Institute of Pathology, University of Regensburg, D-93042 Regensburg, Germany
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ABSTRACT |
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Transforming growth factor-
(TGF-
) is known to induce
-smooth muscle actin (
-SMA) in
fibroblasts and is supposed to play a role in myofibroblast
differentiation and tumor desmoplasia. Our objective was to elucidate
the impact of TGF-
1 on
-SMA expression in fibroblasts in a
three-dimensional (3-D) vs. two-dimensional (2-D) environment. In
monolayer culture, all fibroblast cultures responded in a similar
fashion to TGF-
1 with regard to
-SMA expression. In fibroblast
spheroids,
-SMA expression was reduced and induction by TGF-
1 was
highly variable. This difference correlated with a differential
regulation in the TGF-
receptor (TGF
R) expression, in particular
with a reduction in TGF-
RII in part of the fibroblast types. Our
data indicate that 1) sensitivity to TGF-
1-induced
-SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, such
as a general reduction/loss in TGF-
RIII, contribute to an altered TGF
R expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGF
R types
I and II determine the sensitivity to TGF-
1-induced
-SMA
expression in the 3-D setting. We suggest that fibroblasts that can be
induced by TGF-
1 to produce
-SMA in spheroid culture reflect a
"premyofibroblastic" phenotype.
normal fibroblasts; tumor-derived fibroblasts; multicellular
spheroid; transforming growth factor-1; transforming growth
factor-
receptor;
-smooth muscle actin, ED-A fibronectin
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INTRODUCTION |
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THE RELATIONSHIP BETWEEN tumor cells and their heterologous peritumoral stroma has been of great interest to pathologists since the introduction of microscopic tissue imaging. However, in spite of the diagnostically relevant phenomenon of tumor-associated desmoplasia characterized by enhanced fibroblast proliferation/accumulation and a modified, collagen-rich extracellular matrix (ECM), fibroblasts were long presumed to be passive structural elements and were mainly investigated as a substrate of tumor cell invasion. Studies over the past 10-15 years have demonstrated that not only inflammatory and endothelial cells but also stromal fibroblasts may critically affect malignant growth and progression (for review, see Refs. 13, 14, and 37).
Recently, we established a spheroid coculture model of diverse breast
tumor cell lines and fibroblast types to investigate the multiple
regulatory feedback mechanisms between stromal fibroblasts and breast
tumor cells in a well-defined three-dimensional (3-D) environment in
vitro (12). We examined myofibroblast differentiation and,
in particular, -smooth muscle actin (
-SMA) expression as a
representative potential functional "anomaly" of tumor-associated fibroblasts. Fibroblasts in spheroid culture were in general cell cycle
arrested and immunonegative for
-SMA independent of their origin and
also independent of their
-SMA expression profile in monolayer
culture. We could show that some noninvasive tumor cell types such as
T47D induced
-SMA expression in tumor-derived fibroblasts in the
mesenchymal-epithelial contact zone. Others, e.g., SK-BR-3, induced
-SMA expression in the entire fibroblast population, which may be
due to a diffuse infiltration of tumor cells in these cocultures. BT474
cells reflected a third group of tumor cells that was not capable of
inducing
-SMA expression in any of the fibroblast cocultures investigated.
Interestingly, -SMA expression in stromal fibroblasts was not only
dependent on the interacting tumor cell type. Indeed, some fibroblasts
types outgrown from breast tumor biopsy specimens showed an
-SMA-positive immunohistological staining after tumor cell contact,
whereas normal skin fibroblasts were not induced by the same tumor cell
types in 3-D culture (12). From these in vitro data, we
concluded that some fibroblasts isolated from the reactive environment
of breast lesions may exhibit a "premyofibroblastic" differentiation status that is conserved in vitro and accompanied by a
higher sensitivity to
-SMA-inducing factors.
