Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells

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Abstract

Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited significant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with controls. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was significantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any post-chemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy.

Introduction

Tumor cell invasion is a complex multistep process involving tumor cell attachment to the extracellular matrix (ECM) followed by degradation of host barriers (ECM and basement membranes) by proteolysis and tumor colony formation at distant sites (Duffy, 1992). Several enzyme systems participate in the degradation of ECM and basement membrane (Liotta et al., 1991), among which is cathepsin B, a lysosomal cysteine proteinase (Lah and Kos, 1998). Increased or altered expression of cathepsin B occurs in many types of tumors, including those of the breast (Foekens et al., 1998; Maguire et al., 1998), colon (Hirai et al., 1999; Iacobuzio-Donahue et al., 1997), prostate (Sinha et al., 1998) and lung (Werle et al., 1999), suggesting that cathepsin B may be involved in tumor invasiveness and metastasis. It has also been demonstrated that cathepsin B not only degrades components of the ECM such as laminin, collagen, and fibronectin and structures of basement membranes (Buck et al., 1992), but also activates other proteolytic enzyme systems (Kobayshi et al., 1993; Schmitt et al., 1992). A recent study has shown that inhibition of extracellular cathepsin B inhibits the growth and metastasis of rat colon cancer cells (Van Noorden et al., 1998), providing further evidence for the involvement of cathepsin B in tumor invasion. Cells transfected with antisense cathepsin B cDNA exhibited a decrease in cathepsin B activity, which resulted in a reduced invasion and motility of treated cells (Krueger et al., 1999).

Our interest in gliomas, the most common form of brain tumors, leads us to consider cathepsin B's role in glioma invasiveness. The mechanisms underlying brain tumor cell invasion are not well understood, but it is known that malignant progression of human gliomas is associated with an increase in cysteine proteases (McCormick, 1993; Sivaparvathi et al., 1996). We earlier reported high levels of cathepsin B in tumor and endothelial cells of brain tumor tissues (Sivaparvathi et al., 1995). Strojnik et al. (1999) demonstrated that cathepsin B is localized in tumor cells, macrophages, and endothelial cells of primary tumors of the CNS and its level of expression is a strong prognostic marker for primary tumors of the CNS. Changes in cathepsin B expression and subcellular localization correlated with increased grade, local invasion, and clinical evidence of invasion (Demchik et al., 1999; Rempel et al., 1994), thereby implicating cathepsin B in both glioma progression and invasion. Immunostaining for cathepsin in neovessels has been observed in human gliomas (Mikkelsen et al., 1995) and in rat brain microvascular endothelial cells grown in culture (Keppler et al., 1996), demonstrating the involvement of cathepsin B in brain tumor invasion and angiogenesis. We sought to determine whether down-regulation of cathepsin B expression would affect glioma tumor invasion and tumor growth. We transfected a malignant glioblastoma cell line SNB19 with an expression vector containing cathepsin B cDNA in antisense orientation. This report describes the effects of cathepsin B antisense transfection on glioblastoma invasiveness and tumor formation in in vitro and in vivo models.

Results

Transfection of SNB19 cells with expression constructs

SNB19 cells were transfected with eukaryotic expression vector pH-β-Apr-neo-1 containing the antisense cathepsin B cDNA construct. Control cells were transfected with plasmid vector alone. The recipient cells were isolated by means of their ability to grow in the presence of G418. Clones with a cathepsin B antisense cDNA integrated construct or an empty vector were selected and analysed for the expression of cathepsin B and invasive capabilities in in vitro and in vivo assays.

Northern blot analysis of cathepsin B mRNA levels in transfected glioblastoma clones

To determine the effect of antisense cathepsin B expression on the cathepsin B mRNA level of transfected cells, we analysed G418-resistant clones by Northern blot analysis and compared them with RNA from the parental cell line. Transfection of the vector alone did not alter the levels of cathepsin B mRNA when compared to those of parental cells (Figure 1A). All clones of cell lines transfected with antisense vector showed substantially reduced cathepsin B mRNA (Figure 1a). Cathepsin B hybridization signals were quantitated after normalization with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal by densitometric scanning of the autoradiograms (Figure 1B). Cathepsin B mRNA levels in antisense clones were approximately 10–12-fold decreased (P<0.001) that in the parental cell line and vector clones.

