Download Connective Tissue Growth Factor–Specific

Research Article
Connective Tissue Growth Factor–Specific Monoclonal Antibody
Therapy Inhibits Pancreatic Tumor Growth and Metastasis
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Nadja Dornhöfer, Suzanne Spong, Kevin Bennewith, Ali Salim, Stephen Klaus,
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Neeraja Kambham, Carol Wong, Fiona Kaper, Patrick Sutphin, Rendall Nacalumi,
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Michael Höckel, Quynh Le, Michael Longaker, George Yang,
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Albert Koong, and Amato Giaccia
Departments of 1Radiation Oncology, 2Plastic Surgery, and 3Pathology, Stanford University School of Medicine, Stanford; 4FibroGen, Inc.,
South San Francisco, California; and 5Department of Obstetrics and Gynecology, University of Leipzig, Leipzig, Germany
Abstract
Pancreatic cancer is highly aggressive and refractory to most
existing therapies. Past studies have shown that connective
tissue growth factor (CTGF) expression is elevated in human
pancreatic adenocarcinomas and some pancreatic cancer cell
lines. To address whether and how CTGF influences tumor
growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF
overexpression on cell proliferation was measured in vitro in
monolayer culture, suspension culture, or soft agar, and
in vivo in tumor xenografts. Although there was no effect of
CTGF expression on proliferation in two-dimensional cultures,
anchorage-independent growth (AIG) was enhanced. The
capacity of CTGF to enhance AIG in vitro was linked to
enhanced pancreatic tumor growth in vivo when these cells
were implanted s.c. in nude mice. Administration of a
neutralizing CTGF-specific monoclonal antibody, FG-3019,
had no effect on monolayer cell proliferation, but blocked
AIG in soft agar. Consistent with this observation, anti-CTGF
treatment of mice bearing established CTGF-expressing
tumors abrogated CTGF-dependent tumor growth and
inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate
CTGF as a new target in pancreatic cancer and suggest that
inhibition of CTGF with a human monoclonal antibody may
control primary and metastatic tumor growth. (Cancer Res
2006; 66(11): 5816-27)
Introduction
Pancreatic cancer continues to be one of the most lethal
cancers. More then 30,000 new cases of pancreatic cancer are
diagnosed annually in the U.S. Mortality rates for this disease have
not changed significantly in 30 years and approach 100% within
5 years after diagnosis. Current treatment options are limited due
to the lack of diagnostic biomarkers and a lack of obvious
symptoms until the tumor stage is already advanced. Approximately 10% to 20% of patients have surgically resectable disease
at presentation, but even in these cases, the 5-year survival rate is
only 20% (1). In patients with advanced disease, gemcitabine is
considered to be a first-line option. However, gemcitabine only
Requests for reprints: Amato Giaccia, Division of Radiation and Cancer Biology,
Department of Radiation Oncology, Stanford University School of Medicine, Room
1255, CCSR South, 269 Campus Drive, Stanford, CA 94305. Phone: 650-723-7366;
E-mail: [email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-0081
Cancer Res 2006; 66: (11). June 1, 2006
modestly improves survival with a 1-year survival rate of <20% (1).
Clearly, there exists a need for more effective targeted therapeutics to treat pancreatic cancer by targeting gene products that will
alter the malignant progression of pancreatic cancer or its
response to therapy.
Connective tissue growth factor (CTGF) is a member of the CCN
[CYR61 (cysteine-rich 61) / CTGF / NOV (nephroblastoma overexpressed)] family of secreted proteins, which are characterized
as cysteine-rich matricellular proteins that each contain four
modular domains displaying homology to insulin-like growth
factor–binding proteins (domain 1), a von Willebrand factor type
C repeat (domain 2), a thrombospondin type 1 repeat (domain 3),
and a cysteine knot domain (domain 4), respectively (2). The
cysteine knot domain contains heparin-binding sites that mediate
binding to extracellular matrix and cell surface heparan sulfate
proteoglycans (3). CTGF is an immediate early gene that is potently
induced by a variety of stimuli that regulate extracellular matrix
deposition, tissue remodeling, and neovascularization, including
platelet-derived growth factor, transforming growth factor (TGF)-h,
basic fibroblast growth factor, vascular endothelial growth factor
(VEGF), and hypoxia in fibroblasts or endothelial cells (4–7). CTGF
exhibits a diverse range of cellular functions including cell
adhesion, stimulation of cell migration, and potentiation of growth
factor–induced DNA synthesis (8).
CTGF interacts with integrin receptors including avh3, aIIbh3,
a6h1, and amh2 (3, 9–11) and has been reported to be a ligand for
low-density lipoprotein-related protein 1 (LRP-1); interacts with
LRP-5 to inhibit Wnt signaling (12–14) and can interact directly
with several growth factors including TGF-h (49). Taken together,
past studies indicate that the mechanism of action of CTGF relates
to its capacity to modulate and amplify a variety of biological
processes by binding directly to mitogenic, fibrogenic, and
angiogenic factors that are important in inflammation, fibrosis,
tumor growth, and tumor metastasis.
Elevated CTGF levels have been detected in a number of cancers
including pancreatic (15), breast (16), glioblastoma (17, 18),
esophageal (19), melanoma (20), chondrosarcoma (21), oral
squamous cell cancer (22), acute lymphoblastic leukemia (23),
rhabdomyosarcoma (24), and hepatocellular carcinoma (25), but
its direct role in tumor suppression or progression has not been
investigated in pancreatic cancer nor with therapeutic agents with
the capacity to inhibit CTGF function in vivo. An increase in CTGF
was reported to be associated with decreased survival of patients
with breast cancer (16), glioblastoma (18), or adenocarcinoma of
the esophagus (19), and increased breast cancer bone metastasis
in a mouse model (26). In contrast, high levels of CTGF were
associated with better survival in patients with esophageal
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CTGF in Pancreatic Cancer
squamous cell carcinoma (19) and chondrosarcoma (21), and
decreased metastasis in a colon cancer mouse model (27).
Likewise, in lung adenocarcinoma, reduced CTGF expression was
correlated with advanced disease stage and decreased survival, and
expression of CTGF in lung cancer cell lines suppressed metastasis
in a mouse tail vein injection model (28).
