Functions of Paracrine PDGF Signaling
in the Proangiogenic Tumor Stroma Revealed
by Pharmacological Targeting
Kristian Pietras1,2*, Jessica Pahler1, Gabriele Bergers3, Douglas Hanahan1*
1 Department of Biochemistry and Biophysics, Diabetes Center and Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States of
America, 2 Ludwig Institute for Cancer Research, Karolinska Institutet, Stockholm, Sweden, 3 Department of Neurosurgery, University of California San Franciso, San Francisco,
California, United States of America
Funding: The research was
supported by grants to DH and to
GB from the US National Cancer
Institute, and by an award to DH
from the William F. Bowes Charitable
Foundation. DH is an American
Cancer Society Research Professor.
KP received support from the
Swedish Cancer Society, the Swedish
Society for Medical Research, the
A˚ke Wiberg’s Foundation, the Royal
Swedish Academy of Sciences, and
the Swedish Research Council’s
Linnaeus grant. The funders had no
role in study design, data collection
and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors
have declared that no competing
Academic Editor: Charles L.
Sawyers, Memorial Sloan-Kettering
Cancer Center, United States of
Citation: Pietras K, Pahler J, Bergers
G, Hanahan D (2008) Functions of
paracrine PDGF signaling in the
proangiogenic tumor stroma
revealed by pharmacological
targeting. PLoS Med 5(1): e19. doi:10.
Received: May 29, 2007
Accepted: December 6, 2007
Published: January 29, 2008
Copyright: ? 2008 Pietras et al. This
is an open-access article distributed
under the terms of the Creative
Commons Attribution License, which
permits unrestricted use, distribution,
and reproduction in any medium,
provided the original author and
source are credited.
Abbreviations: CAF, cancer-
associated fibroblast; CIN, cervical
intraepithelial neoplasia; E2, 17b-
estradiol; FACS, fluorescence activated
cell sorting; FGF, fibroblast growth
factor; HPV, human papilloma virus;
K14, keratin 14; MMP, matrix
metalloproteinase; PDGF, platelet-
derived growth factor; rt, room
temperature; RT-PCR, reverse-
transcription polymerase chain
reaction; SCC, squamous cell
carcinoma; VEGF, vascular endothelial
* To whom correspondence should
be addressed. E-mail: kristian.
email@example.com (KP); dh@biochem.
A B S T R A C T
Important support functions, including promotion of tumor growth, angiogenesis, and
invasion, have been attributed to the different cell types populating the tumor stroma, i.e.,
endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells.
Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as
functional participants. The stroma is prominent in cervical carcinoma, and distinguishable
from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated
that pharmacological targeting of putative stromal support functions, in particular those of
cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the
possibility in a pre-clinical setting.
Methods and Findings
We used a genetically engineered mouse model of cervical carcinogenesis to investigate
platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and
pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved
kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and
impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors
reduced proliferation and angiogenesis in cervical lesions through a mechanism involving
suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the
epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing
antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs
impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by
cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs,
promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling
network that elicits functional capabilities in the tumor microenvironment.
This study illustrates the therapeutic benefits in a mouse model of human cervical cancer of
mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs
aimed at stromal fibroblast signals and effector functions may prove complementary to
conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly
including cervical carcinoma, the second most common lethal malignancy in women
worldwide, for which management remains poor.
The Editors’ Summary of this article follows the references.
PLoS Medicine | www.plosmedicine.orgJanuary 2008 | Volume 5 | Issue 1 | e190123
P PL Lo oS S MEDICINE
It is increasingly accepted that cancer results from the
concerted performance of genetically altered tumor cells
interacting with ostensibly normal cell types that together
constitute the tumor microenvironment . In addition to
the endothelial cells forming the tumor vasculature, attention
is now focused on other elements of the stromal compart-
ment, i.e. carcinoma-associated fibroblasts (CAFs), vascular
pericytes, and infiltrating inflammatory cells. These cell types
are being implicated as functionally important for tumori-
genesis, by providing proliferative and antiapoptotic regu-
latory factors, supporting tumor angiogenesis, and
facilitating invasion [2–7]. Recent studies have described
extensive changes in the expression profile of cells within the
stroma compared to their normal counterparts [8–11].
Among the factors implicated in the development of a
reactive stroma, in particular the recruitment and pheno-
typic character of CAFs, are members of the transforming
growth factor-b and the platelet-derived growth factor
(PDGF) families [4,6,12]. The involvement of different PDGF
isoforms in both autocrine and paracrine stimulation of
tumor growth has been extensively studied [13,14]. The
appreciation that PDGFs serve to regulate the reactive
stromal phenotype of tumors has come from studies
demonstrating that expression of PDGF promotes the
establishment of a well-vascularized and prominent stroma
in transplant models of melanoma , breast carcinoma ,
squamous carcinoma , fibrosarcoma , and lung
carcinoma , consequently enhancing tumor growth.
Cervical carcinoma is the second most common malignant
disease among women worldwide and the most common
cause of cancer death in many less-developed countries .
The primary etiologic agents for cervical cancer are human
papilloma viruses (HPV). In particular, invasive cervical and
anogenital tumors are epidemiologically associated with
chronic infection by HPV type 16 (HPV16) and related ‘‘high
risk’’ viral subtypes; the viral genomes contain two tran-
scription units, E6 and E7, encoding proteins that bind to and
inactivate the p53 and pRb tumor suppressors, respectively,
facilitating unchecked cell cycle progression and genomic
instability . Even though cervical cancers are successfully
managed by surgery and chemoradiotherapy if detected at an
early stage, the management of late-stage disease remains
Directed expression of the oncogenes contained in the
early region of HPV16 to their apparent target cell type in
humans, the basal squamous epithelial cell, by way of
expression under control of the keratin 14 (K14) promoter
in transgenic mice, leads to the formation of squamous cell
carcinomas (SCC) of the skin; the skin tumors arise with 50%
penetrance between 6–12 mo of age in the FVB/n mouse
strain . If the normally cyclic estrogen levels (17b-estradiol
[E2]) in young female transgenic mice are maintained by
implantation of slow-release estrogen pellets (HPV/E2mice),
invasive squamous carcinomas of the cervix develop via
transition through distinctive premalignant stages, i.e. cer-
vical intraepithelial neoplasias (CIN) . Following relatively
synchronous progression through CIN1 to CIN2 and CIN3
lesions, cervical carcinomas begin to appear at 3.5 mo of age;
6 wk later, approximately 90% of the HPV/E2mice present
with invasive cervical cancer , well before skin cancers
begin to appear. The HPV16/E2 mouse model of cervical
carcinoma closely resembles the human counterpart with
respect to the progressively intense angiogenic phenotype,
with increased bioavailability of vascular endothelial growth
factor (VEGF) as one feature [24,25]. Studies from our
laboratory have also demonstrated the importance of matrix
metalloproteinase-9 (MMP-9), primarily supplied by infiltrat-
ing macrophages, in the activation of the angiogenic switch in
this model , consistent with the presence of infiltrating
macrophages and MMP-9–expressing cells in human cervical
neoplasias and carcinomas .
The stromal compartment is prominent in cervical
carcinoma, and recent studies have identified numerous
changes in the gene expression pattern of stromal cells in
malignant cervical tissue compared to nonmalignant tissue
[9,27], suggestive of altered functions. An evident question
involves the importance of the stromal compartment for
neoplastic progression, and if important, its roles and
regulation. We sought to address the hypothesis that stromal
support functions in cervical carcinogenesis are a) function-
ally important and b) a potential therapeutic target. To do so
we used a genetically engineered mouse model of cervical
cancer to assess the impact of mechanism-based pharmaco-
logical targeting of stromal support functions, in particular
those of cancer-associated fibroblasts.
Materials and Methods
Animal Care and Generation of HPV/E2Mice
All animal experimentation described herein was approved
by the local Committee for Animal Research. HPV/E2mice
were generated as described previously [22,23,28]. Briefly, at 1
mo of age, virgin, female, heterozygous transgenic K14-
HPV16 mice or nontransgenic FVB/n mice were anesthetized
and slow-release E2pellets (0.05 mg over 60 d; Innovative
Research of America) were implanted subcutaneously in the
dorsal back. Additional pellets were implanted at 3 and 5 mo
of age. Mice were monitored throughout the experiments for
complications due to the dysplastic nature of their skin or to
Tissue Preparation and Histology
Mice were anesthetized with 2.5% Avertin and heart
perfused with PBS followed by ice-cold 10% zinc-buffered
formalin. Subsequently, the vagina, cervix, and uterine horns
were excised and either post-fixed for 1 h at 4 8C followed by
embedding in paraffin or immediately frozen in OTC. The
entire tissue was serially sectioned (10-lm sections) and every
tenth slide was subjected to hematoxylin and eosin staining
for grading in a blinded fashion, as described . Tumor
volume was determined using the formula V ¼ 2/3 3 A 3 Z,
where A is the largest cross-sectional area, as determined by
imaging using a Zeiss Axioskop 2 plus microscope and
OpenLab software (Improvision), and Z is the depth of the
tumor as determined through the serial sections.
Treatment of Mice and Preparation of Adenoviral FGF-Trap
Imatinib (Gleevec; Novartis Pharma) was purchased from
the University of California San Francisco (UCSF) Medical
Center pharmacy. Imatinib was delivered by oral gavage in
100-ll PBS at a dose of 150 mg 3 kg?13 day?1, divided in a
morning dose of 50 mg3kg?13day?1and an afternoon dose
PLoS Medicine | www.plosmedicine.orgJanuary 2008 | Volume 5 | Issue 1 | e190124
PDGF Signaling in the Tumor Stroma
of 100 mg3kg?13day?1. Mice were treated daily either from
3.5–5 mo of age or from 5–6 mo, and closely monitored for
drug-induced side effects. Three hours before they were
humanely killed, mice were injected intraperitoneally with 10
ll/g of a 10 mM bromo-deoxyuridine (BrdU) solution.
Adenovirus FGF-trap was developed and described previously
[30,31], and preparation of high-titer adenovirus was sub-
contracted to Vector Biolabs. The adenovirus was delivered
to 3.5-mo-old HPV/E2mice via tail vein injection at 0.3–1 3
109viral particles/mouse. The mice were humanely killed 14 d
later, and the cervixes were prepared for analysis as described
above. Functional-grade rat monoclonal anti-PDGFR-a
(APA5) was purchased from eBiosciences, and rat monoclonal
anti-PDGFR-b (APB5 ) was produced at high purity from a
hybridoma. The 3.5-mo-old mice were treated with 0.5 mg/
mouse of anti-PDGFR-a and 0.5 mg/mouse anti-PDGFR-b
injected intraperitoneally daily for 3 d. Control mice were
given 1 mg/mouse and day of functional-grade rat IgG
(Jackson ImmunoResearch). Mice were humanely killed 24 h
after the last injection, then tissues collected and flash frozen
in liquid nitrogen for subsequent RT-PCR analysis.
