C H A P T E R
26
Chloride Transport in Glioma Growth
and Cell Invasion
Harald Sontheimer
o u t l i n e
I. Introduction 519
II. Gliomas and their Lineage 520
III. Glioma Migration and Invasion 521
IV. Cl Transport and Cell Volume Regulation
in Glioma Cells 521
V. Changes in Cell Volume of Invading Cell References 529
Require Cl Efflux via Clc Channels 523
VI. Mechanism of Chlorotoxin Action on
Glioma Invasion 524
I.  Introduction
Brain tumors fall into two principal categories, pri-
mary and secondary. Primary tumors are often called
gliomas and originate in the brain. Secondary or meta-
static brain tumors are peripheral cancers that invade
the brain. Together they account for well over 100,000
new cancer cases diagnosed each year in the USA,
of which approximately 40,000 are primary tumors
(according to data from the Central Brain Tumor
Registry of the United States, CBTRUS). In addition
to their dissimilar origin, primary and secondary
brain tumors differ in many aspects of their etiology
and biology. For example, metastatic cancers are eas-
ily distinguishable from normal brain tissue as they
represent the new growth of cancerous tissue with the
Physiology and Pathology of Chloride Transporters and Channels in the Nervous System 519
VII. Clinical Use of Chlorotoxin 525
VIII. Cell Volume Changes Associated with
Cell Proliferation 526
IX. Conclusions 528
Acknowledgements 528
properties of the organ it originated from. Hence, they
present as liver or lung cells growing within brain and
tumors typically grow as confined solid masses. This
is not the case for primary brain tumors which often
lack clear boundaries between normal and malignant
brain tissue. A representative example is illustrated
in Fig. 26.1A, which shows a cerebral glioma with
characteristic diffuse margins. An important differ-
ence between these two cancer types relates to how
the tumors spread and form metastasis. Metastatic
brain tumors disseminate hematogenously throughout
the body and enter the brain through the vasculature.
By comparison, primary brain tumors rarely metasta-
size into the periphery but instead spread within the
brain often reaching distant sites such as the contralat-
eral brain hemispheres or the spinal cord, as illustrated
© 2009, Elsevier Inc.
520 26.  Chloride Transport in Glioma Growth and Cell Invasion
Figure 26.1  Primary glioma at autopsy. A. Poorly defined
margins are characteristic of cerebral gliomas, like the one shown in
this example (arrows). B. Although gliomas rarely metastasize out-
side the brain, they often present secondary tumors in other parts
of the brain, often distant from the site of the primary tumor. These
secondary tumors are highlighted in B by white ovals. Copyrighted
images: University of Alabama at Birmingham, Department of
Pathology.
in the example shown in Fig. 26.1B. These cancers
spread by active cell migration without extravasating
into the vasculature. This is reminiscent of neuronal
and glial cell migration during brain development or
stem cells and microglial cells in the adult brain sug-
gesting that some of the underlying mechanisms of
migration may be shared between these cells.
II.  Gliomas and their lineage
Although primary brain tumors can originate from
a number of growth competent cells in the brain or
spinal cord, the majority of them appear to derive
from glial cells or their precursors. Reflecting this pre-
sumed relationship these tumors are collectively called
gliomas. These include a diverse group of cancers that
may not always be of defined lineage. Among the dis-
tinguishing features are immuno-positivity for cer-
tain glial associated antigens (Kleihues et al., 1995),
for example to glial fibrillary acidic protein (GFAP),
myelin associated glycoprotein (MAG), myelin basic
protein (MBP), S100 beta or vimentin. GFAP and/or
S100 positive cells are frequently termed astrocytomas,
MBP- or MAG-positive cells; Oligodendrogliomas
and cells that stain for both sets of markers are mixed
gliomas. While these names imply a known and well-
defined lineage relationship of these tumors with
normal glial cell type or their progenitor cells, such a
relationship has not actually been demonstrated and
the cell types of origin remain controversial. In stud-
ies addressing this question, investigators have trans-
fected glial progenitor cells with known mutations
in oncogenes and tumor suppressor genes and have
been able to induce a malignant transformation that
yielded tumor growth in mice, suggesting that com-
mitted glial progenitor cells may indeed be the most
likely cell type of origin (Dai et al., 2001).
