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Supplementary Figure 2 shows In vitro scratch migration assay.

Glioma cell cytotoxicity with bevacizumab treatment in vitro

Supplementary Figure 3 shows Full scans of the western blotting shown in the figure 6.

Bevacizumab increased the rate of wound closure

Anti-VEGF treatments such as bevacizumab have demonstrated convincing therapeutic advantage in patients with glioblastoma. However, bevacizumab has also been reported to induce invasiveness of glioma. In this study, we examined the effects of rapid antiangiogenesis mediated by oncolytic virus (RAMBO), an oncolytic herpes simplex virus-1 expressing vasculostatin, on bevacizumab-induced glioma invasion. The effect of the combination of RAMBO and bevacizumab in vitro was assessed by cytotoxicity, migration, and invasion assays. For in vivo experiments, glioma cells were stereotactically inoculated into the brain of mice. RAMBO was intratumorally injected 7 days after tumor inoculation, and bevacizumab was administered intraperitoneally twice a week. RAMBO significantly decreased both the migration and invasion of glioma cells treated with bevacizumab. In mice treated with bevacizumab and RAMBO combination, the survival time was significantly longer and the depth of tumor invasion was significantly smaller than those treated with bevacizumab monotherapy. Interestingly, RAMBO decreased the expression of cysteine-rich protein 61 and phosphorylation of AKT, which were increased by bevacizumab. These results suggest that RAMBO suppresses bevacizumab-induced glioma invasion, which could be a promising approach to glioma therapy.

The combination of bevacizumab with temozolomide and radiotherapy was shown to prolong progression-free survival in newly diagnosed patients with glioblastoma, and this emphasizes the potential of bevacizumab as a glioma treatment. However, although bevacizumab effectively inhibits angiogenesis, it has also been reported to induce invasive proliferation. This study examined gene expression in glioma cells to investigate the mechanisms of bevacizumab-induced invasion. We made a human glioma U87ΔEGFR cell xenograft model by stereotactically injecting these cells into the brain of animals. We administered bevacizumab intraperitoneally three times per week. At 18 days after tumor implantation, the brains were removed for histopathology and mRNA was extracted. In vivo, bevacizumab treatment increased glioma cell invasion. qRT-PCR array analysis revealed upregulation of δ-catenin (CTNND2) and several other factors. In vitro, bevacizumab treatment upregulated δ-catenin expression. A low concentration of bevacizumab was not cytotoxic, but tumor cell motility was increased in scratch wound assays and two-chamber assays. Overexpression of δ-catenin increased the tumor invasion in vitro and in vivo. However, δ-catenin knockdown decreased glioma cell invasiveness. The depth of tumor invasion in the U87ΔEGFR cells expressing δ-catenin was significantly increased compared with empty vector-transfected cells. The increase in invasive capacity induced by bevacizumab therapy was associated with upregulation of δ-catenin expression in invasive tumor cells. This finding suggests that δ-catenin is related to tumor invasion and migration.

WST-1 assay and two-chamber assay

Bevacizumab treatment led to increase in invasive and high expression of Î'-catenin. Silencing of Î'-catenin decreased bevacizumab-induced glioma invasion and Î'-catenin expression. Overexpressed Î'-catenin tumor cells showed more invasiveness and expressed high level ofÎ'-catenin protein in the tumor area in vivo.

Supplementary Figure 1 shows Evaluation of glioma cells infected by oncolytic virus and conditioned medium.

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and its overexpression has been shown to exert anti-tumor effects as a therapeutic target gene in many human cancers. Recently, we demonstrated the anti-glioma effects of an adenoviral vector carrying REIC/Dkk-3 with the super gene expression system (Ad-SGE-REIC). Anti-vascular endothelial growth factor treatments such as bevacizumab have demonstrated convincing therapeutic advantage in patients with glioblastoma. However, bevacizumab could not improve overall survival in patients with newly diagnosed glioblastoma. In this study, we examined the effects of Ad-SGE-REIC on glioma treated with bevacizumab. Treatment of Ad-SGE-REIC resulted in significant reduced numbers of invasion cells treated with bevacizumab.. Western blot analyses revealed increased expression of several endoplasmic reticulum stress markers in cells treated with both bevacizumab and Ad-SGE-REIC and decreased β-catenin protein levels. Expressions of apoptosis markers were also increased in cells with combination therapy. In malignant glioma mouse models, overall survival was extended in the combination therapy group. These results suggest that the combination therapy of Ad-SGE-REIC and bevacizumab exerts anti-glioma effects by suppressing angiogenesis and invasion of tumors. Combined Ad-SGE-REIC and bevacizumab might indicate a promising strategy for the treatment of malignant glioma.