Figure 1
figure1

(A) Northern blot analysis of cathepsin B mRNA in SNB19 and transfected cells. Total RNA was isolated, and 10 μg of RNA was electrophoresed on 1% agarose gels and blotted onto a nylon membrane. The membrane was hybridized with 32P-labeled cathepsin B cDNA. After removal of the radiolabeled probe, the membrane was rehybridized with GAPDH cDNA probe to check the relative amounts of mRNA loaded on to the gel. (B) Levels of Cathepsin B mRNA quantitated by scanning autoradiogram with laser densitometry. Data were shown as mean±s.d. of three different experiments (*P<0.001)

Cathepsin B enzyme activity assay

To determine the effect of antisense cathepsin B cDNA transfection on the cathepsin B enzyme activity, we assayed enzyme activity in the cell lysates of parental, vector and antisense transfected cells using specific substrate. The enzyme activities were found reduced significantly 3–4-fold (P<0.001) in the antisense transfected clones as compared with parental and vector controls (Figure 2).

Figure 2
figure2

Relative cellular cathepsin B activity in parental and transfected human glioblastoma SNB19 cells using fluorogenic cathepsin B-specific substrate as described in Materials and methods. Compared to the parental cell line and vector control, cathepsin B activity was significantly reduced in antisense cDNA transfected clones. Values are mean±s.d. of four different estimations. RFU, relative fluorescence unit (*P<0.001)

Western blot analysis

The antisense cDNA construct of cathepsin B used in glioblastoma cells was designed to suppress the production of cathepsin B protein. The cell lysates of the transfected cells and parental cells were subjected to SDS–PAGE and analysed by Western blotting for expression of cathepsin B protein with a polyclonal antibody. Cell lysates contained a prominent band with few minor bands that cross-reacted with anti-human cathepsin antibodies (Figure 3A). Besides the significantly reduced level of cathepsin B mRNA in antisense-transfected cells, the cathepsin B antigen level was strongly affected. Western blot analysis revealed a considerable decrease in the cathepsin B protein levels in antisense-transfected cell lines compared to parental and vector controls (Figure 3A). Quantitative cathepsin B protein levels were determined after normalization with the β-actin levels by densitometric scanning of the autoradiograms (Figure 3B). Cathepsin B protein levels were approximately 8–10-fold lower in antisense clones (P<0.001) than in parental and vector clones.

Figure 3
figure3

(A) Immunoblot analysis of cathepsin B expression in parental and transfected human glioblastoma SNB19 cells using anti-human cathepsin polyclonal antibody or actin antibody as described in Materials and methods. (B) Quantitative analysis of Cathepsin B protein in the cell extracts by scanning densitometry. The data is shown as mean values ±s.d. three different analysis (*P<0.001)

Invasive potential among transfected clones

Antisense transfectant clones were compared to parental SNB19 cells and vector transfectants to determine the effect of antisense cathepsin B transfection on in vitro invasion in a Matrigel invasion assay. Staining of transwell inserts of antisense stable transfectants of cathepsin B clones that were invaded through the Matrigel was significantly less compared to parental and vector controls (Figure 4A). We found no marked difference in invasion between parental (42%) and vector (40%) transfected clones. However, a significant reduction (P<0.001) in invasive potential was noted with antisense transfected clones (15%, Figure 4B).

