Download Fibroblasts in omentum activated by tumor cells

Carcinogenesis vol.33 no.1 pp.20–29, 2012
doi:10.1093/carcin/bgr230
Advance Access publication October 21, 2011
Fibroblasts in omentum activated by tumor cells promote ovarian cancer growth,
adhesion and invasiveness
Jing Caiy, Huijuan Tangy, Linjuan Xu, Xiaoyi Wang,
Chun Yang, Shasha Ruan, Jianfeng Guo, Sha Hu and
Zehua Wang
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical
College, Huazhong University of Science and Technology, 430022 Wuhan, China
To whom correspondence should be addressed. Tel: þ86 27 85351649;
Fax: þ86 27 85351649;
Email: [email protected]
Omentum metastasis is a common occurrence in epithelial ovarian cancer (EOC), which is often accompanied by ascites that
facilitates the spread of EOC cells. A subpopulation of fibroblasts—the cancer-associated fibroblasts (CAFs) are important
promoters of tumor progression. We have shown previously that
CAFs exist not only in omentum with EOC metastasis but also in
omentum without metastasis. In the present study, using primary
human fibroblasts isolated from normal omentum (NFs) and
omentum with ovarian cancer metastasis (CAFs), we established
in vitro coculture models and a 3D culture model mimicking human omentum structure for investigation of interactions between
fibroblasts and cancer cells. We demonstrate that EOC cells activate NFs and promote their proliferation via transforming growth
factor-b1 (TGF-b1) signaling, and the activated fibroblasts contribute to the invasion and adhesion of EOC cells. Moreover, EOC
cells and NFs coculture led to overexpression of hepatocyte
growth factor (HGF) and matrix metalloproteinase-2 (MMP-2)
and adhesion and invasion of EOC cells could be partially suppressed by blocking the function of HGF or MMP-2. Additionally,
mouse peritoneal dissemination models of EOC confirmed the
activation of fibroblasts by cancer cells and the tumor growthand metastasis-promoting effects of activated fibroblasts in vivo.
Our findings indicate that activated fibroblasts in omentum form
a congenial environment to promote EOC cells implantation. It is
an intriguing concept that targeting the activation of omentum
fibroblast through the inhibition of TGF-b1 signaling can be used
as a new therapeutic strategy against ovarian cancer omentum
metastases, which needs further study.
Introduction
Abdominal peritoneum and the omentum are the most common sites
of ovarian cancer metastasis (1). With the formation of ascites, the
exfoliated tumor cells can spread throughout the peritoneal cavity (2).
Surgery is rarely able to render patients free of disease because of its
diffuse nature, resulting in a 5 years survival of only 20% in patients
with peritoneal microscopic or visible metastatic foci even if postoperative chemotherapy is performed (3). Thus, it is necessary to improve
our understanding of the mechanisms involved in the spread of ovarian
carcinoma to the omentum to identify novel therapeutic targets.
The omentum is a large fatty structure, which literally hangs off the
middle of the colon and drapes over the intestines inside the abdomen.
The morphology of the omentum is simple: a single layer of mesothelial cells covers a submesothelial region composed of connective
Abbreviations: CAF, cancer-associated fibroblast; DMEM, Dulbecco’s
modified Eagle medium; ECM, extracellular matrix; EdU, 5-ethynyl-2#-deoxyuridine; EOC, epithelial ovarian cancer; FBS, fetal bovine serum; HGF,
hepatocyte growth factor; MMP, matrix metalloproteinase; PBS, phosphatebuffered saline; SDF, stromal cell-derived factor; SMA, smooth muscle actin;
TGF, transforming growth factor.
y
These authors contributed equally to this work.
tissue with a few fibroblasts, mast cells, macrophages and blood vessels (4). As the second most numerous cell types in the omentum,
fibroblasts represent a dynamic population of cells that show functional and phenotypic diversity. Among the various fibroblastic phenotypes, activated fibroblasts (which are sometimes referred to as
myofibroblasts) are the most important and characterized by the expression of a-smooth muscle actin (a-SMA) (5). In peritoneal tissues,
activated fibroblasts were initially found in the peritoneal dialysis
patients with simple sclerosis (6). Activated fibroblasts that are found
in association with cancer cells are known as cancer-associated fibroblasts (CAFs) (5). CAFs play an important role in the malignant progression of cancer such as breast, prostate, pancreatic, esophageal and
lung cancer, etc. including the initiation, proliferation and metastasis
of cancer cells (7–10). In addition to secreting growth factors including hepatocyte growth factor (HGF), stromal cell-derived factor-1
(SDF-1) and fibroblast growth factor (FGF) that directly affect cell
motility (9,11), CAFs are the source of extracellular matrix (ECM)degrading proteases such as the MMPs. MMPs probably allow cancer
cells to cross tissue boundaries and escape the primary tumor site (12).
Recent studies have provided evidence that CAFs of different tumor types probably display functional differences (13). To date, little
research has been done to evaluate the role of omentum fibroblasts in
cancer cell omentum metastases. In our previous study, we found
CAFs also existed in omental tissues of ovarian carcinoma patients,
even in some cases of omentum tissue lacking detectable cancer cell
infiltration, and the stroma exhibited increased numbers of CAFs as
the omental metastasis enlarges, but there was no CAFs detected in
omentum tissues from patients with benign ovarian diseases (14).