Whereas elevated levels of CTGF have been detected in the
above tumor types, pancreatic carcinomas are especially noteworthy because a hallmark of their histopathology is desmoplasia. In a
previous study by Wenger et al., 15 of 19 samples from pancreatic
tumors exhibited an average 59-fold enhancement of CTGF mRNA
expression, compared with a 4.5-fold increase in chronic pancreatitis (15). CTGF transcript levels were grossly correlated with the
degree of fibrosis and collagen expression, consistent with known
CTGF bioactivity. In a study of 25 patients with pancreatic cancer,
Hartel et al. reported a 46-fold increase in CTGF mRNA levels in
pancreatic cancer tissue compared with normal tissue (29).
Furthermore, they found a positive correlation between desmoplastic reaction and CTGF mRNA level and concluded that the
desmoplastic reaction might account for better survival of patients
with elevated CTGF expression observed in this study. In contrast,
Ryu and coworkers identified CTGF as a member of a pancreatic
cancer invasion–specific gene cluster (30). In this study, the CTGF
message was localized to pancreatic tumor cells rather than to
stromal or endothelial cells (31). However, CTGF expression has
been seen in both tumor cells and associated fibroblasts,
endothelial cells, pancreatic stellate cells, and vascular smooth
muscle cells (15, 29), not permitting clear insight into the
significance of the cellular origin of CTGF expression. However,
because CTGF has been found to be expressed in both the stromal
and tumoral compartments and is a potential driver of desmoplasia, CTGF represents a unique target in pancreatic tumorigenesis. Although we and others (15) have found that most patients
with pancreatic cancer exhibit elevated levels of CTGF compared
with controls, no study has directly investigated the role of CTGF in
pancreatic cancer growth or the potential of targeting CTGF for
therapy.
In this study, we investigated the influence of overexpressing
CTGF on pancreatic cancer cell growth in vitro and in vivo, and the
effect of inhibiting CTGF to control pancreatic tumor growth. Our
data suggest that tumor cell–derived CTGF enhances pancreatic
tumor growth, whereas inhibition of CTGF with a human
monoclonal antibody (mAb) reduces pancreatic tumor growth
and metastasis. Therefore, CTGF may be a new target for the
treatment of pancreatic cancer.
Materials and Methods
Patient samples. Human tissue from patients with pancreatic cancer
was obtained during Whipple procedures at Stanford University. All
patients signed an informed consent approved by the Stanford Institutional
Review Board (in accord with an assurance filed with and approved by the
U.S. Department of Health and Human Services). Representative paraffin
blocks with carcinoma and normal pancreas were selected from each of
eight cases. The immunohistochemical stains were done using rabbit
polyclonal antibody directed against mouse CCN2/Fisp 12 which crossreacts with human CTGF (provided by L. Lau; ref. 32). Serial sections of
4 Amol/L were obtained from the selected paraffin blocks, deparaffinized in
xylene, and hydrated in a graded series of alcohols. Heat-induced antigen
retrieval was carried out by microwave pretreatment in citric acid buffer
(10 mmol/L, pH 6.0) for 10 minutes. The CCN2 antibody was used at a
dilution of 1:400. The endogenous peroxidase was blocked and the DAKO
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Envision System (DAKO Corporation, Carpinteria, CA) was used for
detection; diaminobenzidine was used as a chromogen.
The intensity of the staining was scored on a scale of 0 to 3+: where 0, no
staining; 1, weak staining; 2, moderate staining; and 3, strong staining. If the
staining in a particular case was variable, the percentage of tumor and
normal tissue with a specific intensity score was also recorded. Scoring was
done separately for normal pancreas, areas of chronic pancreatitis, and
carcinoma; both epithelial elements and stroma were scored.
CTGF ELISA. Cell supernatants were collected in DMEM supplemented
with 1% penicillin/streptomycin, 100 Ag/mL low molecular weight heparin,
and 0.25% bovine serum albumin. CTGF levels in culture supernatants,
blood plasma, and urine were measured using a sandwich ELISA that
detects whole CTGF and the NH2-terminal fragment of CTGF that persists
in body fluids and cell culture supernatants after proteolytic cleavage of the
hinge domain (33).
Cell lines and tissue culture. The human pancreatic cancer cell lines,
MIA PaCa-2 and PANC-1, were grown in DMEM supplemented with 10%
fetal bovine serum (FBS) and 1% penicillin/streptomycin, whereas Su86.86
pancreatic cancer cells were grown in RPMI 1640 containing 10% FBS. To
generate pancreatic cancer cell lines overexpressing CTGF, we stably
transfected MIA PaCa-2 cells with pShuttle-CMV-Puro-CTGF vector
(an adenoviral construct encoding full-length CTGF and the puromycin
antibiotic-resistance gene) using LipofectAMINE 2000 reagent (Invitrogen
Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Control transfections were done with the same vector lacking the
CTGF cDNA sequence. Individual clones were selected based on their
resistance to puromycin, and CTGF mRNA and protein levels were
determined by quantitative real-time PCR (qRT-PCR) and ELISA or Western
blot assay, respectively. A series of CTGF-expressing or vector control clones
were isolated and characterized, and representative clones that exhibited
different levels of CTGF expression and protein production were identified.
Stable transfectants were cultured in DMEM supplemented with 10% FBS,
1% penicillin/streptomycin and 1.5 Ag/mL puromycin. All cell lines were
cultured in a 5% CO2 humidified atmosphere at 37jC.
CTGF mAb. FG-3019 is a fully human IgG1n mAb recognizing domain 2
of human and rodent CTGF, and was obtained from FibroGen, Inc. (South
San Francisco, CA). FG-3019 was purified under cyclic guanosine 3¶,5¶monophosphate conditions and formulated in a 25 mmol/L histidine buffer
(pH 6.0). In some experiments, polyclonal human IgG from Cohn’s fraction
of human serum (Sigma, St. Louis, MO) purified by protein A sepharose was
used as a control.
