A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal–epithelial interactions in prostatic neoplasia

Mukta M. Webber5, Nicholas Trakul, Peter S. Thraves2, Diana Bello-DeOcampo, William W. Chu, Patrick D. Storto1, Thomas K. Huard3, Johng S. Rhim4 and Daniel E. Williams3

Departments of Zoology and Medicine, and
1 Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824-1312,
2 Department of Radiation Medicine, Georgetown University, Washington, DC,
3 Sparrow Regional Cancer Center, Lansing, MI and
4 Laboratory of Biochemical Physiology, National Cancer Institute, Frederick, MD, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle {alpha}-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-ß, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal–epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.

Abbreviations: {alpha}-SMA, smooth muscle {alpha}-actin; bFGF, basic fibroblast growth factor; BPH, benign prostate hyperplasia; EGF, epidermal growth factor; ITS, insulin–transferrin–selenium; MMPs, matrix metalloproteinases; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; PDGF, platelet-derived growth factor; PSA, prostate specific antigen; TGF-ß, transforming growth factor ß.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Much emphasis has been placed on the study of the behavior and characteristics of epithelial cells because the majority of cancers arise from epithelial cells. In order to examine the complex normal interactions between epithelial and stromal cells and their alterations in neoplastic disease, it is necessary to have well characterized cell models. We have already established and characterized a non-tumorigenic, immortalized human prostate epithelial cell line RWPE-1, which mimics normal epithelium in its ability to undergo acinar morphogenesis and to respond to androgen by upregulation of androgen receptor and prostate specific antigen (PSA) expression (13). In addition, six tumorigenic cell lines, derived from RWPE-1 cells by transformation with Ki-ras (2,3) or chemical carcinogens (M.M.Webber and S.Quader, unpublished data), have also been established and characterized. In this paper we describe characterization of a stromal myofibroblast cell line, WPMY-1, which is derived from SV40 large-T antigen-immortalized stromal cells from the same prostate specimen as the above epithelial cell lines, as confirmed by isozyme analysis. Since the epithelial and the stromal cell lines are all derived from the same prostate, they provide novel human prostate cell models for examining stromal–epithelial interactions in benign and malignant tumors of the prostate. While most current strategies for controlling carcinoma cell growth are based on controlling the growth of cancer cells, this unique model provides the opportunity to explore new approaches based on controlling cancer growth indirectly via normalizing stromal cell behavior.

Complex reciprocal interactions between epithelial cells and the underlying stroma are necessary for maintaining homeostasis in normal tissues and are involved in the mutual regulation of growth and differentiation. Transient changes in stromal–epithelial interactions occur in tissue remodeling during development, wound healing and regeneration. However, this homeostasis is disturbed in neoplastic disease. For example, in benign prostatic hyperplasia (BPH), the tissue composition is heterogeneous where the ratio of epithelial to stromal cells may change, in a continuum, from 1:2 to as much as 1:5 (4,5). This represents a change or loss of mutual growth regulation between epithelial and stromal cells.

In their pioneering work on the importance of stromal–epithelial interactions in normal urogenital differentiation, Cunha et al. (6) showed that stromal cells can modulate the differentiation pattern of normal prostatic epithelium. Growth factors produced by epithelial and stromal cell types can reciprocally regulate cell growth (7). The role of stroma in prostate cancer progression has also been suggested (8). In carcinomas, marked changes in stromal–epithelial interactions are frequently seen at the invading front (9). The basement membrane is a boundary which is not breached by normal epithelial cells. However, cancer cells cross this boundary and invade the underlying stroma where a reaction is mounted by stromal cells against the invading cancer cells (9). As a result of this stromal–cancer cell interaction, lasting changes occur in the normal epithelial–stromal interactions. It appears that due to the altered characteristics of cancer cells, such as excessive production and secretion of growth factors and proteases, changes in stromal cell behavior are induced which may actually enhance the motility and invasion by cancer cells (10).

