Effect of Brain- and Tumor-Derived Connective Tissue Growth Factor on Glioma Invasion

Article (PDF Available)inJournal of the National Cancer Institute 103(15):1162-78 · August 2011with30 Reads
DOI: 10.1093/jnci/djr224 · Source: PubMed
Abstract
Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF(200 ng/mL): 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF(200 ng/mL) + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.
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DOI: 10.1093/jnci/djr224 Published by Oxford University Press 2011.
Advance Access publication on July 19, 2011.
The invasive nature of malignant gliomas makes curative treat-
ment impossible and ultimately leads to the patient’s death. The
median survival after diagnosis of patients with the most common
gliomaglioblastomais 14 months (1). Thus, one of the most
challenging issues in glioma biology and developmental therapeu-
tics is the identification of the mechanism(s) responsible for glioma
cell infiltration. Previous work on this topic has largely involved
the use of established glioma cell lines. However, those cell lines
are poor representatives of the molecular and clinical biology of
primary human gliomas (2). Most importantly, few of the estab-
lished glioma cell lines are infiltrative in vivo, making the clinical
relevance of previous findings regarding mechanisms of glioma
invasion uncertain.
We and others have demonstrated that glioma tumor–initiating
or tumor stem cells (TIC/TSCs) represent a subpopulation of
primary human glioblastoma–derived cells that more closely reca-
pitulates the molecular, biological, and clinical behaviors of the
parental tumor (3–5). In particular, the highly infiltrative nature of
these cells in vivo makes them a potentially more clinically relevant
model for studying certain aspects of glioma biology, such as tumor
invasion, compared with the established glioma cell lines. Using this
new model, we now explore the role of a previously implicated
invasion factor (6)connective tissue growth factor (CTGF)in
human glioblastomas.
CTGF (also known as CCN2) is encoded by an immediate early
gene, one of the first genes to be activated in response to various
stimuli (7). The gene is located on human chromosome 6q23.1 and
encodes a highly glycosylated 38-kDa member of connective tissue
growth factor–cysteine-rich angiogenic inducer 61–nephroblastoma
overexpressed (CCN) family of growth factors (8). CTGF has been
ARTICLE
Effect of Brain- and Tumor-Derived Connective Tissue Growth
Factor on Glioma Invasion
Lincoln A. Edwards, Kevin Woolard, Myung Jin Son, Aiguo Li, Jeongwu Lee, Chibawanye Ene, Samuel A. Mantey, Dragan Maric,
Hua Song, Galina Belova, Robert T. Jensen, Wei Zhang, Howard A. Fine
Manuscript received November 16, 2010; revised May 13, 2011; accepted May 23, 2011.
Correspondence to: Howard A. Fine, MD, Neuro-Oncology Branch, National Cancer Institute/National Institute of Neurological Disorders and Stroke,
National Institutes of Health, 10 Center Dr, Bloch Bldg, Number 82, 9030 Old Georgetown Rd, Bethesda, MD 20892-1002 (e-mail: hfine@mail.nih.gov).
Background Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant
gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and inva-
sion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined
potential therapeutic targets.
Methods Highly infiltrative patient-derived glioma tumor–initiating or tumor stem cells (TIC/TSCs) were harvested and
used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immuno-
precipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with
small-molecule inhibitors targeting integrin b1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and
short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of
CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion
assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse
models (n = 30; six mice per group). All statistical tests were two-sided.
Results Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1–TrkA receptor complexes and nuclear
factor kappa B (NF-kB) transcriptional activation as measured by luciferase reporter assays (mean relative lucif-
erase activity, untreated vs CTGF
200 ng/mL
: 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2,
P < .001). NF-kB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP
analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter
region: untreated + ZEB-1 antibody vs CTGF
200 ng/mL
+ ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to
5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the
most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF.
Conclusion A CTGF-rich microenvironment facilitates CTGF–ITGB1–TrkA complex activation in TIC/TSCs, thereby increasing
the invasiveness of malignant gliomas.
J Natl Cancer Inst 2011;103:1162–1178
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implicated in a number of cellular functions, including those
contributing to tumor formation as well as those involved in
tumor suppression suggesting both cell type–specific and context-
dependent properties (9–11).
To date, however, the effects of CTGF on glioma cells and
glioma TIC/TSCs are not fully understood. It is known that
CTGF can bind the cell surface protein beta 1 integrin (ITGB1)
(12) and the tyrosine kinase receptor type A (TrkA) receptor and
co-receptor p75
NTR
(13), both of which have been implicated in
cancer progression (14,15). Furthermore, it has been shown that
CTGF is secreted by reactive astrocytes at sites of traumatic brain
injury (16,17).
The effects of CTGF on glioma cells and glioma TIC/TSCs,
and its precise molecular mechanism(s) of action within a given
context, however, have yet to be fully elucidated. In this study, we
examined the role of tumor- and host-derived CTGF in glioma
invasion.
Materials and Methods
Human Tissues and Glioma TIC/TSC Culture
Tumor samples and glioma TIC/TSCs were harvested as previ-
ously described (5).
Briefly, following signed informed consent, tumor tissue was
obtained from patients (aged 18 years) undergoing medically
indicated resection of malignant gliomas at the National Institutes
of Health as part of a clinical trial approved by the Institutional
Review Board (NCI-02C0140). Tissue sections from tumors path-
ologically identified as glioblastoma based on World Health
Organization criteria (18) were washed and enzymatically disag-
gregated into single cells. Isolated tumor cells were then cultured
in NBE medium consisting of Neurobasal A medium and N2 and
B27 supplements (all from Invitrogen, Carlsbad, CA), and human
recombinant basic fibroblast growth factor and epidermal growth
factor (50 ng/mL each; R&D Systems, Minneapolis, MN). TIC/
TSC nomenclature is based on a random numbering system devel-
oped within the laboratory to maintain patient confidentiality.
Harvested TIC/TSCs were subjected to oligonucleotide precipita-
tion (19) assays, chromatin immunoprecipitation (ChIP) assays
(20), luciferase assays, real-time cell invasion and migration assays,
immunoprecipitation, and immunoblotting and were used in an
intracranial xenograft model.
Cell Lines and Cell Culture
Human glioblastoma U87MG and U251MG cells were obtained
from the American Type Culture Collection (ATCC; Manassas,
VA) and were cultured in Dulbecco’s modified Eagle medium
(DMEM; Gibco, Carlsbad, CA) containing 10% fetal bovine
serum (FBS), 1% L-glutathionine, and 1% penicillin–streptomy-
cin (all from Gibco).
For studies of ligand binding to the TrkA receptor (see
“Competitive Binding Assay” section in “Materials and Methods”),
we used rat pheochromocytoma PC12 cells that stably overexpress
TrkA (PC12-TrkA cells; kindly provided by Dr Moses Chao).
PC12-TrkA cells were compared with the parental PC12 cells for
TrkA expression and verified by immunoblotting. PC12 cells were
cultured in DMEM containing 15% horse serum, 10% FBS, 1%
CO N TEXT AN D CAVEAT S
Prior knowledge
Glioma cell invasionthe main cause of treatment failure and death
among patients with malignant gliomashas been difficult to study
because most established glioma cell lines are neither tumorigenic
nor invasive in vivo. Connective tissue growth factor (CTGF) has
been implicated in metastasis and invasion in a number of cancers.
Study design
Glioma tumor–initiating or tumor stem cells (TIC/TSCs), a highly
infiltrative subpopulation of cells derived from primary human
glioblastomas, were used in in vitro and in vivo assays to examine
the role of tumor- and host-derived CTGF in glioma invasion and
the mechanism of CTGF-mediated glioma cell infiltration.
Contribution
Treatment of TIC/TSCs with CTGF resulted in the formation of a
complex containing CTGF, integrin b1 (ITGB1), and the tyrosine
kinase receptor type A (TrkA), which led to transcriptional activation
of nuclear factor kappa B , induction of the transcriptional repressor
ZEB-1, disruption of cell–cell contacts through loss of E-cadherin,
and glioma cell and TIC/TSC infiltration. Immunohistochemistry
and in situ hybridization revealed that TrkA is selectively expressed
in the most infiltrative glioma cells in situ and that the surrounding
reactive astrocytes secrete CTGF.
Implications
A CTGF-rich microenvironment facilitates CTGF–ITGB1–TrkA
complex activation in TIC/TSCs, thereby increasing the invasive-
ness of malignant gliomas.
Limitation
TIC/TSCs may not be the only population of infiltrative cells within
a given glioma.
From the Editors
L-glutathionine, and 1% penicillin–streptomycin (all from Gibco).
PC12-TrkA cells were maintained in the same medium plus 200
µg/mL G418 (Invitrogen) to select for cells that overexpress TrkA.
Human astrocytes (purchased from Lonza Cambrex, Allendale,
NJ) were cultured in DMEM/F12 medium containing 10% FBS,
1% penicillin–streptomycin, and 1% L-glutathionine (all from
Gibco). Cell line identities were confirmed by immunoblot
analysis of stem cell or astrocytic proteins or immunohistochem-
ical staining for stem cell and astrocytic markers (eg, nestin and
glial fibrillary acidic proteins [GFAPs], respectively).
Chromatin Immunoprecipitation
ChIP assays were carried out with a ChIP assay kit (Active Motif,
Carlsbad, CA) as previously described (20), with slight modifica-
tions. Briefly, 0827 TIC/TSCs were plated in 15-cm plates
(4 × 10
6
cells per plate; three plates per condition), incubated for
12 hours, and then incubated for 1.5 hours in the presence or
absence of purified recombinant CTGF (200 ng/mL; Leinco
Technologies, St Louis, MO). The cells were washed with cold
phosphate-buffered saline (PBS) and treated for 45 minutes at
room temperature with 10 mmol/L of the protein cross-linking
agent, dimethyl adipimidate (Pierce, Rockford, IL), and 0.25%
dimethyl sulfoxide in PBS with shaking. Formaldehyde was added
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to a final concentration of 1.37% (vol/vol), and the mixture was
incubated for 10 minutes at 37°C. The cells were harvested, and
ChIP was carried out using rabbit polyclonal antibodies against
nuclear factor kappa B (NF-kB) or ZEB-1 (4 µg for each; Santa
Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitated
DNA was recovered and analyzed by ChIP real-time polymerase
chain reaction (RT-PCR) using ZEB-1 primers [catalog No.
GPH001541(-)03A and GPH001541(-)02A] obtained from SA
Biosciences (Frederick, MD). ChIP was performed in triplicate
on three different occasions.