In ovarian cancer, Ena Wang et al. found that there were differences
between the transcriptional repertoires of peritoneal tissues lacking
detectable cancer in patients with epithelial ovarian cancer (EOC) and
benign gynecologic disease, and several of the transcripts identified
suggest the presence of gene products that may foster cancer growth
and metastasis (15). This research raises a hypothesis that peritoneal
structures may be affected by the presence of EOC before tumor
implantation. In other cancer tissues such as breast and lung cancers,
researchers found settlement of metastasizing tumor cells is facilitated
by the establishment of special niches in premetastatic organs through
stimulation of bone marrow-derived hematopoietic progenitor cells
and local resident fibroblasts (16). So, we speculated that changes
of fibroblastic phenotypes occurring within omentum tissues also
created a ‘soil’ conductive for ovarian tumor implantation.
In the present study, we found that EOC cells can contribute to
fibroblast activation and hyperplasia through transforming growth
factor-b1 (TGF-b1). We used a novel 3D culture model and an in vivo
assay to investigate the role of fibroblasts in adhesion and invasion of
ovarian cancer cells to the omentum. Evidently, fibroblasts isolated
from the normal omentum tissues of ovarian cyst patients (normal
fibroblasts, NFs) cannot promote the EOC cells invasion, whereas
EOC cells can activate the fibroblasts and foster the ability of fibroblasts to promote the invasion of EOC cells. A83-01 which can block
signaling of type I receptors for cytokines of the TGF-b superfamily
(known as activin receptor-like kinases; ALKs) (17) can reverse both
fibroblasts activation and the followed metastases of EOC cells
promotion.
Materials and methods
Isolation and culture of primary human mesothelial and fibroblasts
Primary human mesothelial cells and normal fibroblasts (NFs) were isolated
from omental tissues of patients undergoing surgery for benign ovarian cysts
and CAFs were obtained from omentum with EOC metastasis as described
(18,19). Briefly, after washings with sterile phosphate-buffered saline (PBS),
several 2–3 cm2 pieces of omentum were incubated with 0.125% trypsin/
Ó The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Fibroblasts in omentum activated by tumor cells
0.01% ethylenediaminetetraacetic acid at 37°C for 20 min. To isolate mesothelial cells, the solution containing cells in suspension was centrifuged at 1500
r.p.m. for 5 min, and the pellet was plated with Dulbecco’s modified Eagle
medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad,
CA). To isolate fibroblasts, the tissue was further digested for two 40 min
periods, the cells obtained in the second period were resuspended in 5 ml
DMEM/F12 supplemented with 20% FBS. Mesothelial cells were verified
by both vimentin and cytokeratin 8-positive staining (20), whereas fibroblasts
were verified by vimentin positive but cytokeratin 8-negative staining (21).
CAFs were further characterized by expression of a-SMA, whereas NFs were
a-SMA negative. Although ovarian epithelial cells also often show vimentinpositive staining, they could be distinguished from fibroblasts by positive
expression of cytokeratin 8 (22). To quantify the purity of primary cells, the
percentage of marker-expressing cells to the 100 cells randomly counted was
calculated. The primary cultures were incubated at 37°C and the medium was
exchanged after 24 h for the first time and every third day thereafter. All
primary fibroblasts used for this study were between passages 2 and 5, and
mesothelial cells were only used in passage 2.
EOC cell lines and culture conditions
Human EOC cell lines SKOV3, CAOV3, OVCAR3 and ES2 were purchased
from China Center for Type Culture Collection. SKOV3, CAOV3 and ES2
were cultured in DMEM supplemented with 10% FBS. OVCAR3 was cultured
in media DMEM supplemented with 10% FBS and 0.01 mg/ml bovine insulin.
All of these cell lines were grown in a humidified 5% CO2 with a temperature
of 37°C. For TGF-b1 stimulation studies, SKOV3 cells were cultured overnight with RPMI-1640 containing 10% FBS and then the supernatants were
replaced and cultured with fresh culture medium RPMI-1640 containing 1%
FBS with recombinant human TGF-b1 of different concentrations (R&D
system, Minneapolis, MN) for 2–48 h.
Direct and indirect coculture
For direct coculture, fibroblasts labeled with a tracking dye (CFDA, V12883;
Invitrogen, Carlsbad, CA) mixed with ovarian cancer cells in a 1:5 ratio
(fibroblasts 0.2 105 cells, cancer cells 1 105 cells) were seeded onto glass
coverslips in six-well plates and cultured in DMEM containing 10% FBS for at
least 96 h. As described in the manual, CFDA can diffuse into cells and cleaved
by intracellular esterases then react with intracellular amines, forming fluorescent conjugates that are well retained and can be fixed with aldehyde fixatives
and permeabilized by ice-cold acetone. For indirect cocultures, transwell plates
(corning) with two compartments separated by a polycarbonate membrane
with 0.4 lm pores were used. Fibroblasts were seeded on the lower compartment (0.2 105 cells per well) and EOC cells in the upper compartment (1 l05cells per well). Fibroblasts and EOC cells could not contact each other, but
the soluble factors derived from the EOC cell lines could reach fibroblasts.
These cells were cultured for 1–5 days together. Fibroblasts alone cultured
were used as controls (0.2 105 cell per well). For TGF-b1 inhibition studies,
5 lM TGF-b type I receptor inhibitor A83-01 (Tocris Bioscience, Ellisville,
MO) was added to the coculture medium.
