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The Journal of Neuroscience, July 15, 1999, 19(14):5942-5954
Modulation of Glioma Cell Migration and Invasion Using
Cl and K+ Ion Channel Blockers
Liliana
Soroceanu,
Timothy
J.
Manning Jr, and
Harald
Sontheimer
Department of Neurobiology, The University of Alabama at
Birmingham, Birmingham, Alabama 35294-0021
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ABSTRACT |
Human malignant gliomas are highly invasive tumors. Mechanisms that
allow glioma cells to disseminate, migrating through the narrow
extracellular brain spaces are poorly understood. We recently demonstrated expression of large voltage-dependent chloride
(Cl ) currents, selectively expressed by human
glioma cells in vitro and in situ
(Ullrich et al., 1998 ). Currents are sensitive to several
Cl channel blockers, including chlorotoxin (Ctx),
(Ullrich and Sontheimer, 1996; Ullrich et al., 1996),
tetraethylammonium chloride (TEA), and tamoxifen (Ransom and
Sontheimer, 1998). Using Transwell migration assays, we show that
blockade of glioma Cl channels specifically
inhibits tumor cell migration in a dose-dependent manner. Ctx (5 µM), tamoxifen (10 µM), and TEA (1 mM) also prevented invasion of human glioma cells into
fetal rat brain aggregates, used as an in vitro model to
assess tumor invasiveness. Anion replacement studies suggest that
permeation of chloride ions through glioma chloride channel is
obligatory for cell migration. Osmotically induced cell swelling and
subsequent regulatory volume decrease (RVD) in cultured glioma cells
were reversibly prevented by 1 mM TEA, 10 µM
tamoxifen, and irreversibly blocked by 5 µM Ctx added to
the hypotonic media. Cl fluxes associated with
adaptive shape changes elicited by cell swelling and RVD in glioma
cells were inhibited by 5 µM Ctx, 10 µM
tamoxifen, and 1 mM TEA, as determined using the
Cl-sensitive fluorescent dye
6-methoxy-N-ethylquinolinium iodide. Collectively, these
data suggest that chloride channels in glioma cells may enable tumor
invasiveness, presumably by facilitating cell shape and cell volume
changes that are more conducive to migration and invasion.
Key words:
chlorotoxin; glioma invasiveness; chloride channel; RVD; MEQ; cell migration
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INTRODUCTION |
A major biological feature of
primary brain tumors, which precludes successful therapy, is their
ability to invade the surrounding healthy brain tissue (Merzak and
Pilkington, 1997). Highly invasive glioma cells are thought to
transiently arrest from the cell cycle during migratory phases and thus
become largely refractory to the various chemotherapy and radiotherapy
regimens currently available. Once these migratory cells have traveled
a certain distance from the primary tumor mass, they re-enter cell
cycle and form recurrent tumors, both adjacent and distant from the
primary neoplasm (Merzak and Pilkington, 1997). Tumor invasion is a
multistep process that includes adhesion of tumor cells to
extracellular matrix (ECM) components, ECM remodeling by proteolytic
enzymes, and cellular mechanisms to generate locomotion and
advance through narrow spaces (Giese et al., 1994; Suigiura et al.,
1998). Thus, migration under spatial constraints requires that glioma
cells undergo major cytoskeletal rearrangements to support shape and
volume changes.
Essentially all cells regulate their cell volume. Multiple mechanisms
are engaged during cellular volume regulation, including transport
across cell membrane of Cl ions, organic
metabolites, and osmolytes. For example, activation of outwardly
rectifying chloride currents is involved in volume regulation of
secretory epithelial cells (Monaghan et al., 1997). Several of the
cloned chloride channels comprising the CLC family, most notably
CLC-2 and CLC-3, are swelling-activated channels, ubiquitously
expressed (Jentsch and Gunther, 1997). We recently identified and
characterized a chloride ion channel that is abundantly expressed in
glioma cell lines (Ullrich et al., 1996) and acute patient biopsies
(Ullrich et al., 1998). Cl currents recorded in
human gliomas showed voltage dependence and were sensitive to bath
application of 1 µM chlorotoxin, a Cl channel-specific peptide purified from
Leiurus quinquestriatus scorpion venom (DeBin et al., 1993).
Currents were inhibited by TEA (1 mM), which has also been
shown to inhibit muscle Cl channels (Sanchez and
Blatz, 1995). Chlorotoxin-sensitive Cl currents
are absent in normal astrocytes (Ullrich et al., 1996). However,
astrocytes do express other Cl channels that
contribute to the regulatory volume decrease (RVD) after hypotonic cell
swelling (Pasantes-Morales et al., 1994). Because the activity of the
glioma, voltage-activated, current could be modulated by perturbations
of the cell cytoskeleton (Ullrich and Sontheimer, 1997), we set out to
investigate whether the glioma-specific Cl
channels may be involved in regulating cell volume.
We hypothesized that cell shrinkage is a requirement for successful
tumor cell invasion through narrow extracellular spaces in the brain
(20 nm). We show that inhibitors of glioma Cl
channels, including Ctx, TEA, and tamoxifen are also effective inhibitors of in vitro tumor cell migration and invasion.
Moreover, using Cl-sensitive fluorescent dyes, we
show that Cl fluxes associated with glioma cell
volume changes are also modulated by Ctx, tamoxifen, and TEA. We
propose that the upregulation of glioma Cl
channels may be a positive adaptation that facilitates cell shape and
volume changes, thereby promoting tumor invasion.
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MATERIALS AND METHODS |
Cell culture
We have used the following glioma cell lines: U251MG and
D54MG, from D. D. Bigner (Duke University, Durham, NC). In
addition, we used SKMEL5 melanoma cell line, Balb3T3 fibroblasts from
the American Tissue Culture Collection and primary cultured rat
astrocytes as control glial cells. Primary culture of rat astrocytes
has been described in detail elsewhere (Macfarlane and Sontheimer, 1997). Glioma cells were maintained in DMEM supplemented with L-glutamine (2 mM) (Life Technologies,
Grand Island, NY) and 10% fetal bovine serum (FBS) (Life
Technologies). Cells were harvested from logarithmic phase growth
cultures by brief exposure to 0.5% trypsin and 0.53 mM
EDTA or 0.2 M EDTA in PBS for invasion assays (see below). Cell lines tested negative for mycoplasma by PCR (Stratagene, La Jolla, CA).
