Spread Of Glioblastoma Research Articles

KCa channel targeting impairs DNA repair and invasiveness of patient-derived glioblastoma stem cells in culture and orthotopic mouse xenografts which only in part is predictable by KCa expression levels.

Prognosis of glioblastoma patients is still poor despite multimodal therapy. The highly brain-infiltrating growth in concert with a pronounced therapy resistance particularly of mesenchymal glioblastoma stem-like cells (GSCs) has been proposed to contribute to therapy failure. Recently, we have shown that a mesenchymal-to-proneural mRNA signature of patient derived GSC-enriched (pGSC) cultures associates with in vitro radioresistance and gel invasion. Importantly, this pGSC mRNA signature is prognostic for patients' tumor recurrence pattern and overall survival. Two mesenchymal markers of the mRNA signature encode for IKCa and BKCa Ca2+-activated K+ channels. Therefore, we analyzed here the effect of IKCa- and BKCa-targeting concomitant to (fractionated) irradiation on radioresistance and glioblastoma spreading in pGSC cultures and in pGSC-derived orthotopic xenograft glioma mouse models. To this end, in vitro gel invasion, clonogenic survival, in vitro and in vivo residual DNA double strand breaks (DSBs), tumor growth, and brain invasion were assessed in the dependence on tumor irradiation and K+ channel targeting. As a result, the IKCa- and BKCa-blocker TRAM-34 and paxilline, respectively, increased number of residual DSBs and (numerically) decreased clonogenic survival in some but not in all IKCa- and BKCa-expressing pGSC cultures, respectively. In addition, BKCa- but not IKCa-blockade slowed-down gel invasion in vitro. Moreover, systemic administration of TRAM-34 or paxilline concomitant to fractionated tumor irradiation increased in the xenograft model(s) residual number of DSBs and attenuated glioblastoma brain invasion and (numerically) tumor growth. We conclude, that KCa-blockade concomitant to fractionated radiotherapy might be a promising new strategy in glioblastoma therapy.

Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro.

Impairing the motility of glioblastoma multiforme (GBM) cells is a compelling goal for new approaches to manage this highly invasive and rapidly lethal human brain cancer. Work here characterized an array of pharmacological inhibitors of membrane ion and water channels, alone and in combination, as tools for restraining glioblastoma spread in human GBM cell lines U87-MG and U251-MG. Aquaporins, AMPA glutamate receptors, and ion channel classes (shown to be upregulated in human GBM at the transcript level and linked to mechanisms of motility in other cell types) were selected as pharmacological targets for analyses. Effective compounds reduced the transwell invasiveness of U87-MG and U251-MG glioblastoma cells by 20-80% as compared with controls, without cytotoxicity. The compounds and doses used were: AqB013 (14 μM); nifedipine (25 µM); amiloride (10 µM); apamin (10 µM); 4-aminopyridine (250 µM); and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 30 µM). Invasiveness was quantified in vitro across transwell filter chambers layered with extracellular matrix. Co-application of each of the ion channel agents with the water channel inhibitor AqB013 augmented the inhibition of invasion (20 to 50% greater than either agent alone). The motility impairment achieved by co-application of pharmacological agents differed between the GBM proneural-like subtype U87-MG and classical-like subtype U251-MG, showing patterns consistent with relative levels of target channel expression (Human Protein Atlas database). In addition, two compounds, xanthurenic acid and caelestine C (from the Davis Open Access Natural Product-based Library, Griffith University QLD), were discovered to block invasion at micromolar doses in both GBM lines (IC50 values from 0.03 to 1 µM), without cytotoxicity, as measured by full mitochondrial activity under conditions matching those in transwell assays and by normal growth in spheroid assays. Mechanisms of action of these agents based on published work are likely to involve modulation of glutamatergic receptor signaling. Treating glioblastoma by the concurrent inhibition of multiple channel targets could be a powerful approach for slowing invasive cell spread without cytotoxic side effects, potentially enhancing the effectiveness of clinical interventions focused on eradicating primary tumors.

