Human Glioma Cells Research Articles

Exosomal miRNA-21 from Toxoplasma gondii-infected microglial cells induces the growth of U87 glioma cells by inhibiting tumor suppressor genes

Toxoplasma gondii is an intracellular protozoan parasite that can modulate the microenvironment of infected hosts and is known to be associated with the incidence of brain tumor growth. In this study, we suggested that the exosomal microRNA-21 derived from Toxoplasma infection would contribute to the growth of brain tumors. Exosomes of BV2 microglial cells infected with Toxoplasma were characterized and confirmed internalization to U87 glioma cells. Exosomal miRNA expression profiles were analyzed using microRNA array and miR-21A-5p associated with Toxoplasma and tumor sorted. We also examined the mRNA level of tumor-associated genes in U87 glioma cells by changing the level of miR-21 within exosomes and the effects of exosomes on the proliferation of human U87 glioma cells. Expression of miRNA-21 was increased and anti-tumorigenic genes (FoxO1, PTEN, and PDCD4) were decreased in exosomes within T. gondii-infected U87 glioma cells. Toxoplasma-infected BV2-derived exosomes induced proliferation of U87 glioma cells. The exosomes induced the growth of U87 cells in a mouse tumor model. We suggest that the increased exosomal miR-21 from Toxoplasma-infected BV2 microglial cells may play an important role as a cell growth promotor of U87 glioma cells through a down-regulation of anti-tumorigenic genes.

Microglia-Based Gene Expression Signature Highly Associated with Prognosis in Low-Grade Glioma

Simple SummaryGliomas make up ~80% of malignant brain tumors in adults and are responsible for the majority of deaths from primary brain tumors. Consequently, a better understanding of the malignant features of the TME in glioma is pertinent. The aim of our study was to evaluate the expression of immune-related genes (IRGs) in glioma and their association with patient prognosis. We utilized several approaches to interrogate the glioma immune microenvironment. We found that immune genes are generally negatively associated with survival and that overall survival was significantly lower in those with a high level of microglia infiltration. The microglia abundance was significantly associated with common genomic aberrations. Lastly, we generated a 23-gene expression signature that is highly associated with patient prognosis, independent of clinical variables. These findings are relevant to investigators in the glioma field, those working in biomarker development, but also to individuals working on glioma therapeutics.Gliomas make up ~80% of malignant brain tumors in adults and are responsible for the majority of deaths from primary brain tumors. The glioma tumor microenvironment (TME) is a dynamic, heterogeneous mixture of extracellular matrix and malignant and non-malignant cells. Several ongoing clinical trials are evaluating the efficacy of therapies that target non-malignant cells, particularly immune cells. Consequently, a better understanding of the TME in glioma is pertinent. We utilized several gene expression datasets to evaluate the relationship between immune-related genes (IRGs) and patient prognosis. We generated microglia signatures using single-cell RNAseq data from human and mouse glioma cells to infer microglia abundance. Lastly, we built a LASSO Cox regression model that predicts patient survival. We found that 428 IRGs were negatively associated with survival in glioma patients. Overall survival was significantly lower in those with a high level of microglia infiltration. In addition, we also found that microglia abundance was significantly associated with several common genomic aberrations, including IDH2 and TP53 mutations. Furthermore, we found that patients with high risk scores had significantly worse overall survival than those with low risk scores in several independent datasets. Altogether, we characterized immune features predictive of overall survival in glioma and found that microglia abundance is negatively associated with survival. We developed a 23-gene risk score that can significantly stratify patients into low- and high-risk categories.

Construction of a novel blood brain barrier-glioma microfluidic chip model: Applications in the evaluation of permeability and anti-glioma activity of traditional Chinese medicine components