Normal rat brain aggregates
Fetal brain aggregates were obtained from 18-d-old fetuses of
Sprague Dawley rats as previously described (Penar et al., 1998). Brains were dissected out under aseptic conditions, the meninges were
removed, and tissue was placed at 37°C, on a rotator, in oxygenated
enzyme solution: 20 U/ml papain and (in mM): NaCl 137, KCl
5.3, Mg Cl2 1, glucose 25, HEPES 10, and
CaCl2 3, supplemented with 0.5 mM EDTA and 0.2 mg/ml L-cysteine. Tissues were then pelleted and triturated
using a fire-polished Pasteur pipette in astrocyte growth medium. Brain
aggregates were produced by seeding 6 × 106
cells in minimum essential media (MEM; Life Technologies) and 10% FBS
in 24 well plates (Fisher Scientific, Houston, TX) base-coated with 1%
medium agar substrate. After 20 d in tissue culture, a three-layered structure was visible, and the cellular differentiation was complete: astrocytes, oligodendrocytes, and neurons could be
identified. Mature aggregates were individually transferred to the
plates used for confrontation assays.
Human glioma spheroids
Brain tumor spheroids were obtained as described elsewhere
(Bjerkvig et al., 1990; Nygaard et al., 1998). Human glioma cell lines
U251MG, D54MGLacZ, and acutely dissociated glioblastoma tissue obtained after surgical resection from the University of Alabama
at Birmingham (UAB) Department of Neurosurgery, were plated on
nonadhesive 0.75% Noble agar-coated 24 multiwell plates (Fisher Scientific) in DMEM-based growth medium. After 2 weeks in culture (37°C; 95% O2 and 5% CO2), tumor
spheroids were formed.
Fluorescent dye labeling
For labeling of tumor spheroids and rat brain
aggregates, we used the carbocyanine fluorescent dyes
1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine (DiI) and
3,3'-dioctadecycloxacarbocyanine perchlorate (DiO) (Molecular Probes,
Eugene, OR), which have maximum absorption at 546 and 489 nm and maximum emission at 563 and 499 nm, respectively. The difference
in fluorescence emissions allows for good separation of the dyes by use
of specific fluorescence filter optics. For cell labeling, we followed
the protocol by Nygaard et al. (1998) and the recommendations from the
manufacturer. A final concentration of 5 µM for DiI and
10 µM for DiO were obtained by diluting the stock (2.5 mg/ml) in the DMEM-based growth media. The loading solution was sterile
filtered through a 5 µm pore; 48 hr before the coculture began,
equally sized tumor spheroids and fetal rat brain aggregates were
incubated for 48 hr in media containing either DiO or DiI, respectively.
Coculture and video time-lapse microscopy
Glioma spheroids (200-300 µm) were transferred under a low
magnification microscope using a sterile Pasteur pipette to 96 multiwell plates (Nunc, Roskilde, Denmark) and 35 mm glass bottom culture dishes (from the MatTek Corporation, Ashland, MA). Rat brain
aggregates of similar size were chosen and individually placed in the
vicinity of the tumor spheroids. Astrocyte growth media with or without
Ctx (5 µM), TEA (1 µM), and tamoxifen (10 µM) was added to the cocultures. After 24 hr from the
initiation of the coculture, the interaction was monitored by means of
fluorescence time-lapse video microscopy. After overnight incubation at
37°C and 5% CO2 and 95% O2, slips
were placed on the bottom of LU-CB-1 tissue culture chamber equipped
with an NP-2 incubator that maintained temperature at 37°C and
atmosphere at 95% O2 and 5% CO2. Cells were
visualized by a Nikon Diaphot inverted microscope with phase contrast
optics at 40× magnification. Images were recorded on a time-lapse VHS
video recorder/player for 24-120 hr. The time-lapse microscope is also
equipped with a mercury fluorescence lamp connected to a Lambda 10-2
filter wheel which controls excitation wavelength; a second
intensifying CCD camera was used to capture images on a computer using
Axon Imaging Workbench (version 2.1, 1997; Axon Instruments, Foster
City, CA) software. In this manner simultaneous phase and fluorescent
recordings were obtained. At the end of the recording period selected
coverslips were further processed for fluorescence microscopy or
transmission electron microscopy as explained bellow.
For time-lapse scrape migration assays U251MG cells were plated on
coverslips as described, mechanically scarred and placed in the time
lapse recording chamber. This technique has been described in detail
elsewhere (MacFarlane and Sontheimer, 1997).
Immunohistochemistry
Processing and staining of the human brain sections for glial
fibrillary acidic protein (GFAP) was done using a monoclonal antibody
from Zymed Laboratories (San Francisco, CA), as previously described.
Staining for Ki-67 antigen was done using the MIB1 antibody available
from Dako (Carpinteria, CA), as described (Shiraishi, 1990; Cavalla and
Schiffer, 1997). Slides were processed using a secondary biotinylated
anti-mouse antibody and developed using an avidin-biotin kit (Elite;
Vector Laboratories, Burlingame, CA) in conjunction with horseradish
peroxidase. Counting of positive cells for 10 microscopic fields for
each case was done independently by two researchers, and tissues were
scored on a scale from negative to highly positive (+++).
Transmission electron microscopy
Cocultures of glioma spheroids and fetal brain aggregates were
prepared for electron microscopy as previously described (Bernstein et
al., 1990; Reuver and Garner, 1998). Thin sections (0.75 µm) were
stained with toluidine blue (1%) and examined by light microscopy. Samples were processed for transmission electron microscopy according to the standard procedures (Weber et al., 1994). Ultra-thin sections (80 nm) were cut on a Ultracut-S ultramicrotome; grids were stained with lead citrate and uranyl acetate and examined on a JEOL 100CX electron microscope equipped with a camera used to obtain photomicrographs.
Invasion assay in brain slices
To be able to quantify the extent to which ion channel blockers
prevent deep invasion of human glioma cells into the normal brain
tissue, we used a brain slice invasion assay adapted after Ohnishi et
al. (1998). An organotypic culture method previously used was modified
as follows: brain slices were prepared from 2-d-old neonatal Sprague
Dawley rats. Whole brains were quickly removed and placed in complete
saline solution (Manning and Sontheimer, 1997), continuously bubbled
with O2 and maintained at <4°C. The brain was mounted on
the stage of a Vibratome (series 1000; Technical Products
International, St. Louis, MO), and 300 µm coronal sections were cut.
The tissue was maintained cold and well oxygenated at all times.
Sections were collected and transferred onto a cell culture insert, 8 µm pore track-etched membrane, fit for six well plates (Becton
Dickinson, Franklin Lakes, NJ) that had been coated with human
vitronectin (10 µg/ml, 24 hr, at 37°C), and maintained moist by
adding 1 ml of PBS in each well. After brain slices were placed on the
membranes, PBS was aspirated and replaced with MEM. Brain slice
cultures were incubated at 37°C under standard conditions (95%
O2 and 5% CO2). Viability of slices was
monitored for 14 d in culture using the vital dye Trypan blue and
propidium iodide staining. After 5 d from initiating the brain
slice culture, DiO-labeled glioma cells were added to the cultured
normal brain, in the presence or absence of Ctx (5 µM),
tamoxifen (10 µM), or TEA (1 mM). DiO-labeled human glioma cells were obtained as described for the confrontation assay. Semiconfluent glioma cells were harvested with 10%
Trypsin-EDTA in PBS, and were resuspended at 1 × 108 cells/ml in migration assay media (see below).