Glioblastoma (GBM) is the most lethal primary brain tumor characterized by significant cellular heterogeneity, namely tumor cells, including GBM stem-like cells (GSCs) and differentiated GBM cells (DGCs), and non-tumor cells such as endothelial cells, vascular pericytes, macrophages, and other types of immune cells. GSCs are essential to drive tumor progression, whereas the biological roles of DGCs are largely unknown. In this study, we focused on the roles of DGCs in the tumor microenvironment. To this end, we extracted DGC-specific signature genes from transcriptomic profiles of matched pairs of in vitro GSC and DGC models. By evaluating the DGC signature using single cell data, we confirmed the presence of cell subpopulations emulated by in vitro culture models within a primary tumor. The DGC signature was correlated with the mesenchymal subtype and a poor prognosis in large GBM cohorts such as The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project. In silico signaling pathway analysis suggested a role of DGCs in macrophage infiltration. Consistent with in silico findings, in vitro DGC models promoted macrophage migration. In vivo, coimplantation of DGCs and GSCs reduced the survival of tumor xenograft-bearing mice and increased macrophage infiltration into tumor tissue compared with transplantation of GSCs alone. DGCs exhibited a significant increase in YAP/TAZ/TEAD activity compared with GSCs. CCN1, a transcriptional target of YAP/TAZ, was selected from the DGC signature as a candidate secreted protein involved in macrophage recruitment. In fact, CCN1 was secreted abundantly from DGCs, but not GSCs. DGCs promoted macrophage migration in vitro and macrophage infiltration into tumor tissue in vivo through secretion of CCN1. Collectively, these results demonstrate that DGCs contribute to GSC-dependent tumor progression by shaping a mesenchymal microenvironment via CCN1-mediated macrophage infiltration. This study provides new insight into the complex GBM microenvironment consisting of heterogeneous cells.

TERT promoter mutations are commonly associated with 1p/19q codeletion in IDH-mutated gliomas. However, whether these mutations have an impact on patient survival independent of 1p/19q codeletion is unknown. In this study, we investigated the impact of TERT promoter mutations on survival in IDH-mutated glioma cases. Detailed clinical information and molecular status data were collected for a cohort of 560 adult patients with IDH-mutated gliomas. Among these patients, 279 had both TERT promoter mutation and 1p/19q codeletion, while 30 had either TERT promoter mutation (n = 24) or 1p/19q codeletion (n = 6) alone. A univariable Cox proportional hazard analysis for survival using clinical and genetic factors indicated that a Karnofsky performance status score (KPS) of 90 or 100, WHO grade II or III, TERT promoter mutation, 1p/19q codeletion, radiation therapy, and extent of resection (90–100%) were associated with favorable prognosis (p TERT promoter mutation had a significantly favorable prognostic impact (hazard ratio = 0.421, p = 0.049), while 1p/19q codeletion did not have a significant impact (hazard ratio = 0.648, p = 0.349). Analyses incorporating patient clinical and genetic information were further conducted to identify subgroups showing the favorable prognostic impact of TERT promoter mutation. Among the grade II-III glioma patients with a KPS score of 90 or 100, those with IDH-TERT co-mutation and intact 1p/19q (n = 17) showed significantly longer survival than those with IDH mutation, wild-type TERT, and intact 1p/19q (n = 185) (5-year overall survival, 94% and 77%, respectively; p = 0.032). Our results demonstrate that TERT promoter mutation predicts favorable prognosis independent of 1p/19q codeletion in IDH-mutated gliomas. Combined with its adverse effect on survival among IDH-wild glioma cases, the bivalent prognostic impact of TERT promoter mutation may help further refine the molecular diagnosis and prognostication of diffuse gliomas.