In vitro growth curve. Cells were plated at a density of 5 104 in 6 cm
dishes. Every 3 or 4 days, cells were trypsinized, counted and 5 104 cells
were replated. To investigate the effect of a CTGF-specific antibody (FG-3019)
on tumor cell growth, growth curves were also determined in the presence of
20 and 40 Ag/mL of FG-3019 or control human IgG. For growth in suspension,
2.5 105 cells were plated on ultra-low cluster plates (Costar, Cambridge,
MA) which have a covalently bound hydrogel layer that effectively inhibits
cellular attachment. Photographs were taken 4 days after cells were plated
using a Leica MZ6 microscope with 10 and 40 objectives. Growth curves
were obtained by plating 2.5 105 tumor cells into each of 12 wells, allowing
the aggregates to form over a 48-hour period, and trypsinizing the tumor cell
aggregates in triplicate wells daily with subsequent cell counting.
Soft agar assay. In duplicate experiments, f3,000 cells from each clone
were resuspended in 2 mL of 0.35% Noble Agar (Difco Laboratories, Detroit,
MI) containing 10% FBS (Life Technologies) and 10% newborn calf serum
(Life Technologies). Each embedded cell mixture was overlaid on 1.5 mL of
0.7% Noble Agar in six-well plates, and a 1.5 mL top layer of 0.7% Noble Agar
was added to each well to prevent evaporation. In some experiments, human
control IgG or FG-3019 was added at a concentration of 100 Ag/mL to the
cell layer containing 1,500 cells in 2 mL. Plates were incubated for 11 days in
a humidified incubator at 37jC, 5% CO2. The number of colonies was
enumerated by counting a 1.5 1.5 cm grid under a microscope. Total colony
counts were extrapolated to the entire plate based on the ratio of the surface
area of each well to the surface area of the grid. Colony morphologies were
captured with a Nikon camera mounted onto an inverted microscope that was
set at 20 magnification.
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qRT-PCR. For confirmation of CTGF mRNA expression, we did qRT-PCR.
We obtained cDNA by reverse transcription of 1 Ag of DNase-treated total
RNA from each sample using random hexamer priming in 50 AL reactions
according to the manufacturer’s recommendations (TaqMan reverse
transcription reagent kit; Applied Biosystems, Foster City, CA). We
proceeded with qRT-PCR using the Applied Biosystems Prism 7900HT
sequence detection system. A nonmultiplexed SYBR Green assay in which
each cDNA sample was evaluated at least in triplicate and 20 AL reactions
was used for all target transcripts. Expression values were normalized to
human glyceraldehyde-3-phosphate dehydrogenase. qRT-PCR primers were
designed using Primer Express version 2.0.0 (Applied Biosystems) and
tested to confirm appropriate product size and optimal concentrations. All
primer sequences are available on request.
Animal experiments/s.c. pancreatic tumor growth. To establish s.c.
xenografts, 6- to 8-week-old male nude mice (nu/nu, f25 g) or severe
combined immunodeficient mice (f28 g; Charles River Breeding
Laboratories, Wilmington, MA) were used. Cells were grown to subconfluency and 2 107 or 1 107 cells in 0.1 mL DMEM + 10% FBS were s.c.
injected into the flank of the animals. Mice were maintained in a pathogenfree environment; food and water were given ad libitum. Housing and
all procedures were done with approval of the Institutional Animal Care
and Use Committee at Stanford University. Tumor size was measured
in three dimensions with a caliper ruler and tumor volume was calculated by multiplication of the three dimensions divided by 2 (volume = a b c / 2). At the end of the experiments, mice were sacrificed using a
CO2 chamber consistent with the 2000 report of the American Veterinary
Medical Association Panel on Euthanasia. Xenografts were then excised
and fixed in 10% neutral buffered formaldehyde or embedded in optimal
cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA)
and stored at 80jC until processing.
Immunohistochemistry (Ki-67 and CD31). Sequential 4 Amol/L
paraffin sections were stained with rabbit anti-Ki-67 antibody (1:50;
Zymed, San Francisco, CA). The detection was done by using biotinylated
secondary antibodies in combination with horseradish peroxidase–
coupled streptavidin (Jackson ImmunoResearch, West Grove, PA) and
the substrate 3,3¶-diaminobenzidine (Research Genetics/Invitrogen). CD31
staining was done on frozen optimal cutting temperature–embedded
tumor sections using rat anti-mouse (platelet/endothelial cell adhesion
molecule 1; 1:50; BD PharMingen, Bedford, MA). All sections were
counterstained with hematoxylin, dehydrated, and mounted using
synthetic nonaqueous mounting medium.
Terminal deoxynucleotidyl transferase–mediated dUTP nick-end
labeling staining for apoptotic cells in tumor sections. Apoptosis
in formalin fixed, paraffin-embedded tumor slides was assessed by the
principle of terminal deoxynucleotidyl transferase–mediated dUTP nick-end
labeling (TUNEL) to detect fragmented DNA in apoptotic cells. The
DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) was used.
Sections were treated according to the manufacturer’s recommendations.
Briefly, sections were deparaffinized and rehydrated, permeabilized in
proteinase K, and treated with terminal deoxynucleotidyl transferase
incubation buffer at 37jC for 60 minutes in the dark. Sections were
counterstained with 4¶-6-diamidino-2-phenylindole (DAPI; Sigma).
Evaluation of immunohistochemical slides. Using the antimouse
Ki-67 antibody and the TUNEL reagent, we determined the percentage of
Ki-67-, and TUNEL-positive tumor cells in relation to all tumor cells. For
this purpose, four representative areas (high-power fields) of tumor sections
were randomly selected. Evaluation was done with a Nikon Eclipse E800
microscope with 400 magnification. The percentage of Ki-67- and TUNELpositive cells was determined by manually counting at least 400 cells per
high power field. Indices of cell proliferation and apoptosis were expressed
Figure 1. Immunohistochemical staining of patient samples. A to D, pancreatic tissue samples were taken from eight patients with pancreatic cancer and were
stained for CTGF by immunohistochemistry. Intensity of the staining was scored on a scale of 0 to 3+, with 3+ being the strongest staining. A, normal pancreas;
B, pancreatitis; C, pancreatic carcinoma; D, overview of sections from all eight patients.
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CTGF in Pancreatic Cancer
as the ratio of Ki-67- and TUNEL-positive cells, respectively, and normalized
to the vector control–transfected cell line, VA2. Using CD31 staining, the
number of vessels was counted in equivalent areas of a low power field
(100) in at least four randomly selected low-power fields per tumor
section. Mean values for all five areas and SDs were calculated.