Since most anti-glioma drug candidates hardly permeate through the blood-brain barrier (BBB), preclinical models that can integrate the complexity of the tumor microenvironment and the structure and function of the BBB is urgently needed for the treatment of glioma. Herein, we constructed an in vitro BBB-glioma microfluidic chip model lined by primary human brain microvascular endothelial cells, pericytes, astrocytes and glioma cells, which could recapitulate the high level of barrier function of the in vivo human BBB and glioma microenvironment. The BBB unit in BBB-glioma microfluidic chip (BBB–U251 chip) displayed selective permeability to fluorescein isothiocyanate isomer-dextran (FITC-dextran) with different molecular weights and three model drugs with different permeability behavior across BBB, which indicated that this glioma model included a functional barrier. Six potential anti-glioma components in traditional Chinese medicine (TCM) were delivered into the blood channel and the permeated amount was quantified by high-performance liquid chromatography combined with ultraviolet (HPLC-UV). The permeated drugs then directly acted on 3D cultured glioma cells (U251) to evaluate the drug efficacy. The results of permeability coefficients of drugs showed that the data were closer to the in vivo data of traditional Transwell model. The effect of the drugs on U251 cells in the BBB-U251 chip was significantly lower due to the existence of BBB. Drug responses on glioma demonstrated the necessity to take BBB into account during the development of anti-glioma new drugs. Therefore, this 3D glioma microfluidic models integrating the BBB functionality can be a useful platform for screening the anticancer drug for brain tumors.

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23.

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

Long non-coding RNA LINC01018 inhibits human glioma cell proliferation and metastasis by directly targeting miRNA-182-5p.

Accumulating evidence suggests that lncRNAs are potential biomarkers and key regulators of tumor development and progression. However, the precise function of most lncRNAs in glioma remains unknown. In this study, we explored the role of long intergenic non-protein coding RNA 1018 (LINC01018) in human glioma. Expression levels of LINC01018 and miR-182-5p in clinical glioma tissues and cell lines were detected by quantitative real-time PCR (qRT-PCR). Cell proliferation, migration, and invasion were determined by Cell Counting Kit-8 (CCK-8) assay and Transwell assay. Epithelial-mesenchymal transition (EMT) related proteins were measured by Western blotting. Direct relationship between LINC01018 and miR-182-5p was tested by dual-luciferase reporter assay, RNA immunoprecipitation assay (RIP), and rescue assays. Lastly, bioinformatics analyses were conducted to predict the downstream factors of LINC01018/miR-182-5p axis in glioma. LINC01018 was significantly down-regulated in glioma tissues and cell lines. Overexpression of LINC01018 dramatically inhibited cell proliferation, migration, and invasion and reverse EMT process in glioma. LINC01018 directly target to miR-182-5p. Forced up-regulation of miR-182-5p reversed the inhibitory effects on proliferative and metastatic abilities of glioma cells with LINC01018 overexpression. Lastly, the bioinformatics analyses revealed that LINC01018/miR-182-5p axis mediated a cluster of downstream genes (ADRA2C, RAB6B, RAB27B, RAPGEF5, STEAP2, TAGLN3, and UNC13C), which were potential key factors in the development of glioma. LINC01018 inhibits cell proliferation and metastasis in human glioma by targeting miR-182-5p, and should be considered as a potential therapeutic target in this cancer.

Effect of glioma-derived immunoglobulin on biological function of glioma cells

IntroductionGlioma is the most common and most invasive primary central nervous system tumour, and it is urgent to develop new specific therapeutic targets. Studies have confirmed that epithelial-derived tumour cells promote tumour cell proliferation and metastasis by secreting a large number of immunoglobulins (Igs), but the role of tumour-derived Igs in glioma has never been reported. MethodsThe Gene Expression Profiling Interactive Analysis and Chinese Glioma Genome Atlas databases were used to analyse the Ig transcription and its correlation with the prognosis of patients with glioma. Immunohistochemistry and immunofluorescence were used to detect the protein expression of IgG and IgM in the glioma tissues of patients and glioma cell lines. When IgG was knocked down by small interfering RNA or knocked out by CRISPR-Cas9, the function of proliferation and migration of glioma cells were analysed by CCK-8, clone formation, wound healing, and transwell assays. Changes in proteins and their phosphorylation in signalling pathways were detected by western blotting. The nude mouse subcutaneous tumour-bearing model was established to analyse the effect of IgG in vivo. ResultsThe transcriptional level of IgG was pretty high in glioma tissues and was positively correlated with high WHO grade, recurrence, and poor prognosis. The expression of IgG and IgM was found in tumour tissues and human glioma cell lines U87 and U251, and the main expression form was secreted. Decreased IgG inhibited the proliferation and migration of glioma cells. Knockout or knockdown of IgG downregulated the phosphorylation of the key molecules in the MAPK and PI3K/Akt pathway through the HGF/SF-Met or FAK/Src pathway. In vivo tumourigenesis mouse model confirmed that reduced IgG expression inhibited glioma growth. ConclusionIg was expressed in glioma tissues and cell lines, and a high expression level predicted a poor prognosis of patients. Glioma-derived IgG promoted glioma cell proliferation and migration through the HGF/SF-Met or FAK/Src pathway.

