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Communications Biology Jun 2024We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a...
We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/μL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.
Topics: Humans; Glioblastoma; Tetraspanin 30; ErbB Receptors; Extracellular Vesicles; Immunoassay; Biomarkers, Tumor; Brain Neoplasms
PubMed: 38830977
DOI: 10.1038/s42003-024-06385-1 -
Oncology Research 2024The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4...
The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory.
BACKGROUND
The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (-promoter methylation, and protein methyltransferase proteins-5 ( activity, with tumor progression has never been described.
METHODS
A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for -promoter methylation and through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated.
RESULTS
Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed -promoter methylation and 12 (54.5%) tumors had unmethylated . All IDH-wildtype tumors had unmethylated . There was a statistically significant relationship between -promoter methylation and IDH in G4 astrocytoma (-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or -methylation status (-value=0.0014). Specifically, IDH-mutant tumors that had upregulated activity and -promoter methylation, who received only TMZ, have exhibited longer PFS.
CONCLUSIONS
The relationship between , -promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated tumors. Thus, using a inhibitor in G4 astrocytomas may help in tumor regression.
Topics: Humans; Protein-Arginine N-Methyltransferases; Tumor Suppressor Proteins; Promoter Regions, Genetic; DNA Repair Enzymes; DNA Modification Methylases; DNA Methylation; Isocitrate Dehydrogenase; Male; Female; Astrocytoma; Disease Progression; Middle Aged; Mutation; Adult; Retrospective Studies; Brain Neoplasms; Neoplasm Grading; Aged; Temozolomide; Gene Expression Regulation, Neoplastic
PubMed: 38827324
DOI: 10.32604/or.2024.051112 -
Neoplasia (New York, N.Y.) Aug 2024Successful treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain neoplasm, mandates the need to develop new therapeutic strategies. In this...
Successful treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain neoplasm, mandates the need to develop new therapeutic strategies. In this study, we investigated the potential of PBI-05204 in targeting GBM stem cells (GSCs) and the underlying mechanisms. Treatment with PBI-05204 significantly reduced both the number and size of tumor spheres derived from patient-derived GSCs (GBM9, GSC28 and TS543), and suppressed the tumorigenesis of GBM9 xenografts. Moreover, PBI-05204 treatment led to a significant decrease in the expression of CD44 and NANOG, crucial markers of progenitor stem cells, in GBM9 and GSC28 GSCs. This treatment also down-regulated GRP78 expression in both GSC types. Knocking down GRP78 expression through GRP78 siRNA transfection in GBM9 and GSC28 GSCs also resulted in reduced spheroid size and CD44 expression. Combining PBI-05204 with GRP78 siRNA further decreased spheroid numbers compared to GRP78 siRNA treatment alone. PBI-05204 treatment led to increased expression of pRIP1K and pRIP3K, along with enhanced binding of RIPK1/RIPK3 in GBM9 and GSC28 cells, resembling the effects observed in GRP78-silenced GSCs, suggesting that PBI-05204 induced necroptosis in these cells. Furthermore, oleandrin, a principle active cardiac glycoside component of PBI-05204, showed the ability to inhibit the self-renewal capacity in GSCs. These findings highlight the potential of PBI-05204 as a promising candidate for the development of novel therapies that target GBM stem cells.
Topics: Endoplasmic Reticulum Chaperone BiP; Humans; Glioblastoma; Neoplastic Stem Cells; Animals; Mice; Heat-Shock Proteins; Cell Line, Tumor; Xenograft Model Antitumor Assays; Plant Extracts; Necroptosis; Brain Neoplasms; Cell Proliferation; Apoptosis; Disease Models, Animal; Hyaluronan Receptors
PubMed: 38823209
DOI: 10.1016/j.neo.2024.101008 -
European Journal of Pharmacology Aug 2024Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) that serves as a receptor for pleiotrophin (PTN) and vascular...
