HSPA4 regulated glioma progression via activation of AKT signaling pathway.

Glioma is still an incurable disease with high invasiveness. Heat shock 70 kDa protein 4 (HSPA4) is a member of the HSP110 family, and is associated with the development and progression of various cancers. In the current study, we assessed the expression of HSPA4 in clinical samples, and found that HSPA4 was up-regulated in glioma tissues and correlated with tumor recurrence and grade. Survival analyses demonstrated that glioma patients with high HSPA4 expression had lower overall survival and disease-free survival times. In vitro knockdown of HSPA4 inhibited glioma cell proliferation, mediated cell cycle arrest at G2 phase and apoptosis, and reduced the migration ability. In vivo, the growth of HSPA4-knockdown xenografts was markedly suppressed compared to the tumors formed by HSPA4-positive control cells. Additionally, Gene set enrichment analyses disclosed that HSPA4 was associated with the PI3K/Akt signaling pathway. The regulatory effect of the AKT activator SC79 on cell proliferation and apoptosis was suppressed by HSPA4 knockdown, indicating that HSPA4 is capable of promoting glioma development. In summary, these data showed that HSPA4 is likely to play a pivotal role in the progression of glioma, and consequently may be a promising therapeutic target for glioma therapy.

Ultra high b-value diffusion weighted imaging enables better molecular grading stratification over histological grading in adult-type diffuse glioma.

PURPOSE: Accurate preoperative radiological staging of adult-type diffuse glioma is crucial for effective prognostic stratification and selection of appropriate therapeutic interventions. The purpose of this study was to compare the effectiveness of apparent diffusion coefficient (ADC) maps generated from ultrahigh b-value diffusion-weighted imaging (DWI) for molecular grading with that for histological grading of adult-type diffuse glioma, and to evaluate the correlation between these ADC maps and molecular and histological biomarkers. METHODS: This study retrospectively enrolled forty adult-type diffuse glioma patients, diagnosed using the 2021 WHO classification criteria. Preoperative imaging data, including multiple b-value DWI and conventional magnetic resonance imaging, were collected. Tumors were graded using both histological and molecular criteria. Histogram analysis was conducted to generate 14 parameters for each tumor. Receiver operating characteristic curves and the area under the curve (AUC) were used to evaluate tumor grading and molecular status differentiation. Analysis of histological biomarkers was performed by calculating the Pearson and Spearman correlation coefficients of continuous and hierarchical variables, respectively. RESULTS: The intensity-related parameters for molecular grading were found to be superior to those for histological grading for the identification of WHO grade 4 (WHO4) adult-type diffuse glioma. The AUC of both grading systems increased with increasing b-values, with ADC8000-based histogram parameters showing the best results (molecular grading, square root: AUC = 0.897; histological grading, median: AUC = 0.737). The intensity-related parameters could also differentiate molecular WHO4 gliomas from histologically lower-grade gliomas (ADC8000-based square root: AUC = 0.919), and different ADC8000-based kurtosis was observed between molecular and histological WHO4 gliomas (AUC = 0.833). Significant correlations between the Ki-67 index and molecular status prediction for IDH, CDKN2A, and EGFR were also demonstrated. CONCLUSION: The histogram parameters derived from high b-value ADC maps were found to be more effective for differentiating molecular grades of WHO4 adult-type diffuse glioma than for differentiating histological grades.

TMEM158 promotes the proliferation and migration of glioma cells via STAT3 signaling in glioblastomas.

Glioblastoma is the most common primary intracranial malignant tumor in adults and has high morbidity and high mortality. TMEM158 has been reported to promote the progression of solid tumors. However, its potential role in glioma is still unclear. Here, we found that TMEM158 expression in human glioma cells in the tumor core was significantly higher than that in noncancerous cells at the tumor edge using bioinformatics analysis. Cancer cells in patients with primary GBMs harbored significantly higher expression of TMEM158 than those in patients with WHO grade II or III gliomas. Interestingly, regardless of tumor grading, human glioma samples that were IDH1-wild-type (IDH1-WT) exhibited higher expression of TMEM158 than those with IDH1-mutant (IDH1-Mut). We also illustrated that TMEM158 mRNA expression was correlated with poor overall survival in glioma patients. Furthermore, we demonstrated that silencing TMEM158 inhibited the proliferation of glioma cells and that TMEM158 overexpression promoted the migration and invasion of glioma cells by stimulating the EMT process. We found that the underlying mechanism involves STAT3 activation mediating TMEM158-driven glioma progression. In vivo results further confirmed the inhibitory effect of the TMEM158 downregulation on glioma growth. Collectively, these findings further our understanding of the oncogenic function of TMEM158 in gliomas, which represents a potential therapeutic target, especially for GBMs.

