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[CANCER RESEARCH 61, 8306–8316, November 15, 2001]
Transforming Growth Factor-�1 Increases Survival of Human
Melanoma throughStroma Remodeling1
Carola Berking, Richelle Takemoto, Helmut Schaider, Louise
Showe, Kapaettu Satyamoorthy, Paul Robbins, andMeenhard Herlyn2
The Wistar Institute, Philadelphia, Pennsylvania 19104 [C. B.,
R. T., H. S., L. S., K. S., M. H.], and the University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania15261 [P.
R.]
ABSTRACT
Transforming growth factor (TGF)-� is growth inhibitory for
normalepithelial cells and melanocytes but can stimulate
mesenchymal cells.Resistance to its inhibitory effects is
characteristic of human melanoma,the growth of which may instead be
promoted by TGF-�, because itsproduction is increased with melanoma
progression. Whether TGF-� hasan autocrine function for melanoma
cells or is important for paracrinestimulation of the tumor stroma
is not known. In this study, TGF-�1 wasexpressed in melanoma cells
via adenoviral gene transfer, and tumorgrowth was analyzed in
vitro, in human skin grafts, and in mixtures withfibroblasts that
were injected s.c. into immunodeficient mice. The TGF-�1produced by
the melanoma cells activated the fibroblasts to producematrix
within and around the tumor mass, whereas control tumorsshowed less
stroma and more cell death. High expression of
collagen,fibronectin, tenascin, and �2 integrin was detected in the
TGF-�1-expressing tumors by immunohistochemistry. Number and size
of lungmetastases were significantly increased. cDNA expression
array analysisof TGF-�1-transduced fibroblasts embedded in type I
collagen and ofTGF-�1-transduced melanoma cells demonstrated
induction of types XV,XVIII, and VI collagens, tenascin,
plasminogen activator inhibitor-I, vas-cular endothelial growth
factor, cysteine-rich fibroblast growth factorreceptor-1, and
platelet-derived growth factor receptor-�, which could belinked to
promotion of growth and survival in melanoma. These datasuggest
that remodeling of the neighboring stroma, which provides
asupporting scaffolding and a positive feedback stimulation of
tumorgrowth, is an important function of TGF-�1 in melanoma.
INTRODUCTION
TGF3-� is an almost ubiquitously expressed protein with
diversefunctions in embryogenesis and adult tissue homeostasis.
There existthree isoforms of TGF-� (TGF-�1, TGF-�2, and TGF-�3) in
mam-mals with 75–80% homology, which arise from proteolytic
cleavageof longer precursors (1, 2). Mature biologically active
TGF-� resultsfrom dissociation of the latent inactive TGF-�
complex, which can bestored in the ECM. TGF-� antagonizes the
mitogenic activities ofmany other growth factors by interfering
with cell cycle progressionand is the most potent growth inhibitor
known for epithelial cells andcells of the immune system. On the
other hand, TGF-� can stimulatemesenchymal cells, such as
fibroblasts, smooth muscle cells, andchondrocytes, and induce the
synthesis of proteins found in the ECM
including collagens, fibronectin, tenascin, thrombospondins,
os-teopontin, osteonectin, and elastin (1, 3, 4). Simultaneously,
TGF-�can reduce the synthesis of proteases, such as collagenases,
andincrease the synthesis of protease inhibitors, such as TIMP-1
andPAI-I (5).
Carcinoma cells of the breast, prostate, lung, and colon
produceTGF-� and are resistant to its growth-inhibitory effects, in
contrastwith their benign precursor cells (6, 7). It has been
hypothesized thatat these advanced stages of transformation, TGF-�
can act as a tumorpromoter. Likewise in melanoma, expression of all
three isoforms ofTGF-� has been found in malignant cells in culture
(8) and in situ(9–11), and an association with progression has been
proposed. Bothhigh-affinity receptors for TGF-�, which form a
heteromeric complexupon activation and transmit signals to the
cytoplasmic SMAD pro-teins (2), are expressed in melanoma (12, 13).
Upon exogenousTGF-� stimulation, melanoma cells display various
degrees of resist-ance to TGF-�-induced inhibition of DNA
synthesis, whereas mela-nocytes are highly sensitive (8, 14).
However, in contrast with whathas been found in some other
TGF-�-resistant carcinomas, no inac-tivating mutations in the TGF-�
receptor system or of the SMADsignaling cascade have been detected
in melanoma, suggesting addi-tional mechanisms for resistance to
the growth-inhibitory functions ofTGF-� in this tumor type
(15).
The biological benefits for melanoma cells to constitutively
pro-duce TGF-� remain unclear. An autocrine TGF-�-mediated
up-regu-lation of integrins and matrix metalloproteinase-9 as well
as a down-regulation of E-cadherin has been described and may
facilitatemelanoma cell migration (16) and adhesion to the
endothelium (13).Paracrine effects of TGF-� on host cells in the
tumor microenviron-ment may also be advantageous for melanoma
cells. Suppressiveeffects on the immune system may allow tumor
cells to escape fromimmune surveillance (17, 18), and angiogenic
properties of TGF-�could support nutrition of the tumor and
facilitate metastasis (3, 19).In addition, stimulation of stromal
cells by TGF-� could lead toincreased production of reciprocally
paracrine-acting growth factorsand to ECM production, which could,
in turn, provide a scaffoldingfor melanoma cells to adhere and
migrate. Finally, the modulation ofproteases and their inhibitors
by TGF-� could facilitate remodeling ofthe stroma and invasion
(20–22).
In this report, we provide evidence that melanoma cells can
mod-ulate their surrounding stroma for their own benefits through
theparacrine activity of TGF-�1. Stimulation of production of
ECMproteins by stromal fibroblasts provided a scaffolding for the
mela-noma cells, which showed increased survival and metastasis
forma-tion compared with the controls. Global gene expression
analyses ofTGF-�1-expressing melanoma cells and fibroblasts in
organotypicculture indicated a complex interplay between matrix
proteins, adhe-sion molecules, and growth factors, providing an
optimal environmentfor tumor growth and progression.
MATERIALS AND METHODS
Cell Culture. Normal human keratinocytes and melanocytes were
isolatedfrom the epidermis, and fibroblasts were isolated from the
dermis of neonatal
Received 6/8/01; accepted 9/19/01.The costs of publication of
this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
advertisement in accordance with18 U.S.C. Section 1734 solely to
indicate this fact.
1 Supported by NIH Grants CA80999, CA25874, and CA10815 (to M.
H.) and apostdoctoral research fellowship BE2189/1-1 from the
Deutsche Forschungsgemeinschaft(to C. B.).
2 To whom requests for reprints should be addressed, at The
Wistar Institute, 3601Spruce Street, Philadelphia, PA 19104. Phone:
(215) 898-3950; Fax: (215) 898-0980;E-mail:
[email protected].
3 The abbreviations used are: TGF, transforming growth factor;
ECM, extracellularmatrix; PAI, plasminogen activator inhibitor;
TIMP, tissue inhibitor of metalloproteinase;VEGF, vascular
endothelial growth factor; CFR, cysteine-rich fibroblast growth
factorreceptor; PDGF, platelet-derived growth factor; PDGFR, PDGF
receptor; SFM, serum-free medium; FBS, fetal bovine serum; SCID,
severe combined immunodeficient;TUNEL, terminal
deoxynucleotidyltransferase-mediated nick end labeling; Ad5,
adeno-virus serotype 5; pfu, plaque-forming unit; VGP, vertical
growth phase; RGP, radialgrowth phase.
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human foreskins. Keratinocytes were cultured in SFM (Life
Technologies,Inc., Rockville, MD) supplemented with human
recombinant epidermal growthfactor and bovine pituitary extract.
Melanocytes were cultured in MCDB153(Sigma Chemical Co., St. Louis,
MO) supplemented with 2% FBS, 10%chelated FBS, 2 mM glutamine
(Mediatech, Herndon, VA), 20 pM choleratoxin(Sigma Chemical Co.),
150 pM recombinant human basic fibroblast growthfactor, 100 nM
endothelin-3 peptide (Peninsula, Belmont, CA), and 10
ng/mlrecombinant human stem cell factor (R&D Systems,
Minneapolis, MN). Fi-broblasts and human embryonic kidney-derived
293 cells used for adenovirusreplication were cultured in DMEM with
glutamine (Life Technologies, Inc.)and 10% FBS (Hyclone, Logan,
UT). Human primary and metastatic mela-noma cells were isolated
from clinically and histologically defined lesions andcultured as
described (23, 24). They were maintained in MCDB153 with
20%Leibovitz’s L-15 medium (Life Technologies, Inc.), 2% FBS, and 5
�g/mlinsulin (Sigma Chemical Co.).