Quantitative real-time polymerase chain reaction
Total RNA was extracted by using TRIzol reagent (Invitrogen) and complementary DNA was synthesized using Reverse Transcription Kit (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. The sequences of primers
used were as follows: a-SMA: 5#-CTGTTCCAGCCATCCTTCATC-3# (sense)
and 5-CCGTGATCTCCTTCTGCATT-3# (antisense); b-actin: 5#-GCCAACACAGTGCTGTCTGG-3#
(sense)
and
5#-GCTCAGGAGGAGCAATGATCTTG-3# (antisense). Polymerase chain reaction was performed as
described by the manufacturer using the SYBR Green PCR Master Mix (Toyobo,
Japan). Amplification protocols were followed: 95°C for 3 min; 40 cycles of
95°C/15 s, 60°C/15 s and 72°C/30 s. After polymerase chain reaction, a melting
curve was constructed by increasing the temperature from 65 to 95°C with
a temperature transition rate of 0.2°C/s. The transcript level of each specific
gene was normalized to the b-actin amplification and was calculated using the
comparative threshold cycle (Ct) method (2DDCt) (23).
Immunofluorescence
For immunofluorescence, the CFDA-labeled fibroblasts mixed with EOC cell
lines were plated on sterilized coverslips in six-well plates. After washing with
PBS, the cells in coverslips were fixed in 4% paraformaldehyde for 30 min and
washed in PBS followed by permeabilizing with ice-cold acetone for 10 min
and blocked in 10% bovine serum albumin and then incubated with primary
antibodies against a-SMA for 30 min at 37°C, anti-mouse IgG-CY3 (Sigma–
Aldrich , Missouri) were used as secondary antibodies (1:100 in a blocking
medium). Following further washing, cells were counterstained with 4#,6diamidino-2-phenylindole (DAPI, 1:1000; Invitrogen, Carlsbad, CA) and
examined under the fluorescence microscope. Total fibroblasts number was
obtained by counting the CFDA-stained cells in five fields at 200 magnification. Percentage of CFDA labeled a-SMA-expressing cells to the total number of the fibroblasts was calculated.
Enzyme-linked immunosorbent assay
EOC cell lines (0.2 105 cells per well) were cultured in six-well plates
overnight with DMEM containing 10% FBS. Supernatants of these cells were
removed and replaced by fresh culture medium DMEM containing 2% FBS.
Cells were cultured for another 24 and 48 h. TGF-b1 in the supernatants was
measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems,
Minneapolis, MN). The assays were performed according to the manufacturer’s instructions.
Western blotting
Equal amount (70 lg) of cell extracts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on a nitrocellulose
filter membrane followed by incubating with primary mouse monoclonal antibody against human a-SMA; rabbit monoclonal antibodies against human
HGF, SDF-1 (1:200 dilution; R&D Systems), smad2, phospho-smad2 (Ser465/
467) (1:500 dilution; Cell Signaling Technology, Beverly, MA), MMP-2 and
MMP-9 (1:500 dilution; Epitomics, Burlingame, CA). Horseradish peroxidaseconjugated secondary antibody (1:5000; Santa Cruz Biotechnology, Santa
Cruz, CA) was used for detection. Proteins were visualized by enhanced
chemiluminescence detection reagents (Pierce, Rockford, IL) and bands were
quantitated by Quantity One Software (Bio-Rad, Hercules, CA).
Cell proliferation analysis
The thymidine nucleotide analog 5-ethynyl-2#-deoxyuridine (EdU) was used
for the in vitro labeling of the nuclei of dividing fibroblasts and EOC cells.
After incubation for 1–5 days, respectively, EdU incorporation assays were
conducted. Briefly, for indirect coculture, half of the media was replaced with
fresh media containing 20 lM EdU and incubated for another 12 h, the cells
in lower compartment were fixed with formaldehyde followed by 0.5% Triton
X-100 permeabilization and then stained by incubating for 30 min with
reaction cocktail which was prepared according to the manufacturer#s instructions. Cells were counterstained with Hoechst33342 and imaged by fluorescence microscopy. For direct coculture, the fibroblasts were prelabeled with
CFDA. The percentage of EdU-positive cells was used to evaluate the cell
proliferation activity.
3D omental culture
To investigate the role of fibroblasts on the adhesion and invasion ability of
ovarian cancer cells, we established a 3D culture model mimicking human
omentum conducted according to the method of Lengyel et al. (24). Briefly,
a 24-well plate was coated with 2 mg neutralized isotonic collagen I mixed
with 1 105 CAFs or NFs. After solidification, the mesothelial cells (5 105)
in 100 ll of growth media were added to the above and incubated at 37°C for
24 h until a confluent layer of mesothelial cells formed and then 5 105 EOC
cells in 100 ll of growth media were plated on top. After incubation for 1, 24,
48 h at 37°C, the 3D omental culture gel was fixed in 10% formalin. Gels were
paraffin embedded, sliced at 4 lm and standard hematoxylin and eosin staining
was performed.
Adhesion and invasion assay
All of the EOC cells used in the adhesion and invasion assay were fluorescently
labeled with CFDA. For adhesion assay, cells were plated in 96-well plates
(5 104 cells per well) which had been precoated with collagen I (5 lg/well),
mesothelial cells (5 104 cells per well), fibroblasts (1 104 cells per well)
including CAFs, NFs, NFs cocultured with SKOV3 cells for 4 days and NFs
treated by TGF-b1 for 2 day. After incubation at 37°C for 0.5 h, cells were
washed three times with PBS and then fixed in 10% formalin. For invasion
assay, 24-well transwell plates (corning) with 8 lm pore size were coated with
15 lg collagen I or collagen I mixed with 2 104 fibroblasts and a confluent
layer of 1 105 mesothelial cells. 1 105 fluorescently labeled EOC cells in
100 ll serum-free media were plated on top. The lower chamber was filled with
600 ll full growth media containing 10% FBS. After incubation for 48 h at
37°C, cells on the top of the membrane were scraped off. The membranes were
fixed in 10% formalin. The fixed cells in adhesion and invasion assays were
visualized by fluorescent microscopy at 200 magnifications, selected five
random regions by two independent observers. The number of cells per field
was quantified by measuring the fluorescence intensity. All assays were run in
triplicate.