Molecular and clinical characterization of Galectin-9 in glioma through 1,027 samples.

In recent years, research on glioma immunotherapy have grown rapidly. However, the autoimmune-like side effects that are caused by blocking immunological checkpoints hinder their clinical application in gliomas currently. Galectin-9, a ligand for T-cell immunoglobulin mucin 3, has shed a new light on the treatment of malignant glioma. However, the potential mechanism of Galectin-9 is still under discussion. In this study, first, we methodically gathered 1,027 glioma patients with RNA-seq and 986 patients with survival data to explore the role and mechanism of Galectin-9 in gliomas. Second, we analyzed glioma samples from 50 patients in the Department of Neurosurgery, Tianjin Medical University General Hospital. Finally, we found that Galectin-9 was strongly upregulated in glioblastoma multiforme compared with normal brain tissues and lower-grade glioma. Patients with Galectin-9 overexpression had a significantly shorter overall survival. Moreover, the tissue microarray data displayed that the expression of Galectin-9 in the core of tumor is higher than that in the border and was correlated with the shorter survival in glioma patients. Galectin-9 is more highly expressed in the mesenchymal subtype of glioblastoma multiforme than in the other subtypes. Simultaneously, Galectin-9 was closely associated with the immune response and lymphocyte activation, especially T-cell activation. To further determine the underlying role of Galectin-9 in the immune response, we selected seven immune metagenes. Through cluster analysis and correlation analysis, we discovered that Galectin-9 was highly correlated with immune checkpoint molecules and M2 tumor-associated macrophages. In summary, Galectin-9 serves as a potential therapeutic target to treat glioblastoma multiforme.

Identification of Key Pathways and Genes in the Orai2 Mediated Classical and Mesenchymal Subtype of Glioblastoma by Bioinformatic Analyses.

BACKGROUND: Ca2+ release-activated Ca2+ channels (CRAC) are the main Ca2+ entry pathway regulating intracellular Ca2+ concentration in a variety of cancer types. Orai2 is the main pore-forming subunit of CRAC channels in central neurons. To explore the role of Orai2 in glioblastoma (GBM), we investigated the key pathways and genes in Orai2-mediated GBM by bioinformatic analyses. METHODS: Via The Cancer Genome Atlas (TCGA), French, Sun, and Gene Expression Omnibus (GEO) (GDS3885) datasets, we collected 1231 cases with RNA-seq data and analyzed the functional annotation of Orai2 by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Univariate and multivariate survival analyses were applied to 823 patients with survival data. RESULTS: We discovered that Orai2 was markedly upregulated in GBM compared to normal brain samples and lower-grade gliomas (LGG). Survival analysis found that higher expression of Orai2 was independently associated with a worse prognosis of patients with the classical and mesenchymal subtypes of GBM. Simultaneously, Orai2 expression was higher in tumors of the classical and mesenchymal subtypes than other subtypes and was significantly correlated with classical- and mesenchymal-related genes. GO and KEGG pathway analysis revealed that genes significantly correlated with Orai2 were involved in the JNK pathway. Through screening transcriptomic data, we found a strong association between Orai2 and apoptosis, stemness, and an epithelial-mesenchymal transition- (EMT-) like phenotype. CONCLUSION: As a prognostic factor, Orai2 is obviously activated in the classical and mesenchymal subtypes of GBM and promotes glioma cell self-renewal, apoptosis, and EMT-like by the JNK pathway. These findings indicate that Orai2 could be a candidate prognostic and therapeutic target, especially for the classical and mesenchymal subtypes of GBM.