Ten microliters of the labeled cells were placed on the surface of
brain slices cultured on Transwell filter chambers as described. Drugs
were added to the lower chamber to the same final concentration as in
the cell suspension. For control experiments, either PBS or vehicle
(DMSO) were used. Cocultures were inspected for 4-6 d every 12 hr by
epifluorescence microscopy with a Nikon Diaphot 200 microscope equipped
with a Nikon FM-2 camera or time-lapse fluorescence video
microscopy (490 nm excitation and 535 nm mission filters) concomitant
with phase-contrast microscopy as described for the confrontation
assays. DiO-labeled tumor cells were visible both on the surface of the
normal brain tissue, as well as on the bottom of the filter chamber
(cells that invaded deeply through the slice). At 96 hr from the
beginning of the coculture, the brain slices and any cells on the
inside of the chamber were cleaned using a cotton swab. Filters were
then cut, placed with the bottom side up on a glass slide, and viewed
under a fluorescence microscope (Leica DMRB, Heerbrugg, Switzerland);
images were captured on a computer using a 3-CCD camera, and the
labeled glioma cells were counted over the entire filter area.
Experiments were repeated three times for each pharmacological condition.
Transwell cell migration assays
We used 5-8 µm polycarbonate Transwell filter chambers
(Costar Corporation, Cambridge, MA) coated on the lower surface with 200 µl of Vitronectin (Life Technologies) (10 µg/ml in PBS) or bovine serum albumin (BSA), 1% in PBS, and incubated at 37°C
overnight. Filters were washed twice with PBS, blocked for 1 hr with
1% BSA in PBS, washed and kept moist in 200 µl of migration assay
buffer (serum-free DMEM, with 1.5 L-glutamine, 0.1% BSA).
Recently subcultured, semiconfluent U251MG cells were harvested with
buffered EDTA (1.5 mM), washed with PBS, and resuspended in
migration assay buffer at 400,000 cells/ml. Cell aliquots of 100 µl
were plated on the upper filter surface. Ctx, TEA, and tamoxifen were
added to the same final concentration to both upper (with cells) and
lower chambers and filters were returned to 37°C and 95%
O2 and 5% CO2 for 3-6 hr for glioma cell
lines and overnight in the case of melanoma cells and rat astrocytes.
Filters were then washed, and cells on the upper surface were removed
using cotton swabs. Cells on the lower filter surface were fixed in 4%
paraformaldehyde (5 min) and stained with 1% crystal violet in 0.2 M sodium borate buffer, pH 9. Ten random 1 mm2 fields were counted to determine the number of
cells that migrated and compared with untreated controls. For ion
replacement experiments, solutions were made in migration assay media
based on DMEM without NaCl (Life Technologies). Osmolality of the final
solution was adjusted using mannitol to 308-312 mOsm/kg after addition
of replacement ionic salts (sodium glutamate, sodium bromide).
Experiments were repeated three times for each filter size (5 and 8 µm) and each of the ion substitution conditions.
Volume measurements
Preparation and loading of the dye. Fura-2 AM
(Teflabs, Austin, TX) was added (5 µM) to U251MG cells
plated on coverslips and incubated in the dark in an atmosphere of 95%
O2 and 5% CO2 for 40 min. Cells were then
rinsed with fresh saline, and the dye was allowed to de-esterify for 15 min before experiments. Coverslips with dye-loaded cells were placed in
a Series 20 Micro-perfusion chamber (Warner Instruments, Hamden, CT) on
the stage of a Nikon Diaphot 200 inverted epifluorescence microscope
and visualized using a 40× oil immersion objective. Cells were kept
under constant perfusion, and all solutions were maintained at 37°C
with a TC-344 Dual Heater Controller and an SH-27A in-line heater
(Warner Instruments, Hamden, CT). Changes between perfusion solutions
were made using a Multi Channel Valve Driver II (General Valve
Corporation, Fairfield, NJ). For some experiments, solutions were
changed using an eight barrel fast application microperfusion system.
Volume measurements were obtained by means of microspectrofluorimetry
that allows simultaneous monitoring of changes in cell water volume and
[Ca2+ ]i in cells loaded with fura-2
AM. When excited at 357 nm (isosbestic point), the fluorescence
intensity of fura-2 AM is insensitive to changes in
[Ca2+ ]i, and thus is directly
proportional with the dye concentration and the intracellular volume.
For simultaneous [Cai]2+ measurements, the
dye was alternately excited at 340 and 380 nm with a single wavelength
monochrometer (Photon Technologies International, South Brunswick, NJ),
and images were obtained every 10 sec. The emitted fluorescence above
520 nm was captured by an intensified CCD camera (Hamamatsu, Tokyo,
Japan) then digitized and saved on a computer for later off-line
analysis using ImageMaster software (Photon Technology International,
South Brunswick, NJ). The ratio of the two images 340:380 was
calculated and converted to absolute
[Ca2+]i concentrations, as previously
described (Manning and Sontheimer, 1997). U251MG cells were perfused
with saline solution consisting of (in mM): NaCl, 122.6;
KCl, 5.0; MgCl2, 1.2; CaCl2, 2.0;
Na2HPO4, 1.6;
NaH2PO4, 0.4; glucose, 10.5; HEPES 5.0;
NaHCO3, 25.0; and Na2SO4, 1.2, pH 7.4. For osmotic
challenges, this solution was modified by removing 50 mM
NaCl in the case of hypo-osmotic solution (200 mOsm/kg) and adding back
mannitol for the isotonic (308 mOsm/kg) solution. In this manner,
solutions were maintained isoionic. Drugs were added to the isotonic
and hypotonic solutions to final concentrations of: 5 µM
Ctx, 10 µM tamoxifen, and 1 mM TEA.
Experiments were repeated three times for each condition, and
recordings from 50 cells/coverslip were averaged.