Figure 4
figure4

Invasion of human glioblastoma SNB19 cells and vector and antisense transfected cells. A suspension of cells was layered on Matrigel-coated transwell clusters and the percentage of invasion was calculated as described in Materials and methods. Cells that invaded through Matrigel-coated transwell inserts were stained, photographed (A, ×100) and the percentage was quantified. Values are mean±s.d. of five determinations, (B) (P<0.001)

Inhibition of SNB19 spheroid invasion into rat-brain aggregates

An in vitro co-culture model in which fetal rat brain cell aggregates were confronted with spheroids of parental, vector, and antisense cathepsin B-transfected SNB19 cells was used to study invasion. Tumor spheroids and fetal rat brain aggregates had been stained with the fluorescent dyes 1,1′ dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) respectively, prior to the coculture. In the coculture assays, spheroids and rat-brain aggregates initially attached each other, and tumor spheroids progressively invaded rat brain aggregates so that only a fraction of the original rat-brain aggregate remained. Cocultures in which fetal rat brain cell aggregates were confronted with spheroids from cathepsin B antisense transfectants failed to attach to one another and remained as two separate spherical groups of cells showing minimal invasion, while the confrontations performed with spheroids of parental or vector alone transfectants showed a normal pattern of invasion, with merging of spheroids and aggregate and occurrence of invasion (Figure 5A). Quantitative analysis of the remaining fetal brain aggregates were 8–12% in parental and vector transfectants compared with 94–96% in antisense clones (Figure 5B). The invasiveness of fetal rat brain aggregates was significantly decreased in antisense clones compared with parental and vector transfected clones (P<0.001).

Figure 5
figure5

(A) Confocal laser scanning images of SNB19 spheroids and rat-brain aggregate co-cultures scanned under laser scanning microscope at a depth of 100 μM from the surface. The images (×40) represented are the rat-brain aggregates (green fluorescence) and either parental or vector- or antisense-transfected tumor cell spheroids (red fluorescence). (B) Quantitation of remaining fetal rat brain aggregates by tumor spheroids as described in Materials and methods. Data shown are the mean±s.d. values from four separate experiments for each group (P<0.001). (C) and (D) Tumor formation by SNB19 and antisense-transfected cells in nude mouse brain. Photomicrograph of tumor section (×200) stained with hematoxylin and eosin (C) or tumor sections from GFP expressing cells (D) demonstrating a decrease in tumor formation in tissues derived from cathepsin B antisense-transfected cells injected into the mouse brain compared to control SNB19 glioblastoma cells. Ten mice each were injected with cells from AS-1 and AS-2 and five mice each were injected with cells from parental, vector-1 and GFP expressing clones. (E) Semiquantitation of tumor volume in parental, vector and antisense clones AS-1 and AS-2 4 weeks after intracranial injection of these cells as described in Materials and methods. Data shown are the ±s.d. values from five animals parental and vector clone and 10 animals from antisense clones (P<0.001)

Intracranial implantation

To examine the importance of reduction of cathepsin B, cells were injected intracerebrally into athymic mice. A total of thirty animals were used, of which five were injected with parental, five with vector transfected cells (SNB19V1), and 10 each with antisense transfected cells (SNB19AS1 and SNB19AS2). Each mouse injected with parental or vector alone transfected cells developed tumors (Figure 5C). In contrast, mice that were injected with antisense cells showed minimal tumor formation at 4 weeks postinjection and a decrease in tumor size (Figure 5C). To visualize tumor cells in vivo without histological treatment of tissue, we injected parental (SNB19) and cathepsin B antisense clones (SNB19AS1) expressing green fluorescent protein (GFP) in nude mice brain. A marked reduction in tumor formation by cathepsin B antisense clones was observed (Figure 5D) demonstrating a reduction in invasiveness and tumor formation by antisense cathepsin B in glioblastoma cells. Quantitation of the fluorescence of GFP revealed that more than 90% reduction in tumor formation by cathepsin B antisense transfectant compared to parental cells (SNB19GFP=100±8.4; SNB19AS1/GFP=9.8±1.2; per cent values as mean±s.d. of five readings). The sections analysed using H & E staining revealed that there was no significant difference in tumor size in mice that received an injection of parental and vector transfected clone. However, the tumor size was significantly reduced (P<0.001) in antisense clones, AS-1 and AS-2 compared with parental and vector clones (Figure 5E).