Predictable Risk Factors of Spontaneous Venous Thromboembolism in Patients Undergoing Spine Surgery.

OBJECTIVE: The purpose of this study was to conduct a meta-analysis to identify the risk factors for formation of venous thromboembolism (VTE) in patients after spine surgery. METHODS: This study retrieved potential academic articles on the related factors for VTE formation in patients after spine surgery from MEDLINE, PubMed, EMBASE, and the Cochrane Library. The reference articles for the identified studies were carefully reviewed to ensure that all available documents were represented in the study. RESULTS: A total of 21 articles (20 retrospective studies and 1 prospective study) involving 2,870,105 patients were identified in the analysis, including 7829 patients who presented with VTE after spine surgery; the incidence of VTE was 0.273%. Our meta-analysis showed that compared with patients who did not have VTE after spine surgery, there was significantly more blood loss (weighted mean difference [WMD], 93.295; 95% confidence interval [CI], 60.521-126.069; P < 0.001), higher age (WMD, 6.011; 95% CI, 3.647-9.376; P < 0.001), thoracolumbar surgery (odds ratio [OR], 0.233; 95% CI, 0.198-0.274; P < 0.001), and longer duration of surgery (WMD, 45.672; 95% CI, 10.433 to -80.911; P = 0.011) among the patients with VTE. Patients with a history of hypertension (OR, 1.785; 95% CI, 1.516-2.103; P < 0.001), diabetes (OR, 1.535; 95% CI, 1.286-1.832; P < 0.001), and preoperative walking disability (OR, 4.882; 95% CI, 2.044-11.663; P < 0.001) showed a significantly higher rate of VTE after spine surgery. However, no significant differences were found in gender (P = 0.289), fusion surgery (P = 0.979), body mass index (P = 0.157), history of heart disease (P = 0.397), and level of D-dimer (P = 0.220). CONCLUSIONS: A higher rate of postoperative VTE is closely associated with the elderly, longer duration of surgery, thoracolumbar surgery, greater blood loss, and patients with a history of hypertension, preoperative walking disability, or diabetes after spinal surgery; these risk factors should be guarded against.

NKCC1 promotes EMT-like process in GBM via RhoA and Rac1 signaling pathways.

Glioblastoma is the most common and lethal primary intracranial tumor. As the key regulator of tumor cell volume, sodium-potassium-chloride cotransporter 1 (NKCC1) expression increases along with the malignancy of the glioma, and NKCC1 has been implicated in glioblastoma invasion. However, little is known about the role of NKCC1 in the epithelial-mesenchymal transition-like process in gliomas. We noticed that aberrantly elevated expression of NKCC1 leads to changes in the shape, polarity, and adhesion of cells in glioma. Here, we investigated whether NKCC1 promotes an epithelial-mesenchymal transition (EMT)-like process in gliomas via the RhoA and Rac1 signaling pathways. Pharmacological inhibition and knockdown of NKCC1 both decrease the expressions of mesenchymal markers, such as N-cadherin, vimentin, and snail, whereas these treatments increase the expression of the epithelial marker E-cadherin. These findings indicate that NKCC1 promotes an EMT-like process in gliomas. The underlying mechanism is the facilitation of the binding of Rac1 and RhoA to GTP by NKCC1, which results in a significant enhancement of the EMT-like process. Specific inhibition or knockdown of NKCC1 both attenuate activated Rac1 and RhoA, and the pharmacological inhibitions of Rac1 and RhoA both impair the invasion and migration abilities of gliomas. Furthermore, we illustrated that NKCC1 knockdown abolished the dissemination and spread of glioma cells in a nude mouse intracranial model. These findings suggest that elevated NKCC1 activity acts in the regulation of an EMT-like process in gliomas, and thus provides a novel therapeutic strategy for targeting the invasiveness of gliomas, which might help to inhibit the spread of malignant intracranial tumors.

Jagged1 is Clinically Prognostic and Promotes Invasion of Glioma-Initiating Cells by Activating NF-κB(p65) Signaling.