Chloride flux measurements using the fluorescent dye
6-methoxy-N-ethylquinolinium iodide
The chloride-sensitive dye
6-methoxy-N-ethylquinolinium iodide (MEQ) was prepared as
recommended by the manufacturer (Molecular Probes, Eugene, OR) and
modified from the protocol by Biwerski and Verkman (1991). Briefly, 16 µM MEQ was reduced by adding a small volume of 12%
sodium borohydride under constant flow of nitrogen. After the reaction
was complete (30 min) the organic phase, which separates as a yellow
oil, was transferred to the DMEM media (final concentration 5 µM) and used to load the cells in the dark, at 37°C for
20 min. The reduced form of MEQ is membrane-permeable; once loaded,
diH-MEQ is converted to the oxidized form (MEQ), which is retained
within the cell. Afterward, cells were rinsed and incubated in
chloride-free DMEM-based media for an additional 10 min, at 37°C, for
uniform distribution of the dye in the cytoplasm. During the recordings
cells were superfused with a solution containing (in mM):
Na gluconate 130, K gluconate, 5.4, MgSO4 0.8, Ca gluconate 1.2, NaH2PO4 1, glucose 5.5, and Tris 5, pH-adjusted to 7.4. To obtain a hypotonic solution, the sodium
gluconate was reduced to 80 mM. Replacement of the chloride
salts with gluconate was necessary to maintain a maximum initial
fluorescence of MEQ, which is quenched by collision with halide ions
(Cl, SCN). Drugs were added
to each solution, as described for volume measurements. Recordings were
obtained using the same equipment as described for fura-2 AM
fluorescence measurements. The dye was excited at 344 nm, and images
were captured above 440 nm every 10 sec. Experiments were repeated at
least three times for each experimental paradigm. Average recordings
from 50 cells per coverslip were analyzed.
Cl flux measurements using a fluorescence
plate reader
To obtain quantitative information regarding the effects of
Cl and K+ channel blockers on
Cl fluxes in glioma cells, a Fluostar 403 fluorescence plate reader was used (BMG LabTechnologies, Durham, NC).
MEQ fluorescence measurements were obtained using 340 nm excitation and
440 emission filters. Glioma cells were plated in 96 multiwell plates
(Fisher Scientific) at 105 cells/ml. After 24 hr
from plating, cells were loaded with the Cl-sensitive fluorescent dye MEQ, as described for
microscope based recordings. Drugs were either added to the cells after
rinsing the dye or applied during the recording using microinjectors
capable of delivering defined volumes to individual wells. Experiments were performed at room temperature in the gluconate based buffers as
described. Multiple readings of the same microplate during 60 min were
obtained. Data were analyzed using Fluostar software that is integrated
with the spreadsheet software Excel 5.0.
Materials and chemicals
All drugs and chemicals were purchased from Sigma (St.
Louis, MO), except when otherwise mentioned. Tamoxifen was
reconstituted in DMSO, which was used as "vehicle" control when
indicated in the experimental results. Chlorotoxin was purchased from
Alomone Laboratories (Jerusalem, Israel) and from Peninsula
Laboratories (Belmont, CA).
Statistical analysis
Results were analyzed using GraphPAD (Instat). Student's
t test (unpaired, two-tailed) was used for data with normal
SD distribution. ANOVAs were used for multiple comparisons.
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RESULTS |
Effects of Cl channel blockers on
glioma cell migration in vitro
Previous data from our laboratory has shown that Ctx selectively
blocks a voltage-activated chloride conductance (Ullrich et al., 1996)
specifically expressed by human glial tumors. These outwardly
rectifying currents are also sensitive to TEA and tamoxifen (Ransom and
Sontheimer, 1998). To investigate the roles that glioma Cl channels play in the invasive behavior of tumor
cells, we used a Transwell migration assay (modified Boyden chamber
assay) frequently used to assess cell chemotaxis and invasiveness.
Glioma cells were plated on the upper side of a filter insert
containing 5-8 µm pores and are attracted to migrate through these
pores toward the extracellular matrix protein vitronectin. Several
glioma cell lines and glioma culture preparations dissociated from
human biopsies (passages 2-3) were subjected to Transwell assays in
the presence or absence of various Cl and
K+ channel blockers. After 3-6 hr migration time,
cells were fixed and stained with crystal violet. Figure
1 shows representative fields of tumor
cells obtained from a glioblastoma multiforme (UAB case no. 98040138)
that have successfully migrated across the chamber filters in the
presence (Fig. 1B) or absence (Fig. 1A) of 5 µM Ctx. Migration of glioma
cells was greatly reduced in the presence of chlorotoxin. Complete
dose-response curves for the inhibitory effects of Ctx were obtained
using the glioma cell line U251MG. As illustrated in Figure
1C, Ctx caused a concentration dependent inhibition of
Transwell migration with maximal inhibition of 54.6% (SD 3.5, n = 15) for concentrations >1 µM, and an
apparent IC50 of 600 nM. Inhibition of
Transwell migration by Ctx appears specific for human gliomas because 5 µM Ctx did not affect the Transwell migration of human
melanoma cells (SKMEL5, 10% inhibition, SD = 2.3, n = 10), Balb3T3 fibroblasts (18.9% inhibition,
SD = 3.4, n = 10), or primary cultures of rat
hippocampal astrocytes (Fig. 1D).
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Figure 1.
A, B, Representative
microscopic fields of human glioblastoma cells that have migrated
across an 8 µm pore size filter in the presence
(B) or absence (A) of 1 µM Ctx. Cells were fixed and stained with crystal violet
(see Materials and Methods). Scale bar, 50 µm.
C, The ability of Ctx to inhibit Transwell
migration of U251MG cells is dose-dependent for both 5 and 8 µm pore
size filters. Half-maximal inhibition (IC50) for Ctx
is ~600 nM (log scale). The y-axis
represents percentage inhibition calculated as decrease in the number
of Ctx-treated cells that migrated across the filters normalized to
control conditions. Data points are mean values from three independent
experiments ± SD. Continuous lines represent a Langmuir-binding
isotherm fitted to data. D, Inhibition of Transwell
invasion by 5 µM chlorotoxin is glioma-specific: U251MG
and D54MG human malignant glioma cell lines are >60% inhibited,
whereas no significant effect is seen on control cell lines. The
y-axis represents percentage inhibition calculated as
decrease in the number of cells that migrated across 8 µm filter in
the presence of 5 µM Ctx compared with control
conditions.
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We investigated the effects of other Cl and
K+ ion channel blockers on Transwell migration of
U251MG cells. These results are summarized in Table
1. Dose-response curves for Transwell
migration of glioma cells were also obtained for TEA concentrations
(Fig. 2A) and tamoxifen
(Fig. 2B) using U251MG cells. Both TEA and tamoxifen have been reported to block K+ and
Cl channels, and in glioma cells tamoxifen may
indeed inhibit both (Chin et al., 1997). We have been able to
effectively inhibit swelling-activated chloride currents in U251MG with
~80% blocked by 10 µM tamoxifen (C. Ransom,
unpublished observations). We tested a wide range of TEA and
tamoxifen concentrations in Transwell assays and have found that doses
<100 µM TEA and 100 nM tamoxifen were
ineffective in preventing glioma cell invasion. The apparent half-maximal inhibition doses for TEA and tamoxifen were 1 mM and 10 µM, respectively (Fig.