Oligonucleotide Precipitation Assays
Oligonucleotide precipitation assays were done as previously
described (19). Briefly, 0827 TIC/TSCs were plated (3 × 10
6
cells
per plate, two plates per condition) and incubated for 12 hours. The
cells were then incubated for 1.5 hours in the presence or absence of
purified recombinant CTGF (200 ng/mL). The cells were lysed, and
the nuclear fraction was obtained with the use of a nuclear Co-IP kit
(Active Motif). We used the MATCH algorithm (http://www.
bioinfo.de/isb/gcb01/poster/goessling.html) and the TFSearch pro-
gram (http://www.cbrc.jp/research/db/TFSEARCH.html) to iden-
tify an NF-kB binding site within the ZEB-1 promoter region. That
information was used to generate a double-stranded biotinylated
oligonucleotide corresponding to the wild-type NF-kB site within
the ZEB-1 promoter (5-AGGGAACTCCCCGG-3; Invitrogen).
In addition, a control biotinylated oligonucleotide corresponding to
a mutant version of the binding site that was predicted not to bind
NF-kB was generated (5-TTAATTGGTTTAAAA-3; Invitrogen).
The nuclear fraction was incubated for 16 hours with 1 µg of either
the wild-type or mutant biotinylated double-stranded oligonucleo-
tides. The mixtures were incubated for 1 hour with streptavidin–
agarose beads (Pierce), and the DNA–protein complexes were
collected by precipitation, washed five times in wash buffer (0.025
M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, and 5% glyc-
erol; pH 7.4), and subjected to immunoblot analysis with a rabbit
polyclonal antibody against NF-kB p65 (Santa Cruz Biotechnology)
at a dilution of 1:1000.
Stable Infections and Transient Transfections
All transfections were performed with the use of an Amaxa
Nucleofector machine (ACEA Biosciences, San Diego, CA)
according to the manufacturer’s protocol. The 0827 TIC/TSCs
and 0206 TIC/TSCs (1 × 10
6
cells per transfection) were tran-
siently transfected with smartpool small interfering RNAs (siRNAs)
targeting E-cadherin (siRNAs targeting E-cadherin designated as
No. 8, 9, 10, and 11) or a nontargeting control siRNA (50 nM of
each siRNA; Dharmacon, Lafayette, CO). These TIC/TSCs were
used in the real-time cell invasion and migration experiments
84 hours after transfection or used for immunoblot analysis of
protein expression by 96 hours after transfection (see below).
To generate TIC/TSCs or human astrocytes that stably express
short hairpin RNAs (shRNAs) that target CTGF or TrkA or
ITGB1, we cotransfected shRNA-CTGF (Openbiosystems,
Huntsville, AL), shRNA-TrkA (Origene, Rockville, MD), shRNA-
ITGB1 (Origene), or a scrambled-sequence control shRNA
(Origene) with a VSV-G expression plasmid (Clontech, Mountain
View, CA) into the GP2-293 packaging cell line (Clontech)
according to the manufacturer’s instructions. The resulting super-
natants containing shRNA-containing retroviral vectors were
used to infect 0206 and 0827 TIC/TSCs or human astrocytes.
Forty-eight hours after infection, the medium was replaced with
complete medium containing 0.1 µg/mL puromycin (Gibco) to
select for shRNA-expressing TIC/TSCs. Cells that were resistant
to puromycin were characterized for CTGF, TrkA, and ITGB1
expression by immunoblotting and in real-time cell invasion and
migration assays.
Luciferase Reporter Assays
To measure transcriptional activity of E-cadherin and NF-kB,
0827 TIC/TSCs or 0206 TIC/TSCs (1 × 10
4
cells per transfection,
three replicates per condition) were transiently transfected with a
NF-kB luciferase reporter plasmid (1 µg; Clontech) or an
E-cadherin [Wt(k1)-Ecad] luciferase reporter plasmid (2 µg;
kindly provided by Dr Eric Fearon) with the use of an Amaxa
Nucleofector machine, seeded in six-well plates (1 × 10
4
cells per
well), and incubated for 46.5 hours. Purified recombinant CTGF
(200, 400, or 600 ng/mL; Leinco Technologies) was added to the
cultures, and the cells were incubated for an additional 1.5 hours.
The cells were harvested and luciferase activity was measured with
the use of a GloMax 20/20 Luminometer (Promega, Madison,
WI). Luciferase activity was expressed relative to that of cells
transfected with a control plasmid containing a minimal luciferase
promoter (pGL3; Promega) or to that of TIC/TSCs transfected
with a luciferase reporter and not treated with CTGF. In parallel,
0827 TIC/TSCs were transiently transfected with 1 µg of the
NF-kB luciferase reporter plasmid as described above, cultured for
46.5 hours, and then treated for 1.5 hours with GW441756 (400,
800, or 1100 nM; Tocris Ellisville, MO), a TrkA inhibitor that
blocks TrkA phosphorylation and subsequent activation, in the
presence of purified recombinant CTGF (200 ng/mL). The cells
were then assayed for NF-kB transcriptional activation by mea-
suring relative luciferase activity using a GloMax 20/20
Luminometer (Promega). These experiments were carried out in
triplicate on three different occasions.
To determine ZEB-1 transcriptional activation via NF-kB or
following treatment with CTGF, 0827 TIC/TSCs (1 × 10
4
cells
per replicate, three replicates per condition) were transiently trans-
fected with a ZEB-1 luciferase reporter plasmid alone (Switchgear
genomics, Menlo Park, CA) or cotransfected with the ZEB-1
luciferase reporter plasmid and an NF-kB expression plasmid
(kindly provided by Dr Warner Greene) and cultured for 48 hours.
In addition, 0827 TIC/TSCs (1 × 10
4
cells per replicate, three
replicates per condition) were transiently transfected with the
ZEB-1 luciferase reporter plasmid (Switchgear genomics) and an
IkB expression plasmid (which expresses an inhibitor of NF-kB;
kindly provided by Dr Keith Brown), cultured for 46.5 hours, and
then treated for 1.5 hours with purified recombinant CTGF (200
ng/mL). The cells were assayed for ZEB-1 transcriptional activa-
tion by measuring luciferase activity using a GloMax 20/20
Luminometer as described above.
To provide further evidence that CTGF mediates decreased
expression of E-cadherin through NF-kB and ZEB-1, 0827 TIC/
TSCs (1 × 10
4
cells per replicate, three replicates per condition)
were transiently transfected with 2 µg of the E-cadherin luciferase
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reporter plasmid alone or in combination with 1 µg of either the
NF-kB expression plasmid or a ZEB-1 expression plasmid (Origene).
The cells were cultured for 48 hours, harvested, and assayed for
luciferase activity using a GloMax 20/20 Luminometer (Promega).
Real-Time Cell Migration and Invasion Assay
Tumor cell invasion and migration were assessed using a transwell
assay that was described previously (21). For this assay, we used
dual-chamber microtiter plates (16 chambers per plate; ACEA
Biosciences): The upper chambers were coated with laminin
(10 ng/mL) to form a barrier coating for invasion of TIC/TSCs
into the lower chamber. Patient glioblastoma–derived 0206 and
0827 TIC/TSCs and human astrocytes (Lonza Cambrex) stably
transfected with shRNA-TrkA, shRNA-ITGB1, or shRNA-CTGF
or transiently transfected with smartpool siRNAs targeting
E-cadherin (Dharmacon) were seeded in the upper chambers at
1 × 10
5
cells per 100 µL of NBE medium per well. Another 100 µL
of NBE containing 200 ng/mL of either purified recombinant full-
length CTGF or a purified recombinant TrkA-binding truncated
form of CTGF (CTGF
182–250
; Prospec Bio, East Brunswick, NJ) or
100 µL of reactive astrocyte–conditioned media (rACM) containing
secreted CTGF or rACM from astrocytes stably expressing a
shRNA-CTGF was added to the upper chambers. The lower cham-
bers contained NBE medium containing or lacking 10% FBS. The
plates were placed into a RT-CIM apparatus (ACEA Biosciences),
and cell invasion and migration were continuously monitored every
30 minutes for 22 hours according to the manufacturer’s protocol;
however, we only used data that were captured during the first
12 hours of monitoring. The level of impedance (arbitrary units)
of an electrical signal then determines the extent of migration and
invasion by the tumor stem cell.
Immunoblotting and Immunoprecipitation Assays
To examine whether ITGB1 is needed for TrkA activation in the
presence of CTGF, 0827 TIC/TSCs or 0827 TIC/TSCs with
stable knockdown of TrkA or ITGB1 were seeded into 10-cm
plates (1 × 10
6
cells per plate, two plates per condition) for 12 hours
and incubated for 1.5 hours with CTGF (200 ng/mL). TIC/TSCs
were then harvested with ice-cold lysis buffer (50 mM Tris, 150
mM NaCl, 2.5 mM EDTA, 0.1% sodium dodecyl sulfate [SDS],
0.5% sodium deoxycholate, 1% Nonidet P-40, and 0.02% sodium
azide) containing Complete-Mini protease inhibitor (one tablet
per 10 mL; Boehringer Mannheim GmBH, Mannheim, Germany).
The samples were incubated for 15 minutes on ice, then
centrifuged at 10 397g for 15 minutes, and the supernatants were
stored at 220°C. Protein content in the lysates was determined
using a detergent-compatible Bradford protein assay (Bio-Rad
Laboratories, Hercules, CA).
Equivalent amounts of protein were resolved by electrophore-
sis on 12% SDS–polyacrylamide gels or premade 4%–15% gra-
dient SDS–polyarcylamide gels (Bio-Rad Laboratories) and
transferred to nitrocellulose membranes (Invitrogen). The mem-
branes were incubated with a rabbit polyclonal anti-ITGB1 anti-
body (1 : 1000 dilution), a rabbit polyclonal anti-CTGF antibody
(1 : 1000 dilution; Santa Cruz Biotechnology), and/or a rabbit
monoclonal anti-TrkA antibody (1 : 500 dilution; Cell Signaling
Technologies, Danvers, MA). The secondary antibodies were
horseradish peroxidase–conjugated anti-mouse IgG and anti-
rabbit IgG (Promega). An anti-human b-actin antibody (1 : 20 000
dilution; Sigma, St Louis, MO) was used to control for equal
protein loading. Proteins were detected with the use of SuperSignal
West Pico Chemiluminescent substrate (Pierce) and visualized
after exposure to Kodak BioMax MS autoradiography films
(Sigma). The 0827 TIC/TSCs and 0206 TIC/TSCs were cultured
in 15-cm plates (2 × 10
6
cells per plate, two replicates per condi-
tion) and were similarly harvested and lysed for immunoprecipita-
tion, which was carried out using a ProFound Mammalian Co-IP
kit (Pierce) according to the manufacturer’s instructions. Briefly,
500 µg of protein was precleared by incubation with protein A/G
beads for 1 hour at 4°C. Aliquots of the precleared TIC/TSC
extracts were incubated for 24 hours at 4°C with 100 µg of
rabbit polyclonal anti-TrkA or rabbit polyclonal anti-ITGB1
(Santa Cruz Biotechnology) attached to a immobilized A/G
slurry or rabbit polyclonal IgG as a negative control. Cell extracts
were resolved on 4%–15% SDS–polyacrylamide gels and trans-
ferred to nitrocellulose membranes, which were probed with a
rabbit monoclonal anti-TrkA antibody (1 : 500 dilution; Cell
Signaling Technologies) or a rabbit polyclonal anti-ITGB1 anti-
body (1 : 1000 dilution; Santa Cruz Biotechnology). Proteins were
detected by SuperSignal West Pico Chemiluminescent substrate
(Pierce) and visualized after exposure to Kodak BioMax MS
autoradiography films (Sigma).