Gliomas exhibit many of the characteristic features
of systemic cancers which include mutations in the
tumor suppressor genes P16 and P53, and amplification
and overexpression of certain oncogenic growth factor
receptors including EGF-R or PDGF-R (Von Deimling
et al., 1995). As with other cancers, angiogenesis or
the induction of new blood vessels in response to the
release of vascular endothelial growth factor is com-
mon (Plate and Risau, 1995). Furthermore, the release
of matrix degrading enzymes that facilitate the remod-
eling of the tumor associated extracellular space is com-
mon and facilitates cell invasion (Giese et al., 1994).
A glioma diagnosis is almost always fatal as current
treatment options are limited (Butowski et al., 2006).
By the time a tumor is detectable, it has frequently
seeded tumor cells throughout the nervous system,
and upon surgery these cells can quickly give rise to
recurrent malignancies. The diffuse pattern of cellular
invasion illustrated in Fig. 26.1 not only makes com-
plete surgical resection impossible, but also limits focal
treatments such as exogenous beam irradiation as cells
remote from the tumor will escape the radiation. Upon
recurrence, many gliomas become even more malig-
nant. Recurrence is believed to result from cells that
have invaded surrounding brain areas. Surprisingly,
little is known about the underlying mechanisms.
For example, pathways of cell migration are poorly
understood as are molecules involved in chemotaxis
and path finding. These aspects of glioma biology are
promising areas for future research as they may yield
more effective therapeutic tools. An important aspect
of tumor biology that has been well studied in recent
years and which will be ­discussed in greater detail in
 IV.  Cl Transport and cell volume regulation in glioma cells
this chapter pertains to biophysical and biomechanical
adaptations that support the migration and invasion
of gliomas into normal brain tissue. Some of these
findings may pertain to other migratory cells in the
brain and even to other cancers.
III.  Glioma migration and
invasion
As illustrated in Fig. 26.1, the boundaries between
a primary glioma and normal brain tissue are often
difficult to delineate at a macroscopic level. At a
microscopic level, thousands of glioma cells will have
diffusely invaded the surrounding areas of brain tissue,
and, over time, they will have spread to very distant
sites. Wherever possible, invading glioma cells appear
to take advantage of other structures in the brain to
migrate. For example, they frequently migrate along
nerve fiber bundles or, as illustrated in Fig. 26.2A,
along blood vessels. Whether the spaces along these
structures are more favorable for migration, or whether
there are other guidance cues, or a more slippery extra-
cellular matrix, is not entirely clear. Without question,
however, the narrowness of the extracellular space pro-
vides a significant impediment to cell migration. At the
electron microscopic level, invading cells appear elon-
gated, wedge shaped, and with an overall shrunken
appearance (Fig. 26.2B). This has led to the hypothesis
that glioma cells may dynamically adjust their cell vol-
ume as they invade. As illustrated in cartoon form in
Fig. 26.2C, and further discussed below, recent findings
support this hypothesis and suggest an important role
for Cl channels and transporters in this context.
