Brain Tumor Invasion Rate Measured In Vitro Does Not Correlate with Ki-67 Expression

The need for more accurate prediction of the biological behavior of brain tumors has lead to the use of immunohistochemical methods for assessment of proliferating cell nuclear antigens such as Ki-67. There is a variable association of glioma Ki-67 labeling index with patient survival. Brain invasion by individual tumor cells also defines biological aggressiveness, and can be assessed in vitro. Further, proliferation and migration seem to be mutually exclusive behaviors for a given cell at a point in time. We studied the relationship between Ki-67 labeling index and invasion rate in a group of 10 gliomas, and 2 meningiomas. Human tumor spheroids obtained from operative speciments were co-cultured with fetal rat brain aggregates, and invasion rate was measured by confocal microscopic observation. There was no correlation between two measures of invasion and Ki-67 labeling. This finding supports the dichotomous nature of glioma proliferation and invasion, and may in part explain the limited usefulness of proliferation marker labeling.     

Effects of Radiation on a Model of Malignant Glioma Invasion

We sought to characterize the effects of radiation alone and in combination with BCNU and dexamethasone on malignant glioma invasion. A model of malignant glioma invasion into a gel matrix of collagen type I was used to characterize response to radiation treatment for four malignant glioma cell lines (C6, U251, U373, A172) and nine primary human glioblastoma explants. A radiation dose dependent inhibition of invasion was noted for the C6 astrocytoma cell line but not the other cell lines or explants. Addition of BCNU and dexamethasone to radiation produced additional inhibition of invasion among the cell lines and explants but could not suppress invasion entirely.     

Expression of ADAMTS-8, a secreted protease with antiangiogenic properties, is downregulated in brain tumours

Angiogenesis and extracellular matrix degradation are key events in tumour progression, and factors regulating stromal-epithelial interactions and matrix composition are potential targets for the development of novel anti-invasive/antiangiogenic therapies. Here, we examine the expression of ADAMTS-8, a secreted protease with antiangiogenic properties, in brain tissues. Using quantitative RT-polymerase chain reaction (PCR), high, equivalent expression of ADAMTS-8 was found in normal whole brain, cerebral cortex, frontal lobe, cerebellum and meninges. ADAMTS-8 expression in 34 brain tumours (including 22 high-grade gliomas) and four glioma cell lines indicated at least two-fold reduction in mRNA compared to normal whole brain in all neoplastic tissues, and no detectable expression in 14 out of 34 (41%) tumours or four out of four (100%) cell lines. In contrast, differential expression of TSP1 and VEGF was seen in nine out of 15 (60%) and seven out of 13 (54%) tumours, with no relationship in the expression of these genes. Immunohistochemistry and Western analysis indicated downregulation of ADAMTS-8 protein in >77% tumours. Methylation-specific PCR analysis of ADAMTS-8 indicated promoter hypermethylation in one out of 24 brain tumours (a metastasis) and three out of four glioma cell lines suggesting an alternative mechanism of downregulation. These data suggest a role for ADAMTS-8 in brain tumorigenesis, warranting further investigation into its role in regulation of tumour angiogenesis and local invasion.     

Serum angiogenic profile of patients with glioblastoma identifies distinct tumor subtypes and shows that TIMP-1 is a prognostic factor

Angiogenesis plays a key role in glioblastoma biology and antiangiogenic agents are under clinical investigation with promising results. However, the angiogenic profiles of patients with glioblastoma and their clinical significance are not well understood. Here we characterize the serum angiogenic profile of patients with glioblastoma, and examine the prognostic significance of individual angiogenic factors. Serum samples from 36 patients with glioblastoma were collected on admission and simultaneously assayed for 48 angiogenic factors using protein microarrays. The data were analyzed using hierarchical cluster analysis. Vessel morphology was assessed histologically after immunostaining for the pan-endothelial marker CD31. Tumor samples were also immunostained for tissue inhibitor of metalloproteinase-1 (TIMP-1). Cluster analysis of the serum angiogenic profiles revealed 2 distinct subtypes of glioblastoma. The 2 subtypes had markedly different tumor microvessel densities. A low serum level of TIMP-1 was associated with significantly longer survival independent of patient age, performance status, or treatment. The serum angiogenic profile in patients with glioblastoma mirrors tumor biology and has prognostic value. Our data suggest the serum TIMP-1 level as an independent predictor of survival.     

