Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model

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Object. Although it is known that malignant astrocytomas infiltrate diffusely into regions of normal brain, it is frequently difficult to identify unequivocally the solitary, invading astrocytoma cell in histopathological preparations or experimental astrocytoma models. The authors describe an experimental system that facilitates the tracking of astrocytoma cells by using nonneoplastic cerebral tissue as the substrate for invasion.

Methods. Cerebral tissue was cut into 1-mm-thick slices and cultured in the upper chamber of a Transwell culture dish on top of a polyester membrane (0.4-mm pore size) that was bathed in medium supplied by the lower chamber. Two astrocytoma cell lines, U-87 MG (U87) and U343 MG-A (U343), were selected because of their differing basal cell motilities in monolayer cultures. The astrocytoma cells were stably transfected with vectors that expressed green fluorescent protein (GFP), either alone or as a fusion protein with the receptor for hyaluronic acid—mediated motility (RHAMM) in either sense or antisense orientations. Stably transfected clones that had high levels of GFP expression were selected using the direct visualization provided by fluorescence microscopy and fluorescence-activated cell-sorter analysis. The GFP-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was measured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing astrocytoma cells could be readily tracked and followed in this model system. Individual astrocytoma cells that exhibited green fluorescence could be readily identified following their migration through the brain slices. The GFP-labeled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma cells also infiltrated farther than the GFP-labeled astrocytoma cells themselves. The expression of antisense RHAMM virtually abrogated the invasion of the brain slices by both astrocytoma cell lines.

Conclusions. The authors believe that this organotypical culture system may be of considerable utility in studying the process of astrocytoma invasion, not only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but also because the GFP tag enables tracking of highly migratory and invasive astrocytoma cells under direct vision.

Astrocytomas are the most common primary human brain tumors. The majority of astrocytomas are histopathologically malignant lesions associated with a poor prognosis. Patients harboring the most malignant form of astrocytoma, GBM, face a median survival time of only 12 months despite having undergone surgery, cranial radiation treatment, and intensive chemotherapy. Malignant astrocytomas rarely metastasize systemically; rather, death results from inexorable local tumor growth and brain invasion.

It can be argued cogently that the infiltration of contiguous and distant regions of normal brain by astrocytoma cells from the primary tumor is a major cause of treatment failure in patients with this disease. To enhance our understanding of the molecular mechanisms underlying astrocytoma cell invasiveness, we previously performed a partial characterization of the ECM of the human brain,26,27,46,47,51 examined the contributions of matrix metalloproteinases and tissue inhibitors of metalloproteinases in astrocytoma invasion models in vitro,5,35,36,49 and studied astrocytoma adhesion to the ECM through an analysis of integrin and focal adhesion kinase expression.50 Work in this field has been hindered by two main factors: first, previously described and currently available in vitro invasion models do not take into consideration the unique representation of ECM macromolecules in the brain; and second, unequivocal identification of tagged and nonperturbed human astrocytoma cells within the selected model of brain tumor invasion has not been consistently achieved.

To overcome these difficulties, we have modified existing organotypical models of cultured rodent brain tissue to establish a reproducible and relevant model for human astrocytoma cell invasion. In addition, we have labeled human astrocytoma cells with GFP, which serves as a reporter molecule for monitoring astrocytoma cell invasion in our model. Finally, to determine whether this model is useful in measuring differential migratory rates among astrocytoma cells, we have cotransfected human astrocytoma cells with a cDNA for RHAMM17,18,56,57 as well as GFP. In this article we show that RHAMM-transfected human astrocytoma cells invade human brain tissue to a greater extent than control tumor cells, and that the degree of this invasiveness is both quantifiable and reproducible.

Materials and Methods

Astrocytoma Cell Lines, Culture Conditions, and Specimens of Tumor and Nonneoplastic Brain

The human malignant astrocytoma cell lines U87 and U343 were grown in monolayer cultures. These two cell lines were selected because of their differing basal motility rates in vitro. The U87 is a highly motile and invasive astrocytoma cell line both in vitro and in vivo,34 whereas the U343 cell line is not49 The cell lines were routinely maintained in α-MEM supplemented with 10% FCS, 100 U/ml penicillin, 100 (µg/ml streptomycin, and 0.25 (xg/ml amphotericin B. The astrocytoma cell lines were grown in a humidified environment containing 5% CO2 at 37°C.

Low- and high-grade human astrocytoma specimens (Grades II and IV according to the World Health Organization classification) were obtained during craniotomy. Specimens of human brain were also obtained during the course of routine craniotomies that were performed to treat arteriovenous malformation, trauma, or epilepsy in children and young adults aged 12 to 18 years. Permission to use these tissues was obtained from the Research Ethics Board of The Hospital for Sick Children. The brain tumor specimens were used in the immunohistochemical experiments described later in this paper. Nonneoplastic brain tissue was used in both immunohistochemical and brain slice experiments. Specimens obtained from surgical cases were either used immediately in fresh form or snap frozen and stored in liquid nitrogen for later use.

Astrocytoma Cell Line Transfections

Enhanced GFP vector was used to transfect U343 and U87 human astrocytoma cells and to label these cells with GFP. Approximately 1.6 × 107 astrocytoma cells were trypsinized in 0.25% trypsin, pelleted, and washed in 5 ml of ice-cold sterile Ca++/Mg++—free PBS before they were repelleted and resuspended in sterile Ca++/Mg++—free PBS. Astrocytoma cells were added to 25 µg of the aforementioned plasmids in a 0.45-;cm electrode-gap electroporation cuvette and incubated on ice for 15 minutes. The specific electroporation voltage and capacitance values required to produce 50% cell death were ascertained and applied.52 For efficient transfection, the U87 and U343 cells required 350 V and 250 mF, and the U343 cells required 300 V and 500 mF. Following electroporation, the cells were incubated on ice for 30 minutes before cells from a single-cell suspension were plated onto 100-mm2 tissue culture dishes. Forty-eight hours after electroporation, the medium was replaced with α-MEM containing 0.6 mg/ml G41. Cell colony formation was observed over time by using phase microscopy. Expression of GFP was detected by direct visualization, Western blot, and FACS analysis. For Western blot analysis, a GFP polyclonal antibody (diluted 1:2000) was used. The FACS analysis was performed on live cells. When individual fluorescent colonies had accumulated 50 to 100 cells (approximately 2 weeks postelectroporation), they were lifted separately from the tissue culture plates by using a P-200 micropipette tip under microscopic guidance. These colonies were then expanded as separate clones of cells in G418-containing media in new tissue culture dishes.

To determine the effects of RHAMM transfection on astrocytoma migration through the brain slice model, we obtained a cDNA probe for the entire coding sequence of the RHAMMv4.18 The RHAMM cDNA was ligated at cohesive ends into pEGFP-C1 in both sense and antisense orientations. Confirmation of sense and antisense orientations of the RHAMM cDNA was confirmed by restriction digest analysis. Transfections were performed as described earlier. Controls for the effects of RHAMM modulation in these cell clones were the transfections of the astrocytoma cell lines with the pEGFP vector by itself.

