Targeting ανβ3 and ανβ5 inhibits photon-induced hypermigration of malignant glioma cells.

Abstract

Sublethal photon irradiation was recently suspected to increase tumor cell motility and promote locoregional recurrence of disease. This study was set up to describe mechanisms underlying increased glioma cell migration through photon irradiation and to analyse the modifiability of photon-altered glioma cell motility by integrin inhibition.
Eight μm pore size membranes were coated with vitronectin (VN), collagen I and collagen IV. U87 and Ln229 glioma cells were analysed in migration experiments with and without radiotherapy (RT), serum stimulation and addition of monoclonal antibodies directed to human integrins ανβ3 and ανβ5. Quantitative FACS analysis of integrins was performed in U87 and Ln229 glioma cells following RT. Statistical analysis was performed using Student's t-test.
Glioma cell migration is serum-dependent and can be increased by photon RT which leads to enhanced expression of Vn receptor integrins. Blocking of either ανβ3 or ανβ5 integrins by antibodies inhibits Vn-based migration of both untreated and photon-irradiated glioma cells.
Peripheral glioma cells are at risk of attraction into the adjacent healthy brain by serum components leaking through the blood brain barrier (BBB). Radiation therapy is associated with upregulation of Vn receptor integrins and enhanced glioma cell migration at sublethal doses. This effect can be inhibited by specific integrin blockade. Future therapeutical benefit may be derived from pharmacological integrin inhibition in combination with photon irradiation.

Full text

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RESEARCH
Targeting aνb3and aνb5inhibits photon-induced
hypermigration of malignant glioma cells
Open Access
Stefan Rieken*, Daniel Habermehl, Angela Mohr, Lena Wuerth, Katja Lindel, Klaus Weber, Jürgen Debus and
Stephanie E Combs
Abstract
Background: Sublethal photon irradiation was recently suspected to increase tumor cell motility and promote
locoregional recurrence of disease. This study was set up to describe mechanisms underlying increased glioma cell
migration through photon irradiation and to analyse the modifiability of photon-altered glioma cell motility by
integrin inhibition.
Methods: Eight μm pore size membranes were coated with vitronectin (VN), collagen I and collagen IV. U87 and Ln229
glioma cells were analysed in migration experiments with and without radiotherapy (RT), serum stimulation and
addition of monoclonal antibodies directed to human integrins aνb3and aνb5. Quantitative FACS analysis of integrins
was performed in U87 and Ln229 glioma cells following RT. Statistical analysis was performed using Student’s t-test.
Results: Glioma cell migration is serum-dependent and can be increased by photon RT which leads to enhanced
expression of Vn receptor integrins. Blocking of either aνb3or aνb5integrins by antibodies inhibits Vn-based
migration of both untreated and photon-irradiated glioma cells.
Conclusions: Peripheral glioma cells are at risk of attraction into the adjacent healthy brain by serum components
leaking through the blood brain barrier (BBB). Radiation therapy is associated with upregulation of Vn receptor
integrins and enhanced glioma cell migration at sublethal doses. This effect can be inhibited by specific integrin
blockade. Future therapeutical benefit may be derived from pharmacological integrin inhibition in combination
with photon irradiation.
Keywords: glioma, radiotherapy, migration, integrin, vitronectin
Introduction
Despite continuously evolving therapy regimes including
extensive neurosurgery, multiagent chemotherapy, and
dose-escalated conformal radiotherapy, primary brain
tumors have not ceased to account for high lethality after
short periods of time in most patients. Deep locoregional
tumor cell infiltration that eludes modern imaging techni-
ques and hampers complete local resection was accounted
responsible for early relapse and spread of disease
throughout the brain. Current glioma therapy involves
surgical tumor resection followed by adjuvant radiotherapy
combined with concomitant and adjuvant chemotherapy
[1].