Growth Factor Detection. Melanoma cells, melanocytes, and
fibroblastswere plated in six-well plates at 1.5 � 105 cells/well
in their culture mediumfor 24 h. They were transduced with TGF-�1
or LacZ via adenoviral vectors,washed with SFM after 24 h, and
cultured in SFM for another 48 h. Cellsupernatants were then
analyzed for the presence of TGF-�1 using a humanTGF-�1 immunoassay
(R&D Systems) that uses TGF-� soluble receptor typeII, which
binds TGF-�1, as the coating reagent and an enzyme-linked
poly-clonal antibody to TGF-�1 as the second reagent. The procedure
followed themanufacturer’s instructions. The measurements represent
the total of bothactive and latent forms of TGF-�1 and are given in
pg/105 cells. For analysisof VEGF protein, an ELISA was used
according to the manufacturer’s instruc-tions (R&D Systems).
Melanoma cells and fibroblasts were transduced withTGF-�1 or LacZ
control, respectively, and conditioned medium was collected72 h
later. Samples were frozen at �70°C and analyzed within the
following1–2 days. Results are expressed as mean � SD ng/ml per 106
cells. Allexperiments were performed in duplicates.
[3H]Thymidine Incorporation Assay. Melanoma cells, fibroblasts,
andmelanocytes were seeded at 2–4 � 104 cells/well in 96-well
plates andinfected with adenoviral vectors. After 2 days, 1 �Ci of
[3H]thymidine wasadded per well, and 18 h later, cells were
harvested, and the activity wascounted with a beta counter.
Experiments were performed twice and intriplicates. Results are
expressed as average difference (in percentages) � SDcompared with
the respective LacZ-transduced control cells.
Adenoviral Vectors for TGF-�1 and LacZ. The adenoviral vector
TGF-�1/Ad5 carrying the gene for the TGF-�1 protein has been
described (25). Thecontrol adenoviral vector LacZ/Ad5 (Vector Core;
University of Pennsylvania,Philadelphia, PA) induces expression of
the reporter gene �-galactosidase fromEscherichia coli. The vectors
were prepared, purified, and titered to1–5 � 1010 pfu/ml.
Melanoma cells were infected with 2 or 20 pfu/cell, and
fibroblasts wereinfected with 40 pfu/cell in serum-free base medium
for 3–4 h. Medium wasthen changed to growth medium, and cells were
used the next day forexperiments.
Human Skin Grafting. Human foreskins from newborns were kept
insterile transport media (HBSS supplemented with antibiotics) and
graftedwithin 48 h of excision as described (26). Female and male
CB-17 SCID micewere bred at the Animal Facility of the Wistar
Institute and housed underpathogen-free conditions in groups of up
to five animals/isolator cage. Graftswere well healed after 4–6
weeks, and mice were then used for the experi-ments. Mice received
injections intradermally with the adenoviral vectorsusing a
26-gauge needle at a concentration of 0.5–5 � 108 pfu in a total
volumeof 100 �l of sterile PBS. Melanoma cells (2–5 � 106 cells)
were injectedintradermally with a 23-gauge needle in 100 �l of cell
culture medium. TheWistar Institutional Animal Care and Use
Committee approved all protocols.
Tumor Growth in Vivo. For in vivo growth studies, 451Lu melanoma
cellswere mixed with normal human fibroblasts in 100 �l of medium
and 100 �lof Matrigel matrix (Collaborative Biomedical Products,
Bedford, MA) andinjected s.c. into SCID mice with a 23-gauge
needle. One day before injection,melanoma cells were transduced
with TGF-�1 or LacZ using adenoviralvectors, respectively, at an
infection dose of 20 pfu/cell. The total injected cellnumber per
mouse was 2.2 � 106 to 4 � 106 cells after mixing melanoma
cellswith fibroblasts at a ratio of 1:10 (2 � 105 melanoma cells
and 2 � 106
fibroblasts), 1:1 (2 � 106 and 2 � 106 cells), and 10:1 (2 � 106
and 2 � 105
cells). Six mice/group received injections and were sacrificed
after 2 weeks,
and 10 additional mice that received injections of TGF-�1- or
LacZ-transducedmelanoma cells mixed with fibroblasts at a ratio of
1:1, respectively, weresacrificed after 5.5 weeks. Tumor growth was
monitored twice weekly.
Western Blot. 451Lu tumors were harvested from the mouse and
mincedwith RIPA buffer (TGF-�1) containing protease inhibitors (1
mM phenylmeth-ylsulfonyl fluoride, 10 �g/ml aprotinin, 10 �g/ml
leupeptin, and 1.8 mg/mliodoacetamide). After centrifugation at 4°C
for 20 min at 12000 � g, proteinin the supernatants was quantified
using the BCA kit (Pierce, Rockford, IL).Equal amounts of total
protein from each sample were resolved in a 15%SDS-polyacrylamide
gel, electroblotted onto a polyvinylidene difluoride mem-brane
(Bio-Rad Laboratories, Richmond, CA), and blocked with a 5%
solutionof dry milk and 0.05% Tween 20 in PBS at room temperature
for 1 h. Themembrane was incubated with rabbit polyclonal
antihuman-TGF-�1 antibody(Santa Cruz Biotechnology, Santa Cruz, CA)
followed by peroxidase-labeledsecondary antibody (Jackson
ImmunoResearch Laboratories, Inc., WestGrove, PA). Immunoreactive
bands were developed using the ECL detectionsystem (Amersham,
Arlington Heights, IL) and exposed to Kodak Biomax film(Eastman
Kodak, Rochester, NY).
Histology and Immunohistochemistry. At the end of each
experiment,mice were sacrificed by CO2 inhalation, and skin grafts
or s.c. tumors wereexcised. Half of the samples were fixed in 10%
neutral-buffered formalin(Fisher Scientific, Pittsburgh, PA) for
6–12 h at room temperature and em-bedded in paraffin. The other
half was dehydrated by increasing concentrationsof sucrose
solutions (5, 10, and 20%) at 4°C overnight, embedded in OCTmedium
(Miles, Elkhart, IN), snap-frozen, and stored at �70°C until
cryosec-tioning at 6–8 �m. Formalin-fixed sections were stained
with H&E forhistopathological evaluation. Masson’s trichrome
stain was used for estimationof the amount and distribution of
collagen in the tissues. The DNA-bindingfluorochrome Hoechst 33258
(Sigma Chemical Co.) was used to distinguishhuman from murine
cells.
Immunohistochemistry was performed on serial sections using an
avidin-biotin-peroxidase system kit (Vector Laboratories,
Burlingame, CA) and 3,3�-diaminobenzidine tetrahydrochloride (Sigma
Chemical Co.) or 3-amino-9-ethylcarbazole (Vector) as chromogens.
Antigens in the formalin-fixed tissueswere retrieved by trypsin
digestion at 37°C or microwave heat treatment incitrate buffer.
Cryostat sections of 6–8 �m were air-dried and fixed in
ice-coldacetone for 10 min. Prior to incubation with the primary
antibodies in ahumidified chamber at 4°C overnight or at room
temperature for 1–2 h,nonspecific binding was blocked with 10%
normal horse or 10% normal goatserum. Primary mouse monoclonal
antibodies against the following humanantigens were used: Ki-67
(Immunotech, Westbrook, ME); HMB45 (Bio-genex, San Ramon, CA); type
IV collagen (hybridoma from ATCC, Manassas,VA); fibronectin
(American Type Culture Collection); tenascin (27); �2 inte-grin
(Chemicon, Temecula, CA); �3 integrin (28); smooth muscle
actin(Zymed, South San Francisco, CA); aminopeptidase N (29); and
CD31 (PE-CAM; Dako Carpinteria, CA). Primary rabbit polyclonal
antibodies used inthis study were rabbit anticow S100 (Dako) and
rabbit antihuman TGF-�1(Santa Cruz Biotechnology). Mouse IgG1
isotype antibody (P3) was used asnegative control for each staining
with mouse monoclonal antibodies and arabbit antihuman involucrin
antibody (Biomedical Technologies, Stoughton,MA) as negative
control for each staining with rabbit polyclonal antibodies.Between
each incubation step, slides were rinsed twice in PBS for 3–5
min.Endogenous peroxidase was quenched with 3% H2O2 in methanol for
20–30min at room temperature. A biotin-labeled antimouse secondary
antibody wasapplied for 30 min at room temperature, followed by
incubation with apreformed avidin-biotinylated enzyme complex for
30 min. After color devel-opment by addition of the chromogen and
counterstaining with Mayer’shematoxylin (Sigma Chemical Co.),
sections were mounted and evaluatedunder a light microscope.