BACKGROUND/AIMS: Jagged1 is the ligands of the Notch signaling and has been shown to promote glioma-initiating cells (GICs) in glioblastoma. The role of Jagged1 in GICs invasion and underlying molecular mechanisms remain unclear. METHODS: Survival data from R2 genomics analysis, the Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA) and visualization platform database were used to evaluate the effects of Jagged1 on overall patient survival. we investigated Jagged1 induced the GICs cells' invasion by matrix degradation assays and Transwell cell invasion assays in vitro, then we further explored the underlying molecular mechanisms using Co-immunoprecipitation (co-IP) analysis. RESULTS: High expression of Jagged1 in human glioma was associated with poor survival. Clinical data analysis showed that the Jagged1 was positively correlated with NF-κB(p65). Jagged1-induced invasion of GICs cells through activation of NF-κB(p65) pathway. In vivo, knockdown of Jagged1 could suppress the tumorigenicity of GICs cells through NF-κB(p65) signaling. CONCLUSION: Insights gained from these findings suggest that Jagged1 plays an important oncogenic role in GICs malignancy by activation of NF-κB(p65) signaling, and Jagged1 could be employed as an effective therapeutic target for GICs.

Autophagy suppresses self-renewal ability and tumorigenicity of glioma-initiating cells and promotes Notch1 degradation.

Autophagy is a vital process that involves degradation of long-lived proteins and dysfunctional organelles and contributes to cellular metabolism. Glioma-initiating cells (GICs) have the ability to self-renew, differentiate into heterogeneous types of tumor cells, and sustain tumorigenicity; thus, GICs lead to tumor recurrence. Accumulating evidence indicates that autophagy can induce stem cell differentiation and increase the lethality of temozolomide against GICs. However, the mechanism underlying the regulation of GIC self-renewal by autophagy remains uncharacterized. In the present study, autophagy induced by AZD8055 and rapamycin treatment suppressed GIC self-renewal in vitro. We found that autophagy inhibited Notch1 pathway activation. Moreover, autophagy activated Notch1 degradation, which is associated with maintenance of the self-renewal ability of GICs. Furthermore, autophagy abolished the tumorigenicity of CD133 + U87-MG neurosphere cells in an intracranial model. These findings suggest that autophagy regulating GICs self-renewal and tumorigenicity is probably bound up with Notch1 degradation. The results of this study could aid in the design of autophagy-based clinical trials for glioma treatments, which may be of great value.

Notch1 is a prognostic factor that is distinctly activated in the classical and proneural subtype of glioblastoma and that promotes glioma cell survival via the NF-κB(p65) pathway.

Glioblastomas (GBMs) are the most prevalent and devastating primary intracranial malignancies and have extensive heterogeneity. Notch1 signaling is a more complex process in the development of numerous cell and tissue types, including gliomagenesis and progression, and is upregulated in glioma-initiating cells. However, the contradictory expression of Notch1 among lower grade gliomas and GBMs confounds our understanding of GBM biology and has made identifying effective therapies difficult. In this study, we validated that Notch1 and NF-κB(p65) are highly expressed in the classical and proneural subtypes of GBM using the data set from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA). DAPT and shRNA targeting Notch1 decreased NF-κB(p65) expression, suppressed cell proliferation, and induced apoptosis of GBM cells in vitro and in vivo. Furthermore, we illustrated that the intracellular Notch could bind with NF-κB(p65) in GBM cells. These findings suggest that the cross-talk between Notch1 signaling and NF-κB(p65) could contribute to the proliferation and apoptosis of glioma, and this discovery could help drive the design of more effective therapies in Notch1-targeted clinical trials.

[Relationship between Golgi apparatus and cell migration direction in vivo and in vitro].

