Protein disulfide isomerase expression is related to the invasive properties of malignant glioma.

By serial transplantation of human glioblastoma biopsies into the brain of immunodeficient nude rats, two different tumor phenotypes were obtained. Initially, the transplanted xenografts displayed a highly invasive phenotype that showed no signs of angiogenesis. By serial transplantation in animals, the tumors changed to a less invasive, predominantly angiogenic phenotype. To identify novel proteins related to the invasive phenotype, the xenografts were analyzed using a global proteomics approach. One of the identified proteins was protein disulfide isomerase (PDI) A6 precursor. PDI is a chaperone protein that mediates integrin-dependent cell adhesion. It is both present in the cytosol and at the cell surface. We show that PDI is strongly expressed on invasive glioma cells, in both xenografts and at the invasive front of human glioblastomas. Using an in vitro migration assay, we also show that PDI is expressed on migrating glioma cells. To determine the functional significance of PDI in cell migration, we tested the effect of a PDI inhibitor, bacitracin, and a PDI monoclonal antibody on glioma cell migration and invasion in vitro. Both tumor spheroids derived from human glioblastoma xenografts in nude rat brain and cell line spheroids were used. The PDI antibody, as well as bacitracin, inhibited tumor cell migration and invasion. The anti-invasive effect of bacitracin was reversible after withdrawal of the inhibitor, indicating a specific, nontoxic effect. In conclusion, using a global proteomics approach, PDI was identified to play an important role in glioma cell invasion, and its action was effectively inhibited by bacitracin.

Opinion: the origin of the cancer stem cell: current controversies and new insights.

Most tumours are derived from a single cell that is transformed into a cancer-initiating cell (cancer stem cell) that has the capacity to proliferate and form tumours in vivo. However, the origin of the cancer stem cell remains elusive. Interestingly, during development and tissue repair the fusion of genetic and cytoplasmic material between cells of different origins is an important physiological process. Such cell fusion and horizontal gene-transfer events have also been linked to several fundamental features of cancer and could be important in the development of the cancer stem cell.

Cryopreservation of alginate-encapsulated recombinant cells for antiangiogenic therapy.

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to -7.5 degrees C and nucleated. Thereafter, the samples were cooled at a rate of 0.25 degrees C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Molecular approaches to brain tumour invasion.

In glioma cells, the stimulatory input of extracellular matrix components and an increased sensitivity to growth factors result in a high proliferative and migratory behaviour. Cell surface receptor interactions play pivotal roles in converging information about conditions in the environment immediately outside the cell. The transduced signal, in turn induces a response within the cell that provokes a specific behaviour. Cellular migration and cell proliferation are interwoven processes that share several common intracellular pathways. The major cross-links are the phosphoinositol phosphate regulating enzymes, PI-3 kinase and PTEN, the focal adhesion kinase (FAK) and the tumour suppressor p53. An understanding of the interaction between the molecular participants involved in migration and proliferation will promote the design of new treatments. A full understanding of the basis of the invasiveness of tumour cells remains elusive. Gene and protein expression are being studied, using modern techniques such as microarray analysis, SAGE and 2-D protein gels. Transient and permanent protein-protein interactions and recruitment of proteins to specialised cellular domains are equally important in regulating cellular invasion and presumably will attract more attention in future.

Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids.

Temozolomide is an alkylating cytostatic drug that finds increasing application in the treatment of melanoma, anaplastic astrocytoma and glioblastoma multiforme. The compound is a prodrug that decomposes spontaneously, independent of an enzymatic activation step. DNA methylation induces futile mismatch repair cycles and depletion of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase should then initiate programmed cell death. We show drug-dependent inhibition of tumour growth in a three-dimensional cell culture model of the glioma cell lines U87MG and GaMG. Migrational behaviour of the glioblastoma cells remained unaltered. However, coincubation of tumour spheroids with primary brain aggregates showed reduced tumour cell invasion into brain tissue in the presence of temozolomide. This was not achieved by slowing cellular migration, as temozolomide-treated cells displayed no reduced motility. By transferase-mediated dUTP nick-end labelling (TUNEL) of apoptotic nuclei, we found that the drug was able to induce apoptosis throughout the tumour cell spheroids. Apoptosis was highest in the core region of the spheroids. Repetitive application of sublethal doses of temozolomide to multicellular spheroids resulted in the development of drug resistance in GaMG cells. We suggest that temozolomide is a strong initiator of apoptosis in glioblastoma tumour cells in a spheroid cell culture system, when cells are already in a stressful environment.