As opposed to the tissue they originate from, most
tumor cells, including malignant glioma cells, possess the
unique ability to migrate and adhere to various surfaces,
displaying polyligand-induced motile phenotypes where
non-malignant cells are subjected to strictly regulated
tissue architecture. Deregulated tumor cell migration is
typically associated with infiltration and dissemination,
resulting in local disease progression and metastases, both
of which account for the majority of cancer-associated
deaths. In gliomas abundant promigratory mediators have
been identified including lipids and peptides, all of which
can be detected in serum reaching the brain via the
tumor-disrupted BBB [2-6].
Besides factors of the microenvironment surrounding
the tumor, also its treatment may effect the migratory
behavior of tumor cells. Radiation therapy, which is
implemented in virtually all concepts of glioma
* Correspondence: Stefan.rieken@med.uni-heidelberg.de
University Hospital of Heidelberg, Department of Radiation Oncology, Im
Neuenheimer Feld 400, 69120 Heidelberg, Germany
Rieken et al. Radiation Oncology 2011, 6:132
http://www.ro-journal.com/content/6/1/132
© 2011 Rieken et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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treatment, was recently observed to increase tumor cell
motility in vitro at sublethal doses < 3 Gray (Gy) [7,8].
Increasing cellular movement in malignant gliomas
would undermine the therapeutical intent and possibly
impose a greater risk of deep locoregional tumor infil-
tration and metastasization in vivo onto the patients
than even without therapy. Furthermore, photon irradia-
tion is kown to modulate the expression of extracellular
matrix proteins and thus alter the motility-determining
environment of malignant gliomas [9].
Depending on their tissue of origin, tumor cells employ
a variety of ECM proteins to adhere to and migrate on.
Primary brain tumors are known to produce and contain
abundant amounts of collagens and other ECM compo-
nents that promote increased motility, induce invasion
and clinically account for poor local control [10,11]. Mole-
cular therapies have long been introduced into the treat-
ment of malignant gliomas and have defined epithelial and
vascular growth factor but also integrin receptors as pro-
mising targets [12-14]. Integrin signalling is known to sig-
nificantly impact glioma cell motility but also survival, and
has therefore emerged as a promising approach to targeted
glioma treatment [15]. To date, only little data exists
addressing the impact that a combination of photon irra-
diation and integrin-inhibition may have on glioma cell
migration. This study was set up in order to characterize
ECM-based motility of U87 and Ln229 glioma cells after
photon irradiation and to analyse the impact of inhibition
of Vn receptor integrins in combination with radiotherapy.
Materials and methods
Cell culture
U87-MG glioma cells were purchased from LGC Promo-
chem (ATTC HTB-14), and kept at 37°C and 5% CO2in
DMEM (FG0415 Biochrom AG) supplemented with 1%
Penicilline/Streptomycine and 10% FCS. Ln229 glioma
cells were purchased from LGC Promochem (ATTC CRL-
2611), and kept at 37°C and 5% CO2in DMEM (FG0415
Biochrom AG) supplemented with 1% Penicillin/Strepto-
mycin and 10% FCS. Twenty-four hours before adhesion
and migration experiments, cells were serum starved in
DMEM containing 1% Penicilline/Streptomycine and 0.5%
FCS. Passaging of cells was performed every week.
Surface coating with extracellular matrix proteins
For migration assays, polycarbonate membranes with 8 μm
pores were coated with 50 ng/cm2vitronectin, 0.5 μg/cm2
collagen I and 0.5 μg/cm2collagen IV over night at 4°C
and washed in twice destillated and salt-free water prior to
the experiments.
Migration assay
Five × 103cells were loaded in the upper chamber of a
48-well modified microchemotaxis chamber (Multiwell
Chemotaxis Chamber, Neuro Probe). The lower well
contained cell culture medium with 0.5% FCS and che-
moattractants as indicated. Lower and upper chambers
were separated by a 8 μm pore size polycarbonate mem-
brane, that had been coated with vitronectin (50 ng/cm2),
collagen I (0.5 μg/cm2) and collagen IV (0.5 μg/cm2)
24 hours before the start of migration. Cells were serum-
starved in medium containing 0.5% FCS 24 hours prior
to the start of migration. Radiation treatments were per-
formed 24 hours before assessment of migration. Before
staining and mounting of Boyden chamber membranes,
non-migrated cells on the upper filter side were removed
by drawing the filter over a wiper blade at least twice.