Frozen sections were air dried and fixed in ice-cold acetone
for 10 min. Paraffin-embedded sections were de-paraffinized,
endogenous peroxidase activity was quenched with 0.3%
H2O2 for 30 min at room temperature (rt). Following
stabilization in PBS for 2 3 5 min, sections were blocked
using a 1:1 mix (blocking solution) of serum-free protein
block (DAKO) and 0.5% blocking reagent in PBS (NEN Life
Science Products) for 1–2 h at rt. After aspiration, antibodies
diluted in blocking solution were applied and incubated at 4
8C overnight. Antibodies and dilutions used were: PDGFR-a
(1:100, APA5; eBiosciences), PDGFR-b (1:100, APB5; eBio-
sciences), NG2 (1:800, AB5320; Chemicon), PDGF-CC (6 lg/ml
), CD31 (1:100, MEC 13.3; Pharmingen), activated caspase-
3 (1:100, Asp175; Cell Signaling Technology), BrdU (1:10,
BrdU labeling and detection kit; Roche), FGF-2 (1:100, sc-79-
G; Santa Cruz Biotechnology). Slides were washed 3310 min
in PBS þ 1% bovine serum albumin (BSA; washing solution)
and subsequently incubated 2 h at rt with appropriate
conjugated secondary antibodies (fluorescently labeled,
1:100; Jackson Immunolaboratories; and HRP-conjugated,
1:100; Vector laboratories) diluted in blocking solution. The
signal from HRP-conjugated secondary antibodies was
amplified using the Vectastain ABC system according to the
manufacturer’s instructions (Vector laboratories). Following
3 3 10 min wash in washing solution, sections were mounted
for microscopic evaluation. The specificity of all stainings was
confirmed by omitting the primary antibody. Moreover, for
immunohistochemical staining of FGF-2, the specificity of the
primary antibody was determined by preincubating the
antibody with a 4-fold molar excess of human FGF-2 for 1 h
at rt prior to the procedure described.
Quantification of Vessel Density, Pericyte Coverage,
Apopotic Index, and Proliferative Index
All measurements were performed using the OpenLab
software (Improvision) on at least ten different sections from
five different mice of similar histological stage in each
treatment group. Vessel density was calculated as percentage
of the stromal cross-sectional surface area that stained
positively for CD31, or as number of CD31-positive vessel
structures per high power field (4003magnification). Pericyte
coverage was calculated as percentage of CD31-positive
vessels that displayed any associated NG2-positive cells. The
apoptotic and proliferative indexes were measured as
percentage of epithelial cells staining positively for activated
caspase-3 or BrdU, respectively.
RNA Extraction and Quantitative RT-PCR Analysis
The cervixes from at least five mice of the same age and
treatment were used to make up a pool of tissue for the
analyses. The mice that were used to make up the various
pools were N/E2, 5-mo-old nontransgenic, estrogen-treated
FVB/n mice; CIN3, 3-mo-old HPV/E2 mice; SCC, 5-mo-old
HPV/E2 mice; sham-treated mice, 4-mo-old HPV/E2 mice;
imatinib-treated mice, 4-mo-old HPV/E2 mice that were
treated as described above from 3.5–4 mo of age. The cervix
was excised, snap frozen in liquid N2, and ground to a powder
using a mortar and pestle. Following further homogenization
using an electrical homogenizer, total RNA was extracted
using the RNeasy mini-kit (Qiagen) under RNase-free con-
ditions. After quantification of yield, 1-lg total RNA was
reverse transcribed into cDNA by incubation at 65 8C for 5
min with random primers (Invitrogen), followed by incuba-
tion at 48 8C for 60 min in a mix of first-strand incubation
buffer (Invitrogen), 10 mM dNTPs (RNase-free; Roche), and
Superscript III reverse transcriptase (Invitrogen) in a total
volume of 40 ll. The yield of total RNA input to cDNA output
was assumed to be 1:1. Taqman analyses of expression levels
were subsequently performed in triplicate for each sample by
the UCSF Genome Analysis core facility using assays from
Applied Biosystems (PDGF-A, Mm00833533_m1; PDGF-B,
Mm00440678_m1; PDGF-C, Mm00480205_m1; PDGF-D,
Mm00546829_m1; PDGFR-a, Mm00440701_m1; PDGFR-b,
Mm00435546_m1; FGF-1, Mm00438906_m1; FGF-2,
Mm00433287_m1; FGF-7, Mm00433291_m1; c-kit,
Mm00445212_m1; SCF, Mm00442972_m1; VEGF,
Mm00437304_m1; angiopoietin-1, Mm00456503_m1; angio-
poietin-2, Mm00545822_m1; ephrin A1, Mm00438660_m1;
ephrin B2, Mm00438670_m1; EGF, Mm00438696_m1; TGF-
a, Mm00446231_m1; IGF-1, Mm00446231_m1; IGF-2,
Mm00439563_m1; and HGF, Mm00690363_m1). Alterna-
tively, analysis of expression of FGF-2 and L19 was performed
using a Rotor Gene RG-3000A (Corbett Research) with SYBR
green mix (Invitrogen) and the following primers: L19 59-GGT
GAC CTG GAT GAG AAG GA-39 (forward); 59-TTC AGC TTG
TGG ATG ATG TGC TC-39 (reverse); FGF-2 59-GGC TGC TGG
CTT CTA AGT GT-39 (forward); 59-CCG TTT TGG ATC CGA
GTT TA-39 (reverse).
Expression levels are expressed as percentage of the
expression level for the housekeeping gene L19, but similar
results were obtained comparing to the housekeeping gene
Immunoprecipitation and Western Blotting
Tissue lysates from the cervixes of 4-mo-old HPV/E2mice
treated or not with imatinib for 2 wk were prepared in a RIPA
buffer (150 mM NaCl, 1% Triton X-100, 0.5% NaDeoxycho-
late, 0.1% SDS, 50 mM Tris-Hcl [pH 8.0]). Immunoprecipi-
tation was performed using a pool of antibodies against FGF-
2 (AB5396, Chemicon; AB-07, Advanced Targeting Systems;
and sc-79-G, Santa Cruz Biotechnology). After collection of
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PDGF Signaling in the Tumor Stroma
immune complexes using Protein A sepharose beads (GE
Healthcare) and SDS-PAGE, western blot analysis was
performed using a 2 lg/ml solution of the sc-79-G antibody
(Santa Cruz Biotechnology).
Flow Cytometry and Qualitative RT-PCR
Six 3.5-mo-old HPV/E2mice were humanely killed and the
cervixes excised and cut into small pieces (1 3 1 mm). The
tissue was subsequently submerged in PBS containing 1%
BSA and 5% cell dissociation buffer (Gibco) (FACS buffer),
supplemented with 0.25% (w/v) type I collagenase (Worthing-
ton), 0.25% (w/v) type II collagenase (Worthington), and
0.05% (w/v) DNAse (Sigma). Digestion was carried out at 37 8C
for 18 min with constant stirring and occasional mechanical
disruption using a plastic pipette. FACS buffer containing
10% fetal bovine serum (FBS; Gibco) was added, and the
solution was filtered through a 70-lm cell strainer. After
centrifugation and aspiration of the buffer, cells were
resuspended in ACK buffer (Cambrex) for 5 min at rt to lyse
red blood cells. Cells were washed once in FACS buffer
containing 10% FBS and diluted to a concentration of 106
cells/ml. Subsequently, Fcreceptors were blocked using a mix
of antibodies towards CD16/CD32 (eBiosciences) for 10 min
on ice. Antibodies for labeling of cells were added for 15 min
on ice at 1:50 dilution, and consisted of PE-labeled anti–c-kit
(ack-45; BD-Pharmingen) and biotinylated anti-PDGFR-a and
-b (APA5, APB5; eBiosciences), followed by FITC-labeled
streptavidin (BD-Pharmingen). After two washes in FACS
buffer, and addition of 1 lg/ml propidium iodide, viable cells
were sorted using a fluorescence activated cell sorting (FACS)
machine. Cells were collected in 350 ll of RLT buffer from
Qiagen’s RNeasy mini kit, and total RNA was purified and
subsequently amplified and transcribed into cDNA using the
Ovation amplification system (Nugene) according to the
manufacturer’s instruction. PCR analysis (Tm¼ 58 8C) was
performed using the following primers: K14 59-TTC CGG
ACC AAG TTT GAG AC-39 (forward); 59-CCT CGT GGT TCT
TCT TCA GG-39 (reverse); vimentin 59-GCA CTA ACG AGT
CCC TGG AG-39 (forward); 59-TCC AGC AGC TTC CTG TAG
GT-39 (reverse); FGF-2 see above.
All measurements are depicted as average 6 standard
error of the mean. Statistical analysis of tumor volume was
performed using a two-tailed Mann-Whitney U test. Statistical
analysis of tumor incidence was performed using a v2test.
Statistical analysis of gene expression and tumor character-
istics, such as vessel density, pericyte coverage, apoptotic and
proliferative index, etc. was performed using a two-tailed,
unpaired Student t-test. A p-value below 0.05 was considered
PDGF Ligands and Receptors Are Up-Regulated during
Cervical Carcinogenesis in HPV/E2Mice
The transformation zone between squamous and columnar
epithelium of the uterine cervix is implicated as the site of
origin of the human cancer . Similarly, in K14-HPV16
female transgenic mice whose estrogen levels are maintained
by time-release implants (HPV/E2mice), incipient neoplasias
first appear in the transformation zone, arising out of the
HPV-16 oncogene-expressing squamous epithelium; the
progressive neoplastic lesions are associated with an aberrant
(‘‘reactive’’) stroma . Motivated by the well-established
association of PDGF signaling with regulation of fibroblast
phenotypes and by our previous observations that vascular
pericytes in tumors are dependent on PDGF signaling [35–
37], we investigated the expression of PDGF ligands and
receptors during cancer progression in HPV/E2 mice. In
normal, estrogen-treated female mice (N/E2mice), as well as
in HPV/E2mice, both PDGF receptor-a and -b were expressed
by cells populating the stroma of the cervical transformation
zone, as revealed by immunostaining (Figure 1A). The
expression of PDGF receptors persisted in the apparently
denser stroma of CIN3 and SCC lesions (Figure 1A).
Immunostaining revealed that stromal cells expressing both
PDGF a- and b-receptors coexpress the mesenchymal cell
marker vimentin, demonstrating together with morpholog-
ical (spindle-shaped cells with indented nuclei) and histo-
logical (patterns of tissue localization ) criteria that these cells
are fibroblasts (Figure 1B and unpublished data). Addition-
ally, PDGF b-receptor was also expressed by pericytes, as
indicated by costaining with the mural cell marker NG2
(Figure 1C). Quantitative PCR analysis revealed that the
cervical expression of both PDGF a- and b-receptors was
increased concomitant with neoplastic progression; the
cervix of HPV/E2mice with SCC displayed a 2.7-fold and
1.6-fold increase in expression of PDGF a- and b-receptor,
respectively, compared to N/E2mice (Figure 1D). Expression
of vimentin was found to be similarly increased (Figure 1D),
indicating that the elevated expression of PDGF receptors in
the tissue largely results from an increased cellularity of the
All four PDGF ligand genes were found to be expressed at
readily detectable levels in the cervixes of control N/E2mice
(Figure 1E). Gene expression of the most abundant PDGF
ligand, PDGF-C, was modestly increased during the course of
tumorigenesis in HPV/E2 mice (Figure 1E). Immunohisto-
chemical analysis confirmed the expression of PDGF-CC, and
revealed that the primary source of PDGF-CC was the
epithelium, both in control N/E2mice and in HPV/E2mice
(Figure 1A). Due to the small mass of the mouse cervix and a
lack of reagents with high sensitivity and specificity, we were
unable to quantitate the level of PDGF-CC protein during the
course of cervical tumor progression.