The prostatic stroma contains many cellular components including smooth muscle cells, fibroblasts, blood vessels and nerve fibers. Due to this heterogeneous nature of the stroma, careful and complete characterization of any stromal cell line is necessary for establishing its cellular origin. To determine the exact nature of WPMY-1 cells, immunocytochemical analysis of marker proteins for various cell types were performed. Epithelial and endothelial cells, respectively, express their marker proteins, cytokeratins and factor VIII, while vimentin and fibronectin are characteristic of most mesenchyme-derived cells. Neuron-specific enolase and chromogranin A are markers for neuroendocrine cells. The presence of smooth muscle {alpha}-actin ({alpha}-SMA) and desmin is indicative of smooth muscle cells, while myofibroblasts express {alpha}-SMA but not desmin. The expression of SV40 large-T antigen, androgen receptor, PSA and the tumor suppressor proteins, p53 and pRb, was examined for further characterization. Response to androgen and growth factors, anchorage-independent growth in soft agar and the development of a serum-free medium for optimum growth were also determined. Since matrix metalloproteinases (MMPs), secreted by stromal cells, may enhance invasion by carcinoma cells, we analyzed the activity of MMPs secreted by WPMY-1 cells. We have developed novel and useful, well characterized cell models to investigate stromal—epithelial interactions in normal and neoplastic tissues. These cell models have applications in the study of etiology, invasion and metastasis, and in the development of new strategies for the prevention and treatment of prostate cancer (11).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
The following materials were used during the course of this study: Dulbecco's Modified Eagle's Medium (DMEM), high glucose, without phenol red; RPMI-1640 medium without phenol red; trypsin–EDTA (0.05% trypsin, 0.53 mM EDTA); insulin–transferrin–selenium (ITS); keratinocyte-SFM and supplement (K-SFM); antibiotic/antimycotic mixture (PSF; 100 U penicillin, 100 µg streptomycin and 25 µg fungizone per ml medium; Gibco BRL, Grand Island, NY); donor calf serum (DCS); fetal bovine serum (FBS; Intergen, Purchase, NY); phosphate-buffered saline (PBS; Pierce, Rockford, IL); tissue culture plasticware (Falcon, Oxnard, CA); 12 mm round coverslips (Fisher, Pittsburgh, PA); 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT); P-iodo-nitrotetrazolium violet (INT); bovine serum albumin (BSA; Sigma, St Louis, MO); Bacto-agar (Difco, Detroit, MI); mibolerone (BIOMOL, Plymouth Meeting, PA); gelatin (Bio-Rad, Richmond, CA), epidermal growth factor (EGF); transforming growth factor-ß (TGF-ß); antibody to human fibronectin (Collaborative Research, Bedford, MA); basic fibroblast growth factor (bFGF); hr platelet-derived growth factor (PDGF) AB; hr PDGF BB (R and D Systems, Minneapolis, MN); polyclonal antibodies to: androgen receptor (Affinity Bioreagents, Neshanic Station, NJ), prostate specific antigen (PSA; BioGenex, San Ramon, CA); monoclonal antibodies to: cytokeratin 8, desmin, factor VIII, vimentin (Sigma); SV40 large-T antigen; p53 (Oncogene Science, Manhasset, NY); Rb, (Pharmingen, San Diego, CA); neuron-specific enolase (NSE; Lipshaw, Pittsburgh, PA); chromogranin A (ChA; BioGenex); Vectastain Elite ABC Peroxidase Kit and 3,3'-diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA) and, for NSE, ChA and PSA, SA Histostain kits (mouse and rabbit; Zymed, San Francisco, CA).

Immortalization and cell culture
Stromal cells from the non-neoplastic prostate of a 54-year-old white male, undergoing cystoprostatectomy, were isolated from the same tissue specimen as that from which the epithelial cell line RWPE-1 was derived (2). Tissue was minced and digested in RPMI-1640 medium containing 5% FBS, PSF and 400 U/ml collagenase. Acini were isolated by gravity sedimentation and stromal cells were collected from the supernates, as described previously (12,13). Stromal cells were plated and maintained in RPMI-1640 containing 10% FBS to establish primary cultures and subsequent passages. Stromal cells, in passages under six, were immortalized with the SV40 large-T antigen, using the pRSVT plasmid construct, according to a method described previously (14). Because of their rapid growth rate, WPMY-1 human prostate stromal cells were maintained in RPMI-1640 or DMEM supplemented with 2.5 or 5% DCS and 1% PSF. Cells from passages 38–40 and 62–78 were used for the experiments. The WPE1-10 human prostate epithelial cell line was maintained in a serum-free medium, K-SFM with supplement. The WPE1-10 cells were cloned from the RWPE-1 cell line (2).