2A,B). Interestingly, the
effectiveness of tamoxifen in this assay is consistent with the
concentration required for channel block (Phillis et al., 1998) but
~100-fold lower than required for its activation of PKC-dependent
apoptosis (Chin et al., 1997). Treatment of cells with vehicle alone
(DMSO) was without effect, moreover, neither of the drugs used showed any cellular toxicity (data not shown) using the Alamar blue (from Accumed, West Lake, Ohio) cell viability assay (Andreanski et al.,
1997). Effects of Ctx, TEA, and tamoxifen on Transwell migration of
acutely dissociated glioma cells from human biopsy tissue were investigated using the same approach as described for cell lines. Results obtained using cells (passages 2-3) from human glioblastoma multiforme (GBM), low grade astrocytoma, and epileptic tissues are
summarized in Table 2. The variability of
response between different cell preparations is not surprising given
the tremendous cellular and physiological heterogeneity of these
tumors. However, it is notable that most of the high-grade glioma
samples exhibited significant sensitivity to Ctx (average inhibitory
effect for GBMs = 34.06%, SD 4.6), and tamoxifen (average
inhibition = 32.4%, SD5.1), whereas cells obtained from
intractable epilepsy biopsy tissue (which we used as nonmalignant
control) were not affected by either of the drugs in their migratory
behavior. Tissue sections from the same specimens were
immunohistochemically stained for GFAP, an astrocytic marker and for
anti-Ki-67 antibody (clone MIB1; Dako), used as a
histological marker, indicative of the proliferative activity (Giese et
al., 1994).
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Figure 2.
Inhibition of Transwell migration of U251MG cells
by TEA (A) and tamoxifen
(B) is dose-dependent. Each data point represents
mean values from three different experiments ± SD. The
y-axis represents percent inhibition calculated as the
decrease in number of cells that migrated through the pores in the
presence of TEA or tamoxifen as compared with vehicle-treated
cells.
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Effects of ion replacement on glioma cell migration
To confirm that the inhibitory effect of Cl
channel blockers on Transwell migration were indeed caused by
inhibition of Cl fluxes across cell
membrane, we repeated the Transwell assays after replacing
Cl ions with other anions that show either similar
or lesser permeability. Electrophysiological recordings indicated the
following permeability sequence for the outwardly rectifying voltage
activated glioma currents:
I>NO3>Br>Cl>acetate>
isethionate>F>glutamate (Ullrich and Sontheimer,
1997). As shown in Figure 3,
substitution of the chloride ions with an equally permeant species,
such as bromide (125 mM NaBr in migration assay buffer based on Cl-free DMEM media) did not affect the
migration rate of U251MG human glioma cells. Furthermore, sensitivity
to ion channel blockers, such as Ctx (1 µM),
4,4'-diisothyanostilbene-2,2'disulfonic acid (200 µM), and Zn2+ (100 mM) was
preserved (Fig. 3). However, Transwell migration of glioma cells was
significantly reduced (>60%inhibition) when NaCl was replaced by
mannitol (120 mM) or Na glutamate, which are both
impermeant to glioma voltage-activated channels (Fig. 3). These results
support the notion that Cl ion fluxes across the
cell membrane are necessary for Transwell migration.
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Figure 3.
Effects of ion substitution on Transwell migration
of U251MG cells. Results are expressed as percentage of cells that
migrated normalized to control conditions (NaCl in migration media).
Results represent mean values from three independent experiments ± SD. Statistics were computed from raw data, using ANOVA.
***p < 0.001; ** p < 0.01.
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Effects of ion channel blockers on U251MG
cell motility
To better understand the mechanisms underlying the inhibitory
effects of ion channel blockers on tumor cell invasion, we first ascertained whether overall cell motility was affected by application of these drugs. Cell movement was monitored by video time lapse microscopy, using a scrape motility assay as previously described (MacFarlane and Sontheimer, 1997). The series of top panels in Figure
4A-D
illustrates the time course of such an assay. U251MG cells readily
migrate into and across an artificially created "scar" (~150
µm) in the presence of 5 µM Ctx
(A-D), and indistinguishable from control
(vehicle alone, data not shown). Similarly, application of 1 mM TEA or 10 µM tamoxifen did not interfere
with the cell motility in a two-dimension scrape motility assay that
lacks the spatial constraints imposed by the Transwell assay.
Rhodamine-conjugated phalloidin staining of Ctx, TEA, or
tamoxifen-treated glioma cells showed integrity of stress fibers (data
not shown), thus indicating that ion channel blockers used herein do
not act by disrupting the actin-myosin molecular motor or interfering
with the integrity of the cell cytoskeleton.
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Figure 4.
A-D represent
time-lapse micrographs of a "scrape-migration" assay. U251MG cells
treated with 5 µM Ctx were scarred and monitored for 24 hr. Glioma cell migration into the empty space results in gradual
closure of the scar, which is complete at 24 hr, unchanged from control
(data not shown). Scale bar, 100 µm. Phase
(E-H) and fluorescent
(I-L) micrographs of a confrontation
assay between DiO-labeled BTs and DiI-labeled FBAs. The interaction was
monitored by video time-lapse microscopy for 72 hr, in the presence or
absence of ion channel blockers. H and L
illustrate significant reduction in the invasion of glioma cells into
the FBA and preservation of a clear border between the two tissue
types; also noticeable is the integrity of normal brain tissue in the
presence of chlorotoxin (5 µM; H,
L) in contrast to massive infiltration of the fetal rat
brain with glioma cells in control conditions (G,
K). Scale bars: A,
E, 100 µm.
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Inhibition of glioma invasion of normal
brain aggregates
To more closely mimic glioma cell invasion into normal brain, we
used a three-dimensional coculture system modified after Bjerkvig et
al. (1990) and Penar et al. (1998). These confrontation assays between
human glioma spheroids (BT) and fetal rat brain aggregates (FBA) were
monitored for up to 120 hr by fluorescence video time-lapse microscopy.
Human glioma spheroids labeled with DiO demonstrated infiltrative
invasion of the FBA (Fig.
4I,J) initiated by single
cell migration around 24 hr after the beginning of the coculture. In
control (vehicle treatment) conditions, the invasion process continues
with a loss of border between the two spheroids and gradual replacement
of the normal brain tissue with tumor cells (Fig.
