Migrating Glioma Cells Research Articles

P13.23 Study of migration characteristics of human glioma cells in vitro

Abstract BACKGROUND Gliomas are still one of the most aggressive human cancers, and even despite modern therapeutic approaches, the prognosis for patients with this disease is not favorable. It is known that glioma cells are capable of local invasiveness, when glioma cells migrate into healthy brain tissue. A lack of any definite markers, characterizing migrating glioma cells and allowing them to be distinguished from healthy brain cells, requires a thorough investigation. In case it would be possible to characterize invasive glioma cells, then a development of targeted therapy could be feasible. MATERIAL AND METHODS Cell cultures of human gliomas Gr II, III and IV were developed with 5 cultures for each Grade. MTT, RT-PCR, Western and Nosern blot, transcriptome analysis were applied. RESULTS Three cultures of human gliomas had a high degree of migration, within the range of 6% - 14%. These cultures were developed from gliomas of Grade III and Grade IV, and with IDH1- (minus) phenotype. Moreover, cell cultures with IDH1 + (plus) phenotype had a low migration rate within 1%. An intensity of migration correlated with the degree of malignancy, and an average rate decreased with a decrease of the Grade. Moreover, an analysis of the proliferative activity of cell cultures of human gliomas of various degrees of malignancy did not reveal a relationship with a migratory properties of cultures. A number of actively proliferating cultures did not show high migration, while cultures with medium proliferative activity could show a high level of migration. The low level of proliferation of cultures of gliomas of Grade II and I at the beginning of cultivation, in some cases, subsequently increased, but an inherent low migration activity did not change. In actively migrating cultures, a significant decrease in the expression of Sox2 and Nestin is detected. A positive correlation was found between migration abilities of human glioma cell culture cells and the marker Ki67, GFAP, Sox2, and Oct4. The difference was statistically significant by the one-sided Mann-Whitney test. CONCLUSION Conclusions: Cell cultures derived from glioma tumor tissue can be used to predict invasive properties of the tumor. High tumor invasiveness is characteristic for Grade III and Grade IV, and with IDH1- (minus) phenotype, and it also correlates with elevated expression of GFAP, Sox2 and Oct4The reported study was funded by RFBR according to the research project № 18-29-01012 and by the Ministry of Science and Higher Education of the Russian Federation, grant number 075-15-2020-809 (13.1902.21.0030).

SWAP70 undergoes dynamic conformational regulation at the leading edge of migrating cells.

Rearrangements of the actin cytoskeleton are regulated in part by dynamic localised activation and inactivation of Rho family small GTPases. SWAP70 binds to and activates the small GTPase RAC1 as well as binding to filamentous actin and PIP3 . We have developed an encoded biosensor, which uses Forster resonance energy transfer to reveal conformational changes in SWAP70 in live cells. SWAP70 adopts a distinct conformation at the plasma membrane, which in migrating glioma cells is enriched at the leading edge but does not always associate with its PIP3 -dependent translocation to the membrane. This supports a role for SWAP70 in positive feedback activation of RAC1 at sites of filamentous actin, PIP3 and active RAC1.

Adaptive adhesion systems mediate glioma cell invasion in complex environments.

Diffuse brain invasion by glioma cells prevents effective surgical or molecular-targeted therapy and underlies a detrimental outcome. Migrating glioma cells are guided by complex anatomical brain structures but the exact mechanisms remain poorly defined. To identify adhesion receptor systems and matrix structures supporting glioma cell invasion into brain-like environments we used 2D and 3D organotypic invasion assays in combination with antibody-, peptide- and RNA-based interference. Combined interference with β1 and αV integrins abolished the migration of U-251 and E-98 glioma cells on reconstituted basement membrane; however, invasion into primary brain slices or 3D astrocyte-based scaffolds and migration on astrocyte-deposited matrix was only partly inhibited. Any residual invasion was supported by vascular structures, as well as laminin 511, a central constituent of basement membrane of brain blood vessels. Multi-targeted interference against β1, αV and α6 integrins expressed by U-251 and E-98 cells proved insufficient to achieve complete migration arrest. These data suggest that mechanocoupling by integrins is relatively resistant to antibody- or peptide-based targeting, and cooperates with additional, as yet unidentified adhesion systems in mediating glioma cell invasion in complex brain stroma.