Targetting Interleukins Involved in Glioblastoma – A New Pharmacological Approach

Brain tumor is unchecked growth of brain cells disrupting many of its important functions and, affecting molecular regulatory and signaling networks. Glioblastoma is one of the most deleterious tumors of human brain. Cytokines are secondary messenger molecules in many of the immunological and other biochemical reactions. With glioblastoma progression, various cytokines are secreted that either up-regulate or down-regulate many others with immunological function. Interleukins are a family of cytokines acting mainly as immune-regulatory proteins. They have been implicated in brain tumor initiation, progression and invasion. Due to their multiple and varied regulatory roles at every stage of tumor, they are now becoming an attractive target for study in glioblastoma. This mini review briefly summarizes the types and roles of major interleukins and their regulation and, effects in glioblastoma that have been studied so far. Immunotherapeutic aspects are particularly emphasized as the promising therapeutic targets to control brain tumors. Main types of interleukins which are implicated in brain tumors are discussed here which includes interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and interleukin-12 (IL-12). Glioblastoma is one of the most invasive tumor and still lack effective control measures clinically. Conventional treatments like chemotherapy and radiation therapy are not only ineffective in controlling the spread of glioblastoma, but also have damaging and unwanted side effects on healthy cells. Immunotherapy by blocking and/or increasing the action of certain interleukins has proven to be effective control measure in laboratory investigations. Future work extending these findings to clinical and human trials is expected to provide effective treatment measures.

Speed Switch in Glioblastoma Growth Rate due to Enhanced Hypoxia-Induced Migration.

We analyze the wave speed of the Proliferation Invasion Hypoxia Necrosis Angiogenesis (PIHNA) model that was previously created and applied to simulate the growth and spread of glioblastoma (GBM), a particularly aggressive primary brain tumor. We extend the PIHNA model by allowing for different hypoxic and normoxic cell migration rates and study the impact of these differences on the wave-speed dynamics. Through this analysis, we find key variables that drive the outward growth of the simulated GBM. We find a minimum tumor wave-speed for the model; this depends on the migration and proliferation rates of the normoxic cells and is achieved under certain conditions on the migration rates of the normoxic and hypoxic cells. If the hypoxic cell migration rate is greater than the normoxic cell migration rate above a threshold, the wave speed increases above the predicted minimum. This increase in wave speed is explored through an eigenvalue and eigenvector analysis of the linearized PIHNA model, which yields an expression for this threshold. The PIHNA model suggests that an inherently faster-diffusing hypoxic cell population can drive the outward growth of a GBM as a whole, and that this effect is more prominent for faster-proliferating tumors that recover relatively slowly from a hypoxic phenotype. The findings presented here act as a first step in enabling patient-specific calibration of the PIHNA model.

Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.

Invasive spread of glioblastoma (GBM) is linked to changes in chondroitin sulfate (CS) proteoglycan (CSPG)-associated sulfated glycosaminoglycans (GAGs) that are selectively up-regulated in the tumor microenvironment (TME). We hypothesized that inhibiting CS-GAG signaling in the TME would stem GBM invasion. Rat F98 GBM cells demonstrated enhanced preferential cell invasion into oversulfated 3-dimensional composite of CS-A and CS-E [4- and 4,6-sulfated CS-GAG (COMP)] matrices compared with monosulfated (4-sulfated) and unsulfated hyaluronic acid matrices in microfluidics-based choice assays, which is likely influenced by differential GAG receptor binding specificities. Both F98 and human patient-derived glioma stem cells (GSCs) demonstrated a high degree of colocalization of the GSC marker CD133 and CSPGs. The small molecule sulfated GAG antagonist bis-2-methyl-4-amino-quinolyl-6-carbamide (surfen) reduced invasion and focal adhesions in F98 cells encapsulated in COMP matrices and blocked CD133 and antichondroitin sulfate antibody (CS-56) detection of respective antigens in F98 cells and human GSCs. Surfen-treated F98 cells down-regulated CSPG-binding receptor transcripts and protein, as well as total and activated ERK and protein kinase B. Lastly, rats induced with frontal lobe tumors and treated with a single intratumoral dose of surfen demonstrated reduced tumor burden and spread compared with untreated controls. These results present a first demonstration of surfen as an inhibitor of sulfated GAG signaling to stem GBM invasion.-Logun, M. T., Wynens, K. E., Simchick, G., Zhao, W., Mao, L., Zhao, Q., Mukherjee, S., Brat, D. J., Karumbaiah, L. Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.

Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments.

The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.

P01.134 Extracranial spread of glioblastoma as the initial manifestation of the disease: case report and literature review

P01.134 Extracranial spread of glioblastoma as the initial manifestation of the disease: case report and literature review

Abstract 931: Extracellular matrix stimuli regulate cancer stem cell population and migratory potential in glioblastoma

Abstract Introduction Glioblastoma (GBM) is the most common and lethal form of brain cancer. Poor survival is linked to diffuse infiltration throughout the brain, suggesting the need to examine the effect of the tissue environment at the margins of the GBM tumor on processes of GBM invasion. Further, the presence of GBM cancer stem cells (GSCs) in primary tumor specimens is believe to play a significant role in GBM spreading, recurrence, and therapeutic resistance. While biophysical properties of the tumor microenvironment, such as spatial gradients in matrix stiffness and hyaluronic acid (HA) content as well as regional hypoxia, may affect GBM invasive phenotypes, limited tools are available to rigorously examine the relationship between tissue microenvironment and tumor growth, invasion, and therapeutic outcomes. This work focuses on the integration of HA-functionalized gelatin hydrogels with 3D cell invasion assays to investigate the impact of tissue microenvironment on GBM invasion and GSC population. Methods Hydrogels are generated from methacrylamide functionalized gelatin (GelMA) and methacrylated HA. Prepolymer mixture was crosslinked under UV light in the presence of a photoinitiator. GBM cells, either U87 cell lines or patient-derived xenografts (PDX), were mixed with prepolymer solution prior to polymerization and collected for up to 7 days in culture. To assess invasion, cells were seeded onto dextran beads overnight, with populational invasion metrics calculated after 3 and 7 days, while live cell tracing via confocal microscopy was employed for single cell invasion metrics. Samples were cultured with 5% CO2 and ~20% O2 (normoxia) or 1% O2(hypoxia). Results The invasion of U87 and U87viii (constitutively active EGFR) cells increased significantly in GelMA hydrogels lacking HA, consistent with our previous findings that compensatory secretion of soluble HA in the absence of matrix-bound HA was associated with upregulated invasion. Heightened U87 invasion was observed in the presence of hypoxia for all hydrogel compositions, and was associated with upregualted markers of invasion (gene expression: MMP-2, VEGF; protein expression: ERK1/2) while PI3K was downregulated, suggesting cells respond to hypoxia by alternating invasion and proliferation behaviors. We subsequently examined the effect of matrix biophysical properties and hypoxia on the expansion of GSC populations within PDX cells (a gift of Jann Sarkaria, Mayo Clinic). Here, the expansion of the GSC population in EGFRviii mutant GBM6 was a function of hypoxia as well as matrix-bound HA. Conclusions This work demonstrates that GBM cells respond dynamically to their matrix biophysical (Stiffness, HA content, hypoxia) properties. Using PDX cells, we provide evidence that the expansion of GSC is related with both metabolic environment (O2%) and matrix composition (+/- HA). Ongoing work seeks to understand how GSCs contribute to the invasive phenotype of GBM. Citation Format: Jee-wei E. Chen, Jan C. Lumibao, H Rex Gaskins, Jann N. Sarkaria, Brendan A. Harley. Extracellular matrix stimuli regulate cancer stem cell population and migratory potential in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2017-931

Glioblastoma on a microfluidic chip: Generating pseudopalisades and enhancing aggressiveness through blood vessel obstruction events.