TUNEL. Detection of apoptosis was done on formalin-fixed,
paraffin-embedded tumor sections with a commercially available
TUNEL kit (Boeh-ringer Mannheim, Indianapolis, IN) according to the
manufacturer’s directionswith modifications. Briefly, after
deparaffinization, rehydration, and quench-ing of endogenous
peroxidase, sections were treated with 0.1% Triton X-100(Sigma
Chemical Co.) and 0.1% citrate buffer for cell permeabilization.
Afterincubation with terminal deoxynucleotidyl transferase and
fluorescein-labelednucleotides for 60 min at 37°C and incubation
with peroxidase-conjugatedanti-fluorescein antibody Fab fragments
for 40 min at 37°C, 3-amino-9-ethylcarbazole substrate and H2O2
were added for color development. Coun-
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terstain was done with Mayer’s hematoxylin. Positive labeled
cells were scoredin a blinded manner in randomly chosen fields at
�200.
Skin Reconstruction. Skin reconstructs were prepared essentially
as de-scribed with modifications (30). Human fibroblasts (FF2441)
were added toneutralized bovine type I collagen (Organogenesis,
Canton, MA) to a finalconcentration of 0.8–1 mg/ml of collagen in
MEM (BioWhittaker, Walkers-ville, MA), 1.66 mM L-glutamine (Life
Technologies, Inc.), 10% FBS, and0.21% sodium bicarbonate
(BioWhittaker). Three ml of fibroblast-containingcollagen (2.5 �
104 cells/ml) were added to each insert of a six-well
tissue-culture tray (Organogenesis) after precoating with 1 ml of
acellular collagen.Mixtures were allowed to constrict in DMEM with
10% FBS for 5–7 days. Theday before seeding, melanoma cells were
infected with TGF-�1/Ad5, andcontrols were infected with LacZ/Ad5
at 20 pfu/cell for 4 h in protein-freeSFM and then incubated
overnight in complete SFM. Keratinocytes weremixed with melanoma
cells at a ratio of 5:1 to 10:1 in low-calcium epidermalgrowth
medium containing DMEM, F-12 Ham’s (Life Technologies, Inc.),
1%newborn calf serum (Hyclone), 4 mM glutamine, 1.48 � 10�6 M
hydrocorti-sone, 4 pM progesterone, 20 pM triiodothyronine, 0.1 mM
O-phosphoryleth-anolamine, 0.18 mM adenine (Sigma Chemical Co.), 5
mg/ml insulin, 5 mg/mltransferrin, 5 mM ethanolamine, 5 g/ml
selenium (BioWhittaker), and 50 �g/mlgentamicin (Mediatech,
Herndon, VA). A total of 5–6 � 105 cells was seededon each
contracted collagen gel. Cultures were maintained submerged in
lowcalcium growth medium for 2 days and in normal calcium (1.88 mM)
growthmedium for another 2 days and then raised to the air-liquid
interface for 10–12days with feeding from below with normal calcium
and high-serum (20%)medium.
RNA Preparation and Labeling. Total RNA was isolated with
TrizolReagent (Life Technologies, Inc.) according to the
manufacturer’s protocol.After DNase I treatment (Roche Diagnostics,
Mannheim, Germany) for 15 minat 37°C and ethanol precipitation, the
RNA quality and quantity was visualizedon a 1% agarose gel with
ethidium bromide. One to 2 �g of total RNA werereverse transcribed
with 300 units of Superscript II reverse transcriptase
(LifeTechnologies, Inc.) in the presence of 2 �g of oligo(dT)15
primer (PromegaCorp., Madison, WI), 1 �l of 10 � decamers (Ambion,
Austin, TX), 1 mMdATP, dGTP, and dTTP (Amersham Pharmacia,
Piscataway, NJ), respectively,0.1 mCi of [33P]dCTP (ICN
Biomedicals, Costa Mesa, CA), and 3.5 mM DTTat 39°C for 90 min. The
labeled cDNA targets were separated from theunincorporated
[33P]dCTP through Sephadex G-50 Quick Spin columns(Roche
Diagnostics, Indianapolis, IN) and denatured at 100°C before
hybrid-ization to the filter arrays. [33P]CTP incorporation was
quantitated by scintil-lation counting.
cDNA Expression Array. Human arrays from the Genomics Core of
theWistar Institute were used. Each array consisted of a nylon
membrane(2.5 � 7.5 cm) spotted with 200–600-bp cDNA fragments from
sequence-validated human clones (Research Genetics, Huntsville, AL)
representing 2280different genes, 9 housekeeping genes, and
negative controls. Arrays wereproduced with a GM417 array station
(Genetic MicroSystems, Bedford, MA)using 300-�m pins with 750-�m
center-to-center spacing.
The labeled cDNA targets were hybridized to the arrays in Church
bufferwith boiled and chilled Cot-1 DNA and sheared salmon sperm
DNA inplastic hybridization bags at 65°C for 18 h. The membranes
were washedat 65°C in 2� SSC/1% lauryl sulfate sodium salt (SDS)
for 30 min 3 timesin 0.1� SSC/0.5% SDS for 30 min once and then
exposed to a Phosphorscreen (Molecular Dynamics, Sunnyvale, CA) for
24 h to 5 days. Duplicatehybridizations were performed with RNA
from two independent experi-ments. Sequential filters from the same
printing were used for the analyses.Phosphorscreens were scanned
with a Storm Phosphorimager (MolecularDynamics) at a resolution of
50 �m. Imagequant files from scans wereimported into ArrayVision
(Imaging Research, St. Catharine, Ontario,Canada) for
quantification. Spot intensities were background
subtracted,globally normalized, and reported as median pixel
densities by the WistarGenomics Core.
Statistics. For ELISA, TUNEL assay, proliferation, and tumor
growthexperiments, the arithmetic mean and SDs were calculated.
Statistical differ-ences to LacZ-treated controls were validated by
the two-sided Student’s t test.P � 0.05 was considered
significant.
RESULTS
Constitutive TGF-�1 Production in Melanoma and Sensitivityto
Induced TGF-�1 Expression. Constitutive TGF-�1 production in21
human melanoma cell lines from different progression stagesranged
between 0 and 248 pg/105 cells with an average of 49 � 70pg/105
cells as measured by ELISA (Fig. 1A). There was a tendencyof higher
TGF-�1 levels in late progression stages but not exclusively.More
than 40 pg of TGF-�1 per 105 cells were found in 3 of 7
(43%)metastatic melanoma lines, 2 of 9 (22%) primary VGP, and 1 of
5(20%) primary RGP melanoma lines, whereas �5 pg/105 cells
werefound in 1 of 7 (14%) metastatic lines, 3 of 9 (33%) primary
VGP, and3 of 5 (60%) primary RGP melanoma lines. Metastatic lines
WM373,WM1617, and 1205Lu secreted higher levels of TGF-�1 than
theirprimary melanoma counterparts WM75, WM278, and WM793
estab-lished from the same patient, respectively. The metastatic
lineWM239A, on the contrary, showed little or no TGF-�1
production(2 � 2 pg/105 cells), whereas the respective primary
tumor lineWM115 produced 36 � 35 pg/105 cells. Normal human
melanocytesproduced negligible levels of TGF-�1 (2 pg/105 cells),
whereas neo-natal foreskin fibroblasts produced 64 pg/105
cells.
The sensitivity of 21 melanoma cell lines to TGF-�1 was
analyzedafter transduction with an adenoviral vector expressing
TGF-�1 (Fig.1B). Cell proliferation was measured by [3H]thymidine
incorporation
Fig. 1. Constitutive production of TGF-�1 and proliferative
response to transductionwith TGF-�1. A, TGF-�1 production in 21
human melanoma cell lines, in foreskinmelanocytes (FM), and in
foreskin fibroblasts (FF) analyzed by ELISA of cell
culturesupernatants 72 h after seeding. Progression stage decreases
from left to right. No., normalcells. Bars, SD. B, growth of the
same melanoma cell lines as above after transductionwith TGF-�1.
Given is the percentage relative to the [3H]thymidine uptake after
trans-duction of each respective line with LacZ control. Bars,
SD.
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and compared with LacZ control vector-transduced cells.