Transforming growth factor- (TGF-
) has been described as one of
the most potent paracrine inducers of myofibroblast differentiation in
vitro and in vivo (5, 27, 33, 36), and not only PDGF but
also TGF-
1 was identified as playing a role in the establishment of
tumor desmoplasia (19, 32). Therefore, the particular aims of the present study were 1) to evaluate whether TGF-
1
differentially induces
-SMA expression in fibroblasts of different
origin according to their behavior in 3-D tumor-fibroblast coculture,
2) to verify whether the 3-D environment affects the
-SMA
inducibility, and 3) to gain deeper insight into the
regulatory mechanism associated with a potentially different behavior
of fibroblasts in 2-D vs. 3-D culture. The experimental design involved
immunohistochemical and Western blot analyses of three fibroblast
types. The effect of TGF-
1 on
-SMA expression in monolayer and
spheroid culture was documented, and the expression pattern of TGF-
receptor (TGF
R) types I, II, and III was examined. In addition,
effects of TGF-
1 on monolayer cell growth were documented, and the
expression of the ED-A fibronectin (ED-A FN) splice variant was
evaluated by immunohistochemistry in monolayer and spheroid cultures
because ED-A FN was recently shown to be involved in the
TGF-
-induced myofibroblast differentiation process (8, 30,
31, 35).
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MATERIALS AND METHODS |
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Fibroblast Types and Routine Cell Culture
PF1, PF28, and PF27 fibroblasts derived from fresh, resected specimens of invasive ductal breast carcinomas as detailed earlier (12). In brief, nontumor tissue was removed after frozen section diagnosis and tumor material was sliced (1-4 mm3) under sterile conditions following extensive washing. Tumor fragments were transferred into culture flasks and covered after attachment with DMEM containing 20% FCS, 25 mM glucose, 1% sodium pyruvate, 1% L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Pan Biotech, Aidenbach, Germany). After sufficient outgrowth, fragments were removed and cells were passaged by using 0.05% trypsin and 0.02% EDTA in PBS (Pan Biotech) and transferred into DMEM with reduced glucose and serum content of 5 mM and 10%, respectively. Morphologically and immunohistochemically characterized stock cultures (12) were frozen with a cumulative population doubling (CPD)All cultures were kept in a humidified atmosphere with 5% CO2 in air at 37°C. Cell counts and cell volumes were routinely recorded with a Casy1 cell analyzer system (Schaerfe, Reutlingen, Germany) for culture quality assessment and to analyze cell growth kinetics as described earlier (16). Experiments were carried out with fibroblasts with a CPD of >30 and <80 to avoid cell senescence phenomena and to guarantee a relatively stable proportion of myofibroblasts in the untreated monolayer culture of about 10% in culture medium containing 10% FCS (12).
Spheroid Culturing
Multicellular spheroids (MCS) were cultured by using the liquid overlay technique (3), agarose-coated 96-well plates (100 µl of 1.5% agarose in serum-free DMEM per well; Sigma-Aldrich), and dissociated subconfluent monolayer fibroblasts. Fibroblast MCS were initiated by inoculating 4 × 103 N1 and 3 × 103 PF1 and PF28 fibroblasts, respectively, per well to reach a defined size of 300-350 µm (MCS volume: ~2 × 107µm3) after 3 days. MCS culturing was performed in supplemented DMEM containing 10% FCS under standard culture conditions. Mean spheroid sizes were routinely recorded by measuring two orthogonal diameters of 12-24 individual MCS quantified in an inverted microscope equipped with a calibrated reticule. Medium was renewed at day 3 and every 48 h thereafter.Experimental Design and TGF-1 Treatment Modalities of
Monolayer Cultures
Experimental Design and TGF-1 Treatment Modalities of Spheroid
Cultures
Immunohistochemistry