[Intraoperative management of unexpected arterial hypotension during lumbar diskectomy]. / Intraoperatives Management eines unerwarteten Blutdruckabfalls bei Bandscheibenoperationen.

An anesthesiological management is difficult in surgical procedures in which the operative situs is not to be seen by the anesthesist. Therefore specific knowledge of the operative procedure and related hazards are mandatory for optimal anesthesiological care. By describing the anesthesiologic proceduces in two lumbar discectomys, the specific problems in recognition and treatment of severe injuries of retroperitoneal vascular structures are explained. In addition, the differential diagnosis of intraoperative arterial hypotension is described. The communication between all disciplines involved is mandatory, especially in cases of severe complications. The management of such complications can only be solved through in-time interdisciplinary cooperation of all involved disciplines.

Effects of DFMO on glioma cell proliferation, migration and invasion in vitro.

The polyamine inhibitor DL-alpha-difluoromethylornithine (DFMO) is a specific irreversible inhibitor of ornithine decarboxylase which is a rate-limiting enzyme in the polyamine bio-synthesis pathway. The present study describes the effects of DFMO on glioma cell proliferation, migration and invasion using multicellular spheroids from three glioma cell lines (GaMg, U-251 Mg and U-87 Mg). 10 mM DFMO reduced cell migration in the three cell lines by about 30-50%. 1 mM putrescine, added together with DFMO inhibited the DFMO effect. A stronger effect was observed in the growth assay where 10 mM DFMO reduced the spheroid growth, for all cell lines, by 90%. This effect was also reversed by adding 1 mM of putrescine. In vitro tumor cell invasion experiments indicated after 3 days of confrontation, an extensive invasion also after 10 mM DFMO treatment. The brain aggregate volumes were reduced to about the same extent as in the absence of drug, suggesting essentially no effects of DFMO on the invasive process. It is concluded that the tumor spheroids retained their ability to invade normal brain tissue even after DFMO exposure. However, DFMO inhibited spheroid growth and cell migration which supports the notion that cell growth, migration and invasion are biological properties that are not necessarily related to each other.

Effects of photodynamic therapy on glioma spheroids.

The present study describes the sensitivity of glioma cells to a haematoporphyrin derivative (Photosan-3) under laser activation (argon-pumped dye laser). The effects of photodynamic therapy (PDT) on cell growth, directional migration and cell invasion were investigated on two human glioma cell lines (GaMg and U-251 Mg). The directional cell migration and spheroid growth was determined for both cell lines exposed to increasing laser energy output (15-35J/cm2) with concentrations of 5 and 7 micrograms/ml of Photosan-3. Both cell lines showed a dose-dependent migratory response to increasing laser irradiation, that was more prominent in the 7 micrograms/ml treatment group. This effect occurred during the first 4 days after drug exposure. Also, spheroids from both cell lines showed a drug and laser output energy dose-dependent inhibition of growth which became apparent after a lag period of 6 days. The lag period was characterized by a decreased growth rate as compared with the control group. During this period the outer cell layers of the spheroids fell apart. The remaining spheroid tissue was not able to migrate and to regrow when exposed to the highest laser energy outputs (30-35J/cm2, 5 and 7 micrograms/ml Photosan-3). These spheroids showed, however, the ability for invasion when confronted with normal brain cell aggregated in vivo. Light microscopic observations of co-cultures between tumour tissue and brain cell aggregates revealed a normal tumour morphology. This indicates that the remaining tumour cells were not dead and could be stimulated to invade the normal tissue when exposed to a normal brain microenvironment.

Proliferation, migration and invasion of human glioma cells exposed to paclitaxel (Taxol) in vitro.