Proliferation of Transfected Astrocytoma Cell Clones and Controls

Parental U87 and U343 astrocytoma cell lines, RHAMM—GFP transfectants, and control cells were seeded at 5 × 105 cells in 60mm2 tissue culture dishes. Cells were harvested and counted daily for 10 days. Growth curves were generated for all astrocytoma clones. The doubling time for each clone was calculated from the slope of the logarithmic portion of the growth curve, as described previously.48

Immunohistochemical Localization of RHAMM in Astrocytomas

Paraffin-embedded sections of human brain and low- and high-grade astrocytomas were deparaffinized, bleached in hydrogen peroxide and methanol, and washed in distilled water. The sections were blocked with 0.5% bovine serum albumin and 0.5% normal goat serum in PBS and incubated with a rabbit polyclonal antibody specific to RHAMM (antibody AP-HV42.1, 0.64 mg/ml, diluted 1:100).18 Peroxidase-labeled secondary antibodies (goat/anti—rabbit/horseradish peroxidase, 1:200 dilution) were then used. The samples were treated with 3.3′diaminobenzidine and 30% hydrogen peroxide. Counterstaining was performed by using the Harris—hematoxylin technique. As a negative control, secondary antibodies were used without addition of primary antibodies.

Brain Slice Model

To establish this model, modifications were made to existing neural organotypical cultures in a manner previously described.26,54,55 For the purposes of this study, only frontal and temporal lobe specimens were used and examined. Specimens of human brain were cut by using sterile scalpel blades in a sterile environment. Gray matter was separated from white matter. Specimens of white matter were cut into 1-mm-thick 8 × 8—mm2 slices by using a brain slicing apparatus. The brain slices were placed in the upper chambers of 24-mm Transwell culture dishes (0.4-mm pore size) and incubated in medium55 containing α-MEM and 10% FCS supplemented with glutamine, insulin/transferrin/splenium-A (× 100), glucose, and 20 nM progesterone (Fig. 1). After allowing the brain slices to equilibrate in medium for 24 hours, a 2-mm hole was made in the center of the brain slice by using a micropipette tip. Following this, 5 × 105 RHAMM—GFP—transfected astrocytoma cells or control cells were placed in the central hole of the brain slice and incubated for different lengths of time. The culture medium was carefully changed every 3 days. After 7 days in culture, the brain slices were fixed in a 4% paraformaldehyde solution12,13,60 (0.1 M phosphate buffer, pH 7.2) and placed on specially adapted microscopy slides. Mounting medium was added to maintain the 3D structure of the brain slices. The samples were then imaged using scanning confocal laser microscopy. A maximum of 250 1-µm-thick optical sections were collected using a GFP barrier filter. The sections were then rendered into a pseudo-3D perspective by using a computer program. The distance from the point of implantation (the fissure in which the cells were implanted) to the point at which the individual GFP-labeled cells migrated was then calculated. Each cell's migration was placed in a distance category by measuring the distance that the cell had traversed and then rounding off the distance value to the closest distance category. The cell density (cells/mm3) for each category and each cell type was expressed as a mean and standard deviation. Significance was then determined using Student's t-test.12,13,60

Fig. 1.
Fig. 1.

Schematic representation of the brain slice model. Specimens of white matter measuring 1 × 8 ×8 mm3 are placed in a Transwell chamber on top of a porous membrane overlying a lower chamber filled with special medium. A central hole in the brain slice is created with a 2-mm sterile pipette tip. The GFP-transfected human astrocytoma cells are then placed in the hole and incubated for 7 days. The extent of astrocytoma cell migration can be measured by confocal laser fluorescence microscopic identification of the GFP-labeled cells.

In separate experiments, we determined the rate of migration of RHAMM-transfected GFP-tagged U87 astrocytoma cells through brain slice preparations derived from temporal and frontal lobe specimens at 3, 5, and 7 days postimplantation.

Preparation of Brain Slice Specimens for TEM

To study the ultrastructural features of astrocytoma invasion into the brain slices and to assess their cytoarchitectural integrity over time, slices of brain that had been cultured for varying time periods were fixed for 4 hours, washed with buffer, and postfixed using phosphate-buffered osmium tetroxide. Samples were dehydrated by treating them with a series of ascending concentrations of ethanol. The samples were embedded in Epon-Araldite by using propylene oxide. Ultrathin sections exhibiting a pale-gold interference color (indicating that there was sufficient brain tissue for TEM analysis) were mounted on grids and stained with uranyl acetate and lead citrate. These specimens were then examined and photographed using a transmission electron microscope.

Sources of Supplies and Equipment

The U-87 MG cells were obtained from the American Type Culture Collection (Rockville, MD) and the U343 MG-A cells from the Brain Tumor Research Center, University of California (San Francisco, CA). The α-MEM, FCS, penicillin, streptomycin, amphotericin B, trypsin, glutamin, and insulin-transferrin/splenium-A were purchased from Gibco BRL (Gaithersburg, MD). Clontech Laboratories, Inc. (Palo Alto, CA), provided the pEGFP-C1 and the GFP polyclonal antibody, and Bio-Rad (Richmond, CA) provided the electrode-gap electroporation cuvette. The tissue culture dishes were manufactured by Corning (Corning, NY) and the Transwell culture dishes by Corning Costar Corp. (Cambridge, MA). Sigma Chemical Co. (St. Louis, MO) provided the G418, bovine serum albumin, and progesterone. The P-200 micropipette tip was obtained from Starstedt (Montreal, PQ, Canada) and the normal goat serum and goat/anti—rabbit/horseradish peroxidase from Dako Corp. (Carpinteria, CA).

We acquired the brain-slicing apparatus from Harvard Instruments (Boston, MA), the scanning confocal laser microscope (model SLM) from Leica (Toronto, ON, Canada) and the Vaytek Voxblast computer program from the University of Iowa (Iowa City, IA). The transmission electron microscope (model 1200 EXII) was purchased from JEOL (Peabody, MA).

The cDNA probe for the coding sequence of the RHAMMv4 and the AP-HV42.1 antibody were generous gifts from Dr. Eva Turley18 (The Hospital for Sick Children).

Results
Isolation of Stable GFP-Transfected Astrocytoma Clones

Following transfections with RHAMM—GFP expression vectors, 60 clones were examined for expression of the RHAMM—GFP fusion protein. Ten clones of the U87 cell line and eight of the U343 cell line were found to express variable levels of RHAMM—GFP. Of these, one cell clone for each astrocytoma cell line that demonstrated stable and high-level expression of GFP according to the results of the FACS (Figs. 2) and Western blot (Fig. 3) analyses were selected for all additional experiments. Transfection of the human astrocytoma cell lines with RHAMM—GFP did not affect the proliferation of these clones, as determined by growth-curve analysis and calculation of doubling times (Fig. 4).

Fig. 2.
Fig. 2.

Results of FACS analysis of astrocytoma cells before and after transfection with pEGFP-C1. The parental cell lines U87 (87P) and U343 (343P) do not demonstrate significant fluorescence. The same cell lines transfected with pEGFP-C1 are clearly identified by their fluorescence peak. 87G3 = GFP-tagged U87 cells; 343G1 = GFP-tagged U343 cells.