Growth in vitro
WPMY-1 cells were plated in triplicate wells, in 96-well plates, at the following densities: 625, 1250, 2500, 5000 and 10 000 cells/well in 200 µl/well of RPMI-1640 plus 2.5% DCS, and the medium was changed every 48 h. Plates were stained on days 2, 4, 6, 8, 10 and 12 using the MTT method (15). Briefly, 50 µl/well of a 1mg/ml MTT solution was added, the plate was incubated at 37°C for 4 h, 30 µl of MTT solution were removed and 150 µl DMSO/well was added for up to 5 min to extract the stain. The absorbance was read immediately at 540 nm using a plate reader (Titertek Multiskan MCC/340 MKII). Results represent one of four experiments.

Serum-free medium
Various media compositions were tested in order to select a serum-free medium for growing WPMY-1 cells. Cells were plated in triplicate wells, in 96-well plates, at a density of 5000 cells/well in DMEM containing 2.5% DCS. After 48 h, cells were rinsed with PBS and the following media were tested in (six wells/medium): DMEM alone or DMEM supplemented with one of the following: 2.5% DCS, 5% DCS, 10 µl/ml ITS, 5 ng/ml EGF and 50 µg/ml bovine pituitary extract (BPE) [supplement 1 (S1)], or S1 + ITS. Media were changed every 48 h. After 5 days of treatment, cells were stained using the MTT assay and absorbance was recorded. Results represent the averages of two experiments.

Androgen and growth factor response
To determine the effects of androgen or growth factors on WPMY-1 cells, dose response assays were performed. Mibolerone, a synthetic non-metabolizable androgen, was used for sustained action and its effects were examined in the absence of serum (1,2). Cells were plated in six wells/treatment at a density of 10 000 cells/well in 96-well plates in RPMI-1640 with 2.5% DCS. After 48 h, cells were rinsed with PBS and fed with RPMI-1640 containing 0.1% BSA and mibolerone at concentrations ranging from 0.01 to 10 nM. Results of one of six experiments are shown. Growth factors tested were EGF, bFGF, PDGF or TGF-ß at 0.1, 1.0 and 10 ng/ml. Cells were plated in triplicate as above with 5000 cells/well. After 48 h, cells were rinsed with PBS and placed on RPMI-1640 medium alone containing the growth factor. The medium for TGF-ß test contained 1% serum (DCS). Medium was changed every 48 h. Plates were stained after 5 days of treatment using the MTT assay and absorbance was read as described earlier. Results shown are for one of three experiments.

Agar assay for anchorage independence and growth in scid mice
For anchorage-independent growth, cells were plated in triplicate 35 mm plates/cell number, in 0.3% agar in RPMI-1640 containing 10% DCS or 10% FBS, as described previously (2), at a density of 12 500 cells/35 mm plate. The plates were incubated at 37°C for 21 days, stained with 1 ml of a 1.0 mg/ml solution of INT for 24 h, counted using a colony counter (Biotran II, New Brunswick Scientific) and the percent colony forming efficiency (CFE) was determined (2). The experiment was conducted three times. Four male scid mice were injected subcutaneously into each flank with 5 million passage 22 cells with Matrigel, and observed for 6 months.

Immunocytochemical analysis
To assay for the cytoskeletal protein expression, and for factor VIII, fibronectin, large-T antigen, p53, pRb and androgen receptor, an indirect immunoperoxidase method was used as described previously (2). WPMY-1 cells were grown on glass coverslips in 24-well plates (20 000 cells/well) in standard medium, except cells used for androgen receptor and PSA staining, which were treated for 6 days with 5 nM mibolerone starting at 48 h after plating (2). Cells were then fixed with a 1:1 acetone:methanol solution and stored at –20°C until staining using a modified avidin–biotin immunoperoxidase vector protocol, which employed either DAB (brown color) or DAB-nickel (gray to black color), as described previously (2). Primary antibody dilutions were: cytokeratin 8, 1:200; desmin, 1:50, factor VIII, 1:1000; {alpha}-SMA, 1:300; vimentin, 1:40; p53, 1:20; pRb, 1:200; large-T antigen, 1:500; fibronectin, 1:50 and androgen receptor, 1:200. Immunostaining was repeated at least three times and in most cases five times using cells in passages 62–72.