Statistical analysis. Statistical evaluation was done using unpaired
Student’s t test for comparison between two values where appropriate. All
statistical tests were two-sided. P < 0.05 was considered statistically
significant.
Results
CTGF is overexpressed in pancreatic cancer tissue. Several
studies have shown CTGF to be elevated in pancreatic cancer
tissue samples. We did immunohistochemical staining of CTGF
expression in noninvolved normal pancreas tissue (Fig. 1A), areas
of chronic pancreatitis (Fig. 1B), and tumor tissue (Fig. 1C) in each
case derived from the same patient. CTGF was overexpressed in all
pancreatic cancer samples. In five of eight patients, CTGF staining
was scored as level 3 (levels 0-3) in pancreatic cancer cells, whereas
staining was scored as levels 1 to 2 in healthy pancreatic tissue or
pancreatitis. Interestingly, the highest CTGF staining was found in
the cytoplasm of pancreatic cancer cells, rather than in the
surrounding stromal cellular and connective tissue elements.
Generation of MIA PaCa-2-derived cell lines overexpressing
CTGF. To understand the effect of CTGF on pancreatic tumor
growth, we used a genetic approach in which we compared
isogenic pancreatic tumor cells that lack constitutive CTGF
expression in vitro with those in which the CTGF gene had been
introduced under the control of a CMV promoter. In this manner,
the effects of CTGF on the same cells could be determined and
differences due to other genetic alterations are minimized. We used
the human MIA PaCa-2 pancreatic cancer cell line to examine
CTGF expression because it does not exhibit TGF-h-responsiveness
(34) and does not constitutively produce endogenous CTGF (15).
Stable clones of MIA PaCa-2 cells overexpressing different levels of
CTGF (CE8, CA9, CD2, and CB4) or vector control cells (VA2, VA3,
VB4, VA6, and VB1) that do not express CTGF in vitro as confirmed
by CTGF-specific ELISA were generated (see Fig. 2A and B). We
also determined VEGF levels produced by the selected clones
because VEGF is known to be overexpressed in many tumor cell
types (35), to enhance tumorigenicity in pancreatic tumor models
(36) and to interact with CTGF (ref. 37; Fig. 2A and B). To rule out
effects of differing VEGF levels on CTGF-dependent tumor cell
growth, we picked three clones with comparably low VEGF
secretion (VA2, CE8, and CA9; Fig. 2A) for further studies and
confirmed expression of CTGF mRNA in these clones by qRT-PCR
(Fig. 2C). As expected, no CTGF mRNA expression was found in the
vector control cell line VA2, whereas there were 40-fold and 180fold inductions of mRNA, respectively, in the clones transfected
with CTGF. MIA PaCa-2 clones with similar VEGF levels but
differing CTGF expression levels were then tested for growth
in vitro and in vivo.
CTGF expression does not influence in vitro (monolayer)
proliferation rate of pancreatic cancer cells. To study the
influence of CTGF expression on cell proliferation in vitro, we
determined the monolayer growth rate of the VEGF-matched, non–
CTGF-expressing cell line (VA2), and the two CTGF-expressing cell
lines (CE8 and CA9). Over 13 days, there were no significant
differences in in vitro cell growth rates (Fig. 3A). Therefore, CTGF
does not have any apparent paracrine or autocrine effect on MIA
PaCa-2 tumor cell proliferation in two-dimensional growth assays.
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Figure 2. MIA PaCa-2–derived clones. A and B, MIA PaCa-2 cells were stably
transfected with an empty vector (VA2, VB4, VA3, VA6, and VB1) or vector
containing human CTGF (CE8, CA9, CD2, and CB4). Clones were analyzed for
levels of secreted CTGF and VEGF by sandwich ELISA of cell culture
supernatants. As expected, CTGF was not detected in supernatants from cell
lines transfected with empty vector and differing levels of CTGF were expressed
by cell lines transfected with human CTGF expression plasmid. Levels of
secreted VEGF varied up to 8-fold between the clones. C, clones with
comparably low VEGF expression were chosen for further studies and analyzed
for relative CTGF mRNA expression by qPCR.
CTGF promotes anchorage-independent tumor cell growth
of MIA PaCa-2 cells. Anchorage-independent growth (AIG) is a
common characteristic of cancer cells. Although unable to induce
AIG independently, CTGF is required for the induction of AIG by
TGF-h (38). MIA PaCa-2 cells reportedly have a colony-forming
efficiency in soft agar of f19% (39). To determine the effect of
CTGF expression on AIG in MIA Paca-2 cells, VEGF-matched pairs
of vector-transfected control cells and CTGF-expressing cells (VA2,
CA9, and VA6, CD2) were analyzed for their ability to grow in
soft agar (Fig. 3B; *, P < 0.001) and in ultra-low attachment plates
(Fig. 3C). The latter have a covalently bound hydrogel layer that
inhibits cell attachment, allowing cells to grow in suspension (40).
Although CTGF had little effect on monolayer growth, both the
number and size of colonies were increased when the same cells
were grown in soft agar (Fig. 3B). Multicellular masses also formed
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in the ultra-low attachment plates after 48 hours of culture, with
the CTGF-expressing tumor cell aggregates displaying increased
proliferation over time (Fig. 3C; *, P < 0.001). These results indicate
that CTGF could increase the AIG of MIA PaCa-2 cells.
CTGF overexpression supports tumor xenograft growth.