Brain meningioma invading and destructing the skull bone: replacement of the missing bone in vivo

Background

Meningiomas are frequently encountered tumours. In those invading locally into the adjacent tissue, reconstructions may pose a problem.

Case report

We report a case of a benign convexity brain meningioma with invasion into the skull bone and subcutaneous tissue. The tumour was removed completely, together with the infiltrated tissue and the defects were successfully closed with in vivo bone reconstruction.

Conclusions

The reconstruction of the skull bone is sometimes needed after the benign meningioma excision. Artificial bone may be a suitable material, allowing fast intraoperative reconstruction with excellent brain protection and cosmetic effect during the one-stage procedure.     

Potential Mechanisms of Benzyl Isothiocyanate Suppression of Invasion and Angiogenesis by the U87MG Human Glioma Cell Line

Glioma is one of the most common tumors in China and chemotherapy is critical for its treatment. Recentstudies showed that benzyl isothiocyanate (BITC) could inhibit the growth of glioma cells, but the mechanisms arenot fully understood. This study explored the inhibitory effect of BITC on invasion and angiogenesis of U87MGhuman glioma cells in vitro and in vivo, as well as potential mechanisms. It was found that BITC could inhibitinvasion and angiogenesis of human glioma U87MG cells by inducing cell cycle arrest at phase G2/M. It alsowas demonstrated that BITC decreased expression of cyclin B1, p21, MMP-2/9, VE-cadherin, CD44, CXCR4and MTH1, the activity of the telomerase and PKCζ pathway. Microarray analysis was thus useful to explorethe potential target genes related to tumorigenic processes. BITC may play important roles in the inhibition ofinvasion and angiogenesis of human glioma cells.     

CD133/prominin1 is prognostic for GBM patient's survival, but inversely correlated with cysteine cathepsins' expression in glioblastoma derived spheroids

Introduction

CD133 is a marker for a population of glioblastoma (GBM) and normal neural stem cells (NNSC). We aimed to reveal whether the migratory potential and differentiation of these stem cells is associated with CD133 expression and with cathepsin proteases (Cats).

Materials and methods.

The invasiveness of normal NNSC, GBM/CD133+ cell lines and GBM spheroids was evaluated in 3D collagen, as well as of U87-MG and normal astrocytes (NHA) grown in monolayers in 2D Matrigel. Expression of Cats B, L and S was measured at mRNA and activity levels and their relation to invasiveness, to CD133 mRNA in 26 gliomas, and to the survival of these patients.

Results

The average yield of CD133+ cells from GBM samples was 9.6 %. Survival of patients with higher CD133 mRNA expression was significantly shorter (p< 0.005). Invasion, associated with proteolytic degradation of matrix, was higher in normal stem cells and GBM spheroids and cells than in isolated GBM CD133+ cells. In glioma samples, there was no correlation between CD133 mRNA expression and Cat mRNAs, but there was an inverse correlation with Cat activities.

Conclusions

The study confirms CD133 as a prognostic marker for the survival of GBM patients. We demonstrated that NNSC have higher invasion potential and invade the collagen matrix in a mode different from that of GBM, initiating stem cell spheres. This result could have implications for the design of new therapeutics, including protease inhibitors that specifically target invasive tumour stem cells. Increased activity of cathepsins in CD133– cells suggests their role in the invasive behaviour of GBM.     