A PDGF Receptor Kinase Inhibitor Delays Progression and
Slows Growth of Cervical Carcinomas
Having documented the expression of PDGF ligands in the
neoplastic epithelia and PDGF receptors in the stroma at all
stages in cervical carcinogenesis, we sought to assess the
functional significance and therapeutic potential of PDGF
signaling by pharmacological inhibition at distinct stages of
cervical carcinogenesis. We chose to disrupt PDGF receptor
signaling with the kinase inhibitor imatinib (Gleevec) ,
which we and others have documented to be effective at
inhibiting PDGF receptor in mice [39,40]. To establish the
efficacy of imatinib treatment in the cervix, we immunopre-
cipitated PDGF receptor-a from cervical tissue lysates from
HPV/E2 mice treated twice daily for 2 wk with imatinib.
Western blotting for activated PDGF receptor-a revealed that
the phosphotyrosine content of the receptor was reduced by
72% following treatment with imatinib (Figure S1). The first
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PDGF Signaling in the Tumor Stroma
therapeutic trial was initiated at the age of 5 mo, when more
than 80% of the mice display overt carcinoma lesions in the
cervix . The trial continued for 1 mo (an Intervention
Trial). The median tumor volume at this temporally defined
endpoint was decreased by 61% following imatinib treatment,
demonstrating that this agent can impair the maintenance
and growth of preexisting cervical tumors (Figure 2A; Mann-
Whitney U test, U ¼ 5, p , 0.05).
We next conducted an earlier stage Prevention Trial aimed
at premalignant disease. Treatment of the HPV/E2mice with
imatinib was initiated at 3.5 mo of age, at which time more
than 90% of the mice harbor CIN3 lesions ; the inhibition
was continued for 6 wk. The incidence of cervical carcinoma
at the defined endpoint in sham-treated HPV/E2mice was
80%, in close agreement with earlier studies (Figure 2B,
untreated cohort; and ). Following treatment with
imatinib, the incidence of cervical carcinomas was signifi-
cantly reduced, to 47% (Figure 2B; v2test, v2¼5.1, p , 0.05).
Figure 1. Expression of the PDGF Receptors during Cervical Carcinogenesis
The cervixes of estrogen-treated normal mice (N/E2), HPV/E2mice with CIN3 lesions (at 3 mo of age), or with cervical squamous cell carcinomas, SCC (at
5 mo of age) were compared. Expression patterns were analyzed in at least five different mice of the same genotype or similar histological stage. Dotted
line marks the epithelium-stroma boundary. E, epithelium; S, stroma; T, tumor.
(A) Immunostaining of PDGFR-a and -b (2003 magnification; PDGFR, red; cell nuclei/DAPI, blue). Immunostaining of PDGF-CC (4003 magnification).
(B) Coexpression of PDGF receptors and the mesenchymal cell marker vimentin in stromal fibroblasts of the transformation zone of HPV/E2mice (2003
magnification; PDGF-a receptors, red; vimentin, green; merge; cell nuclei/DAPI, blue).
(C) Coexpression of PDGFR-b and the pericyte marker NG2 in the cervical stroma of the transformation zone of HPV/E2mice (4003 magnification;
PDGFR-b, red; NG2, green; cell nuclei/DAPI, blue).
(D and E) Quantitative PCR analysis of the expression of PDGF receptors (D) and ligands (E) in the cervixes of estrogen-treated normal or HPV mice with
progressing tumor development. Error bars indicate the standard error of the mean.
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190127
PDGF Signaling in the Tumor Stroma
Moreover, imatinib reduced the median volume of tumors
that did form by 61% (Figure 2C; Mann-Whitney U test, U ¼
46, p , 0.05).
In accordance with the impaired tumor growth in both the
Intervention and the Prevention Trial settings, treatment
with imatinib significantly lowered the cell proliferation
index (Figure 2D; Student t-test, t(CIN3)¼2.3, p , 0.05; t(SCC)
¼3.6, p , 0.01) and increased the apoptotic index (Figure 2E;
Student t-test, t(CIN3)¼3.1, p , 0.01; t(SCC)¼3.6, p , 0.01) of
the transformed squamous epithelial cells in both premalig-
nant CIN3 and invasive cervical carcinomas. Since the PDGF
receptors were not expressed in the cancer cells, we sought
other explanations for these effects on proliferation, apop-
tosis, and neoplastic progression.
Another Imatinib Target, c-kit, Is Not Up-Regulated during
The inhibitory profile of imatinib (with high affinity for the
PDGF a- and b-receptors, c-kit, and abl) raised an additional
possibility, that its activity against c-kit or abl could be a
factor in the observed antiproliferative effects. Indeed, a
putative autocrine loop in squamous epithelial cervical
cancer cells involving c-kit and its ligand (stem cell factor
[SCF]) has been described for a small subset of human cervical
cancers . We therefore analyzed c-kit and SCF expression.
Both were found to be expressed at relatively low levels, and
neither was up-regulated in the stages of neoplastic pro-
gression in HPV/E2 cervix; indeed, c-kit levels declined
despite the substantial expansion in the (neoplastic) epithelial
compartment in CIN3 and cervical carcinomas (Figure S2A).
Immunostaining revealed that c-kit was expressed exclusively
by mast cells in the stromal compartment and at exceedingly
low levels in a subset of squamous epithelial cells in the cell
layers immediately above the basal keratinocytes (Figure S2B).
Taken together, we consider it unlikely that imatinib is acting
directly on the neoplastic basal epithelial cells to produce the
observed growth inhibition. Additional studies with more
selective inhibitors of c-kit or abl will be required to
definitively confirm this conclusion. This consideration
notwithstanding, the functional studies described below
support the inference that imatinib’s inhibition of PDGF
receptors on CAFs forms the predominant basis for its
perturbations of cervical carcinogenesis and the resultant
conclusion that PDGF signaling is instrumental.
Imatinib Reduces Vascular Density and Pericyte Coverage
of Cervical Carcinomas
Since previous studies had shown that imatinib and other
PDGF receptor inhibitors destabilized the tumor vasculature
in different tumor types, we sought to assess the effects of
imatinib-mediated interference of PDGF receptor signaling
on the angiogenic vasculature. The vasculature in CIN 2/3
Figure 2. Effects of Treating HPV/E2Mice with the PDGF Receptor Inhibitor Imatinib
(A) Tumor volume of carcinomas in the uterine cervixes of cohorts of mice treated (or not) with imatinib for 4 wk in an intervention trial, ending at 6 mo
of age. Mann-Whitney U test, U ¼ 5, p , 0.05.
(B and C) Incidence (v2test, v2¼5.1, p , 0.05) (B) and tumor volume (Mann-Whitney U test, U¼46, p , 0.05) (C) of invasive tumors in the uterine cervix
following a 6-wk-long prevention trial ending at 5 mo of age. Mice without carcinomas had CIN2–3 lesions.
(D and E) Analysis of the proliferative (Student t-test, t(CIN3)¼2.3, p , 0.05; t(SCC)¼3.6, p , 0.01) (D) and apoptotic index (Student t-test, t(CIN3)¼3.1,
p , 0.01; t(SCC)¼3.6, p , 0.01) (E) of CIN and SCC lesions in the cervixes of HPV/E2mice treated in the prevention trial with vehicle or imatinib. Error
bars indicate the standard error of the mean.
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190128
PDGF Signaling in the Tumor Stroma
lesions and cervical carcinomas in the HPV/E2mice shows
signatures of chronic angiogenesis similar to those evident in
the cognate human lesions, including an increased density of
dilated and tortuous microvessels proximal to the hyper-
proliferative epithelium. As seen in Figure 3A and 3B,
imatinib caused a reduction in lesional neovascularization;
the blood vessel density was diminished by 45% and 52% in
CIN3 lesions and SCC, respectively, following treatment with
imatinib (Figure 3B; Student t-test, t(CIN3) ¼ 4.6, p , 0.001,
t(SCC)¼3.8, p , 0.01). Since PDGF receptors are expressed by
pericytes in the cervixes of HPV/E2mice (Figure 1C), and
since inhibition of PDGF receptor signaling by imatinib and
other drugs is known to dissociate pericytes from blood
vessels in tumors [35–37,42], we also analyzed the pericyte
coverage of capillaries. Immunohistochemical staining for the
pericyte marker NG2 revealed that blood vessels within the
transformation zone of the normal cervix are associated with
pericytes to a comparatively low degree; only 21% of blood
vessels displayed any attached pericytes (Figure 3C and 3D).
This observation was confirmed using desmin as an alter-
native pericyte marker (unpublished data). In accordance
with results from other cancer models [35–37,42], imatinib
reduced the fraction of capillaries associated with pericytes in
cervical premalignant and SCC lesions of HPV/E2mice by
42% and 39%, respectively (Student t-test, t(CIN3) ¼ 4.5, p ,
0.001, t(SCC) ¼ 3.6, p , 0.01). Thus imatinib affected the
angiogenic vasculature in two ways: it was antiangiogenic,
reducing the number of vessels, and it reduced the coverage
of those vessels by pericytes.
Activated macrophages have been reported to express
Figure 3. The Angiogenic Phenotype Is Impaired by Imatinib Therapy
(A) Representative immunostaining of blood vessels (CD31, green) in the cervical transformation zone of HPV/E2mice treated with vehicle or imatinib.
Vessel density was analyzed in at least ten sections from five different mice of similar histological stage. Magnification is 2003; cell nuclei/DAPI, blue;
dotted line marks the epithelium-stroma boundary. E, epithelium; S, stroma.
(B) Quantification of vessel density in the cervical transformation zone of HPV/E2mice treated in a prevention trial from 3.5 mo to 5 mo of age or in an
intervention trial from 5 mo to 6 mo with vehicle or imatinib. Student t-test, t(CIN3) ¼ 4.6, p , 0.001, t(SCC) ¼ 3.8, p , 0.01.
(C) Visualization of pericytes and endothelial cells, using the markers NG2 (green) and CD31 (red), respectively. Pericyte coverage was analyzed in at
least ten sections from five different mice of similar histological stage. Magnification is 2003; cell nuclei/DAPI, blue; dotted line marks the epithelium-
stroma boundary; arrows indicate coexpression. E, epithelium; S, stroma.
(D) Quantification of pericyte coverage in the cervical transformation zone of HPV/E2mice following a Prevention Trial or an Intervention Trial. Student
t-test, t(CIN3) ¼ 4.5, p , 0.001, t(SCC) ¼ 3.6, p , 0.01.