To determine the effect of activation of fibroblasts in EOC cells adhesion and
invasion, A83-01 was added to the pretreating step (fibroblasts culture before
they were added into collagen) or the 3D culture system directly (in the collagen
gel and media). To investigate the role of HGF and MMP-2 in EOC cell adhesion
and invasion in 3D culture, the neutralization HGF antibody (1 lg/ml) (R&D
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J.Cai et al.
Systems, Minneapolis, MN) and MMP-2 selective inhibitor (IC50 5 12 nM)
(ARP 100; Tocris Bioscience, Ellisville, MO) which is a kind of N-arylsulfonylN-alkoxyaminoacetohydroxamic acids designed as oxa-analogs of MMP2 were
added to the 3D culture (25).
Animal models
All studies in vivo were done according to the guidelines for animal care
established by the Tongji Medical College’s Animal Care and Use Committee.
Female BALB/c nude mice (Beijing Vital River, China) at 4–6 weeks of age
were used. SKOV3 cells (1 107 cells/body), NFs (2 106 cells/body) and
A83-01 (150 lg/body/day) were injected intraperitoneal. Twenty-four mice
were divided into four groups and they were injected with SKOV3 cells,
SKOV3 cells and NFs, SKOV3 cells and A83-01 or SKOV3 cells, NFs and
A83-01, respectively. A83-01 was administered thrice per week from first
week after the inoculation to death. Mice were killed 8 weeks after tumor
inoculation or if showing signs of distress. After killing, location of macroscopic lesions was recorded, and the tumor colonies were dissected, collected
and weighed. The tumor tissues were used for hematoxylin–eosin staining and
histopathological study for a-SMA, HGF and MMP-2. Image Proplus 5.1
software was used to analyze the area and intensity of positive staining in five
random regions (200 magnification) and then the average value per field was
used for evaluation of protein expression level.
Statistical analysis
All data are expressed as mean ± standard deviation, all means were calculated
at least from three independent experiments. Statistical significance of differences was analyzed by two-tailed Student’s t-test or one-way analysis of variance. A value of P ,0.05 was considered to be statistically significant.
Results
EOC cells induce the activation of NFs
Primary human fibroblasts were isolated from omentum tissues with
ovarian cancer cell implant (CAFs) or from nomal omentum tissues
(NFs) followed by morphological and immunocytochemical characterization. The primary fibroblasts initially showed bi- and/or multi-polar
morphology (Figure 1A) and then assumed a uniform spindle-shape
appearance and formed parallel arrays and whorls at confluence (Figure
1B). By immunocytochemical staining, they were positive for vimentin
but negative for cytokeratin 8 (Figure 1C and D). The purity of second
generation fibroblasts was 98%. Moreover, CAFs were identified by
their expression of a-SMA (Figure 1E), whereas NFs were a-SMA
negative (Figure 1F). In order to investigate the role of EOC cells in
the activation of local fibroblasts, we set EOC cells-NFs coculture
systems. NFs were directly cocultured with EOC cell lines SKOV3,
OVCAR3, CAOV3 and ES2, respectively, for 4 days and then the
a-SMA expression was assayed by immunofluorescence staining
(Figure 2A). NFs cultured alone were used as control. On average
37.6–70.0% of cocultured NFs expressed a-SMA, whereas only
1.72% of the control NFs expressed this CAF marker (Figure 2B). In
addition, increased a-SMA expression in NFs indirectly cocultured
with EOC cell lines was detected by polymerase chain reaction and
western blot (Figure 2C,D). Among the four EOC cell lines studied,
SKOV3 showed the strongest ability to activate fibroblasts in direct as
well as indirect coculture models. Thus, SKOV3 was chosen for subsequent experiments in vitro and in vivo. Using western blot, the
a-SMA levels in NFs physically separated from SKOV3 cells (indirectly cocultured) for up to 5 days were determined. We found that the
a-SMA expression increased time dependently, and the difference was
significant from day 4 (P , 0.01, Figure 2E). These results indicate that
fibroblasts can be activated by EOC cells even if a direct cancer cell
fibroblast contact is not present. Besides, the original phenotype of
primary fibroblasts remained after culturing was confirmed by the fact
that the CAFs were a-SMA positive and NFs were a-SMA negative in
the control group. However, 3 days after the end of coculture, the
expression of a-SMA in NFs reduced to the level in control NFs (Figure
2F), suggesting stable a-SMA expression in fibroblasts needs long-term
stimulation by cancer cells.
In addition, the effect of EOC cells on proliferation of NFs was
evaluated by EdU incorporation assay. It was showed that hyperproliferation of NFs began to be evident after indirect coculture with
SKOV3 cells for 96 h (P 5 0.0004) (Figure 2G, H, J). Similar proproliferation effect of SKOV3 cells on NFs was observed in direct
coculture assay (P 5 0.039) (Figure 2I and J). But neither CAFs nor
NFs could promote the proliferation of SKOV3 cells (data not shown).
Paracrine TGF-b1 from cancer cells is responsible for NFs activation
Having demonstrated the activating effect of EOC cells on fibroblasts,
we were interested in the underlying molecular mechanisms. ELISA
showed notably increased secretion of TGF-b1 in routinely cultured
EOC cell lines at 48 h (P , 0.05, Figure 3A), and during the coculture
with SKOV3 cells, in NFs, the phosphorylation level of SMAD2,
which is a cytoplasmic signaling molecule for TGF-b1 began to be
elevated at 2 days (Figure 3B). It implies that the EOC cells-induced
expression of a-SMA may be mediated by TGF-b1. To verify this
Fig. 1. Characterization of primary fibroblasts. Phase-contrast image of primary human fibroblasts before confluence (A), and after confluence (B), fibroblasts
stain for vimentin (C), but are negative for cytokeratin 8 (D). CAFs (E) are a-SMA positive, NFs (F) are a-SMA negative. Magnification 200.