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.

Transcriptome sequencing analysis of echovirus 30 infection reveals its potential pathogenesis.

Echovirus 30 (E30) causes various diseases, such as viral encephalitis; aseptic meningitis; hand, foot, and mouth diseases; and acute flaccid paralysis. Related neurological infections are most concerning. However, the molecular mechanisms of E30 pathogenesis are not fully understood. There is a growing research interest in E30 as a cause of neurological disease. The aim of this study was to describe E30 infection, especially the changes in differential factor expressions after infection, in human glioma (U251) cells and mice brains using transcriptome sequencing analysis. Clear changes in the gene expression of factors associated with the defense response to viruses, inflammation-related signaling pathways, and neurological complication-related pathways were observed. Our results suggest that after E30 infection, the genes related to immune response were induced in the human glioma cells and mice brains, whereas genes functioning in the development and function of neural tissue were inhibited. Overall, this study successfully established E30 infection of U251 and mouse brain tissue, profiled the infection-induced changes in cellular and organizational transcriptomes, and revealed the molecular level changes during E30 infection.

OS10.4.A A macrophage-based drug delivery platform for glioma treatment

Abstract Background There is an urgent need for more effective treatment strategies against gliomas. At present, even though various drugs have potent anti-tumor activity in vitro, their application in vivo is limited by ineffective delivery and systemic toxicity. Therefore, novel strategies are needed to deliver these drugs effectively and safely to the tumor site. Here, we developed an adoptive transfer strategy against malignant brain tumors utilizing macrophages that are loaded with ferritin-protein cages containing drugs or other proteins and transfer these nanocarriers to cancer cells in vitro and in vivo. Material and Methods Live-time imaging, microscopy and flow cytometry were utilized to investigate the transfer of ferritin cages from loaded macrophages to human or mouse glioma cells. Co-cultures of glioma cells and macrophages loaded with ferritin-drug cages were used to study the anti-glioma activity in vitro and orthotopic immunocompetent mouse glioma models were used to study the anti-glioma activity in vivo. Affinity purification-mass spectrometry (AP-MS) was used to elucidate the mechanisms of transfer by characterizing the interactome of ferritin cages within macrophages and cancer cells. Results We observed a high transfer efficiency of ferritin-cages from loaded macrophages into human and mouse glioma cells in vitro in co-culture assays and confirmed the transfer from macrophages to glioma cells also in vivo upon intravenous or intratumoral treatment of GL-261 or CT-2A glioma-bearing mice. To study the anti-glioma activity with therapeutically active payloads, we loaded murine/human macrophages with ferritin cages carrying cytotoxic payloads. Co-culture of these loaded macrophages with murine or human glioma cells in vitro revealed a time- and concentration-dependent cytotoxicity to glioma cells. In vivo, intravenous or intratumoral administration of ferritin-drug protein cages was tolerated without toxicities and conferred a survival benefit in two orthotopic murine glioma models (GL-261 and CT-2A). Interactome studies of ferritin-cage-binding proteins revealed phagocytic and cytoskeleton re-arrangement pathways to be involved in uptake and transfer of ferritin-cages from macrophages to glioma cells. Conclusion This ‘Trojan Horse’ approach constitutes a promising platform to deliver cytotoxic drugs effectively and safely to gliomas and provides a rationale for clinical translation.

OS10.7.A Activation of the CCR8-ACP5 axis by human microglia/macrophage derived CCL18 promotes glioma growth