Error bars indicate the standard error of the mean.
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190129
PDGF Signaling in the Tumor Stroma
PDGF receptors . In previous studies, we have implicated
macrophage-supplied MMP-9 as a factor in the angiogenic
switch in premalignant lesions in the cervixes of HPV/E2mice
. Therefore, we investigated whether the cervixes from
HPV/E2mice treated with imatinib displayed a reduction of
MMP-9–expressing cells. Immunohistochemical analysis re-
vealed no difference in the number of cells expressing MMP-9
or in the number of macrophages in the cervixes from
imatinib-treated mice (Figure S3A and S3B). Additionally, no
change in the abundance of other constituent cell types of the
neoplastic cervix, such as leukocytes, mast cells, NK cells, or
dendritic cells, were observed following treatment with
imatinib (Figure S3B).
A Screen for Molecular Effectors of Imatinib’s Effects
Revealed FGF-7 and FGF-2
The evidently paracrine impairment, both of the cancer
cell growth rate and of the angiogenic phenotype, in the
neoplastic cervix following inhibition of PDGF signaling,
prompted us to investigate possible changes in growth and
angiogenic regulatory signals. The expression of a panel of
candidate genes encoding proliferative and antiapoptotic
signaling molecules, based on a literature search for factors
known to be of importance for cervical cancer growth, as well
as prototypical angiogenic factors, was analyzed by quantita-
tive RT-PCR in normal mouse cervix, and in CIN3 and SCC
lesions, as depicted in Figure 4A. The genes coded with white
bars indicate there was no change in expression during tumor
progression, whereas the red bars symbolize the genes up-
regulated during tumor progression, and green bars those
down-regulated during tumor progression. The data are
presented in more depth in Table S1. To assess changes in
gene expression effected by treatment with imatinib, cervical
tissues from HPV/E2 mice treated for 2 wk with vehicle
control or with imatinib were examined. The analysis
revealed that expression of fibroblast growth factor (FGF)
type-7 (FGF-7; keratinocyte growth factor) was elevated
throughout the progression of cervical neoplasias in HPV/E2
mice (Figure 4B; Student t-test, t ¼ 15.9, p , 0.001), and its
expression was decreased by 35% in imatinib-treated versus
control tumors (Figure 4A and 4B; Student t-test, t¼10.9, p ,
0.001); down-regulation of FGF-7 may therefore contribute to
the observed antiproliferative effect of imatinib. The small
mass of the mouse cervix and a lack of reagents with high
sensitivity and specificity precluded biochemical quantiation
of the protein levels of FGF-7 during the course of cervical
tumor progression. Conspiciously, a large proportion of
human cervical SCC lesions reportedly express FGF receptor-
2IIIB, the receptor for FGF-7, in contrast to normal cervical
epithelium . Increased expression of insulin-like growth
factors ?1 and ?2, and hepatocyte growth factor, was
observed during neoplastic progression; notably, however,
no substantive alterations in the expression of these factors,
or of other candidate tumor growth factors and receptors,
resulted from the treatment with imatinib (Figure 4A and
A similar survey of angiogenic signaling factors revealed
that VEGF-A, FGF-1, angiopoietin-1 and ?2, and Ephrin A1
and B2 were similarly expressed in the cervical neoplasias of
imatinib-treated versus untreated mice (Figure 4A and Table
S1). Among these, only angiopoietin-1 was appreciably up-
regulated during tumor progression (Figure 4A and Table
S1). In contrast, FGF-2 (basic FGF) was not only appreciably
up-regulated in the neoplastic cervix of HPV/E2 mice,
compared to control N/E2mice (Figure 4A and 4C; Student
t-test, t ¼ 13.7, p , 0.001), but its mRNA levels were reduced
by 65% in CIN3 lesions from HPV/E2 mice treated with
imatinib (Figure 4A and 4C; Student t-test, t ¼ 18.7, p ,
0.0001). The down-regulation of FGF-2 upon treatment with
imatinib was further confirmed at the protein level by
immunoprecipitation of FGF-2 from lysates of cervical tissue
followed by western blotting (Figure 4D).
FGF-2 Functionally Contributes to the Angiogenic
We next assessed expression of the principal mitogenic
signaling receptor for FGF-2, FGF Receptor-1 (FGFR1), by
immunohistochemical staining. FGFR1 was found to be
predominantly expressed by endothelial cells in the tissue
underlying the dysplastic and invasive epithelium (Figure 4E),
supporting the proposition that down-regulation of FGF-2 in
response to treatment with imatinib is serving to inhibit
angiogenesis driven in part by FGFR1 signaling in tumor
To address the hypothesis that FGF-2 is causally involved in
the angiogenic phenotype of HPV/E2 mice, we employed
adenoviral delivery of a soluble form of an FGF receptor-2-Fc
fusion protein (FGF-trap ). Typically, the adenovirus will
be highly produced in the liver of infected mice, from where
it will sustain high levels of production of the neutralizing
FGF-trap into the blood stream for approximately 2 wk. HPV/
E2 mice were humanely killed 2 wk after injection of
adenovirus at the age of 4 mo. Treatment with FGF-trap
produced a similar reduction in blood vessel density of
cervical lesions to that resulting from treatment with imatinib
(Figure 4F; Student t-test, t ¼ 5.8, p , 0.001). Thus, we
conclude that the down-regulation of FGF-2 expression in the
cervixes of HPV/E2mice is in large part responsible for the
impaired angiogenesis produced by treatment with imatinib.
However, we cannot exclude indirect effects on the angio-
genic phenotype from inhibition of FGF-7 by FGF-trap.
FGF-2 Is Expressed by Stromal Fibroblasts in HPV/E2Mice
Having functionally implicated FGF-2 in the angiogenic
phenotype, we sought to identify the cell type in cervical
neoplasias and cancer that expressed FGF-2, by using FACS.
Total RNA was extracted from the pools of cells labeled
positively for either of the imatinib receptor tyrosine kinase
targets, i.e., for PDGF receptor-a plus PDGF receptor-b, or
for c-kit, and subjected to semiquantitative RT-PCR analysis
to establish the identity of the cells. The pool of cells sorted
for expression of c-kit was positive for expression of the basal
squamous epithelial gene K14, but not for the mesenchymal
gene vimentin, indicating that c-kit is expressed by a subset of
cervical keratinocytes (Figure 5A). In contrast, the pool of
cells sorted for expression of PDGF receptors contained the
transcripts for the mesenchymal gene vimentin, but not K14,
in accordance with earlier experiments showing the PDGF
receptors were selectively expressed in CAFs and pericytes
(Figures 1A and 5A). The transcript for FGF-2 was predom-
inant in the pool sorted for expression of PDGF receptors,
indicating that FGF-2 was largely produced by PDGF
receptor-expressing cells (Figure 5A). Next, we employed
immunostaining of tissue sections to visualize the cells
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190130
PDGF Signaling in the Tumor Stroma
Figure 4. Expression of FGF-2 Is Repressed by Imatinib in Preclinical Trials, and Treatment with FGF-Trap Impairs Angiogenesis in the Neoplastic Cervix
(A) Quantitative RT-PCR analysis evaluating expression of a set of growth and/or angiogenic regulatory factors in the neoplastic cervixes of 4-mo-old
HPV/E2mice treated with imatinib for 2 wk. The results are expressed as the ratio of expression (as percentage of the ribosomal protein gene L19) of
imatinib-treated mice versus vehicle-treated mice. Bar colors indicate relative expression levels during the neoplastic progression (green ¼ down-
regulated expression compared with normal estrogen-treated cervix; red¼up-regulated expression compared with normal estrogen-treated cervix; and
white ¼ unchanged expression compared with normal estrogen-treated cervix).
(B and C) Quantitative RT-PCR analysis of FGF-7 (Student t-test, t¼15.9, p , 0.001) (B) and FGF-2 (Student t-test, t¼13.7, p , 0.001) (C) expression in the
cervixes of: estrogen-treated normal mice (N/E2); HPV/E2mice with CIN3 lesions (3 mo) or SCC (5 mo) and HPV/E2mice treated from 3.5 mo to 4 mo of
age with vehicle or imatinib.
(D) Western blot (WB) analysis of FGF-2 expression following immunoprecipitation (IP) of FGF-2 from tissue lysates of neoplastic cervixes of mice
untreated or treated with imatinib for 2 wk. Two individual tissue lysates are shown for each treatment, and every lysate for each treatment group
consisted of the combined cervixes from five mice. Omission of the immunoprecipitating antibody was used as a negative control (No Ab-lane), and 50
ng of recombinant mouse FGF-2 was used as a positive control. Densitometric quantification is shown normalized to lane 1.
(E) Immunostaining for the mitogenic signaling receptor for FGF-2, i.e., FGF receptor-1 (FGFR-1), in CIN3 lesions of the uterine cervix from HPV/E2mice.
Expression of FGFR-1 (green) was predominantly detected in the stroma and colocalized with a marker for endothelial cells (red, CD31). The expression
pattern was analyzed in at least five different mice of similar histological stage. Parameters: 2003magnification; cell nuclei/DAPI, blue; dotted line marks
epithelium–stroma boundary. Similar results were seen in analysis of cervical carcinoma lesions (unpublished data). Note that the scattered punctate
shapes distal from the vasculature are non–cell-associated debris derived from the secondary antibody, as revealed by evaluation at high magnification
and analysis of tissue sections in which the primary antibody was omitted. E, epithelium; S, stroma.
(F) Quantification of vessel density in the cervical transformation zone of HPV/E2mice at 4 mo of age following a 2-wk treatment with imatinib or 2 wk
after a single treatment with adenoviral delivery of FGF-trap or control GFP. Student t-test, t ¼ 5.8, p , 0.001.
Error bars indicate the standard error of the mean.
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190131
PDGF Signaling in the Tumor Stroma
expressing FGF-2 in the cervixes of HPV/E2mice. FGF-2 was
found to be predominantly expressed by stromal cells, in
particular the subset of cells in proximity to the basal
keratinocytes (Figure 5B). In accordance with the diminished
FGF-2 mRNA levels seen following treatment with imatinib,
the staining intensity for FGF-2 was reduced in tissue sections
from imatinib-treated mice (Figure 5B). To ascertain which
stromal cell type expressed FGF-2, we performed coimmu-
nostaining of FGF-2 and markers for various prevalent cell
types within the cervix. FGF-2 expression colocalized with
expression of PDGF receptor-a and vimentin, indicating that
FGF-2 is expressed by CAFs, but not with markers for
endothelial cells, leukocytes, macrophages, mast cells, NK
cells, or dendritic cells (Figure S4).
