Alf Giese

DICHOTOMY OF ASTROCYTOMA MIGRATION AND PROLIFERATION

Astrocytomas often show high rates of local invasion that lead to local recurrence of the disease. Histologically, the most highly invasive astrocytoma cells are detected in isolation rather than as nests of tumor. Our study attempted to determine whether the migratory response to extracellular substrates influences the proliferative behavior of these highly invasive cells. The preferential and specific migratory response of human astrocytoma cells to extracellular matrix proteins was assessed by a microliter scale migration assay. Growth curve studies on protein ligands permissive (merosin) for cell migration indicated that the lag phase was protracted compared with cells seeded on non‐permissive proteins (vitronectin). Once a certain cell density was reached, logarithmic proliferation was indistinguishable on the different proteins. The proliferation index of populations of cells migrating on merosin and vitronectin was measured by both BrdU incorporation and MIB‐I immunocytochemistry labeling. Cells seeded on vitronectin showed higher proliferation throughout the population than cells seeded on merosin. On merosin, the more migratory cells at the periphery were less proliferative than non‐migratory cells in the central region of that population. The integrin‐associated signal transduction protein, p125FAK, was heavily localized in the membrane of non‐migrating cells and largely absent in migrating astrocytoma cells. We conclude that temporally, proliferation and migration are mutually exclusive behaviors. Cell density or non‐permissive substrates that inhibit cell motility favor a more proliferative phenotype. Conversely, active migration suppresses cell proliferation.

Glioma cell motility is associated with reduced transcription of proapoptotic and proliferation genes: A cDNA microarray analysis

Microarray analysis of complementary DNA (cDNA) allows large-scale, comparative, gene expression profiling of two different cell populations. This approach has the potential for elucidating the primary transcription events and genetic cascades responsible for increased glioma cell motility in vitro and invasion in vivo. These genetic determinants could become therapeutic targets.We compared cDNA populations of a glioma cell line (G112) exposed or not to a motility-inducing substrate of cell-derived extracellular matrix (ECM) proteins using two sets of cDNA microarrays of 5700 and 7000 gene sequences. The data were analyzed considering the level and consistency of differential expression (outliers) and whether genes involved in pathways of motility, apoptosis, and proliferation were differentially expressed when the motility behavior was engaged. Validation of differential expression of selected genes was performed on additional cell lines and human glioblastoma tissue using quantitative RT-PCR.Some genes involved in cell motility, like tenascin C, neuropilin 2, GAP43, PARG1 (an inhibitor of Rho), PLCγ, and CD44, were over expressed; other genes, like adducin 3γ and integrins, were down regulated in migrating cells. Many key cell cycle components, like cyclin A and B, and proliferation markers, like PCNA, were strongly down regulated on ECM. Interestingly, genes involved in apoptotic cascades, like Bcl-2 and effector caspases, were differentially expressed, suggesting the global down regulation of proapoptotic components in cells exposed to cell-derived ECM. Overall, our findings indicate a reduced proliferative and apoptotic activity of migrating cells. cDNA microarray analysis has the potential for uncovering genes linking the phenotypic aspects of motility, proliferation, and apoptosis.

MicroRNAs in cerebrospinal fluid identify glioblastoma and metastatic brain cancers and reflect disease activity