The Role of Matrix Metalloproteinase Genes in Glioma Invasion: Co-dependent and Interactive Proteolysis

Matrix metalloproteinases (MMPs) are cation-dependent endopeptidases which have been implicated in the malignancy of gliomas. It is thought that the MMPs play a critical role in both metastasis and angiogenesis, and that interference with proteases might therefore deter local tumor dissemination and neovascularization. However, the attempt to control tumor-associated proteolysis will rely on better definition of the normal tissue function of MMPs, an area of study still in its infancy in the central nervous system (CNS). Understanding the role of MMP-mediated proteolysis in the brain relies heavily on advances in other areas of molecular neuroscience, most notably an understanding of extracellular matrix (ECM) composition and the function of cell adhesion molecules such as integrins, which communicate knowledge of ECM composition intracellularly. Recently, protease expression and function has been shown to be strongly influenced by the functional state and signaling properties of integrins. Here we review MMP function and expression in gliomas and present examples of MMP profiling studies in glioma tissues and cell lines by RT-PCR and Western blotting. Co-expression of MMPs and certain integrins substantiates the gathering evidence of a functional intersection between the two, and inhibition studies using recombinant TIMP-1 and integrin antisera demonstrate significant inhibition of glioma invasion in vitro. Use of promising new therapeutic compounds with anti-MMP and anti-invasion effects are discussed. These data underline the importance of functional interaction of MMPs with accessory proteins such as integrins during invasion, and the need for further studies to elucidate the molecular underpinnings of this process.     

Inhibition of angiogenesis and invasion in malignant gliomas

Malignant gliomas confer a dismal prognosis. As the molecular events that underlie tumor angiogenesis are elucidated, angiogenesis inhibition is emerging as a promising therapy for recurrent and newly diagnosed tumors. Data from animal studies suggest that angiogenesis inhibition may promote an invasive phenotype in tumor cells. This may represent an important mechanism of resistance to antiangiogenic therapies. Recent studies have begun to clarify the mechanisms by which glioma cells detach from the tumor mass, remodel the extracellular matrix and infiltrate normal brain. An array of potential therapeutic targets exists. Combination therapy with antiangiogenic and novel anti-invasion agents is a promising approach that may produce a synergistic antitumor effect and a survival benefit for patients with these devastating tumors.     

The Expression and Activation of Matrix Metalloproteinase-2 in Rat Brain After Implantation of C6 Rat Glioma Cells

It has been reported that matrix metalloproteinases (MMPs) are highly expressed in malignant glioma cells and that this increased expression may facilitate the invasiveness of tumor cells. The authors investigated the expression and enzymatic activity of MMPs in rat brain during the growth of malignant gliomas at different time intervals. C6 rat glioma cells were unilaterally implanted into rat cerebral hemispheres. After 7 or 14 days, these brain tissues were prepared for SDS-PAGE zymography, Western blotting, immunohistochemistry, and in situ zymography. SDS-PAGE zymography and Western blotting revealed that the expression of proMMP-2 in rat brains with C6 glioma cells was significantly higher than that in normal or the sham-operated rat brains, and that the activated form of MMP-2 was detected only in the former but not in the latter. On immunohistochemistry, C6 glioma cells presenting invasive growth into the rat brain parenchyma and vessels demonstrated MMP-2 immunoreactivity. On in situ zymography, foci of invasive C6 glioma cells in rat brain tissue showed gelatinolytic activity. These results suggest that expression and activation of MMP-2 may be one of the crucial steps for glioma cell invasion into the brain parenchyma in vivo.     

Angiogenesis and Blood Vessel Invasion as Prognostic Indicators for Node-Negative Breast Cancer