PDGF Signaling Regulates FGF-2 Expression in Stromal
We performed two experiments aimed to further assess the
hypothesis that a signaling pathway involving PDGF receptors
regulates expression of the angiogenic factor FGF-2 in
stromal fibroblasts. In one approach, we treated HPV/E2
mice with neutralizing antibodies to both PDGF receptors
, in a molecular efficacy (target modulation) trial. We first
determined that a brief 3-d treatment with imatinib was
sufficient to suppress expression of FGF-2 similarly to that of
2–6 wk of daily imatinib treatment (Figure 5C; Student t-test,
t¼3.5, p , 0.05). Then we treated HPV/E2mice for 3 d with a
cocktail of two function-blocking antibodies for PDGF
receptor-a and PDGF receptor-b, which reduced levels of
FGF-2 mRNA in the neoplastic cervix to a level comparable to
that produced by treatment with imatinib (Figure 5C; Student
t-test, t ¼ 3.1, p , 0.05). In contrast, treatment with control
IgG for 3 d had no effect. Similarly, the expression of FGF-7
was reduced in the cervixes from mice treated with
neutralizing PDGF-receptor antibodies, compared with con-
trol IgG (unpublished data). Thus, we conclude that imatinib
modulates the expression of FGF-2 and?7 through inhibition
of PDGF receptors. In a second approach, we stimulated
cultured fibroblasts with PDGF ligands. Both PDGF-AA (a
PDGF receptor-a ligand) and PDGF-BB (a PDGF receptor-b
Figure 5. FGF-2 Is Expressed by CAFs and Repressed by Specific Inhibition of PDGF Receptor Signaling
(A) Analysis of cells isolated by FACS from the cervixes of 3.5-mo-old HPV/E2mice by sorting for expression of PDGFR-a and -b or c-kit. RT-PCR was
performed to assess the expression of FGF-2, the squamous epithelial marker K14, the fibroblast cell marker vimentin, and the housekeeping gene L19
as a loading control.
(B) Representative immunohistochemical staining of FGF-2 in the transformation zone of the uterine cervixes of HPV/E2mice that had or had not been
treated with imatinib displaying CIN3 lesions. The expression pattern was analyzed in at least five different mice of similar histological stage from each
treatment group. Parameters: 4003magnification; dotted line marks epithelium-stroma boundary. As a control for specificity, the primary antibody was
pre-blocked by incubation with recombinant mouse FGF-2 . E, epithelium; S, stroma.
(C) Expression of FGF-2 analyzed by quantitative RT-PCR following a long (14 d) and a short (3 d) treatment with imatinib (Student t-test, t ¼ 3.5, p ,
0.05). Expression of FGF-2 analyzed by quantitative RT-PCR following a 3-d treatment with control IgG or inhibitory antibodies against PDGFR-a and
PDGFR-b (Student t-test, t ¼ 3.1, p , 0.05). Note that for technical reasons, a different primer set was used in this experiment compared to the
experiment shown in Figure 4C, yielding different absolute values of expression (for details, see Materials and Methods). Error bars indicate the standard
error of the mean.
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190132
PDGF Signaling in the Tumor Stroma
ligand) resulted in up-regulated expression of FGF-2 in
fibroblasts (Figure S5).
In regard to possibly broader effects of suppressing PDGF
receptor signaling in CAFs, we did not observe significant
reductions in the cellularity of the neoplastic stroma, as
evidenced histologically or by vimentin expression (Figure S6
and unpublished data), although there was a modestly
increased incidence of apoptosis (unpublished data). Thus
we infer that imatinib was primarily interfering with CAF
effector functions dependent on PDGF signaling, but not
with CAF viability in CIN or SCC lesions per se. This result
contrasts with reports [18,19] in which CAF function and
survival was reported to be PDGF-dependent in subcutaneous
tumor xenotransplant models. Notably, in such cell trans-
plant models, CAFs must be recruited from the ectopic
subcutaneous microenvironment. By contrast, in many
organized epithelia, including the squamous epithelia of the
cervix, an abundant fibroblastic stroma is normally present.
Thus, during tumorigenesis, we infer that the normal stromal
fibroblasts in the cervix become activated CAFs in response
to PDGF signaling, but do not strictly depend on it for
survival, suggestive of significant differences in the regulation
of CAFs populating distinct tumor microenvironments.
Collectively, our data indicate that PDGF receptor signaling
in cervical CAFs dictates the up-regulation of FGF-2 and
FGF-7 during cervical carcinogenesis, such that PDGF
receptor inhibitors suppress their expression with conse-
quent functional impairment of angiogenic and neoplastic
PDGFR and FGF-2 Are Similarly Up-Regulated in Human
FGF-2 and its mRNA are reportedly elevated in cervical
cancers in humans [26,45,46]. Moreover, a study of human
cervical carcinomas employing in situ hybridization previ-
ously reported that FGF-2 was expressed by stromal fibro-
blasts . Extending upon these studies, we performed
immunohistochemical staining of high-grade dysplastic le-
sions (HSIL/CIN3) or carcinomas of the human cervix,
revealing prominent expression of FGF-2 in stromal cells
(Figure 6; and unpublished data). Parallel analysis of the
expression pattern of PDGF receptors in human cervical
lesions revealed an expression pattern in the stroma similar
to that of FGF-2 (Figure 6). Similar to the HPV/E2 mice,
PDGF-CC was abundantly expressed by the neoplastic
epithelium, consistent with a paracrine signaling circuit
operating between the tumor cells and CAFs (Figure 6).
Additionally, we analyzed normal human cervical samples
derived from hysterectomies. Much as in the normal mouse
cervix, the expression of the PDGF receptors was detectable
in stromal fibroblasts and pericytes, and PDGF-CC was
expressed by the epithelial compartment (Figure 6). Recog-
nizing the qualitative nature of the analysis of archival human
samples, the expression of both PDGF receptors and FGF-2
appears to be lower in normal versus cancerous stromal
fibroblasts, consistent with the clear evidence in the mouse
that PDGF signaling up-regulates FGF-2 in the neoplastic and
cancerous stroma. Independent validation of our immuno-
histochemical analyses of human cervical cancer specimens
comes from recently published data on the Web site of the
Human Protein Atlas project  (http://www.proteinatlas.
org/) (Figure S7). Notably, FGF-2, PDGF receptor-a, and
PDGF receptor-b were found to be expressed in the stroma of
9/11, 12/12, and 9/9 cervical cancers, respectively, whereas
expression in the overt squamous cancer cells was not
consistently detected, the exception being a single case that
expressed PDGF receptor-b (Figure S7 and Table S2).
Collectively, these results and conclusions contradict a recent
study of human cervical cancer cell lines and biopsy speci-
mens, based solely on immunohistochemical analysis using
antibodies with poorly characterized specificity. That report
concluded that the PDGF receptors were expressed in a series
of cultured human cervical cancer cell lines and in the tumor
cell compartment of approximately half of the human
cervical cancer specimens analyzed . In contrast, we have
been unable to detect expression of either PDGF receptor by
RT-PCR or western blot analysis using validated antibodies,
nor growth inhibition by imatinib, during careful analysis of
three of the cervical cancer cell lines used in this study 
(Figure S8). Moreover, and consistent with our analyses, meta-
analysis of the gene expression profile of human cervical
cancer cell lines in the National Center for Biotechnology
Information (NCBI) GEO database revealed no detectable
transcripts for either of the PDGF receptors in three separate
studies of HeLa cells (unpublished data), one of the cell lines
stated to express PDGFR in the report in question . Our
data, as well as that from the Human Protein Atlas project,
from analysis of human cervical cancer cell lines and human
tissues are consistent with the more definitive analysis in the
mouse, indicating that PDGF receptors are predominantly
expressed by stromal fibroblasts and pericytes. We leave open
the possibility that certain human cervical carcinomas, for
example ones that have progressed from the common
squamous cell carcinoma state through an epithelial-mesen-
chymal transition (EMT) to a spindle cell stage, might express
the PDGF receptors, a possibility that deserves future analysis
with well-validated reagents. Collectively, the data suggest
commonality between our genetically engineered mouse
model and the prevalent form of human cervical cancer,
and encourage the possibility that our findings have transla-
tional relevance to mechanisms and therapeutic interven-
tions in human cervical neoplasia and cancer.
Herein we demonstrate the functional role of PDGF
receptor signaling in cancer-associated fibroblasts and
pericytes for cancer of the uterine cervix using a mouse
model of HPV16-induced cervical carcinoma. We assessed the
functional importance of PDGF receptor signaling via
preclinical trials with the selective PDGF receptor inhibitor
imatinib, which slowed the progression of lesions from
premalignant to invasive, and impaired the growth of existing
tumors. Moreover, the angiogenic phenotype of both
premalignant cervical neoplasias and invasive carcinomas
was affected: the treated lesions exhibited diminished blood
vessel density and reduced pericyte coverage. In seeking the
mechanism behind the impaired tumor growth and angio-
genesis, we found that the production by CAFs of FGF-7, an
epithelial cell growth factor, and of FGF-2, an angiogenic
factor, was substantively diminished by imatinib. Recognizing
that imatinib inhibits multiple kinases, we used neutralizing
antibodies to the PDGF receptors to demonstrate that
suppression of FGF-2 and FGF-7 expression was a specific
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190133
PDGF Signaling in the Tumor Stroma
consequence of PDGFR blockade. Additionally, we used an
FGF ligand trap (an FGFR2-Fc fusion) to demonstrate that
FGF-2 (and possibly FGF-7) signaling was indeed functionally
involved in regulating angiogenesis in the neoplastic cervix,
in that neovascularization was markedly reduced in mice
treated with this FGF inhibitor. Notably, human cervical
neoplasias and carcinomas coexpressed FGF-2 and PDGF
receptors in stromal cells, revealing a close similarity between
this mouse model and the human disease. We have, therefore,
demonstrated the utility of pharmaceutical targeting of CAFs,
using a PDGF receptor inhibitor, to impair the progression to
and subsequent growth of cervical carcinomas using a
genetically engineered mouse model.
PDGF ligands are expressed in a variety of tumor types, as
well as by epithelial cells during embryogenesis [13,14,50]. Our
study employing the PDGF receptor inhibitor imatinib
indicates that PDGF plays a dual role in the angiogenic
phenotype of cervical SCC. A model for the angiogenic
regulatory circuits contributing to neoplastic progression and
tumor growth in the cervix of HPV/E2mice, as elucidated by
Figure 6. PDGF Receptors and FGF-2 Are Expressed in Human Normal Cervix and in Cervical SCC
Expression of PDGF receptors and FGF-2 was assessed in human hysterectomy samples and in human cervical SCC lesions by immunohistochemistry.
Representative pictures from analysis of a total of three separate human normal cervixes and SCC lesions are shown. PDGF receptor-a was exclusively
expressed by stromal cells underlying the epithelium, whereas PDGF receptor-b was expressed by stromal cells and pericytes. Similarly, FGF-2 was
expressed by stromal cells. Although qualitative, the staining intensity of FGF-2 is clearly lower in all of the normal human samples than in comparable
analyses of neoplastic human cervixes, and consistent with the results from the mouse (Figures 4C and 5B, and unpublished data), where FGF-2 is
expressed in the normal cervical stroma at low levels and clearly up-regulated in the neoplastic cervix. As in the mouse model, PDGF-CC was
predominantly expressed by the cervical epithelium. Parameters: 4003magnification; dotted line marks epithelium-stroma boundary. E, epithelium; S,
stroma; T, tumor.
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190134
PDGF Signaling in the Tumor Stroma
our studies (this work, and ), is shown in Figure 7.
