Glioma Tissues Research Articles

SUMF1 overexpression promotes tumorous cell growth and migration and is correlated with the immune status of patients with glioma.

Glioma is a prevalent type of malignant tumor. To date, there is a lack of literature reports that have examined the association between sulfatase modifying factor 1 (SUMF1) and glioma. The levels of SUMF1 were examined, and their relationships with the diagnosis, prognosis, and immune microenvironment of patients with glioma were investigated. Cox and Lasso regression analysis were employed to construct nomograms and risk models associated with SUMF1. The functions and mechanisms of SUMF1 were explored and verified using gene ontology, cell counting kit-8, wound healing, western blotting, and transwell experiments. SUMF1 expression tended to increase in glioma tissues. SUMF1 overexpression was linked to the diagnosis of cancer, survival events, isocitrate dehydrogenase status, age, and histological subtype and was positively correlated with poor prognosis in patients with glioma. SUMF1 overexpression was an independent risk factor for poor prognosis. SUMF1-related nomograms and high-risk scores could predict the outcome of patients with glioma. SUMF1 co-expressed genes were involved in cytokine, T-cell activation, and lymphocyte proliferation. Inhibiting the expression of SUMF1 could deter the proliferation, migration, and invasion of glioma cells through epithelial mesenchymal transition. SUMF1 overexpression was significantly associated with the stromal score, immune cells (such as macrophages, neutrophils, activated dendritic cells), estimate score, immune score, and the expression of the programmed cell death 1, cytotoxic T-lymphocyte associated protein 4, CD79A and other immune cell marker. SUMF1 overexpression was found to be correlated with adverse prognosis, cancer detection, and immune status in patients with glioma. Inhibiting the expression of SUMF1 was observed to deter the proliferation, migration, and invasion of cancer cells. The nomograms and risk models associated with SUMF1 could predict the prognosis of patients with glioma.

DNA methylation heterogeneity attributable to a complex tumor immune microenvironment prompts prognostic risk in glioma

ABSTRACT Gliomas are malignant tumours of the human nervous system with different World Health Organization (WHO) classifications, glioblastoma (GBM) with higher grade and are more malignant than lower-grade glioma (LGG). To dissect how the DNA methylation heterogeneity in gliomas is influenced by the complex cellular composition of the tumour immune microenvironment, we first compared the DNA methylation profiles of purified human immune cells and bulk glioma tissue, stratifying three tumour immune microenvironmental subtypes for GBM and LGG samples from The Cancer Genome Atlas (TCGA). We found that more intermediate methylation sites were enriched in glioma tumour tissues, and used the Proportion of sites with Intermediate Methylation (PIM) to compare intertumoral DNA methylation heterogeneity. A larger PIM score reflected stronger DNA methylation heterogeneity. Enhanced DNA methylation heterogeneity was associated with stronger immune cell infiltration, better survival rates, and slower tumour progression in glioma patients. We then created a Cell-type-associated DNA Methylation Heterogeneity Contribution (CMHC) score to explore the impact of different immune cell types on heterogeneous CpG site (CpG ct ) in glioma tissues. We identified eight prognosis-related CpG ct to construct a risk score: the Cell-type-associated DNA Methylation Heterogeneity Risk (CMHR) score. CMHR was positively correlated with cytotoxic T-lymphocyte infiltration (CTL), and showed better predictive performance for IDH status (AUC = 0.96) and glioma histological phenotype (AUC = 0.81). Furthermore, DNA methylation alterations of eight CpG ct might be related to drug treatments of gliomas. In conclusion, we indicated that DNA methylation heterogeneity is associated with a complex tumour immune microenvironment, glioma phenotype, and patient’s prognosis.