4E-G, I-K);
local areas of tissue loss were noticeable in the FBA. Cells from the
normal brain aggregate were also seen migrating toward the
tumor spheroid, probably because of chemotaxic effects exerted by
growth factors and cytokines released by glioma cells. Addition of 5 µM Ctx to the coculture significantly reduced the
invasion of tumor cells into normal brain tissue. As shown in
Figure 4, fluorescently labeled tumor cells migrate toward,
surround the FBA, and attach to the normal tissue. However, no deep
invasion of glioma cells is seen, and the border between the tissues
remains clearly defined (Fig.
4H,L, arrows).
Furthermore, treatment with 1 mM TEA or 10 µM
tamoxifen significantly diminished tumor invasion (data not shown).
Experiments for each of the drugs were repeated three times with
similar results. Although we did not attempt to quantitate the degree
of tumor invasion inhibition by directly measuring the extent FBA
destruction, this assays allowed for a qualitative sequential analysis
of glioma invasion and modulation of invasion by Ctx, TEA, and
tamoxifen. "Sister" cultures for the treated spheroids were
maintained in 37°C, 95% O2 and 5% CO2
simultaneously with those subjected to video recording, and cell
viability was confirmed by staining with calcein and propidium iodide
as previously described (Nygaard et al., 1998). None of the drugs
exhibited cellular toxicity at the doses used in these experiments.
Inhibition of glioma cell invasion into brain slices
To quantify the effects of ion channel blockers on glioma cell
invasion we used a modified organotypic culture system (Ohnishi et al.,
1998). Viability of cultured rat brain slices was monitored for 14 d by propidium iodide staining and demonstrated cellular integrity,
organotypic organization and preservation of laminar structure of the
cortex, (although the thickness of the slice was reduced to ~150 µm
from the initial 300 µm). After 5 d in culture, fluorescently
(DiO) labeled 106 glioma cells (U251MG cells and
acutely dissociated cells from patient biopsy) were placed on the
surface of the slice. Movement of tumor cells within the slice was
monitored by epifluorescence microscopy; representative fields of
glioma cells on the surface and within the brain slice are shown in
Figure 5B (bright
regions indicated by arrow). No significant
differences in the number of tumor cells between preparations were
noticed in the initial 24 hr after plating; we thus assumed that an
equivalent number of glioma cells have become attached to the rat brain
slice. After 96 hr in coculture, cells that migrated through the brain
slice and across the 8 µm membrane were counted (entire filter area was counted in six filters per condition, in three independent experiments). As shown in Figure 5A, Ctx (5 µM) application reduced by 30% (SD= 4.6, n = 18, p < 0.05) the number of glioma
cells that invaded through the brain slice, compared with
vehicle-treated cells. TEA (1 mM) had the most prominent
inhibitory effect on glioma cell invasion (45% decrease in the number
of cells compared to control), suggesting that multiple TEA-sensitive
transport mechanisms (including Cl and
K+ ion channels) are involved in mediating glioma
cell shape and volume changes that take place during tumor invasion.
Using the brain slice invasion assay we could best mimic the
tridimensional environment of the healthy brain tissue, while also
accurately count the distinctively labeled human tumor cells.
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Figure 5.
Slice invasion assay. A, Ten
microliters of DiO-labeled glioma cells (106) were
plated atop each of the brain slices placed in the top compartment of a
two-well culture chamber. After 4-6 d, fluorescently marked tumor
cells that had migrated through the neonatal rat brain slice and across
the membrane were retrieved on the bottom of the filter and counted
under the fluorescence microscope. Entire filter areas were counted in
each condition (six filters per condition). Results represent mean
values from three independent experiments. Percentage inhibition was
calculated as the decrease in the number of drug-treated cells
normalized to control. Bonferroni p values were obtained
using ANOVA. **p < 0.01. B,
Photomicrograph showing a representative field of a tissue culture
insert with DiO-labeled glioma cells that successfully invaded the
brain slice (arrows). Scale bar, 150 µm.
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Effects of ion channel blockers on RVD in human
glioma cells
Volume adaptive changes are essential for any cell survival. In
the case of spatial constraints imposed by the skull, migratory tumor
cells that disseminate from primary tumor mass are faced with a very
narrow extracellular space, further reduced by edema present in the
tumor environment. As such, glioma cells first shrink, reduce their
volume by secreting water and ions, after which they undergo volume
recovery. To investigate whether inhibition of Cl
channels by the drugs used for invasion assays perturb the tumor cells' ability to regulate cell volume, we experimentally altered the
cell volume of glioma cells by changes in bath osmolality and evaluated
the effects of Cl channel blockers on volume
regulation. We subjected glioma cells to a brief hypo-osmotic swelling
and monitor their gradual volume decrease toward the original cell
size. This was accomplished using the isosbestic point of fura-2 AM
(357 nm) where fluorescence changes are a good indicator of variations
in cell volume. Furthermore simultaneous [Ca2+
]i measurements can be obtained (Altamirano et al., 1998).
Exposure of cultured glioma cells to a hypotonic solution (200 mOsm)
induced rapid cell swelling measured as a fura-2 fluorescence intensity decrease when excited at the isosbestic point wavelength (357 nm)
because of dilution of the dye. This was followed by a slow recovery up
to 85% of the initial volume within the next 13.5 min; this volume
recovery phase has been termed regulatory volume decrease (RVD)
(Chamberlin and Strange, 1989). A recording obtained by averaging
responses from 50 glioma cells is shown in Figure 6A (bottom,
thin trace). The volume recovery mechanisms in human glioma cells were Ca2+ independent, because the
presence of EGTA (2 mM) in the hypotonic media did not
alter the volume recovery rates (data not shown). Pretreatment with and
inclusion in the perfusion solutions of 2 µM cytochalasin
D (an actin-depolimerizing drug) did not prevent RVD, although glioma
cells were swollen to a lesser extent compared with the control, under
same hypotonic conditions (data not shown). Interestingly, application
of hypotonic media induced [Ca2+ ]i
oscillations with a transient increase in intracellular
Ca2+ concentration of 400 nM above its
levels in isotonic conditions (data not shown). The time course and
characteristics exhibited by glioma cell RVD (rate, extracellular
calcium independence) are in agreement with previous studies that
explored volume regulatory mechanisms in astrocytes (Pasantes-Morales
et al., 1994). Pretreatment with 5 µM Ctx, which was also
included in the hypotonic solution, resulted in 40% less cell swelling
and virtually no recovery of the cell volume (Fig.