Abstract Background Glioblastoma multiforme is a highly malignant primary brain tumor with an average survival of 14 months and very limited therapeutic options. Glioma associated microglia and macrophages (GAMs) foster tumor growth by releasing several cytokines, which have only partly been identified. Here, we studied the chemokine (C-C motif) ligand 18 (CCL18), a chemokine which is only expressed in human, but not rodent GAMs, in a novel ex-vivo brain slice model including transplantation of human induced pluripotent stem cells (iPSC) derived human microglia (iMGL) and human glioma cells in to murine brain slices, which had been depleted of intrinsic murine microglia before. Material and Methods After establishing the humanized ex-vivo brain slice model, we performed immunohistochemical analysis (IHC) of growth and invasiveness, qrtPCR on glioma cells isolated by magnetic-activated cell sorting (MACS), functional assays measuring invasiveness, proliferation, migration and colony formation of glioma cells in vitro and in slice experiments. Corresponding studies on tumor growth and invasiveness were performed after treatment with a CCL18 neutralizing antibody, a CCR8 neutralizing antibodies and knockdown of CCR8, ACP5 (Acid Phosphatase 5) and PITPNM3 with small interfering RNA (siRNA) and short hairpin RNA (shRNA). QrtPCR, IHC and Westernblot analysis were performed on primary glioma specimens. We also conducted bioinformatic analyses, based on the TCGA GBM, GLIOVIS and GEPIA databases. Results We observed that CCL18 was highly expressed in GAMs, whereas CCR8 was only expressed in glioma cells. We identified the chemokine (C-C motif) receptor 8 (CCR8) as a functional receptor for CCL18 and ACP5 as an important down-stream signaling component in glioma cells. Activation of the CCL18/CCR8/ACP5 signaling pathway in human glioblastoma was associated with enhanced tumor growth and invasiveness. Conclusion GAMs derived CCL18 promoted glioma growth by activation of the CCR8/ACP5 axis in human glioma cells and therefore is a potential therapeutic target.

P10.19.B An immunotoxin targeting CD317 for the treatment of glioblastoma

Abstract Background CD317 is an interferon-inducible cell surface receptor expressed in several solid cancer types. HM1.24-ETA’ is a small immunotoxin with a CD317 single-chain variable fragment (svFv) antibody fused to a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’) that is explored as a novel therapeutic approach in CD317-expressing tumors. Material and Methods CD317 mRNA expression in human gliomas and its association with survival was analyzed using the database of the Cancer Genome Atlas (TCGA). CD317 protein levels in human gliomas were assessed by immunohistochemistry. CD317 mRNA expression was assessed by RT-PCR and CD317 protein levels by flow cytometry in 13 human glioma cell lines in vitro. Efficacy of HM1.24-ETA’ was analyzed in acute cytotoxicity assays in vitro. Finally, HM1.24-ETA’ was evaluated in the intracranial human LN-229 glioma xenograft nude mouse model after intravenous injection. Results Interrogation of the TCGA database showed that increased CD317 mRNA expression correlated with grade of malignancy among isocitrate dehydrogenase (IDH) wildtype and IDH-mutant gliomas. Enhanced CD317 mRNA expression was associated with inferior survival in glioblastoma and astrocytoma, IDH-mutant, WHO grade 4. Immunohistochemistry confirmed CD317 overexpression in human glioblastoma compared to lower grade astrocytomas. CD317 was expressed heterogeneously on mRNA and protein levels in glioma cell lines in vitro. HM1.24-ETA’ induced acute cytotoxicity in CD317-positive glioma cells in vitro. CD317 expression and susceptibility to HM1.24-ETA’-induced cell death were enhanced by interferon-β. HM1.24-ETA’ prolonged survival in the LN-229 xenograft nude mouse model. Conclusion These data define CD317 as a novel target for treatment of human gliomas with immunoconjugates.

CASP6 predicts poor prognosis in glioma and correlates with tumor immune microenvironment.

BackgroundGlioma is an aggressive tumor of the central nervous system. Caspase-6 (CASP6) plays a crucial role in cell pyroptosis and is a central protein involved in many cellular signaling pathways. However, the association between CASP6 and prognosis of glioma patients remains unclear.MethodsFour bioinformatic databases were analyzed to identify differentially expressed genes (DEGs) between glioma and healthy tissues. Eighty-one protein-coding pyroptosis-related genes (PRGs) were obtained from the GeneCards database. The pyroptosis-related DEGs (PRDEGs) were extracted from each dataset, and CASP6 was found to be aberrantly expressed in glioma. We then investigated the biological functions of CASP6 and the relationship between CASP6 expression and the tumor microenvironment and immunocyte infiltration. The half maximal inhibitory concentration of temozolomide and the response to immune checkpoint blockade in the high- and low-CASP6 expression groups were estimated using relevant bioinformatic algorithms. Quantitative real-time reverse transcription PCR and western blotting were carried out to confirm the different expression levels of CASP6 between human astrocytes and glioma cell lines (U251 and T98G). We determined the role of CASP6 in the tumorigenesis of glioma by knocking down CASP6 in U251 and T98G cell lines.ResultsWe found that CASP6 was overexpressed in glioma samples and in glioma cell lines. CASP6 expression in patients with glioma correlated negatively with overall survival. In addition, CASP6 expression correlated positively with the degree of glioma progression. Functional analysis indicated that CASP6 was primarily involved in the immune response and antigen processing and presentation. Patients with high CASP6 levels responded more favorably to temozolomide, while patients with low expression of CASP6 had a better response to immunotherapy. Finally, in vitro experiments showed that CASP6 knockdown inhibited glioma proliferation.ConclusionsThe pyroptosis-related gene CASP6 might represent a sensitive prognostic marker for patients with glioma and might predict their response of immunotherapy and temozolomide therapy. Our results might lead to more precise immunotherapeutic strategies for patients with glioma.