Glioma-targeted oxaliplatin/ferritin clathrate reversing the immunosuppressive microenvironment through hijacking Fe2+ and boosting Fenton reaction

Glioma is easy to develop resistance to temozolomide (TMZ). TMZ-resistant glioma secretes interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), recruiting regulatory T cell (Treg) and inhibiting the activity of T cells and natural killer cell (NK cell), subsequently forming an immunosuppressive microenvironment. Oxaliplatin (OXA) greatly inhibits the proliferation of TMZ-resistant glioma cells, but the ability of OXA to cross blood–brain barrier (BBB) is weak. Thus, the therapeutic effect of OXA on glioma is not satisfactory. Transferrin receptor 1 (TfR1) is highly expressed in brain capillary endothelial cells and TMZ-resistant glioma cells. In this study, OXA was loaded into ferritin (Fn) to prepare glioma-targeted oxaliplatin/ferritin clathrate OXA@Fn. OXA@Fn efficiently crossed BBB and was actively taken up by TMZ-resistant glioma cells via TfR1. Then, OXA increased the intracellular H2O2 level and induced the apoptosis of TMZ-resistant glioma cells. Meanwhile, Fn increased Fe2+ level in TMZ-resistant glioma cells. In addition, the expression of ferroportin 1 was significantly reduced, resulting in Fe2+ to be locked up inside the TMZ-resistant glioma cells. This subsequently enhanced the Fenton reaction and boosted the ferroptosis of TMZ-resistant glioma cells. Consequently, T cell mediated anti-tumor immune response was strongly induced, and the immunosuppressive microenvironment was significantly reversed in TMZ-resistant glioma tissue. Ultimately, the growth and invasion of TMZ-resistant glioma was inhibited by OXA@Fn. OXA@Fn shows great potential in the treatment of TMZ-resistant glioma and prospect in clinical transformation.Graphical

Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis

Temozolomide (TMZ) represents a standard-of-care chemotherapeutic agent in glioblastoma (GBM). However, the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma. Although specific innovative approaches, such as immunotherapy, have shown favorable clinical outcomes, the inherent invasiveness of most gliomas continues to make them challenging to treat. Consequently, there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development. This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors. A total of 648 proteins exhibiting significant differential expression were identified. Gene Set Enrichment Analysis (GSEA) unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups. Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate (IP3) kinase B (ITPKB) in the recurrence group, correlating with poor survival in glioma patients. In TMZ-resistant cells, the depletion of ITPKB led to an increase in reactive oxygen species (ROS) related to NADPH oxidase (NOX) activity and restored cell sensitivity to TMZ. Mechanistically, the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination. This, in turn, elevated ITPKB stability and impaired ROS production. Furthermore, ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model. These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target for TMZ-resistant GBM.

Expression and Prognostic Role of PANK1 in Glioma.

Malignant gliomas are the most common type of primary malignant brain tumors. Pantothenate kinase 1 (PANK1) mRNA is highly expressed in several metabolic processes, implying that PANK1 plays a potential role in metabolic programming in cancers. However, the role of PANK1 in glioma has not been fully explored. Public datasets (The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), Gravendeel and Rembrandt) and validation cohort were used to explore the expression of PANK1 in glioma tissues. Kaplan-Meier and Cox regression analyses were used to explore the relationship between PANK1 and prognosis in glioma. Cell proliferation and invasion were determined using Cell Counting Kit-8 (CCK8) and transwell invasion in vitro assays. Analysis using the four public datasets and the validation cohort showed that PANK1 expression was significantly downregulated in glioma tissues compared with non-tumor tissues (P<0.01). PANK1 expression was negatively correlated with World Health Organization (WHO) grade, 1p/19q non-codeletion and isocitric dehydrogenase 1/2 (IDH1/2) wildtype. Furthermore, high expression of PANK1 was correlated with significantly better prognosis of glioma patients compared to patients with low expression of PANK1 (all P<0.01 in the four datasets). Besides, both lower-grade glioma (LGG) and glioblastoma multiform (GBM) patients with high expression of PANK1 had a significantly better prognosis than those with low expression of PANK1 in TCGA, Gravendeel and Rembrandt datasets (all P <0.01). Multivariate Cox regression analysis revealed that low PANK1 expression was an independent risk factor associated with a worse prognosis of glioma patients. Moreover, overexpression of PANK1 significantly inhibited the proliferation and invasion of U87 and U251 cells. PANK1 expression is downregulated in glioma tissues and is a novel prognostic biomarker in glioma patients.