Conditioned medium (CM) crossover experiments
CM was collected from WPMY-1 and WPE1-10 cells, and maintained in T-75 flasks, in the following manner: after cells reached 80–90% confluence, cultures were rinsed with PBS and 5 ml of the medium to be conditioned was added. WPMY-1 stromal cells received basal K-SFM (no EGF, no BPE), and WPE1-10 epithelial cells received DMEM alone. CM was collected after 48 h, centrifuged to remove cell debris and frozen at –70°C until needed. A dose–response assay was performed to determine the effects of crossover CM. For this, WPMY-1 cells (5000 cells/well) and WPE1-10 cells (10 000 cells/well) were plated in 96-well plates, in complete medium (DMEM plus 2.5% DCS for WPMY-1 cells, and K-SFM plus supplement for WPE1-10 cells). Different cell numbers for the two cell lines were used because of their different growth rates. After 48 h, medium was replaced with their own respective media without serum but which contained the following concentrations of the appropriate CM: 0 (control), 25, 50 and 75%, using six wells/medium. Media were changed every 48 h, cells were stained using the MTT method after 5 days of treatment, and the absorbance was read and plotted. Results shown represent the averages of two experiments.

Gel electrophoresis and zymography
To analyze the presence of gelatinases (MMP-2 and MMP-9) in WPMY-1 and WPE1-10 cell lines, SDS–PAGE gelatin zymography was performed using 10% minigels containing 1% gelatin in three independent experiments (16). One million cells were plated in 60 mm plates in their respective growth media until ~80% confluence was reached. Cells were rinsed twice with PBS and fed with 2.2 ml medium (RPMI-1640 medium without serum for WPMY-1 cells, and K-SFM without supplement for WPE1-10 cells). CM was collected at 48 h and centrifuged to remove cell debris. CM samples containing 1 µg protein were loaded per lane. Minigels were run at 125 V for 2.5 h at 4°C, then gently rocked in two changes of 2.5% Triton X-100 for 1 h at room temperature to remove SDS, and incubated at 37°C for 18 h in a Tris–HCl buffer (50 mM Tris–HCl, 5 mM CaCl2, 0.2 M NaCl and 0.02% Brij 35, pH 7.6) to allow renaturing of enzymes. Gels were stained with Coomassie blue and destained in methanol:acetic acid:water (3:1:6). The presence of MMP activity is indicated by bands of lysis against a dark background.

Statistical analysis
All of the results in this study were obtained from two to six independent experiments. Results are expressed as means ± SEM. The differences between means were considered significant if P < 0.05. Data were analyzed using one-way analysis of variance (ANOVA) for growth factor and hormone effects. Response to individual concentrations was compared with the control using the Tukey-Kramer multiple comparison test or a paired t-test, as appropriate. GraphPad InStat v3 was used for these analyses.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Morphology and immunocytochemical analysis
WPMY-1 cells have an elongated, spindle-shaped morphology which is intermediate between fibroblasts and smooth muscle cells (Figure 1aGo). By immunocytochemical analysis for a variety of antigens (Figure 1Go), it was determined that these cells do not express ChA (Figure 1cGo), NSE (Figure 1eGo), cytokeratin 8 (Figure 1fGo), factor VIII (Figure 1gGo) or desmin (Figure 1jGo), thus eliminating the possibility of their being neuroendocrine, epithelial, endothelial or smooth muscle cells, respectively. WPMY-1 cells show positive staining for {alpha}-SMA (Figure 1kGo) and vimentin (Figure 1lGo). On the basis of this co-expression of {alpha}-SMA and vimentin and the absence of desmin, it is concluded that WPMY-1 cells exhibit characteristics of myofibroblasts. Additionally, these cells express fibronectin (Figure 1mGo), SV40 large-T antigen (Figure 1nGo), as well as nuclear androgen receptor (Figure 1pGo). RWPE-1 human prostatic epithelial cells were used as a positive control for nuclear androgen receptor expression (Figure 1oGo). WPMY-1 cells show positive but heterogeneous staining for the tumor suppressor proteins p53 and pRb (Figure 1q and rGo). WPMY-1 cells do not express PSA (not shown), a marker for prostatic epithelial cells. The immunocytochemical profile of WPMY-1 cells is summarized in Table IGo. We used a dichotomous dendrogram (Figure 2Go) to establish that WPMY-1 cells are myofibroblastic in nature under the culture conditions used in this study.