Resistanceor a �30% inhibition upon TGF-�1 transduction was found
in 12 of21 lines (57%). Inhibition defined as a �30% reduction
comparedwith the controls was observed in 7 of 21 lines (33%), and
stimulationwas found in 2 of 21 lines (10%). Melanocytes were
inhibited, andfibroblasts were resistant. In general, cell lines
with high constitutiveTGF-�1 levels were resistant to TGF-�1
transduction, and cell lineswith low or no endogenous production of
TGF-�1 showed eitherstimulation, resistance, or inhibition. The
strongest inhibition wasobserved for WM239A, which was found to be
the lowest TGF-�1producer among the metastatic lines analyzed.
The dose-dependent protein production of TGF-�1 after
adenoviraltransduction was analyzed in six selected melanoma cell
lines byELISA (Fig. 2A). An infection dose of 2 pfu/cell led to a
1.2–4-foldincrease in TGF-�1 protein production in five of six cell
lines and a153-fold increase in cell line WM239A. An infection dose
of 20 pfuled to a 9–23-fold increase in TGF-�1 protein production
in four ofsix cell lines and an 115-fold and 333-fold increase in
the cell lines1205Lu and WM239A, respectively. This dose was
therefore used inthe following in vivo studies.
The effect of TGF-�1 on cell morphology after adenoviral
trans-duction was tested in 24 melanoma cell lines. Three of the 24
celllines, WM1552c, WM793, and 1205Lu, which had an
epitheloidmorphology, displayed a more spindle-shaped,
fibroblastoid morphol-ogy starting 3 days after transduction (Fig.
2B). It should be noted thatthe VGP primary melanoma cell line
WM793 and the metastaticmelanoma cell line 1205Lu were derived from
the same patient (31).
Stroma Activation and Decreased Tumor Cell Death throughTGF-�1
in Melanoma. To analyze melanoma-stroma interactionsmediated by
TGF-�1, we used an in vivo model of close proximity of
human melanoma cells with fibroblasts. Melanoma cell line
451Lu(32) was chosen, because the cells showed low constitutive
levels ofTGF-�1, were resistant to TGF-�1 transduction (see Fig.
1), produced23-fold higher levels of TGF-�1 than controls after
transduction (seeFig. 2), and were highly tumorigenic in
immunodeficient mice (33).After transduction with TGF-�1 in vitro,
451Lu melanoma cells weremixed with normal human skin fibroblasts
in ratios of 1:10, 1:1, and10:1 and injected together with Matrigel
matrix s.c. into SCID mice.After 2 weeks, solid tumors were
palpable in all groups with nosignificant difference in tumor
volume from controls (data notshown). Increased protein expression
of TGF-�1 in tumors of TGF-�1-transduced melanoma cells was
confirmed by Western blot anal-ysis (Fig. 3). Histologically, thick
stroma septae were found through-out and around the
TGF-�1-expressing tumors (Fig. 4A), whereasthere was only little
stroma in the control tumors (Fig. 4B). Controltumors were
characterized by a higher proportion of necrosis withblood
extravasation (Fig. 4B), and TUNEL assay demonstrated
asignificantly higher number of apoptotic cells (Fig. 4D)
comparedwith the TGF-�1-expressing tumors (Fig. 4C). Decreased cell
death inmelanoma by TGF-�1 was also observed in organotypic
cultures.Melanoma cell line WM793, which showed low constitutive
levels ofTGF-�1 and growth resistance as well as spindle-shaped
morphologyafter TGF-�1 transduction, was incorporated into human
skin recon-structs after TGF-�1 and LacZ transduction, respectively
(Fig. 4, Eand F). In this organotypic culture model, fibroblasts
embedded intype I collagen form the dermis, and keratinocytes
seeded on top forma stratified epithelium, i.e., epidermis. After 2
weeks, melanoma cellclusters were found in the epidermis and upper
dermis in both groups;however, much less cell death was observed in
the tumors formed byTGF-�1-transduced melanoma cells (Fig. 4E)
compared with thecontrols (Fig. 4F).
Paracrine stroma activation by TGF-�1 expression in
melanomacells was also detected in an orthotopic melanoma model.
Primarymelanoma cell line WM3248, which was stimulated by TGF-�
trans-duction in vitro, was transduced with TGF-�1 or LacZ in vitro
andthen injected intradermally into human foreskins grafted to
SCIDmice. After 2 weeks, solid dermal tumors were palpable in both
theTGF-�1 and LacZ groups without a significant difference in
volumeuntil sacrifice 3–4 weeks after injection. A capsule-like
stromal ma-trix had formed around the TGF-�1-expressing melanoma
with in-creased numbers of fibroblasts (Fig. 4G) when compared with
thecontrols (Fig. 4H). The stromal cells produced high levels of
collagenas demonstrated by Masson’s trichrome stain (not shown),
and stromacapsules directly surrounding tumor cell clusters
contained type IVcollagen, which was not seen in the controls (not
shown). Numerous
Fig. 3. Detection of TGF-�1 precursor forms by Western blot
analysis of 2-week-old451Lu melanomas after transduction of
melanoma cells with TGF-�1 or LacZ in vitro andinjection s.c. into
SCID mice together with human fibroblasts in a ratio 1:1. Equal
amountsof protein were loaded.
Fig. 2. Induction of TGF-�1 production by adenoviral gene
transfer and morphologicaleffect on melanoma. A, TGF-�1 production
in six different melanoma cell lines aftertransduction with TGF-�1
at 2 and 20 pfu, LacZ control vector (20 pfu), or
withouttransduction. Bars, SD. B, morphology of 1205Lu melanoma
cells 3 days after transduc-tion with LacZ control (A) or TGF-�1
(B). Note the TGF-�1-induced, elongated spindleshape of the
cells.
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vessels were detected in this stroma; however, there seemed to
be nosignificant difference in vascularity compared with the LacZ
controls.
Induction of ECM Proteins by TGF-�1 in Melanoma. Thestroma
activation by TGF-�1 produced by melanoma cells was
furthercharacterized in 2-week-old tumors of TGF-�1-transduced
451Lumelanoma cells, which had been mixed with fibroblasts 1:1 and
s.c.injected into SCID mice. Masson’s trichrome stain revealed an
in-crease in collagens (Fig. 5A) when compared with LacZ controls
(Fig.5B). Immunohistochemical analyses demonstrated an induction
offibronectin (Fig. 5C) and �2 integrin (Fig. 5E) expression by
TGF-�1,
whereas they were only weak or undetectable in the controls
(Fig. 5,D and F). Tenascin expression was not stronger in intensity
but morewidely distributed throughout the TGF-�1-expressing tumors
(notshown). Detection of CD13 (aminopeptidase N) as a human
fibroblastmarker revealed high positivity in the interstitium of
the TGF-�1-expressing tumors (Fig. 5G) in contrast with the
controls (Fig. 5H),suggesting the close proximity of human
fibroblasts to the melanomacells.
The described remodeling of stroma was obviously mediated
byparacrine effects of TGF-�1 produced by melanoma cells.
Therefore,
Fig. 4. Stroma activation and decreased tumor celldeath through
TGF-�1 in melanoma. A–D, 451Lumelanoma 19 days after s.c. injection
into SCID mice.Before injection, melanoma cells were transduced
withTGF-�1 (A and C) or LacZ (B and D) and mixedtogether with
normal human fibroblasts in Matrigel ata ratio 1:1. A and B,
increase in stroma around theTGF-�1-transduced (A) versus
LacZ-transduced (B)melanoma cells (H&E, �100). The control
tumors (B)show more necrosis and blood extravasation. C and D,TUNEL
assay for evaluation of apoptosis (red) showsonly few positive
cells in the TGF-�1-transduced mel-anoma cells (C) but high
positivity in the controltumors (D; �100). E and F, histological
section of16-day-old human skin reconstructs with normal hu-man
fibroblasts, keratinocytes, and WM793 primarymelanoma cells
(arrowheads) transduced withTGF-�1 (E) or LacZ control (F). Note
the higherdegree of cell death in the control tumor (F;
H&E,�200). G and H, WM3248 melanoma after transduc-tion with
TGF-�1 (G) or LacZ control (H) and intra-dermal injection into
human skin grafts. Shown is anH&E-stained histological section
of a sample 3 weeksafter injection (�200). Note the
capsule-formingstroma reaction around the tumor WM3248
transducedwith TGF-�1 (G).
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we tested direct expression of TGF-�1 in human skin by
intradermalinjection of the adenoviral vectors into human skin
xenografts, whichresults in a highly efficient gene transduction of
fibroblasts in thedermis.4 As early as 1 week, a strong thickening
of the skin wasvisible and palpable, whereas there was no change in
LacZ-injected
skins. TGF-�1 production in the dermis led to the formation of
astrong collagen fiber network, as determined by H&E (not
shown) andMasson’s trichrome stain (Fig. 5I), which was not seen in
the controls(Fig. 5J).