Shift of microRNA profile upon glioma cell migration using patient-derived spheroids and serum-free conditions

Glioblastoma multiforme (GBM) is the most frequent malignant primary brain tumor. A major reason for the overall median survival being only 14.6 months is migrating tumor cells left behind after surgery. Another major reason is tumor cells having a so-called cancer stem cell phenotype being therefore resistant towards traditional chemo- and radiotherapy. A group of novel molecular targets are microRNAs (miRNAs). MiRNAs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. The aim of this study was to identify differentially expressed miRNAs in migrating GBM cells using serum-free stem cell conditions. We used patient-derived GBM spheroid cultures for a novel serum-free migration assay. MiRNA expression of migrating tumor cells isolated at maximum migration speed was compared with corresponding spheroids using an OpenArray Real-Time PCR System. The miRNA profiling revealed 30 miRNAs to be differentially expressed. In total 13 miRNAs were upregulated and 17 downregulated in migrating cells compared to corresponding spheroids. The three most deregulated miRNAs, miR-1227 (up-regulated), miR-32 (down-regulated) and miR-222 (down-regulated), were experimentally overexpressed. A non-significantly increased migration rate was observed after miR-1227 overexpression. A significantly reduced migration rate was observed after miR-32 and miR-222 overexpression. In conclusion a shift in microRNA profile upon glioma cell migration was identified using an assay avoiding serum-induced migration. Both the miRNA profiling and the functional validation suggested that miR-1227 may be associated with increased migration and miR-32 and miR-222 with decreased migration. These miRNAs may represent potential novel targets in migrating glioma cells.

STMC-20. MIGRATING GLIOMA CELLS EXPRESS STEM CELL MARKERS AND GIVE RISE TO NEW TUMORS UPON XENOGRAFTING

Glioblastoma (GBM) is the most frequent and malignant brain tumor with an overall survival of only 14.6 months. Although these tumors are treated with surgery, radiation and chemotherapy recurrence is inevitable. A critical population of tumor cells in terms of therapy, the so-called cancer stem cells (CSCs), has been identified in gliomas and many other cancers. These tumor cells have a stem cell-like phenotype and are suggested to be responsible for tumor growth, chemo- and radio-resistance as well as recurrence. However, functional evidence for migrating glioma cells having a stem cell-like phenotype is currently lacking. In the present study, the aim was to characterize the phenotype of migrating tumor cells using a novel migration assay based on serum-free stem cell medium and patient-derived spheroid cultures. The results showed pronounced migration of five different GBM spheroid cultures, but not of the commercial cell line U87MG. An in vitro limiting dilution assay showed preserved but reduced spheroid formation capacity of migrating cells. Orthotopic xenografting in mice showed preserved but reduced tumorigenic capacity. Profiling of mRNAs revealed no significant deregulation of 16 predefined CSC-related genes and the HOX-gene list in migrating cells compared to spheroids. Determination of GBM molecular subtypes revealed that subtypes of spheroids and migrating cells were identical. In conclusion, migrating tumor cells preserve expression of stem cell markers and functional CSC characteristics. Since CSCs are reported to be highly resistant to therapy, these results emphasize that the CSC phenotype should be taken into consideration in future treatment of GBMs.

Migrating glioma cells express stem cell markers and give rise to new tumors upon xenografting.

Glioblastoma (GBM) is the most frequent and malignant brain tumor with an overall survival of only 14.6 months. Although these tumors are treated with surgery, radiation and chemotherapy, recurrence is inevitable. A critical population of tumor cells in terms of therapy, the so-called cancer stem cells (CSCs), has been identified in gliomas and many other cancers. These tumor cells have a stem cell-like phenotype and are suggested to be responsible for tumor growth, chemo- and radio-resistance as well as recurrence. However, functional evidence for migrating glioma cells having a stem cell-like phenotype is currently lacking. In the present study, the aim was to characterize the phenotype of migrating tumor cells using a novel migration assay based on serum-free stem cell medium and patient-derived spheroid cultures. The results showed pronounced migration of five different GBM spheroid cultures, but not of the commercial cell line U87MG. An in vitro limiting dilution assay showed preserved but reduced spheroid formation capacity of migrating cells. Orthotopic xenografting in mice showed preserved but reduced tumorigenic capacity. Profiling of mRNAs revealed no significant deregulation of 16 predefined CSC-related genes and the HOX-gene list in migrating cells compared to spheroids. Determination of GBM molecular subtypes revealed that subtypes of spheroids and migrating cells were identical. In conclusion, migrating tumor cells preserve expression of stem cell markers and functional CSC characteristics. Since CSCs are reported to be highly resistant to therapy, these results emphasize that the CSC phenotype should be taken into consideration in future treatment of GBMs.

