA, and has improved the individualized treatment of many cancers, but relatively little progress has been made in validating circulating biomarkers for brain malignancies. So far, data on circulating tumor cells about glioma are limited, the application of circulating tumor cells as biomarker for glioma patients has only just begun. This article reviews the research status and application prospects of circulating tumor cells in gliomas. Several detection methods and research results of circulating tumor cells about clinical research in gliomas are briefly discussed. The wide application prospect of circulating tumor cells in glioma deserves further exploration, and the research on more sensitive and convenient detection methods is necessary.

Immune Checkpoint Targeted Therapy in Glioma: Status and Hopes.

Glioma is the most malignant primary tumor of the central nervous system and is characterized by an extremely low overall survival. Recent breakthroughs in cancer therapy using immune checkpoint blockade have attracted significant attention. However, despite representing the most promising (immunotherapy) treatment for cancer, the clinical application of immune checkpoint blockade in glioma patients remains challenging due to the "cold phenotype" of glioma and multiple factors inducing resistance, both intrinsic and acquired. Therefore, comprehensive understanding of the tumor microenvironment and the unique immunological status of the brain will be critical for the application of glioma immunotherapy. More sensitive biomarkers to monitor the immune response, as well as combining multiple immunotherapy strategies, may accelerate clinical progress and enable development of effective and safe treatments for glioma patients.

Weighted gene correlation network analysis identifies microenvironment-related genes signature as prognostic candidate for Grade II/III glioma.

Glioma is the most common malignant tumor in the central nervous system. Evidence shows that clinical efficacy of immunotherapy is closely related to the tumor microenvironment. This study aims to establish a microenvironment-related genes (MRGs) model to predict the prognosis of patients with Grade II/III gliomas. Gene expression profile and clinical data of 459 patients with Grade II/III gliomas were extracted from The Cancer Genome Atlas. Then according to the immune/stromal scores generated by the ESTIMATE algorithm, the patients were scored one by one. Weighted gene co-expression network analysis (WGCNA) was used to construct a gene co-expression network to identify potential biomarkers for predicting the prognosis of patients. When adjusting clinical features including age, histology, grading, IDH status, we found that these features were independently associated with survival. The predicted value of the prognostic model was then verified in 440 samples in CGGA part B dataset and 182 samples in CGGA part C dataset by univariate and multivariate cox analysis. The clinical samples of 10 patients further confirmed our signature. Our findings suggested the eight-MRGs signature identified in this study are valuable prognostic predictors for patients with Grade II/III glioma.

RND2 attenuates apoptosis and autophagy in glioblastoma cells by targeting the p38 MAPK signalling pathway.

BACKGROUND: Inhibition of p38 MAPK signalling leads to glioblastoma multiform (GBM) tumourigenesis. Nevertheless, the molecular mechanism that induces p38 MAPK signalling pathway silencing during GBM genesis has yet to be determined. Identifying new factors that can regulate p38 MAPK signalling is important for tumour treatment. METHODS: Flow cytometry, TUNEL assays, immunofluorescence, JC-1 assays, and western blot analyses were used to detect the apoptosis of GBM cells. The specific methods used to detect autophagy levels in GBM cells were western blot analysis, LC3B protein immunofluorescence, LC3B puncta assays and transmission electron microscopy. The functions of these critical molecules were further confirmed in vivo by intracranial xenografts in nude mice. Tumour tissue samples and clinical information were used to identify the correlation between RND2 and p62 and LC3B expression, survival time of patients, and tumour volumes in clinical patients. RESULTS: By summarizing data from the TCGA database, we found that expression of the small GTPase RND2 was significantly increased in human glioblastomas. Our study demonstrated that RND2 functions as an endogenous repressor of the p38 MAPK phosphorylation complex. RND2 physically interacted with p38 and decreased p38 phosphorylation, thereby inhibiting p38 MAPK signalling activities. The forced expression of RND2 repressed p38 MAPK signalling, which inhibited glioblastoma cell autophagy and apoptosis in vitro and induced tumour growth in the xenografted mice in vivo. By contrast, the downregulation of RND2 enhanced p38 MAPK signalling activities and promoted glioma cell autophagy and apoptosis. The inhibition of p38 phosphorylation abolished RND2 deficiency-mediated GBM cell autophagy and apoptosis. Most importantly, our study found that RND2 expression was inversely correlated with patient survival time and was positively correlated with tumour size. CONCLUSIONS: Our findings revealed a new function for RND2 in GBM cell death and offered mechanistic insights into the inhibitory effects of RND2 with regard to the regulation of p38 MAPK activation.

AKT Inhibitor SC66 Inhibits Proliferation and Induces Apoptosis in Human Glioblastoma Through Down-Regulating AKT/ß-Catenin Pathway.

Glioblastoma multiforme (GBM) is the most common intracranial malignancy in adults with the highest degree of malignancy and mortality. Due to its nature of diffuse invasiveness and high migration, GBM lacks an effective treatment strategy and is associated with poor prognosis. SC66 is a novel AKT inhibitor that has been reported to exert antiproliferative activity in many types of cancer cells. However, it remains unclear whether SC66 has antitumor effects in GBM. In this study, we found SC66 obviously suppressed U87 and U251 cell proliferation and EMT- mediated cell migration and invasion. Moreover, SC66 induced GBM cells apoptosis and arrested cell cycle in G0/G1 phase. Furthermore, SC66 also downregulated AKT signaling pathway in a concentration dependent manner. We also found the level of ß-catenin nuclear translocation was prominently downregulated after SC66 treatment. Meanwhile, TCF/LEF luciferase report assay indicated that the activity of TCF/LEF was remarkably suppressed. Elevating ß-catenin activity by using IM12 rescued SC66 inhibition-mediated GBM cell proliferation and metastasis. In addition, SC66 showed significantly suppressed the tumorigenicity compared to the control group in the xenograft mouse model. In conclusion, our study demonstrated that SC66 exerts prominently antitumor efficiency in GBM cells in vivo and in vitro by downregulated AKT/ß-catenin pathway.