The TGF-�1/Ad5-treated animals suffered from systemic effects
inthe second week after injection. They became weak and apathic
andeventually died. Autopsy showed normal lung and liver tissue
but4 C. J. Gruss, K. Satyamoorthy, C. Berking, J. Lininger, M.
Nesbit, H. Schaider, Z-J.
Liu, M. Oka, M-Y. Hsu, T. Shirakawa, G. Li, P. Carmeliet, W.
El-Deiry, S. L. Eck, J. S.Rao, A. H. Baker, J. Bennett, T.
Crombleholme, J. Karmacharya, D. J. Margolis, J. M.Wilson, S.
Werner, M. Detmar, M. Skobe, P. D. Robbins, C. Johnson, D. Carbone,
C.Buck, and M. Herlyn. Re-modeling of the human skin architecture
in vivo by adenovirus-
mediated gene transfer of growth factors, adhesion molecules,
proteolytic enzymes,oncogenes and tumor suppressor genes, submitted
for publication.
Fig. 5. Induction of ECM proteins by TGF-�1 inmelanoma. A–H,
451Lu melanoma 19 days after s.c.injection into SCID mice. Before
injection, melanomacells were transduced with TGF-�1 (A, C, E, and
G) orLacZ (B, D, F, and H) and mixed together with normalhuman
fibroblasts in Matrigel at a ratio 1:1. A and B,Masson’s trichrome
stain illustrates an increase incollagen (blue) induced by
TGF-�1-expressing mela-noma cells (A) compared with the controls
(B; �100).C and D, immunohistochemical detection of fibronec-tin
(red) reveals an increase in the stroma septaearound the
TGF-�1-transduced (C) compared with theLacZ-transduced (D) melanoma
cells (�50). E and F,�2 integrin expression in the interstitium of
TGF-�1-transduced melanoma cells (E) is not seen in the
LacZ-transduced controls (F). G and H, immunohistochem-ical
detection of human fibroblasts by CD13(aminopeptidase N; red) in
the TGF-�1-transduced(G) compared with the LacZ-transduced (H)
controlgroup (�100). I and J, human foreskin graft 10 daysafter
intradermal injection of 5 � 108 pfu of TGF-�1/Ad5 (I) or LacZ/Ad5
control vector (J) in 100 �l ofsterile PBS. Masson’s trichrome
stain reveals an in-crease in collagen in blue (I) compared with
the con-trol (J; �100).
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myeloid metaplasia in the spleen and acute tubular necrosis in
thekidney. Lethal effects of the TGF-�1/Ad5 treatment were also
ob-served with 10-fold lower injection doses. The systemic serum
levelsof circulating TGF-�1 were at ng/ml levels, as analyzed by
ELISA.
Increased Metastasis Development by TGF-�1-transducedMelanoma
Cells. For metastasis studies, SCID mice were injecteds.c. with
TGF-�1- or LacZ-transduced 451Lu melanoma cells mixedwith normal
human skin fibroblasts in Matrigel matrix in a ratio of 1:1and
observed for 39–41 days. Tumor volume was not
significantlydifferent between groups during the first 32 days. At
day 39, TGF-
�1-transduced tumors were 1.7-fold larger than LacZ
controls(1.46 � 0.95 cm3) versus 0.8 � 0.55 cm3). S100-positive
microme-tastases in the lungs were found in 8 of 10 mice with
TGF-�1-transduced melanomas (Fig. 6A) and in 7 of 10 mice with
LacZ-transduced control melanomas (Fig. 6B). Metastases in 10
randomlychosen fields at �100 were counted in each lung. The
average numberof micrometastases/microscopic lung field was
significantly(P � 0.03) higher in the TGF-�1 group (4.1 � 4.7)
compared with thecontrols (0.4 � 0.7), and the average size of each
metastasis wasbigger as well. The average diameter of each
metastasis was in the
Fig. 6. Increased metastasis formation by TGF-�1-transduced
melanoma cells. A and B, S100-positivemicrometastases in lungs from
SCID mice 39 daysafter s.c. injection of TGF-�1-transduced (A) or
LacZ-transduced (B) 451Lu melanoma cells mixed in a ratio1:1 with
normal human fibroblasts in Matrigel matrix.Number and size of
metastases were significantly in-creased in the TGF-� group.
Metastases stained posi-tive for TGF-�1 (C), whereas controls
showed no oronly weak stain for TGF-�1 (D; �100). E and F,smooth
muscle actin expression (red) in the lung invessel walls
(arrowheads) and around melanoma me-tastases (arrows) after TGF-�1
(E) and LacZ (F) trans-duction of s.c.-injected 451Lu melanoma
cells (�200).G and H, smooth muscle actin expression
(immuno-histochemical detection in brown) is induced by injec-tion
of adenoviral vectors for TGF-�1 (G) in humanskin grafts but not by
injection of LacZ control vectors(H; �50).
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TGF-�1 group (85 � 44 �m), 1.8-fold larger than in the LacZ
group(47 � 29 �m; Table 1). The metastases were found to
expressTGF-�1 (Fig. 6C), whereas the controls showed no or only
weakstaining for TGF-�1 (Fig. 6D). Smooth muscle actin, which
wasdetected in the vessel walls of the murine lungs, was
commonlyexpressed around the micrometastases in the TGF-�1 group
(Fig. 6E)but only rarely in the control group (Fig. 6F).
Induction of smooth muscle actin expression was also observed
infibroblasts in human skin grafts when injected with adenoviral
vectorsfor TGF-�1 (Fig. 6G). This indicated that TGF-�1 can induce
trans-differentiation of fibroblasts in the dermis into
myofibroblasts, sug-gesting that they are also induced by
TGF-�1-derived from melanomacells.
Gene Expression Profiling of TGF-�1-transduced Fibroblastsand
Melanoma Cells. The mRNA expression of 2280 different genesin
melanoma cells and fibroblasts after transduction with TGF-�1
wasanalyzed by cDNA microarray. Melanoma lines 1205Lu, 451Lu,
andWM793 were tested 3 days after adenoviral infection with 20 pfu
ofTGF-�1/Ad5 or LacZ/Ad5 per cell. At this harvesting time
point,1205Lu and WM793 displayed in 40–50% of all cells a
TGF-�1-induced fibroblastoid phenotype, which was not seen in the
LacZ-transduced or nontransduced controls. Normal human
foreskinfibroblasts were analyzed after adenoviral infection with
40 pfu ofTGF-�1/Ad5 or LacZ/Ad5 per cell and organotypic culture in
type Icollagen for 4 days. Table 2 summarizes the expression
results ofselected genes encoding for ECM proteins, adhesion
receptors,growth factors, their binding proteins and receptors, as
well as pro-tease inhibitors.5
Among the different collagen subtypes, expression of the �1
chainof type XVIII and type XV was increased up to 12-fold in
bothmelanoma cells and fibroblasts. Tenascin and osteonectin
expressionincreased 5- and 6-fold, respectively, in
TGF-�1-transduced fibro-blasts, whereas it was unchanged or 2-fold
reduced in melanoma cells.Integrins �1, �5, �V, �3, and �6 showed a
2–6-fold reduction inTGF-�1-transduced fibroblasts. In at least 2
melanoma cell lines, a
2–4-fold increase in �v and �3 integrin after TGF-�1
transductionwas detected, whereas other subtypes were not changed
in more thanone of the three analyzed lines. VEGF-A increased after
TGF-�1transduction up to 35-fold in all analyzed cell lines and was
thereforethe growth factor that exhibited the strongest induction.
The inductionof VEGF was confirmed at the protein level by ELISA
(Fig. 7).Although WM793 melanoma cells and dermal fibroblasts
werestrongly induced to secrete VEGF by TGF-�1 transduction,
VEGFlevels in 451Lu melanoma cells were already relatively high in
thecontrols (1.53 ng/ml/106 cells) and stayed at similar levels
afterTGF-�1 transduction.
PDGF receptor-� expression was induced by TGF-�1 transduction5-
and 21-fold in two of three analyzed melanoma lines, and CFR-1was
increased up to 6-fold in TGF-�1-transduced melanoma cells
and4-fold in fibroblasts. PA1-I was, with 14- to 43-fold increase
inexpression in all analyzed TGF-�1-transduced cell lines, the
highestinduced protease inhibitor.