Because the transfected clones showed different levels of VEGF
expression, we next investigated the influence of VEGF expression
on tumor growth in our system. We implanted three vector control
clones (VA2, VA3, and VB4) expressing different VEGF levels in
nude mice. As shown in Fig. 4A, the growth of MIA PaCa-2 vector
control clones was VEGF-dependent in the absence of CTGF. These
data indicate that CTGF-deficient tumor cells such as MIA PaCa-2
grow in a VEGF-dependent manner. To determine the effects of
elevated CTGF expression on tumor cell growth in vivo, three low
VEGF-expressing MIA PaCa-2 clones with differing CTGF expression levels were injected s.c. into the flanks of nude mice. As shown
in Fig. 4B, there was little growth of the vector control clone that
does not express CTGF. Clones expressing CTGF exhibited
significantly enhanced tumor growth that was directly related to
CTGF protein levels (*, P V 0.01). Representative photographs
of tumors are shown in Fig. 4C. Similar results were obtained
with severe combined immunodeficient mice (data not shown)
indicating no difference in CTGF-enhanced tumor growth with the
host strain. To determine whether the expression levels of CTGF
observed in vitro translated into higher circulating levels of CTGF
in vivo, CTGF levels were measured in the plasma (41) and urine
(33) of hosts bearing tumors derived from MIA PaCa-2 clones
expressing different levels of CTGF. The level of CTGF present in
both urine and plasma of tumor-bearing hosts paralleled the
in vitro CTGF expression levels observed in culture supernatants of
the respective MIA PaCa-2 clones (Fig. 4D). Animals implanted
with high CTGF-expressing clones exhibited high levels of
circulating and urinary CTGF. CTGF levels were undetectable in
hosts implanted with the non–CTGF-expressing vector control
cells. Taken together, these data suggest that CTGF promotes
tumor growth in immunodeficient mice, and that CTGF produced
by pancreatic cancer cells could be detected in the plasma and
urine of pancreatic tumor–bearing mice.
CTGF increases proliferation and decreases apoptosis in
pancreatic tumors. We examined tumor sections for effects on
proliferation using Ki-67 staining, apoptosis by TUNEL staining,
and neovascularization by CD31 staining. We found that the levels
of Ki-67-positive cells correlated with tumor growth and CTGF
levels (Fig. 4E). The percentage of Ki-67-positive cells in tumors
Figure 3. Effect of CTGF expression on in vitro monolayer and AIG of MIA PaCa-2–derived clones. A, influence of CTGF on monolayer growth rate of MIA
PaCa-2–derived clones. Vector-transfected (VA2) and CTGF-expressing (CE8 and CA9) clones were tested for their proliferation rates in vitro. Cells were plated in
triplicate at the same starting cell density (5 104) and counted in regular intervals over a period of 13 days. B, effect of CTGF expression on tumor cell growth in
soft agar. Vector control (VA2 and VA6) and CTGF-expressing (CA9 and CD2) MIA PaCa-2 cells were plated in soft agar and allowed to form colonies over an
11-day period before counting the colonies. The VA2-CA9 and VA6-CD2 pairs are matched for similar VEGF expression (see Fig. 2A and B). Bars, SE; *, P < 0.001
(Student’s t test). C, effect of CTGF expression on AIG of cells plated in ultra-low attachment plates. Photographs were taken after 72 hours with 10 and 40
magnification. Tumor cell aggregates were trypsinized and counted daily for the growth curves; points, mean from triplicate wells; bars, FSE; *, P < 0.001
(Student’s t test).
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Figure 4. Influence of CTGF expression on tumor xenograft growth. A, s.c. tumor xenograft growth of MIA PaCa-2 clones transfected with empty vector–expressing
different levels of VEGF. B, s.c. tumor xenograft growth of MIA PaCa-2 transfectants expressing either empty control vector (VA2) or human CTGF (CE8 and CA9) in
nude mice. Tumor volume was calculated at weekly intervals with caliper measurements. Points, mean; bars, FSE; *, P V 0.01 (Student’s t test). C, macroscopic
photographs of vector-transfected (VA2) and CTGF-expressing (CE8 and CA9) s.c. xenografts excised from nude mice. D, CTGF levels detected in urine and
plasma samples from mice implanted with non–CTGF-expressing (VA2) or CTGF-expressing (CE8 and CA9) xenografts. CTGF levels were determined by sandwich
ELISA. E, Ki-67 staining of paraffin-embedded tumor sections from vector control (VA2) and CTGF-expressing (CE8) xenografts were used to assess tumor cell
proliferation (brown, proliferating cells; 200 magnification). Columns, mean percentage of proliferating cells; bars, FSE; *, P < 0.01 (Student’s t test). F, TUNEL and
DAPI staining of paraffin-embedded tumor sections of vector control (VA2) and CTGF-expressing (CE8) xenografts were used to assess apoptosis (green, 200
magnification). Columns, mean percentage of apoptotic cells; bars, F SE; *, P < 0.01 (Student’s t test). G, CD31 staining of paraffin-embedded tumor sections of vector
control (VA2) and CTGF-expressing (CE8 and CA9) xenografts were used to assess blood vessel density (brown, endothelial cells; 100 magnification). Columns,
mean number of blood vessels per field of view; bars, FSE.
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generated with the non–CTGF-expressing cell line (VA2) was 11%
compared with 18% (P < 0.01) in tumors of the CTGF-expressing
cell line CE8. The percentage of apoptotic cells in tumors generated
with the VA2 cell line was 2.2%, whereas for tumors generated with
CE8, the percentage of apoptotic cells was significantly lower at
0.7% (Fig. 4F; P < 0.01). Thus, the ratio of proliferation to apoptosis
for VA2 is 5 (11/2.2) and for CE8 is 26 (18/0.7). No difference in
neovascularization was found between the three cell lines (Fig. 4G).
Taken together, CTGF expression induces a significant increase in
proliferation and a significant inhibition of apoptosis in pancreatic
tumor xenografts, leading to a nearly 4-fold increase in the
proliferation/apoptosis ratio. These data suggest that the tumor
growth advantage of CTGF-overexpressing cells is due to enhanced
cell growth and diminished cell death.
The neutralizing CTGF-specific mAb FG-3019 does not
influence monolayer tumor cell proliferation in vitro. To
confirm that increased tumor growth and progression in vivo are
CTGF-dependent, we investigated the effects of CTGF inhibition
in vitro and in vivo using a CTGF-specific human mAb FG-3019
(FibroGen). We first determined the monolayer proliferation rate
of the three cell lines we had previously assessed in xenograft
transplant studies (VA2, CE8, and CA9) in the presence of FG-3019
or control human IgG. As shown in Fig. 5A, FG-3019 did not exhibit
a significant effect on monolayer tumor cell growth over a time
period of 10 days in all three cell lines, consistent with the finding
that CTGF overexpression had little effect on cell growth on tissue
culture plates.