Consistent with previous studies in other tumor types
[35,51,52], the data suggest that PDGF helps maintain pericyte
support of the tumor vasculature. In addition, we have
identified PDGF receptor signaling in CAFs as a mediator of
the angiogenic response in tumors, by virtue of up-regulating
expression of the proangiogenic factor FGF-2. We infer that
both cellular targets, CAFs and pericytes, are contributing to
the angiogenic phenotype and to its impairment by imatinib.
In a model of islet cell carcinogenesis, imatinib was not
antiangiogenic when used as monotherapy; rather it destabi-
lized pericyte association and rendered coadministered
endothelial cell inhibitors more effective in vessels lacking
pericyte coverage [35–37]. In notable contrast, imatinib is
directly antiangiogenic in the cervix as monotherapy, a result
we have functionally attributed to FGF signaling from PDGFR-
expressing CAFs (which are rare in the islet carcinoma model).
Additionally, PDGF signaling elevates expression of FGF-7,
which we infer may directly stimulate the cervical cancer cells,
a possibility that deserves future investigation. Our findings
are in agreement with earlier studies demonstrating produc-
tion of both FGF-2 and FGF-7 by CAFs in response to PDGF
. We do not exclude the possibility that there will prove to
be additional PDGF- and/or imatinib-regulated cross-talk
between CAFs, carcinoma cells, and the other cell types
constituting the tumor microenvironment, potentially embel-
lishing the critical pathway we have elucidated.
The notion of targeting CAFs as a means to interfere with
the growth of tumors is attractive, since the stromal
compartment is a rich contributor of various growth- and
invasion-promoting activities [3,4,7]. The capability of CAFs
to produce proangiogenic factors, including FGFs and VEGF,
has been demonstrated in several recent studies [18,53–58].
We have now implicated FGF-2 produced by CAFs in
response to PDGF signaling as a new proangiogenic regu-
latory axis in the neoplastic cervix. It will be of interest to
assess expression of FGF-2 and its concordance with PDGF
receptor expression in CAFs of other tumor types. Indeed,
FGF-2 was found to be highly up-regulated and exclusively
expressed by stromal fibroblasts in breast carcinomas,
compared to adjacent normal tissue . Despite indications
of functional and molecular genetic heterogeneity in differ-
ent tissues, CAFs are likely to have common determinants,
including a dependence on PDGF signaling [18,19]. PDGF
receptors are expressed by CAFs in a majority of tumor types
(J. Paulsson, T. Sjo ¨blom, C-H. Heldin, A. O¨stman et al.,
unpublished data; and [60–63]), and as such, our results may
have broad clinical implications for targeting of stromal cells
as a therapeutic strategy.
The entry of targeted therapeutics into the clinic has been
much anticipated. However, despite successful preclinical
trials, the results from clinical trials with targeted agents as
monotherapy have not in general produced enduring
responses. Increasingly, clinical trial designs involving combi-
nations of drugs targeting different critical components or
compartments of a tumor are being discussed to enhance
efficacy and to reduce the likelihood of emergence of
resistance to treatment. Our findings encourage confirmatory
studies in other mouse models of cervical cancer, as well as
concomitant discussions of cervical cancer clinical trials
involving treatment with imatinib, or other Federal Drug
Administration–approved drugs incorporating inhibitory
action against the PDGF receptors, e.g. the multitargeted
tyrosine kinase inhibitors sunitinib, sorafenib, or dasatinib.
Moreover, combinatorial treatment regimens with imatinib
and standard-of-care modalities, such as topotecan chemo-
therapy, or as an adjuvant to radiation therapy could be
considered, perhaps buoyed by further preclinical trials in
mouse models such as the one employed herein. Interestingly,
several phase II studies investigating the utility of sunitinib or
sorafenib in combination with chemotherapy and/or radia-
tion therapy are currently recruiting cervical cancer patients.
Importantly, rationale for such trials integrating treatment
with VEGF and PDGF receptor inhibitors and chemotherapy,
either in concomitant treatment strategies, or in sequential
‘‘chemo-switch’’ regimens, has been provided by several
recent studies involving preclinical trials in mouse models
[36,39,64]. Additional multitargeting strategies are likely to be
forthcoming from mechanism-based preclinical trials. For
example, using the HPV/E2 transgenic mice, we have
previously demonstrated functional inhibition of VEGF
receptor signaling by treatment with zoledronate (Zometa)
as a strategy to impair angiogenesis, and thereby tumor
growth, in cervical cancer. Zoledronate acts by reducing the
expression of MMP-9 by infiltrating macrophages and by
inhibiting the residual proteolytic activity of MMP-9, con-
sequently reducing the bioavailability of VEGF and impairing
angiogenesis and neoplastic progression . We envision
that combined treatment with imatinib and zoledronate, in
Figure 7. Schematic Model of the Angiogenic Circuitry Operative during
Malignant Progression in the Cervical Transformation Zone of HPV/E2Mice
The transformed epithelial cells (purple) secrete VEGF, which becomes
sequestered in the matrix until released by the action of MMP-9,
produced by infiltrating macrophages (blue); the bioavailable VEGF then
acts directly on endothelial cells (red) to stimulate angiogenesis. PDGF
ligands are predominantly produced by the squamous epithelium and
stimulate production of FGF-2 and FGF-7 by carcinoma-associated
fibroblasts (brown) that express PDGF-receptors. FGF-2 stimulates
angiogenesis by direct action on endothelial cells, and PDGF further
promotes the angiogenic process by inducing pericyte (green) recruit-
ment and association with newly formed blood vessels. FGF-7 may signal
to the cervical carcinoma cells. Imatinib acts by inhibiting PDGF ligand-
dependent PDGF receptor signaling (solid red lines), thereby repressing
the production of FGF-2 and FGF-7 by carcinoma-associated fibroblasts
(dotted red lines) and additionally reducing the pericyte coverage on
tumor blood vessels. The clinically approved bisphosphonate zoledro-
nate (zoledronic acid, Zometa, ZA) has been shown to suppress the
expression of MMP-9 by macrophages (dotted red line), as well as to
directly inhibit the proteolytic action of MMP-9 (solid red line).
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190135
PDGF Signaling in the Tumor Stroma
effect targeting two distinct angiogenic circuits within the
cervical tumor (Figure 7), might further weaken the angio-
genic response and limit tumor formation and growth.
Indeed, we have conducted a pilot study combining the two
drugs that encourages this line of reasoning and motivates
further investigation (unpublished data).
The realization that imatinib, which inhibits PDGF
receptor, but not VEGF receptor signaling, and yet is
demonstrably antiangiogenic (via suppressing FGF-2) and
antiproliferative (likely via suppressing FGF-7), might foster
consideration of clinical trial designs not feasible with the
aforementioned multi-targeted inhibitors. In particular,
there is increasing discussion about ‘‘sequencing’’ of alter-
nating or layering on drugs with different specificities, aiming
to improve efficacy and reduce toxicity. We suggest that
imatinib and other selective PDGF receptor inhibitors (e.g.,
dasatinib) might prove beneficial when combined with VEGF
receptor inhibitors and/or with chemotherapy in more
flexible combinations than strictly simultaneous dosing,
involving evolutions of the chemo-switch regimen ; thus
we might imagine first normalizing the tumor vasculature
with VEGF pathway inhibitors , then applying high-dose
(standard-of-care) chemotherapy, and finally introducing
PDGF receptor inhibitors, alone or in combination with
VEGF inhibitors or low-dose metronomic chemotherapy.
Although the number of possible combinations in drugs and
regimens is challenging, we speculate that preclinical trials in
mouse models as illustrated by this study could help evaluate
and prioritize the possibilities.
Figure S1. Systemic Treatment with Imatinib Inhibits PDGF Receptor
Activation in the Neoplastic Cervix
Immunoprecipitation (IP) of PDGF receptor-a from a pool of three
cervical tissue lysates derived from mice treated for 2 wk with twice
daily administrations of imatinib (total dose 150 mg 3 kg?13 day?1).
Parallel membranes were probed for the abundance of PDGF
receptor-a and calnexin to demonstrate equal loading and amount
of starting material.
Found at doi:10.1371/journal.pmed.0050019.sg001 (1.2 MB TIF).
Figure S2. Expression of the Imatinib Target c-kit and Its Ligand Is
Unaltered during Cervical Carcinogenesis in the Mouse
(A) Quantitative RT-PCR analysis of expression of c-kit receptor and
its ligand SCF in the cervixes of estrogen-treated normal mice (N/E2)
or HPV/E2mice with CIN3 lesions (3 mo) or SCC (5 mo).
(B) Immunostaining of neoplastic cervix for c-kit (red) revealed
coexpression with markers for mast cells (mast cell tryptase, green) in
the stromal compartment, as well as very weak staining of a subset of
epithelial cells above the layer of basal keratinocytes. Arrow points
out a mast cell for comparison of expression levels. Magnification is
4003; cell nuclei/DAPI, blue; dotted line marks epithelial-stromal
E, epithelium; S, stroma.
Found at doi:10.1371/journal.pmed.0050019.sg002 (3.1 MB TIF).
Figure S3. The Abundance of MMP-9–Expressing Cells, or Other
Constituent Cell Types of the Neoplastic Cervix, Is Not Altered by
(A) Immunostaining of cells expressing MMP-9 (green) in the cervical
transformation zone of HPV/E2 mice. Magnification is 2003; cell
nuclei/DAPI, blue; dotted line marks epithelial-stromal boundary. E,
epithelium; S, stroma.
(B) Immunostaining of the neoplastic cervix using cell-type–specific
markers (green) revealed no differences in abundance following
treatment with imatinib. The cell-type markers were F4/80, macro-
phages; CD45, leukocytes; mast cell tryptase, mast cells; CD69, NK
cells; and CD11c, dendritic cells) Magnification is 4003; cell nuclei/
DAPI, blue. Quantifications were performed using five mice per
Found at doi:10.1371/journal.pmed.0050019.sg003 (14 MB TIF).
Figure S4. FGF-2 Is Produced by CAFs Expressing PDGF Receptor-a
Immunostaining of the stromal compartment of the neoplastic cervix
for FGF-2 (red) and for cell type specific markers (green). The
markers used to identify the particular cell types are PDGF receptor-
a for fibroblasts, vimentin for CAFs; CD31 for endothelial cells; F4/80
for macrophages; CD45 for leukocytes; mast cell tryptase for mast
cells; CD69 for NK cells; and CD11c for dendritic cells). Magnification
is 4003; cell nuclei/DAPI, blue.
Found at doi:10.1371/journal.pmed.0050019.sg004 (21 MB TIF).
Figure S5. Induction of FGF-2 by PDGF in Cultured Fibroblasts
FGF-2 transcription was assessed following stimulation of NIH-3T3
mouse fibroblasts with PDGF-AA (100 ng/ml for 6 h in 37 8C) or
PDGF-BB (100 ng/ml for 6 h in 37 8C). The analysis revealed that
fibroblasts up-regulate expression of FGF-2 in response to PDGF.
Expression of the housekeeping gene GAPDH was used as a control.
Found at doi:10.1371/journal.pmed.0050019.sg005 (418 KB TIF).