Cytoseal XYL mounting medium was used for filter pre-
servation (Richard-Allan Scientific). Transmigrated cells
were stained with DiffQuick®and counted with a Leica
DC300F microscope. Integrin blockade was performed
using monoclonal antibodies directed against aνb3-
(MAB3050, R&D) and aνb5-integrins (MAB 2528, R&D).
All assays were done in at least triplicates and wells were
counted by an investigator blinded to experimental set-
up. Cell numbers are expressed as multiples of controls
or as proportion of inputs.
FACS analysis
Twenty-four hours after irradiation, cells were fixed with
70% ethanol and stained with a PE-labelled anibody
directed against aνb3(555505, BD) and a FITC-labelled
antibody directed against aνb5(FAB2528P, R&D). Ln229
and U87 glioma cells were analysed with a three-colour
FACScan flow cytometer and CellQuestPro software
(BD Biosciences). Results are displayed with histogram
plots and subsequent quantitative analyses.
Radiation treatment
Photon radiation was performed using with a 6 MeV lin-
ear accelerator (Siemens, Erlangen, Germany). We
applied single doses of 2, 5, and 10 Gy 24 hours prior to
migration experiments.
Statistics
All migration experiments and FACS analyses were per-
formed at least three times. Modified Boyden Chamber
assays were set up in triplicates and analysed by an investi-
gator blinded to experimental setup. Data are displayed as
means ± standard deviation (SD). Comparisons between
two groups were performed with Student’s t-test.
Results
Glioma cell migration is promoted by serum exposition
Modified Boyden chamber assays were performed to ana-
lyse transmigration of U87 glioma cells through 8 μm
pore size polycarbonate membranes coated with Vn and
collagen I and IV. In order to mimic a disturbed BBB
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with leakage of blood-borne mediators, such as often
detected in brain tumors, cells were attracted by serum.
This gradient clearly increased the number of transmi-
grated glioma cells (Figure 1). Compared to unstimulated
conditions, serum exposition using a 10% FCS gradient
increased chemotactic transmigration by a factor of 2.91
(p < 0.0001), 2.90 (p = 0.0004) and 2.89 (p < 0.0001)
through Vn-, collagen I- and collagen IV-coated filters
(Figure 1).
Sublethal photon irradiation enhances glioma cell
migration
Since photon irradiation is implemented in most glioma
treatment protocols, we irradiated U87 glioma cells with
single photon doses of 2 Gy and analysed transmigration
24 hours afterwards. Single photon doses of 2 Gy pro-
moted glioma cell transmigration (Figure 2). On col-
lagen I-coated surfaces, the fraction of transmigrated
cells was increased from 15.3% to 22.9% (p = 0.0002);
on collagen IV transmigration was raised from 11.6% to
20.1% (p = 0.01). The highest photon stimulation of
migration was detected on Vn-coated membranes,
where an increase from 11.1% to 32.3.% was observed
(p < 0.0001).
Photon irradiation increases expression of vitronectin
receptor integrins aνb3and aνb5
Quantitative FACS analyses using PE- and FITC-labelled
antibodies directed against the Vn receptor integrins aνb3
and aνb5on cell surfaces were performed in order to
investigate the effects of photon RT on integrin expres-
sion. We found that single doses of 2 and 10 Gy caused a
rightshift in the FACS histograms (Figure 3A) and
increased the expression of both aνb3and aνb524 hours
after irradiation (Figure 3B). This rightshift was consistent
for all doses tested and for both aνb3and aνb5in U87
cells. However, statistical significance was only reached for
expression of aνb5after 10 Gy (p < 0.05).