Discussion

In this study we stably transfected the glioblastoma cell line SNB19 with an expression vector containing cathepsin B cDNA in the antisense orientation. Successful transfection led to decreased cathepsin B mRNA transcript and protein levels, with the potential of profound biological consequences. In parental and vector transfected clones there were no changes in cathepsin B enzyme activity, protein and mRNA levels, suggesting that antisense construct is responsible for the observed decrease in transcripts and protein. The mechanism by which antisense RNA affects the expression of cathepsin B in antisense-transfected cells is unclear, although it may be related to interference with mRNA transport or hybridization with cytoplasmic cathepsin B mRNA, which would presumably interfere with translation.

Tumor cell invasiveness involves cell attachment, proteolysis of extracellular matrix components, and migration of cells through the disrupted matrix (Aznavoorian et al., 1993). It is hard to sufficiently explain with a single mechanism the biological processes of invasion, since the degradation of the ECM in the vicinity of a tumor by tumor cells is thought to be among the most important of the initial steps (Schmitt et al., 1992). A well-defined basal lamina covers the glial externa and is composed of ECM macromolecules type IV collagen, laminin, and fibronectin (Goldbrunner et al., 1998). Among the cysteine proteinases, cathepsin B expression has been particularly well documented in a variety of tumor cell types (Friedrich et al., 1999; Heidtmann et al., 1997) including brain tumors (Demchik et al., 1999; Rempel et al., 1994). It has been suggested that cathepsin B can mediate dissemination of cancer cells by degrading ECM (Spiess et al., 1994) and/or activating other proteinases capable of degrading the matrix (Kobayashi et al., 1993). Buck et al. (1992) have shown that cathepsin B can degrade the major components of basement membranes, i.e. type IV collagen, laminin, and fibronectin. Studies by Kobayashi et al. (1992) have established that cathepsin B can activate receptor-bound uPA in ovarian tumor cells, a proteolytic cascade responsible for increased invasion through Matrigel. Malignant brain tumors were found that had an increase of several fold in cathepsin B enzyme activity and an abundance of transcripts (Rempel et al., 1994; Sivaparvathi et al., 1995) compared with levels seen in normal brain, suggesting that cathepsin B contributes to the malignant invasive phenotype. Alterations in subcellular localization and the expression of cathepsin B at the invading edges are suggestive of a role for this enzyme in local glioma invasion (Mikkelsen et al., 1995). Therefore the high degree of diffuse local invasion of normal nervous tissue observed in human brain tumors could be due, in part, to the increased levels of cathepsin B expression. If the abundant expression of cathepsin B is responsible for the aggressive invasive behavior of gliomas, then down-regulation of cathepsin B could reduce cathepsin B-mediated invasiveness in glioblastomas.

Strategies such as expression of antisense RNA enables cell surface events to be bypassed such that the antisense sequence can directly influence the expression of a given gene of interest. Expression of DNA constructs resulting in antisense RNA provides a direct and unambiguous experimental approach for studying the involvement of cathepsin B in brain tumor progression. It prompted us to use a strong constitutive promoter to drive the expression of cathepsin B in long-term transfection experiments, and we selected the β-actin promoter since it has proven useful in promoting expression of antisense RNA in other cell systems (Gunning et al., 1987).

Antisense-transfected clones showed a marked reduction in in vitro invasiveness when compared with controls, suggesting that the reduction of cathepsin B levels in SNB19 cells altered their invasive potential in this experimental model system. The findings obtained in the in vitro invasion assays are in accordance with the results obtained by the selective cysteine protease inhibitors in murine squamous carcinoma cells (Coulibaly et al., 1999) and glioblastoma cells (Demchik et al., 1999), demonstrating that cathepsin B can mediate the degradation of ECM components. Kolkhorst et al. (1998) found that invasiveness of tumor cell lines expressing high levels of cathepsin B are efficiently inhibited by CA074, a selective cathepsin B inhibitor. In a recent study, cathepsin B antisense-transfected human osteosarcoma cells showed a markedly lower invasiveness and motility in Matrigel invasion assays than did controls (Krueger et al., 1999). To further confirm the effect of antisense cathepsin B cDNA on invasion, we utilized a three-dimensional coculture model that closely mimics in vivo invasion into a complex and intact ECM synthesized by fetal rat brain aggregates by confocal microscopy (Nygaard et al., 1995). This model gives the added advantage of observing tumor-cell invasion at different depths by optical sectioning. As seen in the Matrigel assays, SNB19 spheroids from cathepsin B antisense construct transfected clones failed to invade fetal rat brain aggregates significantly. The results obtained with both systems present functional evidence of reduced invasiveness of high-grade glioma cells by cathepsin B antisense cDNA transfection.