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Fig. 1. Characterization of WPMY-1 cells on the basis of cellular proteins using avidin–biotin immunoperoxidase staining. (a) Cell morphology, hematoxylin and eosin; (b) ChA, adrenal medulla positive control (red stain), counter-stained with hematoxylin; (c) WPMY-1 cells are negative for ChA; (d) NSE, nerve fiber positive control (red stain). WPMY-1 cells are negative for (e) NSE; (f) cytokeratin 8 and (g) factor VIII. (h) WPMY-1 cells, negative control lacking primary antibody; (i) desmin, a smooth muscle cell from the human prostate, used as a positive control for desmin; (j) WPMY-1 cells are negative for desmin. WPMY-1 cells are positive for (k) {alpha}-SMA; (l) vimentin and (m) fibronectin. (n) WPMY-1 cells show positive nuclear staining for large-T antigen; (o) positive control for androgen receptor, RWPE-1 human prostate epithelial cells; (p) WPMY-1 cells show positive nuclear staining for androgen receptor; (q) p53 and (r) heterogeneous nuclear staining for pRb. (a and f–r) x625; (b–e) x450.

 

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Table 1. Immunocytochemical profile of WPMY-1 cells
 


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Fig. 2. A dendrogram showing stepwise characterization of WPMY-1 cells.

 
Isozyme and chromosome analysis
WPMY-1 cells have an isozyme profile which is identical to that of RWPE-1 epithelial cells (2), confirming the origin of both cell lines from the same prostate. The profile is: LDH, human; G6PD, B; PGM1, 2; PGM3, 1; ESD, 2; Me-2, 0, AK-1, 1; GLO-1, 1–2. The frequency of this phenotype was calculated to be 0.00012, which means that <0.1% of cell lines might be expected to have an identical isozyme phenotypic profile. The cell line is hyperdiploid, X–Y, with chromosome numbers varying from 58 to 68.

Growth characteristics and response to androgen
Growth curves for WPMY-1 cells are shown in Figure 3AGo. Cells plated at low densities (625 and 1250 cells/well) show a long lag phase as compared with those plated at higher cell densities (2500 cells/well and higher). To date, these cells have undergone 79 passages. WPMY-1 cells show a dose-dependent stimulation of growth in response to treatment with the synthetic androgen mibolerone at concentrations ranging from 0.01 to 10 nM, with maximum stimulation to 145% of control at 10 nM when treated for 5 days (Figure 3BGo). This growth response is highly significant (ANOVA, P < 0.0001).



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Fig. 3. (A) Growth curves for WPMY-1 cells. Cells were plated in 96-well plates at densities varying from 625 to 10 000 cells/well. (B) Effects of the synthetic androgen mibolerone on the growth of WPMY-1 cells. Cells were plated in 96-well plates at a density of 10 000 cells/well and treated with mibolerone concentrations ranging from 0.01 to 10 nM for 5 days. Absorbance values were measured at 540 nm and plotted, ±SEM. For one-way ANOVA, P < 0.0001. Tukey–Kramer multiple comparison is shown as *P < 0.01 and **P < 0.001.