To confirm that this phenomenon was not limited to
U87 glioma cells (Figure 3B, upper row), we analysed
Ln229 glioma cells and found the same phenotype of
enhanced integrin expression following photon RT
Figure 1 FCS stimulation of U87 glioma cell migration. Transmigration through DiffQuik®-stained 8 μm pore size polycarbonate membranes
coated with Vn, collagen I and IV with and without serum stimulation (magnification, x20). Induction of transmigration by a 10% FCS gradient
displaying a 2.91-fold increase on Vn (p < 0.0001), a 2.90-fold increase on collagen I (p = 0.0004), and a 2.89-fold increase on collagen IV (p <
0.0001). Display of mean value ± standard deviation (SD); statistical analysis using Student’s t-test (*, indicating significance p < 0.05).
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(Figure 3B, lower row). Photon-induced upregulation of
integrins was particularly strong for aνb5in Ln229 cells,
where statistical significance was reached for all photon
doses tested (p < 0.05).
Inhibition of aνb3and aνb5significantly impairs photon-
induced hypermigration
In order to analyse the effect of the integrin-disruption,
we used Vn receptor-targeting anti-aνb3- and -aνb5-
antibodies in Vn-based migration experiments and
found that concentrations of 50 ng/ml impaired glioma
cell migration. Both single inhibition of either aνb3and
aνb5caused significantly reduced transmigration in both
U87 (Figure 4) and Ln229 (Figure 5) cells. Besides in
untreated cells, also photon-stimulated cells were signifi-
cantly inhibited from migrating through Vn-coated
membranes. Strongest inhibition was achieved when
both anti-aνb3- and anti-aνb5-antibodies were added.
Addition of isotype controls did not affect migration.
Discussion
In the present manuscript we investigated a repeatedly
described phenomenon of photon-induced tumor cell
migration and found that promotion of glioma cell che-
motaxis by photon doses of 2 Gy was particularly strong
on Vn-coated surfaces. Providing a molecular explana-
tion for this phenotype, we detected a consistent trend
of increased expression of the Vn receptor integrins
aνb5and aνb3following photon RT. Pharmacological
disruption of these integrins reversed the phenomenon
of photon-triggered migration and may therefore serve
as novel approach for combination treatments.
Tumor cell exposition to serum components at the dis-
turbed BBB in glioma patients is generally accepted to
account for locoregional infiltration and early tumor
relapse. Hartman et al have shown that in 75% of all
recurrent malignant glioma, relapse occurs within the
initial edema conformation [3]. Tumor-associated brain
edema represents breakdown of the BBB and allows the
entry of chemotactic serum compounds into the widened
interstitial spaces thus facilitating cell motility and pro-
moting clinical tumor infiltration [16,17]. We supplemen-
ted cell culture medium with 10% FCS, thus, exposing the
cells to a physiological combination of serum compounds
and found that migration was significantly enhanced on
Vn- and collagen-coated surfaces. Our findings support
previously established theories of tumor cell dissemination
along serum gradients and relapses of disease occurring in
environments with a promigratory milieu. Many models
were established in order to explain the deregulation of
tumor cell motility, including tumor cell specific adhesion,
migration, and infiltration. In malignant gliomas, distur-
bance of the BBB allows exposure of peripheral tumor
cells to serum components which the healthy brain tissue
is physiologically separated from [18]. Independently of
prior treatments, the site of exposure to serum in the
tumor periphery is known to represent the typical location
for tumor recurrence [3]. Various authors have found
glioma cells to migrate towards serum proteins, such as
albumin [2], and phospholipids, such as lysophosphatic
acid [4]. Also, blood-bourne chemokines such as CXCL-
12 are known to attract tumor cells and contribute to
their wide spread within the brain [5]. Due to continuous
ligand exposition, peripheral glioma cells are at highest
risk of chemotactic distraction.