Exosome-transmitted podoplanin promotes tumor-associated macrophage-mediated immune tolerance in glioblastoma.

Glioblastoma is the most frequent and aggressive primary brain tumor, characterized by rapid disease course and poor treatment responsiveness. The abundance of immunosuppressive macrophages in glioblastoma challenges the efficacy of novel immunotherapy. Bulk RNA-seq and single-cell RNA-seq of glioma patients from public databases were comprehensively analyzed to illustrate macrophage infiltration patterns and molecular characteristics of podoplanin (PDPN). Multiplexed fluorescence immunohistochemistry staining of PDPN, GFAP, CD68, and CD163 were performed in glioma tissue microarray. The impact of PDPN on macrophage immunosuppressive polarization was investigated using a co-culture system. Bone marrow-derived macrophages (BMDMs) and OT-II T cells isolated from BALB/c and OT-II mice respectively were co-cultured to determine T-cell adherence. Pathway alterations were probed through RNA sequencing and western blot analyses. Our findings demonstrated that PDPN is notably correlated with the expression of CD68 and CD163 in glioma tissues. Additionally, macrophages phagocytosing PDPN-containing EVs (EVsPDPN ) from GBM cells presented increased CD163 expression and augmented secretion of immunoregulatorycytokine (IL-6, IL-10, TNF-α, and TGF-β1). PDPN within EVs was also associated with enhanced phagocytic activity and reduced MHC II expression in macrophages, compromising CD4+ T-cell activation. This investigation underscores that EVsPDPN derived from glioblastoma cells contributes to M2 macrophage-mediated immunosuppression and is a potential prognostic marker and therapeutic target in glioblastoma.

UBE2C enhances temozolomide resistance by regulating the expression of p53 to induce aerobic glycolysis in glioma.

UBE2C is overexpressed in gliomas, and its overexpression has been reported to be correlated with the drug resistance of gliomas to some extent. In this study, we explore the role of UBE2C in regulating temozolomide (TMZ) resistance in glioma and investigate the underlying mechanisms involved. Twenty normal brain tissues and 100 glioma tissues from 50 TMZ-resistant patients and 50 TMZ-sensitive patients are included in this study. TMZ-resistant cell lines are constructed to explore the role of UBE2C in regulating glioma cell viability and TMZ resistance. Our results show that both the mRNA and protein levels of UBE2C are significantly elevated in the brain tissues of glioma patients, especially in those of TMZ-resistant patients. Consistently, UBE2C expression is markedly upregulated in TMZ-resistant cell lines. Overexpression of UBE2C rescues glioma cells from TMZ-mediated apoptosis and enhances cell viability. In contrast, downregulation of UBE2C expression further enhances TMZ function, increases cell apoptosis and decreases cell viability. Mechanistically, UBE2C overexpression decreases p53 expression and enhances aerobic glycolysis level by increasing ATP level, lactate production, and glucose uptake. Downregulation of p53 level abolishes the role of UBE2C downregulation in inhibiting TMZ resistance and aerobic glycolysis in glioma cells. Moreover, an animal assay confirms that downregulation of UBE2C expression further suppresses tumor growth in the context of TMZ treatment. Collectively, this study reveals that downregulation of UBE2C expression enhances the sensitivity of glioma cells to TMZ by regulating the expression of p53 to inhibit aerobic glycolysis.

Molecularly targeted protease-activated probes for visualization of glioblastoma: a comparison with 5-ALA.