The 0827 TIC/TSCs and 0206 TIC/TSCs (1 × 10
6
cells per
transfection) were transfected with 50 nM of smartpool siRNA
targeting E-cadherin (Dharmacon), as described above. The
TIC/TSCs were analyzed 96 hours after transfection and resolved
on 4%–15% SDS–polyacrylamide gels and transferred to nitrocel-
lulose membranes, which were probed with a rabbit polyclonal
anti-E-cadherin antibody (1 : 500 dilution, Santa Cruz
Biotechnology) and a mouse monoclonal anti-b-Actin antibody
(1 : 20 000 dilution; Sigma). Proteins were detected as described
above. This experiment was repeated at least three times on three
different occasions.
The 0827 TIC/TSCs and 0206 TIC/TSCs stably infected with
shRNA-CTGF, shRNA-TrkA, or shRNA-ITGB1 were seeded
into three 10-cm plates (1 × 10
6
cells per plate, three plates per
condition) and harvested 12 hours after seeding with ice-cold lysis
buffer as described above. Proteins were resolved on 4%–15%
SDS–polyacrylamide gels and transferred to nitrocellulose mem-
branes, which were probed with a rabbit monoclonal anti-TrkA,
a rabbit polyclonal anti-ITGB1, or an anti-CTGF antibody as
described above. Proteins were detected as described above.
Intracranial Tumor Model
We used a stereotactic device (Stoelting Co, Wood Dale, IL) to
inject neonatal SCID mice (approximately 2 days old; Jackson labs,
Bar Harbor, MI) into the left lateral ventricle with the following
TIC/TSCs (10
5
cells per injection; n = 6 mice per group): wild-type
0827 TIC/TSCs, 0827 TIC/TSCs stably infected with one of two
shRNAs targeting TrkA (shRNA-TrkA58; shRNA-TrkA59), 0827
TIC/TSCs infected with scrambled-sequence control shRNA, or
wild-type 0206 TIC/TSCs. Following injection, the mice were
returned to their mother and monitored for tumor growth by
checking for changes in body weight, lethargy, dehydration, and/or
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labored breathing. The mice were killed when one or more of the
above criteria were met as per the National Cancer Institute Animal
Care and Use Committee policy with a mixture of ketamine (150
mg/kg body weight) and xylazine (10 mg/kg body weight). Mouse
brains were either perfused with 4% paraformaldehyde via cardiac
perfusion after the mice were killed and then sectioned and stained
with hematoxylin–eosin or perfused and washed with PBS, followed
by incubation for 1 hour in 10% sucrose in PBS, followed by incu-
bation for 1 hour in 20% sucrose in PBS, and storage overnight at
4°C in 30% sucrose in PBS. The latter tumors were then frozen in
isopentane solution (Sigma) that had been cooled in dry ice and
serially sectioned on a CM1850 cryostat (Leica, Richmond Hill,
ON) into 7-µm (for in situ hybridization) or 10m (for immunoflu-
orescence labeling and hematoxylin–eosin staining) sections. Sections
were mounted on silanized slides (Superfrost slides; Thermo Fisher
Scientific, Pittsburgh, PA). All procedures involving mice were in
adherence with NIH Animal Care and Use Committee protocols.
Immunofluorescence, Fluorescence-activated Cell Sorting
Analysis, and Confocal Microscopy
Patient-derived glioma TIC/TSCs spontaneously form neuro-
spheres when cultured in NBE medium (22). The neurosphere is a
ball of proliferating cells generated by a single-cell clone and is a
hallmark feature of TIC/TSCs (23). The 0827 TIC/TSCs, 0822
TIC/TSCs, and 0206 TIC/TSCs were cultured in 10-cm plates
(2 × 10
5
cells per plate, two plates per condition) for 12 hours to allow
neurospheres to form, followed by incubation for 1.5 hours in the
presence or absence of purified recombinant CTGF (200 ng/mL;
Leinco Technologies). The cells were collected, washed with PBS,
and fixed for 1 hour at room temperature in 4% paraformaldehyde.
The cells were washed three times with PBS, resuspended in a
mixture of PBS and Trypan blue (ratio of 50 : 1) for subsequent
visualization of the neurospheres during cyrosectioning, then incu-
bated for 1 hour in 10% sucrose in PBS, followed by incubation for
1 hour in 20% sucrose in PBS, followed by storage overnight at 4°C
in 30% sucrose in PBS. The sucrose solution was removed from the
tube, and approximately 500 µL of Tissue Tek Optimal Cutting
Temperature (OCT) solution (Thermo Fisher Scientific) was added
to the cells. The sample was allowed to solidify by placing the tube
in a Petri dish containing isopentane cooled with dry ice. The solidi-
fied OCT-embedded neurospheres were stored at 280°C. Sections
(10-µm thick) of this material were cut on a cryostat and placed on
silanized slides. The sections were incubated in 0.1% Triton X-100,
followed by incubation with Image iT-FX Signal Enhancer (Invitrogen,
Molecular Probes) to block nonspecific staining. The sections were
then incubated with a mouse monoclonal anti-E-cadherin antibody
(1 : 400 dilution; BD Biosciences, San Diego, CA), washed with PBS,
followed by incubation with an Alexa 488–conjugated anti-mouse IgG
antibody (1 : 500 dilution; Invitrogen, Molecular Probes).
The sections were washed and mounted in Vectashield
mounting medium containing 4’,6-damidino-2-phenylindole
(Vector Labs, Burlingame, CA). The sections were examined with
the use of a Zeiss LSM 510 confocal imaging system (Zeiss,
Heidelgberg, Germany). For flow cytometric analysis, neuro-
spheres were stained for E-cadherin, then dissociated into single-
cell suspensions, and analyzed on a fluorescence-activated cell
sorting Vantage SE flow cytometer (BD Biosciences).
Intracranial xenograft tumors derived from U87MG and
U251MG glioma cell lines and from 0827 TIC/TSCs underwent
the same procedure as neurospheres for embedding and sectioning
(described above) and were cut on a cryostat (Leica) in serial sec-
tions at a thickness of 10 µm. Tumor sections were processed for
immunohistochemistry and stained using either a mouse monoclo-
nal antibody against GFAP (anti-GFAP, 1 : 400 dilution; Sigma)
or a rabbit monoclonal anti-TrkA antibody (1 : 500 dilution; Cell
Signaling Technologies). The sections were washed with PBS and
stained with either an Alexa 488–conjugated anti-mouse IgG anti-
body or an Alexa 594–conjugated anti-rabbit IgG antibody (each at
1 : 500 dilution; Invitrogen, Molecular Probes). The sections were
washed with PBS and mounted in Vectashield mounting medium
containing 4’,6-damidino-2-phenylindole (Vector Labs). The
xenograft sections were examined using a Zeiss LSM 510 confocal
imaging system (Zeiss).
Imaging and Fluorescence Resonance Energy Transfer
Analysis
Fluorescence resonance energy transfer analysis (24) allows for the
detection of a protein–protein interaction based on the transfer of
energy of a fluorescent dye conjugated to one protein (the donor
protein) to another protein that is conjugated to a different fluo-
rescent dye (the acceptor protein). Light excites the donor protein
and triggers the energy transfer to the acceptor protein; if the two
proteins are in close proximity, that is, if they are interacting (or if
the conjugated fluorescent dyes are in close proximity), the energy
transfer allows the acceptor protein to emit light and produce an
image similar to that of the donor protein when viewed by confocal
microscopy because the energy transferred to the acceptor protein
is coming from the donor protein.
Mouse xenograft tumors derived from intracranial injection of
0827 TIC/TSCs were fixed and serially sectioned (as described
above). The sections were incubated with a mouse monoclonal
anti-ITGB1 antibody (1 : 500 dilution; Santa Cruz Biotechnology),
washed with PBS, and incubated with Alexa 488–conjugated anti-
mouse IgG (1 : 500 dilution; Invitrogen, Molecular Probes). The
Alexa 488–labeled ITGB1 served as the donor protein. Subsequent
serial sections were incubated with a rabbit polyclonal anti-TrkA
antibody (1 : 400 dilution; Santa Cruz Biotechnology), washed
with PBS, and incubated with Alexa 594–conjugated anti-rabbit
IgG (1 : 500 dilution; Invitrogen, Molecular Probes). The Alexa
594–labeled TrkA served as the acceptor protein. The sections
were imaged using a confocal microscope (Zeiss LSM 510). The
efficiency of fluorescence resonance energy transfer was determined
by using an algorithm in Zeiss LSM software that indicates the likeli-
hood that ITGB1 and TrkA interact. To avoid the possibility of
spectral overlap or a false positive (ie, one emitting protein appearing
in the spectral emission of the other protein), image excitation and
detection were corrected using single images of TrkA and ITGB1 and
the background or spectral emission is corrected. This correction
normalizes for fluorescence resonance energy transfer and was desig-
nated normalized-fluorescence resonance energy transfer (N-FRET).
Radioiodination of Nerve Growth Factor (NGF)
Preparation of
125
I-labeled NGF was performed as previously
described (25) with a slight modification. Briefly, 0.8 µg of IOD-GEN
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solution (0.01 µgL 1,3,4,6-tetrachloro-3a, 6a-diphenylglycouril
[Pierce] in chloroform) was added to a 5-mL plastic test tube, dried
under nitrogen, and washed with 100 µL of 0.5 M potassium
phosphate solution (pH 7.4). To this tube we added 20 µL of
potassium phosphate (0.5 M, pH 7.4), NGF (8 µg in 4 µL of water;
Promega), and 2 mCi (20 µL) of Na
125
I (PerkinElmer, Waltham,
MA), and the mixture was incubated for 6 minutes at room tempera-
ture. The reaction was stopped by adding 300 µL of water. The
radiolabeled NGF proteins were separated from unlabeled protein
by using a Sep-Pak Column (Waters Associates, Milford, MA) and
further purified by reverse-phase high-performance liquid chroma-
tography on a C18 column (Sigma). The fractions with the highest
radioactivity and binding to PC12-TrkA cells were neutralized with
0.2 M Tris buffer (pH 9.5) and stored in 0.5% bovine serum albumin
(wt/vol) at 220°C. The specific activity of the
125
I-NGF was 2200
Ci/mmol.