Glioblastoma (GBM) is characterized by extensive tumor cell invasion, angiogenesis, and proliferation. We previously established subclones of GBM cells with distinct invasive phenotypes and identified annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we further explored the role of ANXA2 in regulating phenotypic transition in GBM. We identified oncostatin M receptor (OSMR) as a key ANXA2 target gene in GBM utilizing microarray analysis and hierarchical clustering analysis of the Ivy Glioblastoma Atlas Project and The Cancer Genome Atlas datasets. Overexpression of ANXA2 in GBM cells increased the expression of OSMR and phosphorylated signal transducer and activator of transcription 3 (STAT3) and enhanced cell invasion, angiogenesis, proliferation, and mesenchymal transition. Silencing of OSMR reversed the ANXA2-induced phenotype, and STAT3 knockdown reduced OSMR protein expression. Exposure of GBM cells to hypoxic conditions activated the ANXA2–STAT3–OSMR signaling axis. Mice bearing ANXA2-overexpressing GBM exhibited shorter survival times compared with control tumor-bearing mice, whereas OSMR knockdown increased the survival time and diminished ANXA2-mediated tumor invasion, angiogenesis, and growth. Further, we uncovered a significant relationship between ANXA2 and OSMR expression in clinical GBM specimens, and demonstrated their correlation with tumor histopathology and patient prognosis. Our results indicate that the ANXA2–STAT3–OSMR axis regulates malignant phenotypic changes and mesenchymal transition in GBM, suggesting that this axis is a promising therapeutic target to treat GBM aggressiveness.

Background The prevalence of programmed death-ligand 1 (PD-L1) and PD-L2 expression on tumor cells and tumor-infiltrating immune cells in primary central nervous system lymphoma (PCNSL) remains unclear. In the present study, we analyzed needle biopsy and craniotomy specimens of patients with PCNSL to compare the PD-L1 and PD-L2 levels in the tumor and surrounding (peritumoral) tissue. We also assessed the correlation between biological factors and the prognostic significance of PD-L1 and PD-L2 expression. Methods We retrospectively analyzed the cases of 70 patients histologically diagnosed with PCNSL (diffuse large B-cell lymphoma). Immunohistochemistry for CD20, CD68, PD-L1, and PD-L2 was performed. In cases with specimens taken by craniotomy, the percentages of PD-L1- and PD-L2-positive macrophages were evaluated in both tumor and peritumoral tissue. The Kaplan-Meier method with log-rank test and Cox proportional hazard model were used for survival analysis. Results The tumor cells expressed little or no PD-L1 and PD-L2, but macrophages expressed PD-L1 and PD-L2 in most of the patients. The median percentage of PD-L2-positive cells was significantly higher among peritumoral macrophages (32.5%; 95% CI: 0–94.6) than intratumoral macrophages (27.5%; 95% CI: 0–81.1, p = 0.0014). There was a significant correlation between the percentages of PD-L2-positive intratumoral macrophages and PD-L2-positive peritumoral macrophages (p = 0.0429), with very low coefficient correlation (ρ = 0.098535). PD-L1 expression on macrophages was significantly associated with biological factors (intratumoral macrophages: better KPS, p = 0.0008; better MSKCC score, p = 0.0103; peritumoral macrophages: low proportion of LDH elevation, p = 0.0064) and longer OS (for intratumoral macrophages: high PD-L1 = 60 months, 95% CI = 30–132.6; low PD-L1 = 24 months, 95% CI = 11–48; p = 0.032; for peritumoral macrophages: high PD-L1 = 60 months, 95% CI = 30.7–NR; low PD-L1 = 14 months, 95% CI = 3–26). PD-L1 expression on peritumoral macrophages was strongly predictive of a favorable outcome (HR = 0.30, 95% CI = 0.12–0.77, p = 0.0129). Conclusions Macrophages in intratumoral and peritumoral tissue expressed PD-L1 and PD-L2 at a higher rate than tumor cells. PD-L1 expression, especially on peritumoral macrophages, seems to be an important prognostic factor in PCNSL. Future comprehensive analysis of checkpoint molecules in the tumor microenvironment, including the peritumoral tissue, is warranted.