OBJECTIVE: To explore the relationship between Golgi apparatus and the direction of tumor cell migration in vivo and in vitro. METHODS: Cell migration assays were conducted with rat C6 glioma cells, human U251 and SNB19 glioma cells respectively. Then immunofluorescence was used to detect the position of Golgi apparatus in migrating cells. The percentage of cells with Golgi apparatus facing towards wound edge was calculated. Cell pseudopodium was stained with TRITC-phalloidin and the relationship between Golgi apparatus and pseudopodium detected. Immunohistochemistry was used to reveal the Golgi apparatus in tumor tissue samples. And the percentage of cells with Golgi apparatus facing opposite to the necrotic zones was calculated. RESULTS: In cells located at wound edge, the Golgi apparatus was found facing towards the wound in the vast majority of cells (C6 83% ± 6%, U251 80% ± 7%, SNB19 82% ± 6%). In U251 and SNB19 cells, the golgi apparatus was located in the same direction with cellular pseudopodium. Immunohistochemical staining showed that in cells located around the necrotic zone, the Golgi apparatus faced opposite to the necrotic zones in most cells (rat tissue samples 80% ± 7%, human tissue samples 82% ± 6%). CONCLUSIONS: The Golgi apparatus is closely correlated with cell migration and it may be considered as a direction indicator of cell migration. And it provides an important index for the study of tumor cell invasion both in vivo and in vitro.

Expression of the Arp2/3 complex in human gliomas and its role in the migration and invasion of glioma cells.

A hallmark of directional cell migration is localized actin polymerization at the leading protrusions of the cell. The Arp2/3 complex nucleates the formation of the dendritic actin network (lamellipodia) at the leading edge of motile cells. This study was designed to investigate the role of the Arp2/3 complex in the infiltrative behavior of glioma cells. Immunofluorescence and western blotting showed a positive correlation between the expression of Arp2/3 and the malignancy of glioma specimens (r=0.686, P=0.02) and confocal microscopy demonstrated localization of the Arp2/3 complex in lamellipodia of glioma cells. Furthermore, we examined the effects of Arp2/3 complex inhibition in U251, LN229 and SNB19 glioma cells using CK666, an Arp2/3 complex inhibitor. Glioma cells lost lamellipodia and cell polarity after treatment with CK666. Inhibition of the Arp2/3 complex significantly affected the ability of glioma cells to migrate and invade. In the wound-healing assay, CK666 markedly inhibited cell migration, U251 cell migration was inhibited to 38.73±3.45% of control, LN229 cells to 57.40±2.16% of control and SNB19 cells to 34.17±3.82% of control. Also, CK666 significantly impaired Transwell chamber invasion capability of U251, LN229 and SNB19 cells compared with DMSO control by 72.70±4.86, 39.12±8.42 and 41.41±4.66%, respectively. The Arp2/3 complex is, therefore, likely to be a crucial participant in glioma cell invasion and migration, and may represent a target for therapeutic intervention.

Rac1+ cells distributed in accordance with CD 133+ cells in glioblastomas and the elevated invasiveness of CD 133+ glioma cells with higher Rac1 activity.

BACKGROUND: Recent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells. METHODS: A series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133 - U87 cells. The migration and invasive ability of CD133+ and CD133 - U87 cells were determined by cell migration and invasion assays in vitro. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study. RESULTS: In the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14 ± 4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378 ± 0.007), compared to CD133- cells (0.195 ± 0.004) (t = 27.81; P < 0.05). CD133+ cells had stronger ability to migrate (74.34 ± 2.40 vs. 38.72 ± 2.60, t = 42.71, P < 0.005) and invade (52.00 ± 2.28 vs. 31.26 ± 1.82, t = 30.76, P < 0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay. CONCLUSIONS: These data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.

[Glioma stem cells enhanced angiogenesis and its relationship with microvessel].