Fig. 3.
Fig. 3.

Western blot comparing parental astrocytoma cell lines with RHAMM-GFP—transfected astrocytoma cell lines. The U87 and U343 astrocytoma cell lines transfected with pEGFP-C1 and RHAMM express an expected fusion protein at 99 kD.

Fig. 4.
Fig. 4.

Bar graphs demonstrating doubling times of astrocytoma cell lines. The doubling times of U-87 (upper) and U343 (lower) astrocytoma cell lines were 35 and 37 hours, respectively. These times were not significantly affected by transfection of cells with the RHAMM—GFP expression vector.

Expression of RHAMM by Human Astrocytoma Cells

Ten different specimens each of nonneoplastic brain, low-grade astrocytoma, and GBM were examined for RHAMM expression by using immunohistochemical analysis. Weak expression of RHAMM was found in nonneoplastic brain tissue and low-grade astrocytomas. In contrast, RHAMM was more strongly expressed in higher-grade astrocytic neoplasms such as GBM (Fig. 5).

Fig. 5.
Fig. 5.

Photomicrographs demonstrating immunohistochemical localization of RHAMM in human brain and astrocytomas. In contrast to the negative control (a) and a nonneoplastic brain specimen (b), which do not display any staining pattern for RHAMM, a low-grade astrocytoma (c) demonstrates minimal RHAMM immunostaining (brown staining) and a GBM displays moderate immunostaining for RHAMM (d). Original magnification × 250.

Analysis of GFP-Labeled Astrocytoma Invasion of Brain Slices

Hematoxylin and eosin staining of organotypical cultures of human brain slices demonstrated histological integrity 1 week after placement in culture. Results of previous studies performed using unlabeled astrocytoma cells demonstrated the ability of these cells to penetrate the brain slices;26 however, the unequivocal identification of the solitary invading astrocytoma cell was not possible. Using GFP-transfected astrocytoma cells, we were able to show that astrocytoma invasiveness is readily detected by confocal laser microscopy (Fig. 6). The RHAMM-transfected astrocytoma cells exhibited a spindle-shaped, bipolar structure compared with antisense RHAMM-transfected cells within the brain slice. Several RHAMM-transfected astrocytoma cells acquired long cell processes, which reached forward into the parenchyma of the brain slice. Antisense RHAMM-transfected U87 astrocytoma cells, in particular, displayed a round, globular phenotype within the brain slice (Fig. 6D).

Fig. 6.
Fig. 6.

Confocal laser photomicrographs obtained 7 days postimplantation, identifying GFP-expressing astrocytoma cells in brain-slice cultures. A: The RHAMM-GFP—transfected U343 astrocytoma cells migrate readily into the brain slice, with many cells adopting a bipolar phenotype. B: Antisense RHAMM—transfected U343 astrocytoma cells are effectively blocked from invading the brain slices. C: The RHAMM-GFP—transfected U87 astrocytoma cells have migrated away from the site of implantation as solitary invading cells. D: Antisense RHAMM-transfected U87 astrocytoma cells are confined to the site of implantation. Many cells have adopted a large, round, globular phenotype compared with the RHAMM transfectants seen in C. These cells have been prevented from invading the brain slice. Asterisks indicate sites of implantation of astrocytoma cells. Original magnification × 400.

The distance that the GFP-labeled astrocytoma cells migrated from the site of implantation in the fissure into the depths of the brain slice was measured. The control U87 astrocytoma cells migrated farther than the U343 astrocytoma cells (Fig. 7). The RHAMM-transfected U87 and U343 astrocytoma cells migrated farther into the brain slices than the controls. In contrast, antisense RHAMM-transfected astrocytoma cells displayed poor migration into the brain slice and were found predominantly within 1 mm of the site of implantation.

Fig. 7.
Fig. 7.

Bar graphs depicting the extent of invasion of brain slices by human astrocytoma cells, as shown using quantitative morphometric analysis at 7 days postimplantation The extent of migration by GFP-expressing astrocytoma cells was measured from the implantation site (fissure) into the substance of the brain slice in millimeters. The U343 astrocytoma cells migrated less readily into the brain slice than the U87 cells (mean 1.5 mm compared with 2.5 mm, p < 0.05). The pEGFP antisense RHAMM—transfected cells were virtually prevented from entering the brain slices. The RHAMM-transfected U87 and U343 astrocytoma cells were capable of traveling farther within the brain slice than the astrocytoma cell lines transfected with GFP alone.

At 3, 5, and 7 days, we observed that the RHAMMtransfected U87 astrocytoma cells had penetrated the brain slice preparations to mean distances (± standard deviations) of 0.5 ± 0.2 mm, 1.8 ± 0.4 mm, and 3 ± 0.5 mm, respectively. There was no significant difference in the rate of migration of RHAMM-transfected U87 astrocytoma cells in specimens derived from either temporal or frontal lobe locations (data not shown).

Brain Slice Specimens Shown to Have Cytoarchitectural Preservation by TEM

Examination of brain slice specimens containing implanted astrocytoma cells incubated for 3, 5, 7, and 14 days revealed astrocytoma cells with rich filamentous structures within intact neuropil containing readily identifiable myelinated axons (Fig. 8). However, at 30 days postimplantation the neuropil was found to be degenerating, and necrosis of astrocytes and neurons was observed (data not shown).

Fig. 8.
Fig. 8.

Low-power electron micrograph showing a U87 astrocytoma cell in the brain slice following examination by TEM at 7 days postimplantation. The cell nucleus (N) as well as filament-rich processes (asterisks) are easily discriminated. Relatively intact myelinated axons (arrowheads) are seen throughout the brain slice. Bar = 1 µm.

Discussion

We have shown that the invasive potential of GFPlabeled human astrocytoma cells can be assessed by using organotypical brain slices derived from nonneoplastic cerebral tissue as a barrier to astrocytoma invasion. The migratory behavior of infiltrating human astrocytoma cells was readily tracked by using confocal laser microscopy. Astrocytoma cells transfected with a RHAMMv4 cDNA were stimulated to migrate faster than nonRHAMM-transfected astrocytoma cells. Interestingly, antisense RHAMM-transfected astrocytoma cells showed little if any migratory potential when placed within the implantation site of the brain slice model. Together, these results suggest that our model may have utility as an in vitro system with which we can further our understanding of the process of astrocytoma invasion.