IV.  Cl Transport and cell
volume regulation in is replaced by halide ions such as I or Br with known
glioma cells
As extensively discussed in Chapter 15 in this book,
all eukaryotic cells have developed powerful mecha-
nisms to maintain a constant cell volume even when
extracellular osmotic conditions change. Glioma cells are
not an exception; when exposed to a 50% hyposmotic
challenge they regulate their volume back to baseline
within just a few minutes. As illustrated in Fig. 26.3A,
this regulatory volume decrease (RVD) is inhibited by
drugs known to block Cl channels including NPPB,
Cd2 and DIDS with almost complete inhibition by
NPPB and Cd2 (Ernest et al., 2005). The remaining
volume regulatory response is inhibited by drugs that
521
Figure 26.2  Invading glioma cells in situ. A. Confocal images
of invading D54MG cells stably expressing EGFP (green). The
invading glioma cells adhere to blood vessels (red). Cells often
exhibit an elongated wedge-shaped appearance as shown in the
lower panel. B. The elongated shape is quite apparent in this elec-
tron micrograph that captured an invading glioma cell (*, recog-
nized because of the abundance of ribosomes and other organelles
that incorporate lead citrate and give a darker appearance) extend-
ing between normal brain cells, likely astrocytes (based on their
large nuclei and abundance of electron dense, glycogen deposits
throughout the cytoplasm). C. Cell shrinkage requires water efflux
which is driven by the concomitant efflux of Cl and K through
their respective ion channels. Glutamate is shown as a possible
motogenic stimulus acting via AMPA receptors that raise intracel-
lular [Ca2] which may in turn activate BK channels. (Panel B is
reproduced with permission from Soroceanu et al., 1999.)
block the K-Cl cotransporters (Ernest et al., 2005).
Furthermore, volume regulation is supported when Cl
permeability to Cl channels, but not when gluconate
substitutes for Cl (Ernest, 2007). These studies suggest
that RVD in glioma cells utilizes Cl as osmolyte, which
is released from the cell through Cl channels.
An important question is whether Cl may simi-
larly act as an osmolyte during cell volume changes
associated with cell invasion, a process that is very
different from cell volume changes elicited by osmotic
challenges. Under hyposmotic conditions, a gradi-
ent for Cl efflux is favored by the dilution of extra-
cellular ions with water, whereas under isosmotic
conditions, this is not the case, unless the cell has a
sufficiently high [Cl]i. Hence, the hypothesized cell
shrinkage of invading cells requires that intracellu-
lar Cl be accumulated so that an outward directed
522 26.  Chloride Transport in Glioma Growth and Cell Invasion

Figure 26.3  Volume regulation in glioma cells. A. On exposure to a 50% hypotonic challenge, glioma cells swell and regulate their vol-
ume back to baseline or even below the baseline level. This regulatory volume decrease is partially inhibited by NPPB or DIDS and is almost
completely inhibited by NPPB and Cd2. B. Glioma cells maintain an elevated intracellular [Cl] which is accumulated via the bumetanide-
sensitive Na-K-Cl cotransporter, NKCC1. Pharmacological inhibition of the cotransporter by 20 μM bumetanide causes a decrease in [Cl]i.
Exposure to 40 μM DIOA causes an increase in [Cl]i above control levels, presumably by inhibition of KCC mediated Cl efflux. C. Western
blot of lysates from two glioma cell-lines D54-MG and U251, and from samples obtained from one patient (GBM50), show prominent expres-
sion of NKCC1 but absence of NKCC2. Rat kidney lysates were used as control for NKCC2. D. Immunostaining also shows prominent mem-
brane associated labeling for NKCC1 in representative U251 glioma cells. Antibodies directed against NKCC1 and NKCC2 were from Alpha
Diagnostics, and were used at a dilution of 1:500. (Panels A and B are reproduced with permission from Ernest et al. (2005), and panel C is
reproduced with permission from Ernest and Sontheimer (2007).)

electrochemical gradient for Cl is maintained. Using
the Cl sensitive fluorescence indicator SPQ, intracel-
lular [Cl] was measured in glioma cells and found to
be around 100 mM (Ernest, 2007), a value far greater
than that typically observed in mature central neurons
(7–10 mM) , glial cells (30–40 mM) or mature primary
afferent neurons (45 mM) (see Chapters 7, 19 and
22 in this volume). These findings were recently con-
firmed by patch-clamp studies at the single cell level
in which the reversal potential of Cl currents was
used as an indirect indicator of [Cl]i. Since glioma
cells in culture lack ligand-gated Cl channels, such
as the GABAA receptor-channel widely expressed in
neurons, recombinant ligand-gated non-inactivating
Cl channels (GABA-rho) were introduced into glial
cells, and stable cell-lines expressing GABA-gated
Cl channels were created. Gramicidin-perforated
patch recordings allowed determination of the rever-
sal potential of the GABA-induced currents (EGABA).