 
Effects of growth factors
WPMY-1 cells show a dose-dependent stimulation of growth when treated with EGF, bFGF (Figure 4AGo), PDGF-AB and PDGF-BB (Figure 4BGo). Maximum stimulation to 143% of control with EGF (ANOVA, P = 0.01) and 146% with bFGF (ANOVA, P = 0.0001) was achieved at 10 nM. Of the two PDGF isoforms, PDGF-BB was more effective (138% of control; ANOVA, P = 0.006) in stimulating growth than PDGF-AB (125% of control; ANOVA, P = 0.01). WPMY-1 cells show a dose-dependent inhibition of growth in response to TGF-ß (Figure 4AGo). However, this inhibition was only significant when 1% serum was included in the medium. Reduction in growth with TGF-ß to only 89% of control was seen at 10 ng/ml in the absence of serum (not shown) but in the presence of serum, growth was reduced to 65% of control (ANOVA, P = 0.0003) at 10 ng/ml (Figure 4AGo). TGF-ß also caused a dose-dependent inhibition of bFGF-induced growth when bFGF was included at 0.1, 1.0 or 10 ng/ml level. At 10 ng/ml TGF-ß, growth was reduced to 30% of control (P < 0.001) at all bFGF concentrations (Figure 4CGo).



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Fig. 4. Effects of growth factors on the growth of WPMY-1 cells. Cells were plated in 96-well plates at a density of 5000 cells/well and treated with growth factors for 5 days. Absorbance values were measured at 540 nm and plotted, ±SEM. (A) Effects of EGF, bFGF and TGF-ß. One-way ANOVA for EGF, P = 0.01; for bFGF, P = 0.0001 and for TGF-ß, P = 0.0003. Tukey–Kramer is shown as *P < 0.01, **P < 0.001. (B) Effects of PDGF-AB and PDGF-BB. One-way ANOVA for PDGF-AB, P = 0.01 and for PDGF-BB, P = 0.006. Tukey–Kramer is shown as *P < 0.05, **P < 0.01. (C) Effects of TGF-ß on bFGF-induced growth. P-values for one-way ANOVA for all curves are <0.007. By Tukey–Kramer, all values at 1.0 and 10 ng/ml TGF-ß are significant (P < 0.05). For curve 4 (10 ng/ml bFGF), significance values are shown as *P < 0.01, **P < 0.001.

 
Serum-free medium for WPMY-1 cells
Results show that WPMY-1 cells can be maintained in a serum-free medium. The basic medium in all test media consisted of DMEM. Using growth in DMEM plus 2.5% serum as the 100% control, treatment groups with DMEM alone or with 10 µl/ml ITS showed reduced growth at 35 (P = 0.02) and 50% (P = 0.002) of control, respectively (Figure 5Go). However, cells grown in DMEM with a supplement (S1) of 50 µg/ml BPE and 5 ng/ml EGF showed 100% growth, whereas those given 10 µl/ml ITS, in addition to supplement S1, showed an increase in growth to 141% of control (P = 0.06), apparently exceeding that in medium containing 5% serum (106%).



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Fig. 5. Development of a serum-free medium for optimum growth of WPMY-1 cells. Cell growth in different serum-free media was compared with that in medium containing serum. The basal medium DMEM was supplemented with one of the following: 2.5% DCS, 5% DCS; ITS, S1 (a supplement containing 5 ng EGF and 50 µg BPE per ml medium) or S1 + ITS. Cells were plated in 96-well plates at 5000 cells/well and treated with test media for 5 days. Absorbance values were measured at 540 nm and plotted ±SEM. Using a paired t-test and taking DMEM with 2.5% DCS as the control (100%), *P = 0.02, **P = 0.002, S1 + ITS, P = 0.06.

 
Anchorage-independent growth and growth in scid mice
WPMY-1 cells are capable of anchorage-independent growth as demonstrated by their ability to form colonies in soft agar. The percent cloning efficiency for cells plated at 12 500 cells/35 mm plate was 0.7% when 10% DCS was used, as compared with 4.6% when 10% FBS was used. During the 6 month observation period, none of the eight injected sites in scid mice showed any tumors.

Paracrine interactions between myofibroblasts and epithelial cells
CM from WPMY-1 cells caused a marked inhibition (ANOVA, P < 0.0001) of the growth of WPE1-10 epithelial cells in a dose-dependent manner. Maximum and highly significant (P < 0.001) reduction in growth of WPE1-10 cells, to ~60% of control, was seen at 75% WPMY-1-CM (Figure 6Go). However, CM from WPE1-10 cells caused only a marginal stimulation in the growth of WPMY-1 cells to 115% of control (P = 0.07), which was not statistically significant at P < 0.05 (Figure 6Go).