Since glioblastomas express high cathepsin B protein as well as transcripts, and since abundant expression of cathepsin B correlates with tumor progression, we reasoned that the efficient reduction of cathepsin B would be a therapeutically feasible approach to inhibiting the invasiveness and possibly the growth of such tumors in nude mice. Indeed cells expressing antisense cathepsin B construct exhibited reduced tumorigenic capacity after injection into nude mice when compared to cells transfected with a control plasmid, which had typical invasive behavior and exhibited progressive tumor growth. Cysteine proteinase inhibitors effectively prevent metastasis of tumor cell lines in animal models. For example, pretreatment of H-59 Lewis lung carcinoma cells with E-64, a cysteine-proteinase inhibitor, significantly inhibited and in a dose-dependent manner the ability of these cells to colonize the liver of C57BL6 mice (Navab et al., 1997), and overexpression of the cysteine proteinase inhibitor cystatin in B16 melanoma cells inhibited its capacity to metastasize to the lungs in C57BL6 mice (Cox et al., 1999). These observations consolidate the notion that tumor cells overexpressing cathepsin B are more invasive in normal brain, suggesting that cathepsin B plays a major role in invasion and growth of glioblastoma. The mechanism by which the antisense suppression of cathepsin B expression decreased the invasive potential of SNB19 glioblastoma cells in our study is not yet clear because the reduction in protein levels could reduce tumor growth by multiple mechanisms. We are currently examining our antisense cathepsin B clones to see if their decreased invasiveness relates to reduction in cathepsin B-mediated activation of plasminogen activators, the proteolytic cascade involved in the degradation of extracellular matrix components. A recent study suggested that besides directly participating in tissue destruction, cathepsin B enhances the activity of matrix metalloproteinases by degrading their inhibitors tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 in human articular chondrocytes (Kostoulas et al., 1999). However, whether similar mechanisms are operative in the glioblastoma cells we used in our study is not known. Our findings do demonstrate that a reduction in cathepsin B activity in glioblastoma cells suppresses tumor growth in vivo. The antisense cathepsin B strategy offers a new avenue of adjuvant treatment for gliomas.

Materials and methods

Cell culture

Glioblastoma cells SNB19 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS at 37°C in a humidified CO2 incubator and subcultured every 3–4 days.

Plasmid construction

To express antisense cathepsin B in SNB19 cells, the plasmid pLC343 (kindly provided by Dr B Slone, Wayne State University, Detroit, MI, USA) containing human Cathepsin B cDNA was cut with EcoRI and the 1.15 kb cDNA fragment coding for cathepsin B (Cao et al., 1994) was blunt end cloned in the antisense orientation into the HindIII site of the eukaryotic expression vector pH-β-Apr-neo-1 (Gunning et al., 1987), which contains the neomycin selectable marker gene and the human β-actin promoter driving the transcription of the inserted cDNA. This construct and the parental plasmid pH-β-Apr-neo-1 were used in the transfection of SNB19 cells.