Bioinformatic Profiling of Prognosis-Related Genes in Malignant Glioma Microenvironment.

BACKGROUND Gliomas are the most common primary tumors of the brain and spinal cord. The tumor microenvironment (TME) is the cellular environment in which tumors exist. This study aimed to identify the role of the TME and the effects of genes involved in the TME of malignant glioma. MATERIAL AND METHODS The ESTIMATE algorithms in the R package were used to calculate the immune and stromal scores of samples in the TCGA and GSE4290 datasets. The associations of stromal and immune scores with clinicopathological characteristics and overall survival of malignant glioma patients were assessed by analysis of variance and Kaplan-Meier analysis. Differentially expressed genes (DEGs) were obtained through the median immune and stromal score using the R package "limma". Functional enrichment analysis and the PPI network MCODE were used to analyze DEGs. RESULTS Increased immune and stromal scores were closely related with advanced glioma grade and poor prognosis (all P<0.01). In total, 558 DEGs were found and most were related to tumor prognosis. Functional enrichment analysis showed that DEGs were associated with cell-matrix regulation and immune response. Four hub modules related to tumor angiogenesis, collagen formation, and immune response were found and analyzed. Previously overlooked microenvironment-related genes such as LAMB1, FN1, ACTN1, TRIM, SERPINH1, CYBA, LAIR1, and LILRB2 showed prognostic values in malignant glioma patients. CONCLUSIONS The glioma stromal/immune scores are closely related to glioma grade, histology, and survival time. Some glioma microenvironment-related genes including LAMB1, FN1, ACTN1, TRIM6, SERPINH1, CYBA, LAIR1, and LILRB2 show prognostic values in malignant gliomas and serve as potential biomarkers.

Downregulation of LUZP2 Is Correlated with Poor Prognosis of Low-Grade Glioma.

BACKGROUND: LUZP2 is a protein limitedly expressed in the brain and spinal cord, while there are few studies on it in brain tumors. Low-grade glioma (LGG) is one of the most common brain tumors. However, the biological behavior of LGG is not very clear at present. This study was aimed at exploring the role of LUZP2 in LGG. METHODS: By data mining in The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA), the expression, clinical characteristics, and potential regulatory mechanism of LUZP2 in LGG were assessed. The regulatory miRNAs of LUZP2 were predicted using miRDB, TargetScan, and miRTarBase. Meanwhile, the potential biological function of coexpressed genes was investigated by GO and KEGG analyses. RESULTS: LUZP2 expression was downregulated with the increase of tumor grade (p < 0.05). Low LUZP2 expression independently predicted poor OS in LGG in TCGA cohort and the CGGA part B and part C cohorts (all p < 0.001). Additionally, LUZP2 was targeted by miR-142-5p according to 2 prediction databases and 1 validated database, which was negatively related to LUZP2 mRNA expression (p < 0.001). Kaplan-Meier analyses demonstrated that low miR-142-5p expression was significantly associated with poor OS (p < 0.001). Furthermore, coexpression genes of LUZP2 were significantly involved in nervous system development and metabolic pathways. CONCLUSIONS: LUZP2 may be crucial for nervous system extracellular matrix development and serve as an important clinical biomarker for LGG patients. miR-142-5p upregulation could be the upstream regulator that contributed to LUZP2 downregulation.

HHLA2 is a novel prognostic predictor and potential therapeutic target in malignant glioma.

Glioma is the most common and aggressive tumor type of the central nervous system and is associated with poor prognosis. To date, novel emerging immunotherapies have significantly improved outcomes for patients with various cancer types. Human endogenous retrovirus­H long terminal repeat­associating protein 2 (HHLA2), a newly discovered immune checkpoint molecule, has demonstrated its potential as a novel therapeutic target. Therefore, the present study aimed to investigate the clinical prognostic value of HHLA2 in gliomas and its mechanistic role. A systematic review of datasets from The Cancer Genome Atlas was performed. The RNA­seq data of a total of 669 cases were analyzed and the biological function of HHLA2 was predicted by Gene Ontology (GO) and pathway enrichment analysis. Immunohistochemistry labelling images for HHLA2 was obtained from the Human Protein Atlas. xCell was used to comprehensively analyze the model of tumor­infiltrating immune cell in glioma. The Cox proportional hazards regression model was used to predict outcomes for glioma patients. The results revealed that the expression levels of HHLA2 were significantly lower in high­grade glioma, as well as glioma with wild­type isocitrate dehydrogenase, no deletion of 1p/19q and telomerase reverse transcriptase promoter mutation. Receiver operating characteristic analysis revealed that HHLA2 was a predictor of the neural subtype. The tumor­infiltrating immune cell model indicated that HHLA2 was negatively associated with tumor­associated macrophages. GO analysis and pathway enrichment analysis revealed that HHLA2­associated genes were functionally involved in inhibition of neoplasia­associated processes. HHLA2 was significantly negatively correlated with certain genes, including interleukin­10, transforming growth factor­ß, vascular endothelial growth factor and δ­like canonical Notch ligand 4, and other immune checkpoint molecules, including programmed cell death 1, lymphocyte activating 3 and CD276. Survival analysis indicated that high expression of HHLA2 predicted a favorable prognosis. In conclusion, the present study revealed that upregulation of HHLA2 is significantly associated with a favorable outcome for patients with glioma. Targeting HHLA2 as an immune stimulator may become a valuable approach for the treatment of glioma in clinical practice.