Photon irradiation has a substantial role in modern
interdisciplinary cancer therapies. It is implemented in
most glioma treatment regimes and improves local con-
trol rates and survival [19]. However, several reports
have described sublethal photon doses lower than 3 Gy
to trigger tumor cell migration [7,8,20]. These doses are
commonly applied in fractionated RT and may therefore
impose a risk of enhanced tumor cell invasion and dis-
semination onto the patients. Wick et al showed that
single photon doses as high as 6 Gy increased chemo-
tactic glioma cell migration along with upregulated
integrin expression and enhanced activity of matrix
metalloproteinases [8]. Knowing that peripheral tumor
cells are at highest risk of ligand-induced distraction
and may at the same time be triggered to migrate by
sublethal photon RT requires either conceptual changes
in RT, such as wider safety margins or use of particle
irradiation or administration of additional motility-sup-
pressive agents.
We demonstrated that glioma cell migration was signif-
icantly enhanced by serum exposition on collagen I and
Figure 2 Photon stimulation of U87 glioma cell migration.
Graphical analysis of induction of transmigration by a single photon
doses of 2 Gy through membranes coated with collagen I (15.3% vs.
22.9% [p = 0.0002]), collagen IV (11.6% vs. 20.1% [p = 0.01]) and Vn
(11.1% vs. 32.3% [p < 0.0001]). Display of mean value ± standard
deviation (SD); statistical analysis using Student’s t-test (*, indicating
significance p < 0.05)
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collagen IV. Collagen I is detected abundantly in the
outer lining of the brain parenchyma, the glia limitans
externa. Collagen IV is typically found in the basement
membrane of blood vessels and critically contributes to
pathological vessel formation in malignant disease. Both
are known to represent preferred substrates for invasive
glioma cells which have been shown to migrate along dis-
tinct anatomical interfaces [21-23]. In our experiments,
we detected robust - both serum- and photon-responsive
- glioma cell interaction with both collagen I and IV.
However, due to the large family of collagen-binding
integrins and their presence in multitudinous tissues
essentially contributing to organ architecture, targeting
collagen-cell-interaction does not appear to be safely fea-
sible [24].
Vn and its main binding integrins, aνb3and aνb5, are
found in high density at the infiltrating invasion front of
high grade gliomas [25,26] and inhibition of aνb3was
demonstrated to slow down glioma cell migration [27].
Furthermore, soluble Vn was identified as one of the
major factors in serum and cerebrospinal fluid to induce
glioma cell migration [28]. Depletion of Vn in these
fluids caused significant decrease in migration. Vn there-
fore contributes to the malignant phenotype of gliomas
and also affects their sensitivity to treatment. The integ-
rins aνb3and aνb5represent physiological Vn receptors,
and they were described to significantly influence the
radiosensitivity of gliomas. Their downstream signalling
involves integrin-linked kinases and GTPases, and their
ligand-induction resulted in enhanced radioresistance in
vitro [29]. Pharmacological disruption of these signalling
cascades would, therefore, be an auspicious strategy in
glioma treatments and permit additional radiosensitiza-
tion. Our results support the hypothesis of Vn as a
radioprotective ECM protein, because the highest
photon-induced migration was detected on Vn-covered
Figure 3 Photon-induced stimulation of integrin expression. A: FACS analysis of aνb3(upper row) and aνb5(lower row) expression with
(red) and without (black) photon irradiation with single doses of 2 Gy (left) and 10 Gy (right). B: Graphical analysis of integrin aνb3(gray bars)
and aνb5(black bars) expression with (bars) and without (red line) irradiation with single photon doses of 2, 5, and 10 Gy in U87 (upper chart)
and Ln229 (lower chart) glioma cells. Display of mean value ± standard deviation (SD); statistical analysis using Student’s t-test (*, indicating
significance p < 0.05).
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membranes, where sublethal RT yielded an almost two-
fold increase.