The highly infiltrative growth of glioblastoma (GBM) makes distinction between the tumor and normal brain tissue challenging. Therefore, fluorescence-guided surgery is often used to improve visual identification of radiological tumor margins. The aim of this study was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to visualize GBM tissue and to compare the most promising candidate with the gold standard, 5-aminolevulinic acid (5-ALA). Single-substrate probes 6QC-ICG and 6QC-Cy5 (cysteine cathepsin cleavable), double-substrate probes AG2-FNIR and AG2-Cy5 (cysteine cathepsin and caspase 3 cleavable), and 5-ALA were administered intravenously to mice with orthotopic tumors. Activation of the probes was also evaluated in cell cultures in vitro and in biopsy material from patients with GBM ex vivo. The tumor to normal brain tissue fluorescence ratio (TNR) was quantified in brain sections using preclinical and clinical visualization platforms, and in tissue homogenates and cell suspensions using spectrofluorimetry. Subcellular localization of the fluorophores was visualized by confocal microscopy. In vitro, the single-substrate probe 6QC-ICG was cleaved in glioma cells and macrophages, and the resulting fluorophore accumulated intracellularly. In experimental GBMs, both single- and double-substrate probes visualized tumor tissue, while in healthy brain tissue the signal was minimal. TNR was highest for 6QC-ICG and AG2-FNIR, but the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, as well as human GBM biopsy material ex vivo, the authors confirmed the ability of 6QC-ICG to specifically visualize the glioma tissue using preclinical and clinical visualization platforms. Finally, a comparison with 5-ALA in animals coadministered with both compounds revealed a higher TNR for 6QC-ICG in experimental GBMs. The cysteine cathepsin-cleavable probe 6QC-ICG is activated by glioma cells and tumor-associated macrophages, leading to a high contrast between tumor and nontumorous brain tissue that is superior to that of the current standard, 5-ALA. In addition to a well-defined mechanism of action, protease-activated probes that use NIR fluorophores (e.g., indocyanine green) have the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. These results suggest that 6QC-ICG has the potential to become the targeted agent in intraoperative detection of GBM tissue using fluorescence imaging.

Homeostatic status of thyroid hormones and brain water movement as determinant factors in biology of cerebral gliomas: a pilot study using a bioinformatics approach.

The expression and localization of the water channel transporters, aquaporins (AQPs), in the brain are substantially modified in gliomas during tumorigenesis, cell migration, edema formation, and resolution. We hypothesized that the molecular changes associated with AQP1 and AQP4 in the brain may potentially be anticancer therapeutic targets. To test this hypothesis, a bioinformatics analysis of publicly available data from international consortia was performed. We used RNA-seq as an experimental strategy and identified the number of differential AQP1 and AQP4 transcript expressions in glioma tissue compared to normal brain tissue. AQPs genes are overexpressed in patients with glioma. Among the glioma subtypes, AQP1 and AQP4 were overexpressed in astrocytoma (low-grade glioma) and classical (high-grade glioma). Overall survival analysis demonstrated that both AQP genes can be used as prognostic factors for patients with low-grade glioma. Additionally, we observed a correlation between the expression of genes involved in the tyrosine and thyroid hormone pathways and AQPs, namely: PNMT, ALDH1A3, AOC2, HGDATP1B1, ADCY5, PLCB4, ITPR1, ATP1A3, LRP2, HDAC1, MED24, MTOR, and ACTB1 (Spearman's coefficient = geq 0.20 and p-value = ≤ 0.05). Our findings indicate that the thyroid hormone pathways and AQPs 1 and 4 are potential targets for new anti-tumor drugs and therapeutic biomarkers for malignant gliomas.

Deep Learning Glioma Grading with the Tumor Microenvironment Analysis Protocol for Comprehensive Learning, Discovering, and Quantifying Microenvironmental Features.