[Cl]i was estimated from EGABA (Habela et al., 2009).
These studies indicated an intracellular [Cl] of
105 mM in glioma cells, a value close to that deter-
mined using SPQ. Hence, glioma cells maintain a
steep outward directed gradient for Cl. In most cells,
Cl is actively accumulated via the Na-K-2Cl
cotransporter (NKCC1), which is a widely expressed
Cl importer (Chapters 2, 16 and 19 in this volume).
Western blot and immunostaining analyses of sev-
eral glioma cell-lines, including those obtained from
acute patient biopsies, demonstrated prominent
expression of NKCC1 and absence of NKCC2 (see
Fig. 26.3C and 26.3D as well as Ernest and Sontheimer,
2007). Gliomas also express KCC1 and KCC3 (Ernest
et al., 2005). Consistent with NKCC1 being princi-
pally responsible for the accumulation of intracellu-
lar Cl above electrochemical equilibrium in gliomas,
pharmacological inhibition of the cotransporter with
bumetanide causes a significant drop in intracellular
 V.  Changes in cell volume of invading cell require Cl efflux via clc channels
Cl (Fig. 26.3B). As we will be discussing below, high
intracellular [Cl] is possibly required for immature
neurons and glioma cells to migrate. High Cl might
facilitate water extrusion and cell shrinkage, processes
necessary for migrating cells to navigate through con-
fined extracellular spaces.
V.  Changes in cell volume of
invading cell require Cl efflux
via clc channels
As illustrated in Fig. 26.2C migratory glioma cells
appear to undergo profound changes in cell volume as
they invade surrounding tissues. We hypothesize that
these spontaneously occurring changes in cell volume
are driven by efflux of Cl and obligatory water. The
notion that a favorable outward Cl gradient is estab-
lished by NKCC1 was experimentally tested in a recent
study (Habela et al., 2009). Glioma cells were stably
transfected with GABA-rho channels, and subjected
to volume measurements using a Coulter Counter.
Application of GABA induced opening of Cl chan-
nels which caused progressive cell volume decrease.
This cell shrinkage was not observed in untransfected
cells, or in the absence of GABA. This suggests that
opening of Cl channels causes efflux of Cl associated
with obligatory water loss, and cell shrinkage. These
experiments also suggest that Cl efflux is sufficient to
induce a volume decrease in glioma cells. Interestingly,
these studies were made by inserting a ligand-gated
Cl channel which could be activated on demand, but
glioma cells express a significant resting Cl conduc-
tance (Ransom et al., 2001). Indeed, when recorded
using perforated patch-clamp technique to avoid dis-
turbing cytosolic Cl, glioma cells exhibit a resting Cl
conductance sensitive to NPPB and DIDS. These cur-
rents are outwardly rectifying, show time-dependent
inactivation at positive potentials and exhibit the fol-
lowing anion permeability sequence: IBrCl.
However, although the currents could be potentiated
by cell swelling, this was not required for current acti-
vation. Because Cl channel inhibitors still lack speci-
ficity, attributing the inhibitory effect of NPPB and
DIDS to a specific ion channel is not yet possible. As
a first step towards the molecular identification of the
Cl channels expressed in glioma cells, Western blots
using lysates obtained from gliomas isolated from
patients were probed with antibodies directed against
epitopes of cloned Cl channels. These studies dem-
onstrated the presence of ClC-2, ClC-3 and ClC-5
proteins in all gliomas examined (Olsen et al., 2003).
Further, immunolabeling studies showed that ClC-3
523
staining was predominant in invading processes of
isolated ­glioma cells. In an effort to further identify the
Cl channels functioning in gliomas, cells were treated
with antisense oligonucleotides to known members
of the ClC Cl channels’ super family. These stud-
ies showed prominent expression of currents attrib-
utable to ClC-2 and ClC-3, respectively (Olsen et al.,
2003). ClC-2 currents, known to be sensitive to Cd2,
inwardly rectifying, and potentiated by a negative pre-
pulse to 120 mV, were selectively eliminated in
glioma cells treated with ClC-2 antisense oligonucle-
otides (Fig. 26.4A). As expected, these currents were
unaltered by ClC-3 antisense oligonucleotides.