Transfection

Cells were seeded in 35 mm culture dishes grown overnight, transfected with 5 μg DNA using Lipofectin (Mohanam et al., 1997) and then cultured for 48 h in complete medium before transfer to petridishes containing complete medium and 800 μg/ml G418. After 3–4 weeks in culture, G418-resistant colonies were isolated by means of cloning cylinders and characterized for the reduction in production of cathepsin B protein. Cells transfected with the expression vector alone served as control. To identify individual tumor cells in vivo and to establish a way to monitor tumor cell migration during tumor formation, we transfected parental SNB 19 cells and cathepsin B antisense stable transfectants (SNB19AS1) with the expression vector pEGFP-N1 (Clontech Laboratories, Palo Alto, CA, USA). To obtain cells with high-level GFP expression, cells were plated in limiting dilution and bright green fluorescent colonies were visually selected, lifted, expanded and subcloned by serial dilution to ensure purity. Cells were checked for G418 resistance and for reduction in cathepsin B protein.

Northern blotting analysis

Total cellular RNA was extracted from confluent cultures as described earlier (Mohanam et al., 1997). Aliquots of 10 μg of RNA was separated by electrophoresis on 1% agarose formaldehyde gels, capillary transferred to a nylon membrane overnight and the filters were hybridized at 65°C with 32P-radiolabelled cathepsin B cDNA probe (Sivaparvathi et al., 1995). Following hybridization with cathepsin B, blots were stripped and rehybridized with GAPDH to check loading equalities in transfer errors and the results were normalized to GAPDH RNA levels.

Cathepsin B enzyme activity assay

Cells were washed, lysed in assay buffer (50 mM acetate buffer (pH 5.5) 2.5 mM DTT, 2.5 mM EDTA) with 1% Triton X-100, centrifuged and supernatants were collected. Protein assay was done using Bio-rad protein assay reagent (Bio-rad, Hercules, CA,USA). The cathepsin B activity in cell lysates was assayed in 96-well microtiter plates using the specific substrate Z-Arg-Arg-AMC (Bachem, King of Prussia, PA, USA) and fluorescence was measured in a Fluoroskan Ascent Fluorimeter (Labsystems, Franklin, MA, USA). Control assays were carried out using 10 μM of cysteine proteinase inhibitor E-64. The assays were done in quadruplicate.

Immunoblotting analysis

Confluent cell cultures were washed twice with phosphate- buffered saline and harvested by scraping. The cells were collected by centrifugation, homogenized in RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM EDTA, and 50 mM Tris Ph 7.4) with protease inhibitors and centrifuged at 18 000×g for 15 min. The supernatant was boiled in SDS sample buffer under reducing conditions and applied on a 12.5% polyacrylamide gel prepared as described earlier (Mohanam et al., 1997). Separated proteins were electroblotted onto a nitrocellulose membrane, and nonspecific binding was blocked by incubation with 5% milk powder in washing buffer. After being washed, the membrane was incubated with rabbit antibodies against human cathepsin B (Athens Research and Technology Inc., Athens, GA, USA) diluted 1 : 5000 in washing buffer, and protein was localized by enhanced chemiluminescence-Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For loading control, membranes were probed with antibodies for actin.

Matrigel invasion assay

Invasion of glioma cells and stable transfectants in vitro was measured by the invasion of cells through Matrigel-coated transwell inserts (Becton Dickinson, Bedford, MA, USA). Briefly, transwell inserts with 8 μm pore size were coated with a final concentration of 1 mg/ml of Matrigel in cold serum-free DMEM. Cells were trypsinized, and 200 μl of cell suspension (3×105 cells per ml) were added in triplicate wells. After 24 h incubation, the cells that passed through the filter into lower wells were quantitated as described earlier (Mohanam et al., 1997) and expressed as a percentage of the sum of the cells in the upper and lower wells. Cells on the lower side of the membrane were fixed, stained with Heme-3 and photographed.

Glioma spheroids

Multicellular glioma spheroids were cultured in 25 cm culture flasks, base coated with 0.75% Noble agar prepared in DMEM. Briefly, 3×106 cells (parental, vector, and antisense transfectants) were suspended in 10 ml of medium, seeded onto 0.75% agar-coated plates and cultured until a spheroid formed. Spheroids about 200 μm in diameter were selected for the experiments. Tumor spheroids were stained with the fluorescent dye Dil and confronted with a fetal rat brain aggregates that were stained with DiO (Pedersen et al., 1993).