Competitive Binding Assay
The
125
I-labeled NGF competitive binding assay was performed as
previously described (25) with a slight modification. Briefly,
PC12-TrkA cells (2 × 10
5
cells) were harvested, washed in PBS,
resuspended in 1.5 mL binding buffer (24.5 mM HEPES [pH
7.4], 98 mM NaCl, 6 mM KCl, 5 mM MgCl
2
, 2.5 mM NaH
2
PO
4
,
5 mM sodium pyruvate, 5 mM sodium fumarate, 0.01% [wt/vol]
soybean trypsin inhibitor, 1% amino acid mixture, 0.2% [wt/vol]
bovine serum albumin, and 0.05% [wt/vol] bacitracin), and incu-
bated for 1 hour at 22°C with 50 pM
125
I-labeled NGF in the
presence or absence of increasing concentrations (up to 1 µM) of
unlabeled purified recombinant NGF (Promega), CTGF (Leinco
Technologies), or neurotrophin-3 (NT-3) (R&D Systems).
Aliquots (100 µL) were removed and centrifuged through 300
µL of binding buffer in 400-µL microfuge tubes at 10 000g for
1 minute in a Beckman Microcentrifuge B. The pellets were then
washed twice with binding buffer and counted for radioactivity in
a gamma counter. The amount of recovered radioactivity from
each of the experimental conditions with increasing concentra-
tions of unlabeled purified NGF or NT-3 was then expressed as a
ratio to the maximal amount of TrkA binding (ie, saturation
binding) as determined by the amount of
125
I-NGF ligand (2200
Ci/mmol) binding to PC12-TrkA cells in the absence of unlabeled
ligand. The nonspecific binding was the amount of radioactivity
associated with cells incubated with 50 pM
125
I-NGF ligand (2200
Ci/mmol) and 1 µM unlabeled ligand. Nonspecific binding turned
out to be less than 10% of total binding in all the experiments. To
quantify the binding of CTGF, NGF, or NT-3, a binding concen-
tration curve was made, and the concentration that defines
the binding halfway between the baseline and maximal binding
(EC
50
), was determined using KaleidaGraph software (Synergy,
Reading, PA). This experiment was carried out four times on four
different occasions.
In Situ Hybridization
Intracranial xenograft tumors derived from 0206 TIC/TSCs
were processed as described above, serially sectioned (7-µm
thick), and mounted on silanized slides. In situ hybridization was
carried out with the use of a HybriProbe In Situ Hybridization
Assay kit and proprietary oligonucleotide probe specific for
mouse CTGF (Boca Scientific, Boca Raton, FL) according to the
manufacturer’s instructions. Briefly, Hybribuffer ISH was heated
to 95°C and then cooled to 30°C–40°C and 25 µL was added to
tumor sections. The slides were incubated in a humid box for 4
hours at 30°C. The hybridization was carried out by adding the
fluorescein isothiocyanate (FITC)–labeled mouse-specific CTGF
hybriprobe (Boca Scientific) at a final concentration of 60 pmol
per 1000 µL of Hybribuffer ISH to the tumor sections and incu-
bating them for 12 hours at 30°C. The sections were rinsed
twice in 1 × Saline and Sodium Citrate (SSC) (Invitrogen) at
room temperature for 10 minutes, then twice in 0.1 × SSC at
39°C–41°C for 15 minutes. The sections were washed two times
for 10 minutes in wash buffer (100 mM Tris–HCl, 0.1% Tween
20) and incubated at room temperature in blocking buffer
(1× PBS, 0.1% Triton-X, 2% bovine serum albumin). Bound
probe was detected by immunochemical staining using an alka-
line phosphatase–conjugated F(ab) antibody fragment to FITC
(1 : 800 dilution; Dako, Carpinteria, CA), which produced a
purple reaction product at the site of hybridization that can be
visualized by light microscopy.
Statistical Analysis
The Student t test was used to compare continuous variables
between two groups. P values less than .05 were considered statis-
tically significant. All statistical tests were two-sided. Data are
presented as mean values with 95% confidence intervals (CIs).
Results
TrkA and Glioma Invasion
We first examined the expression of the CTGF binding
receptor, TrkA, in mouse xenografts generated by intracranial
injection with human glioma–derived 0827 TIC/TSCs.
Immunohistochemistry of xenograft sections revealed consider-
able TrkA expression at the tumor margin (Figure 1, B) and in the
infiltrative cells extending from the tumor mass (Figure 1, C)
compared with the noninvasive bulk tumor (Figure 1, A), as shown
by immunofluorescence staining. An independent human glioma–
derived TIC/TSC (0206 TIC/TSCs) demonstrated the same
pattern of TrkA staining with greater TrkA expression at the in-
vasive front of intracranial xenograft tumor compared with cells in
the bulk region of the tumor (Supplementary Figure 1, A, available
online). By contrast, xenograft tumors derived from injection with
the established glioma cell lines U87MG (Figure 1, D) and
U251MG (Figure 1, E) showed no invasion and no TrkA staining.
These data indicate that TrkA expression is relegated largely to
glioma cells and TIC/TSCs at the infiltrating front of the xeno-
graft tumors in vivo. Furthermore, immunohistochemical locali-
zation of CTGF in sections of xenograft tumors established by
intracranial injection of 0827 TIC/TSCs that stably express red
fluorescent protein (RFP) suggests that gliomas derived from
injection of 0827-RFP TIC/TSCs secrete CTGF (Supplementary
Figure 1, B, available online): Measurement of the fluorescence
intensity of CTGF within the tumor and the surrounding paren-
chyma is consistent with an increasing gradient of CTGF from
the center of the tumor out to the infiltrative front (Supplementary
Figure 1, C, available online).
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Identification of a Complex Between CTGF,
ITGB1, and TrkA
ITGB1 can partner with different membrane protein receptors,
such as platelet-derived growth factor receptor and epidermal
growth factor receptor, to form a trimolecular complex between
ITGB1, the growth factor receptor, and the ligand to the receptor
(26). To test the hypothesis that ITGB1 and TrkA form a receptor
complex for CTGF binding, we first examined whether we could
obtain evidence for an interaction between endogenous ITGB1
and TrkA in TIC/TSCs via coimmunoprecipitation. Lysates pre-
pared from 0206 TIC/TSCs and 0827 TIC/TSCs were subjected
to an immunoprecipitation assay. ITGB1 and TrkA coimmuno-
precipitated from lysates of both TIC/TSCs, regardless of whether
an antibody to TrkA or ITGB1 was used for immunoprecipitation
(Figure 2, A and B). To provide further support for an in vivo in-
teraction between ITGB1 and TrkA, we performed immunohisto-
chemistry on sections of 0827 TIC/TSC–derived intracranial
xenografts using an Alexa 488 green fluorescent dye–labeled anti-
body to ITGB1 and an Alexa 594 red fluorescent dye–labeled
antibody to TrkA and looked for areas of antibody colocalization.
ITGB1 and TrkA colocalized in infiltrating TIC/TSCs in vivo
(Figure 2, C).
To further verify the in vivo interaction of ITGB1 with TrkA,
xenograft tumors derived from intracranial injection of 0827
TIC/TSCs were serially sectioned and labeled in the same manner
as the colocalization studies and used for fluorescence resonance
energy transfer experiments. Fluorescence resonance energy trans-
fer analysis can be used to detect a protein–protein interaction
based on the transfer of energy (a photon) of a fluorescent dye
conjugated to one protein (the donor protein) to the conjugated
fluorescent dye of the other protein (the acceptor protein). When
two proteins are in close proximity (ie, when the proteins interact
with each other), light excites the donor protein and triggers the
energy transfer to the acceptor protein. The energy transfer will
Figure 1. Tyrosine kinase receptor type A
(TrkA) expression in sections of glioma-
derived tumor-initiating or tumor stem cell
(TIC/TSC) intracranial xenograft tumors.
Sections of xenograft tumors derived from
intracranial injection of neonatal SCID mice
with glioma-derived 0827 TIC/TSCs were dou-
ble immunostained for GFAP (a marker for
reactive astrocytes; green) and TrkA (red); DNA
was stained with 4’,6-diamidino-2-phenylindole
(DAPI; blue). White boxes (left) identify magni-
fied images to the right scale bars = 100 µm (at
×40 magnification) on right images. A)
Noninvasive bulk tumor. B) Tumor margin. C)
Intracranial tumor infiltration. TrkA immunos-
taining of the established glioma cell lines
U87MG (D) or U251MG (E); scale bars = 100 µm.
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allow the acceptor to emit light and produce an image that can be
visualized with a confocal microscope, indicating the transfer of
fluorescence resonance energy. However, if the two proteins are
not close enough for energy transfer (ie, if they do not physically
interact) no image is produced. As shown in Figure 2, D, fluores-
cence resonance energy transfer was achieved in the labeled
xenograft sections, suggesting a physical interaction between
ITGB1 and TrkA. Results of the coimmunoprecipitation experi-
ments, the colocalization studies, and the fluorescence resonance
energy transfer analysis indicate that ITGB1 and TrkA interact in
TIC/TSCs in gliomas in vivo.
We next conducted a competitive binding assay to examine
whether CTGF binding to TrkA could compete with TrkA
binding to NGF or NT-3, two known ligands for TrkA (27,28).
Rat pheochromocytoma PC12 cells that overexpress TrkA were
incubated with
125
I-labeled NGF, then washed with increasing
concentrations of unlabeled NGF, NT-3, or CTGF, and the
125
I-labeled NGF that remained bound to cells was quantified using
a gamma counter. The amount of
125
I-labeled NGF that remained
bound to cells decreased when cells were washed with increasing
concentrations of unlabeled NGF and NT-3 but not when they
were washed with increasing concentrations of unlabeled CTGF
Figure 2. In vitro and in vivo evidence for a
TrkA–ITGB1–CTGF complex. Lysates of glioma
TIC/TSCs (0206 and 0827) were subjected to
immunoprecipitation with a control IgG or anti-
TrkA antibody (A) or anti-ITGB1 antibody (B).
The immunoprecipitates were immunoblotted
with anti-ITGB1 (A) or anti-TrkA antibody (A).
Loading controls are shown in the lower panels
(inputs). C) Immunofluorescence localization of
ITGB1 and TrkA in a section of a TIC/TSC-
derived intracranial tumor. ITGB1 (green) was
stained with Alexa 488–conjugated antibody
and TrkA (red) was stained with Alexa 594–
conjugated antibody. Merged images of ITGB1
and TrkA (yellow) are also shown. The white
crosshair in the merged figure marks an area of
antibody colocalization as does the boxed
region. Scale bars = 15 µm. D) Fluorescence
resonance energy transfer analysis. Sections of
xenograft tumors derived from 0827 TIC/TSCs
were stained with a Alexa 488 green fluorescent
dye–labeled antibody to ITGB1 and an Alexa 594
red fluorescent dye–labeled antibody to TrkA.
Fluorescence resonance energy transfer effi-
ciency was normalized for possible spectral
overlap (designated N-FRET) Scale bars = 50 µm.