This study was undertaken to determine the value of angiogenesis and blood vessel invasion (BVI) using both Factor VIII-related antigen and elastica van Gieson staining in predicting 20-year relapse-free survival (RFS) and 20-year overall survival (OS) rates in Japanese patients with node-negative breast cancer. Two hundred and sixty patients were studied. We investigated nine factors, including angiogenesis (average microvessel count (AMC)), BVI, proliferating cell nuclear antigen (PCNA), p53, c-erbB-2, clinical tumor size (T), histological grade, tumor necrosis, and lymphatic vessel invasion (LVI). Twenty-five patients (9.6%) had recurrence and 17 patients (6.5%) died of breast cancer. Univariate analysis showed that BVI, AMC, T, histological grade, PCNA, p53, and tumor necrosis were significantly predictive of RFS or OS. Multivariate analysis showed that AMC, BVI, and T were significant independent factors for RFS or OS. Moreover, the combination of AMC/BVI was an especially significant factor for RFS or OS (P<0.0001, P=0.0003, respectively). When stratified by T, a significant impact of AMC or BVI on RFS was seen in patients with T1, T2, and T3 carcinomas. Multivariate analysis in patients with T2 carcinoma showed that both AMC and BVI were significant independent factors for RFS (P=0.0231, P=0.0388, respectively) and OS (P=0.0331 and P=0.0479, respectively). AMC, BVI, and T were independent prognostic indicators. As the combined impact of AMC/BVI is especially strong, AMC/BVI is useful in selecting high-risk node-negative breast cancer patients who may be eligible to receive aggressive adjuvant chemotherapy.     

Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas

Neuroblastoma is the most common type of cancer in infants. In children this tumor is particularly aggressive; despite various new therapeutic approaches, it is associated with poor prognosis. Given the importance of endosomal-lysosomal proteolysis in cellular metabolism, we hypothesized that inhibition of lysosomal protease would impact negatively on neuroblastoma cell survival. Treatment with E-64 or CA074Me (2 specific inhibitors of cathepsin B) or with pepstatin A (a specific inhibitor of cathepsin D) was cytotoxic for 2 neuroblastoma cell lines having different degrees of malignancy. Cell death was associated with condensation and fragmentation of chromatin and externalization of plasma membrane phosphatidylserine, 2 hallmarks of apoptosis. Concomitant inhibition of the caspase cascade protected neuroblastoma cells from cathepsin inhibitor-induced cytotoxicity. These data indicate that prolonged inhibition of the lysosomal proteolytic pathway is incompatible with cell survival, leading to apoptosis of neuroblastoma cells, and that the cathepsin-mediated and caspase-mediated proteolytic systems are connected and cooperate in the regulation of such an event. Since modern antitumor chemotherapy is aimed at restoring the normal rate of apoptosis in neoplastic tissues, the demonstration that endosomal-lysosomal cathepsins are involved in this process may constitute a basis for novel strategies that include cathepsin inhibitors in the therapeutic regimen.     

Invasion and proliferation kinetics in enhancing gliomas predict IDH1 mutation status

Background

Glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than gliomas with wild-type IDH1.

Methods

Here we compare the IDH1 mutational status in 172 contrast-enhancing glioma patients with the invasion profile generated by a patient-specific mathematical model we developed based on MR imaging.

Results

We show that IDH1-mutated contrast-enhancing gliomas were relatively more invasive than wild-type IDH1 for all 172 contrast-enhancing gliomas as well as the subset of 158 histologically confirmed glioblastomas. The appearance of this relatively increased, model-predicted invasive profile appears to be determined more by a lower model-predicted net proliferation rate rather than an increased model-predicted dispersal rate of the glioma cells. Receiver operator curve analysis of the model-predicted MRI-based invasion profile revealed an area under the curve of 0.91, indicative of a predictive relationship. The robustness of this relationship was tested by cross-validation analysis of the invasion profile as a predictive metric for IDH1 status.

Conclusions

The strong correlation between IDH1 mutation status and the MRI-based invasion profile suggests that use of our tumor growth model may lead to noninvasive clinical detection of IDH1 mutation status and thus lead to better treatment planning, particularly prior to surgical resection, for contrast-enhancing gliomas.     