FG-3019 inhibits AIG. Because CTGF expression supports AIG,
we further examined the influence of CTGF inhibition by FG-3019
on cells growing in soft agar. Soft agar assays were therefore done
in the presence of FG-3019 or control human IgG over 10 days. As
shown in Fig. 5B (right columns), colony formation of a CTGFoverexpressing cell line can be inhibited down to the level of non–
CTGF-expressing clones by treatment with FG-3019 compared with
control human IgG (*, P < 0.001). No effect of FG-3019 was
observed on the growth of vector controls that do not express
CTGF (left columns). This further supports the hypothesis that
CTGF promotes AIG and shows that this effect could be inhibited
by blocking CTGF using a neutralizing human mAb.
FG-3019 has a cytostatic effect on MIA PaCa-2-derived
tumor xenografts. As the expression of CTGF promotes tumor
xenograft growth, and the in vitro soft agar experiments showed an
inhibition of AIG with FG-3019, we further sought to investigate the
effect of CTGF inhibition on tumor growth in vivo. Based on
previous pharmacokinetic data generated with FG-3019 in normal
mice (data not shown), we gave FG-3019 twice a week to animals
that had been implanted with CTGF-expressing MIA PaCa-2 cells
(CE8). When mean tumor size reached 150 to 200 mm3, animals
were stratified on the basis of tumor volume (such that the mean
tumor volume in each group was uniform) to either a treatment or
vehicle control group, and antibody treatment was started.
Antibody or neutral-buffered vehicle was injected i.p. twice a week
at a dose of 40 mg/kg. As shown in Fig. 5C, animals dosed with FG3019 exhibited decreased tumor growth after f2 weeks of therapy
in xenografts derived from a CTGF-expressing MIA PaCa-2 clonal
cell line, whereas a continuous increase in tumor volume was seen
in the vehicle control groups. These experiments show that
neutralization of CTGF in CTGF-expressing xenografts inhibits
tumor growth in vivo. They further show that enhanced tumor
growth of CTGF-expressing MIA PaCa-2 clones is CTGF-dependent
and is not due to other genetic lesions.
Cancer Res 2006; 66: (11). June 1, 2006
FG-3019 treatment inhibits tumor growth and increases
apoptosis, but does not alter proliferation. To analyze the
potential mechanisms for the inhibitory effect of FG-3019, we
examined whether antibody treatment alters the proliferative,
apoptotic, or angiogenic status of treated tumors. We excised
tumors from animals implanted with CTGF-expressing MIA PaCa-2
cells that were treated or not treated with FG-3019. Tumor sections
were analyzed for effects on apoptosis or proliferation as before,
and for neoangiogenesis by staining for the endothelial cell marker
CD31. No significant differences were seen in proliferation markers
or CD31 staining in tumor xenografts from mice treated with or
without FG-3019 (Fig. 5D and F). However, the percentage of
apoptotic cells increased 3-fold in the FG-3019 treatment group
(Fig. 5E; P < 0.01). The ratio of proliferation to apoptosis was 3-fold
higher in the control versus the FG-3019 group [30 (20.7/0.7)
compared to 10 (23.5/2.4)]. Taken together, these data indicate that
antibody treatment inhibits tumor growth by decreasing the ratio
of proliferation to apoptosis by 67%, mainly by inducing apoptosis.
FG-3019 inhibits the pancreatic cancer tumor growth of two
endogenous CTGF-expressing pancreatic tumor cell lines.
Thus far, all experiments described have been done with genetically
engineered tumor cell clones originating from the pancreatic cancer
cell line MIA PaCa-2. We next investigated the effect of inhibiting
CTGF on the wild-type pancreatic tumor cell lines Su86.86 and
PANC-1, both of which endogenously express CTGF (Fig. 6A) in a
TGF-h-inducible manner (ref. 15; data not shown). As seen
previously with MIA PaCa-2 clones that overexpress CTGF, the
in vitro monolayer growth rate of PANC-1 cells was not affected
using CTGF antibody concentrations up to 100 Ag/mL (Fig. 6B). For
in vivo studies, PANC-1 or Su86.86 cells were s.c. injected into the
flanks of nude mice. After a mean tumor size of 150 to 200 mm3 was
reached, animals were stratified on the basis of tumor volume (such
that the mean tumor volume in each group was uniform) into a
treatment or a control group such that the starting tumor volume in
each group was uniform. The treatment group was injected i.p. with
FG-3019 (40 mg/kg) twice per week. As shown in Fig. 6C, PANC-1
tumor growth was significantly decreased when treated with
neutralizing CTGF-specific antibody. After 6 weeks of treatment,
the mean tumor size of the treatment group was reduced by
>50% relative to the control group treated with control human IgG
(*, P < 0.01). A tumor growth delay of 2.5 weeks was also observed
when comparing the growth curves at the half-maximal control
tumor size. Su86.86 tumor growth was even more affected by FG3019 treatment (Fig. 6D), with treated tumors 75% smaller than
control tumors after 6 weeks of treatment, and a tumor growth
delay of 5 weeks at the half-maximal control tumor size. The delay
of Su86.86 tumor growth induced by inhibition of CTGF was of
comparable magnitude to that induced by three injections of 100
mg/kg of gemcitabine in a separate experiment (Fig. 6E).
FG-3019 inhibits lymph node metastasis of PANC-1 tumor
cells. It is noteworthy that in mice implanted with s.c. PANC-1
tumors, five of six mice in the control group developed macroscopically visible inguinal and/or axillary lymph node metastases,
whereas only one of five mice in the antibody treatment group
developed macroscopically visible metastases (Fig. 7A). Histologic
examination of excised lymph nodes showed that the macroscopic
enlargement in control groups was due to tumor cell infiltration
(Fig. 7B). These data suggest that inhibition of CTGF is growthinhibitory for two wild-type pancreatic tumor types with endogenous CTGF expression, and that CTGF inhibition is also
antimetastatic.