An accurate, nonsurgical diagnostic test for brain tumors is currently unavailable, and the methods of monitoring disease progression are not fully reliable. MicroRNA profiling of biological fluids has recently emerged as a diagnostic tool for several pathologic conditions. Here we tested whether microRNA profiling of cerebrospinal fluid (CSF) enables detection of glioblastoma, discrimination between glioblastoma and metastatic brain tumors, and reflects disease activity. We determined CSF levels of several cancer-associated microRNAs for 118 patients diagnosed with different types of brain cancers and nonneoplastic neuropathologies by quantitative reverse transcription PCR analysis. The levels of miR-10b and miR-21 are found significantly increased in the CSF of patients with glioblastoma and brain metastasis of breast and lung cancer, compared with tumors in remission and a variety of nonneoplastic conditions. Members of the miR-200 family are highly elevated in the CSF of patients with brain metastases but not with any other pathologic conditions, allowing discrimination between glioblastoma and metastatic brain tumors. Quantification of as few as 7 microRNAs in CSF enables differential recognition of glioblastoma and metastatic brain cancer using computational machine learning tools (Support Vector Machine) with high accuracy (91%-99%) on a test set of samples. Furthermore, we show that disease activity and treatment response can be monitored by longitudinal microRNA profiles in the CSF of glioblastoma and non-small cell lung carcinoma patients. This study demonstrates that microRNA-based detection of brain malignancies can be reliably performed and that microRNAs in CSF can serve as biomarkers of treatment response in brain cancers.

Pattern of recurrence following local chemotherapy with biodegradable carmustine (BCNU) implants in patients with glioblastoma

AbstractObjective: Recently a randomized placebo-controlled phase III trial of biodegradable polymers containing carmustine has demonstrated a significant survival benefit for patients treated with local chemotherapy. A local chemotherapy applied directly to the resection cavity may act directly on residual tumor cells in adjacent brain possibly leading to a local control of the tumor and increased survival. Methods: We have analyzed the pattern of recurrence using serial MRI studies of 24 patients treated with GLIADEL® Wafers or placebo wafers following resection of glioblastomas. Results: Of 24 patients 11 received carmustine wafers and 13 placebo. The age distribution and Karnowsky performance scores of the two populations were not different. However, the median survival (14.7 versus 9.5 months; P = 0.007) and the time to neurological deterioration (12.9 ± 4.85 vs. 9.4 ± 2.73 months; P = 0.035) was significantly longer in the treatment group versus the placebo treated control. Preoperative and follow up MRI studies were evaluated in a blinded fashion. Out of 24 patients that entered the analysis 11 showed clearance of all contrast enhancement following resection of glioblastomas. Seventeen tumors progressed locally and 7 showed different patterns of distant failure. Within the carmustine treated group 8 patients showed a local treatment failure with recurrent tumors immediately adjacent to the resection cavity or progression form a residual tumor. Three patients showed a multifocal distant and local pattern of failure after complete or subtotal removal. In no case the local chemotherapy resulted in a distant recurrence only. However, the time to radiographic progression was 165.1 ± 80.75 days for the GLIADEL® Wafer group and 101.9 ± 43.06 days for the placebo group (P = 0.023). Conclusion: In this subgroup analysis of a phase III trial population both the clinical progression and radiological progression were significantly delayed in patients treated with local chemotherapy, resulting in an increased survival time. Local chemotherapy with carmustine containing wafer implants did not result in an altered pattern of recurrence and did not promote multifocal patterns of recurrence.

Safety profile of carmustine wafers in malignant glioma: a review of controlled trials and a decade of clinical experience.