Paclitaxel (Taxol), an anti-cancer drug derived from Taxus species, was tested for its anti-migrational, anti-invasive and anti-proliferative effect on two human glioma cell lines (GaMg and D-54Mg) grown as multicellular tumour spheroids. In addition, the direct effect of paclitaxel on glioma cells was studied using flow cytometry and scanning confocal microscopy. Both cell lines showed a dose-dependent growth and migratory response to paclitaxel. The GaMg cells were found to be 5-10 times more sensitive to paclitaxel than D-54Mg cells. Paclitaxel also proved to be remarkably effective in preventing invasion in a co-culture system in which tumour spheroids were confronted with fetal rat brain cell aggregates. Control experiments with Cremophor EL (the solvent of paclitaxel for clinical use) in this study showed no effect on tumour cell migration, cell proliferation or cell invasion. Scanning confocal microscopy of both cell lines showed an extensive random organization of the microtubules in the cytoplasm. After paclitaxel exposure, the GaMg and the D-54Mg cells exhibited a fragmentation of the nuclear material, indicating a possible induction of apoptosis. In line with this, flow cytometric DNA histograms showed an accumulation of cells in the G2/M phase of the cell cycle after 24 h of paclitaxel exposure. After 48 h, a deterioration of the DNA histograms was observed indicating nuclear fragmentation.

Glioma cell invasion visualized by scanning confocal laser microscopy in an in vitro co-culture system.

Confrontation cultures between glioma spheroids and brain cell aggregates are well established in glioma research, and the model reflects several similarities to the in vivo brain tumour invasive process. The lipid-binding fluorescent carbocyanine dyes DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) and DiI (1,1'-dioctadecyl-3,3,3,'3,'-tetramethylinocarbocyanine perchlorate) are widely used in cell biology as tracers for studying cell movement. Mature brain cell aggregates grown from fetal rat brain cells, and spheroids initiated from two glioma cell lines (GaMg and D-54Mg) were stained with DiO and DiI, respectively. Penetration of DiI and DiO into the tumour spheroids and brain aggregates was studied by confocal laser scanning microscopy (CLSM). After 48 h of dye exposures, the tracers had almost completely penetrated the tumour spheroids and brain aggregates. Light-microscopic sections of the specimens indicated that the dye incorporation had little effect on cellular morphology. Cell migration from DiI stained D-54Mg and GaMg spheroids was similar to that observed from unstained spheroids. Growth was also unaffected after 48 h of DiI exposure. Gioma cell invasion was assessed by CLSM using co-cultures of DiI -stained spheroids and DiO-stained brain cell aggregates. Optical sections revealed a gradual decrease in remaining brain volume, indicating a progressive invasive process. Single tumour cells were identified deep within the brain aggregates. In addition normal brain cells were also identified in the tumour spheroids. It is concluded that vital staining can be used to identify both normal cells and tumour cells during tumour cell invasion in vitro. The method may provide the possibility for studying the kinetics of single normal and tumour cell movement in individual tumour/brain co-cultures.

Leptomeningeal tissue: a barrier against brain tumor cell invasion.

BACKGROUND: Primary brain tumors are characterized by an extensive infiltrative growth into the surrounding brain tissue. This process is confined to the central nervous system, and tumor cell metastasis to other organs is rare. However, other tumors of non-neural origin may frequently metastasize to the central nervous system. PURPOSE: The purpose of the present study was to examine the invasive behavior of different glioma cells into tissues of neural (brain aggregates) as well as non-neural origin (leptomeningeal tissue). Using the same target tissues, the invasive characteristics of two neural metastatic tumors (one malignant melanoma and one small-cell lung carcinoma) were also studied. This direct comparison of the invasive behavior between tumors of neural and non-neural origin provides valuable information regarding the mechanisms of glioma cell dissemination in the central nervous system. METHODS: The in vitro invasive behavior of human tumors of the central nervous system into human leptomeningeal tissue as well as into normal rat brain tissue was studied. For this purpose, a co-culture system consisting of tumor biopsy specimens, human leptomeningeal cell aggregates, and brain cell aggregates was established. Three glioblastomas, one oligodendroglioma, one meningioma, one small-cell lung carcinoma, and one malignant melanoma were studied. RESULTS: In co-cultures of gliomas and leptomeningeal cell aggregates, a well-defined border between the two tissues was observed. The brain cell aggregates, in contrast, were consistently invaded by the glioma cells. The brain metastases showed a different invasion pattern. The metastatic cells invaded and progressively destroyed leptomeningeal cell aggregates, whereas they did not invade the brain cell aggregates. Upon confrontation of the leptomeningeal tissue with the meningioma, a fusion of the two tissues was observed. Immunostaining of the leptomeningeal tissue showed a strong expression of the basement membrane components fibronectin, collagen type IV, and laminin with no expression of glial fibrillary acidic protein, neuron-specific enolase, or S-100 protein. CONCLUSIONS: The present study indicates that there may be important biologic differences between the invasive behavior of gliomas and non-neuroepithelial tumors. Our co-culture experiments suggest that leptomeningeal cells and associated acellular components may constitute a barrier against glioma cell invasion. However, this barrier may not be functional for metastatic tumors to the brain. The presence of glioma cells within the leptomeninges should not necessarily be taken as evidence of aggressive growth or as an indicator of malignancy.