CBIO-25NEW HYPOXIA-INDUCED INVASION FACTOR IN HUMAN GLIOBLASTOMA INVASION

Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.

Connexin 43-targeted T1 contrast agent for MRI diagnosis of glioma.

Glioblastoma multiforme is the most aggressive form of brain tumor. Early and accurate diagnosis of glioma and its borders is an important step for its successful treatment. One of the promising targets for selective visualization of glioma and its margins is connexin 43 (Cx43), which is highly expressed in reactive astrocytes and migrating glioma cells. The purpose of this study was to synthesize a Gd-based contrast agent conjugated with specific antibodies to Cx43 for efficient visualization of glioma C6 in vivo. We have prepared stable nontoxic conjugates of monoclonal antibody to Cx43 and polylysine-DTPA ligands complexed with Gd(III), which are characterized by higher T1 relaxivity (6.5 mM(-1) s(-1) at 7 T) than the commercial agent Magnevist® (3.4 mM(-1) s(-1)). Cellular uptake of Cx43-specific T1 contrast agent in glioma C6 cells was more than four times higher than the nonspecific IgG-contrast agent, as detected by flow cytometry and confocal analysis. MRI experiments showed that the obtained agents could markedly enhance visualization of glioma C6 in vivo after their intravenous administration. Significant accumulation of Cx43-targeted contrast agents in glioma and the peritumoral zone led not only to enhanced contrast but also to improved detection of the tumor periphery. Fluorescence imaging confirmed notable accumulation of Cx43-specific conjugates in the peritumoral zone compared with nonspecific IgG conjugates at 24 h after intravenous injection. All these features of Cx43-targeted contrast agents might be useful for more precise diagnosis of glioma and its borders by MRI.

Treatment of poorly differentiated glioma using a combination of monoclonal antibodies to extracellular connexin-43 fragment, temozolomide, and radiotherapy.

Antitumor efficiencies of monoclonal antibodies to connexin-43 second extracellular loop (MAbE2Cx43), temozolomide, and fractionated γ-irradiation in the monotherapy mode and in several optimized combinations were studied in Wistar rats with induced C6 glioma. The survival of animals with glioma and the dynamics of intracerebral tumor development were evaluated by MRT. Temozolomide monotherapy (200 mg/m(2)) and isolated radiotherapy in a total dose of 36 Gy shifted the survival median from 28 days (no therapy) to 34 and 38 days, respectively; 100% animals died under conditions of temozolomide monotherapy and radiotherapy. Monotherapy with MAbE2Cx43 in a dose of 5 mg/kg led to significant regression of the tumor (according to MRT data), cure of 19.23% animals with glioma, and prolongation of the survival median to 39.5 days after tumor implantation. Combined therapy with MAbE2Cx43 and temozolomide completely abolished the antitumor effect (survival median 29 days). Treatment with MAbE2Cx43 in combination with radiotherapy was associated with mutual boosting of the therapeutic efficiencies, leading to a significant inhibition of tumor development and prolongation of the survival median to 60 days. The mechanism of tumorsuppressive activity of the antibodies could be due to connexon blockade in Cx43-positive glioma cells in the peritumor invasion zone. Higher efficiency of combined therapy was presumably due to the increase in blood-brain barrier permeability for antibodies after irradiation of the brain and to additional inhibitory effect of antibodies towards radioresistant migrating glioma cells. The results suggested that MAbE2Cx43 could be effective as the first-line drug in combined therapy for poorly differentiated gliomas.

Cyclin G2 Promotes Hypoxia- Driven Local Invasion of Glioblastoma by Orchestrating Cytoskeletal Dynamics

Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.

Abstract 5336: Targeting glioma growth and invasiveness by microRNA-1.