Several in vitro invasion assays have been developed that present various ECM macromolecules as barriers to tumor invasion, including natural tissues such as amnion and eye lens, or ECM gels such as type I collagen gels or the gelled basement membrane extract Matrigel.19,24,29,32,38,45 The Matrigel invasion assay has been the most widely used because it is quick, reliable, easy to quantitate, and available commercially.2,6,25 There has been a consistent correlation between the malignancy of a cell and its ability to infiltrate through Matrigel.2,6,25,38 Matrigel is a soluble basement membrane extract of the Engelbreth-Holm-Swarm tumor, which gels to form a reconstituted basement membrane, as shown by its composition, structure, and physical properties.28 The major components of Matrigel are laminin, collagen IV, entactin, and heparan sulfate proteoglycan.58 Although some of these ECM macromolecules are found within the defined basal laminae at the glial limitans externa or around cerebral blood vessels in the central nervous system, invasive astrocytoma cells do not frequently breach these structures. Instead, astrocytoma cells are characterized by their diffuse infiltration of brain parenchyma, especially within and along white matter tracts. As such, the Matrigel invasion assay does not provide an adequate modeling of the ECM normally encountered by infiltrating astrocytoma cells.

The limited applicability of Matrigel and other conventional membrane-based systems (in which single tumor cells invade a nonviable matrix) to the process of brain tumor invasion has spawned the development of a tumor cell—invasion model in which the interactions of both malignant and normal cells can be studied within a 3D framework. In this model, tumor cell fragments are placed side by side with normal tissue in a confrontational assay system.3,14,15 To make this model relevant to brain tumor invasion, Bjerkvig and colleagues7,8,16,41,42 used fetal rat brain cell aggregates as targets for brain tumor spheroids obtained from permanent glioma cell lines and primary human brain tumor biopsy specimens. In their model, brain tumor cell invasion was assessed after 72 to 120 hours of coculture. These investigators reported that spheroids obtained from biopsy specimens of primary intracranial tumors with different histological characteristics and malignancy grades demonstrated the same growth and invasiveness that these tumors displayed in vivo.16,41,42 Although such confrontation systems are advantageous in the study of tumor invasion, widespread acceptance has been limited for two main reasons. First, the fetal rat brain, which is used as a target for human brain tumor cells, has a distinctly different ECM than the adult rat brain or, for that matter, the human brain.46 Second, it has proven difficult to quantify the degree to which brain tumor cells invade normal brain. Some of these difficulties have been overcome by labeling human glioma cells with fluorescent vital dyes before placing them in the coculture confrontation system.33

Because the Matrigel and coculture-confrontation in vitro systems do not provide the normal cytoarchitectural pattern of differentiated neural tissue or the biochemical and electrophysiological functions of normal neurons, we sought to use brain organotypical cultures to study human astrocytoma invasion.26,37 Brain organotypical cultures have long played an important role in elucidating mechanisms related to fiber outgrowth, myelin formation, and the development of synaptic networks.22,23 The characteristic cytoarchitecture and distinct organization of brain tissue can be maintained in this system for several weeks to months.22,23,55 Ohnishi, et al.,37 used rat brain slices obtained from the hippocampus or cortical regions of 2-dayold rats and maintained the brain slices in culture at the interface between air and the culture medium. These cultures could be maintained with adequate tissue preservation for extended periods of observation, frequently longer than 1 month. The C6 rat glioma spheroids were labeled with a PKH2 fluorescent marker before placement on the brain slices. In addition, these authors used L1-expressing fibroblasts in coculture with the brain slice preparations to stimulate C6 glioma spheroid migration.37 In fact, stimulation with L1, a neural cell adhesion molecule, was absolutely required for marked C6 glioma migration.37 In contrast to the model described by Ohnishi, et al., we used human tissue derived from surgical specimens obtained in patients undergoing temporal lobectomies for seizure disorders. To simulate the clinical situation in which astrocytoma cells disperse most frequently along white matter tracts, we used white matter freshly dissected from these specimens as the barrier to human astrocytoma cell infiltration. Confocal laser microscopy was then used to track and identify astrocytoma cells within the brain tissue.

However, single-cell infiltration and micrometastasis have often been difficult to study in experimental tumor models and in histopathological preparations because of a lack of suitable and sensitive markers that can discriminate individual tumor cells from normal cell populations. Recently, vital dyes such as fast blue, FluoroGold, 1,1′dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, and 3,3′-diaoctadecyloxacarbocyanine perchlorate have been developed to track neural cell populations in complex tissues.4,20,33 Even though several of these vital dyes are regarded as nontoxic, it remains unclear how and to what extent these compounds affect cellular behavior. To overcome the deficiencies of vital dye markers, several reporter genes have been used to study the migration of human astrocytomas in vitro and in vivo.4,10,11,20,30,33,39,40 The bacterial Escherichia coli lacZ gene has been the most common reporter gene used in numerous transfection studies and gene therapy experiments.44 The LacZ gene can be stably transfected into most tumor cell lines by using both liposomes and viral vectors as vehicles for gene delivery.31 Problems with the use of bacterium-derived lacZ as a reporter gene arise from the fact that intracellular β-gal has antigenic properties when inserted into nonimmunogenic glioma cell lines, with the result that transfer of this gene into a highly tumorigenic cell line may reduce the cell's tumorigenicity.1,30 Furthermore, analysis of lacZ-transfected cells in tissues requires the sacrifice either of the animal or of a portion of the tissue, because fixation at a neutral pH is required to view expression of the blue product.

Another marker for gene expression that has become popular in recent times is GFP. This protein is naturally produced by the bioluminescent jellyfish Aequorea victoria in which calcium binds to the phosphoprotein aqueorin. The GFP is a 238—amino acid peptide that absorbs blue light and emits green light without the need for additional cofactors or substrates.9 This protein has been used to monitor gene expression and protein localization in living organisms, as well as to infect neurons in frog brains59 and to visualize cancer invasion and metastasis.12,13 In our study, transfection of human astrocytoma cells with a GFP expression vector enabled us to establish a heritable, stable volume marker for the unequivocal identification of invasive astrocytoma cells in our brain slice model. Both U87 and U343 astrocytoma cell lines penetrated the brain slice to different degrees. Following 5 days of culture, GFP-positive U87 astrocytoma cells were found at distances of 2.5 mm from the primary implantation site, whereas U343 astrocytoma cells were limited to distances of 1.5 mm. This difference in invasiveness between these two cell lines correlates well with their known tumorigenicity: U87 astrocytoma cells are highly tumorigenic in athymic mice, whereas U343 cells are not. Interestingly, migratory GFP-positive human astrocytoma cells were capable of changing their structure from that of round flat cells (as seen in vitro) to that of bipolar spindle-shaped cells within the brain slices. The GFP transfection of astrocytoma cells did not alter astrocytoma proliferation or growth rates.

To determine whether the migration of human astrocytoma cells through the brain slices could be stimulated, we transfected the cell lines with an RHAMMv4 cDNA. A well-described hyaluronic acid receptor, RHAMM has been shown to be critical for cell locomotion.56 We have shown by immunohistochemical analysis that RHAMM is minimally expressed by astrocytes in normal human brain. However, with increasing astrocytic anaplasia, RHAMM expression increases. Following RHAMMv4 transfection, we found that U343 and U87 human astrocytoma cells were capable of penetrating brain slices to a significantly greater degree than control cells, as measured by the detection of GFP-positive cells by using confocal laser microscopy. Interestingly, invasion was virtually abolished in our brain slice system by the use of antisense RHAMM transfections of the astrocytoma cell lines. These data indicate not only that RHAMM may be an important mediator of motility in the 3D context of our brain-slice model, but also that differences in degrees of invasion in a given cell line can be measured accurately and reproducibly by using our model.