Glioblastoma (GBM) is one of the most lethal tumor types. Hypercellular regions, named pseudopalisades, are characteristic in these tumors and have been hypothesized to be waves of migrating glioblastoma cells. These "waves" of cells are thought to be induced by oxygen and nutrient depletion caused by tumor-induced blood vessel occlusion. Although the universal presence of these structures in GBM tumors suggests that they may play an instrumental role in GBM's spread and invasion, the recreation of these structures in vitro has remained challenging. Here we present a new microfluidic model of GBM that mimics the dynamics of pseudopalisade formation. To do this, we embedded U-251 MG cells within a collagen hydrogel in a custom-designed microfluidic device. By controlling the medium flow through lateral microchannels, we can mimic and control blood-vessel obstruction events associated with this disease. Through the use of this new system, we show that nutrient and oxygen starvation triggers a strong migratory process leading to pseudopalisade generation in vitro. These results validate the hypothesis of pseudopalisade formation and show an excellent agreement with a systems-biology model based on a hypoxia-driven phenomenon. This paper shows the potential of microfluidic devices as advanced artificial systems capable of modeling in vivo nutrient and oxygen gradients during tumor evolution.

High Precision Imaging of Microscopic Spread of Glioblastoma with a Targeted Ultrasensitive SERRS Molecular Imaging Probe

The dismal prognosis of patients with malignant brain tumors such as glioblastoma multiforme (GBM) is attributed mostly to their diffuse growth pattern and early microscopic tumor spread to distant regions of the brain. Because the microscopic tumor foci cannot be visualized with current imaging modalities, it remains impossible to direct treatments optimally. Here we explored the ability of integrin-targeted surface-enhanced resonance Raman spectroscopy (SERRS) nanoparticles to depict the true tumor extent in a GBM mouse model that closely mimics the pathology in humans. The recently developed SERRS-nanoparticles have a sensitivity of detection in the femtomolar range. An RGD-peptide-conjugated version for integrin-targeting (RGD-SERRS) was compared directly to its non-targeted RAD-SERRS control in the same mice via Raman multiplexing. Pre-blocking with RGD peptide before injection of RGD-SERRS nanoparticles was used to verify the specificity of integrin-targeting. In contrast to the current belief that the enhanced permeability and retention (EPR) effect results in a baseline uptake of nanoparticles regardless of their surface chemistry, integrin-targeting was shown to be highly specific, with markedly lower accumulation after pre-blocking. While the non-targeted SERRS particles enabled delineation of the main tumor, the RGD-SERRS nanoparticles afforded a major improvement in visualization of the true extent and the diffuse margins of the main tumor. This included the detection of unexpected tumor areas distant to the main tumor, tracks of migrating cells of 2-3 cells in diameter, and even isolated distant tumor cell clusters of less than 5 cells. This Raman spectroscopy-based nanoparticle-imaging technology holds promise to allow high precision visualization of the true extent of malignant brain tumors.

Accelerated tumor invasion under non-isotropic cell dispersal in glioblastomas

Glioblastomas are highly diffuse, malignant tumors that have so far evaded clinical treatment. The strongly invasive behavior of cells in these tumors makes them very resistant to treatment, and for this reason both experimental and theoretical efforts have been directed toward understanding the spatiotemporal pattern of tumor spreading. Although usual models assume a standard diffusion behavior, recent experiments with cell cultures indicate that cells tend to move in directions close to that of glioblastoma invasion, thus indicating that a biased random walk model may be much more appropriate. Here we show analytically that, for realistic parameter values, the speeds predicted by biased dispersal are consistent with experimentally measured data. We also find that models beyond reaction–diffusion–advection equations are necessary to capture this substantial effect of biased dispersal on glioblastoma spread.

TLR4 signal transduction pathways neutralize the effect of Fas signals on glioblastoma cell proliferation and migration

The Fas pathway is described as an activator of the glioblastoma proliferation by increasing the pathogenicity of this tumour. The lipopolysaccharide (LPS) pathway depending on Toll-like receptor 4 (TLR4) could limit the glioblastoma spreading. Here, Fas and TLR4 pathways were activated in glioblastoma cell lines by an agonist antibody and/or LPS treatment. Activation of the Fas pathway or of the TLR4 pathway induced cell proliferation. However, simultaneous treatment with agonist antibody and LPS decreased proliferation. This anti-proliferative effect was caspase dependent, and a decreased cell migration and matrix metalloproteinase (MMP)-9 expression were also observed. Both TLR4 and MMP-9 were highly expressed in human glioblastoma tissues. These data suggest that TLR4 signal transduction pathways neutralize proliferation and migration induced by Fas pathway activation in glioblastoma cell lines.