Figure S6. Stromal Cell Density Is Not Altered in the Neoplastic
Cervix following Treatment with Imatinib
Quantification of stromal cell density in the stroma of the trans-
formation zone of the cervixes from groups of five mice treated, or
not, with imatinib for 2 wk.
Found at doi:10.1371/journal.pmed.0050019.sg006 (405 KB TIF).
Figure S7. PDGF Receptors and FGF-2 Are Expressed in the Stromal
Cell Compartment of an Extended Set of Cervical Cancers
Representative images of immunohistochemical stainings obtained
from the Human Protein Atlas project (http://www.proteinatlas.org/)
demonstrate stromal expression of FGF-2 and PDGF receptors.
Found at doi:10.1371/journal.pmed.0050019.sg007 (12 MB TIF).
Figure S8. Lack of PDGF Receptor Expression or Functionality on
Human Cervical Cell Lines
(A) PCR analysis of expression of PDGFR-a and PDGFR-b by human
cervical cancer cell lines (SiHa, C33-A, HeLa) demonstrated that the
PDGF-receptor genes were not transcribed. Normal human fibro-
blasts (NF) were used as a positive control.
(B) Western blot analysis of immunoprecipitated PDGFR-a  using
cell lysates from human cervical cancer cell lines stimulated or not
with PDGF-CC demonstrated that PDGFR-a was not expressed.
Normal human fibroblasts (NF) were used as a control.
(C) In vitro analysis of the growth rate of the cervical cancer cell line
HeLa grown in the presence or absence of the PDGF-receptor
inhibitor imatinib showed that the growth rate of HeLa cells is not
altered by the presence of 4.4 lM imatinib, corresponding to the peak
plasma concentration of imatinib delivered to patients at the
standard dose of 400 mg/day. Porcine aortic endothelial (PAE) cells
transfected with the PDGFR-a were used as a positive control for the
action of imatinib.
Found at doi:10.1371/journal.pmed.0050019.sg008 (3.0 MB TIF).
Table S1. Expression Level of Growth Factors in the Normal and
Total RNA was extracted from the cervixes of 5-mo-old FVB/n mice
treated with estrogen (N/E2), from 3-mo-old HPV/E2mice (CIN3), or
from 5-mo-old HPV/E2mice (SCC). A pool consisting of five mice
from each group was assessed for gene expression using quantitative
RT-PCR. The data shown represent the mean from two separate
experiments, and are depicted as percent expression of the reference
Found at doi:10.1371/journal.pmed.0050019.st001 (29 KB DOC).
Table S2. Expression of FGF-2 and PDGF-Receptors in Cervical
Data obtained from meta-analysis of immunohistochemical stainings
performed by the Human Protein Atlas project (http://www.
proteinatlas.org). Images were scored for expression in the tumor
stroma or neoplastic compartment following careful examination of
each specimen displayed on the Web site.
Found at doi:10.1371/journal.pmed.0050019.st002 (30 KB DOC).
PLoS Medicine | www.plosmedicine.orgJanuary 2008 | Volume 5 | Issue 1 | e19 0136
PDGF Signaling in the Tumor Stroma
We thank Thea Tlsty, Karen Smith-McCune, Enrico Giraudo, and
Arne O¨stman for helpful comments on the manuscript; Cherry
Concengco, Joanna Berrocal, Dale Milfay, and Ehud Drori for
technical assistance; Christopher Chiu for experimental advice;
Shin-Ichi Nishikawa for donating the hybridoma for the APB5
antibody; Ulf Eriksson for the PDGF-C antibody; and Karen Smith-
McCune and Akiko Kobayashi for tissue sections representing the
stages in human cervical carcinogenesis. We appreciate access to the
UCSF Diabetes Center’s (DERC-funded) Microscopy Core as well as to
core facilities maintained by the UCSF Comprehensive Cancer Center.
Author contributions. KP and DH designed the study. KP and JP
performed research. KP, GB, and DH analyzed the data and
contributed to writing the paper. GB contributed new reagents.
1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57–70.
2. De Wever O, Mareel M (2003) Role of tissue stroma in cancer cell invasion. J
Pathol 200: 429–447.
3. Tlsty TD, Hein PW (2001) Know thy neighbor: stromal cells can contribute
oncogenic signals. Curr Opin Genet Dev 11: 54–59.
4. Bhowmick NA, Neilson EG, Moses HL (2004) Stromal fibroblasts in cancer
initiation and progression. Nature 432: 332–337.
5. Bhowmick NA, Chytil A, Plieth D, Gorska AE, Dumont N, et al. (2004) TGF-
beta signaling in fibroblasts modulates the oncogenic potential of adjacent
epithelia. Science 303: 848–851.
6. Micke P, Ostman A (2005) Exploring the tumour environment: cancer-
associated fibroblasts as targets in cancer therapy. Expert Opin Ther
Targets 9: 1217–1233.
7. Joyce JA (2005) Therapeutic targeting of the tumor microenvironment.
Cancer Cell 7: 513–520.
8. Allinen M, Beroukhim R, Cai L, Brennan C, Lahti-Domenici J, et al. (2004)
Molecular characterization of the tumor microenvironment in breast
cancer. Cancer Cell 6: 17–32.
9. Chen Y, Miller C, Mosher R, Zhao X, Deeds J, et al. (2003) Identification of
cervical cancer markers by cDNA and tissue microarrays. Cancer Res 63:
10. Moinfar F, Man YG, Arnould L, Bratthauer GL, Ratschek M, et al. (2000)
Concurrent and independent genetic alterations in the stromal and
epithelial cells of mammary carcinoma: implications for tumorigenesis.
Cancer Res 60: 2562–2566.
11. St Croix B, Rago C, Velculescu V, Traverso G, Romans KE, et al. (2000)
Genes expressed in human tumor endothelium. Science 289: 1197–1202.
12. Park CC, Bissell MJ, Barcellos-Hoff MH (2000) The influence of the
microenvironment on the malignant phenotype. Mol Med Today 6: 324–
13. Pietras K, Sjoblom T, Rubin K, Heldin CH, Ostman A (2003) PDGF
receptors as cancer drug targets. Cancer Cell 3: 439–443.
14. Board R, Jayson GC (2005) Platelet-derived growth factor receptor
(PDGFR): a target for anticancer therapeutics. Drug Resist Updat 8: 75–83.
15. Forsberg K, Valyi-Nagy I, Heldin C-H, Herlyn M, Westermark B (1993)
Platelet-derived growth factor (PDGF) in oncogenesis: development of a
vascular connective tissue stroma in xenotransplanted human melanoma
producing PDGF-BB. Proc Natl Acad Sci U S A 90: 393–397.
16. Shao Z-M, Nguyen M, Barsky SH (2000) Human breast carcinoma
desmoplasia is PDGF initiated. Oncogene 19: 4337–4345.
17. Skobe M, Fusenig NE (1998) Tumorigenic conversion of immortal human
keratinocytes through stromal cell activation. Proc Natl Acad Sci U S A 95:
18. Dong J, Grunstein J, Tejada M, Peale F, Frantz G, et al. (2004) VEGF-null
cells require PDGFR alpha signaling-mediated stromal fibroblast recruit-
ment for tumorigenesis. EMBO J 23: 2800–2810.
19. Tejada ML, Yu L, Dong J, Jung K, Meng G, et al. (2006) Tumor-driven
paracrine platelet-derived growth factor receptor alpha signaling is a key
determinant of stromal cell recruitment in a model of human lung
carcinoma. Clin Cancer Res 12: 2676–2688.
20. Waggoner SE (2003) Cervical cancer. Lancet 361: 2217–2225.
21. Wolf JK, Ramirez PT (2001) The molecular biology of cervical cancer.
Cancer Invest 19: 621–629.
22. Arbeit JM, Munger K, Howley PM, Hanahan D (1994) Progressive squamous
epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice.
J Virol 68: 4358–4368.
23. Arbeit JM, Howley PM, Hanahan D (1996) Chronic estrogen-induced
cervical and vaginal squamous carcinogenesis in human papillomavirus
type 16 transgenic mice. Proc Natl Acad Sci U S A 93: 2930–2935.
24. Giraudo E, Inoue M, Hanahan D (2004) An amino-bisphosphonate targets
MMP-9-expressing macrophages and angiogenesis to impair cervical
carcinogenesis. J Clin Invest 114: 623–633.
25. Smith-McCune K, Zhu YH, Hanahan D, Arbeit J (1997) Cross-species
comparison of angiogenesis during the premalignant stages of squamous
carcinogenesis in the human cervix and K14-HPV16 transgenic mice.
Cancer Res 57: 1294–1300.
26. Van Trappen PO, Ryan A, Carroll M, Lecoeur C, Goff L, et al. (2002) A
model for co-expression pattern analysis of genes implicated in angio-
genesis and tumour cell invasion in cervical cancer. Br J Cancer 87: 537–
27. Gius D, Funk MC, Chuang EY, Feng S, Huettner PC, et al. (2007) Profiling
microdissected epithelium and stroma to model genomic signatures for
cervical carcinogenesis accommodating for covariates. Cancer Res 67:
28. Elson DA, Riley RR, Lacey A, Thordarson G, Talamantes FJ, et al. (2000)
Sensitivity of the cervical transformation zone to estrogen-induced
squamous carcinogenesis. Cancer Res 60: 1267–1275.
29. Riley RR, Duensing S, Brake T, Munger K, Lambert PF, et al. (2003)
Dissection of human papillomavirus E6 and E7 function in transgenic
mouse models of cervical carcinogenesis. Cancer Res 63: 4862–4871.
30. Compagni A, Wilgenbus P, Impagnatiello MA, Cotten M, Christofori G
(2000) Fibroblast growth factors are required for efficient tumor angio-
genesis. Cancer Res 60: 7163–7169.
31. Casanovas O, Hicklin DJ, Bergers G, Hanahan D (2005) Drug resistance by
evasion of antiangiogenic targeting of VEGF signaling in late-stage
pancreatic islet tumors. Cancer Cell 8: 299–309.
32. Sano H, Sudo T, Yokode M, Murayama T, Kataoka H, et al. (2001)
Functional blockade of platelet-derived growth factor receptor-beta but
not of receptor-alpha prevents vascular smooth muscle cell accumulation
in fibrous cap lesions in apolipoprotein E-deficient mice. Circulation 103:
33. Li X, Ponte ´n A, Aase K, Karlsson L, Abramsson A, et al. (2000) PDGF-C is a
new protease-activated ligand for the PDGF a-receptor. Nature Cell Biol 2:
34. Castle PE (2004) Beyond human papillomavirus: the cervix, exogenous
secondary factors, and the development of cervical precancer and cancer. J
Low Genit Tract Dis 8: 224–230.
35. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D (2003) Benefits
of targeting both pericytes and endothelial cells in the tumor vasculature
with kinase inhibitors. J Clin Invest 111: 1287–1295.
36. Pietras K, Hanahan D (2005) A multitargeted, metronomic, and maximum-
tolerated dose ‘‘chemo-switch’’ regimen is antiangiogenic, producing
objective responses and survival benefit in a mouse model of cancer. J
Clin Oncol 23: 939–952.