Interactions between fetal rat brain cells and mature brain tissue in vivo and in vitro.

Fetal as well as mature neural cells were homografted into the right cerebral hemisphere of adult BD-IX rats. The animals were sacrificed 7 d after implantation, and the localization of implanted cells was visualized by fluorescence and light microscopy. The cell implants were prestained with the fluorescent vital dye 1,1'-Dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) to discriminate between implanted cells and host brain tissue. At the implantation site, the fetal brain cells as well as the cells from immature brain cell aggregates showed diffuse infiltration into the surrounding host brain tissue of up to 0.5 mm. Extensive cell migration along the corpus callosum for up to 5 mm in the coronal and to a lesser extent in the sagittal plane was also observed. In addition, fetal cells were distributed in the subarachnoid space of both cerebral hemispheres and showed a distinct association with larger blood vessels. Cells from mature brain aggregates did not migrate as far as fetal cells and showed only a local infiltration into the host neuropil. Fluorescent microspheres as well as fixed fetal brain cells were implanted, either alone or in combination with vital cells to distinguish between active cell migration and passive cell displacement. The microspheres and the fixed cells were found either localized to the implantation pathway or distributed in the corpus callosum for up to 2 mm in the coronal plane without any dispersion in the sagittal plane. The microspheres also showed an extensive displacement in the subarachnoid space. In vitro co-culture experiments between two immature aggregates showed a complete fusion of the two aggregates during a 96 h culture period. In co-cultures between two mature aggregates complete fusion was not prominent, although the confrontation zone appeared diffuse. Confrontations between a mature and an immature aggregate showed the same pattern of interaction as seen for the two mature aggregates. It is concluded that carbocyanine dyes may be used as a tracer for transplanted cells. Cells from fetal rat brain cell aggregates, opposed to those from mature aggregates, showed extensive migration along well defined anatomical structures in the mature along well defined anatomical structures in the mature brain. Some of the spread of cells following implantation is probably due to passive movement since inert microspheres will spread into certain areas of the CNS.

Induced hyperthermia in brain tissue in vivo.

Concerning hypothermia treatment, knowledge of time-temperature and of temperature distributions within tumor volumes is essential in order to obtain the maximal therapeutic effect. New techniques are being developed to overcome these difficulties. Two different heat sources, a contact Nd:YAG laser system and an automatically controlled high-frequency current system were investigated on 15 rabbits. Changes of the intracerebral temperature were registered at 4 different distances from the energy source. The intracerebral temperature was increased to 42.5 degrees C at a distance of 5 mm to the heat source and maintained at this level for a period of 60 min. The contact Nd:YAG laser system reached 42.5 degrees C at 3 W of output power. Using higher laser output power, brain tissue herniation (brain edema) through the burrhole was observed. The automatically controlled high-frequency current system reached 42.5 degrees C at 18.75 W of output current. A very small herniation of brain tissue could be observed using higher output current. Both heat sources presented an exponential decrease of the temperature profile depending on the distance. The tissue heat clearance was compensated for by intermittent laser or high-frequency current application. Both systems proved efficient for inducing hyperthermia as needed for antitumoral therapy.