Although our brain slice culture system has several limitations, such as abrogation of vascular supply to the tissue, finite lifespan of the tissue in vitro, and lack of directed immune responsiveness to the implanted tumor cells, we have observed that the cytoarchitecture of the brain tissue remains viable and intact for 14 days without exhibiting obvious histopathological signs of necrosis. As such, our human brain slice model has several distinct advantages over the conventional basement membrane—based assays and the confrontational assay system for the study of human astrocytoma invasion. Using the confocal laser microscopic images, we were able to observe invasion patterns in different axial planes and, finally, to create a 3D image demonstrating the relationship between tumor cells and normal brain. Tumor spread within the brain slice was quite obvious within the first several days of incubation.

Of course, the ideal model to study brain invasion would be homologous, immunocompetent, and in vivo. The superiority of animal models over purely in vitro experimentation consists in the fact that in vivo studies of chemosensitivity and anticancer drug pharmacology can take advantage of the growth of glioma cells as a 3D tumor within a living organism. However, human astrocytomas that are xenotransplanted into the brain of the athymic mouse grow predominantly by expansion rather than diffuse filtration, due to their high proliferative rates.43,53 Spontaneous brain tumors in animals may provide the cleanest, most natural model with which to work, but these tumors occur far too infrequently to allow consistent study, and the problem of identifying solitary infiltrating tumor cells remains.

Recently, Farina and associates21 described the tracking of GFP-labeled rat mammary adenocarcinoma cells orthotopically implanted into the mammary fat pad of live, immunocompetent, and homologous female rats. Movement of metastatic tumor cells in live rats was depicted by means of intravital imaging of the primary tumor in situ performed with the aid of a scanning confocal laser microscope. These investigators showed that only a small fraction of primary tumor cells in a tumor mass are actively motile; tumor cells become actively motile as they disseminate from the primary site. Although this model may be construed as the ultimate design for studying tumorcell motility and invasion, it is technically demanding and labor intensive, requiring anesthetization of the animal, surgical exposure of an accessible tumor, and intravital imaging over extended time periods. As such, its applicability to brain-tumor invasion, in which the tumors are not readily accessible and amenable to intravital imaging, will be limited.

Conclusions

We have described an organotypical human brain slice model to be used for studying human astrocytoma invasion. Using this model, GFP-labeled human astrocytoma cells can be tracked reliably and reproducibly. In this study, RHAMMv4-stimulated human astrocytoma cells migrated at faster rates through the brain slice than control tumor cells. We believe this model has merit compared with existing models as a means of studying patterns of human astrocytoma invasion. Studies are underway in which the unique features of this model are being implemented to determine whether inhibitors of invasion can be developed to help in the treatment of these surgically and medically recalcitrant tumors.

Acknowledgment

We thank Eva Turley for the RHAMMv4 cDNA.

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    Bjerkvig RTønnesen ALaerum ODet al: Multicellular tumor spheroids from human gliomas maintained in organ culture. J Neurosurg 72:4634751990J Neurosurg 72:

  • 9.

    Chalfie MTu YEuskirchen Get al: Green fluorescent protein as a marker for gene expression. Science 263:8028051994Science 263:

  • 10.

    Chicoine MRSilbergeld DL: Assessment of brain tumor cell motility in vivo and in vitro. J Neurosurg 82:6156221995in vivo and in vitro. J Neurosurg 82:

  • 11.

    Chicoine MRSilbergeld DL: Invading C6 glioma cells maintaining tumorigenicity. J Neurosurg 83:6656711995J Neurosurg 83:

  • 12.

    Chishima TMiyagi YWang Xet al: Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res 57:204220471997Cancer Res 57:

  • 13.

    Chishima TYang MMiyagi Yet al: Governing step of metastasis visualized in vitro. Proc Natl Acad Sci USA 94:11573115761997in vitro. Proc Natl Acad Sci USA 94:

  • 14.

    Easty DMEasty GC: Measurement of the ability of cells to infiltrate normal tissues in vitro. Br J Cancer 29:36491974Br J Cancer 29:

  • 15.

    Easty GCEasty DM: An organ culture system for the examination of tumour invasion. Nature 99:110411051963Nature 99:

  • 16.

    Engebraaten OBjerkvig RLund-Johansen Met al: Interaction between human brain tumour biopsies and fetal rat brain tissue in vitro. Acta Neuropathol 81:1301401990Acta Neuropathol 81:

  • 17.

    Entwistle JHall CLTurley EA: HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem 61:5695771996J Cell Biochem 61:

  • 18.

    Entwistle JZhang SYang Bet al: Characterization of the murine gene encoding the hyaluronan receptor RHAMM. Gene 163:2332381995Gene 163:

  • 19.

    Erkell LJSchirrmacher V: Quantitative in vitro assay for tumor cell invasion through extracellular matrix or into protein gels. Cancer Res 48:693369371988in vitro assay for tumor cell invasion through extracellular matrix or into protein gels. Cancer Res 48:

  • 20.

    Espinosa de los Monteros ABernard RTiller Bet al: Grafting of fast blue labeled glial cells into neonatal rat brain: differential survival and migration among cell types. Int J Dev Neurosci 11:6256391993Int J Dev Neurosci 11:

  • 21.

    Farina KLWyckoff JBRivera Jet al: Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. Cancer Res 58:252825321998Cancer Res 58:

  • 22.

    Gähwiler BH: Organotypic cultures of neural tissue. Trends Neurosci 11:4844891988Gähwiler BH: Organotypic cultures of neural tissue. Trends Neurosci 11:

  • 23.

    Gähwiler BH: Slice cultures of cerebellar, hippocampal, and hypothalamic tissue. Experimentia 40:2352431984Experimentia 40:

  • 24.

    Hendrix MJCSeftor EASeftor REBet al: Comparison of tumor cell invasion assays: human amnion versus reconstituted basement membrane barriers. Invasion Metastasis 9:2782971989Invasion Metastasis 9:

  • 25.

    Janiak MHashmi HRJanowska-Wieczorek A: Use of the Matrigel-based assay to measure the invasiveness of leukemic cells. Exp Hematol 22:5595651994Exp Hematol 22:

  • 26.

    Jung SHinek ATsugu Aet al: Astrocytoma cell interaction with elastin substrates: implications for astrocytoma invasive potential. Glia 25:1791891999Glia 25:

  • 27.

    Jung SRutka JTHinek A: Tropoelastin and elastin degradation products promote proliferation of human astrocytoma cell lines. J Neuropathol Exp Neurol 57:4394481998J Neuropathol Exp Neurol 57:

  • 28.

    Kleinman HKMcGarvey MLLiotta LAet al: Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:618861931982Biochemistry 21:

  • 29.

    Kramer RHBensch KGWong J: Invasion of reconstituted basement membrane matrix by metastatic human tumor cells. Cancer Res 46:198019891986Cancer Res 46:

  • 30.