Glioblastoma has the poorest prognosis, and is characterized by excessive invasion and angiogenesis. To determine the invasive mechanisms, we previously used two glioma cell lines (J3T-1 and J3T-2) with different invasive phenotypes. The J3T-1 showed abundant angiogenesis and tumor cell invasion around neovasculature, while J3T-2 showed diffuse cell infiltration into surrounding healthy parenchyma. Microarray analyses were used to identify invasion-related genes in J3T-2 cells, and the expressed genes and their intracellular and intratumoral distribution patterns were evaluated in J3T-2 cell lines, human glioma cell lines, human glioblastoma stem cells and human glioblastoma specimens. To determine the role of the invasion-related genes, invasive activities were evaluated in vitro and in vivo. Fibroblast growth factor 13 (FGF13) was overexpressed in J3T-2 cells compared to J3T-1 cells, and in human glioma cell lines, human glioblastoma stem cells and human glioblastoma specimens, when compared to that of normal human astrocytes. Immunohistochemical staining and the RNA-seq (sequencing) data from the IVY Glioblastoma Atlas Project showed FGF13 expression in glioma cells in the invasive edges of tumor specimens. Also, the intracellular distribution was mainly in the cytoplasm of tumor cells and colocalized with tubulin. Overexpression of FGF13 stabilized tubulin dynamics in vitro and knockdown of FGF13 decreased glioma invasion both in vitro and in vivo and prolonged overall survival of several xenograft models. FGF13 was negatively regulated by hypoxic condition. Silencing of FGF13 also decreased in vivo bevacizumab-induced glioma invasion. In conclusion, FGF13 regulated glioma cell invasion and bevacizumab-induced glioma invasion, and could be a novel target for glioma treatment.

BACKGROUND Malignant glioma has a poor prognosis and is characterized by excessive proliferation, invasion, and angiogenesis. Previously, we established subclones of glioma cell lines with different invasive phenotypes to investigate the mechanisms of invasion in this malignancy. That study revealed annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we investigated the molecular mechanism of the phenotypic shift in glioma that is induced by ANXA2. METHODS We identified ANXA2 target genes using microarray analyses of our four established cell lines and RNA-seq data from the IVY Glioblastoma Atlas Project. Co-expression was then confirmed in human glioma cells. Additionally, qRT-PCR, immunoblotting, invasion assays, proliferation assays, and tube formation assays were performed to uncover the specific roles of these angiogenic invasion-related genes. Furthermore, we examined survival times and phenotypic shifts in vivo. RESULTS We identified oncostatin M receptor (OSMR) as an ANXA2-target gene using microarray and RNA-seq, and confirmed its expression correlated with ANXA2 in glioma cells. ANXA2-overexpressing glioma cells showed enhanced OSMR expression and STAT3 phosphorylation, while STAT3 knockdown reduced OSMR expression. When ANXA2 was overexpressed in glioma cells, invasion, angiogenesis, proliferation, and epithelial-mesenchymal transition were promoted; however, silencing OSMR attenuated the ANXA2-induced phenotypic shift. In vivo, ANXA2-overexpressing glioma cells shortened the survival time of tumor-bearing mice, whereas OSMR knockdown increased survival times. CONCLUSIONS We analyzed genes whose expression was regulated by ANXA2 in glioma using invasive glioma models. Through this analysis, we identified that ANXA2 and OSMR regulate a phenotypic shift, suggesting that OSMR could be a promising target to treat and prevent glioma invasion.