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Median survival is about 14 months, even with the most effective regimen of surgery followed by radiation and chemotherapy. GBM is characterized by the hallmarks of cellular heterogeneity, rapid proliferation, angiogenesis and extensive invasion. The dual feature of high proliferation and infiltration constitutes the major challenge for glioma therapy, as even extensive resection of the primary tumor mass leaves a significant number of tumor cells in the brain and leads to almost unavoidable recurrence. Despite abundance of data concerning glioma growth/angiogenesis, the mechanisms underlying the regulation of glioma cell motility and invasiveness are comparatively poorly understood. Established anti-glioma therapeutical modalities (radiation, chemotherapy) target preferentially rapidly proliferating rather than infiltrating glioma cells. Moreover invading cells are resistant to conventional therapies, and anti-angiogenic treatment was shown to induce invasiveness. Thus novel therapies aiming at blockade of glioma invasion, while curbing their proliferative potential are needed. MicroRNAs (miRs) are small, single stranded, non-coding RNAs that act as key regulators of gene expression, by reducing translation of target mRNAs with partial complementarity in their 3′UTRs. MiRs deregulation is a general feature of cancer, including GBM, and was associated with tumor suppressor and oncogenic activities. A number of studies have identified critical roles for miRs in the regulation of tumor cell invasion, migration and metastasis. In this study we report that miR-1 is down-regulated in glioma patients (relatively to adjacent tissue) and that it is down-regulated in migrating glioma cells in vitro (relatively to non-migrating glioma cells). Its forced expression reduces glioma cell growth and angiogenesis in vivo. MiR-1 inhibits migration in vitro and invasiveness in vivo. Additionally, we demonstrated impaired cell-cell adhesion and aggregation in glioma cells expressing miR-1. We identified Annexin A2, recognized oncogene in glioma, as a direct target of miR-1. Annexin A2 is up-regulated in various tumor types, including glioma. In vivo studies revealed a critical role for Annexin A2 in driving glioma growth, angiogenesis, progression and invasion. Our results show that the effects of miR-1 on glioma cells are consistent with Annexin A2 down-regulation, including a reduction in glioma cell proliferation and invasiveness, and re-expression of Annexin A2 rescues the effect of miR-1 expression. When glioma cells stably expressing miR-1 were injected intracranially, we observed significantly improved survival when compared to control cells. Our results suggest that miR-1 inhibits simultaneously glioma growth and invasiveness in vivo and as such may be a candidate for the therapeutic targeting of glioma cells. Citation Format: Jakub A. Godlewski, Agnieszka Bronisz, Yan Wang, E. Antonio Chiocca. Targeting glioma growth and invasiveness by microRNA-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2013-5336

Stopping Cancer in its Tracks: Using Small Molecular Inhibitors to Target Glioblastoma Migrating Cells

Glioblastoma multiforme (GBM) represents one of the most common aggressive types of primary brain tumors. Despite advances in surgical resection, novel neuroimaging procedures, and the most recent adjuvant radiotherapy and chemotherapy, the median survival after diagnosis is about 12-14 months. Targeting migrating GBM cells is a key research strategy in the fight against this devastating cancer. Though the vast majority of the primary tumor focus can be surgically resected, these migrating cells are responsible for its universal recurrence. Numerous strategies and technologies are being explored to target migrating glioma cells, with small molecular inhibitors as one of the most commonly studied. Small molecule inhibitors, such as protein kinase inhibitors, phosphorylation site inhibitors, protease inhibitors, and antisense oligonucleotides show promise in slowing the progression of this disease. A better understanding of these small molecule inhibitors and how they target various extra- and intracellular signaling pathways may eventually lead to a cure for GBM.

Abstract 306: TWEAK functions as chemotactic factor for glioma cells via Lyn activation

Abstract The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK using the Modified Boyden chamber assay. Because TWEAK has been shown to be expressed in normal astrocytes, microglia, and endothelial cells, we neutralized TWEAK using a recombinant soluble murine Fn14-Fc decoy receptor and examined the effect on glioma cell invasion in the ex vivo rat brain slice model. Incubation of the brain slice with the Fn14-Fc decoy receptor reduced glioma cell invasion as compared to control IgG treated brain slice. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of Src as detected by Western blot analysis using a pan-Src phosphorylation antibody. To determine which SFK member is phosphorylated by TWEAK, we applied the multiplex Luminex assay and identified Lyn as a candidate SFK. Immunoprecipitation of Lyn from TWEAK-treated cells verified that Lyn is phosphorylated as determined by phosphotyrosine immunoblot analysis. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 306. doi:1538-7445.AM2012-306

Abstract 3994: Loss of miR-23b enhances Pyk2-induced glioma cell migration and invasion