    Lampson LALampson MADunne AD: Exploiting the lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZ gene product as a tumor antigen, for evaluation of antigenic modulation, and to facilitate image analysis of tumor growth in situ. Cancer Res 53:1761821993lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZin situ. Cancer Res 53:

  • 31.

    Lin WPretlow TPPretlow TG IIet al: Bacterial lacz gene as a highly sensitive marker to detect micrometastasis formation during tumor progression. Cancer Res 50:280828171990lacz gene as a highly sensitive marker to detect micrometastasis formation during tumor progression. Cancer Res 50:

  • 32.

    Liotta LALee WCMorakis DJ: A new method for preparing large surfaces of intact human basement membrane for tumor invasion studies. Cancer Lett 11:1411471980Cancer Lett 11:

  • 33.

    Marienhagen KPedersen PHTerzis AJAet al: Interactions between fetal rat brain cells and mature brain tissue in vivo and in vitro. Neuropathol Appl Neurobiol 20:1301431994in vivo and in vitro. Neuropathol Appl Neurobiol 20:

  • 34.

    Martuza RLMalick AMakert JMet al: Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science 252:8548561991Science 252:

  • 35.

    Matsuzawa KFukuyama KDirks PBet al: Expression of stromelysin 1 in human astrocytoma cell lines. J Neurooncol 30:1811881996J Neurooncol 30:

  • 36.

    Matsuzawa KFukuyama KHubbard SLet al: Transfection of an invasive human astrocytoma cell line with a TIMP-1 cDNA: modulation of astrocytoma invasive potential. J Neuropathol Exp Neurol 55:88961996J Neuropathol Exp Neurol 55:

  • 37.

    Ohnishi TMatsumura HIzumoto Set al: A novel model of glioma cell invasion using organotypic brain slice culture. Cancer Res 58:293529401998Cancer Res 58:

  • 38.

    Parish CRJackobsen KBCoombe DR: A basement membrane permeability assay which correlates with the metastatic potential of tumour cells. Int J Cancer 52:3783831992Int J Cancer 52:

  • 39.

    Pedersen PHEdvardsen KGarcia-Cabrera Iet al: Migratory patterns of lac-z transfected human glioma cells in the rat brain. Int J Cancer 62:7677711995lac-z transfected human glioma cells in the rat brain. Int J Cancer 62:

  • 40.

    Pedersen PHMarienhagen KMørk S: Migratory pattern of fetal rat brain cells and human glioma cells in the adult rat brain. Cancer Res 53:515851651993Cancer Res 53:

  • 41.

    Pedersen PHNess GOEngebraaten Oet al: Heterogeneous response to the growth factors (EGF, PDGF (bb), TGF-α, bFGF, IL-2) on glioma spheroid growth, migration and invasion. Int J Cancer 56:2552611994Int J Cancer 56:

  • 42.

    Pedersen PHRucklidge GJMørk SJet al: Leptomeningeal tissue: a barrier against brain tumor cell invasion. J Natl Cancer Inst 86:159315991994J Natl Cancer Inst 86:

  • 43.

    Pilkington GJDarling JLLantos PLet al: Tumorigenicity of cell lines(VMDk) derived from a spontaneous murine astrocytoma: histology, fine structure and immunocytochemistry of tumours. J Neurol Sci 71:1451641985J Neurol Sci 71:

  • 44.

    Ram ZCulver KWWalbridge Set al: In situ retroviral-mediated gene transfer for the treatment of brain tumors in rats. Cancer Res 53:83881993Cancer Res 53:

  • 45.

    Repesh LA: A new in vitro assay for quantitating tumor cell invasion. Invasion Metastasis 9:1922081989Repesh LA: A new in vitro assay for quantitating tumor cell invasion. Invasion Metastasis 9:

  • 46.

    Rutka JTApodaca GStern Ret al: The extracellular matrix of the central and peripheral nervous systems: structure and function. J Neurosurg 69:1551701988J Neurosurg 69:

  • 47.

    Rutka JTGiblin JRApodaca Get al: Inhibition of growth and induction of differentiation in a malignant human glioma cell line by normal leptomeningeal extracellular matrix proteins. Cancer Res 47:351535221987Cancer Res 47:

  • 48.

    Rutka JTGiblin JRDougherty DYet al: Establishment and characterization of five cell lines derived from human malignant gliomas. Acta Neuropathol 75:921031987Acta Neuropathol 75:

  • 49.

    Rutka JTMatsuzawa KHubbard SLet al: Expression of TIMP-1, TIMP-2, 72- and 92-kDa type IV collagenase transcripts in human astrocytoma cell lines: correlation with astrocytoma invasiveness. Int J Oncol 6:8778841995Int J Oncol 6:

  • 50.

    Rutka JTMuller MHubbard SLet al: Astrocytoma adhesion to extracellular matrix: functional significance of integrin and focal adhesion kinase expression. J Neuropathol Exp Neurol 58:1982091999J Neuropathol Exp Neurol 58:

  • 51.

    Rutka JTMyatt CAGiblin JRet al: Distribution of extracellular matrix proteins in primary human brain tumours: an immunohistochemical analysis. Can J Neurol Sci 14:25301987Can J Neurol Sci 14:

  • 52.

    Rutka JTSmith SL: Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity. Cancer Res 53:362436311993Cancer Res 53:

  • 53.

    Saggu HPilkington GJ: Immunocytochemical characterization of the A15 A5 transplantable brain tumour model in vivo. Neuropathol Appl Neurobiol 12:2913031986in vivo. Neuropathol Appl Neurobiol 12:

  • 54.

    Stoppini LBuchs PAMuller D: A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:1731821991J Neurosci Methods 37:

  • 55.

    Tanaka MTomita AYoshida Set al: Observation of the highly organized development of granule cells in rat cerebellar organotypic cultures. Brain Res 641:3193271994Brain Res 641:

  • 56.

    Turley EA: Hyaluronan and cell locomotion. Cancer Metastasis Rev 11:21301992Turley EA: Hyaluronan and cell locomotion. Cancer Metastasis Rev 11:

  • 57.

    Turley EABelch AJPoppema Set al: Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes. Blood 81:4464531993Blood 81:

  • 58.

    Vukicevic SKleinman HKLuyten FPet al: Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res 201:181992Exp Cell Res 201:

  • 59.

    Wu GYZou DJKoothan Tet al: Infection of frog neurons with vaccinia virus permits in vivo expression of foreign proteins. Neuron 14:6816841995Neuron 14:

  • 60.

    Zhuo LSun BZhang CLet al: Live astrocytes visualized by green fluorescent protein in transgenic mice. Dev Biol 187:36421997Dev Biol 187:

This work was supported by the Canadian Institutes of Health Research (CIHR), Brainchild, and the Research Institute, The Hospital for Sick Children. Dr. Rutka is a recipient of a Scientist Award from the CIHR. Stacey Ivanchuk is a recipient of a fellowship award from the National Cancer Institute of Canada. Dr. Jung was supported by a research grant from the Korean Research Foundation.