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Fibroblasts in omentum activated by tumor cells
Fig. 2. EOC cells increase expression of a-SMA in NFs. (A) Immunofluorescent staining for a-SMA expression (red) are observed in CAFs, NFs directly
cocultured with EOC cells SKOV3, OVCAR3, CAOV3 or ES2, but not in NFs cultured alone. Fibroblasts are fluorescently labeled (green), nucleuses are labeled
with 4#,6-diamidino-2-phenylindole (blue). (B) Quantified results of immunofluorescence. The percentage of a-SMA-positive cells to the total number of the
fibroblasts is increased in 4 days cocultured NFs compared with control. (C and D) Result of real-time polymerase chain reaction and western blot analysis for
a-SMA expression in CAFs, NFs cultured alone and NFs indirect cocultured with EOC cell lines SKOV3, OVCAR3, CAOV3, ES2 for 4 days. Graphs show
relative expression levels, normalized to b-actin. (E) Western blot analysis for a-SMA expression in NFs cocultured with SKOV3 cells for indicated time periods.
The CAFs and NFs cultured alone were used as control. Quantification of western blot is shown below. (F) Real-time polymerase chain reaction show decreased
a-SMA expression in NFs separated from coculture system for 1–4 days. (G) Growth rates of NFs were determined by EdU incorporation assay each day for
5 days. The growth rates of SKOV3 treated NFs were slightly increased on days 4 and 5 compared with control NFs. (H) Representative results of EdU assay of
NFs indirectly cultured. After 12 h exposure to EdU, dividing NFs cultured alone (NF) or indirectly cocultured with SKOV3 cells (NF SKOV3) are labeled with
EdU (red). All of the NFs are counterstained with Hoechst33342 (blue). (I) Representative result of EdU assay of cells in direct coculture models. NFs are labeled
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Fig. 3. TGF-b1 secreted by EOC cells induces the activation of NFs. (A) ELISA assay shows the total TGF-b1 secreted by EOC cell lines SKOV3, OVCAR3,
CAOV3 and ES2. These cell lines produce a basal level of TGF-b1 in the first day, and this was significantly increased in the second day. (B) Western blot analysis
for the phosphorylation levels of SMAD2 in NFs cocultured with SKOV3 cells for 1–4 days. Quantification of western blot is shown below. (C) Real-time
polymerase chain reaction show exogenous TGF-b1 at 1–10 ng/ml (96 h) increases a-SMA messenger RNA in NFs. (D)Western blot analysis show exogenous
TGF-b1 at 1–10 ng/ml (96 h) induces a-SMA expression and SMAD2 phosphorylation in NFs. Quantification of western blot is shown below. (E) NFs were treated
with 1 ng/ml TGF-b1 for indicated time periods. The expressions of a-SMA and phosphorylated SMAD2 were determined by western blotting. Quantification of
western blot is shown below. (F) EdU incorporation analysis was performed to detect the proliferation of NFs activated by exogenous TGF-b1 (1 ng/ml, 96 h).
(G) Western blot. A83-01 (5 lmol/l) inhibits a-SMA overexpression and SMAD2 phosphorylation in NFs stimulated by EOC and TGF-b1. Quantification of
western blot is shown on the right. (H) EdU incorporation assay. A83-01 reduced the number of dividing cells in NFs simulated by TGF-b1 and SKOV3 cells
treated. Error bars show mean ± standard deviation (of experiments performed in triplicate). P , 0.05 P , 0.01 P , 0.001.
hypothesis, we examined a-SMA expression in NFs treated with different concentrations of TGF-b1 (0.1, 0.5, 1, 5, 10 ng/ml) for 2 days
using quantitative real-time polymerase chain reaction as well as
western blotting. As shown in Figures 3C and 3D, the expression level
of a-SMA and phospho-SMAD2 in NFs were significantly increased
by treatment with TGF-b1 of 1 ng/ml or higher concentrations
(P , 0.01). To find out the chronological order of the changes in
expression of a-SMA and phospho-SMAD2, we assessed their expression levels at five time points (0, 2, 12, 24 and 48 h) during
treatment with 1 ng/ml TGF-b1 using western blotting. The expression of a-SMA as well as phospho-SMAD2 in NFs increased time
dependently and significant increase of phospho-SMAD2 occurred at
with CFDA (green); dividing cells including NFs and SKOV3 are labeled with EdU (red); dividing fibroblasts are labeled with both the EdU (red) and CFDA (green).
(J) Quantitative analysis of the EdU assay shows that the number of dividing NFs increased when cocultured with SKOV3 cells. Magnification 200. Error bars
show mean ± standard deviation (of experiments performed in triplicate). P , 0.05 P , 0.01 P , 0.001.
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Fibroblasts in omentum activated by tumor cells
2 h (P , 0.001) while a-SMA expression started to rise until after
TGF-b1 stimulation for 48 h (P , 0.001, Figure 3E), confirming the
role of TGF-b1 signaling pathway in activation of fibroblasts from
another angle. In addition, similar to coculture with EOC cells,
exogenous TGF-b1 stimulation could also promote proliferation of
fibroblasts (P 5 0.002, Figure 3F).