E) Competitive binding assay. Rat pheochromo-
cytoma PC12 cells that overexpress TrkA were
incubated with
125
I-labeled nerve growth factor
(NGF) and washed with increasing amounts of
unlabeled CTGF, NGF, or NT-3. Radioactivity
that remained bound to the cells was deter-
mined using a gamma counter and presented as
a percentage of control (defined as cells sub-
jected to the same procedure minus unlabeled
ligand, therefore representing the maximum
binding capacity or a 100% bound
125
I-NGF).
Error bars represents 95% confidence intervals
for the means of three independent experiments
performed in triplicate. F) Immunoblot analysis
of TrkA activation in TIC/TSCs exposed to short
hairpin RNA-mediated knockdown of TrkA or
ITGB1 (shTrkA or shITGB1) in the presence of
exogenous purified recombinant CTGF (200 ng/
mL). TrkA activation was detected with phos-
photyrosine-specific antibody (p-Tyr). Lane 1,
untreated cells; lane 2, targeted TrkA knock-
down in the presence of CTGF; lane 3, targeted
ITGB1 knockdown in the presence of CTGF; lane
4, TIC/TSCs treated with CTGF. Immunoblotting
for actin was used as a control for equal sample
loading. CTGF = connective tissue growth factor;
ITGB1 = integrin b1; NT-3 = neurotrophin-3; NGF =
nerve growth factor; TIC/TSC = tumor-initiating
or tumor stem cell; TrkA = tyrosine kinase re-
ceptor type A.
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(Figure 2, E). These data suggest that CTGF may bind to TrkA at
a site different from that of NGF and NT-3. However, we cannot
rule out the possibility that CTGF binds to TrkA with very low
affinity.
Mechanism of CTGF Signaling
To identify downstream effectors of CTGF signaling in
TIC/TSCs, we first performed coimmunoprecipitation experi-
ments, which demonstrated that CTGF binds to both TrkA and to
ITGB1 (Supplementary Figure 2, A and B, available online).
Furthermore, CTGF binding to TrkA induces its activation
through TrkA phosphorylation (Supplementary Figure 2, A and C
available online).
To further elucidate the necessary components for CTGF acti-
vation of the TrkA receptor via phosphorylation, we selectively
knocked down expression of the components of the putative
receptor complex by stably transfecting 0827 TIC/TSCs with
shRNAs targeting either TrkA or ITGB1 and examined TrkA
phosphorylation in cells cultured in the presence or absence of
purified recombinant CTGF (200 ng/mL). CTGF-mediated TrkA
phosphorylation was reduced by knockdown of either TrkA or
ITGB1 (Figure 2, F). These data demonstrate that CTGF
induces TrkA phosphorylation in 0827 TIC/TSCs that express
TrkA and ITGB1.
Effect of TrkA Activation on NF-kB Activity
Given that NF-kB has been previously implicated in tumor cell
migration and metastasis (29), and TrkA activation can lead to
NF-kB activation (30), we examined the effect of TrkA activation
on NF-kB activity in TIC/TSCs. The 0827 TIC/TSCs were tran-
siently transfected with an NF-kB luciferase reporter plasmid and
then incubated in the absence or presence of increasing concentra-
tions of recombinant purified CTGF. NF-kB transcriptional acti-
vation was then assessed by measuring the relative luciferase
activity (compared with a minimal luciferase promoter plasmid
that served as a baseline for luciferase activity).
Increasing concentrations of CTGF resulted in increasing
NF-kB luciferase reporter activity in 0827 TIC/TSCs (mean rela-
tive luciferase activity [arbitrary units], untreated vs CTGF
200 ng/mL
:
0.42 vs 2.35, difference = 1.93, 95% CI = 1.53 to 2.33, P < .001;
untreated vs CTGF
400 ng/mL
: 0.42 vs 4.5, difference = 4.08, 95%
CI = 3.82 to 4.34, P < .001; untreated vs CTGF
600 ng/mL
: 0.42 vs 8.3,
difference = 7.8, 95% CI = 5.3 to 10.5, P < .001). To determine if
TrkA was required for CTGF-mediated NF-kB transcriptional
activity, 0827 TIC/TSCs were transiently transfected with an
NF-kB luciferase reporter plasmid and then treated with
increasing concentrations of GW441756, a TrkA inhibitor that
blocks TrkA phosphorylation and subsequent activation, in the
presence of a constant concentration of purified recombinant
CTGF (200 ng/mL). The cells were then assayed for NF-kB tran-
scriptional activation by measuring relative luciferase activity.
Treatment of cells with the TrkA inhibitor reversed NF-kB tran-
scriptional activity (mean relative luciferase activity, GW
400 nM
vs
CTGF
200 ng/mL
: 2.6 vs 2.35, difference = 0.25, 95% CI = 20.25 to
0.75, P = .12; GW
800 nM
vs CTGF
200 ng/mL
: 2 vs 2.35, difference =
0.35, 95% CI = 20.17 to 0.87, P = .5; GW
1100 nM
vs CTGF
200 ng/mL
:
1.2 vs 2.35, difference = 1.15, 95% CI = 0.67 to 1.63, P = .19).
Similar results were seen in 0206 TIC/TSCs (relative luciferase
activity, untreated vs CTGF
200 ng/mL
: 0.53 vs 1.87, difference = 1.34,
95% CI = 0.69 to 2, P < .001). These data suggest that TrkA signals
through NF-kB. Therefore, NF-kB might be a potential down-
stream activator of CTGF-mediated tumor cell infiltration.
Importantly, addition of up to 250 ng/mL of CTGF to 0827 TIC/
TSCs or 0206 TIC/TSCs did not result in any cytotoxic effects or
a noticeable increase in cell proliferation as determined by cell via-
bility assays using Alamar blue (Supplementary Figure 2, D and E,
available online).
Identification of Effector Molecules Downstream of NF-kB
The observation that TrkA activation is associated with a trimo-
lecular complex containing ITGB1, TrkA, and the CTGF ligand
with subsequent NF-kB activation led us to explore possible
effector molecules downstream of NF-kB. Given the role of the
ZEB-1 transcription factor in tumor cell migration and its plau-
sible relationship with NF-kB (31), we investigated whether
NF-kB signaled through ZEB-1.
To determine if CTGF-mediated NF-kB activation induced
the transcription factor ZEB-1, 0827 TIC/TSCs were transiently
transfected with a ZEB-1 promoter luciferase reporter or a control
minimal promoter reporter plasmid and then incubated with
recombinant purified CTGF. ZEB-1 transcriptional activation was
then determined by measuring the difference in luciferase activity
between the ZEB-1 promoter and the control promoter reporter.
Similarly, the luciferase activity of recombinant CTGF–treated
0827 TIC/TSCs cotransfected with the ZEB-1 luciferase reporter
plasmid, and an NF-kB expression plasmid was compared with the
luciferase activity of recombinant CTGF–treated 0827 TIC/TSCs
cotransfected with the ZEB-1 luciferase reporter plasmid and an
IkB (an inhibitor of NF-kB) expression plasmid. Overexpression of
NF-kB p65 or exposure of 0827 TIC/TSCs to purified recombi-
nant CTGF resulted in induction of the ZEB-1 luciferase reporter
(mean relative luciferase activity, untreated vs CTGF
200 ng/mL
: 1.0 vs
5.00, difference = 4, 95% CI = 3.4 to 4.6, P < .001; untreated vs
NF-kB overexpression: 1.0 vs 4.6, difference = 3.6, 95% CI = 3.4
to 3.8, P < .001) (Figure 3, A). By contrast, CTGF treatment of
0827 TIC/TSCs transiently transfected with a plasmid expressing
the NF-kB inhibitor IkB resulted in a decrease in ZEB-1 promoter
activation compared with TIC/TSCs treated with CTGF or trans-
fected with NF-kB p65 expression plasmid (mean relative lucif-
erase activity, CTGF
200 ng/mL
vs CTGF/IkB: 5.00 vs 2.8, difference
= 2.2, 95% CI = 1.69 to 2.71, P = .11; NF-kB overexpression vs
CTGF/IkB: 4.6 vs 2.8, difference = 1.8, 95% CI = 1.71 to 1.89,
P = .035).
We used two approaches to examine whether NF-kB could
bind directly to the ZEB-1 promoter. We first performed an oli-
gonucleotide precipitation assay (19) in which nuclear lysates made
from 0827 TIC/TSCs (untreated or treated with 200 ng/mL puri-
fied recombinant CTGF) were incubated with biotinylated
double-stranded oligonucleotides containing a putative NF-kB
binding sequence located within the ZEB-1 promoter region
(Figure 3, B, top).
Immunoblot analysis of DNA–protein complexes precipitated
with streptavidin–agarose beads revealed the p65 subunit of
NF-kB bound to the oligonucleotides corresponding to the ZEB-1
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promoter to a much greater extent when the assay was conducted
with lysates made from CTGF-treated 0827 TIC/TSCs compared
with lysates made from untreated 0827 TIC/TSCs, or when a
NF-kB mutant double-stranded oligonucleotide control sequence
was used in the precipitation assay (Figure 3, B, bottom).
We next used an antibody to NF-kB in a ChIP assay to exam-
ine the direct binding of NF-kB to the ZEB-1 promoter in 0827
TIC/TSCs that were treated with CTGF. CTGF-treated 0827
TIC/TSCs displayed an increase in NF-kB binding at the
ZEB-1 promoter region compared with untreated 0827 TIC/
TSCs as shown by ChIP RT-PCR (fold increase in NF-kB
binding to the ZEB-1 promoter region: Untreated + NF-kB
antibody vs CTGF
200 ng/mL
+ NF-kB antibody: 0.375 vs 2.45, dif-
ference = 2.075, 95% CI = 2.04 to 2.11, P < .001) (Figure 3, C,
left panel). Taken together, these data demonstrate that exposure
of TIC/TSCs to CTGF results in increased transcriptional
Figure 3. Identification of effector
molecules downstream of nuclear
factor kappa B (NF-kB). A) ZEB-1 lu-
ciferase reporter assay. The 0827
tumor-initiating or tumor stem cells
(TIC/TSCs) were transfected with a
ZEB-1 luciferase reporter alone or in
combination with purified recombi-
nant CTGF
200 ng/mL
or cotransfected
with pNF-kB can be used through-
out a pNF-kB expression plasmid or
cotransfected with a pIkB (I kappa
Beta, an inhibitor of NF-kB) expres-
sion plasmid and treated with puri-
fied recombinant CTGF (200 ng/mL).
The 0827 TIC/TSCs were harvested
and processed for measurement of
luciferase activity. Data represent
mean values of three independent
experiments performed in triplicate;
error bars = 95% confidence inter-
vals. All P values are two-sided
(Student t test). B) Oligonucleotide
precipitation assay. Nuclear extracts
from untreated 0827 TIC/TSCs or
0827 TIC/TSCs treated with purified
recombinant CTGF (200 ng/mL)
were incubated with biotinylated
double-stranded oligonucleotides
corresponding to the wild-type
NF-kB binding site in the ZEB-1 pro-
moter or a mutant version of that
site. The DNA–protein complexes
were precipitated with streptavidin
agarose beads and subjected to
immunoblot analysis with an anti-
NF-kB p65 antibody. Top, schematic
representation of the NF-kB binding
site located in the ZEB-1 promoter.