Article Information

Address reprint requests to: James T. Rutka, M.D., Ph.D., F.R.C.S.(C), Division of Neurosurgery, The Hospital for Sick Children, 555 University Avenue, Suite 1502, Toronto, Ontario M5G 1X8, Canada. email: rutka@sickkids.on.ca.

© AANS, except where prohibited by US copyright law."

Headings

Figures

  • View in gallery

    Schematic representation of the brain slice model. Specimens of white matter measuring 1 × 8 ×8 mm3 are placed in a Transwell chamber on top of a porous membrane overlying a lower chamber filled with special medium. A central hole in the brain slice is created with a 2-mm sterile pipette tip. The GFP-transfected human astrocytoma cells are then placed in the hole and incubated for 7 days. The extent of astrocytoma cell migration can be measured by confocal laser fluorescence microscopic identification of the GFP-labeled cells.

  • View in gallery

    Results of FACS analysis of astrocytoma cells before and after transfection with pEGFP-C1. The parental cell lines U87 (87P) and U343 (343P) do not demonstrate significant fluorescence. The same cell lines transfected with pEGFP-C1 are clearly identified by their fluorescence peak. 87G3 = GFP-tagged U87 cells; 343G1 = GFP-tagged U343 cells.

  • View in gallery

    Western blot comparing parental astrocytoma cell lines with RHAMM-GFP—transfected astrocytoma cell lines. The U87 and U343 astrocytoma cell lines transfected with pEGFP-C1 and RHAMM express an expected fusion protein at 99 kD.

  • View in gallery

    Bar graphs demonstrating doubling times of astrocytoma cell lines. The doubling times of U-87 (upper) and U343 (lower) astrocytoma cell lines were 35 and 37 hours, respectively. These times were not significantly affected by transfection of cells with the RHAMM—GFP expression vector.

  • View in gallery

    Photomicrographs demonstrating immunohistochemical localization of RHAMM in human brain and astrocytomas. In contrast to the negative control (a) and a nonneoplastic brain specimen (b), which do not display any staining pattern for RHAMM, a low-grade astrocytoma (c) demonstrates minimal RHAMM immunostaining (brown staining) and a GBM displays moderate immunostaining for RHAMM (d). Original magnification × 250.

  • View in gallery

    Confocal laser photomicrographs obtained 7 days postimplantation, identifying GFP-expressing astrocytoma cells in brain-slice cultures. A: The RHAMM-GFP—transfected U343 astrocytoma cells migrate readily into the brain slice, with many cells adopting a bipolar phenotype. B: Antisense RHAMM—transfected U343 astrocytoma cells are effectively blocked from invading the brain slices. C: The RHAMM-GFP—transfected U87 astrocytoma cells have migrated away from the site of implantation as solitary invading cells. D: Antisense RHAMM-transfected U87 astrocytoma cells are confined to the site of implantation. Many cells have adopted a large, round, globular phenotype compared with the RHAMM transfectants seen in C. These cells have been prevented from invading the brain slice. Asterisks indicate sites of implantation of astrocytoma cells. Original magnification × 400.

  • View in gallery

    Bar graphs depicting the extent of invasion of brain slices by human astrocytoma cells, as shown using quantitative morphometric analysis at 7 days postimplantation The extent of migration by GFP-expressing astrocytoma cells was measured from the implantation site (fissure) into the substance of the brain slice in millimeters. The U343 astrocytoma cells migrated less readily into the brain slice than the U87 cells (mean 1.5 mm compared with 2.5 mm, p < 0.05). The pEGFP antisense RHAMM—transfected cells were virtually prevented from entering the brain slices. The RHAMM-transfected U87 and U343 astrocytoma cells were capable of traveling farther within the brain slice than the astrocytoma cell lines transfected with GFP alone.

  • View in gallery

    Low-power electron micrograph showing a U87 astrocytoma cell in the brain slice following examination by TEM at 7 days postimplantation. The cell nucleus (N) as well as filament-rich processes (asterisks) are easily discriminated. Relatively intact myelinated axons (arrowheads) are seen throughout the brain slice. Bar = 1 µm.

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Bjerkvig RTønnesen ALaerum ODet al: Multicellular tumor spheroids from human gliomas maintained in organ culture. J Neurosurg 72:4634751990J Neurosurg 72:

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Chalfie MTu YEuskirchen Get al: Green fluorescent protein as a marker for gene expression. Science 263:8028051994Science 263:

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Chicoine MRSilbergeld DL: Assessment of brain tumor cell motility in vivo and in vitro. J Neurosurg 82:6156221995in vivo and in vitro. J Neurosurg 82:

11.

Chicoine MRSilbergeld DL: Invading C6 glioma cells maintaining tumorigenicity. J Neurosurg 83:6656711995J Neurosurg 83:

12.

Chishima TMiyagi YWang Xet al: Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res 57:204220471997Cancer Res 57:

13.

Chishima TYang MMiyagi Yet al: Governing step of metastasis visualized in vitro. Proc Natl Acad Sci USA 94:11573115761997in vitro. Proc Natl Acad Sci USA 94:

14.

Easty DMEasty GC: Measurement of the ability of cells to infiltrate normal tissues in vitro. Br J Cancer 29:36491974Br J Cancer 29:

15.

Easty GCEasty DM: An organ culture system for the examination of tumour invasion. Nature 99:110411051963Nature 99:

16.

Engebraaten OBjerkvig RLund-Johansen Met al: Interaction between human brain tumour biopsies and fetal rat brain tissue in vitro. Acta Neuropathol 81:1301401990Acta Neuropathol 81:

17.

Entwistle JHall CLTurley EA: HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem 61:5695771996J Cell Biochem 61:

18.

Entwistle JZhang SYang Bet al: Characterization of the murine gene encoding the hyaluronan receptor RHAMM. Gene 163:2332381995Gene 163:

19.

Erkell LJSchirrmacher V: Quantitative in vitro assay for tumor cell invasion through extracellular matrix or into protein gels. Cancer Res 48:693369371988in vitro assay for tumor cell invasion through extracellular matrix or into protein gels. Cancer Res 48:

20.

Espinosa de los Monteros ABernard RTiller Bet al: Grafting of fast blue labeled glial cells into neonatal rat brain: differential survival and migration among cell types. Int J Dev Neurosci 11:6256391993Int J Dev Neurosci 11:

21.

Farina KLWyckoff JBRivera Jet al: Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. Cancer Res 58:252825321998Cancer Res 58:

22.

Gähwiler BH: Organotypic cultures of neural tissue. Trends Neurosci 11:4844891988Gähwiler BH: Organotypic cultures of neural tissue. Trends Neurosci 11:

23.

Gähwiler BH: Slice cultures of cerebellar, hippocampal, and hypothalamic tissue. Experimentia 40:2352431984Experimentia 40:

24.

Hendrix MJCSeftor EASeftor REBet al: Comparison of tumor cell invasion assays: human amnion versus reconstituted basement membrane barriers. Invasion Metastasis 9:2782971989Invasion Metastasis 9:

25.

Janiak MHashmi HRJanowska-Wieczorek A: Use of the Matrigel-based assay to measure the invasiveness of leukemic cells. Exp Hematol 22:5595651994Exp Hematol 22:

26.