18F-FDG PET detection of spinal leptomeningeal metastases from cerebral glioblastoma multiforme

A 59-year-old man who presented with weight loss and fatigue showed no pathological findings on the contrastenhanced thorax/abdominal CT scan, but a single frontal nodular lesion on the contrast-enhanced cranial CT scan (a) that was considered to represent a single cerebral metastases from an unknown primary. The F-FDG PET scan was performed for staging to exclude a primary tumour, and also a cerebral-MR scan to complete the diagnostic imaging work-up. The PET/CT scan showed intense FDG uptake (b) by the cerebral lesion, but no evidence of any primary tumour. On the basis of the MR imaging findings, a frontal glioblastoma multiforme, well seen on the T1-weighted image (c), was diagnosed. MR imaging also showed additional multiple small meningeal foci surrounding the cisternal areas. PET/CT also showed heterogeneous FDG uptake at the cervical and dorsal spinal cord on the sagittal view (d), which was more easily detectable by increasing the contrast on the same PET scan sagittal view as multiple foci of FDG uptake distributed along the spinal leptomeningeal area (e, arrows) and also on the PET/CT sagittal fusion image (f, arrows). A spinal MRI study confirmed the leptomeningeal infiltration. Although the frontal lesion was surgically treated, the extensive disease led to the patient’s death within 30 days. Leptomeningeal metastases from previously untreated cerebral glioblastoma spreading through the cerebrospinal fluid is an uncommon diagnostic finding (more commonly detected on necropsy studies) [1]. Although, to date no report of leptomeningeal metastases from glioblastoma detected by F-FDG PET is available in the literature, there are some cases reports of the ability of FDG PET to detect leptomeningeal metastases [2, 3].

Infiltrative patterns of glioblastoma spread detected via diffusion MRI after treatment with cediranib

To evaluate the role of apparent diffusion coefficient (ADC) imaging in assessing tumor cell infiltration after treatment with the antivascular endothelial growth factor (anti-VEGF) agent, cediranib, we prospectively analyzed diffusion MRI scans from 30 patients participating in a Phase II trial of cediranib for recurrent glioblastoma. A patient-specific threshold was selected below which ADC values were determined to be abnormally low and suggestive of tumor. We determined the percent of low ADC in the FLAIR hyperintensity surrounding the enhancing tumor and then visualized the location of these low ADC voxels. The percent volume of the FLAIR hyperintensity comprised by low ADC increased significantly from baseline (2.3%) to day 28 (2.9%), day 56 (5.0%), and day 112 (6.3%) of treatment with cediranib suggesting increasing infiltrative tumor in some patients. Visualization of the location of the low ADC voxels suggested regions of tumor growth that were not visible on contrast-enhanced MRI. ADC maps can be used to suggest regions of infiltrative tumor cells with anti-VEGF therapy and should be validated in future studies.

Transdural Spread of Glioblastoma through the Foramen Ovale with Presentation as a Masticator Space Mass

Direct extension of a glioblastoma through the dura at the skull base is an uncommon occurrence. We report an unusual case of recurrent glioblastoma that spread transdurally through the foramen ovale and presented primarily as a masticator space mass. There was mandibular denervation and a relative paucity of intracranial disease.

Migration of human malignant astrocytoma cells in the mammalian brain: Scherer revisited

Fresh suspensions of human glioblastoma multiforme were preincubated in the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) and implanted into cortical pockets in adult rat brain. Brains were investigated periodically over 30 postoperative days and the migration of the human glioblastoma cells was traced with anti-PHAL immunofluorescence or the overexpression of human specific p185c-neu a specific marker of a class of human malignant astrocytoma cells. The principal pathway of migration of the implanted human cells in the rat brain was ventrally through cortical gray matter and into the corpus callosum, with rapid lateral distribution in this and other parallel and intersecting white matter fascicles. Human glioblastoma cells also migrated on basement membrane lined blood vessels, pia-glia membrane and spaces of Virchow-Robin, as well as the subependymal space of the ventricles. These paths of migration of human glioblastoma cells in the rat brain are consistent with the pathways of spread of glioblastoma in the human brain as described by Scherer over 50 years ago, indicating that multifocal malignant astrocytomas have common migratory pathways in mature mammalian brain.