NF-kB (N) is the wild-type binding
site at position 2387 to 2353 at
which NF-kB binds ZEB-1; mutNF-
kB (M) is a mutant NF-kB binding
site. Bottom, immunoblot of binding
assay. C) Chromatin immunopreci-
tation (ChIP) assays. The 0827 TIC/
TSCs were incubated with or with-
out CTGF (200 ng/mL) and sub-
jected to ChIP with antibody against
NF-kB (left panel) or against ZEB-1
(right panel). The y-axis indicates
the fold increase in the binding of
the NF-kB transcription factor to the
promoter region of ZEB-1 (left) or
the fold increase in the binding of
ZEB-1 transcription factor to the promoter region of E-cadherin (right)
with CTGF treatment (200 ng/mL) compared with no CTGF treatment.
Data represent mean values of three independent experiments per-
formed in triplicate; error bars = 95% confidence intervals. All P values
are two-sided (Student t test). D) E-cadherin promoter luciferase assay.
The 0827 TIC/TSCs were transfected with the E-cadherin luciferase
reporter plasmid Wt(k1)-E-cad alone (unreated) or in combination with a
pNF-kB or pZEB-1 expression plasmid. Luciferase activity was measured
with the use of a luminometer and presented as relative luciferase
activity (relative to standard transfection control containing a minimal
luciferase promoter with baseline luciferase activity). Data represent
mean values of three independent experiments performed in triplicate;
error bars = 95% confidence intervals. All P values are two-sided
(Student t test). CTGF = connective tissue growth factor.
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activation of ZEB-1 and the direct binding of NF-kB to the
ZEB-1 promoter.
CTGF-Dependent NF-kB Activation and E-cadherin
Expression
The development of invasive tumors is often associated with the
decreased expression of E-cadherin, which plays an important role
in epithelial cell adhesion and cell mobility. Interactions among
integrins, the extracellular matrix, and cell surface receptors are also
involved in regulating cell-to-cell contacts and cell mobility (32).
It has been reported (31) that NF-kB can affect E-cadherin
expression, possibly via the transcriptional repressor ZEB-1. Given
that ZEB-1 has been shown to bind to E-box regions of E-cadherin
(E-box regions are found within promoter regions and can either
increase or decrease transcriptional activity depending on which
specific transcription factor binds to the E-box region), we sur-
mised that CTGF binding to a TrkA–ITGB1 complex would
induce NF-kB activation and subsequent ZEB-1 activation, ulti-
mately leading to decreased expression of E-cadherin (33,34). We
used a ChIP assay with an antibody to ZEB-1 to examine whether
CTGF signaling could result in ZEB-1 binding to the E-box
region of the E-cadherin promoter in glioma 0827 TIC/TSCs.
CTGF-treated 0827 TIC/TSCs displayed increased binding of
ZEB-1 to the E-box region of the E-cadherin promoter compared
with untreated 0827 TIC/TSCs as shown by ChIP RT-PCR (fold
increase in ZEB-1 binding to the E-cadherin promoter region:
Untreated + ZEB-1 antibody vs CTGF
200 ng/mL
+ ZEB-1 antibody: 1.5
vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001) (Figure 3, C,
right panel).
To provide further evidence that CTGF mediates decreased
expression of E-cadherin through NF-kB and ZEB-1, we exam-
ined E-cadherin promoter activity in 0827 TIC/TSCs that were
transfected with an E-cadherin luciferase reporter plasmid alone or
in combination with an NF-kB or ZEB-1 expression plasmid.
Cells that overexpressed either NF-kB or ZEB-1 had decreased
E-cadherin luciferase activity compared with cells that overex-
pressed neither protein (Figure 3, D). Conversely, transfection of
0206 TIC/TSCs with the E-cadherin luciferase reporter plasmid
and either of two shRNAs targeting ZEB-1 resulted in transcrip-
tional activation of the E-cadherin promoter (Supplementary
Figure 3, available online). Together, these data indicate that
exposure to CTGF leads to decreased expression of E-cadherin
through a signaling pathway involving the activation of TrkA,
which leads to activation of NFkB and subsequent ZEB-1-
mediated transcriptional repression of E-cadherin.
Effect of CTGF on E-cadherin Expression in Glioma
TIC/TSCs
We examined the effects of CTGF on E-cadherin expression in
TIC/TSCs obtained from three adult glioblastoma patients
because these cells more closely mimic the infiltrative nature of
primary human glioblastomas compared with established glioma
cell lines (5). Exposure of the three TIC/TSC lines to purified
recombinant CTGF resulted in a considerable decrease in the
number of E-cadherin–positive cells as demonstrated by immuno-
fluorescence staining with an antibody to E-cadherin (Figure 4, A,
left) and quantitative fluorescence-activated cell sorting analysis
(Figure 4, A, right). Similarly, CTGF exposure resulted in a con-
siderable decrease in E-cadherin luciferase reporter activity in
0827 TIC/TSCs and 0206 TIC/TSCs (Supplementary Figure 4, A,
available online). RT-PCR further confirmed decreased expression
of E-cadherin mRNA in 0827 TIC/TSCs treated with CTGF
(Supplementary Figure 4, B, available online). By contrast, CTGF
exposure of 0827 TIC/TSCs and 0206 TIC/TSCs that were
transfected with an shRNA-targeting TrkA resulted in increased
E-cadherin luciferase activity compared with cells transfected with
a control shRNA (Supplementary Figure 4, C, available online).
In addition, 0827 TIC/TSCs treated with CTGF and either a
TrkA inhibitor (GW441756) or an ITGB1 inhibitor (MAB225Z)
showed no CTGF-mediated inhibition of E-cadherin protein
expression compared with cells treated with CTGF alone (Figure 4, B,
right; Supplementary Figure 4, D, available online), further con-
firming the involvement of a ITGB1–TrkA complex in E-cadherin
regulation. It is of interest to note that exposure of 0206 TIC/TSCs
to pharmacological concentrations (ie, 10 ng/mL) of either NGF
or NT-3 did not affect E-cadherin protein expression as detected
by immunofluorescence staining, indicating the specificity of the
CTGF–TrkA complex for decreasing expression of E-cadherin
(Figure 4, C).
Effect of the CTGF–ITGB1–TrkA Complex on Tumor Cell
Migration and Invasion
Although the effects of CTGF have been previously studied in a
number of established cancer cell lines, to our knowledge, the
effects on patient-derived primary cancer stem cells have yet to be
explored. Thus, we evaluated the contribution of each member of
the CTGF–ITGB1–TrkA complex to glioma TIC/TSC invasion
by using an automated in vitro real-time cell migration and inva-
sion assay. The 0206 TIC/TSCs and 0827 TIC/TSCs stably
expressing shRNA-TrkA, shRNA-ITGB1, or shRNA-CTGF
were seeded in the upper chamber of a 16-chamber microtiter plate
containing purified recombinant full-length CTGF or a truncated
CTGF
182–250
(200 ng/mL) in NBE. The upper chamber was coated
with laminin to form a barrier for invasion of TIC/TSCs into the
lower chamber. The lower chamber contained NBE with no growth
factors (serum free). The 16-chamber microtiter plates were placed
into a RT-CIM apparatus for measurement of cell invasion and
migration. Treatment of 0827 TIC/TSCs and 0206 TIC/TSCs with
purified recombinant full-length CTGF or CTGF
182–250
(a TrkA-
binding truncated form of CTGF) induced a statistically significant
increase in the amount of tumor cell migration compared with
untreated cells (mean electrical impedance [arbitrary units],
0827 TIC/TSCs, untreated vs full-length CTGF: 3.88 vs 10.32,
difference = 6.44, 95% CI = 6.2 to 6.7, P < .001; 0206 TIC/TSCs,
untreated vs full-length CTGF: 1.38 vs 4.1, difference = 2.72, 95%
CI = 2.5 to 3, P < .001; 0206 TIC/TSCs, untreated vs CTGF
182–250
:
1.38 vs 6.26, difference = 4.88, 95% CI = 4.6 to 5.1, P < .001).
By contrast, shRNA-mediated knockdown of endogenously
produced CTGF decreased cell migration and invasiveness of
TIC/TSCs compared with untransfected (wild-type) cells, suggesting
a potential paracrine and/or autocrine mechanism of CTGF-mediated
TIC/TSC invasion and migration (mean electrical impedance
[arbitrary units], 0827 TIC/TSCs, WT vs shCTGF: 5.3 vs 1.2, dif-
ference = 4.1, 95% CI = 3.9 to 4.3, P < .001; 0206 TIC/TSCs,
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WT vs shCTGF: 1.4 vs 0.38, difference = 1.02, 95% CI = 1.01 to
1.03, P < .001).
To examine the importance of TrkA and ITGB1 in transducing
the CTGF promigratory signal, we assayed migration of TIC/
TSCs transfected with shRNAs that target TrkA or ITGB1 or a
scrambled-sequence control shRNA (Scr). shRNA-mediated
knockdown of either TrkA or ITGB1 resulted in a decrease in
CTGF-induced cell migration compared with CTGF-induced Scr
control (mean electrical impedance [arbitrary units], 0827 TIC/
TSCs, WT vs Scr: 4.3 vs 7.7, difference = 3.4, 95% CI = 3.1 to 3.6, P
< .001; 0827 TIC/TSCs, Scr vs shTrkA59: 7.7 vs 4.6, difference = 3.1,
95% CI = 2.97 to 3.23, P < .001; 0206 TIC/TSCs, WT vs Scr: 0.88
vs 10.6, difference = 9.72, 95% CI = 9.71 to 9.73, P < .001; 0206 TIC/
TSCs, Scr vs shTrkA59: 10.6 vs 0.82, difference = 9.78, 95% CI =
9.77 to 9.79, P < .001; 0827 TIC/TSCs, WT vs shITGB1: 5.0 vs 1.1,
difference = 3.9, 95% CI = 3.8 to 4.0, P < .001; 0206 TIC/TSCs,
Figure 4. Effect of connective tissue growth
factor (CTGF) on E-cadherin expression in
tumor-initiating or tumor stem cells (TIC/TSCs).
A) Indirect immunofluorescence detection (left)
and Fluorescence-activated cell sorting analysis
(right) of E-cadherin expression in TIC/TSC
neurospheres. Three TIC/TSC lines (0206, 0822,
and 0827) were incubated in the presence or
absence of purified recombinant CTGF (left)
and stained with an Alexa 488–conjugated anti-
body to E-cadherin (green); DNA was stained
with DAPI (blue). Scale bars = 150 µm.