A specific inhibitor of TGF-b1 (A83-01) was used to further verify
the role of TGF-b1 in fibroblast activation. The a-SMA expression
and SMAD2 activation in NFs induced by SKOV3 cells or exogenous
TGF-b1 could be attenuated by administration of 5 lmol/l A83-01
(Figure 3G). Besides, we found TGF-b1 inhibition also reduced the
proproliferation effect of TGF-b1 (P 5 0.003) and SKOV3 cells (P ,
0.001) on NFs (Figure 3H). We therefore conclude that TGF-b1 is
a key factor mediating fibroblasts activating effect of ovarian cancer
cells.
The activated fibroblasts play key roles in ovarian cancer cell
adhesion and invasion
To examine the role of fibroblasts in omental metastasis of EOC, we
established a 3D culture model consisted of primary human omentum
fibroblasts in a collagen matrix and a confluent layer of mesothelial
cells. The primary mesothelial cells were isolated from normal human
omentum. During the initial growth phase in culture, the subconfluent
mesothelial cells showed multipolar morphology, and a classic cobblestone appearance was present after cell confluence. In addition to
morphology, they were further identified by immunohistochemical
expression of the cell type-specific markers cytokeratin 8 and vimentin (Figure 4A). The mesothelial character of these cells was clear and
the purity of second generation mesothelial cells was 95%.
In the omental 3D culture model with CAFs, SKOV3 cells efficiently attached to the surface of the artificial omentum at 1 h and
formed a dense and adherent layer of SKOV3 cells coated the mesothelial cells at 24 h and then they began to invade into the collagen at
48 h. But when we replaced CAFs with NFs, the adhesion and
invasion of SKOV3 cells were significantly delayed (Figure 4B).
To further determine whether the activity level of fibroblasts is
associated with adhesion and invasion of EOC cells, we performed
adhesion and invasion assays for SKOV3 cells in omental 3D culture
models with CAFs, NFs and activated NFs, respectively. Model without fibroblasts was used as blank control. The presence of CAFs, NFs
cocultured with SKOV3 cells and NFs exposed to TGF-b1 increased
the number of adhered SKOV3 cells 4.77 (P 5 0.007), 3.08 (P 5
0.022) and 3.14-fold (P 5 0.018), respectively, and augmented the
number of invaded SKOV3 cells 6.81 (P 5 0.0001), 3.80 (P 5 0.03)
and 6.06-fold (P 5 0.0005), respectively, compared with the control.
However, there was no significant difference between naive NFs 3D
model and blank control, indicating unactivated NFs are insufficient
to induce the adhesion and invasion of SKOV3 cells to the omentum.
The adhesion- and invasion-promoting effects of NFs stimulated by
SKOV3 cells and exogenous TGF-b1 could be partially (44.9 and
61.6% in adhesion, 58.5 and 54.8% in invasion, respectively) attenuated by addition of A83-01 in the culture period of NFs before they
were added into collagen, i.e. pretreating step while A83-01 directly
added into the gel of 3D culture system did not influence SKOV3 cell
adhesion (Figure 4C and D). These results suggest that TGF-b1 promotes adhesion and invasion of EOC cells through regulating activity
of fibroblasts, and A83-01 can inhibit the activation of NFs mediated
by TGF-b1 but can neither restrain SKOV3 cells adhesion or invasion
nor influence activity of CAFs per se.
Activated fibroblasts increase the adhesion and invasion of EOC cells
through regulating HGF expression
To explore the molecular mechanisms underlying promotion of EOC
metastasis by activated fibroblasts, we examined two important
growth factors HGF and SDF-1 in the tumor microenvironment,
which were previously identified in melanoma and oral cancers as
fibroblast-secreted factors that promote tumor progression (26,27).
The NFs activated by SKOV3 cells or exogenous TGF-b1 expressed
significantly higher levels of HGF compared with primary NFs (3.6fold, P 5 0.008; 4.3-fold, P 5 0.002). Both of these increases could
be attenuated by adding A83-01 to the coculture systems (78.8 and
66.9%, P 5 0.005) (Figure 5A). To determine whether HGF participates in adhesion and invasion of EOC cells, we used inhibitory
antibody to block its function in the 3D coculture system. Figures
5B and 5C demonstrates that inactivation of HGF significantly reduced the adhesion and invasion of SKOV3 cells promoted by NFs
cocultured with SKOV3 (56.9 and 30.7%, respectively) as well as NFs
treated with TGF-b1 (57.9 and 27.5%, respectively) but did not
decrease to the level produced by SKOV3 cells cultured alone, suggesting that promoting effect of activated NFs was only partly mediated by HGF. No increase of SDF-1 was detected in activated NFs
compared with control (data not shown).
Inhibition of MMP-2 reduces adhesion and invasion of SKOV3 cells
mediated by activated fibroblasts
Both epithelial cells and stromal fibroblasts can produce enzymes that
degrade the mesenchymal ECM, such as matrix metalloproteinases
(MMPs) (28). Generally, MMP-2 and MMP-9 are involved in tumor
metastasis. Therefore, we were interested in whether if they regulate
adhesion and invasion of EOC cells mediated by activated fibroblasts.
Using western blot, the MMP-2 and MMP-9 expression in fibroblasts
and SKOV3 cells in different culture models were determined. When
cultured alone, NFs expressed small amount of MMP-2, and its expression in SKOV3 cells was even fainter. Enhanced expression of
MMP-2 was found after 3 days of direct coculture (P , 0.001),
whereas neither NFs nor SKOV3 cells expressed increased MMP-2
up to 5 days in indirect coculture system (Figure 5D), suggesting that
cell–cell contact between EOC cells and fibroblasts is essential for the
enhancement of MMP-2 expression. This speculation was further
confirmed by the fact that exogenous TGF-b1 could not induce the
MMP-2 expression in NFs either. We next used specific inhibitor to
demonstrate the function of MMP-2. The MMP-2 inhibitor decreased
the adhesion and invasion ability of SKOV3 cells promoted by activated fibroblasts in a dose-dependent fashion in the 3D culture models
(Figure 5E and F). These results suggest that MMP-2 is involved in
omental metastasis of EOC cells mediated by fibroblasts, although
MMP-2 expression is not regulated by TGF-b1. However, no change
in MMP-9 expression was detected in different coculture systems
described above (data not shown).