Effects of octadecadienoic acid on proliferation and apoptosis of glioma cells and its mechanisms

To study the effects of octadecadienoic acid (ODA) on the proliferation and apoptosis of glioma cells and its mechanisms. Cultured human glioma cells (cell density 2×106 cells/L) were divided into solvent control group (DMSO, 30 μl/L), 5-FU group (10 mg/L) and octadecadienic acid groups (0.3, 0.6 and 1.2 mg/L groups). The toxicity of ODA on glioma cells was detected by trypan blue and thiazolium blue (MTT). The expression levels of P53, PI3K, P21, PKB/Akt and Caspase-9 in glioma cells were determined by enzyme-linked immunosorbent assay (ELISA). ① Cell count under optical microscope showed that the inhibition rate of cell proliferation in ODA low, medium and high dose groups and 5-FU group was significantly higher than that in the solvent control group (P＜0.01), but there was no statistical significance compared with the 5-FU group (P＞0.05). ② MTT assay showed that the inhibition rate of cell proliferation was increased significantly in ODA low, medium and high dose groups and 5-FU groups (P＜0.01), compared with the solvent control group. Compared with 5-FU group, the inhibition rate of cell proliferation was increased significantly only in ODA high dose group (P＜0.01). ③ The number of G0/G1 phase cells in ODA low, medium and high dose groups and 5-FU group were increased significantly (P＜0.05, P＜0.01), the number of G2/M phase cells were decreased significantly (P＜0.01), and the apoptosis rate was increased significantly (P＜0.01),compared with the solvent control group. Compared with the 5-FU group, the number of cells in G2/M phase was decreased significantly (P＜0.01) and the apoptosis rate was increased significantly (P＜0.01) in ODA high dose group. ④ ELISA test results showed that the protein expression levels of P53, PI3K and PKB/Akt in ODA low , medium and high dose groups and 5-FU group were significantly lower than those in solvent control group (all P＜0.01), but the protein expression levels in ODA high dose group were significantly lower than those in 5-FU group (P＜0.01). The protein expression levels of P21 and caspase-9 in ODA low , medium and high dose groups and 5-FU group were significantly higher than those in solvent control group (P＜0.05, P＜0.01), but the protein expression levels in ODA high dose group were significantly higher than those in 5-Fu group (P＜0.01). ODA can significantly inhibit the proliferation and promote apoptosis of glioma cells. The mechanisms are related to up-regulating the levels of P21 and caspase-9 to promote apoptosis, down-regulating the levels of P53, PI3K and PKB/Akt to inhibit the cell division cycle, and reducing the activity of PI3K-Akt signal transduction pathway.

Collagen Matrices Mediate Glioma Cell Migration Induced by an Electrical Signal.

Glioma cells produce an increased amount of collagen compared with normal astrocytes. The increasing amount of collagen in the extracellular matrix (ECM) modulates the matrix structure and the mechanical properties of the microenvironment, thereby regulating tumor cell invasion. Although the regulation of tumor cell invasion mainly relies on cell–ECM interaction, the electrotaxis of tumor cells has attracted great research interest. The growth of glioma cells in a three-dimensional (3D) collagen hydrogel creates a relevant tumor physiological condition for the study of tumor cell invasion. In this study, we tested the migration of human glioma cells, fetal astrocytes, and adult astrocytes in a 3D collagen matrix with different collagen concentrations. We report that all three types of cells demonstrated higher motility in a low concentration of collagen hydrogel (3 mg/mL and 5 mg/mL) than in a high concentration of collagen hydrogel (10 mg/mL). We further show that human glioma cells grown in collagen hydrogels responded to direct current electric field (dcEF) stimulation and migrated to the anodal pole. The tumor cells altered their morphology in the gels to adapt to the anodal migration. The directedness of anodal migration shows a field strength-dependent response. EF stimulation increased the migration speed of tumor cells. This study implicates the potential role of an dcEF in glioma invasion and as a target of treatment.