We performed flow cytometry analyses and found a
trend toward increased expression of aνb3and aνb5fol-
lowing photon RT, while expression of b1-integrins
remained stable (data not shown). Our results are in
line with previously published results data on photon-
induced integrin upregulation in endothelial cells [30],
lung cancer cells [31], colon carcinoma cells and also
glioma cells [20]. At low photon doses, integrin expres-
sion corresponds to the significantly increased migration
of glioma cells on Vn-coated membranes following RT,
whereas increased integrin expression following 10 Gy
was associated with unaltered migration. This indicates
that at higher photon doses, further mechanism must
influence tumor cell motility. We confirm data
previously published by Goetze et al., who concluded
that radiotherapy may affect tumor cell migration in
partially opposing ways [20].
Since integrin signalling is susceptible to pharmacolo-
gical disruption, we added Vn receptor antibodies in
order to analyse the modifiability of glioma cell migra-
tion. The addition of either anti-aνb3- and -aνb5-antibo-
dies reduced transmigration of both untreated and
irradiated U87 and Ln229 cells. Blocking aνb3yielded
slightly stronger inhibition than did sole blocking of
aνb5. This is in line with several previous works that
have identified aνb3to be both predominant in Vn-asso-
ciated migration and also mediate signalling effects of
furtherECMcomponents,
[26,32-34]. However, the effect of integrin inhibition
could still be enhanced when anti-aνb3- and -aνb5-anti-
bodies were combined, thus fully abrogating Vn signal-
ling effects. Our experiments show that a photon-
derived induction of aνb3and aνb5expression with con-
secutively increased Vn-based glioma cell migration at
sublethal doses, can be successfully counteracted by
combining RT with targeted therapies.
such as fibronectin
Conclusion
Photon RT with single doses of 2 Gy increases glioma cell
migration via integrin-induction and may, therefore,
enhance the risk of tumor cell spread and infiltration.
Integrin-targeting antibodies effectively antagonize this
photon-induced increased migration. Therefore, they
represent a novel and promising approach to combination
treatments with fractionated photon RT especially during
early fractions when lethal doses have not been reached,
yet. Higher photon doses do not promote cell migration
despite integrin upregulation, and must therefore be inter-
preted with caution. Further studies are needed to evaluate
the clinical impact of these in vitro findings in a clinical
setting.
List of abbreviations
RT: radiotherapy; FCS: fetal calf serum; Vn: vitronectin; ECM: extracellular
matrix; BBB: blood brain barrier; Gy: Gray
Authors’ contributions
SR conceived of the study design, performed all experiments and wrote the
manuscript. DH and AM helped to analyse migration experiments. LW was
responsible for irradiation of the cells and for FACS analysis of integrin
expression. KL and KW supervised irradiation experiments. JD contributed
with regard to content, scientific context and financial support. SC
conceived of the study and helped to write and finalize the manuscript. All
authors helped with the interpretation of the data, read and approved the
final manuscript.
Competing interests
There are no conflicts of interest to declare. Stefan Rieken was supported by
the medical faculty of Heidelberg (PostDoc grant).
Received: 31 May 2011 Accepted: 6 October 2011
Published: 6 October 2011
Figure 4 Inhibiton of U87 glioma cell migration by integrin
inhibition. Quantitative analysis of Vn-based transmigration of U87
glioma cells following single photon doses of 0, 2, and 10 Gy
without and with the addition of 50 ng/ml anti-aνb3- and -aνb5-
antibodies and corresponding isotype controls.
Figure 5 Inhibiton of Ln229 glioma cell migration by integrin
inhibition. Quantitative analysis of Vn-based transmigration of
Ln229 glioma cells following single photon doses of 0, 2, and 10 Gy
without and with the addition of 50 ng/ml anti-aνb3- and -aνb5-
antibodies and corresponding isotype controls. Display of mean
value ± standard deviation (SD); statistical analysis using Student’s t-
test (*, indicating significance p < 0.05).
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doi:10.1186/1748-717X-6-132
Cite this article as: Rieken et al.: Targeting aνb3and aνb5inhibits
photon-induced hypermigration of malignant glioma cells. Radiation
Oncology 2011 6:132.
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