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Fig. 6. Effects of CM from WPMY-1 cells on the growth of prostatic epithelial cells WPE1-10 ({blacksquare}) and of CM from WPE1-10 cells on the growth of WPMY-1 cells ({circ}). Cells were plated in 96-well plates at 5000 cells/well and treated with test media for 5 days. Absorbance values were measured at 540 nm and plotted ±SEM. One-way ANOVA for WPE1-10 cells, P < 0.0001 and for WPMY-1 cells, P = 0.07. Tukey–Kramer is shown as *P = 0.01, **P = 0.001.

 
Zymography
The zymogram in Figure 7Go shows gelatinase activity of MMP-2 (72 kDa) and MMP-9 (92 kDa) in CM from both the epithelial WPE1-10 and the myofibroblastic WPMY-1 cells. WPMY-1 cells secrete high levels of MMP-2 and detectable levels of MMP-9. The secreted MMP-2 activity by WPMY-1 cells is markedly greater than that produced by epithelial cells.



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Fig. 7. Serum-free CM from WPMY-1 cells was analyzed and compared with that from WPE1-10 prostatic epithelial cells by SDS–PAGE zymography for matrix metalloproteinase activity. For WPMY-1 cells the CM was DMEM and for WPE1-10 cells, it was basal K-SFM without the supplement.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the context of BPH and prostate cancer, questions that arise are: what is the significance of myofibroblasts in the normal, benign and malignant prostate and what is their origin? Fibroblasts are an important component of connective tissue in most tissues. In some normal tissues (e.g. oviduct, ovary, testes, renal glomerulus) myofibroblasts are another important stromal cellular element of mesenchymal origin (8). These cells exhibit some characteristics of both fibroblasts and smooth muscle cells (SMCs), such as co-expression of {alpha}-SMA and vimentin; however, {alpha}-SMA, a contractile form of actin, can be used as a marker for myofibroblasts. Because myofibroblasts are not easily identified in routine hematoxylin and eosin stained sections, their presence in normal tissues, in the absence of trauma, has not received much attention (17). Myofibroblasts in the prostate have also not been well studied.

We have established a myofibroblast cell line WPMY-1 by immortalization of stromal cells, derived from a non-neoplastic human prostate, using SV40 large-T antigen. Although at the passage used, WPMY-1 cells did not form tumors in nude mice, they did form colonies in agar. WPMY-1 cells showed a higher CFE in agar cultures when FBS was used as compared with DCS. We also observed differences in CFE in FBS obtained from different sources. It is, therefore, important to take note of the serum type and source, as all sera are not the same. The WPMY-1 cells co-express {alpha}-SMA and vimentin but do not express desmin. This leads us to conclude that these cells are myofibroblasts. WPMY-1 cells show strong nuclear staining for androgen receptor and a growth response to the synthetic androgen mibolerone. Androgen responsiveness was examined in serum-free, growth factor-free medium so that the growth response would not be masked by exogenous growth factors (1,2,18). Growth stimulation of prostate myofibroblasts by EGF, bFGF and PDGF, and inhibition by TGF-ß, were observed. Although cells derived from the mesenchyme are generally thought to show growth stimulation by TGF-ß, this effect may depend on the cell type and the concentration of TGF-ß relative to that of other growth factors. Inhibition of prostate stromal cell growth by TGF-ß, in agreement with our observations, has also been observed by others (7,19,20). Growth inhibition with TGF-ß was seen in the presence of serum because one must have a growing and not a static culture before growth inhibition can occur. TGF-ß also counteracted growth stimulatory effects of bFGF. A serum-free medium, which supports growth of WPMY-1 cells at a level at least as good, and perhaps a little better, as that in a medium containing 5% DCS has been developed. Using crossover CM between stromal and epithelial cells, we show that paracrine interactions do take place between these two cell types. The growth of WPE1-10 epithelial cells was significantly inhibited by CM from WPMY-1 stromal cells, whereas results suggest only a slight stimulation of growth of WPMY-1 cells by CM from WPE1-10 epithelial cells. Our results are supported by the observation that stromal cell CM inhibited the growth of PC-3 human prostate carcinoma cells (21). Taken together, these results provide the basis for further studies on stromal–epithelial interactions.