Mouse model of ovarian cancer metastasis
To determine the interactions between fibroblasts and EOC cells in
vivo, SKOV3 cells were injected intraperitoneally into BALB/c nude
mice either alone or admixed with NFs with/without subsequent A8301 administration, respectively (n 5 6 for each group). SKOV3 cells
coinjected with NFs produced many tumor nodules in the peritoneal
cavity covering and invading the body wall, mesentery and diaphragm
in the mice. The SKOV3/NFs tumor weight was 4.12-fold higher than
SKOV3 tumor (P , 0.001) and A83-01 could impair the growth of
SKOV3/NFs tumors (P , 0.001) but not the growth of SKOV3 tumors
(P 5 0.11) (Figure 6A). There were no differences in median survival
time among these four groups (data not shown). Microscopically,
hematoxylin and eosin staining revealed high abundance of fibrotic
tissue within SKOV3/NFs tumors (Figure 6B). Immunostaining assay
for a-SMA showed abundant CAFs in SKOV3/NFs tumors and also
a few activated fibroblasts present in SKOV3 tumors, which mainly
intermingled with cancer cells at margin. They may be recruited from
host by tumor cells (Figure 6C) and their inadequate numbers is likely
to be the major cause leading to ineffectiveness of A83-01 on SKOV3
tumors. Besides, upregulation of MMP-2 was found in SKOV3/NFs
xenografts (Figure 6D). A83-01 administration could reduce the fibrosis, a-SMA expression and MMP-2 expression in SKOV3/NFs
tumors. There was no difference of HGF expression between SKOV3
tumors and SKOV3/NFs tumors (data not shown), it may be because
HGF is expressed mainly in fibroblasts, which were very few in
xenografts.
25
J.Cai et al.
Fig. 4. The activated fibroblasts promote ovarian cancer cell adhesion and invasion in omental 3D culture. (A) Characterization of primary mesothelial cells.
Representative phase-contrast pictures of primary human mesothelial cells before confluence and after confluence. All mesothelial cells express vimentin and
cytokeratin 8. (B) Hematoxylin and eosin stain of cross-sections of 3D omentum culture gel composed of mesothelial cells (arrow) and CAFs or NFs with SKOV3
cells (arrow head) cultured for 1, 24 and 48 h. T, tumor cell side; S, stroma cell side. Adhesion (C) and invasion (D) of SKOV3 cells to the 3D omentum culture
composed of mesothelial cells and collagen I with CAFs, NFs cultured alone, NFs cocultured with SKOV3 cells, NFs exposed to TGF-b1 or without fibroblasts
(blank). The adhesion and invasion of SKOV3 cells to the 3D omentum culture containing A83-01 or fibroblasts pretreated with A83-01 were also quantified.
Fluorecent images of CDFA-labeled adherent or invading cells are shown below the corresponding graphs. Magnification 200. Error bars show mean ± standard
deviation (of experiments performed in triplicate). P , 0.05 P , 0.01 P , 0.001.
Discussion
The surrounding tumor microenvironment seems to be an important
determinant in the metastases of cancer (12,29). Our previously research indicated that CAFs exist not only in omentum with EOC
26
metastasis but also in omentum of patients without metastasis (14),
prompting us to explore the mechanisms underlying activation of
fibroblasts in omentum and the role of fibroblasts in cancer metastasis.
In the present study, we showed the bidirectional interactions between
fibroblasts and EOC cells and explored the key factors contributing to
Fibroblasts in omentum activated by tumor cells
Fig. 5. Activated NFs promote the invasion and adhesion of SKOV3 cells through elevating expression of HGF and MMP-2. (A) Western blot analysis show
enhanced HGF expression in NFs sitimulated by either exogenous TGF-b1 or EOC cells. A83-01 can block the increases. Quantification of western blot is shown
below. (B and C) Adhesion and invasion analysis show HGF antibodies significantly inhibit the adhesion and invasion ability of SKOV3 cells promoted by SKOV3
cells and TGF-b1 treated NFs but did not influence SKOV3 cells per se. (D) Western blot analysis show MMP-2 expression in NFs and SKOV3 cells and cells
cultured in different models. Quantification of western blot is shown below. (E and F) Adhesion and invasion experiments. The bars show a dose-dependent effect
of MMP-2 inhibition on the adhesion and invasion ability of SKOV3 cells cocultured with control NFs and NFs activated by TGF-b1 or SKOV3 cells. Error bars
show mean ± standard deviation (of experiments performed in triplicate). P , 0.05 P , 0.01 P , 0.001.
fibroblasts activation and fibroblasts mediated metastasis of cancer
cells.
It has been shown that CAFs might originally derive from several
types of cells, such as pre-existing normal fibroblasts, nearby epithelial cells through an epithelial-to-mesenchymal transition, bone marrow-derived mesenchymal stem cells and endothelial cells (30). One
of the best-known factors responsible for the phenotypic emerge
of CAFs is TGF-b (30). Many studies have identified that tumorcell-autonomous TGF-b expression is often increased in human carcinoma including EOC (31), which was confirmed by our results that all
of the four EOC cell lines studied secreted high level of TGF-b1.