Quantitative analysis of E-cadherin expression
was by flow cytometry. Percentages indicate
proportions of E-cadherin–positive cells. B)
Immunofluorescence (left) and Fluorescence-
activated cell sorting analysis (right) of
E-cadherin expression in 0206 TIC/TSC neuro-
spheres treated with CTGF and either GW441756
(a tyrosine kinase receptor type A [TrkA] inhib-
itor) or MAB225Z (an integrin b1 [ITGB1] inhib-
itor) in the presence of CTGF. Scale bars = 50
µm. C) Immunofluorescence labeling of
E-cadherin in TIC/TSC 0206 neurospheres
treated with basic fibroblast growth factor and
epidermal growth factor (bFGF + EGF), nerve
growth factor (NGF), or neurotrophin-3 (NT-3).
Scale bars = 150 µm.
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WT vs shITGB1: 1.5 vs 0.78, difference = 0.72, 95% CI = 0.71 to
0.73, P < .001).
Finally, to examine the effect of a different type of knockdown
of E-cadherin expressionsiRNA-mediated gene silencingon
TIC/TSC invasion and migration in vitro, we assayed migration of
TIC/TSCs that were transfected with four siRNAs targeting
E-cadherin (designated No. 8, 9, 10, and 11) or a nontargeting
siRNA as a negative control. siRNA-mediated knockdown of
E-cadherin expression increased TIC/TSC invasion and migration
in vitro compared with nontargeting siRNA negative control
transfected TIC/TSCs (mean electrical impedance [arbitrary units],
0827 TIC/TSCs, control vs E-cadherin siRNA No. 8: 0.6 vs 3.8,
difference = 3.2, 95% CI = 3.1 to 3.3, P < .001; 0827 TIC/TSCs,
control vs E-cadherin siRNA No. 9: 0.6 vs 12, difference = 11.4,
95% CI = 11.39 to 11.4, P < .001; 0827 TIC/TSCs, control vs
E-cadherin siRNA No. 10: 0.6 vs 7.6, difference = 7, 95% CI = 6.8
to 7.2, P < .001; 0827 TIC/TSCs, control vs E-cadherin siRNA
No. 11: 0.6 vs 8.6, difference = 8, 95% CI = 7.5 to 8.5, P < .001;
0206 TIC/TSCs, control vs E-cadherin siRNA No. 8: 1.0 vs 7.0,
difference = 6.0, 95% CI = 5.9 to 6.1, P < .001; 0206 TIC/TSCs,
control vs E-cadherin siRNA No. 9: 1.0 vs 5.1, difference = 4.1,
95% CI = 3.5 to 4.7, P < .001; 0206 TIC/TSCs, control vs
E-cadherin siRNA No. 10: 1.0 vs 6.7, difference = 5.7, 95% CI =
5.69 to 5.71, P < .001; 0206 TIC/TSCs, control vs E-cadherin
siRNA No. 11: 1.0 vs 6.4, difference = 5.4, 95% CI = 5.1 to 5.6, P <
.001). This finding supports our hypothesis that the promigratory
effect of CTGF is transduced downstream through its repression of
E-cadherin.
Effect of TrkA on Glioma TIC/TSC Infiltration In Vivo
To directly evaluate the importance of TrkA expression for TIC/
TSC invasion in vivo, we created 0827 TIC/TSCs that stably
express one of two shRNAs targeting TrkA (sh58 or sh59) or a con-
trol scrambled-sequence shRNA. Stable expression of sh58 and sh59
resulted in considerable knockdown of TrkA protein expression by
immunoblot analysis compared with uninfected 0827 TIC/TSCs or
0827 TIC/TSCs expressing a scrambled-sequence shRNA (more so
for sh59 than for sh58; Figure 5, A). However, shRNA-mediated
knockdown of TrkA did not affect the proliferation or clonogenicity
of the TIC/TSCs in vitro (data not shown).
To examine the effect of shRNA-mediated knockdown of TrkA
on TIC/TSC infiltration in vivo, we studied intracranial xenograft
tumors generated by injection of mice with patient-derived glioblas-
toma 0827 TIC/TSCs stably expressing scrambled sequence
shRNA, sh58, sh59, or wild-type cells. The tumors were sectioned
and stained with hematoxylin and eosin for examination of tumor
cell infiltration. We observed extensive tumor cell infiltration of
the cerebral cortex in mice injected with wild-type 0827 TIC/
TSCs or scrambled sequence shRNA-expressing 0827 TIC/TSCs
(Figure 5, B, upper left and right panels, respectively). By contrast,
mice injected with sh58-expressing 0827 TIC/TSCs had considerably
less tumor cell invasion in vivo, and those injected with sh59-
expressing 0827 TIC/TSCs, which showed the greatest TrkA
knockdown by immunoblot analysis, had no tumor cell invasion into
normal cortex (Figure 5, B, lower left and right panels, respectively).
These data demonstrate the requirement for TrkA expression in
glioma invasion in this model system.
Characterization of Reactive Astrocytes in an Orthotopic
Glioma Model
A profound glial reaction is often seen within and around the in-
vading front of malignant gliomas in situ. The glial reaction is
characterized by the presence of reactive astrocytes that are likely
to be responding to an endogenous brain injury signal and/or to
the release of cytokine(s) by glioma cells and associated inflamma-
tory cells. Given that injured brain can release CTGF (16), and
given the intimate proximity of reactive astrocytes to the invading
glioma front, we first examined whether reactive astrocytes provide
Figure 5. Effect of tyrosine kinase re-
ceptor type A. TrkA expression on inva-
siveness of glioma tumor–initiating or
tumor stem cell (TIC/TSC)–derived xeno-
grafts. The 0827 TIC/TSCs were stably
transfected with one of two short hairpin
RNA (shRNA) constructs targeting TrkA
(sh58 or sh59) or a scrambled-sequence
shRNA (SC) as a negative control; wild-
type (WT) cells were not transfected. TIC/
TSCs that stably expressed the shRNAs
were subjected to immunoblot analysis
of TrkA expression and were used to
generate xenograft tumors by intracra-
nial injection of neonatal SCID mice. A)
Immunoblot analysis of TrkA expression
in TIC/TSC transfected with shRNA con-
structs. Immunoblotting for actin was the
control for equal protein loading. B)
Intracranial tumor histology. Tumor sec-
tions (10 µm) were stained with hematox-
ylin and eosin and examined for evidence
of tumor cell invasion (arrows) at low and
high magnification (left and right panels,
respectively, of each pair). Scale bar left
panels for WT, SC, sh58, and sh59 is 500
µm. Scale bar right panels for WT, SC,
sh58, and sh59 is 50 µm.
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a promigratory stimulus to glioma cells through the release of
CTGF. Intracranial xenograft tumors generated by injection of
mice with patient-derived glioblastoma 0206 TIC/TSCs were
serially sectioned and stained for GFAP, a marker of reactive
astrocytes. We observed large numbers of GFAP-positive reactive
host astrocytes surrounding the growing tumor mass, as is typically
seen in human gliomas in situ (Figure 6, A, left panel). We used a
mouse-specific probe to detect mouse CTGF mRNA by in situ
hybridization on serial mouse brain sections containing the intra-
cranial human glioma xenograft. There were very high levels of
expression of mouse-specific CTGF mRNA in reactive astrocytes
adjacent to the infiltrating tumor front (Figure 6, A, middle panel).
To confirm that reactive astrocytes not only produce CTGF
mRNA but also secrete biologically active CTGF protein, we cul-
tured human reactive astrocytes in vitro, collected the culture
medium, and used a CTGF antibody to immunodeplete CTGF
from this rACM. First, we demonstrated by immunoblot analysis
of control IgG–treated rACM (lane 1) that CTGF was present in
rACM. This was confirmed by the selective depletion of CTGF in
the CTGF antibody–treated rACM (lane 2). Finally, total rACM,
Figure 6. Characterization of reactive astro-
cytes in orthotopic gliomas derived from intra-
cranial injection of mice with 0206
tumor-initiating or tumor stem cells (TIC/TSCs).
Xenograft tumors derived from intracranial
injection of SCID mice with 0206 TIC/TSCs
were harvested and serially sectioned. A)
Immunohistochemistry and in situ hybridiza-
tion. Serial sections were subjected to immu-
nohistochemical (IHC) detection of the reactive
astrocyte marker GFAP (brown, left panel), in
situ hybridization (ISH) detection of mouse
CTGF mRNA using a mouse-specific CTGF
probe (purple, middle panel), and ISH using
scrambled-sequence probe (control, right
panel). Tu indicates central tumor mass. Scale
bars = 50 µm. B) Immunodepletion of reactive
astrocyte–conditioned medium (rACM) with
either an IgG (lane 1) or CTGF-specific anti-
body (lane 2). Lane 3 represents total rACM
with no antibody depletion that was probed
for CTGF to indicate that CTGF is present in
rACM. An equal amount of protein was run in
each lane. C) Effect of rACM on the in vitro
migratory and invasive activity of 0206 TIC/
TSC. The effects of conditioned medium (ACM)
from either mock-transfected or CTGF shRNA-
transfected astroctyes on 0206 TIC/TSCs inva-
sion and migration was assessed using an in
vitro assay. P value from two-sided Student t
test. Data represent the mean values of three
independent experiments performed in tripli-
cate; error bars = 95% confidence intervals. D)
Summary of CTGF signal transduction path-
way. CTGF = connective tissue growth factor;
Ikk = IkB Kinase; Ub = Ubiquitin; P = phosphor-
ylation; IkB = Inhibitor of NF-kB; p65 = NF-kB
p65 subunit; TrkA = tyrosine kinase receptor,
type A; ZEB-1 = zinc finger E-box binding
homeobox 1.
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with no antibody treatment, demonstrated that CTGF is secreted
by rACM (lane 3, Figure 6, B). Next, to verify the biological
activity of the immunoblot-detected CTGF, we examined the
effect of rACM collected from cultures of human reactive astro-
cytes transfected with CTGF shRNA and from mock-transfected
human reactive astrocytes on the in vitro migratory and invasive
activity of 0206 TIC/TSCs. The 0206 TIC/TSCs exposed to
rACM from mock-transfected reactive astrocytes had more
migrating cells compared with 0206 TIC/TSCs exposed to rACM
from CTGF shRNA–infected reactive astrocytes (mean electrical
impedance: 0206 TIC/TSC, mock-transfected vs CTGF shRNA–
tranfected: 9.75 vs 1.25, difference = 8.5, 95% CI = 7.7 to 9.3,
P < .001) (Figure 6, C). Together, these results demonstrate that
reactive astrocytes secrete biologically active CTGF, which poten-
tiates the proinfiltrative phenotype of the glioma cells via a novel
host-tumor signaling pathway (Figure 6, D).