OBJECTIVES: To dynamically observe how glioma stem cells promote the tumor formation and angiogenesis, and to study the correlation between the distribution of glioma stem cells and microvessels within different growth stages of subcutaneous tumor. METHODS: Stem cell medium culture and magnetic activated cell sorting were carried out to obtain CD133+ cells from C6 rat glioma cell line. Sprague Dawley (SD) rat ears model were established to observe glioma stem cells promoting blood vessel formation. Subcutaneous glioma model of C6 and immunohistochemical staining of hypoxia inducible factor-1α (HIF-1α) and CD133 were used to investigate the relationship between distribution of glioma stem cells and microvessels. Expressions of CD133 protein in each stage of the subcutaneous tumor were detected by Western blot. RESULTS: Isolation and identification of glioma stem cells deprived from C6 glioma cell line successfully, the establishment of ears model showed real-time dynamic observation of CD133+ cells involved in angiogenesis and tumor formation. SD rat model of subcutaneous glioma showed the initial of tumor formation, CD133+ cells scattered. With tumor growth, CD133+ cells began to tend to capillaries, in late distributed clusters in perivascular. Meanwhile as tumor growth, CD133 protein expression was gradually increased: the values of Western blot analysis of CD133 expression on 6, 9, 12, 15, 20 d were 0.208±0.004, 0.282±0.003, 0.360±0.004, 0.564±0.135, 0.756±0.007, the differences were significant between different groups (F=2601.681, P<0.01). At a high magnification, the CD133 scores with immunohistochemical staining on 6, 9, 12, 15 d were 0.8±0.4, 2.4±0.5, 4.0 ± 0.7, 6.0±0.7; HIF-1α scores were 0.8±0.4, 2.8±0.8, 5.0±0.7, 6.8±0.4. By Spearman rank correlation analysis found that the relationship between CD133 and HIF-1α expression was positively correlated (r=0.921, P<0.01). CONCLUSIONS: Glioma stem cells promote angiogenesis more than non-stem cells; HIF-1α and its downstream gene product might mediate the distribution of glioma stem cells around the perivascular.

[Role of Rac1 in the SDF-1-induced migration and invasion of human glioma cell line U251].

OBJECTIVE: To explore the role of Rac1 in the SDF-1-induced migration and invasion of glioma cells with a specific Rac1 inhibitor. METHODS: Human glioma cell lines U251 treated with SDF-1 or/and specific Rac1 inhibitor were used. The migration and invasion capacities of cells in 2D cell migration/3D invasion assay were assessed. Western blot was employed to detect the levels of Rac1 and GAPDH in cell lysates and the Rac1 activity measured by Rac1 activation assays. Immunofluorescence was used to identify the expression and intracellular location of Rac1 in U251 cells. RESULTS: SDF-1 significantly increased the migration and invasion capacities of U251 cells (P < 0.05). The stimulation of SDF-1 boosted the activity of Rac1 versus the unstimulated cells (P < 0.05). And Rac1 was recruited to protruding edge in SDF-1-stimulated cells. Inhibition of Rac1 with specific Rac1 inhibitor decreased the migration and invasion capacities of SDF-1-induced U251 cells (P < 0.05). In comparison with the SDF-1 treated group, the activity of Rac1 significantly decreased (P < 0.05) and the recruitment of Rac1 to protruding edge significantly decreased in the NSC23766 pre-treated group. CONCLUSIONS: This study provides novel evidence that Rac1 modulates the SDF-1-induced migration and invasion of glioma cells. It suggests that the inhibition of Rac1 activation may be a new therapeutic target for glioma.

Enhanced invasion in vitro and the distribution patterns in vivo of CD133+ glioma stem cells.

BACKGROUND: Recent studies have suggested that cancer stem cells cause tumor recurrence based on their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma cells is also implicated in the failure of current therapies, it is not clear whether cancer stem cells are involved in invasiveness. This study aimed to assess invasive ability of glioma stem cells (GSCs) derived from C6 glioma cell line and the distribution patterns of GSCs in Sprague-Dawley (SD) rat brain tumor. METHODS: Serum-free medium culture and magnetic isolation were used to gain purely CD133(+) GSCs. The invasive ability of CD133(+) and CD133(-) C6 cells were determined using matrigel invasion assay. Immunohistochemical staining for stem cell markers and luxol fast blue staining for white matter tracts were performed to show the distribution patterns of GSCs in brain tumor of rats and the relationship among GSCs, vessels, and white matter tracts. The results of matrigel invasion assay were estimated using the Student's t test and the analysis of Western blotting was performed using the one-way analysis of variance (ANOVA) test. RESULTS: CD133(+) GSCs (number: 85.3 ± 4.0) were significantly more invasive in vitro than matched CD133(-) cells (number: 25.9 ± 3.1) (t = 14.5, P < 0.005). GSCs invaded into the brain diffusely and located in perivascular niche of tumor-brain interface or resided within perivascular niche next to white fiber tracts. The polarity of glioma cells containing GSCs was parallel to the white matter tracts. CONCLUSIONS: Our data suggest that CD133(+) GSCs exhibit more aggressive invasion in vitro and GSCs in vivo probably disseminate along the long axis of blood vessels and transit through the white matter tracts. The therapies targeting GSCs invasion combined with traditional glioblastoma multiforme therapeutic paradigms might be a new approach for avoiding malignant glioma recurrence.