ClC-3 channels giving rise to outwardly rectifying
currents that show time-dependent inactivation and
are sensitive to NPPB were greatly reduced in glioma
cells treated with ClC-3 antisense oligonucleotides
(Fig. 26.4B). These data are consistent with both ClC-2
and ClC-3 channels being functionally expressed in
gliomas. However, functional expression of ClC-3
channels in the plasma membrane is controversial, as
discussed in detail in Chapter 12 of this volume. ClC-
3 knockout mice primarily show CNS pathology asso-
ciated with loss of synaptic vesicles in hippocampal
neurons (Stobrawa et al., 2001). Thus, whether ClC-3
protein is able to generate functional channels in the
plasma membrane has been questioned. Immuno-
gold electron microscopy of human gliomas, however,
shows immunoreactivity associated with both plasma
membrane and intracellular vesicles (Fig. 26.4C).
Further, ClC-3 in cultured glioma cells colocalizes to
the -subunit of cholera-toxin, which binds to lipid
raft domains, arguing for membrane localization of
the Cl channel (see merged signals in Fig. 26.4D).
Figure 26.5A shows that currents with the biophysi-
cal signature of ClC-3 are inhibited in a dose-dependent
fashion by chlorotoxin (Cltx), a peptide isolated from
the venom of the scorpion Leiurus quinquestriatus
(DeBin et al., 1993). This toxin might inhibit Cl chan-
nels with some specificity (McFerrin and Sontheimer,
2005). Importantly, as shown in Fig. 26.5B–C, when
cells were challenged to cross a transwell barrier that
mimics the spatial constraints of the extracellular
space in the brain, cell migration across the barrier was
inhibited when the Cl conductance was blocked with
NPPB (Ransom et al., 2001), Cd2 or Cltx (Soroceanu
et al., 1999). Of all these drugs, NPPB and Cltx were
the most effective inhibitors of cell migration in the
transwell assays. Cltx in both its native and recom-
binant form inhibited transwell migration in a dose-
dependent fashion (Deshane et al., 2003). Further, a
fluorescently labeled Cltx showed binding to the cell
surface of human malignant glioma cells in patient
biopsies. Based on these data we proposed ClC-3 as a
524 26.  Chloride Transport in Glioma Growth and Cell Invasion

Figure 26.4  Glioma cells express functional ClC-2 and ClC-3 channels. Using specific antisense oligonucleotides to ClC-2 (A) and ClC-
3 (B), it was possible to identify currents attributable to these channels, respectively. The Western blots illustrate effective reduction in cor-
responding protein expression following antisense treatment, demonstrating specificity of the effects observed in the membrane currents.
C. Immuno-gold EM with antibodies to ClC-3 show clusters of channels at the cell surface (thin white arrow) as well as in intracellular vesicles
(thick white arrow). D. Merged image of triple immunolabeling of cultured D54-MG (human glioma cell line). ClC-3 antibody (labeled with
alexa 546) shows that this protein colocalizes with lipid rafts which are identified by immunolabeling of the beta subunit of cholera-toxin
(fluorescein-conjugated cholera-toxin  subunit). Nuclei were counterstained blue with DAPI. (Panels A and B were modified from Olsen et al.
(2003); C is an unpublished image; and D is reproduced with permission from McFerrin and Sontheimer (2006).)

prime candidate for mediating the Cl fluxes required
to accomplish the cell shrinkage needed for glioma
cell migration. The data further suggest that ClC-3
may be a biological target of Cltx and that the latter
might be a potent inhibitor of glioma cell migration.
VI.  Mechanism of chlorotoxin
action on glioma invasion
The finding that Cltx inhibited Cl channels (Fig.