Gliomas are primary brain tumors that arise from neural stem cells, or glial precursors. Diagnosis of glioma is based on histological evaluation of pathological cell features and molecular markers. Gliomas are infiltrated by myeloid cells that accumulate preferentially in malignant tumors, and their abundance inversely correlates with survival, which is of interest for cancer immunotherapies. To avoid time-consuming and laborious manual examination of images, a deep learning approach for automatic multiclass classification of tumor grades was proposed. As an alternative way of investigating characteristics of brain tumor grades, we implemented a protocol for learning, discovering, and quantifying tumor microenvironment elements on our glioma dataset. Using only single-stained biopsies we derived characteristic differentiating tumor microenvironment phenotypic neighborhoods. The study was complicated by the small size of the available human leukocyte antigen stained on glioma tissue microarray dataset - 206 images of 5 classes - as well as imbalanced data distribution. This challenge was addressed by image augmentation for underrepresented classes. In practice, we considered two scenarios, a whole slide supervised learning classification, and an unsupervised cell-to-cell analysis looking for patterns of the microenvironment. In the supervised learning investigation, we evaluated 6 distinct model architectures. Experiments revealed that a DenseNet121 architecture surpasses the baseline's accuracy by a significant margin of 9% for the test set, achieving a score of 69%, increasing accuracy in discerning challenging WHO grade 2 and 3 cases. All experiments have been carried out in a cross-validation manner. The tumor microenvironment analysis suggested an important role for myeloid cells and their accumulation in the context of characterizing glioma grades. Those promising approaches can be used as an additional diagnostic tool to improve assessment during intraoperative examination or subtyping tissues for treatment selection, potentially easing the workflow of pathologists and oncologists.

WSCD2 Expression: Its Relevance to Tumor-Infiltrating Immune Cells and Glioma Prognosis.

Patients with glioma have limited treatment options and experience poor prognoses. Therefore, it is urgently needed to explore new diagnostic and therapeutic targets. This study aimed to investigate the relevance of WSC domain-containing 2 (WSCD2) expression to glioma, clinicopathological characteristics, tumor-infiltrating immune cells (TILs), and patient prognosis. We analyzed WSCD2 mRNA expression in glioma tissues and patient survival using the Gene Expression Profiling Interactive Analysis database. Furthermore, the relationship between the expressions of WSCD2 mRNA and TILs in gliomas was evaluated utilizing the Tumor Immune Estimation Resource database. Lastly, we employed multiplex immunohistochemistry to detect the protein expressions of WSCD2 and TILs in glioma tissues. WSCD2 mRNA expression in glioma tissues was lower than that in tissues of benign brain disease. High WSCD2 mRNA expression was also significantly associated with a favorable outcome. Additionally, WSCD2 mRNA expression was correlated with TIL expression in glioma; however, no such relationship was detected between the protein expressions of WSCD2 and TILs in glioma tissues. Cox regression multivariate analysis and Kaplan-Meier survival analysis showed that WSCD2 expression in glioma tissues could be an independent prognostic factor. This study highlights the correlation between WSCD2 expression and TILs and demonstrates the prognostic significance of WSCD2 in glioma. Furthermore, our results suggest that WSCD2 may be a potential immunotherapy target in glioma.

A custom next-generation sequencing panel for 1p/19q codeletion and mutational analysis in gliomas.

The World Health Organization has updated their classification system for the diagnosis of gliomas, combining histological features with molecular data including isocitrate dehydrogenase 1 and codeletion of chromosomal arms 1p and 19q. 1p/19q codeletion analysis is commonly performed by fluorescence in situ hybridization (FISH). In this study, we developed a 57-gene targeted next-generation sequencing (NGS) panel including 1p/19q codeletion detection mainly to assess diagnosis and potential treatment response in melanoma, gastrointestinal stromal tumor, and glioma patients. Loss of heterozygosity analysis was performed using the NGS method on 37 formalin-fixed paraffin-embedded glioma tissues that showed 1p and/or 19q loss determined by FISH. Conventional methods were applied for the validation of some glioma-related gene mutations. In 81.1% (30 of 37) and 94.6% (35 of 37) of cases, 1p and 19q were found to be in agreement whereas concordance for 1p/19q codeletion and no 1p/19q codeletion was found in 94.7% (18 of 19) and 94.4% (17 of 18) of cases, respectively. Overall, comparing NGS results with those of conventional methods showed high concordance. In conclusion, the NGS panel allows reliable analysis of 1p/19q codeletion and mutation at the same time.

Protoporphyrin IX in serum of high-grade glioma patients: A novel target for disease monitoring via liquid biopsy.