ABSTRACT Background: Carmustine (1,3-bis [2-chloroethyl]-1-nitrosourea, or BCNU) wafers are approved for recurrent glioblastoma and newly diagnosed malignant glioma (MG). Based on considerable clinical experience and use in multimodal regimens, the safety of BCNU wafers needs a re-evaluation. Scope: A review of literature from 1996 to February 2008 was conducted on the safety of BCNU wafer in MG patients using search criteria in Medline, EMBASE, and BIOSIS. Abstracts from relevant US and European meetings were also evaluated. Three Phase III (two were pivotal) and 26 non-Phase III studies met inclusion criteria. Overall incidence was estimated for each adverse event (AE), and data from individual studies were summarised as median (range) rates. Comparisons were based on consistent similarities or differences across overall incidence, median rate and range. Findings: BCNU wafer group AE rates from the two pivotal Phase III trials ranged from 4–23% for cerebral oedema, 4–9% for intracranial hypertension, 14–16% for healing abnormalities, 5% for CSF leaks, 4–5% for intracranial infection, 19–33% for seizures, 10% for deep vein thrombosis, and 8% for pulmonary embolus. There were no notable differences in AE rates between the two pivotal Phase III and 26 non-Phase III studies. For the non-pivotal studies, the overall incidence of AEs was low, ranging from 0.2% for intracranial hypertension to 9.6% for healing abnormalities. Healing abnormalities, intracranial infection, and seizures were the most consistently reported AEs, having been observed in 16, 12, and 11 studies, respectively. Rates of healing abnormalities appeared higher in recurrent than in newly diagnosed disease. There were no notable differences between BCNU wafer plus adjuvant treatment (e.g., temozolomide) and BCNU wafer alone, with the exception of haematologic toxicity. Conclusion: This review of safety data for BCNU wafers provides reassurance that the AE rates reported in current treatment strategies including multimodal treatment approaches are comparable to those observed in the initial registration studies. The broad range of AE rates may reflect differences in the perioperative and postoperative management. Clinical experience suggests that strategies may exist to reduce the risk of complications.

Multiphoton excitation of autofluorescence for microscopy of glioma tissue.

OBJECTIVE:Intraoperative detection of residual tumor tissue in glioma surgery remains an important challenge because the extent of tumor removal is related to the prognosis of the disease. Multiphoton excited fluorescence tomography of living tissues provides high-resolution structural and photochemical imaging at a subcellular level. In this conceptual study, we have used multiphoton microscopy and fluorescence lifetime imaging (4D microscopy) to image cultured glioma cell lines, solid tumor, and invasive tumor cells in an experimental mouse glioma model and human glioma biopsy specimens. MATERIAL AND METHODS:A laser imaging system containing a mode-locked 80 MHz titanium:sapphire laser with a tuning range of 710 to 920 nm, a scan unit, and a time correlated single photon counting board was used to generate autofluorescence intensity images and fluorescence lifetime images of cultured cell lines, experimental intracranial gliomas in mouse brain, and biopsies of human gliomas. RESULTS:Multiphoton microscopy of native tumor bearing brain provided structural images of the normal brain anatomy at a subcellular resolution. Solid tumor, the tumor-brain interface, and single invasive tumor cells could be visualized. Fluorescence lifetime imaging demonstrated significantly different decay of the fluorescent signal in tumor versus normal brain, allowing a clear definition of the tumor-brain interface based on this parameter. Distinct fluorescence lifetimes of endogenous fluorophores were found in different cellular compartments in cultured glioma cells. The analysis of the relationship between the laser excitation wavelength and the lifetime of excitable fluorophores demonstrated distinct profiles for cells of different histotypes. CONCLUSION:Multiphoton excited fluorescence of endogenous fluorophores allows structural imaging of tumor and central nervous system histo-architecture at a subcellular level. The analysis of the decay of the fluorescent signal within specific excitation volumes by fluorescent lifetime imaging discriminates glioma cells and normal brain, and the excitation/lifetime profiles may further allow differentiation of cellular histotypes. This technology provides a noninvasive optical tissue analysis that may potentially be applied to an intraoperative analysis of resection plains in tumor surgery.

Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells

Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy.