Hybrid Membrane-Derived Nanoparticles for Isoliquiritin Enhanced Glioma Therapy.

Due to the obstruction and heterogeneity of the blood-brain barrier, the clinical treatment of glioma has been extremely difficult. Isoliquiritigenin (ISL) exhibits antitumor effects, but its low solubility and bioavailability limit its application potential. Herein, we established a nanoscale hybrid membrane-derived system composed of erythrocytes and tumor cells. By encapsulating ISL in hybrid membrane nanoparticles, ISL is expected to be enhanced for the targeting and long-circulation in gliomas therapy. We fused erythrocytes with human glioma cells U251 and extracted the fusion membrane via hypotension, termed as hybrid membrane (HM). HM-camouflaged ISL nanoparticles (ISL@HM NPs) were prepared and featured with FT-IR, SEM, TEM, and DLS particle analysis. As the results concluded, the ISL active pharmaceutical ingredients (APIs) were successfully encapsulated with HM membranes, and the NPs loading efficiency was 38.9 ± 2.99% under maximum entrapment efficiency. By comparing the IC50 of free ISL and NPs, we verified that the solubility and antitumor effect of NPs was markedly enhanced. We also investigated the mechanism of the antitumor effect of ISL@HM NPs, which revealed a marked inhibition of tumor cell proliferation and promotion of senescence and apoptosis of tumor cells of the formulation. In addition, the FSC and WB results examined the effects of different concentrations of ISL@HM NPs on tumor cell disruption and apoptotic protein expression. Finally, it can be concluded that hybridized membrane-derived nanoparticles could prominently increase the solubility of insoluble materials (as ISL), and also enhance its targeting and antitumor effect.

MicroRNA-542 suppressed the proliferation of human glioma cells by targeting talin-2 (TLN2)

Purpose: To investigate the effect of miR-542 in the development of human glioma.
 Methods: The expressions of miR-542 and TLN2 in glioma cells and normal human astrocytes were determined using qRT-PCR, while MTT and colony formation assays were used to determine cell proliferation. Western blotting was used to determine protein expression.
 Results: It was revealed that miR-542 was significantly downregulated in glioma cells. Overexpression of miR-542 inhibited the proliferation and clonogenicity of glioma cells via induction of apoptosis. The percentage of apoptotic U87 cells increased from 5.32 in control to 26.76 upon miR-542 overexpression. Moreover, TLN2 was identified as the functional regulatory target of miR542 in glioma. The expression of TLN2 was markedly upregulated in human glioma cells. However, overexpression of miR-542 suppressed TLN2 expression. Silencing of TLN2 mimicked the tumor-suppressive effects of miR-542 in glioma cells, but this effect was blocked by TLN2 over-expression.
 Conclusion: These results suggest that miR-542 exerted glioma-suppressive effect, with TLN2 as its functional regulatory target.
 Keywords: Glioma; Proliferation; Micro-RNA; Tumorigenesis; MiR-542; Apoptosis; Prognosis; talin-2; Oncogene

Hetero-bivalent agents targeting FAP and PSMA.