TERT promoter mutation is associated with 1p/19q codeletion and favorable prognosis in IDH-mutated gliomas. Prognostic and diagnostic significance of TERT promoter mutation is well-recognized in IDH-wildtype glioblastomas, but not in IDH-mutated gliomas. We investigated prognostic efficacy of TERT mutation in a cohort of 560 Japanese IDH-mutated adult gliomas. The molecular status of IDH, TERT and 1p/19q and patient clinical data including Karnofsky performance status (KPS) were collected in all cases. TERT mutations and 1p/19q codeletions were found in 303 and 285 cases, respectively. The patient cohort was divided into four groups by a combination of the 1p/19q and TERT status. The characteristics of 1p/19q intact-TERT mutated group (Astro-TERT group, n=24) were compared with those of 1p/19q intact-TERT wild (Astro-group, n=251) or 1p/19q codeleted-TERT mutated (Oligo-group, n=279) cases. Astro-TERT group with any grade showed intermediate overall survival between the Oligo-group and Astro-group although the survival differences were not statistically significant (median overall survival (OS) not reached (NR) versus NR, and 106 months, respectively. p >0.05). We further conducted subgroup analysis by adjusting KPS and WHO grade as Cox regression analysis for survival indicated the unfavorable survival impact of KPS < 90 and WHO grade IV. In the subgroup with favorable KPS (90–100) and grade II-III (n=438), The OS of Astro-TERT group (median NR) was significantly longer survival than that of Astro-group (median 120.2 months, p=0.032), and was comparable with that of the Oligo-group (median NR, p >0.05). On the other hand, OS of none of the molecular groups significantly differ in poorer KPS subgroups (p >0.05). In grade IV tumors, the OS of the Astro-TERT group (NR) was comparable with that of Astro-group (29 months, p=0.19) rather than Oligo-group (NR, p=0.051). Thus, TERT promoter status provides a valuable prognostic information for IDH-mutated grade II-III gliomas in the current molecular diagnostic system.

INTRODUCTION Expression of the gene encoding reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) was reported to be reduced in a variety of human cancer cells. We previously examined the antitumor effect of an adenoviral vector carrying REIC/Dkk-3 gene (Ad-CAG-REIC). Recently, a novel adenoviral vector expressing REIC/Dkk-3 has also been developed based on the cytomegalovirus promoter-driven super gene expression system (Ad-SGE-REIC). We evaluated the anti-glioma effect of the Ad-SGE-REIC and planned a clinical trial of Ad-SGE-REIC for malignant glioma. MATERIALS AND METHODS We evaluated a cytotoxicity assay to treatments with Ad-SGE-REIC, Ad-CAG-REIC, or Ad-LacZ (control) using malignant glioma cells. The survival of mice in each group was analyzed by the Kaplan–Meier method. We also performed Good Laboratory Practice (GLP) toxicology tests and prepared a protocol for this clinical trial. RESULTS In the cytotoxicity assay, after treatment with Ad-SGE-REIC, the number of malignant glioma cells attached to the bottom of culture wells was significantly reduced in a time-dependent manner. Mice treated with Ad-SGE-REIC survived significantly longer than those treated with other vectors. We also performed GLP toxicology tests using intracranial injection of higher doses of Ad-SGE-REIC in rats at Shin Nippon Biomedical Laboratories (SNBL Japan) to determine the injection dose of Ad-SGE-REIC for this clinical trial. After finishing the consultation with Pharmaceuticals and Medical Devices Agency (PMDA), we prepared a protocol for a phase I/IIa clinical trial of Ad-SGE-REIC for the treatment of recurrent malignant glioma supported by Japan Agency for Medical Research and Development (AMED). This protocol was reviewed by institution review board (IRB) in March 2019. We submitted a notification of this trial in April 2019. CONCLUSIONS We demonstrated the anti-glioma effect of Ad-SGE-REIC. We will start a phase I/IIa clinical trial of Ad-SGE-REIC for the treatment of recurrent malignant glioma.