Wild-type p53 in cancer cells: When a guardian turns into a blackguard

The tumor suppressor p53 controls a broad range of cellular responses. Induction of a transient (cell cycle arrest) or a permanent (senescence) block of cell proliferation, or the activation of cell death pathways in response to genotoxic stress comprise the major arms of the survival-death axis governed by p53. Due to these biological properties, inactivation of p53 is a crucial step in tumor development and progression, reflected by the high incidence of TP53 mutations in different types of human cancers. The remarkable potency of p53 in suppressing tumorigenic outgrowth has promoted the expectation that tumor cells expressing wild-type p53 (wtp53) should be more prone to elimination by cytotoxic treatments than tumor cells expressing mutant p53 (mutp53) with defunct wtp53 activities. However, recent findings yielded somewhat unexpected insights concerning the preponderance of the survival-promoting effects of wtp53 in cancer cells, a rather undesired property from the therapeutic point of view. In this commentary we will discuss the possibility that the developmentally established distinct patterns of wtp53 mediated responses in different tissues are an important factor in determining the ultimate outcome of cellular responses mediated by wtp53 in different types of tumor cells, with a particular focus on the divergent impact of wtp53 in malignant tumors of the central nervous system. We infer that a selective gain of pro-survival functions of wtp53 in cancer cells will confer a survival advantage that counteracts tumor therapy.

Untreated Glioblastoma Multiforme: Increased Myo-inositol and Glutamine Levels in the Contralateral Cerebral Hemisphere at Proton MR Spectroscopy

PURPOSE To use localized in vivo proton magnetic resonance (MR) spectroscopy of the contralateral hemisphere in patients with glioblastoma multiforme (GBM) to detect alterations in cerebral metabolites as potential markers of infiltrating GBM cells. MATERIALS AND METHODS The study was approved by the ethics committee, and written informed consent was obtained. Twenty-two patients with newly diagnosed and untreated GBM underwent in vivo single-voxel short echo time proton MR spectroscopy with a 3-T MR imaging system. Absolute metabolite concentrations in the hemisphere contralateral to the tumor were compared with data from five patients with low-grade gliomas (LGGs) and from a group of 14 age-matched control subjects by using analysis of variance and subsequent t tests or corresponding nonparametric tests. RESULTS In the contralateral hemisphere, MR spectroscopy revealed increased concentrations of myo-inositol and glutamine. Mean myo-inositol levels were significantly increased in patients with GBM (3.6 mmol/L +/- 0.8 [standard deviation]) relative to levels in control subjects (3.1 mmol/L +/- 0.6; P = .03) and tended to be higher relative to levels in patients with LGG (2.7 mmol/L +/- 0.8; P = .09). Mean glutamine concentrations in patients with GBM (3.4 mmol/L +/- 0.9) differed significantly from those in control subjects (2.7 mmol/L +/- 0.7; P = .01); mean concentrations in patients with GBM differed from those in patients with LGG (2.4 mmol/L +/- 0.5; P = .01). There were no significant differences between data in patients with LGG and in control subjects. CONCLUSION Increased concentrations of myo-inositol and glutamine in the contralateral normal-appearing white matter of GBM patients are consistent with mild astrocytosis and suggest the detectability of early neoplastic infiltration by using proton MR spectroscopy in vivo.

Thromboxane synthase regulates the migratory phenotype of human glioma cells

The capacity of glial tumor cells to migrate and diffusely infiltrate normal brain compromises surgical eradication of the disease. Identification of genes associated with invasion may offer novel strategies for anti-invasive therapies. The gene for TXsyn, an enzyme of the arachidonic acid pathway, has been identified by differential mRNA display as being overexpressed in a glioma cell line selected for migration. In this study TXsyn mRNA expression was found in a large panel of glioma cell lines but not in a strain of human astrocytes. Immunohistochemistry demonstrated TXsyn in the parenchyma of glial tumors and in reactive astrocytes, whereas it could not be detected in quiescent astrocytes and oligodendroglia of normal brain. Glioma cell lines showed a wide range of thromboxane B2 formation, the relative expression of which correlated with migration rates of these cells. Migration was effectively blocked by specific inhibitors of TXsyn, such as furegrelate and dazmegrel. Other TXsyn inhibitors and cyclooxygenase inhibitors were less effective. Treatment with specific inhibitors also resulted in a decrease of intercellular adhesion in glioma cells. These data indicate that TXsyn plays a crucial role in the signal transduction of migration in glial tumors and may offer a novel strategy for anti-invasive therapies.