Abstract Glioblastoma is the most common and lethal type of primary brain tumor. Despite recent therapeutic advances in other cancers, the treatment of glioblastomas remains ineffective and essentially palliative due the highly invasive nature of the disease. To discern molecular mechanisms that drive GBM migration, we investigated the potential role of differential expression of microRNAs (miRNA) by comparing the matched large-scale, genome-wide miRNA expression profiles of migrating and migration-restricted human glioma cells. Using a radial migration assay, migrating and non-migrating populations from seven well-established glioma cell lines were isolated and utilized for miRNA expression analysis. Statistical analyses revealed 22 miRNAs common to all seven glioma cell lines were down-regulated in the migrating cell population (p < 0.0005) relative to cells in the non-migratory population. Specifically, we report here that the expression level of miR-23b was markedly down-regulated in migrating glioma cells as compared to migration-restricted cells. Over-expression of miR-23b significantly inhibited glioma cell migration and invasion. A bioinformatics search revealed a conserved target site within the 3’untranslated region (UTR) of Pyk2, a non-receptor tyrosine kinase that has been previously implicated in the regulation of glioma cell migration and invasion. Increased expression of miR-23b reduced the expression level of Pyk2 in glioma cells indicating that miR-23b targets Pyk2. Notably, increased expression of miR-23b did not alter the expression level of the related focal adhesion kinase FAK indicating that miR-23b specifically inhibits the expression Pyk2. Taken together, our study demonstrates that reduced expression of miR-23b facilitates the enhancement of glioma cell migration and invasion via modulation of Pyk2 expression. Thus, these data suggest that certain miRNAs may regulate glioma migration and invasion to influence the progression of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3994. doi:10.1158/1538-7445.AM2011-3994

Frizzled 4 Regulates Stemness and Invasiveness of Migrating Glioma Cells Established by Serial Intracranial Transplantation

One of the most detrimental hallmarks of glioblastoma multiforme (GBM) is cellular invasiveness, which is considered a potential cause of tumor recurrence. Infiltrated GBM cells are difficult to completely eradicate surgically and with local therapeutic modalities. Although much effort has focused on understanding the various mechanisms controlling GBM invasiveness, its nature remains poorly understood. In this study, we established highly serial intracranial transplantation. U87R4 cells were highly invasive and displayed stem cell-like properties, as compared to noninvasive but proliferative U87L4 cells. Microarray analysis during serial transplantation revealed that apoptosis-inducing genes (caspase3 and PDCD4) were downregulated whereas several cancer stem cell-relevant genes [Frizzled 4 (FZD4) and CD44] were upregulated in more invasive cells. U87R4 cells were resistant to anticancer drug-induced cell death, partly due to downregulation of caspase3 and PDCD4, and they retained activated Wnt/β-catenin signaling due to upregulation of Frizzled 4, which was sufficient to control neurosphere formation. We also found that FZD4 promoted expression of the epithelial to mesenchymal transition regulator SNAI1, along with acquisition of a mesenchymal phenotype. Taken together, our results argue that Frizzled 4 is a member of the Wnt signaling family that governs both stemness and invasiveness of glioma stem cells, and that it may be a major cause of GBM recurrence and poor prognosis.

Abstract 5128: Identification of RhoGEFs within the TWEAK-R (Fn14)-Rac1 signaling pathway that mediate glioblastoma cell invasion

Abstract Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. Despite efforts at surgical resection of the glioma mass, invasive cells are always left behind and the tumor will inevitably recur and kill the patient. As such, novel therapeutics targeting pro-invasive factors could improve neurological outcomes and survival for these patients. This requires a detailed understanding of the mechanisms driving glioma migration and invasion. We have been investigating whether the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) regulate glioma cell invasive activity. We have previously demonstrated that Fn14 expression was elevated in the invasive rim of GBM specimens and migrating glioma cells in vitro. The Fn14 signaling axis is known to drive glioma invasion via Rac1. We have previously reported that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, including Rac1 and Cdc42, is overexpressed in GBM, and that overexpression of Ect2 correlates directly with tumor grade and inversely with patient survival. In this study we show that Ect2 regulates Rac1 activation downstream of Fn14. Depletion of Ect2 by siRNA duplexes abrogates Fn14-induced Rac1 activation and subsequent glioma cell migration and invasion. We also found that Cdc42 activation by TWEAK is directly mediated by Ect2. Interestingly, depletion of Cdc42 expression impairs TWEAK-induced Rac1 activation and also results in a significant reduction of glioma cell migration and invasion. This suggests that Cdc42 is, in part, important for Rac1 activation downstream of the TWEAK-Fn14 signaling pathway and argues that another GEF(s) may be involved in the Fn14-Rac1 signaling axis. Recently, we have identified Trio as an additional GEF that activates Rac1 downstream of Cdc42. It is also known that Trio expression correlates directly with brain tumor grade and inversely with patient survival. siRNA-mediated depletion of Trio inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Therefore, delineating the mechanisms of Fn14-RhoGEF-RhoGTPase signaling pathway, may lead to identification of novel targets that can serve as possible points of therapeutic intervention. (Supported by NIH R01-CA130940) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5128.