Spheroid Preparation from Hanging Drops: Characterization of a Model of Brain Tumor Invasion

BACKGROUND: The use of three-dimensional in vitro models of brain tumor invasion has provided a system for reconstructing some of the cellular microenvironments present in the tumor mass. While spheroids of murine and human astrocytoma cells can be prepared using spinning cultures, spheroid preparation using many cell lines is not amenable to this method. We have developed a reproducible system of creating implantable spheroids that is applicable to different cell lines, and is independent of cell line characteristics. METHODS: For murine and human brain tumor cell lines, 20 microl drops containing predetermined cell concentrations were suspended from the lids of culture dishes and the resulting aggregates were transferred to culture dishes base-coated with agar. The two-dimensional aggregates formed three-dimensional spheroids on the non-permissive agar substrate, and were then implanted into three-dimensional collagen I gels and the invasive activity assessed. The invasive activity of C6 and U251 spheroids prepared by hanging drops was compared to spheroids of similar size prepared by spinner culture. RESULTS: The hanging drop method produced implantable spheroids capable of sustained invasion using all cell lines tested. Most cell lines required initial hanging drop cell concentrations of 45,000 cells/drop, suspension times of 48, and 72 h on agar. C6 spheroids had the same invasive capacity regardless of the model utilized, however U251 spheroids produced by hanging drops had significantly increased invasion compared to those prepared by spinner culture. Only spheroids prepared by spinner culture showed histological evidence of central necrosis. CONCLUSIONS: This model represents a reproducible approach to the preparation of implantable spheroids with invasive potential that compares with those produced using spinner culture. The use of hanging drops broadens the applicability of three-dimensional in vitro assays examining brain tumor invasiveness.     

Suppression of Cell Invasion on Human Malignant Glioma Cell Lines by a Novel Matrix-metalloproteinase Inhibitor SI-27: In Vitro Study

Matrix metalloproteinase (MMP) has come to be highlighted by its close relation to the cell invasion of gliomas. Suppression of MMP activity in malignant glioma cells would be meriting to local delivery of genes or chemotherapeutic agents. In this study, we employed a novel MMP inhibitor, SI-27 to investigate inhibition of cell invasiveness in human malignant glioma cell lines, U87MG, U251MG, and U373MG. We evaluated with zymogram, reverse zymogram, and cell invasion assay after exposure of SI-27 for 24h followed by preliminary MTT assay to find non-cytotoxic dose range, 51050100g/ml compared with non-treatment group as the control. Common to three glioma cell lines, zymogram disclosed that expressions of MMP-2 and -9 were suppressed in a dose-dependent fashion, meanwhile those of tissue inhibitor of MMP (TIMMP) in reverse zymogram were not. The numbers of invading cells through Boyden chamber were significantly reduced in a dose-dependent manner, while those with 5g/ml were not diminished common to those three lines. In conclusion, dose concentration ranging 10–100g/ml of SI-27 inhibited MMP-2 and -9 mediated cell invasiveness in malignant glioma cell lines. This is the first report for chemotherapeutic effect of SI-27 on glioma cells.     

Biological Mechanisms of Glioma Invasion and Potential Therapeutic Targets

The current understanding of glioma biology reveals targets for anti-invasive therapy which include manipulations of extracellular matrix and receptors, growth factors and cytokines, proteases, cytoskeletal components, oncogenes and tumor suppressor genes. A better understanding of the complex regulation and the signalling molecules involved in glioma invasion is still needed in order to design new and effective treatment modalities towards invasive tumor cells. Representative and valid in vitro experimental systems and animal models of gliomas are necessary for the characterization of the invasive phenotype and further development of anti-invasive therapy. In the future, it will probably be important to move from comparative genomic modelling through protein characterization based on advanced proteomic techniques to analyse tissue samples, where the aim for gliomas should be to compare invaded and non-invaded tissue. This will hopefully render promising new therapeutic targets for gliomas.     