Discussion
The migratory and invasive capacity of glioma cells is a hallmark
of glioblastoma and all but assures ultimate treatment failure and
patient death (35). Our studies provide one of the first molecular
analyses of glioma cell invasion using patient-derived TIC/TSCs,
and the data demonstrate that CTGF binds to an ITGB1- and
TrkA-containing complex. CTGF binding to the ITGB1–TrkA
complex causes NF-kB-mediated activation of the ZEB-1 pro-
moter with subsequent induction of the ZEB-1 transcriptional
repressor resulting in decreased expression of E-cadherin. This
decreased expression of E-cadherin results in enhanced glioma cell
invasion and migration. Consistent with these observations, inhi-
bition of CTGF, ITGB1, or TrkA activation resulted in decreased
tumor cell invasion and migration in vitro and in vivo. Finally, our
demonstration of a host-derived CTGF-mediated contribution to
a proinvasive microenvironment within the brain indicates a
potential paracrine- and/or autocrine-signaling mechanism for
glioma cell infiltration.
Studies of glioma cell invasion that have been published over
the last two decades have largely used established glioma cell lines
that have been passaged in vitro for years or decades. The clinical
relevance of that model system for looking at the mechanisms that
are operative in human glioma invasion in situ is questionable given
that most established glioma cell lines are neither tumorigenic nor
invasive in vivo (5). Even the few glioma lines that are tumorigenic
in vivo are not infiltrative (5). By contrast, we (5) and others (3,36)
have shown that xenograft tumors derived from human glioma
TIC/TSCs more closely recapitulate human glioblastomas at the
genetic, cellular, and biological levels than do xenograft tumors
derived from established human glioma cell lines. Specifically, we
have shown that glioma TIC/TSCs are as highly invasive in vivo as
were the tumors from which they were derived.
Glioma TIC/TSCs are therefore likely to represent a more
reliable and more biologically and clinically relevant model system
for investigating the mechanistic basis for human glioma cell infil-
tration. To our knowledge, this study is one of the first to use this
more clinically relevant model system to explore the molecular
mechanisms operative in the invasive behavior of primary human
glioblastomas.
Our data demonstrate that glioma TIC/TSCs require the cell
surface protein receptors ITGB1 and TrkA for CTGF-mediated
reduction of E-cadherin expression and tumor cell infiltration.
Whether this is a common mechanism for glioma cell invasion in
patients raises the question of how often ITGB1, TrkA, and
CTGF are found associated with each other in a complex in
gliomas in situ. Although ITGB1 has been reported to be ubiqui-
tously expressed in gliomas, the frequency of glioma-associated
TrkA is less consistent, with a report (37) finding that it is
expressed and another report (38) finding that it is not. Our data
suggest a potential explanation for this discrepancy in the litera-
ture: We found that TrkA is strongly expressed in the subpopula-
tion of highly infiltrating glioma cells in vivo and not in the glioma
cells that remain within the bulk of the tumor. Thus, TrkA expres-
sion is dependent on both the cell type and the location within the
tumor.
What remains unclear is whether the TrkA-expressing highly
infiltrative glioma cells represent a genetically or epigenetically
distinct subpopulation of cells within a given tumor, whether TrkA
expression is a stochastic event that allows a subpopulation of
TrkA-expressing cells to infiltrate, and/or whether there exist dis-
tinct host-induced signals within a proinfiltrative and TrkA-
inducing “niche” within the heterogeneous microenvironment of a
given glioma. Given previous work from our group (39) and others
(40) showing that suppression of NF-kB results in the enhance-
ment of glioma cell death, NF-kB could be a promising thera-
peutic target for inhibiting glioma cell invasion and survival.
CTGF may also turn out to be a promising therapeutic target.
For example, we found that addition of purified recombinant
CTGF
182–250
, a truncated form of the CTGF protein that essen-
tially removes the carboxyl-terminal region and the cysteine knot
(41) and does not bind TrkA, caused increased TIC/TSC invasion
and migration in vitro. The carboxyl-terminal region of CTGF
has previously been linked to enhanced cell adhesion in several
cell types (42). Therefore, we predicted that the loss of this car-
boxyl-terminal region would result in an increase in TIC/TSC
infiltration, which is what we observed in vitro. Thus, CTGF as
well as NF-kB may be potential therapeutic targets to alleviate
tumor cell infiltration.
A potential limitation of this study is the use of glioma TIC/
TSCs as a clinically relevant model to explore glioma invasion. It
is possible that TIC/TSCs are not the only population of infiltra-
tive cells within a given glioma. Indeed, our studies were not
designed to address whether TIC/TSCs are the only infiltrative
cells or whether subpopulations of their progeny are also infiltra-
tive. Given that TIC/TSCs are defined functionally (ie, by their
tumor-initiating ability), the prospective identification of such cells
in situ is currently impossible.
Nevertheless, the fact that many of the infiltrative tumor cells
in our in vivo model did not express glioma clonogenic (ie, CD133
and CD15) or stem cell (ie, Sox2 and nestin) markers (5) but do
express markers of partial differentiation (ie, GFAP and Tuj1)
suggests that it is highly likely that both the clonogenic glioma
stem cell population as well as their partially or fully differentiated
TIC/TSC progeny have infiltrative properties in vivo. A definitive
answer about the relative invasiveness of TIC/TSCs compared
with their more differentiated progeny requires further studies.
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We examined whether the reactive astrocytosis (“gliosis”) that is
invariably associated with malignant gliomas contributes to the
invasive phenotype of gliomas through the local secretion of
CTGF. Our data clearly demonstrate that tumor-associated reac-
tive astrocytes secrete high levels of CTGF in vitro and in vivo
(Figure 6, A and B; Supplementary Figure 1, B, available online)
and thereby establish a permissive microenvironment for glioma
cell invasion. Thus, a provocative and potentially worrisome ques-
tion raised by these data is whether the injury response of normal
brain tissue to standard glioma treatments, such as surgery and
radiation, contribute to a proinvasive microenvironment for the
surviving fraction of tumor cells through the induction of host-
derived CTGF (Supplementary Figure 5, available online).
In summary, this study provides one of the first, to our knowl-
edge, molecular analyses of glioma cell invasion using primary
patient-derived TIC/TSCs. We elucidate a cell signaling pathway
that requires binding of CTGF to a novel ITGB1–TrkA cell surface
receptor complex, which leads to NF-kB activation, induction of
the transcriptional repressor ZEB-1, disruption of cell–cell contacts
through loss of E-cadherin, and glioma cell and TIC/TSC infiltra-
tion. Finally, the demonstration of a host-derived CTGF-mediated
contribution to a proinvasive microenvironment within the brain
both raises potential concerns regarding the effects of current ther-
apeutic modalities on the invasive characteristic of glioblastoma
while at the same time offering a number of potentially new thera-
peutic targets.
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Funding
This work was supported by the intramural programs of National Cancer
Institute and the National Institute of Neurological Disorders and Stroke of the
National Institutes of Health.
Notes
The authors had full responsibility for the design of the study; the collection,
analysis, and interpretation of the data; the decision to submit the article for
publication; and the writing of the article.
Affiliations of authors: Neuro-Oncology Branch, National Cancer Institute/
National Institute of Neurological Disorders and Stroke, National Institutes of
Health, Bethesda, MD (LAE, KW, MJS, AL, JL, CE, HS, GB, WZ, HAF);
National Institute of Neurological Disorders and Stroke, National Institutes of
Health, Bethesda, MD (DM); Digestive Diseases Branch, NIDDK, National
Institutes of Health, Bethesda, MD (SAM, RTG).
    • "[42] Consistently, co-cultured astrocytes display increased expression levels of a number of growth factors and cytokines and enhance invasiveness of glioblastoma stem-like cells. [43] Another decisive input could stem from astrocytes activated by the neoplastic lesion and the consequent up-regulation of matricellular proteins such as secreted protein acidic and rich in cys-teins (SPARC) in astrocytoma [44] and medulloblastoma [45] or connective tissue growth factor in glioma, [46] which jointly with additional matricellular proteins remodel neuronal tissue during development or after brain injury. [28] Significantly, the concept of reciprocal stimulation of tumor cells and astrocytes was recently also identified in metastatic melanoma, which elicits an inflammatory cytokine response in astrocytes that facilitates brain metastasis. "
    [Show abstract] [Hide abstract] ABSTRACT: Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system (CNS) tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors. A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes, to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness. These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments. However, the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel, physiologically more relevant assay systems including organotypic brain slice culture. In this review, using publicly available peer-reviewed primary research and review articles, we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors. Furthermore, we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors. We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.
    Full-text · Article · May 2016
    • "CTGF has previously been identified as a fibrogenic cytokine that is highly expressed in wound healing and fibrotic lesions [12]. In human cancers, the pleiotropic functions of CTGF have been investigated, including the function as an oncoprotein in glioma [13] and melanoma [14], but a tumor-suppressor in lung cancer and colon cancer [15, 16]. In breast cancer, studies have shown that CTGF cooperates with other genes to mediate osteolytic metastasis, and high expression of CTGF mRNA in the bulk tumor correlated with advanced tumor stages [17], however, the mechanistic origin of CTGF has rare been explored. "
    [Show abstract] [Hide abstract] ABSTRACT: Interactions among tumor cells, stromal cells, and extracellular matrix compositions are mediated through cytokines during tumor progression. Our analysis of 132 known cytokines and growth factors in published clinical breast cohorts and our 84 patient-derived xenograft models revealed that the elevated connective tissue growth factor (CTGF) in tumor epithelial cells significantly correlated with poor clinical prognosis and outcomes. CTGF was able to induce tumor cell epithelial-mesenchymal transition (EMT), and promote stroma deposition of collagen I fibers to stimulate tumor growth and metastasis. This process was mediated through CTGF-tumor necrosis factor receptor I (TNFR1)-IκB autocrine signaling. Drug treatments targeting CTGF, TNFR1, and IκB signaling each prohibited the EMT and tumor progression.
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    • "In medulloblastoma, the methylation frequency of E-cadherin gene was not high (8%) [60]. In the context of ZEB suppression, binding of ZEB1 to the E-cadherin promoter was dependent on the activation of NF-κB in glioblastoma [61]. In the glioblastoma cell lines, it has been demonstrated that high ZEB2 levels could suppress E-cadherin, thereby regulating cancer cell differentiation [62]. "
    [Show abstract] [Hide abstract] ABSTRACT: Downregulation of E-cadherin in solid tumors with regional migration and systematic metastasis is well recognized. In view of its significance in tumorigenesis and solid cancer progression, studies on the regulatory mechanisms are important for the development of target treatment and prediction of clinical behavior for cancer patients. The vertebrate zinc finger E-box binding homeobox (ZEB) protein family comprises 2 major members: ZEB1 and ZEB2. Both contain the motif for specific binding to multiple enhancer boxes (E-boxes) located within the short-range transcription regulatory regions of the E-cadherin gene. Binding of ZEB1 and ZEB2 to the spaced E-cadherin E-boxes has been implicated in the regulation of E-cadherin expression in multiple human cancers. The widespread functions of ZEB proteins in human malignancies indicate their significance. Given the significance of E-cadherin in the solid tumors, a deeper understanding of the functional role of ZEB proteins in solid tumors could provide insights in the design of target therapy against the migratory nature of solid cancers.
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