We developed a theranostic radiopharmaceutical that engages two key cell surface proteases, fibroblast activation protein alpha (FAP) and prostate-specific membrane antigen (PSMA), each frequently overexpressed within the tumor microenvironment (TME). The latter is also expressed in most prostate tumor epithelium. To engage a broader spectrum of cancers for imaging and therapy, we conjugated small-molecule FAP and PSMA-targeting moieties using an optimized linker to provide 64Cu-labeled compounds. We synthesized FP-L1 and FP-L2 using two linker constructs attaching the FAP and PSMA-binding pharmacophores. We determined in vitro inhibition constants (Ki) for FAP and PSMA. Cell uptake assays and flow cytometry were conducted in human glioma (U87), melanoma (SK-MEL-24), prostate cancer (PSMA + PC3 PIP and PSMA - PC3 flu), and clear cell renal cell carcinoma lines (PSMA + /PSMA - 786-O). Quantitative positron emission tomography/computed tomography (PET/CT) and tissue biodistribution studies were performed using U87, SK-MEL-24, PSMA + PC3 PIP, and PSMA + 786-O experimental xenograft models and the KPC genetically engineered mouse model of pancreatic cancer. 64Cu-FP-L1 and 64Cu-FP-L2 were produced in high radiochemical yields (> 98%) and molar activities (> 19MBq/nmol). Ki values were in the nanomolar range for both FAP and PSMA. PET imaging and biodistribution studies revealed high and specific targeting of 64Cu-FP-L1 and 64Cu-FP-L2 for FAP and PSMA. 64Cu-FP-L1 displayed more favorable pharmacokinetics than 64Cu-FP-L2. In the U87 tumor model at 2h post-injection, tumor uptake of 64Cu-FP-L1 (10.83 ± 1.02%ID/g) was comparable to 64Cu-FAPI-04 (9.53 ± 2.55%ID/g). 64Cu-FP-L1 demonstrated high retention 5.34 ± 0.29%ID/g at 48h in U87 tumor. Additionally, 64Cu-FP-L1 showed high retention in PSMA + PC3 PIP tumor (12.06 ± 0.78%ID/g at 2h and 10.51 ± 1.82%ID/g at 24h). 64Cu-FP-L1 demonstrated high and specific tumor targeting of FAP and PSMA. This compound should enable imaging of lesions expressing FAP, PSMA, or both on the tumor cell surface or within the TME. FP-L1 can readily be converted into a theranostic for the management of heterogeneous tumors.

Chalcone 9X Contributed to Repressing Glioma Cell Growth and Migration and Inducing Cell Apoptosis by Reducing FOXM1 Expression In Vitro and Repressing Tumor Growth In Vivo.

Objective Natural and synthetic chalcones played roles in inflammation and cancers. Chalcone 9X was an aromatic ketone that was found to inhibit cell growth of hepatic cancer and lung cancer cells. In this study, we wanted to investigate the functions of Chalcone 9X in glioma. Materials and Methods Chemical Chalcone 9X was added in human glioma cell lines (U87 and T98G cells) and normal astrocyte cell lines (HA1800) with various concentrations (0 μmol/L, 20 μmol/L, 50 μmol/L, and 100 μmol/L). CCK-8 assay was used to measure cell viability. Flow cytometric assay was used to measure cell apoptotic rates. Wound healing assay and transwell assay were used to measure cell invasion. RT-PCR was used to detect relative mRNA expressions, and the protein expressions were detected by western blot (WB) and immunohistochemical staining (IHC). Finally, nude mouse xenograft assay was performed to prove the effects of Chalcone 9X in vivo. Results Results revealed that Chalcone 9X treatment suppressed cell viability and cell migration capacity; it could also induce cell apoptosis in U87 and T98G cells with dose dependence. However, it had little cytotoxicity to normal astrocyte HA1800 cells. Moreover, Chalcone 9X treatment could repress the mRNA and protein expressions of FOXM1 in human glioma cell lines, which was an oncogene that could promote the progression and malignancy of glioma. In addition, FOXM1 overexpression dismissed the Chalcone 9X effects on cell proliferation, apoptosis, and migration in human glioma cell lines. Finally, in vivo assay showed that Chalcone 9X treatment repressed the expression of FOXM1, which inhibited the tumor growth of a xenograft model injected with U87 in nude mice. Conclusions In all, we found that Chalcone 9X could suppress cell proliferation and migration and induce cell apoptosis in human glioma cells, while it has little cytotoxicity to normal astrocyte cells. Therefore, we uncovered a novel way that Chalcone 9X could inhibit FOXM1 expression and repress the progression and biofunctions of glioma cells, which might be a potential therapeutic drug for treating human glioma.

Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 μM), TMZ (155 μM), and NGF (7.55 × 10−3 μM) inhibited 43.9%–60.3%, 73.5%–81.3%, 66.2% the clonogenicity of glioma U251 cells for 1–2 days, respectively. LL-37 (4 μM), and NGF (7.55 × 10−3 μM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1–3 days. In contrast, PG-1 (16 μM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

Interferon-γ resistance and immune evasion in glioma develop via Notch-regulated co-evolution of malignant and immune cells.

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of Tcells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled byupregulation of oncogenes anddownregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control.