Interaction between human medulloblastomas and foetal rat brain aggregates in vitro.

The invasiveness of three medulloblastoma permanent cell lines (D-283, D-341, and DAOY), a human medulloblastoma biopsy, and in addition, a human rhabdomyosarcoma permanent cell line (TE-671), which previously had been regarded as a human medulloblastoma, was studied in an organ co-culture assay. All the four cell lines and the biopsy were co-cultured with normal rat brain cell aggregates for up to six days in vitro. The medulloblastoma biopsy, the D-283 and the D-341 cells invaded the brain tissue by diffuse single cell infiltration. The medulloblastoma cell line (DAOY) showed an invasive pattern similar to that observed earlier for most glioblastoma cell lines. This was characterized by massive cell replacement and destruction of normal brain tissue. The rhabdomyosarcoma cell line (TE-671) presented a solid invasive pattern with a fairly well defined border between normal brain and tumour tissue. Thus, the organ co-culture assay system in vitro seems to mimic several aspects of the in situ invasive behaviour of medulloblastomas. It may, therefore, provide new perspectives for pretreatment investigations with chemotherapy and radiotherapy of these malignancies.

Proliferation, migration and invasion of human glioma cells exposed to antifolate drugs.

The present study describes the effects of 2 folate antagonists, methotrexate (MTX) and the lipophilic antifolate trimetrexate (TMX) on 2 permanent human glioma cell lines (GaMg and D-54Mg) grown as monolayers and as multicellular tumor spheroids. In addition, the effects of drug exposure on tumor cell invasion was studied using a three-dimensional organ co-culture system. In monolayer cultures, TMX was a more potent inhibitor of cell growth than MTX, especially towards the GaMg cell line. The 2 drugs, however, showed similar cytotoxicity as assessed by the plating efficiency assay. Reduced ability of directional migration of cells on a plastic surface was seen by either antifolate usually at concentrations to 10-fold higher than those exerting a cytotoxic effect in the plating efficiency assay. TMX was somewhat more potent than MTX as an inhibitor of spheroid growth. When tumor spheroids were exposed to MTX or TMX at concentrations that caused 65 to 70% inhibition of cell migration, there was a latent period of 4 to 5 days before inhibition of spheroid growth ensued. Invasion was investigated in a co-culture system, where tumor spheroids were confronted with fetal rat brain cell aggregates. Neither drug reduced tumor cell invasion, although histological examination revealed toxic effects both in GaMg and in D-54Mg spheroids. We conclude that spheroids from human glioma cells were less sensitive to the antifolates than monolayers. For both drugs a latency period was observed before inhibition of spheroid growth. The spheroids retained their ability to invade normal brain tissue when exposed to levels of folate antagonists inhibiting spheroid growth.

Induced hyperthermia in brain tissue: comparison between contact Nd:YAG laser system and automatically controlled high frequency current.

Concerning hyperthermia treatment, knowledge of time-temperature and of temperature distributions within tumour volumes is essential in order to obtain the maximal therapeutic effect. New techniques are developed to overcome these difficulties. Two different heat sources, the contact Nd:YAG laser system and the automatically controlled high frequency current are investigated. In a defined volume of 1 cm3, the laser system reaches 45 degrees C after 1.8 s exposure with 15 W output power. The high frequency current reaches 45 degrees C with 48 s exposition with 18.75 W output current. Both heat sources present an exponential decrease of the temperature profile depending on the distance and prove efficient for inducing anti-tumoural hyperthermia. The tissue heat clearance is compensated for by intermittent laser and high frequency current application.

A new system for cutting brain tissue preserving vessels: water jet cutting.

The water jet cutting system allows transaction and dissection of biological structures with little bleeding. Structures of higher tissue rigidity remain unchanged while softer tissues are mechanically dissected. In brain tissue, all vessels larger than 20 microns are left intact after the passage of the jet stream with a pressure of up to 5 bar, and therefore vessels can be isolated selectively from the surrounding tissue. Oedema is present adjacent to the cut and no increase of temperature occurs.