Protein tyrosine phosphatase receptor zeta 1 (PTPRZ1) is a transmembrane tyrosine phosphatase (TP) that serves as a receptor for pleiotrophin (PTN) and vascular endothelial growth factor A 165 (VEGFA) to regulate endothelial cell migration. In the present work, we identify a PTN peptide fragment (PTN) that inhibits the interaction of PTN and VEGFA with PTPRZ1 but not VEGF receptor 2. This peptide abolishes the stimulatory effect of PTN and VEGFA on endothelial cell migration, tube formation on Matrigel, and Akt activation in vitro. It also partially inhibits VEGFA-induced VEGF receptor 2 activation but does not affect ERK1/2 activation and cell proliferation. In vivo, PTN inhibits or dysregulates angiogenesis in the chick embryo chorioallantoic membrane and the zebrafish assays, respectively. In glioblastoma cells in vitro, PTN abolishes the stimulatory effect of VEGFA on cell migration and inhibits their anchorage-independent growth, suggesting that this peptide might also be exploited in glioblastoma therapy. Finally, in silico and experimental evidence indicates that PTN and VEGFA bind to the extracellular fibronectin type-III (FNIII) domain to stimulate cell migration. Collectively, our data highlight novel aspects of the interaction of PTN and VEGFA with PTPRZ1, strengthen the notion that PTPRZ1 is required for VEGFA-induced signaling, and identify a peptide that targets this interaction and can be exploited for the design of novel anti-angiogenic and anti-glioblastoma therapeutic approaches.
Topics: Vascular Endothelial Growth Factor A; Humans; Animals; Receptor-Like Protein Tyrosine Phosphatases, Class 5; Cell Movement; Cytokines; Carrier Proteins; Human Umbilical Vein Endothelial Cells; Neovascularization, Physiologic; Chick Embryo; Zebrafish; Protein Binding; Cell Proliferation; Cell Line, Tumor; Endothelial Cells; Neovascularization, Pathologic; Glioblastoma; Angiogenesis
PubMed: 38821164
DOI: 10.1016/j.ejphar.2024.176692 -
Journal of Translational Medicine May 2024Primary malignant brain tumours are more than one-third of all brain tumours and despite the molecular investigation to identify cancer driver mutations, the current...
BACKGROUND
Primary malignant brain tumours are more than one-third of all brain tumours and despite the molecular investigation to identify cancer driver mutations, the current therapeutic options available are challenging due to high intratumour heterogeneity. In addition, an immunosuppressive and inflammatory tumour microenvironment strengthens cancer progression. Therefore, we defined an immune and inflammatory profiling of meningioma and glial tumours to elucidate the role of the immune infiltration in these cancer types.
METHODS
Using tissue microarrays of 158 brain tumour samples, we assessed CD3, CD4, CD8, CD20, CD138, Granzyme B (GzmB), 5-Lipoxygenase (5-LOX), Programmed Death-Ligand 1 (PD-L1), O-6-Methylguanine-DNA Methyltransferase (MGMT) and Transglutaminase 2 (TG2) expression by immunohistochemistry (IHC). IHC results were correlated using a Spearman correlation matrix. Transcript expression, correlation, and overall survival (OS) analyses were evaluated using public datasets available on GEPIA2 in Glioblastoma (GBM) and Lower Grade Glioma (LGG) cohorts.
RESULTS
Seven out of ten markers showed a significantly different IHC expression in at least one of the evaluated cohorts whereas CD3, CD4 and 5-LOX were differentially expressed between GBMs and astrocytomas. Correlation matrix analysis revealed that 5-LOX and GzmB expression were associated in both meningiomas and GBMs, whereas 5-LOX expression was significantly and positively correlated to TG2 in both meningioma and astrocytoma cohorts. These findings were confirmed with the correlation analysis of TCGA-GBM and LGG datasets. Profiling of mRNA levels indicated a significant increase in CD3 (CD3D, CD3E), and CD138 (SDC1) expression in GBM compared to control tissues. CD4 and 5-LOX (ALOX5) mRNA levels were significantly more expressed in tumour samples than in normal tissues in both GBM and LGG. In GBM cohort, GzmB (GZMB), SDC1 and MGMT gene expression predicted a poor overall survival (OS). Moreover, in LGG cohort, an increased expression of CD3 (CD3D, CD3E, CD3G), CD8 (CD8A), GZMB, CD20 (MS4A1), SDC1, PD-L1, ALOX5, and TG2 (TGM2) genes was associated with worse OS.
CONCLUSIONS
Our data have revealed that there is a positive and significant correlation between the expression of 5-LOX and GzmB, both at RNA and protein level. Further evaluation is needed to understand the interplay of 5-LOX and immune infiltration in glioma progression.