6A, top bold trace). The
swelling decrease suggests a reduction in water influx. The inhibition
of RVD suggests that Ctx blocks Cl fluxes involved
in water extrusion during cell shrinking. Ctx treatment did not alter
the changes seen in [Ca2+]i during
hypotonic media application. The effects of TEA (1 mm) and tamoxifen
(10 µM) on glioma cell RVD were tested in the same manner
as explained for chlorotoxin. Tamoxifen inhibited the rate of RVD by
60% (measured as difference in the slopes of recovery). Tamoxifen
regulation of RVD has been described in other cell types, including
neuroblastoma (Diaz, 1996). One millimolar TEA application inhibited by
50% RVD in osmotically challenged U251MG cells.
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Figure 6.
A, Swelling-induced volume changes
in glioma cells were measured using fura-2 dye excited at its 357 nm
isosbestic point. Hypo-osmotic shock results in a rapid decrease in
fluorescence intensity that corresponds to the dye dilution after water
entry, followed by a slow recovery termed RVD initiated while hypotonic
conditions were maintained (bottom thin line). A sharp
fluorescence increase above the initial levels was triggered by
isotonic wash. Exposure to 5 µM Ctx before and during the
hypotonic shock resulted in a marked decrease in cell swelling and
virtually abolished RVD (top bold line). Both traces
were obtained by averaging recordings from 50 cells. The bar
above the traces denotes the duration of exposure to hypotonic
solution. B, The degree of recovery was calculated by
fitting the fluorescence plot to an exponential function (from microcal
Origin version 5.0 software); mean values from three independent
experiments for each of TEA (1 mM), Ctx (5 µM), and tamoxifen (10 µM) were normalized
to their respective control. Results are presented as percentage
inhibition of RVD. Error bars indicate SD. Statistics were computed
using ANOVA; ***p < 0.001.
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Modulation of Cl fluxes in glioma
cells by ion channel blockers
To better understand the mechanisms of Ctx, TEA, and tamoxifen
action during hypo-osomotic challenge and volume recovery in glioma
cells, we used the chloride-sensitive fluorescent indicator MEQ.
Through fluorescence imaging of MEQ-loaded human glioma cells, we
measured Cl ion movement across the cell membrane
during challenges with hypo-osmotic solutions and
Cl "pulses", as described previously for
epithelial cells, fibroblasts (Woll et al., 1996), synaptosomes, and
brain slice (Yu and Schwartz, 1995). U251MG plated on 22 mm square
coverslips were loaded with MEQ as described and perfused with a
gluconate-based solution; in this low Cl ion
concentration environment MEQ fluorescence is maximum. Quenching of the
MEQ signal (48% from resting conditions) was triggered by addition of
a hypotonic (200 mOsm/kg) solution with 30 mM NaCl. DiH-MEQ
fluorescence levels were completely restored by isotonic wash with no
Cl (Fig.
7A, bottom thin
trace). One millimolar TEA significantly reduced
glioma cell permeability for Cl. The MEQ signal
decreased only by 5% from control and recovered after restoring of
iso-osmotic conditions (Fig. 7A, top bold
trace). We tested Ctx, TEA, and tamoxifen over a wide range
of concentrations in their ability to impede Cl
fluxes across glioma cells plated in 96 multiwell dishes. The use of
fluorescence plate reader allowed us to obtain multiple readings for 30 min. In U251MG cells pretreated with Ctx (1 µM) the
swelling-induced Cl influx was reduced by 60% as
compared with the control conditions (Fig. 7B). Tamoxifen
(10 µM) treatment diminished by 35% the quenching of
the Cl-sensitive dye in glioma cells during
challenge by hypotonic solution, whereas 1 mM TEA
application resulted in an almost complete block of
Cl influx (Fig. 7B). These results,
together with fura-2 volume measurements, demonstrate that chlorotoxin,
TEA, and tamoxifen are able to significantly reduce swelling induced
chloride flux across glioma cell membrane, thus possibly interfering
with the shape and volume changes required for tumor cell invasion.
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Figure 7.
A, Osmotically induced chloride
fluxes in human glioma cells were measured using the
Cl-sensitive dye MEQ. Application of a hypotonic
solution containing 30 mM NaCl resulted in a rapid
fluorescence intensity decrease because of dye quenching, followed by
recovery to initial levels after the restoration of iso-osmotic
conditions (bottom thin line). In the presence of 1 mM TEA (both in the isotonic and hypotonic media),
Cl entry into the cells was significantly
prevented (top bold line). Traces
represent average responses from 50 cells. The bar above
corresponds to the duration of exposure to hypotonic media and 30 mM NaCl. B, Glioma cells plated in 96-well
plates (5000 cells per well) were pretreated with TEA (1 mM), Ctx (5 µM), or tamoxifen (10 µM) and loaded with the Cl-sensitive
dye MEQ. Fluorescence measurements were done using a plate reader. A
"chloride pulse" in hypotonic media was administered to glioma
cells via microinjectors (100 µl/well, arrow). MEQ
fluorescence intensity sharply decreases as a result of dye quenching
by Cl ions entering the cell in control conditions
( , bottom trace). In the presence of ion channel
blockers, the dye quenching is significantly decreased, 60% in the
case of Ctx ( ), 35% by tamoxifen ( ), and >85% by TEA
pretreatment ( ). The decrease in MEQ fluorescence quenching
indicates reduced permeability of the glioma cell membrane to
Cl. Traces represent average recordings from four
wells (20,000 cells) obtained at nine time intervals after the initial
challenge. Error bars indicate SD.
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DISCUSSION |
Unlike tumors that form elsewhere in the body or tumors that are
metastatic to brain, primary brain tumors are characterized by a
relentless drive to invade surrounding healthy tissues. This invasive
behavior has long been recognized as a major impediment in the
treatment of gliomas. Mechanisms that contribute to the invasiveness of
glioma cells have been studied by a number of laboratories and have
identified alterations in cell adhesion molecules (Edvardsen et al.,
1994), extracellular matrix molecules, and their cell surface
receptors in gliomas (Giese et al., 1994; Haughland et al., 1997).
Specifically these studies show upregulation of fibronectin, laminin,
vitronectin, and collagen types I, III, and IV (Rutka et al., 1988) and
de novo expression of a novel hyaluronan-binding protein
BEHAB, believed to be a glioma-specific extracellular matrix element
(Jaworski et al., 1996; Zhang et al., 1998). A neural cell adhesion
molecule termed L1 has recently been implicated in promoting glioma
cell adhesion and invasion along neuronal fibers (Izumoto et al.,
1996). To date, a new candidate "invasion suppressor" gene for the
CNS tumors has been proposed, Nm23, previously implicated in
microtubule disruption and modulation of the metastatic potential of
melanomas (Martin and Pilkington, 1998). Clearly, migration and
invasion of cells are complex processes that require adhesion of cells
to tissue structures, mechanisms to generate locomotion, and the
ability to alter the space into which cells move (Liotta et al.,
1991).