PD-L1 and PD-L2 expression on tumor cells and tumor-infiltrating immune cells in primary central nervous system lymphoma (PCNSL) remains unclear. In the present study, we investigated the expressions of PD-L1 and PD-L2 in surgical specimens from needle biopsies and craniotomies to compare tumor tissue with surrounding tumor tissue (peritumoral tissue) and analyzed the correlation between expression of PD-L1/PD-L2 and survival in patients with PCNSL. We retrospectively analyzed the cases of 70 patients histologically diagnosed with PCNSL (diffuse large B-cell lymphoma). Immunohistochemistry for CD20, CD68, PD-L1, and PD-L2 was performed. In cases with specimens taken by craniotomy, the percentages of PD-L1- and PD-L2-positive macrophages were evaluated in both tumor and peritumoral tissue. The Kaplan-Meier method with log-rank test and Cox proportional hazard model were used for survival analysis. The tumor cells did not express very much PD-L1 and PD-L2, but macrophages expressed PD-L1 and PD-L2 in most of the patients. The median percentage of PD-L2-positive cells was significantly higher among peritumoral macrophages (32.5%; 95%CI: 0–94.6) than intratumoral macrophages (27.5%; 95%CI: 0–81.1, p=0.0014). There was a significant correlation between the percentages of PD-L2-positive intratumoral macrophages and PD-L2-positive peritumoral macrophages (p=0.0429), with very low coefficient correlation (ρ=0.098535). PD-L1 expression on macrophages was significantly associated with biological factors (intratumoral macrophages: better KPS, p=0.0008; better MSKCC score, p=0.0103; peritumoral macrophages: low proportion of LDH elevation, p=0.0064) and longer OS (for intratumoral macrophages: high PD-L1=60 months, 95%CI=30–132.6; low PD-L1=24 months, 95%CI=11–48; p=0.032; for peritumoral macrophages: high PD-L1=60 months, 95%CI=30.7-NR; low PD-L1=14 months, 95%CI=3–26). PD-L1 expression on peritumoral macrophages was strongly predictive of a favorable outcome (HR=0.30, 95%CI=0.12–0.77, p=0.0129). Macrophages in intratumoral and peritumoral tissue expressed PD-L1 and PD-L2 at a higher rate than tumor cells. PD-L1 expression, especially on peritumoral macrophages, seems to be an important prognostic factor in PCNSL.

INTRODUCTION: Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. We have investigated the anti-glioma effect of the adenovirus vector carrying REIC/Dkk-3 (Ad-CAG-REIC). Recently an Ad-REIC vector with the super gene expression (SGE) system (Ad-SGE-REIC) has been developed for higher protein expression and therapeutic effects than the conventional adenoviral vector (Ad-CAG-REIC). In this study, we evaluated the anti-glioma effect of the Ad-SGE-REIC against malignant glioma. MATERIALS AND METHODS: Transcriptome analysis of the differential (exome sequencing-derived) expression levels of REIC was conducted based on using The Cancer Genome Atlas (TCGA) GBM patient dataset (n=349) using the Project Betastasis web platform (http://www.betastasis.com/glioma/tcga_gbm/). To evaluate the anti-glioma effect of the Ad-SGE-REIC, we conducted a cytotoxicity assay to assess treatments with Ad-SGE-REIC, Ad-CAG-REIC, or Ad-LacZ (control) using malignant glioma cells (U87ΔEGFR or GL261) and normal human astrocytes (NHAs). Seven days after implantation of glioma cells into the brain of mice, Ad-SGE-REIC, Ad-CAG-REIC, or Ad-LacZ (control) was injected stereotactically at the tumor inoculation site. The survival of mice in each group was analyzed by the Kaplan–Meier method. RESULTS: Analysis of the TCGA GBM patient dataset revealed that differential expression levels of REIC were lower in all subgroups, including Proneural, Neural, Classical, and Mesenchymal groups, than in the control group. In the cytotoxicity assay, after treatment with Ad-SGE-REIC, the number of malignant glioma cells attached to the bottom of culture wells was significantly reduced in a time-dependent manner. The survival time of the mice treated with Ad-SGE-REIC was significantly longer than those treated with Ad-LacZ or Ad-CAG-REIC. CONCLUSIONS: We demonstrated the anti-glioma effect of Ad-SGE-REIC. We are now planning an investigator-initiated clinical trial (phase I/IIa) of Ad-SGE-REIC for the treatment of recurrent malignant glioma.