26.5A) and was effective in preventing cell invasion
in transwell assays (Fig. 26.5B) prompted further studies
on its mechanism of action; Cltx has a potential clini-
cal use as an anti-invasive drug. While biophysical
studies suggested that Cltx inhibits Cl channels in
glioma cells, it took up to 15 minutes to achieve its
maximal effect. This long delay questioned a direct
action on the channels; channel-specific toxins typi-
cally act almost instantaneously. Using a His-tagged
recombinant Cltx, Deshane and collaborators were
able to isolate a protein complex and analyzed it by
mass-spectroscopy (Deshane et al., 2003). These stud-
ies showed that matrix-metalloproteinase-2 (MMP-2),
a 72 kD protein that is highly expressed on the surface
of invading glioma cells, could be the primary binding
site for Cltx. However, the isolated protein complex
 VII.  Clinical use of chlorotoxin 525

Figure 26.5  Inhibition of Cl channels with chlorotoxin retards glioma cell migration. A. Representative currents in response to volt-
age steps, recorded before (control) and 15 minutes after application of 1 M chlorotoxin. Outwardly rectifying, inactivating Cl− currents were
recorded by whole-cell patch-clamp in D54-MG glioma cells using 20 mV voltage steps ranging from −120 to 160 mV. B. To show that a chlo-
rotoxin-sensitive Cl− conductance is required for migration across a spatial barrier, D54-MG glioma cells were plated on the upper surface of a
Transwell insert with 8 μm pores and allowed to migrate for 4 hours towards vitronectin coated on the bottom of the filter insert (top left). Under
control conditions, most cells migrated successfully, indicated by crystal violet staining of cells (control). In the presence of 5 μM chlorotoxin only
a few cells migrated through the filter. C. Chlorotoxin inhibits glioma cell migration. Dose–response curve of D54-MG cells treated with His-Cltx
or commercial Cltx peptide (Alomone) and analyzed by matrigel invasion assay at 24 h post-treatment. Half maximal inhibition (IC50) for Cltx was
184 nM. Percent inhibition was calculated as the decrease in the number of migrated cells normalized to control. (Panel A reproduced with per-
mission from McFerrin and Sontheimer (2006); panels B and C are reproduced with permission from Deshane et al. (2003).)

also contained ClC-3 channels and several regulators

of MMP-2. To further investigate how Cltx may have
decreased Cl channel function, cell surface expres-
sion was examined by biotinylation. These studies
showed that upon application of Cltx, membrane
associated ClC-3 channels gradually disappeared and
were almost undetectable at the surface after 30 min-
utes (McFerrin and Sontheimer, 2005). Further, it was
observed that the plasma membrane channels ended
up in intracellular caveolar vesicles. In the presence of
filipin, a sterol-binding drug that prevents the forma-
tion of caveolar vesicles, Cltx lost its effect on ClC-3
channel internalization. This suggested that binding of
the toxin induces the internalization of ClC-3 channels
together with Cltx into caveolar raft vesicles. These
findings explain the intracellular trapping of Cltx
observed in other studies, including those in humans,
as discussed below.
VII.  Clinical use of chlorotoxin
In light of the specific binding of Cltx to cultured
glioma cells, it was logical to explore the biological
activity of this molecule in animal models of malig-
nant glioma. Using a radiolabeled peptide we dem-
onstrated its specific binding to human gliomas
xenografted into the cerebrum of immunocompro-
mised mice (Soroceanu et al., 1998). These studies were
followed by screening of human tissues searching
for specific binding of Cltx (Lyons et al., 2002). These
studies, which examined over 100 samples, revealed
binding of Cltx to gliomas of all malignancy grades,
as well as to tumors that share an embryological rela-
tionship with them. The latter includes primarily can-
cers originating from neuroectodermally derived
tissues such as melanoma or small lung cell carcinomas.
526 26.  Chloride Transport in Glioma Growth and Cell Invasion
In contrast, non-malignant tissues were ­universally neg-
ative. In 2002, a synthetically manufactured Cltx was
approved by the US Food and Drug Administration
(FDA) to be examined in 18 patients in a phase I study.