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CTGF in Pancreatic Cancer
Figure 5. Influence of neutralizing CTGF-specific mAb on CTGF-expressing cells in vitro and in vivo. A, vector-transfected (VA2) and CTGF-expressing
(CE8 and CA9) clones were grown in the presence of the CTGF-specific mAb FG-3019 (20 or 40 Ag/mL) or human IgG (Control IgG) and the in vitro proliferation
rates were determined over 10 days. Antibody concentrations up to 100 Ag/mL were used, showing comparable results. B, vector-transfected (VA6) and
CTGF-expressing MIA PaCa-2 cells (CD2) were plated in soft agar in the presence of the CTGF-specific mAb FG-3019 (100 Ag/mL) or control human IgG and allowed
to form colonies over a 10-day period before counting the colonies. Columns, mean number of tumor cell colonies; bars, FSE; *, P < 0.001 (Student’s t test).
C, CTGF-expressing MIA PaCa-2 clone CE8 were s.c. inoculated into nude mice. At a mean tumor size of 150 to 200 mm3, animals were stratified on the basis of tumor
volume (such that the mean starting tumor volume in each group was uniform) into treatment and control groups, and i.p. injections of the neutralizing CTGF-specific
mAb FG-3019 or vehicle control were done twice a week (40 mg/kg). Tumor volume was calculated at weekly intervals with caliper measurements. Points, mean;
bars, FSE; *, P < 0.05 (Student’s t test). D, Ki-67 staining of paraffin-embedded tumor sections of CTGF-expressing (CE8) xenografts treated with either CTGF-specific
mAb FG-3019 or vehicle were used to assess the influence of CTGF antibody treatment on tumor cell proliferation (brown, proliferating cells; 200 magnification).
Columns, mean percentage of proliferating cells; bars, FSE. E, TUNEL of paraffin-embedded tumor sections of CTGF-expressing (CE8) xenografts treated with either
CTGF-specific mAb FG-3019 or vehicle were used to assess the influence of CTGF antibody on apoptosis (green, TUNEL-positive apoptotic cells; 200 magnification).
Columns, mean percentage of apoptotic cells; bars, FSE; *, P < 0.01 (Student’s t test). F, CD31 staining of paraffin-embedded tumor sections of CTGF-expressing
(CE8) xenografts treated with either CTGF-specific mAb FG-3019 or vehicle, were used to assess blood vessel density (brown-green, endothelial cells, 100
magnification). Columns, mean number of blood vessels per field of view; bars, FSE.
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Figure 6. Influence of CTGF-specific antibody FG-3019 on the growth of xenografts derived from the pancreatic cancer cell lines, PANC-1 and Su86.86. A, the
pancreatic cancer cell line PANC-1 expresses CTGF endogenously. B, the CTGF-specific mAb FG-3019 does not influence in vitro monolayer growth of PANC-1 cells
(concentrations up to 100 Ag/mL). C, nude mice were s.c. inoculated with 107 PANC-1 cells, a pancreatic cancer cell line with endogenous CTGF expression. At a
mean tumor size of 150 to 200 mm3, animals were stratified on the basis of tumor volume (such that the mean starting tumor volume in each group was uniform)
into treatment and control groups and i.p. injections of the neutralizing, CTGF-specific mAb FG-3019, or control IgG were done twice a week (40 mg/kg). Tumor volume
was calculated at weekly intervals with caliper measurements. Points, mean; bars, FSE; *, P < 0.01 (Student’s t test). D, nude mice were s.c. implanted with
107 Su86.86 cells, a second pancreatic cancer cell line with endogenous CTGF expression. At a mean tumor size of 150 to 200 mm3, animals were randomized
into treatment and control groups and i.p. injections of FG-3019 (40 mg/kg) or vehicle control were done twice per week. Tumor volume was calculated at weekly
intervals with caliper measurements. Points, mean; bars, FSE; *, P < 0.05 (Student’s t test). E, nude mice bearing Su86.86 tumors were given 100 mg/kg of
gemcitabine on days 1, 5, and 9 of the experiment. The growth delay induced by gemcitabine was of comparable magnitude to that obtained with 40 mg/kg of FG-3019.
Tumor volume was calculated twice weekly with caliper measurements. Points, mean; bars, FSE; *, P < 0.05 (Student’s t test).
Discussion
The most extensive literature to date regarding CTGF defines its
role in wound-healing and fibrotic disease. However, several recent
studies implicate CTGF in tumor development and progression
(16–19, 25, 26) and tumor cell survival (24). Our results show that
CTGF promotes anchorage-independent pancreatic cancer cell
growth, which translates in vivo to enhanced tumor growth.
Furthermore, anti-CTGF treatment with FG-3019 inhibited AIG
in vitro, primary tumor growth in vivo, and the development of
macroscopic lymph node metastases. Thus, CTGF may represent a
novel target in pancreatic cancer and blocking its activity may also
inhibit the growth of distant metastasis. This is especially relevant
because patients are frequently diagnosed with advanced metastatic disease and there are few effective therapies for pancreatic
cancer metastasis.
These results with CTGF in the context of pancreatic adenocarcinomas are particularly noteworthy because a hallmark of their
histopathology is desmoplasia, and overexpression of CTGF is
associated with increased tissue fibrosis. To date, only one small
clinical study has examined the relationship between CTGF,
desmoplasia, and prognosis of pancreatic cancer (29). In this
Cancer Res 2006; 66: (11). June 1, 2006
study, CTGF mRNA was overexpressed mostly in connective tissue
cells and high CTGF levels in tumor samples were associated with
increased tumor differentiation and patient survival. The authors
hypothesized that elevated CTGF resulted in a better prognosis by
inducing fibrosis and inhibiting metastasis. However, the fact
remains that >90% of pancreatic tumors have prominent
desmoplasia (30), and the aggressive nature of these tumors
suggests that this reaction may facilitate invasion rather than
prevent it (31, 42). In situ hybridization studies by IacobuzioDonahue et al. have shown CTGF mRNA expression within the
tumor cells in one pancreatic cancer tumor specimen (31).
Although our immunohistochemical and transplanted tumor data
suggest that tumor cell–derived CTGF is important for tumor
growth and metastasis, other reports suggest that CTGF produced
by stromal fibroblasts mediate tumor growth (15, 29). Our
xenograft studies indicate that CTGF overexpression could
promote tumor growth and metastasis, but does not exclude the
possibility that stromal CTGF may have a significant role in tumor
progression. Although the production of CTGF by the stroma could
explain why pancreatic tumors which possess low levels of CTGF
still grow and metastasize, our data suggest that elevated levels of
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CTGF in Pancreatic Cancer
Figure 7. Influence of CTGF-specific
antibody FG-3019 on the development of
macroscopic lymph node metastases
derived from s.c. PANC-1 xenografts.