DISCUSSION
Interactions of tumor cells with their microenvironment and
theinfluence of stroma on tumor and vice versa have been
increasinglyrecognized to be essential for tumor survival and
progression and havebeen primarily studied in carcinomas of the
breast (33), pancreas (34),prostate (35), skin (36), and cervix
(37). In melanoma, a wide varietyof different cytokines and growth
factors are expressed (38), whichoften act in an autocrine way, but
also may influence the tumorenvironment via paracrine loop (39).
Most studies have hereby fo-cused on the induction of angiogenesis
by VEGF, basic fibroblastgrowth factor, PDGFs, and IL-8 (40). In
this study, it is shown thatstroma can be remodeled by the
paracrine effects of TGF-�1 producedby melanoma cells, which
results in an increased deposition of ECMproteins in the
interstitium of the tumor. The previously describedinduction of
collagen, fibronectin, tenascin, and �2 integrin by TGF-�(41–43)
could be demonstrated in the human fibroblast-containingstroma
surrounding TGF-�-producing melanoma cells in an in vivomodel.
Microarray studies of TGF-�1-transduced fibroblasts in orga-notypic
culture, in which tenascin and types VI, XV, and XVIIIcollagen were
up to 12-fold increased, partly mirrored the in vivo data.However,
other collagen subtypes, �2 integrin, or fibronectin precur-sor
were not increased, which might be because of limitations of thein
vitro culture system, degradation of RNA, missed transient
timepoints of induction, or the fact that fibroblasts were
transduced withTGF-� and not stimulated by exogenous TGF-�.
Concomitant with the stroma reaction, the absence of larger
ne-crotic areas and the fewer number of apoptotic cells in the
TGF-�1-transduced tumors were the most striking differences to the
controls.The fact that this did not result in a greater volume of
the ECM-richmelanomas in the first 4 weeks was most likely
attributable to in-creased edema and blood content in the more
necrotic control tumors,which, however, at later time points were
found to be smaller than theTGF-�1-transduced tumors, possibly as a
consequence of resorptionof the edema and necrotic cells. Decreased
death attributable toTGF-�1 transduction was also seen in human
melanoma skin recon-structs. TGF-� obviously conferred a selective
survival advantage tothe melanoma cells, which culminated in the
significant increase innumber and size of lung metastases in mice
injected with TGF-�1-transduced 451Lu melanoma cells. No growth
induction by TGF-�1was seen in 451Lu melanoma cells in vitro,
indicating that directautocrine effects of TGF-�1 were not
responsible for this phenome-non, but that neighboring stroma cells
in vivo were a prerequisite forthese beneficial effects of TGF-�1
on the tumor. The stroma forma-tion might have provided the
melanoma cells a scaffolding, to which
5 The detailed results of all 2280 genes can be found on our Web
site, http://www.wistar.upenn.edu/herlyn.
Table 1 Lung metastases of TGF-�1-transduced melanoma in SCID
mice
451Lu melanoma cells were injected s.c. into SCID mice after
transduction withTGF-�1 or LacZ and after mixing with normal human
fibroblasts in a ratio 1:1. After39–41 days, lungs were harvested
and analyzed microscopically. Micrometastases werecounted in 10
randomly chosen fields at �100, and the largest diameter of each
metastasiswas measured.
Experimental group Mouse
Metastases
Number(mean � SD)
Diameter (mm)(mean � SD)
TGF-�1 1 13.1 � 2.47 0.11 � 0.182 10.7 � 2.58 0.11 � 0.093 6.5 �
3.89 0.15 � 0.184 4.6 � 3.81 0.12 � 0.025 4.08 � 3.8 0.06 � 0.066
0.73 � 1.22 0.05 � 0.057 0.5 � 0.53 0.01 � 0.018 0.3 � 0.67 0.08 �
0.059 0 0
10 0 0LacZ 1 2.3 � 2.19 0.04 � 0.03
2 0.9 � 0.99 0.05 � 0.063 0.4 � 0.7 0.04 � 0.044 0.23 � 0.44
0.03 � 0.025 0.2 � 0.42 0.05 � 06 0.2 � 0.42 0.11 � 0.137 0.15 �
0.38 0.01 � 08 0 09 0 0
10 0 0
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they can adhere and along which they can migrate. The
stimulation offibroblasts might have induced in turn other growth
factors, whichpositively regulate survival and growth of melanoma.
Microarray andELISA analyses demonstrated VEGF to be strongly
induced byTGF-�1 in both fibroblasts and melanoma cells, confirming
previousin vitro data (44). However, angiogenesis was not
significantly in-creased in tumors from TGF-�1-transduced 451Lu
melanoma cells.This may have been attributable to the already high
constitutiveproduction of VEGF protein in 451Lu cells and the
already highvascularization of 451Lu control tumors. On the other
hand, TGF-�1-induced VEGF in fibroblasts might have contributed to
the survivaladvantage of the TGF-�1-transduced tumors by acting as
an antiapo-ptotic factor both for the fibroblasts themselves and
for the melanomacells via reciprocal paracrine routes.
Microarray analyses further revealed an induction of PDGF
recep-tor-� by TGF-�1 in melanoma cells, which normally do not
expressthis receptor (45). Also an increase in CFR-1 was detected,
indicatingthat melanoma cells might have become increasingly
responsive togrowth factors produced by stromal cells through
up-regulation ofgrowth factor receptors. Increased survival and
motility were addi-tionally observed by an increase in systemic
metastases. This mighthave been either attributable to an altered
expression of adhesionmolecules, proteases, or plasminogen
activators on the melanomacells themselves (13, 16, 21, 22) or to
the altered microenvironmentenhancing the chance of surviving
melanoma cells to migrate alongthe stromal septae and enter the
lymphatic or blood system.
Although TGF-�1 had stimulatory effects in melanoma in vivo,
itdoes not seem to be essential for tumor growth and progression
ingeneral. This is reflected in the heterogeneous pattern of
endogenousTGF-�1 production in melanoma, which included advanced
stages ofmelanoma that showed no or low levels of TGF-�1. It cannot
beexcluded that the ratio between the active and the latent form
ofTGF-�1 differed among the tested cell lines, because only the
total
levels of TGF-�1 were measured in the ELISA used. However,
inselected cases, constitutive TGF-�1 production was associated
withprogression with highest concentrations found in single
metastatic andadvanced primary cell lines (WM373, WM1617, and
WM902B) andhigher concentrations in advanced cell lines when
compared with theirprimary counterparts derived from the same
patient (WM373-WM75,WM278-WM1617, and 1205Lu-WM793). The levels of
TGF-�1 pro-duction in transduced melanoma cells were within the
range of con-stitutive TGF-�1 levels in selected “high producer”
melanoma celllines.
Induced expression of TGF-�1 in melanoma cells had
differenteffects on their phenotype and proliferation capability.
Many mela-noma cell lines were resistant to the growth-inhibitory
effects ofTGF-�1, which was in line with previously reported
effects of exog-
Fig. 7. Production of VEGF protein by human melanoma cells
(WM793 and 451Lu)and dermal fibroblasts (FF2441) 72 h after
transduction with TGF-�1 (f) or LacZ control(o) via adenoviral
vectors. Results are expressed in average ng/ml per 106 cells;
bars, SD.
Table 2 Modulation of gene expression by TGF-�1 in fibroblasts
and melanoma cells
Fibroblasts and melanoma cell lines were transduced with either
TGF-�1 or LacZ using adenoviral vectors. Fibroblasts were then
embedded in type 1 collagen gels for 72 h, whereasmelanoma cells
were maintained in monolayers. Microarray analysis of 2280
different human clones was performed. Genes coding for matrix
proteins, adhesion receptors, growthfactors, their binding proteins
and receptors, as well as protease inhibitors are shown. Detailed
results can be found at Internet address
http:/www.wistar.upenn.edu/herlyn.
Gene group
Fibroblasts (F) and melanoma cells (M)a
F1b M1 F2 M2
Matrix proteins Collagen XV �1c Collagen XVIII �1c Collagen III
�1 Thrombospondin 2Collagen XVIII �1d Collagen XV �1d Collagen IV
�2 OsteonectinOsteonectind Collagen IV �2 Collagen IX �3Tenascind
Syndecan1Collagen VI �3 VitronectinSyndecan 4 Thrombospondin 1
Thrombospondin 2Thrombospondin 4
Integrins �V �1d
�3 �5�V�3�6
Growth factors VEGF-Ac VEGF-Ac NGFe-�c
TGF-�1d TGF-�1d IGF-1BMP-6 BMP-6d IGF-2VEGF-B IGF-2 PDGF-B
BMP-7Growth factor receptors TGF-�RII PDGFR-�c IGF-R1
CFR-1 IGF-R2d PDGFR-�TGF-�RIICFR-1
Protease inhibitors PAI-Ic PAI-Ic TIMP-IPAI-II PAI-IId
TIMP-3 TIMP-3a Melanoma cell lines WM793, 1205Lu, and 451Lu were
used. Results indicated were found in at least two of the three
cell lines.b Results are expressed as at least 2-fold higher (1) or
lower (2) than LacZ controls.c Expression was more than 10-fold
higher or lower than in LacZ controls.d Expression was 5–10-fold
higher or lower than in LacZ controls.e NGF, nerve growth factor;
BMP, bone morphogenetic protein; IGF, insulin-like growth
factor.