Like in the previous preclinical studies, this trial used
a radiolabeled form of the peptide which was intro-
duced through an intrathecal catheter. The radiolabel
could then be detected by whole-body gamma cam-
era scans (Fig. 26.6A), or with greater resolution, by
SPEC imaging (Fig. 26.6B). In this study, fluid sam-
ples were collected to determine the pharmacokinet-
ics of the molecule. The data from this clinical study
were published in 2006 (Mamelak et al., 2006). Sample
images like those shown in Fig. 26.6 were published in
2005 (Hockaday et al., 2005). The safety and localiza-
tion data gathered in the phase I trial justified further
use of Cltx in 59 patients, in a phase II clinical study
which concluded recently. Preliminary data released
from this trial showed a significant increase in mean
Figure 26.6  The Cl channel inhibitor chlorotoxin localizes to
gliomas in vivo. A. A single dose of 131I-chlorotoxin given to a patient
in a phase I clinical study shows tumor-specific localization in whole-
body scans performed over a 5 day period (modified from Shen et al.,
2005). B. Overlay of MRI and SPECT images showing tumor-specific
retention of chlorotoxin, 8 days after administration of the drug.
Axial view of T1-Wc (left), coregistered (middle), and SPECT (right).
(Reproduced with permission from Hockaday et al., 2005.)
s­ urvival, following administration of three or six doses
of Cltx. Importantly, imaging studies such as those
illustrated in Fig. 26.6 suggest that Cltx is retained at
the tumor for 5–8 days. This observation is in good
agreement with the internalization of Cltx together
with ClC-3 and MMP-2 into caveolar vesicles. The
therapeutic efficacy of Cltx is therefore, in all likeli-
hood, due to (1) the internalization of ClC-3 channels
and decreased cell migration and (2) the trapping of
the radiolabel toxin which could have its own effect on
cellular DNA.
VIII.  Cell volume changes
associated with cell
proliferation
In addition to being highly invasive, primary brain
tumors also exhibit relentless growth, with mitotic
indices suggesting that over 30% of high-grade glio-
mas are in the active process of cell division. As cells
divide, they give rise to two daughter cells of approx-
imately half the volume of the parent cell. Yet, within
just a few hours, cell size and volume are restored
in both daughter cells. Surprisingly, little is known
about cell volume changes occurring in dividing cells
in general (see Chapter 27 in this volume). In a recent
study, we imaged complete cycles of cell division
using three-dimensional time-lapsed video micros-
copy following individual cells from birth through
to the next cell division giving rise to new daughter
cells (Fig. 26.7A, B). In this study, cell volume was
obtained from 200 to 400 serial sections at each time
point, allowing relatively accurate cell volume mea-
surements for the entire cell cycle (Fig. 26.7C). We
demonstrated a reduction in cell volume prior to the
M-phase of the cell cycle (Fig. 26.7D), a phenomenon
which we termed ‘‘pre-mitotic volume condensation’’
(Habela and Sontheimer, 2007). Regardless of the
cell volume that a cell maintains during interphase,
it condenses to a volume of approximately 6000-fL
prior to entering into M-phase, approximately 6 h
before giving rise to two daughter cells of approxi-
mately 3000-fL volume (Fig. 26.7D). The condensed
cells have already synthesized the cell membrane of
the two daughter cells, as this is readily visible by the
thickened membrane (Fig. 26.8A). This finding was
entirely unexpected, as the common assumption has
been that cells grow in size continuously until divi-
sion occurs. A contraction of the cytoplasmic volume
was not expected. Furthermore, the fact that the cell
 VIII.  Cell volume changes associated with cell proliferation 527

Figure 26.7  3D time-lapse imaging of glioma cells division allows accurate determination of cell volume throughout the cell cycle pro-
cess. A. 3D projections created from image z-stacks computed from 200 optical sections such as those shown in B, and rendered in 3D using
ImagePro. This program also computed volumes in fL for each 3D rendered cell. B. Sections from the z-stack used to generate the correspond-
ing projections shown in A. Images are in chronological order from 1 to 5. C. Volume measurements (in fL) at specific time points relative to
division are shown for four cells including the cell in A (green triangle symbols). Time points 1 through 4 correspond to projections 1–4 in B.