37. Song S, Ewald AJ, Stallcup W, Werb Z, Bergers G (2005) PDGFRbetaþ
perivascular progenitor cells in tumours regulate pericyte differentiation
and vascular survival. Nat Cell Biol 7: 870–879.
38. Capdeville R, Buchdunger E, Zimmermann J, Matter A (2002) Glivec
(STI571, imatinib), a rationally developed, targeted anticancer drug. Nat
Rev Drug Discov 1: 493–502.
39. Pietras K, Rubin K, Sjo ¨blom T, Buchdunger E, Sjo ¨quist M, et al. (2002)
Inhibition of PDGF receptor signaling in tumor stroma enhances
antitumor effect of chemotherapy. Cancer Res 62: 5476–5484.
40. Greco A, Roccato E, Miranda C, Cleris L, Formelli F, et al. (2001) Growth-
inhibitory effect of STI571 on cells transformed by the COL1A1/PDGFB
rearrangement. Int J Cancer 92: 354–360.
41. Inoue M, Kyo S, Fujita M, Enomoto T, Kondoh G (1994) Coexpression of
the c-kit receptor and the stem cell factor in gynecological tumors. Cancer
Res 54: 3049–3053.
42. Erber R, Thurnher A, Katsen AD, Groth G, Kerger H, et al. (2004)
Combined inhibition of VEGF and PDGF signaling enforces tumor vessel
regression by interfering with pericyte-mediated endothelial cell survival
mechanisms. Faseb J 18: 338–340.
43. Krettek A, Ostergren-Lunden G, Fager G, Rosmond C, Bondjers G, et al.
(2001) Expression of PDGF receptors and ligand-induced migration of
partially differentiated human monocyte-derived macrophages. Influence
of IFN-gamma and TGF-beta. Atherosclerosis 156: 267–275.
44. Kurban G, Ishiwata T, Kudo M, Yokoyama M, Sugisaki Y, et al. (2004)
Expression of keratinocyte growth factor receptor (KGFR/FGFR2 IIIb) in
human uterine cervical cancer. Oncol Rep 11: 987–991.
45. Fujimoto J, Ichigo S, Hori M, Hirose R, Sakaguchi H, et al. (1997)
Expression of basic fibroblast growth factor and its mRNA in advanced
uterine cervical cancers. Cancer Lett 111: 21–26.
46. Sliutz G, Tempfer C, Obermair A, Dadak C, Kainz C (1995) Serum
evaluation of basic FGF in breast cancer patients. Anticancer Res 15: 2675–
47. Turner MA, Darragh T, Palefsky JM (1997) Epithelial-stromal interactions
modulating penetration of matrigel membranes by HPV 16-immortalized
keratinocytes. J Invest Dermatol 109: 619–625.
48. Uhlen M (2007) Mapping the human proteome using antibodies. Mol Cell
Proteomics 6: 1455–1456.
49. Taja-Chayeb L, Chavez-Blanco A, Martinez-Tlahuel J, Gonzalez-Fierro A,
Candelaria M, et al. (2006) Expression of platelet derived growth factor
family members and the potential role of imatinib mesylate for cervical
cancer. Cancer Cell Int 6: 22.
50. Heldin C-H, Westermark B (1999) Mechanism of action and in vivo role of
platelet-derived growth factor. Physiol Rev 79: 1283–1316.
51. Furuhashi M, Sjoblom T, Abramsson A, Ellingsen J, Micke P, et al. (2004)
Platelet-derived growth factor production by B16 melanoma cells leads to
increased pericyte abundance in tumors and an associated increase in
tumor growth rate. Cancer Res 64: 2725–2733.
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190137
PDGF Signaling in the Tumor Stroma
52. Gerhardt H, Betsholtz C (2003) Endothelial-pericyte interactions in
angiogenesis. Cell Tissue Res 314: 15–23.
53. Thijssen VL, Brandwijk RJ, Dings RP, Griffioen AW (2004) Angiogenesis
gene expression profiling in xenograft models to study cellular inter-
actions. Exp Cell Res 299: 286–293.
54. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, et al. (1998) Tumor
induction of VEGF promoter activity in stromal cells. Cell 94: 715–725.
55. Williams CS, Tsujii M, Reese J, Dey SK, DuBois RN (2000) Host
cyclooxygenase-2 modulates carcinoma growth. J Clin Invest 105: 1589–
56. Ikeda Y, Hayashi I, Kamoshita E, Yamazaki A, Endo H, et al. (2004) Host
stromal bradykinin B2 receptor signaling facilitates tumor-associated
angiogenesis and tumor growth. Cancer Res 64: 5178–5185.
57. Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, et al.
(2005) Stromal fibroblasts present in invasive human breast carcinomas
promote tumor growth and angiogenesis through elevated SDF-1/CXCL12
secretion. Cell 121: 335–348.
58. Amano H, Hayashi I, Endo H, Kitasato H, Yamashina S, et al. (2003) Host
prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis
and tumor growth. J Exp Med 197: 221–232.
59. Smith K, Fox SB, Whitehouse R, Taylor M, Greenall M, et al. (1999)
Upregulation of basic fibroblast growth factor in breast carcinoma and its
relationship to vascular density, oestrogen receptor, epidermal growth
factor receptor and survival. Ann Oncol 10: 707–713.
60. Bhardwaj B, Klassen J, Cossette N, Sterns E, Tuck A, et al. (1996)
Localization of platelet-derived growth factor b receptor expression in
the periepithelial stroma of human breast carcinoma. Clin Cancer Res 2:
61. Lindmark G, Sundberg C, Glimelius B, Pa ˚hlman L, Rubin K, et al. (1993)
Stromal expression of platelet-derived growth factor b-receptor and
platelet-derived growth factor B-chain in colorectal cancer. Lab Invest
62. Sundberg C, Branting M, Gerdin B, Rubin K (1997) Tumor cell and
connective tissue cell interactions in human colorectal adenocarcinoma.
Transfer of platelet-derived growth factor-AB/BB to stromal cells. Am J
Pathol 151: 479–492.
63. Kawai T, Hiroi S, Torikata C (1997) Expression in lung carcinomas of
platelet-derived growth factor and its receptors. Lab Invest 77: 431–436.
64. Uehara H, Kim SJ, Karashima T, Shepherd DL, Fan D, et al. (2003) Effects of
blocking platelet-derived growth factor-receptor signaling in a mouse
model of experimental prostate cancer bone metastases. J Natl Cancer Inst
65. Jain RK (2005) Normalization of tumor vasculature: an emerging concept in
antiangiogenic therapy. Science 307: 58–62.
66. Eriksson A, Siegbahn A, Westermark B, Heldin C-H, Claesson-Welsh L
(1992) PDGFa- and b-receptors activate unique and common signal
transduction pathways. EMBO J 11: 543–550.
Background. Cancers—disorganized, life-threatening masses of cells—
develop when cells acquire genetic changes that allow them to divide
uncontrollably and to move into (invade) other tissues. Interactions with
ostensibly normal cells in the tissue surrounding the tumor (the stroma)
support the growth of these abnormal cells. The stroma contains
endothelial cells and pericytes (which line the inside and coat the
outside, respectively, of blood vessels), cancer-associated fibroblasts, and
some immune system cells. Together, these cells support angiogenesis
(the formation of a blood supply, which feeds the tumor), produce
factors that stimulate tumor cell growth, and facilitate tumor cell
invasion into surrounding tissues. One type of tumor with a prominent
stromal compartment is cervical cancer. Precancerous changes in the
epithelial cells lining the cervix (the structure that connects the womb to
the vagina) are usually triggered by infection with human papillomavirus.
Some of these early lesions, which are known as cervical intraepithelial
neoplasias (CINs), develop into invasive cervical cancer, which is treated
by surgery followed by chemotherapy or radiotherapy.
Why Was This Study Done? The outlook for women whose cervical
cancer is detected early is good but only 15%–30% of women whose
cancer has spread out of the cervix survive for five years. If, as researchers
believe, the stromal compartment is important in the development and
growth (neoplastic progression) of cervical cancer, it might be possible
to help these women by specifically targeting the cells in the stroma.
However, relatively little is known about the role that the stroma plays in
the neoplastic progression of cervical cancer or how it is regulated other
than that a protein called platelet-derived growth factor (PDGF), which is
made by the tumor cells, might be involved in its formation. In this study,
the researchers have used a mouse model of cervical cancer (HPV/E2
mice) to investigate PDGF signaling in the tumor stroma. HPV/E2mice
develop CINs before they are three months old; by five months of age,
90% of them have invasive cervical cancer.
What Did the Researchers Do and Find? The researchers report that
PDGF was expressed in the cervixes of normal and HPV/E2mice, mainly
by epithelial cells, and that PDGF receptors (cell-surface proteins that
bind PDGF and send a message into the cell that alters the expression of
other proteins) were expressed on cells within normal stroma and in
fibroblasts and pericytes in the stroma surrounding CINs and tumors (but
not on the cancer cells). The expression of PDGF and its receptors
increased slightly during tumor progression. Treatment of the HPV/E2
mice with imatinib, an inhibitor of PDGF signaling, slowed the
progression of precancerous lesions, impaired the growth of invasive
cancers, and reduced the number of blood vessels formed in the tumors
and the coverage of these vessels with pericytes. Other experiments
indicate that imatinib had these effects because its inhibition of stromal
PDGF receptors suppressed the expression of FGF-7 (a factor that
encourages epithelial cell division) and FGF-2 (a proangiogenic factor) by
cancer-associated fibroblasts. Finally, as in HPV/E2mice, FGF-2 and PDGF
receptors were expressed in the stroma of human cervical cancers
whereas PDGF was expressed in the cancer cells.
What Do These Findings Mean? These findings suggest that PDGF
receptor signaling in the stromal cells associated with cervical tumors in
mice has a functional role during tumor progression. More specifically,
they suggest that PDGF released by the tumor cells triggers PDGF
signaling in the stromal cells, which increases the expression of factors
that both directly and indirectly stimulate the growth of the tumor cells.
Confirmation of this scheme will require additional experiments in
mouse models of cervical cancer and the careful examination of more
human material. Importantly, although approaches that work in mice do
not always work in people, the current findings suggest that targeted
therapeutics that prevent the stromal support of tumor growth (such as
inhibitors of PDGF receptor signaling) might provide a complementary
approach to conventional treatments that target the cancer cells
Additional Information. Please access these Web sites via the online
version of this summary at http://dx.doi.org/10.1371/journal.pmed.
? The US National Cancer Institute provides information on all aspects of
cancer, including information about cervical cancer (in English and
? The UK charity Cancerbackup also provides information on all aspects
of cancer, including information on cervical cancer and on imatinib
? Wikipedia has pages on platelet-derived growth factor, on PDGF
receptors, and on imatinib (note that Wikipedia is a free online
encyclopedia that anyone can edit; available in several languages)
PLoS Medicine | www.plosmedicine.org January 2008 | Volume 5 | Issue 1 | e190138
PDGF Signaling in the Tumor Stroma