High-grade gliomas (HGG) carry a dismal prognosis. Diagnosis comprises MRI followed by histopathological evaluation of tissue; no blood biomarker is available. Patients are subjected to serial MRIs and, if unclear,surgery for monitoring of tumor recurrence, which is laborious. MRI provides only limited diagnostic information regarding the differentiation of true tumor progression from therapy-associated side effects. 5-aminolevulinic acid (5-ALA)is routinely used for induction of protoporphyrin IX (PpIX) accumulation in malignant glioma tissue, enablingimproved tumor visualizationduring fluorescence-guided resection (FGR). We investigated whether PpIX can also serve as a serum HGG marker to monitor relapse. Patients (HGG: n = 23 primary, pHGG; n = 5 recurrent, rHGG) undergoing FGR received 5-ALA following standard clinical procedure. The control group of eight healthy volunteers (HCTR) also received 5-ALA. Serum was collected before and repeatedly up to 72h after drug administration. Significant PpIX accumulation in HGG was observed after 5-ALA administration (ANOVA: p = 0.005, post-hoc: HCTR vs. pHGG p = 0.029, HCTR vs. rHGG p = 0.006). Separation of HCTR from pHGG was possible when maximum serum PpIX levels were reached (CI95% of tMax). ROC analysis of serum PpIX within CI95% of tMax showed successful classification of HCTR and pHGG (AUCROC 0.943, CI95% 0.884-1.000, p < 0.001); the optimal cut-off for diagnosis was 1275pmolPpIX/ml serum, reaching 87.0% accuracy, 90.5% positive predictive and 84.0% negative predictive value. Baseline PpIX level was similar in patient and control groups. Thus, 5-ALA is required for PpIX induction, which is safe at the standard clinical dosage. PpIX is a new target for liquid biopsy in glioma. More extensive clinical studies are required to characterize its full potential.

PBX3 as a biomarker for the early diagnosis and prediction of prognosis of glioma.

Increasing evidence have elucidated that PBX3 played a crucial role in cancer initiation and progression. PBX3 was differentially expressed in many cancer types. However, PBX3 potential involvement in gliomas remains to be explored. The expression level of PBX3 in glioma tissues and glioma cells, and its correlation with clinical features were analyzed by data from TCGA, GEPIA, CGGA and CCLE. Univariable survival and Multivariate Cox analysis was used to compare several clinical characteristics with survival. We also analyzed the correlation between PBX3 expression level and survival outcome and survival time of LGG and GBM patients by using linear regression equation. GSEA was used to generate an ordered list of all genes related to PBX3 expression and screening of genes co-expressed with PBX3 mRNA by "limma" package. The results showed that PBX3 was highly expressed in gliomas and its expression increased with the increase of malignancy. Survival analysis found that PBX3 is more valuable in predicting the OS and PFI of LGG patients than that of GBM. For further study, TCGA and CGGA data were downloaded for univariate Cox analysis and multivariate Cox analysis which showed that the expression of PBX3 was independent influencing factors for poor prognosis of LGG patients. Meanwhile, Receiver operating characteristic (ROC) curve showed that PBX3 was a predictor of overall survival rate and progression-free survival rate of LGG. Linear regression model analysis indicated that the higher expression of PBX3 the higher the risk of death of LGG patients, and the higher expression of PBX3 the higher the risk of disease progression of LGG patients. Next, TCGA data were downloaded for GSEA and Co-expression analyses, which was performed to study the function of PBX3. PBX3 may be involved in the occurrence and development of glioma, and has potential reference value for the early diagnosis and prediction of prognosis of glioma.