Jung SHinek ATsugu Aet al: Astrocytoma cell interaction with elastin substrates: implications for astrocytoma invasive potential. Glia 25:1791891999Glia 25:

27.

Jung SRutka JTHinek A: Tropoelastin and elastin degradation products promote proliferation of human astrocytoma cell lines. J Neuropathol Exp Neurol 57:4394481998J Neuropathol Exp Neurol 57:

28.

Kleinman HKMcGarvey MLLiotta LAet al: Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:618861931982Biochemistry 21:

29.

Kramer RHBensch KGWong J: Invasion of reconstituted basement membrane matrix by metastatic human tumor cells. Cancer Res 46:198019891986Cancer Res 46:

30.

Lampson LALampson MADunne AD: Exploiting the lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZ gene product as a tumor antigen, for evaluation of antigenic modulation, and to facilitate image analysis of tumor growth in situ. Cancer Res 53:1761821993lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZin situ. Cancer Res 53:

31.

Lin WPretlow TPPretlow TG IIet al: Bacterial lacz gene as a highly sensitive marker to detect micrometastasis formation during tumor progression. Cancer Res 50:280828171990lacz gene as a highly sensitive marker to detect micrometastasis formation during tumor progression. Cancer Res 50:

32.

Liotta LALee WCMorakis DJ: A new method for preparing large surfaces of intact human basement membrane for tumor invasion studies. Cancer Lett 11:1411471980Cancer Lett 11:

33.

Marienhagen KPedersen PHTerzis AJAet al: Interactions between fetal rat brain cells and mature brain tissue in vivo and in vitro. Neuropathol Appl Neurobiol 20:1301431994in vivo and in vitro. Neuropathol Appl Neurobiol 20:

34.

Martuza RLMalick AMakert JMet al: Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science 252:8548561991Science 252:

35.

Matsuzawa KFukuyama KDirks PBet al: Expression of stromelysin 1 in human astrocytoma cell lines. J Neurooncol 30:1811881996J Neurooncol 30:

36.

Matsuzawa KFukuyama KHubbard SLet al: Transfection of an invasive human astrocytoma cell line with a TIMP-1 cDNA: modulation of astrocytoma invasive potential. J Neuropathol Exp Neurol 55:88961996J Neuropathol Exp Neurol 55:

37.

Ohnishi TMatsumura HIzumoto Set al: A novel model of glioma cell invasion using organotypic brain slice culture. Cancer Res 58:293529401998Cancer Res 58:

38.

Parish CRJackobsen KBCoombe DR: A basement membrane permeability assay which correlates with the metastatic potential of tumour cells. Int J Cancer 52:3783831992Int J Cancer 52:

39.

Pedersen PHEdvardsen KGarcia-Cabrera Iet al: Migratory patterns of lac-z transfected human glioma cells in the rat brain. Int J Cancer 62:7677711995lac-z transfected human glioma cells in the rat brain. Int J Cancer 62:

40.

Pedersen PHMarienhagen KMørk S: Migratory pattern of fetal rat brain cells and human glioma cells in the adult rat brain. Cancer Res 53:515851651993Cancer Res 53:

41.

Pedersen PHNess GOEngebraaten Oet al: Heterogeneous response to the growth factors (EGF, PDGF (bb), TGF-α, bFGF, IL-2) on glioma spheroid growth, migration and invasion. Int J Cancer 56:2552611994Int J Cancer 56:

42.

Pedersen PHRucklidge GJMørk SJet al: Leptomeningeal tissue: a barrier against brain tumor cell invasion. J Natl Cancer Inst 86:159315991994J Natl Cancer Inst 86:

43.

Pilkington GJDarling JLLantos PLet al: Tumorigenicity of cell lines(VMDk) derived from a spontaneous murine astrocytoma: histology, fine structure and immunocytochemistry of tumours. J Neurol Sci 71:1451641985J Neurol Sci 71:

44.

Ram ZCulver KWWalbridge Set al: In situ retroviral-mediated gene transfer for the treatment of brain tumors in rats. Cancer Res 53:83881993Cancer Res 53:

45.

Repesh LA: A new in vitro assay for quantitating tumor cell invasion. Invasion Metastasis 9:1922081989Repesh LA: A new in vitro assay for quantitating tumor cell invasion. Invasion Metastasis 9:

46.

Rutka JTApodaca GStern Ret al: The extracellular matrix of the central and peripheral nervous systems: structure and function. J Neurosurg 69:1551701988J Neurosurg 69:

47.

Rutka JTGiblin JRApodaca Get al: Inhibition of growth and induction of differentiation in a malignant human glioma cell line by normal leptomeningeal extracellular matrix proteins. Cancer Res 47:351535221987Cancer Res 47:

48.

Rutka JTGiblin JRDougherty DYet al: Establishment and characterization of five cell lines derived from human malignant gliomas. Acta Neuropathol 75:921031987Acta Neuropathol 75:

49.

Rutka JTMatsuzawa KHubbard SLet al: Expression of TIMP-1, TIMP-2, 72- and 92-kDa type IV collagenase transcripts in human astrocytoma cell lines: correlation with astrocytoma invasiveness. Int J Oncol 6:8778841995Int J Oncol 6:

50.

Rutka JTMuller MHubbard SLet al: Astrocytoma adhesion to extracellular matrix: functional significance of integrin and focal adhesion kinase expression. J Neuropathol Exp Neurol 58:1982091999J Neuropathol Exp Neurol 58:

51.

Rutka JTMyatt CAGiblin JRet al: Distribution of extracellular matrix proteins in primary human brain tumours: an immunohistochemical analysis. Can J Neurol Sci 14:25301987Can J Neurol Sci 14:

52.

Rutka JTSmith SL: Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity. Cancer Res 53:362436311993Cancer Res 53:

53.

Saggu HPilkington GJ: Immunocytochemical characterization of the A15 A5 transplantable brain tumour model in vivo. Neuropathol Appl Neurobiol 12:2913031986in vivo. Neuropathol Appl Neurobiol 12:

54.

Stoppini LBuchs PAMuller D: A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:1731821991J Neurosci Methods 37:

55.

Tanaka MTomita AYoshida Set al: Observation of the highly organized development of granule cells in rat cerebellar organotypic cultures. Brain Res 641:3193271994Brain Res 641:

56.

Turley EA: Hyaluronan and cell locomotion. Cancer Metastasis Rev 11:21301992Turley EA: Hyaluronan and cell locomotion. Cancer Metastasis Rev 11:

57.

Turley EABelch AJPoppema Set al: Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes. Blood 81:4464531993Blood 81:

58.

Vukicevic SKleinman HKLuyten FPet al: Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res 201:181992Exp Cell Res 201:

59.

Wu GYZou DJKoothan Tet al: Infection of frog neurons with vaccinia virus permits in vivo expression of foreign proteins. Neuron 14:6816841995Neuron 14:

60.

Zhuo LSun BZhang CLet al: Live astrocytes visualized by green fluorescent protein in transgenic mice. Dev Biol 187:36421997Dev Biol 187:

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