[Correlation between the distribution of CD133-positive cells and the proliferation of microvessels in glioblastoma multiforme].

OBJECTIVE: To investigate the expression of CD133 and CD34 in different parts of glioblastoma and its margin and explore the invasive path of glioma stem cells within the tumor and surrounding tissue. METHODS: The surgical specimens were collected from the core of mass, junctional zones between tumor and peritumoral edematous areas and edematous areas in 52 patients with glioblastoma. Immunohistochemical cell staining and Western blot were employed to evaluate the expression of CD133 in different specimens while immunohistochemistry was used to detect the CD34-microvessel postforming. A correlation analysis was performed between them. RESULTS: The expression of CD133 was not detected in the control groups while the scores were 7.3 ± 1.4, 5.2 ± 1.1, 2.7 ± 1.0 in junctional zones, tumor cores and edematous areas with immunohistochemistry and 0.79 ± 0.03, 0.38 ± 0.01, 0.20 ± 0.04 with Western blot respectively. There were significant differences between different groups (P < 0.05). Under light microscope, the CD133-positive cells frequently forming perivascular pseudorosettes were dense in junctional zones and mostly clustered near the microvessels in tumor cores and scattered in edematous areas. At a high magnification (200×), the CD34-MVD (/HP) values were 31.3 ± 4.0, 21.8 ± 2.6, 15.3 ± 2.4, 4.7 ± 1.5 respectively in junctional zones, tumor cores, edematous areas and control tissues. Significant differences were also found in these groups (P < 0.05). The expression level of CD133-positive cell was positively correlated with the distribution of CD34-microvessels (r = 0.948, P < 0.05). CONCLUSION: Glioma stem cells tend to assemble in the junctional zones where the microvessels are enriched. There is probably an intimate nourishing relationship with the microvessels. The distribution of glioma stem cells may be related with the invasiveness within glioblastoma.

Expression and Distribution Characteristics of Human Ortholog of Mammalian Enabled (hMena) in Glioma.

OBJECTIVE: To investigate the utility of hMena, a family of enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), we sought to characterize the expression profile and distribution characteristics of hMena in a large panel of glioma samples and determine whether hMena expression levels might correlate with the pathological grade of glioma. METHODS: Sixty-five specimens of glioma with different pathological grades and five control brain tissues were collected. In 6 of the 21 glioblastoma patients, multi-specimens were obtained respectively from the main tumor mass, the junction zone between the tumor and the normal tissue, and adjacent brain tissue 1.5 cm away from the tumor boundary under assistance of neuronavigation system during the operation. Immunohistochemistry was used to detect the expression and distribution characteristics of hMena. hMena expression was analyzed by Western blot in 20 specimens. RESULTS: The hMena expression was negative in control brain tissue but positive in different grades of glioma. The expression rate of hMena was positively correlated with the increasing grade of the World Health Orgnization (WHO) classification (r(s)=0.682, P=0.000). hMena was located in cytoplasm. Positive cells only distributed around the vessels within the tumor mass in low grade glioma, while in high grade glioma, these cells were able to be detected not only in the tumor but also in the boundary zone and adjacent brain parenchyma. In the tumor mass, hMena expressed highly and diffusedly. In the junction zone, hMena positive cells formed radiolitic pattern around the vessels. In adjacent brain parenchyma, single positive cell was scattered. hMena expression was markedly elevated in Grade III and IV glioma compared with Grade II and I. CONCLUSION: Our data suggested that the expression of hMena is closely related to malignant grade of glioma. hMena can label the migrating cells, and indicate the migrating path of glioma cells from the tumor to adjacent tissue along with the vascular basement membranes and tracts of white matter.