Basic Transcription Factor 3 Like 4 Enhances Malignant Phenotypes through Modulating Tumor Cell Function and Immune Microenvironment in Glioma

Recent investigations into the tumor microenvironment have provided insights into the limited response of glioma progression to immunotherapy. However, the specific involvement of Basic Transcription Factor 3 Like 4 (BTF3L4) in glioma progression and its correlation with immune cell infiltration remained areas of uncertainty that require further exploration. In the present study, BTF3L4 expression was delineated using gene-expression-profiling-interactive-analysis and multiplex-immunohistological staining of tissue microarrays. Then, the prognostic value of BTF3L4 was assessed using Cox regression models and Kaplan-Meier methods and conducted in vitro experiments to investigate how BTF3L4 protein impacts the proliferation, migration, and invasion capabilities of glioma cells. Furthermore, the Cibersort and Estimate methods were employed to quantify immune cells that correlate to BTF3L4 expression and applied multiplex-immunohistological staining to investigate its correlation with infiltrated immune cells in glioma tissues. These findings revealed a higher BTF3L4 expression in glioma tissues compared to non-tumor brain tissues, which correlated with clinical characteristics and worse patient prognosis. Furthermore, the downregulation of BTF3L4 protein in glioma cell line had a detrimental effect on cell migration, invasion, and proliferation. Additionally, the association between BTF3L4 and key immune molecules in glioma, particularly with the infiltration of CD66B+ neutrophils and PD-L1 expression was identified. These results highlight the prognostic significance of BTF3L4 and propose BTF3L4 as a potential target for glioma immune therapy.

Characterization of the m6A/m1A/m5C/m7G-related regulators on the prognosis and immune microenvironment of glioma by integrated analysis of scRNA-seq and bulk RNA-seq data.

Proliferation, metabolism, tumor occurrence and development in gliomas are greatly influenced by RNA modifications. However, no research has integrated the four RNA methylation regulators of m6A, m1A, m5C and m7G in gliomas to analyze their relationship with glioma prognosis and intratumoral heterogeneity. Based on three in-house single-cell RNA-sequencing (scRNA-seq) data, the glioma heterogeneity and characteristics of m6A/m1A/m5C/m7G-related regulators were elucidated. Based on publicly available bulk RNA-sequencing (RNA-seq) data, a risk-score system for predicting the overall survival (OS) for gliomas was established by three machine learning methods and multivariate Cox regression analysis, and validated in an independent cohort. Seven cell types were identified in gliomas by three scRNA-seq data, and 22 m6A/m1A/m5C/m7G-related regulators among the marker genes of different cell subtypes were discovered. Three m6A/m1A/m5C/m7G-related regulators were selected to construct prognostic risk-score model, including EIFA, NSUN6 and TET1. The high-risk patients showed higher immune checkpoint expression, higher tumor microenvironment scores, as well as higher tumor mutation burden and poorer prognosis compared with low-risk patients. Additionally, the area under the curve values of the risk score and nomogram were 0.833 and 0.922 for 3 year survival and 0.759 and 0.885 for 5 year survival for gliomas. EIF3A was significantly highly expressed in glioma tissues in our in-house RNA-sequencing data (p < 0.05). These findings may contribute to further understanding of the role of m6A/m1A/m5C/m7G-related regulators in gliomas, and provide novel and reliable biomarkers for gliomas prognosis and treatment.

LncRNA CAI2 Contributes to Poor Prognosis of Glioma through the PI3K-Akt Signaling Pathway.

We aim to explore new potential therapeutic targets and markers in human glioma. Gliomas are the most common malignant primary tumor in the brain. In the present research, we evaluated the effect of CAI2, a long non-coding RNA, on the biological behaviors of glioma and explored the related molecular mechanism. The expression of CAI2 was analyzed using qRT-PCR in 65 cases of glioma patients. The cell proliferation was determined with MTT and colony formation assays, and the PI3K-AKt signaling pathway was analyzed using western blot. CAI2 was upregulated in human glioma tissue compared with the matched, adjacent nontumor tissue and was correlated with WHO grade. Survival analyses proved that the overall survival of patients with high CAI2 expression was poor compared to that of patients with low CAI2 expression. High CAI2 expression was an independent prognostic factor in glioma. The absorbance values in the MTT assay after 96 h were .712 ± .031 for the si-control and .465 ± .018 for the si- CAI2-transfected cells, and si-CAI2 inhibited colony formation in U251 cells by approximately 80%. The levels of PI3K, p-AKt, and AKt in si-CAI2-treated cells were decreased. CAI2 may promote glioma growth through the PI3K-AKt signaling pathway. This research provided a novel potential diagnostic marker for human glioma.