Little attention has been paid to the spatial constraints in which
cells move and mechanisms by which cells adapt to such constraints.
We propose here, as illustrated in Figure
8A, that glioma cells
can adjust their cell shape and cell volume to facilitate invasion into
narrow spaces. These changes require secretion of Cl ions along with either K+ or
Na+ to allow water loss and cell shrinkage. We thus
hypothesize that upregulation of Cl channels
represent an adaptive feature in human glioma cells. Light microscopy
analysis of toluidine blue-stained sections obtained from cocultures of
glioma spheroids with fetal rat brain aggregates (Fig.
8B) and electron microscopy analysis of the same
preparation (Fig. 8C) support the notion that invading
glioma cells assume a pronounced elongated cells shape that is
consistent with a loss in cell volume. Similar cell shape and volume
changes have been recognized by computer reconstruction of electron
microscopic sections of ascites carcinoma cells (Parsons et al., 1982)
after penetration of the perineum. Cell shape and volume changes that facilitate migration of glioma cells within the brain are clearly not
sufficient to account for long distance movements through narrow
extracellular spaces. Brain tumor cells further enlarge this space by
secretion of proteolytic enzymes (Sugiura et al., 1998) and show
alterations in their interactions with extracellular matrix proteins. A
role for Cl and K+ channels and
changes in cell morphology have been documented for various cell types
including muscle cells, lymphocytes, melanocytes, and astrocytes
(Cornett et al., 1993; Haussler et al., 1994; Kauranen et al.,
1995). In the latter, chloride currents can be activated by changes
in cell morphology or cell volume (Lascola and Kraig, 1996). Moreover,
mitogen-activated protein and tyrosine kinases in astrocytes are
activated by astrocytic volume changes (Crépel et al., 1998).
Data presented here clearly support the notion that pharmacological
blockade of glioma Cl currents retards the ability
of the cells to migrate across Transwell filters, without interfering
with the overall motility.
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Figure 8.
A, Model representing glioma cell
shape and volume-adaptive changes that occur during invasion in
spatially restricted conditions. These changes, accompanied by water
loss and cytoskeletal rearrangements, are mediated by ion fluxes
through Cl and K+ ion channels
and other ion transport mechanisms. B, Semithin section
through a coculture of tumor spheroids and fetal rat brain aggregates,
stained with toluidine blue. Glioma cells are seen advancing through
two normal rat brain cells (arrows). Scale bar, 20 µm.
C, Area of detail of the same preparation as in
B, analyzed by transmission electron microcopy. Glioma
cells are easily recognized because of the abundance of ribosomes and
other organelles that incorporate lead citrate and give a darker
appearance. Arrows indicate area of contact between an
elongated tumor cell and two other membranes, presumably of the fetal
rat brain. Scale bar, 1 µm.
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We currently speculate that Ctx, tamoxifen, and TEA each impede cell
shrinkage by inhibiting Cl fluxes that are
obligatory for the movement of water across cell membranes. Indeed,
this conclusion is supported by our ion replacement studies in which
Transwell migration is undisturbed if Cl is
replaced by a permeant anion but greatly reduced if substituted by an
impermeant anion. Moreover, direct measurements of
Cl fluxes and glioma cell volume using fluorescent
techniques show that these Cl channel blockers
reduce transmembrane Cl fluxes and greatly reduced
osmotically induced cell volume changes. A recent study in another
human glioma cell line (U138MG) showed the ability of halide and alkyl
phenols to block volume-sensitive chloride channels (Roy et al., 1998).
Studies by others further support the notion that ion channel function
may be associated with certain cancers. Swelling-activated outwardly
rectifying chloride currents were selectively expressed in
carcinoma cells in situ and invasive cervical cancer cell
lines in vitro that were absent in normal tissue or low
grade tumor cells (Chou et al., 1995; Shen et al., 1996). Inhibition of
voltage-activated Na+ channels can decrease the
invasiveness of human prostate cancer cells (Laniado et al., 1997), and
tamoxifen-sensitive Cl channels support migration
and invasion of human thyroid cancer cells (Hoelting et al., 1995).
Although our in vitro experimental conditions could not
completely mimic the spatial constraints and the complexity of the
extracellular environment of the brain through which glioma cells
migrate in vivo, results from confrontation assays and the
slice invasion experiments further indicate that blockade of glioma
ionic currents can modulate tumor invasiveness.
Expression and functional roles of the above described
Cl channels appear particularly enhanced in tumor
cells. Interestingly, while absent in normal rat astrocytes, large,
outwardly rectifying voltage-activated currents were described in
embryonic rat glial cells (Ransom, unpublished observations). It is
conceivable that such Cl channels are also
expressed during early brain developmental where neurogenesis and
gliogenesis are associated with cell migration, because similar
requirements for cell shape and cell volume exist under these
conditions. Indeed, an earlier report documents the presence of a
chlorotoxin-sensitive Cl channel in
preparations from embryonic fetal rat brain growth cones (DeBin et al.,
1994). This "secretory" Cl channel is
hypothetically involved in plasmallemma expansion and membrane
recycling occurring during neurite outgrowth (DeBin et al., 1994).
Expression by glioma cells of a current with similar gating and
pharmacological characteristics may recapitulate a phenotype that is
expressed earlier in development. Our findings, corroborated with
previous studies showing enhanced channel expression in highly
malignant brain tumors propose that glioma ion currents may confer a
growth advantage and represent a tumor-specific adaptive feature.
Although other cancerous cells do not display the degree of
invasiveness observed in gliomas, their initial penetration into organ
tissues necessitate similar shape and volume changes in these cells
(Parsons et al., 1982). It is conceivable that Cl
channels with related properties to the conductance described in this
report are ubiquitously expressed in migrating cells. Targeting
Cl channels may thus provide a novel way to
inhibit invasion and cell migration within tissues. Specifically,
inhibition of glioma chloride ion currents may restrain
dissemination of primary brain tumor cells and render these
malignancies more amenable to surgical intervention.
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FOOTNOTES |
Received Feb. 23, 1999; revised May 6, 1999; accepted May 6, 1999.
This work was supported by Grants NS36692 from the National Institutes
of Health and RPG-97-083-01CDD from the American Cancer Society. We
thank Dr. Steven Rosenfeld for advice on cell migration assays, Dr.
Yancey Gillespie for providing glioma biopsy tissue, Ed Phillips for
technical support with electron microscopy, and Chris Ransom for
critical comments.
Correspondence should be addressed to Dr. Harald Sontheimer, 1719 6th
Avenue South CIRC 545, Birmingham, AL 35294-0021.
| |
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ABSTRACT
INTRODUCTION
Compound via MeSH