To determine theWestern Blotting
Monolayer cultures and pellets of MCS were washed with PBS and lysed under addition of 24 mM Tris · HCl (pH 7.6), 1 mM EDTA, 1 mM PMSF, 1% DTT, and 1% SDS. Cell lysates were transferred into Eppendorf cups for a 30-min incubation on ice. Protein concentrations were determined via the BCA protein assay reagent kit (Pierce), adjusted by adding appropriate amounts of lysis buffer, subsequently mixed with 5× loading buffer (50 mM Tris · HCl, pH 6.8, 2% SDS, 0.1% bromphenol blue, 10% glycerin, and 5%Proteins were separated by SDS-PAGE [10% polyacrylamide
(PAA):bis-acrylamide (bis-AA) 38:1] with 5 mM Tris, 38.4 mM glycin, and 0.02% SDS as running buffer in a MiniproteanIII-electrophoresis system (Bio-Rad, Munich, Germany) at 120 V for 90 min. A routine semidry blotting technique (transfer buffer: 25 mM Tris, 150 mM glycin,
10% methanol; 2 h, 4-5 mA/cm2) was used to
transfer protein to PVDF membrane (Boehringer). Membranes were blocked
with 5% milkpowder in AP/T-buffer (0.1 M
Tris · HCl, pH 7,4, 0.1 M NaCl, 2.5 mM
MgCl2, and 0.05% Tween-20). Proteins were detected by
indirect labeling using the monoclonal mouse anti-human -SMA
antibody (1:600, final concentration: 0.08 µg/ml; Boehringer),
polyclonal rabbit antibodies against TGF
R types I and II (clone
V-22, 1:50-1:100, final concentration: 2-4 µg/ml, and clone
L-21, 1:100-1:200, final concentration: 1-2 µg/ml), or a
polyclonal goat anti-TGF
R type III (clone C-20, 1:200, final concentration: 1 µg/ml; all from Santa Cruz Biotechnology, Santa Cruz, CA). Incubation was carried out for 1 h at room temperature except for the goat IgG that required incubation overnight at 4°C.
After subsequent washing, horseradish peroxidase (HRP)-conjugated secondary anti-mouse, anti-rabbit, and anti-goat IgG
(1:500-1:1,000; all from Dako Diagnostika) were applied for 1 h at 22°C. Peroxidase activity was recorded on a Hyperfilm-ECL
(Amersham, Buckinghamshire, UK) using the Nowa-Western blotting
detection kit (EnerGene, Regensburg, Germany). If possible, membranes
were stripped in 0.1% glycin (pH 2.5) and stained for a different
antibody. This allowed for a parallel detection of all antigens on one
membrane if
-actin or total actin was not stained as an internal
protein control. Membranes were routinely stained with Coomassie blue.
All experiments were performed at least three times. In some control
experiments, actin (
-actin and/or total actin-nonsmooth muscular)
was determined as a protein control using monoclonal rabbit-anti-human
or mouse-anti-human
-actin antibodies (Sigma-Aldrich, Deisenhofen,
Germany). In some cases, relative signal intensities were analyzed by
densitometry. Unspecified chemicals and antibodies were obtained from
Sigma-Aldrich or Merck (Darmstadt, Germany).
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RESULTS |
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-SMA Expression in Tumor-Associated Fibroblasts in Situ and in
3-D Coculture
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-SMA expression in tumor-associated fibroblasts was highly variable
not only for different ductal invasive breast carcinomas but also
within the individual tumor, reflecting its histomorphological heterogeneity (Fig. 1A). Thus application of different tumor
cell lines in the coculture system may, to some extent, reflect the situation within one tumor. However, it remains unsolved why some fibroblast types, e.g., most tumor-derived fibroblast types, seem to be
more sensitive to tumor-induced
-SMA expression in the coculture
model than others, such as normal skin fibroblasts. Therefore,
experiments to verify the impact of TGF-
1 on
-SMA expression were
performed with two representative fibroblast types: N1 normal skin
fibroblasts that were immunonegative for
-SMA in tumor-fibroblast
cocultures and breast tumor-derived PF1 fibroblasts that clearly
expressed
-SMA following contact with tumor cells in vitro (Fig.