For each cell, M-phase was set at t  0 minutes. Note the convergence in volumes immediately before M-phase, where volumes are tightly
clustered around 6000 fL. D. Cells assume a volume of 6000 fL as they reach M-phase, regardless of their volume during interphase (n  14
cells). (Reproduced with permission from Habela and Sontheimer, 2007.)

membrane thickens as the cell volume condenses sug-
gests that, at this stage, cells have membrane folds
ready to be unfolded once a cell division and separa-
tion of two daughter cells has occurred. Upon divi-
sion, to achieve normal volume, each daughter cell
only needs to reaccumulate water through the uptake
of Na and Cl, presumably via NKCC1. The vol-
ume changes that may occur through the cell cycle
are illustrated in Fig. 26.8B. Importantly, studies that
directly compared intracellular [Cl] in M-phase cells
versus the bipolar interphase cells showed a 40%
reduction in [Cl]i in the condensed M-phase cells,
suggesting that Cl efflux is mechanistically linked
to the cell volume reduction (Habela et al., 2009).
Closer examination also showed that cytoplasmic
condensation is accompanied by condensation of
nuclear chromatin and indeed, the two processes
appear to occur in close synchrony (Habela and
Sontheimer, 2007). The initial condensation of the
cytoplasm and hence the chromosomal condensa-
tion are mediated by the efflux of Cl through the
same ClC-3 channels that are involved in cell volume
decreases associated with invading cells since shRNA
knock-down of ClC-3 impaired cell condensation
(Habela et al., 2008). While pharmacological studies
have long suggested a role for Cl channels in cell
division, these studies are the first to ascribe a mech-
anistic role to these channels in cell division; they
mediate cytoplasmic condensation through water loss,
a necessary step for cells to enter the M-phase.
528 26.  Chloride Transport in Glioma Growth and Cell Invasion

Figure 26.8  A. Membrane thickening of M-phase cells following volume condensation are displayed by labeling cells expressing cytosolic
eGFP (green) with membrane bound DiI (red). A. In interphase, the cell membrane associated DiI is a thin membrane layer surrounding the
cytoplasm. In M-phase, this area is thickened suggesting a ruffled membrane. B. Changes in cell volume during the cell cycle illustrated in
cartoon form. As cells progress through G1/S, they increase their plasma membrane area and overall cell volume. As they progress to the M-
phase they condense their cytoplasmic volume but maintain their cell membrane area which becomes thickened and folded. Cell division into
two daughter cells divides membrane and cytoplasm equally between daughter cells. The acquisition of new membrane is accompanied by
uptake of Na, Cl and water through NKCC1, which establishes the normal cell size/volume. (Reproduced with permission from Habela
and Sontheimer, 2007.)

IX.  Conclusions
Taken together, the data discussed in this chapter
suggest that Cl channels and transporters cooperate
to support dynamic changes in cell volume that gov-
ern cell proliferation and cell migration/invasion. The
outward electrochemical gradient for Cl is estab-
lished by the Na-K-2Cl cotransporter, NKCC1,
and this gradient permits rapid Cl efflux through
Cl channels and obligatory water movement
across the plasma membrane, ultimately leading to
cell volume reduction. It is conceivable that similar
mechanisms operate in other migratory cells, e.g.
developing neurons, stem cells, and other cell types
which migrate during development prior to settling
down, maturing and forming tissues. Furthermore,
a similar role for Cl channels in the control of cell
growth and proliferation may widen the therapeu-
tic potential for Cl channel blockers as anti-cancer
reagents.
Acknowledgements
The author is grateful for the continued support
by grants from the National Institutes of Health RO1
NS-31234, RO1 NS-52634, NS-36692 and P50-CA97247.
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