However, EOC cells frequently escape the influence of TGF-b through
the acquisition of missense mutations in TGF-b receptor I (32).
In the present study, we showed that TGF-b1 play important roles
in induction of the activated phenotype of fibroblasts through stimulating SMAD signaling and promoting the proliferation of NFs in vitro
and in vivo. However, the activation of NFs induced by EOC cells was
not perpetual in vitro. The activated phenotype was vanished 3 days
after the end of coculture. This result suggests that EOC cells induce
activation of NFs in vitro but not to fully differentiated CAFs which
remain perpetually activated even when the initial insult has regressed
(5,11).
Ernst Lengyel et al. showed that normal omental mesothelial cells
inhibit, whereas normal omental fibroblasts enhance the attachment
and invasion of ovarian cancer cells to the omentum (24). In our study,
CAFs could induce EOC cells metastases but the primary NFs could
not. The activated NFs obtained the ability to promote the adhesion
and invasion of EOC cells in vitro and in vivo, which could be abrogated by inhibition of fibroblast activation. Using an organotypic
culture model, recent studies showed that employment of fetal esophageal fibroblasts within the esophageal cancer ECM resulted in a more
aggressive invasion, whereas fetal skin fibroblasts which are less activated produce little evidence of invasion of tumor cells (9,12). These
data combined with our studies provide compelling evidence that the
activation level of fibroblasts is critical in fostering the extent of tumor
invasion.
The ascites of ovarian cancer contained different kinds of cells and
protein including TGF-b (33). We imagine that during the course of
tumor progression, the normal omentum fibroblasts activation is initially triggered by TGF-b released in significant quantities by carcinoma cells and from ascites and then the activated fibroblasts formed
a ‘pre-metastatic niche’ favor EOC cells implantation which promote
more fibroblasts activation and hyperproliferation then induce more
EOC cells implant to the omentum.
HGF is expressed ubiquitously and plays an important role in the
tumor stromal microenvironment (34). Secreted by mesenchymal
cells, HGF acts on epithelial and endothelial cells. Many types of
cancer cells secrete molecules that enhance HGF production in fibroblasts while fibroblast-derived HGF, in turn, is a potent stimulator of
the invasion of cancer cells (35). In ovarian cancer, HGF was found to
stimulate the migration of the ovarian carcinoma cell line SKOV3
(36). Here, we show that activated fibroblasts expressed higher HGF
than primary NFs. The neutralization HGF antibody attenuates
SKOV3 invasion and adhesion induced by activated NFs provided
the fact that HGF is a significant factor responsible for cancer cell
metastasis mediated by tumor–stromal interactions. However, this
does not preclude the likely involvement of other growth factors
and/or cytokines such as fibroblast growth factor and epidermal
growth factor, which contribute to tumor growth and metastases
(26,37).
The MMP-2 can degrade many ECM components and often associated with cancer cell metastasis (38). The submesothelial ECM of
both the peritoneum and omentum is essentially composed of collagen type I, vitronectin and fibronectin (39), whereas the first two
components were previously reported as MMP-2 substrates (40). In
this paper, we show that cell–cell interaction is a major factor of
enhanced MMP-2 expression in vitro, and stable upregulation of
MMP-2 was found in SKOV3 cells and NFs inoculation xenografts.
27
J.Cai et al.
Fig. 6. The effect of fibroblasts on the peritoneal dissemination of EOC in vivo. At 8 weeks after inoculation of the SKOV3 cells, mice were killed for macroscopic
examination to determine distribution of the dissemination. (A) Macroscopical morphology of xenografts. Only a few metastatic nodules (arrow) were observed in
the mesentery of mice injected with SKOV3 cell alone. In contrast, inoculation of NFs and SKOV3 cells together significantly increases the metastatic nodules and
A83-01 administration attenuates these increases. The weight of the metastatic nodules was quantified. (B) The hematoxylin and eosin staining of xenografts. The
nodule in the NFs and SKOV3 coinjected mouse shows diffuse infiltration of cancer cells with fibrous stroma (arrow) in the NFs and SKOV3 cells coinjected
group. This fibrosis was reduced by A-83-01 administration. (C and D) Representative immunostaining for a-SMA and MMP-2 in SKOV3/NFs tumor xenografts
and SKOV3 tumor xenografts. The a-SMA-positive cells (arrow) and MMP-2 expression in SKOV3/NFs tumors was more than that in SKOV3 tumors, A83-01
attenuated these increases. Magnification 200. Quantifications are shown on the right. Error bars show mean ± standard deviation (of experiments performed in
triplicate). P , 0.05 P , 0.01 P , 0.001.
Attenuate MMP-2 activity suppressed EOC cells attached and invaded
to the 3D culture, which underscored a direct role for MMP-2 activation in EOC metastases. So, we hypothesized that cancer cells break
off the surface of the ovarian mass adhere to and disaggregate on
omentum, subsequently forming invasive foci (41). In these foci,
hyperproliferated and activated stroma fibroblasts interacted with cancer cells resulted in hyper-expression of MMP-2 which could degrade
the ECM, mediate metastasis of neoplastic cells (42,43).
In summary, our results partly explain why ovarian cancer cells
have a clear predilection for the omentum. Since the high metastases
rate of ovarian cancer, the role of CAFs in tumor cell invasion and
adhesion makes targeting the omentum fibroblast activation through
the inhibition of TGF-b1 signaling an appealing therapeutic option.
Acknowledgements
We would like to thank the Department of Obstetrics and Gynecology, Union
Hospital, Wuhan, China, for providing tissue samples.
Conflict of Interest Statement: None declared.
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Received June 2, 2011; revised October 9, 2011; accepted October 16, 2011
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