Migration Pathways of Human Glioblastoma Cells Xenografted into the Immunosuppressed Rat Brain

Diffuse invasion of the brain by tumor cells is a hallmark of human glioblastomas and a major cause for the poor prognosis of these tumors. This phenomenon is only partially reproduced by rodent models of gliomas that display a very high rate of proliferation and limited cell migration. We have analyzed the development of human glioblastoma cells (GL15) xenografted into the brain of immunosuppressed rats, in order to define the characteristics of tumor cell invasion. As identified by the specific immunolabeling of the tumor cells for the human HLA-ABC antigen, GL15 tumors reproduced the three types of intraparenchymal invasion observed in patients. First, a majority of multipolar tumor cells intermingled rapidly and profusely with host neural cells in the margin of the injection site. This progressively enlarging area was principally responsible for the tumor growth over time. Second, in the gray matter, columns of thin bipolar tumor cells aligned along capillary walls. Third, in the white matter, elongated bipolar isolated tumor cells were observed scattered between axonal fibers. The maximum migration distances along white matter fibers remained significantly higher than the maximum migration distances along blood vessels, up to two months after injection. Development of the tumor was associated with a significant increase of vascularization in the area of tumor spread. Xenografting of human GL15 glioblastoma cells into the immunosuppressed rat brain allowed to differentiate between the three classical types of invasion identified in the clinic, to quantify precisely the distances of migration, and to evaluate cell morphology for each of these routes. The present results support the existence of host/tumor cells interactions with specific characteristics for each type of invasion.     

A phase I trial of erlotinib in patients with nonprogressive glioblastoma multiforme postradiation therapy,and recurrent malignant gliomas and meningiomas

The objective of this phase I study was to determine the maximal tolerated dose (MTD) of erlotinib in patients with recurrent malignant gliomas (MGs) or recurrent meningiomas on enzyme-inducing antiepileptic drugs (EIAEDs). Dose escalation was by a standard 3 × 3 design. The initial starting dose of erlotinib was 150 mg daily. If no dose-limiting toxicity (DLT) was observed, then dose escalation occurs as follows: 200 mg/day, 275 mg/day, and then increased in 125 mg increments until the MTD was reached. The MTD was defined as the dose where ≤1 of 6 patients experienced a DLT and the dose above had 2 or more DLTs. The MTD was 650 mg/day; the observed DLTs were grade 3 rash in 2 patients at 775 mg/day. Pharmacokinetic analysis showed a significant influence of EIAEDs on the metabolism of erlotinib when compared with our phase II data published separately. Primary toxicities were rash and diarrhea. The MTD of erlotinib in patients receiving EIAEDs is substantially higher than the standard dose of 150 mg. This has important implications for further development of this drug in the treatment of MG as well as the optimal management of patients with other malignancies such as NSCLC who are on enzyme-inducing drugs.     

Discoidin Domain Receptor-1a (DDR1a) Promotes Glioma Cell Invasion and Adhesion in Association with Matrix Metalloproteinase-2

Summary Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix (ECM), disruption of ECM components, and subsequent cell penetration into adjacent brain structures. Discoidin domain receptor 1 (DDR1) tyrosine kinases constitute a novel family of receptors characterized by a unique structure in the ectodomain (discoidin-I domain). These cell surface receptors bind to several collagens and facilitate cell adhesion. Little is known about DDR1 expression and function in glioblastoma multiforme. In this study we demonstrate that DDR1 is overexpressed in glioma tissues using cDNA arrays, immunohistochemistry and Western blot analysis. Functional comparison of two splice variants of DDR1 (DDR1a and DDR1b) reveal novel differences in cell based glioma models. Overexpression of either DDR1a or DDR1b caused increased cell attachment. However, glioma cells overexpressing DDR1a display enhanced invasion and migration. We also detect increased levels of matrix metalloproteinase-2 in DDR1a overexpressing cells as measured by zymography. Inhibition of MMP activity using MMP inhibitor suppressed DDR1a stimulated cell-invasion. Similarly, an antibody against DDR1 reduced DDR1a mediated invasion as well as the enhanced adhesion of DDR1a and DDR1b overexpressing cells. These results suggest that DDR1a plays a critical role in inducing tumor cell adhesion and invasion, and this invasive phenotype is caused by activation of matrix metalloproteinase-2.