1B) (12). PF28 cells were included as a second breast cancer-derived fibroblast type.
TGF-1 Induced
-SMA Expression in 2-D Fibroblast
Cultures
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The observation of an induction of -SMA expression in fibroblast by
10 ng/ml TGF-
1 was confirmed for confluent monolayers in three
independent Western blot analyses (Fig.
3A). The induction ranged
between two- and threefold (n = 3 per fibroblast type) as verified by densitometric analysis but did not reflect the proportion of
-SMA-positive fibroblasts documented in Fig. 2. This
phenomenon will be discussed below (see DISCUSSION,
Technical Considerations).
TGF-1 and Fibroblast Monolayer Growth
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In parallel, cell volume was determined throughout growth. No
systematic and reproducible alteration in the cell volume of normal
skin N1 and tumor-derived PF1 or PF28 fibroblasts following TGF-1
treatment was observed. Average cell volumes ranged between 5,500-7,500 µm3 for N1 and 8,000-12,000
µm3 for PF1 and PF28 fibroblasts, respectively.
-SMA and TGF
R Expression in 2-D and 3-D Fibroblast Cultures
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Comparison of the receptor expression of fibroblasts in monolayer and
spheroid cultures, taking into account at least three Western blot
analyses for each TGFR type, demonstrated the following.
TGFR type I.
The TGF
RI level in N1 and PF1 fibroblasts in spheroid cultures is
potentially higher than in the corresponding monolayers, which does not
correlate with the respective discrepancy in the
-SMA inducibility.
However, in 3-D culture, TGF
RI is highest in PF1 fibroblasts that
are characterized by the most prominent
-SMA induction by TGF-
1;
PF28 and N1 fibroblasts did not reproducibly differ in their TGF
RI
content in spheroids. TGF-
1 treatment of fibroblast spheroids is
(frequently but not always) accompanied by an increase in the TGF
RI level.
TGFR type II.
This serine-threonine kinase receptor is reduced in N1 normal skin
fibroblasts when grown in spheroid culture. This reduction is moderate
but reproducible and in contrast to PF1 and PF28 tumor-derived fibroblasts with constant TGF
RII levels. As a result, TGF
RII expression in spheroids is lower in N1 than in PF1 or PF28 fibroblasts.
TGFR type III.
TGF
RIII is downregulated in 3-D cultures of all fibroblast types and
is undetectable in the Western blot analyses of spheroid cultures with
and without TGF-
1 treatment. Representative Western blots
demonstrating expression of
-SMA and TGF-
receptors in monolayer
vs. TGF-
1-treated and untreated spheroid cultures are shown in Fig.
5.
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Fibronectin/ED-A Fibronectin Distribution in 2-D and 3-D Fibroblast Cultures
All fibroblasts produce a dense ECM in monolayer and spheroid culture. As documented earlier, fibronectin is one of the major ECM compounds in spheroids of all fibroblast types, including normal skin fibroblasts N1 (12). Immunohistochemical staining for the oncofetal ED-A FN variant showed that monolayer fibroblasts of normal skin and breast tumor origin in general express and secrete ED-A FN. There was no difference between the various types of fibroblasts based on semiquantitative immunohistochemical evaluation (data not shown). However, if fibroblast spheroids highly positive for fibronectin were stained with the ED-A FN-specific antibody, a striking difference between N1 and tumor-derived PF1 and PF28 fibroblasts was observed. N1 spheroids showed only poor staining, whereas spheroids of tumor-derived PF1 and PF28 stained strongly (Fig. 7A). The fibroblast type-specific difference in the ED-A FN distribution/expression was preserved in spheroid cocultures of fibroblasts of different origin and breast tumor cell lines, as documented in Fig. 7B. No induction of ED-A FN in fibroblasts following tumor cell contact was observed.