An agent-based model identifies MRI regions of probable tumor invasion in a patient with glioblastoma

We present an application of a previously developed agent-based glioma model (Chen et al 2009 Biosystems 95 234–42) for predicting spatio-temporal tumor progression using a patient-specific MRI lattice derived from apparent diffusion coefficient (ADC) data. Agents representing collections of migrating glioma cells are initialized based upon voxels at the outer border of the tumor identified on T1-weighted (Gd+) MRI at an initial time point. These simulated migratory cells exhibit a specific biologically inspired spatial search paradigm, representing a weighting of the differential contribution from haptotactic permission and biomechanical resistance on the migration decision process. ADC data from 9 months after the initial tumor resection were used to select the best search paradigm for the simulation, which was initiated using data from 6 months after the initial operation. Using this search paradigm, 100 simulations were performed to derive a probabilistic map of tumor invasion locations. The simulation was able to successfully predict a recurrence in the dorsal/posterior aspect long before it was depicted on T1-weighted MRI, 18 months after the initial operation.

A coculture assay to visualize and monitor interactions between migrating glioma cells and nerve fibers

Glioma-cell migration is usually assessed in dissociated cell cultures, spheroid cultures, acute brain slices and intracranial implantation models. However, the interactions between migrating glioma cells and neuronal tracts remain poorly understood. We describe here a protocol for the coculture of glioma cells with myelinated axons in vitro. Unlike other methods, this protocol allows the creation of in vitro conditions that largely mimic the complex in vivo environment. First, long retinal axons from embryonic chicken are formed in an organotypic culture. Glioma cells are then positioned in the vicinity of the explants to allow them to contact the axons, interact with them and eventually migrate along them. High-resolution video microscopy and confocal microscopy can be used to monitor the migratory behavior. This protocol, which takes about 5 days to complete, could be applied to different types of tumor cells that interact with neurites, and is suitable for pharmacological and genetic approaches aimed at elucidating mechanisms underlying tumor migration.

Targeted therapy of glioblastomas: a 5-year view

The treatment of glioblastomas requires a multidisciplinary approach that takes the presently incurable nature of the disease into consideration. Treatments are multimodal and include surgery, radiotherapy and chemotherapy. Current recommendations are that patients with glioblastoma should undergo maximum surgical resection followed by concurrent radiation and chemotherapy with the novel alkylating drug temozolomide, followed subsequently by additional adjuvant temozolomide for a period of up to 6 months. We describe here the major signaling pathways that can be constitutively activated in migrating glioma cells, and which render these cells resistant to pro-apoptotic insults such as conventional chemotherapies. In light of this resistance, we therefore describe the molecular therapies and local drug delivery systems that could be used to complement conventional treatments. We have reviewed more than 400 ongoing clinical trials with respect to these new targeted therapy approaches alone or in combination for glioblastoma therapy.

Rocks are expressed in brain tumors and are required for glioma‐cell migration on myelinated axons

The interactions between migrating glioma cells and myelinated fiber tracts are poorly understood. We identified that C6 glioma cells can migrate along myelinated chicken retinal axons in a novel coculture, thereby expressing small GTPases of the Rho family and serine/threonine Rho-associated kinases (ROCKs). We found that the ROCK1 isoform is also highly expressed in native human high-grade gliomas. Glioma cells migrated faster in vitro along myelinated axons than on laminin-1, with the former but not the latter being specifically and reversibly blocked by the ROCK inhibitor Y27632. These data suggest that the mechanisms underlying the migration of glioma cells on myelinated axons differ from those underlying the migration on extracellular matrix molecules such as laminin-1.