Anti-VEGF treatments such as bevacizumab have demonstrated convincing therapeutic advantage in patients with glioblastoma. However, bevacizumab has also been reported to induce invasiveness of glioma. In this study, we examined the effects of rapid antiangiogenesis mediated by oncolytic virus (RAMBO), an oncolytic herpes simplex virus-1 expressing vasculostatin, on bevacizumab-induced glioma invasion. The effect of the combination of RAMBO and bevacizumab in vitro was assessed by cytotoxicity, migration, and invasion assays. For in vivo experiments, glioma cells were stereotactically inoculated into the brain of mice. RAMBO was intratumorally injected 7 days after tumor inoculation, and bevacizumab was administered intraperitoneally twice a week. RAMBO significantly decreased both the migration and invasion of glioma cells treated with bevacizumab. In mice treated with bevacizumab and RAMBO combination, the survival time was significantly longer and the depth of tumor invasion was significantly smaller than those treated with bevacizumab monotherapy. Interestingly, RAMBO decreased the expression of cysteine-rich protein 61 and phosphorylation of AKT, which were increased by bevacizumab. These results suggest that RAMBO suppresses bevacizumab-induced glioma invasion, which could be a promising approach to glioma therapy.

The combination of bevacizumab with temozolomide and radiotherapy was shown to prolong progression-free survival in newly diagnosed patients with glioblastoma, and this emphasizes the potential of bevacizumab as a glioma treatment. However, although bevacizumab effectively inhibits angiogenesis, it has also been reported to induce invasive proliferation. This study examined gene expression in glioma cells to investigate the mechanisms of bevacizumab-induced invasion. We made a human glioma U87ΔEGFR cell xenograft model by stereotactically injecting these cells into the brain of animals. We administered bevacizumab intraperitoneally three times per week. At 18 days after tumor implantation, the brains were removed for histopathology and mRNA was extracted. In vivo, bevacizumab treatment increased glioma cell invasion. qRT-PCR array analysis revealed upregulation of δ-catenin (CTNND2) and several other factors. In vitro, bevacizumab treatment upregulated δ-catenin expression. A low concentration of bevacizumab was not cytotoxic, but tumor cell motility was increased in scratch wound assays and two-chamber assays. Overexpression of δ-catenin increased the tumor invasion in vitro and in vivo. However, δ-catenin knockdown decreased glioma cell invasiveness. The depth of tumor invasion in the U87ΔEGFR cells expressing δ-catenin was significantly increased compared with empty vector-transfected cells. The increase in invasive capacity induced by bevacizumab therapy was associated with upregulation of δ-catenin expression in invasive tumor cells. This finding suggests that δ-catenin is related to tumor invasion and migration.

In spite of the development of treatments, long-term survival in patients with glioblastoma is very rare. Recently, studies about clarifying prognostic markers for glioblastoma have been growing. Previously we reported that median progression-free survival and overall survival (OS) of patients with high cysteine-rich protein 61 (CCN1) expression were significantly shorter than those of patients with low CCN1 expression. To investigate the molecular mechanisms that regulate CCN1 expression, we analysed 147 tumor samples from 80 glioblastoma patients and 67 lower grade glioma patients using next-generation and Sanger sequencing. Sequencing analysis showed that CCN1 high expression group includes PIK3R1Met326Ile more frequently (10/37 cases, 27.0%) than CCN1 low expression group (3/38 cases, 7.9%) in glioblastoma. We also found this mutation in corresponding blood samples. And multivariate analysis indicated that, high CCN1 expression and PIK3R1Met326Ile in glioblastoma patients were prognostic factors for OS ([HR=2.488 (1.298–4.769), p=0.006] and [HR=2.089 (1.020–4.277), p=0.0439], respectively. According to our results, the PIK3R1Met326Ile mutation related to CCN1 expression and poor prognosis in glioblastoma.

Despite therapeutic advances, glioblastoma represents a lethal brain tumor. Recently, research to identify prognostic markers for glioblastoma has intensified. Our previous study demonstrated that median progression-free survival (PFS) and overall survival (OS) of patients with high cysteine-rich protein 61 (CCN1) expression was significantly shorter than that of patients with low CCN1 expression. To understand the molecular mechanisms that regulate CCN1 expression, we examined 147 tumour samples from 80 patients with glioblastoma and 67 patients with lower grade glioma. Next-generation and Sanger sequencing showed that PIK3R1Met326Ile was more frequent in the CCN1 high expression group (10/37 cases, 27.0%) than the CCN1 low expression group (3/38 cases, 7.9%) in glioblastoma. This mutation was also detected in corresponding blood samples. In multivariate analysis, high CCN1 expression and PIK3R1Met326Ile in glioblastoma patients were prognostic factors for OS [HR = 2.488 (1.298–4.769), p = 0.006] and [HR = 2.089 (1.020–4.277), p = 0.0439], respectively. Thus, the PIK3R1Met326Ile germline appears to be correlated with CCN1 expression and poor prognosis in glioblastoma.