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DISCUSSION |
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Myofibroblastic Phenotype in Vivo and in Vitro
Myofibroblasts were first described by G. Majno and G. Gabbiani and were mainly investigated in chronic inflammatory diseases and during wound healing (for review, see Refs. 20, 25, 29, and 31). The expression ofTGF-1 Induced
-SMA Expression and Myofibroblastic
Phenotype
Our data show that TGF-1-induced
-SMA expression in fibroblasts
in vitro is critically affected by the culture conditions and, in
particular, by a tissue-like 3-D environment. In monolayer culture,
-SMA expression is induced in all fibroblast types independent of
their origin. This behavior correlates with a comparable expression pattern of the TGF-
receptors and the ED-A FN variant in the different fibroblast types. In contrast, cultivation of fibroblast spheroids is accompanied by a considerable reduction in the
-SMA expression in all fibroblast types and in a reduced sensitivity to
TGF-
1, in particular in normal skin N1 fibroblasts but also in one
of the two tumor-derived fibroblast types (PF28) studied in detail.
-SMA, TGF
R Types I and II, and ED-A FN in Normal
Skin Fibroblasts
-SMA, TGF
R Types I and II, and ED-A FN in Tumor-Derived
Fibroblasts
The tumor-derived fibroblast type PF27 behaved analogous to normal skin
fibroblasts, with loss of sensitivity correlating with a reduction in
TGFRII. These results indicate that regulation of
-SMA in normal
skin- and breast tumor-derived fibroblasts does not necessarily differ.
Also, it is in accordance with the observation that only fibroblast
subpopulations, but not all stromal fibroblasts in breast tumors, may
show myofibroblast differentiation (Fig. 1) and supports the hypothesis
of a premyofibroblastic phenotype.
In a model of myofibroblastic differentiation presented by Serini and
Gabbiani (31), TGF- is released as a paracrine inducer from platelets and macrophages as a consequence of activation through
granulocyte macrophage colony-stimulating factor
(38). Our previous observations with spheroid
cocultures of tumor cells and fibroblasts showed that tumor-associated
induction of
-SMA expression in fibroblasts does not require immune
cell commitment (12). Data presented further indicate that
TGF-
and/or the activation of the TGF-
signal transduction
pathway via TGF
R types I and II play a potential role in this process.
TGF-1-Induced
-SMA Expression and the TGF
R Type III
Technical Considerations and Future Directions
Growth curves andWe have avoided cell senescence, which accompanies long-term cell
quiescence in fibroblast monolayer cultures. However, future investigation is needed to show whether long-term cell cycle-arrested fibroblasts in 2-D culture still differ from those in a 3-D environment with regard to TGF-1-induced
-SMA expression. To interpret these data, one needs to consider that cell cycle in fibroblasts may be
differentially regulated in 2-D and 3-D culture as indicated by LaRue
et al. (18), who showed a divergent regulation of diverse cyclin-dependent kinases and their inhibitors in a rat fibroblast model.
No striking effect of TGF-1 exposure on fibroblast spheroid size was
observed. However, studies to evaluate proliferative activity and
viability of different fibroblasts in spheroid coculture with tumor
cells and in spheroid monocultures with TGF-
1 are recommended to
verify whether the model system not only reflects late stages but also
the onset of tumor desmoplasia.
Conclusions
Our data clearly show that the TGF- ![]() |
ACKNOWLEDGEMENTS |
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We thank F. Van Rey and M. Hoffmann for excellent technical assistance.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (Grants Ku 917/2-1 to 2-4) and by the Bayerische Staatsministerium für Wissenschaft, Forschung, und Kunst.
Address for reprint requests and other correspondence: L. A. Kunz-Schughart, Institute of Pathology, Univ. of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany (E-mail: leoni.kunz-schughart{at}med.uni-regensburg.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 11, 2002;10.1152/ajpcell.00557.2001
Received 5 December 2001; accepted in final form 3 September 2002.
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