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enous TGF-� on melanoma cells (8, 14). For a more detailed
analysisof resistance of melanoma cells to TGF-�1, both the active
and thelatent form of TGF-�1 need to be determined separately
before andafter TGF-�1 transduction of each cell line and different
infectiondoses need to be tested to be able to determine the
threshold concen-tration of sensitivity, which was, however, not
the aim of this study.Resistance to TGF-� has been linked in
several tumors to mutationsin genes involved in the TGF-� signaling
pathway, such as TGF-�receptors I and II, Smad2, or Smad4 (6);
however, none of thesemutations could be demonstrated in melanoma
(15).
Three of 24 cell lines displayed a morphological
transdifferentia-tion toward a spindle cell-like fibroblastoid
phenotype. This has beenreported previously after addition of
exogenous TGF-� and wasassociated with increased metastatic
capacity (16). Metastatic capacityof the transdifferentiated cell
lines was not tested in this study;however, in skin
reconstructions, increased tumor cell survival wasfound.
Transdifferentiation was also detected in fibroblasts, whenTGF-�1
was overexpressed in human skins. The widespread detectionof smooth
muscle actin in the dermis indicated a differentiation of theskin
fibroblasts into myofibroblasts, which has been described only
invitro (46) and not yet in human skin in vivo. The
myofibroblastphenotype has been discussed to be the main source of
increased ECMdeposition in fibrosis of the kidney, liver, and lung
(47), and the dataof this study suggest that the same holds true
for fibrosis of the skin.A strong collagen fiber network was
induced by TGF-�1, which wasstronger than for any other growth
factor studied.6
In summary, we have shown that TGF-�1 expression in
humanmelanoma cells can lead to stimulation of the neighboring
stroma cellswith increased production and deposition of ECM
proteins. Theactivation of stroma in turn leads to a survival
advantage and in-creased metastasis formation of the melanoma
cells. A TGF-�1-triggered complex interplay between matrix
proteins, proteases, inte-grins, and growth factors is suggested by
global gene expressionstudies and excludes attempts to limit the
characterization of melano-ma-stroma interactions to just single
genes or a single gene group.
ACKNOWLEDGMENTS
We thank Sylvia Major, Katerina Chruma, Adrien Jarvis, and Dr.
Ling Lifor technical assistance with the in vitro cell and
immunohistochemistrystudies; Dr. Jonathan Garlick for invaluable
support for the skin reconstructionmodel; Emma DeJesus and Rena
Finko for technical assistance with cellcultures and skin
reconstruction; Elsa Aglow for excellent histological proc-essing
of the samples; Dr. Dirk Ruiter for helpful discussions about
thehistopathological sections; and Wen Hwai Horng for
biostatistical analysis ofthe microarray data.
REFERENCES
1. Roberts, A. B., McCune, B. K., and Sporn, M. B. TGF-�:
regulation of extracellularmatrix. Kid. Int., 41: 557–559,
1992.
2. Massagué, J. TGF-� signal transduction. Annu. Rev. Biochem.,
67: 753–791, 1998.3. Roberts, A. B., Sporn, M. B., Assoian, R. K.,
Smith, J. M., Roche, N. S., Wakefield,
L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H.,
et al. Transforming growthfactor type �: rapid induction of
fibrosis and angiogenesis in vivo and stimulation ofcollagen
formation in vitro. Proc. Natl. Acad. Sci. USA, 83: 4167–4171,
1986.
4. Verrechia, F., Chu, M-L., and Mauviel, A. Identification of
novel TGF-�/Smad genetargets in dermal fibroblasts using a combined
cDNA microarray/promoter transac-tivation approach. J. Biol. Chem.,
276: 17058–17062, 2001.
5. Lund, L. R., Riccio, A., Andreasen, P. A., Nielsen, L. S.,
Kristensen, P., Laiho, M.,Saksela, O., Blasi, F., and Dano, K.
Transforming growth factor-� is a strong and fastacting positive
regulator of the level of type-1 plasminogen activator inhibitor
mRNAin WI-38 human lung fibroblasts. EMBO J., 6: 1281–1286,
1987.
6. Reiss, M. Transforming growth factor-� and cancer: a
love-hate relationship? Oncol.Res., 9: 447–457, 1997.
7. Gold, L. I. The role of transforming growth factor-� (TGF-�)
in human cancer. Crit.Rev. Oncog., 10: 303–360, 1999.
8. Krasagakis, K., Kruger-Krasagakes, S., Fimmel, S., Eberle,
J., Tholke, D., von derOhe, M., Mansmann, U., and Orfanos, C. E.
Desensitization of melanoma cells toautocrine TGF-� isoforms. J.
Cell. Physiol., 178: 179–187, 1999.
9. Reed, J. A., McNutt, N. S., Prieto, V. G., and Albino, A. P.
Expression of transform-ing growth factor-�2 in malignant melanoma
correlates with the depth of tumorinvasion. Implications for tumor
progression. Am. J. Pathol., 145: 97–104, 1994.
10. Van Belle, P., Rodeck, U., Nuamah, I., Halpern, A. C., and
Elder, D. E. Melanoma-associated expression of transforming growth
factor-� isoforms. Am. J. Pathol., 148:1887–1894, 1996.
11. Moretti, S., Pinzi, C., Berti, E., Spallanzani, A.,
Chiarugi, A., Boddi, V., Reali, U. M.,and Giannotti, B. In situ
expression of transforming growth factor � is associated
withmelanoma progression and correlates with Ki67, HLA-DR and �3
integrin expres-sion. Melanoma Res., 7: 313–321, 1997.
12. Schmid, P., Itin, P., and Rufli, T. In situ analysis of
transforming growth factor-�s(TGF-�1, TGF-�2, TGF-�3), and TGF-�
type II receptor expression in malignantmelanoma. Carcinogenesis
(Lond.), 16: 1499–1503, 1995.
13. Teti, A., De Giorgi, A., Spinella, M. T., Migliaccio, S.,
Canipari, R., Onetti Muda, A.,and Faraggiana, T. Transforming
growth factor-� enhances adhesion of melanomacells to the
endothelium in vitro. Int. J. Cancer, 72: 1013–1020, 1997.
14. Rodeck, U., Bossler, A., Graeven, U., Fox, F. E., Nowell, P.
C., Knabbe, C., and Kari,C. Transforming growth factor � production
and responsiveness in normal humanmelanocytes and melanoma cells.
Cancer Res., 54: 575–581, 1994.
15. Rodeck, U., Nishiyama, T., and Mauviel, A. Independent
regulation of growth andSMAD-mediated transcription by transforming
growth factor � in human melanomacells. Cancer Res., 59: 547–550,
1999.
16. Janji, B., Melchior, C., Gouon, V., Vallar, L., and Kieffer,
N. Autocrine TGF-�-regulated expression of adhesion receptors and
integrin-linked kinase in HT-144melanoma cells correlates with
their metastatic phenotype. Int. J. Cancer, 83: 255–262, 1999.
17. Letterio, J. J., and Roberts, A. B. Regulation of immune
responses by TGF-�. Annu.Rev. Immunol., 16: 137–161, 1998.
18. Conrad, C. T., Ernst, N. R., Dummer, W., Brocker, E. B., and
Becker, J. C.Differential expression of transforming growth factor
�1 and interleukin 10 inprogressing and regressing areas of primary
melanoma. J. Exp. Clin. Cancer Res., 18:225–232, 1999.
19. Iruela-Arispe, M. L., and Sage, E. H. Endothelial cells
exhibiting angiogenesis invitro proliferate in response to TGF-�1.
J. Cell. Biochem., 52: 414–430, 1993.
20. Festuccia, C., Angelucci, A., Gravina, G. L., Villanova, I.,
Teti, A., Albini, A.,Bologna, M., and Abini, A. Osteoblast-derived
TGF-�1 modulates matrix degradingprotease expression and activity
in prostate cancer cells. Int. J. Cancer, 85: 407–415,2000.