Despite advances in treatment, glioblastoma remains a lethal brain tumor. Efforts to identify prognostic molecular biomarkers for glioblastoma have intensified, and we previously reported that the median progression-free survival (PFS) and overall survival (OS) of patients with high levels of cysteine-rich protein 61 (CCN1/CYR61) are significantly shorter compared with those with low levels of CCN1. To identify the molecular mechanisms that regulate the expression of CCN1 in glioblastomas, we analyzed tumor samples from 42 patients with glioblastoma. Next-generation and Sanger sequencing techniques revealed that the PIK3R1Met326Ile mutation was significantly more frequent in patients with high vs low levels of CCN1 (9/22, 40.9% vs 2/20, 10.0%; respectively). Moreover, this mutation was identified in the corresponding blood samples. Univariate analysis of Kaplan-Meier survival data revealed that high levels of CCN1 and the presence of the PIK3R1Met326Ile mutation were significantly associated with PFS (p=0.0003 and 0.0191, respectively) and that age, high levels of CCN1, and the presence of the PIK3R1Met326Ile mutation were significantly associated with OS (p=0.0016, 0.0019 and 0.0057, respectively). Multivariate analysis identified age >65 years and the presence of the PIK3R1Met326Ile mutation as prognostic factors for OS (HR=3.533 [1.519-8.220], p=0.003) and (HR=2.712 [1.187-6.198], p=0.018, respectively). We conclude that the germline mutation PIK3R1Met326Ile correlates with the levels of CCN1 and poor prognosis of glioblastoma.

Introduction: Malignant gliomas are known to secrete high levels of vascular endothelial growth factor (VEGF). Bevacizumab, which is a monoclonal antibody to VEGF, has demonstrated convincing therapeutic benefits in patients with glioblastoma. However, several reports have shown that anti-VEGF therapy induced invasive proliferation. Materials and Methods: We examined the effects of bevacizumab-induced invasive changes in glioma xenografts. U87ΔEGFR and Gli36Δ5 cells were stereotactically injected into the brain of nude mice or rats. Five days after tumor cell implantation, bevacizumab was administered intraperitoneally three times a week. At 18 days after tumor implantation, the brains were removed and observed histopathologically by an immunohistochemical assay and a transmission electron microscope. We analyzed the pathological changes which were treated with bevacizumab compared to the untreated group. Results: The bevacizumab treatment led to increased tumor cell invasion in spite of decreased angiogenesis and tumor size. Both immunohistochemical staining by collagen type I and transmission electron microscopic analysis demonstrated the remarkable increase of extracellular components in contrast to the decrease of angiogenesis. Conclusions: This study showed that the anti-angiogenic treatment with bevacizumab was effective to decrease vessels and tumor mass of malignant glioma, however it changed the microenvironment, including extracellular components around malignant glioma. The elucidation of this mechanism might contribute to the treatment of bevacizumab-refractory glioma.

We report here a case of cervical carotid artery aneurysm. This 37-year-old male suffered from acute neck swelling when he was taking lunch after physical work. Ultrasonography demonstrated a cervical pseudoaneurysm and a jet flow, which was blowing into the dome from the carotid artery. Angiogram revealed an aneurysm with a diameter of 3 cm at the bifurcation of the common carotid artery. Coil embolization using double-catheter technique was performed and complete occlusion of the aneurysm was obtained without any complications. The patient returned to his normal life. Cervical carotid artery aneurysms are rare and they induce pain by swelling in the neck, hoarseness, swallowing disturbance, hemorrhage, and cerebral ischemia due to embolism. In case of a cervical carotid artery aneurysm, safe and effective treatments are required and endovascular treatment should be considered. Although stent-assisted coil embolization or covered-stent placement were reported as an effective treatment for cervical aneurysms, coil embolization without using a stent was performed in this particular patient who is a young, blue-collar worker because the avoidance of long-term anti-platelet therapy was desirable. Preoperative evaluation is important to select adequate treatment.