Topics: Humans; Brain Neoplasms; Male; Inflammation; Female; Middle Aged; Aged; Gene Expression Regulation, Neoplastic; Adult; Lymphocytes, Tumor-Infiltrating; Tumor Microenvironment; Immunohistochemistry; Cohort Studies; Survival Analysis
PubMed: 38816839
DOI: 10.1186/s12967-024-05309-1 -
PLoS Biology May 2024Glioblastoma, the most aggressive and prevalent form of primary brain tumor, is characterized by rapid growth, diffuse infiltration, and resistance to therapies.... (Review)
Review
Glioblastoma, the most aggressive and prevalent form of primary brain tumor, is characterized by rapid growth, diffuse infiltration, and resistance to therapies. Intrinsic heterogeneity and cellular plasticity contribute to its rapid progression under therapy; therefore, there is a need to fully understand these tumors at a single-cell level. Over the past decade, single-cell transcriptomics has enabled the molecular characterization of individual cells within glioblastomas, providing previously unattainable insights into the genetic and molecular features that drive tumorigenesis, disease progression, and therapy resistance. However, despite advances in single-cell technologies, challenges such as high costs, complex data analysis and interpretation, and difficulties in translating findings into clinical practice persist. As single-cell technologies are developed further, more insights into the cellular and molecular heterogeneity of glioblastomas are expected, which will help guide the development of personalized and effective therapies, thereby improving prognosis and quality of life for patients.
Topics: Humans; Glioblastoma; Single-Cell Analysis; Brain Neoplasms; Transcriptome; Animals
PubMed: 38814900
DOI: 10.1371/journal.pbio.3002640 -
Scientific Reports May 2024Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and...
Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization.
Topics: Humans; Glioblastoma; Tumor Suppressor Proteins; DNA Repair Enzymes; Melanoma; DNA Modification Methylases; Cell Line, Tumor; Radiation Tolerance; Neoplastic Stem Cells; Promoter Regions, Genetic; DNA Methylation; DNA Repair; DNA Breaks, Double-Stranded; Gene Expression Regulation, Neoplastic; Temozolomide; Brain Neoplasms; Purines
PubMed: 38811596
DOI: 10.1038/s41598-024-61240-x -
Asian Pacific Journal of Cancer... May 2024Glioblastoma Multiforme (GBM) poses a significant challenge due to its high aggressiveness and unfavorable prognosis, with existing treatments demonstrating limited...
OBJECTIVE
Glioblastoma Multiforme (GBM) poses a significant challenge due to its high aggressiveness and unfavorable prognosis, with existing treatments demonstrating limited efficacy in prolonging survival rates. This study aimed to assess the anticancer properties of Aaptos suberitoides extracts and fraction on the U87 cell line, serving as a representative model for GBM.
METHODS
U87 cells were treated with ethanol extracts derived from Aaptos suberitoides, specifically two extracts (OAA-1 and OAA-2) and one ethyl acetate fraction (EA) isolated from specimens collected on Pramuka Island and Tinjil Island. The evaluation encompased microscopic observation and MTT assay to determine the IC50. Subsequently, antiproliferative effects were investigated through apoptosis and cell cycle assays.
RESULTS
The extract demonstrated cytotoxic activity against U87 cells, with OAA-1 and OAA-2 exhibiting IC50 values of 35.78 μg/mL and 25.38 μg/mL, respectively. OAA-1 notably induced apoptosis at 50 μg/mL and induced cell cycle arrest. On other hand, OAA-2, while also inducing apoptosis significantly, had a lesser impact on cell cycle arrest. In contrast, EA induced significant apoptosis at a concentration of 100 μg/mL.
CONCLUSION
The ethanol extracts and the ethyl acetate fraction of Aaptos suberitoides emerged as a promising candidate for Glioblastoma Multiforme cancer therapy, showing potential in inhibiting cell proliferation and inducing apoptosis.
Topics: Humans; Glioblastoma; Apoptosis; Cell Proliferation; Plant Extracts; Tumor Cells, Cultured; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Brain Neoplasms
PubMed: 38809654
DOI: 10.31557/APJCP.2024.25.5.1815 -
Aging May 2024Glioblastoma multiforme (GBM) is the most prevalent and lethal primary intracranial neoplasm in the adult population, with treatments of limited efficacy. Recently,...
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 and . In conclusion, bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.
Topics: Glioblastoma; Humans; Apoptosis; Mitochondria; Proto-Oncogene Proteins c-akt; Signal Transduction; Bufanolides; Cell Line, Tumor; Animals; Reactive Oxygen Species; Cell Proliferation; Mice; Brain Neoplasms; Epithelial-Mesenchymal Transition
PubMed: 38809514
DOI: 10.18632/aging.205883 -
The EMBO Journal Jul 2024Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and...
Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.
Topics: Glioblastoma; Animals; Neutrophils; Humans; Mice; Phagocytosis; Ferroptosis; Cell Line, Tumor; Microtubule-Associated Proteins; Necrosis
PubMed: 38806658
DOI: 10.1038/s44318-024-00130-4