Identification and validation of a novel 9-gene signature of non-specific classification to predict prognosis in glioma patients.

This study aimed to identify and validate a 9-gene signature for predicting overall survival (OS) in glioma patients. Analysis of multiple gene expression datasets led to the identification of 135 candidate genes associated with OS in glioma patients. Further analysis revealed that IGFBP2, PBK, NRXN3, TGIF1, DNAJA4, and LGALS3BP were identified as risk factors for OS, while ENAH, PPP2R2C, and SPHKAP were found to be protective factors. Multifaceted validation using different databases confirmed their differential expression patterns in glioma tissues compared to normal brain tissue. By utilizing LASSO regression and multivariate Cox regression analysis, a risk score was developed based on the expression levels of the 9 crucial genes. The risk score showed a significant correlation with OS in both training and validation cohorts and yielded superior predictive accuracy compared to individual gene expression. Moreover, a predictive nomogram incorporating the risk score, WHO grade, age, IDH mutation, and 1p/19q co-deletion was constructed and validated, which exhibited high predictive capabilities for survival rates at different time points. Enrichment analysis revealed the involvement of extracellular matrix-related pathways and immune system signaling in glioma prognosis. Furthermore, the risk score showed a strong correlation with immune cell infiltration and immune checkpoint expression, suggesting its potential role in the tumor immune microenvironment. In conclusion, our study provides a robust 9-gene signature and a predictive nomogram for evaluating the prognosis of glioma patients, offering valuable insights into personalized treatment strategies.

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

MAPK-activated protein kinase 2 is associated with poor prognosis of glioma patients and immune inhibition in glioma.

An effective therapeutic method to noticeably improve the prognosis of glioma patients has not been developed thus far. MAPK-activated protein kinase 2 (MAPKAPK2) is a serine/threonine kinase, which is involved in tumorigenesis, tumor growth, metastasis, and the inflammatory process. The clinical significance and molecular function of MAPKAPK2 in glioma remain unclear. MAPKAPK2 expression in human glioma tissues was detected by immunohistochemistry and analyzed from the transcriptome sequencing data in TCGA and CGGA. Prognostic nomogram was constructed to predict the survival risk of individual patients. GO and KEGG enrichment analyses were performed to analyze the function and pathways MAPKAPK2 involved. Single-cell RNA sequencing data was used to analyze the cell types in which MAPKAPK2 was enriched. Flow cytometry was used for cell cycle and apoptosis detection. The ability of cell proliferation and migration was analyzed by CCK8 and cell migration assay, respectively. Correlation analyses were performed to analyze the relationship of MAPKAPK2 with immune infiltration, immune regulators, chemokine, and chemokine receptors. MAPKAPK2 was not only aberrantly upregulated in glioma tissues but also correlated with poor clinical characteristics. Moreover, MAPKAPK2 was prevalent in isocitrate dehydrogenase (IDH) wild-type and 1p/19q non-codeletion glioma cohorts and predicted poor prognosis of glioma patients. MAPKAPK2 may be involved in cell proliferation, cell migration, DNA damage repair, and immune regulation in glioma. MAPKAPK2 was enriched in microglia/macrophages and malignant tumor cells. Further investigation into cellular function revealed that inhibiting MAPKAPK2 suppressed the proliferation and migration of glioblastoma multiforme (GBM) cells in vitro. The inhibition of MAPKAPK2 significantly induced the G1 cell cycle arrest and cell apoptosis of GBM cells. Consistent with the enriched function of MAPKAPK2 in immune regulation, MAPKAPK2 was correlated with immune cell infiltration in glioma tissues. Mechanistically, a series of immune regulators, immunomodulatory chemokine, and chemokine receptors were positively correlated with MAPKAPK2 expression. Our findings provide evidence of the clinical relevance of MAPKAPK2 in prognosis evaluation of glioma patients and highlight the underlying significance of MAPKAPK2 in glioma therapy.