Fetal rat brain aggregates

Fetal rat brain aggregates were obtained from 18-day-old fetuses of Sprague-Dawley rats. The brains were aseptically removed, minced, and dissociated by serial trypsinization. A single-cell suspension was obtained and plated into agar-coated multiwell plates. After 48 h, aggregates were transferred into new plates and cultured for 16 days. Aggregates 100–200 μm in diameter were used in further experiments.

Confocal laser scanning microscopy

Invasion of the tumor spheroids into fetal rat-brain aggregates was analysed by confocal laser scanning microscopy (Nygaard et al., 1995). Briefly Dil-stained tumor spheroids and DiO-stained fetal rat-brain aggregates were washed in the medium and transferred in quadruplicate to individual wells of a 96 well plate, base coated with agar. Using a sterile syringe and stereomicroscope, we placed tumor spheroids and fetal rat-brain aggregates in close contact to each other. At different time intervals, serial 1 μm thick optical sections were obtained from the surface to the center of the co-cultures by a confocal laser-scanning microscope. Detection of Dil and DiO fluorescence was done by using an argon laser at 488 nm with a band-pass filter 520–560 nm and a helium/neon laser at 543 nm with a long-pass filter of 590 nm, respectively. The remaining volume of the brain aggregate or tumor spheroid during cocultures at 24, 48 and 72 h was quantitated using image analysis software.

Intracranial implantation

Mice were anesthetized with an i.p.injection of a 0.35–0.45 ml solution consisting of 0.06 M 2,2,2-tribromoethanol (Aldrich Chemical, Milwaukee, WI, USA) 1.25% isoamyl alcohol, and 98.5% bacteristatic saline. A midline scalp incision was made, and a burr hole located 2.5 mm lateral to the sagittal suture and at the coronal suture was made using an electric drill with a 1.5 mm engraving cutter (Dremel MotoTool, Racine, WI, USA). A cell suspension of 10 μl containing 2×106 parental and transfected SNB19 cells in serum-free DMEM was injected using a Hamilton syringe with a cone-tipped 0.7 mm needle attached to a stereiotactic frame (David Kopf, Tujunga, CA, USA) at a depth of 3 mm. After the cells were injected, the needle was left intracerebrally for 10 min to minimize the outflow of the solution. The needle was then withdrawn, the burr hole sealed with sterile bone wax, and the scalp closed (Go et al., 1996).

Tissue preparation and histochemical staining

The mice were anesthetized and killed at 4 weeks after injection by intracardiac perfusion with phosphate-buffered saline followed by 4% paraformaldehyde in saline for in situ fixation of the tumor. The brains were removed, placed in 4% paraformaldehyde, and allowed to stand at 4°C. After 4 h, the brains were transferred to a solution of 0.5 M sucrose in PBS and incubated overnight at 4°C. The following day, the brain was sliced into sections and embedded in microscopic slides and was frozen by placing at −80°C. Cryostat sections were stained with hematoxylin and eosin to reveal the tumor growth (Go et al., 1996). The sections were blindly reviewed and scored for the size of the tumor in each case semi-quantitatively. The average of cross-sectional diameter measured in sections of each tumor was used to measure tumor size and compared between controls and antisense transfectants. The variation between the sections in each group was less than 10%. Fluorescence microscopy was performed on tumor sections from GFP expressing cells without any chemical treatment and quantitated using Fluoview 2.1 software

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Acknowledgements

We thank Lydia Soto for preparing and Walter J Pagel for reviewing the manuscript. This work was supported by National Cancer Institute Grants CA 76350 (to JS Rao) and P30 CA-16672.

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Correspondence to Jasti S Rao.

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Mohanam, S., Jasti, S., Kondraganti, S. et al. Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells. Oncogene 20, 3665–3673 (2001) doi:10.1038/sj.onc.1204480

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Keywords

  • glioblastoma
  • invasion
  • cathepsin B

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