21. Santibánez, J. F., Frontelo, P., Iglesias, M., Martı́nez,
J., and Quintanilla, M. Uroki-nase expression and binding activity
associated with transforming growth factor�1-induced migratory and
invasive phenotype of mouse epidermal keratinocytes.J. Cell.
Biochem., 74: 61–73, 1999.
22. Farina, A. R., Coppa, A., Tiberio, A., Tacconelli, A.,
Turco, A., Colletta, G., Gulino,A., and Mackay, A. R. Transforming
growth factor-�1 enhances the invasiveness ofhuman MDA-MB-231
breast cancer cells by up-regulating urokinase activity. Int.
J.Cancer, 75: 721–730, 1998.
23. Herlyn, M., Thurin, J., Balaban, G., Bennicelli, J. L.,
Herlyn, D., Elder, D. E., Bondi,E., Guerry, D., Nowell, P., Clark,
W. H., et al. Characteristics of cultured humanmelanocytes isolated
from different stages of tumor progression. Cancer Res.,
45:5670–5676, 1985.
24. Hsu, M-Y., Elder, D. E., and Herlyn, M. The Wistar melanoma
(WM) cell lines. In:J. R. W. Masters and B. Palsson (eds.), Human
Cell Culture, Vol. 3, Solid Cancers,pp. 259–274. Norwell, MA:
Kluwer Academic Publishers, 1999.
25. Lee, W. C., Zhong, C., Qian, S., Wan, Y., Gauldie, J., Mi,
Z., Robbins, P. D.,Thomson, A. W., and Lu, L. Phenotype, function,
and in vivo migration and survivalof allogeneic dendritic cell
progenitors genetically engineered to express
TGF-�.Transplantation, 66: 1810–1817, 1998.
26. Berking, C., and Herlyn, M. Experimental induction of
atypical melanocytic lesionsand melanoma in ultraviolet-irradiated
human skin grafted to immunodeficient mice.In: B. Nickoloff (ed.),
Melanoma Techniques and Protocols. Molecular Diagnosis,Treatment,
and Monitoring, Methods in Molecular Medicine, pp. 71–84. Totowa,
NJ:Humana Press, 2001.
27. Herlyn, M., Graeven, U., Speicher, D., Sela, B. A.,
Bennicelli, J. L., Kath, R., andGuerry, D. Characterization of
tenascin secreted by human melanoma cells. CancerRes., 51:
4853–4858, 1991.
28. Hsu, M. Y., Shih, D. T., Meier, F. E., Van Belle, P., Hsu,
J. Y., Elder, D. E., Buck,C. A., and Herlyn, M. Adenoviral gene
transfer of �3 integrin subunit inducesconversion from radial to
vertical growth phase in primary human melanoma. Am. J.Pathol.,
153: 1435–1442, 1998.
29. Menrad, A., Speicher, D., Wacker, J., and Herlyn. M.
Biochemical and functionalcharacterization of aminopeptidase N
expressed by human melanoma cells. CancerRes., 53: 1450–1455,
1993.
30. Meier, F., Nesbit, M., Hsu, M. Y., Martin, B., Van Belle,
P., Elder, D. E.,Schaumburg-Lever, G., Garbe, C., Walz, T. M.,
Donatien, P., Crombleholme, T. M.,and Herlyn, M. Human melanoma
progression in skin reconstructs: biological sig-nificance of bFGF.
Am. J. Pathol., 156: 193–200, 2000.
31. Juhasz, I., Albelda, S. M., Elder, D. E., Murphy, G. F.,
Adachi, K., Herlyn, D.,Valyi-Nagy, I. T., and Herlyn, M. Growth and
invasion of human melanomas inhuman skin grafted to immunodeficient
mice. Am. J. Pathol., 143: 528–537, 1993.6 Unpublished data.
8315
TGF-�1 IN MELANOMA
on June 7, 2021. © 2001 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/
-
32. Herlyn, D., Iliopoulos, D., Jensen, P. J., Parmiter, A.,
Baird, J., Hotta, H., Adachi, K.,Ross, A. H., Jambrosic, J.,
Koprowski, H., et al. In vitro properties of humanmelanoma cells
metastatic in nude mice. Cancer Res., 50: 2296–2302, 1990.
33. Shekhar, M. P., Werdell, J., Santner, S. J., Pauley, R. J.,
and Tait, L. Breast stromaplays a dominant regulatory role in
breast epithelial growth and differentiation:implications for tumor
development and progression. Cancer Res., 61: 1320–1326,2001.
34. Lohr, M., Schmidt, C., Ringel, J., Kluth, M., Muller, P.,
Nizze, H., and Jesnowski, R.Transforming growth factor-�1 induces
desmoplasia in an experimental model ofhuman pancreatic carcinoma.
Cancer Res., 61: 550–555, 2001.
35. Harding, M. A., and Theodorescu, D. Prostate tumor
progression and prognosis.Interplay of tumor and host factors.
Urol. Oncol., 5: 258–264, 2000.
36. Lengyel, E., Gum, R., Juarez, J., Clayman, G., Seiki, M.,
Sato, H., and Boyd, D.Induction of Mr 92,000 type IV collagenase
expression in a squamous cell carcinomacell line by fibroblasts.
Cancer Res., 55: 963–967, 1995.
37. Turner, M. A., Darragh, T., and Palefsky, J. M.
Epithelial-stromal interactionsmodulating penetration of Matrigel
membranes by HPV 16-immortalized keratino-cytes. J. Investig.
Dermatol., 109: 619–625, 1997.
38. Moretti, S., Pinzi, C., Spallanzani, A., Berti, E.,
Chiarugi, A., Mazzoli, S., Fabiani,M., Vallecchi, C., and Herlyn,
M. Immunohistochemical evidence of cytokine net-works during
progression of human melanocytic lesions. Int. J. Cancer, 84:
160–168,1999.
39. Lazar-Molnar, E., Hegyesi, H., Toth, S., and Falus, A.
Autocrine and paracrineregulation by cytokines and growth factors
in melanoma. Cytokine, 12: 547–554,2000.
40. Rofstad, E. K., and Halsor, E. F. Vascular endothelial
growth factor, interleukin 8,platelet-derived endothelial cell
growth factor, and basic fibroblast growth factor
promote angiogenesis and metastasis in human melanoma
xenografts. Cancer Res.,60: 4932–4938, 2000.
41. Varga, J., Rosenbloom, J., and Jimenez, S. A. Transforming
growth factor � (TGF �)causes a persistent increase in steady-state
amounts of type I and type III collagen andfibronectin mRNAs in
normal human dermal fibroblasts. Biochem. J., 247:
597–604,1987.
42. Ignotz, R. A., Endo, T., and Massagué, J. Regulation of
fibronectin and type Icollagen mRNA levels by transforming growth
factor-�. J. Biol. Chem., 262: 6443–6446, 1987.
43. Raghow, R., Postlethwaite, A. E., Keski-Oja, J., Moses, H.
L., and Kang, A. H.Transforming growth factor-� increases steady
state levels of type I procollagen andfibronectin messenger RNAs
posttranscriptionally in cultured human dermal fibro-blasts. J.
Clin. Investig., 79: 1285–1288, 1987.
44. Dolecki, G. J., and Connolly, D. T. Effects of a variety of
cytokines and inducingagents on vascular permeability factor mRNA
levels in U937 cells. Biochem. Bio-phys. Res. Commun., 180:
572–578, 1991.
45. Barnhill, R. L., Xiao, M., Graves, D., and Antoniades, H. N.
Expression of platelet-derived growth factor (PDGF)-A, PDGF-B and
the PDGF-� receptor, but not thePDGF-� receptor, in human malignant
melanoma in vivo. Br. J. Dermatol., 135:898–904, 1996.
46. Vaughan, M. B., Howard, E. W., and Tomasek, J. J.
Transforming growth factor-�1promotes the morphological and
functional differentiation of the myofibroblast. Exp.Cell Res.,
257: 180–189, 2000.
47. Fan, J-M., Ng, Y-Y., Hill, P. A., Nikolic-Paterson, D. J.,
Mu, W., Atkins, R., and Lan,H. Y. Transforming growth factor-�
regulates tubular epithelial-myofibroblast trans-differentiation in
vitro. Kidney Int., 56: 1455–1467, 1999.
8316
TGF-�1 IN MELANOMA
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Research. cancerres.aacrjournals.org Downloaded from
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-
2001;61:8306-8316. Cancer Res Carola Berking, Richelle Takemoto,
Helmut Schaider, et al. Melanoma through Stroma Remodeling
1 Increases Survival of HumanβTransforming Growth Factor-
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