A, macroscopic lymph node metastases
were observed in mice bearing s.c.
PANC-1 tumor xenografts. Axillary and/or
inguinal lymph node metastases were
macroscopically visible in five of six mice
treated with control antibody and one of
five mice in the FG-3019-treated group.
B, lymph nodes were removed and
paraffin-embedded sections were stained
with H&E (100 and 400 magnification).
Tumor cell infiltration into lymph node
tissue is evident.
VEGF could compensate for a lack of CTGF production by the
tumor. Consistent with this idea, preclinical studies have shown
that VEGF inhibition with a modified soluble VEGF receptor
(VEGF-Trap) inhibited both s.c. and orthotopic pancreatic tumor
growth and metastasis (43). CTGF may also act as a biostore for
angiogenic factors, e.g., CTGF can bind to VEGF and inhibit its
signaling, but VEGF function is restored upon cleavage of CTGF by
matrix metalloproteases. Thus, a combination of anti-VEGF
therapy (43) with FG-3019 could be a highly effective treatment
strategy.
Our data suggest that CTGF mediates both cell survival and
proliferation; in vitro in AIG assays and in vivo in pancreatic
tumors. This is consistent with earlier reports which showed that
CTGF mediates the effects of TGF-h on AIG of NRK fibroblasts
(38). However, the role of CTGF in regulating cell survival or
proliferation is likely to be highly cell type–specific and dependent
on what other signaling pathways are also activated in the target
cell. For example, CTGF is co-mitogenic for fibroblast proliferation
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in combination with epidermal growth factor or basic fibroblast
growth factor (32), but will induce differentiation into myofibroblasts in the absence of epidermal growth factor (44). In
rhabdomyosarcoma-derived cell lines that secrete CTGF, inhibition
of CTGF results in increased apoptosis, suggesting that CTGF
establishes an essential autocrine loop in these cells that is
necessary for cell survival (24). This is consistent with the
increased levels of pancreatic tumor cells undergoing apoptosis
in vivo after FG-3019 administration (Fig. 5E). Furthermore, the
increase of soft agar colony number, colony size, and apparent cell
density indicates that CTGF expression may lead to enhanced
deregulation of cell-cell contact inhibition of growth.
CTGF may also modulate the expression of metastasis by
mechanisms dependent on the microenvironment and growth
factor availability. CTGF was one of four genes significantly upregulated in bone metastatic populations of mammary gland
tumors (26, 45), but was differentially necessary according to the
expression levels of other metastatic genes such as osteopontin,
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interleukin-11, and CXCR4 that increase bone metastases. CTGF
has also been shown to be elevated in breast cancer patients with
positive lymph nodes compared to patients with negative nodes
(16, 46).
Several studies have shown a role for the TGF-h pathway in
pancreatic cancer growth and metastasis (47). Domain 2 of CTGF
has also been shown to bind and potentiate TGF-h effects,
suggesting that one possible mechanism for CTGF is through
amplifying the protumorigenic actions of TGF-h (48). However, this
may not entirely explain how CTGF promotes tumor growth and
metastasis in pancreatic tumor cells like MIA PaCa-2 that are
defective in TGF-h signaling. Therefore, it is likely that other
domains of CTGF may affect pancreatic tumor growth. The domain
structure of CTGF has been associated with various interactions
with other growth factors and cell receptors. Domain 1 contains
homology to insulin-like growth factor (IGF)–binding proteins and
could bind to IGF-I and IGF-II; domain 3 possesses thrombospondin type 1–like repeats and could bind VEGF, whereas domain 4
could bind to heparan sulfate proteoglycans and integrins (9, 23, 37,
49, 50). Thus, there are a number of possible mechanisms whereby
CTGF interactions with other growth factors and cell surface
receptors might affect pancreatic tumorigenesis.
Overall, this study supports the clinical investigation of antiCTGF therapy. However, it will first be important to test the effects
of combining FG-3019 with standard treatments like gemcitabine
and radiotherapy to determine the potential additive or synergistic
effects on primary tumor growth inhibition. We have already
shown that FG-3019 treatment alone produces an inhibition of
Su86.86 tumor growth that is similar to gemcitabine (Fig. 6E), and
recent clinical studies have indicated that combining targeted
agents with chemotherapy or radiation therapy are resulting in
improved outcomes for patients. Thus, targeting CTGF with FG3019 in combination with a cytotoxic agent such as gemcitabine or
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Acknowledgments
Received 1/10/2006; revised 3/2/2006; accepted 4/4/2006.
Grant support: Else Kröner-Fresenius-Foundation (N. Dornhöfer) and from the
National Cancer Institute (A. Giaccia). K. Bennewith is supported by a fellowship from
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Cancer Res 2006; 66: (11). June 1, 2006
Downloaded from cancerres.aacrjournals.org on June 25, 2015. © 2006 American Association for Cancer
Research.
Correction
CTGF in Pancreatic Cancer
In the article on CTGF in pancreatic cancer in the June 1, 2006
issue of Cancer Research (1), the correct spelling of the tenth
author’s name is Randall Nacamuli.
1. Dornhöfer N, Spong S, Bennewith K, Salim A, Klaus S, Kambham N, Wong C, Kaper
F, Sutphin P, Nacamuli R, Höckel M, Le Q, Longaker M, Yang G, Koong A, Giaccia A.
Connective tissue growth factor-specific monoclonal antibody therapy inhibits
pancreatic tumor growth and metastasis. Cancer Res 2006;66:5816–27.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-66-15-COR2
Cancer Res 2006; 66: (15). August 1, 2006
7832
www.aacrjournals.org
Connective Tissue Growth Factor−Specific Monoclonal
Antibody Therapy Inhibits Pancreatic Tumor Growth and
Metastasis
Nadja Dornhöfer, Suzanne Spong, Kevin Bennewith, et al.
Cancer Res 2006;66:5816-5827.
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