Myofibroblasts, which have contractile ability, have most commonly been recognized in granulation tissue during wound healing where they are considered to be a reactive and transient cell type arising in response to tissue damage (17). Experimental evidence suggests that myofibroblasts can arise from other myofibroblasts, fibroblasts, SMCs and undifferentiated mesenchyme (17). This demonstrates the plasticity of these different phenotypes and their cytoskeletal heterogeneity (9,22).

There is evidence to suggest that myofibroblasts may be an important component of the normal human prostate stroma. Stromal cells isolated from normal and benign human prostate tissue expressed {alpha}-SMA and vimentin but did not express desmin (5). In histological sections of BPH, {alpha}-SMA-positive cells occupied 2-fold more area than desmin-positive cells, suggesting that myofibroblasts are a dominant phenotype in BPH stroma (23). We have made similar observations using immunofluorescene double staining for {alpha}-SMA and desmin (preliminary observation). It has been suggested that cells (myofibroblasts?) in BPH stromal nodules are similar to those seen during fetal development, wound healing and tissue repair (24). These observations lend support to McNeal's hypothesis that BPH reflects a reawakening and recapitulation of some fetal prostate characteristics (25).

In cancer, a common stromal response at the invasion front involves the appearance of {alpha}-SMA-positive but desmin-negative myofibroblasts (9,26). In the early stages of invasion, this reaction is similar to wound healing. It may progress to desmoplasia due to increased proliferation of myofibroblasts and matrix deposition, and ultimately, it may become fibrotic. Such lesions containing {alpha}-SMA-positive myofibroblasts have been observed in lung, colon, breast and in prostate carcinomas (9,10,27). The observation, that stromal cells may actually enhance the ability of cancer cells to invade, is supported by the fact that in an in vitro invasion assay, the presence of myofibroblasts, which secreted the 72 kDa MMP-2, increased the invasive ability of colon cancer cells (27). Zymographic analysis of secreted MMPs showed that WPMY-1 cells produce considerably higher 72 kDa MMP-2 activity than WPE1-10 epithelial cells. Our stromal and epithelial cell lines can be used to examine the role of proteases secreted by stromal cells during invasion of the stroma by carcinoma cells. Myofibroblasts are also common in precancerous conditions of the colon and breast (9,10). The dominance of myofibroblasts in benign or malignant lesions may alter the growth factor micro-environment, resulting in changes in stromal–epithelial interactions. Conversely, cancer cells that secrete growth factors could also modulate the differentiation pattern of stromal cells (9). Myofibroblasts have also been identified in diseases involving inflammation and neoplasia showing fibroblastic proliferation of unknown etiology (17). The nature of the stimuli responsible for inducing a myofibroblast phenotype in vivo may depend on the local micro-environment resulting from inflammation and tissue remodeling as seen in wound healing.

It is interesting to note that prostatitis and associated inflammation are frequently seen in the prostate (5,28). In one study, 98% of BPH specimens showed chronic inflammation (29). The complex process of wound healing also includes inflammation and remodeling. Cancer has been referred to as a wound that does not heal, because it shows similar characteristics including the presence of myofibroblasts (30,31). The inflammatory response involves infiltration by platelets, fibroblasts and inflammatory cells which release cytokines that can induce hyperplasia in responsive cells. In addition to these growth factors, extracellular matrix and proteases are also involved.

Stromal cells not only respond to paracrine growth factors produced by cancer and inflammatory cells, but they also produce growth factors (e.g. bFGF, TGF-ß) to which they respond in an autocrine manner (7,20). A complex mixture of growth factors present in an inflammatory micro-environment in the prostate may include EGF, TGF-{alpha}, FGF, IGF, KGF, NGF, PDGF, TGF-ß and other cytokines (7,9,32). Many of these growth factors are ubiquitous. Our results show that EGF, bFGF and PDGF are mitogenic for myofibroblasts, whereas TGF-ß inhibits their growth. PDGF is a key growth factor in wound healing. Since it is released during inflammation, it may be involved in the etiology of BPH (29). The desmoplastic response seen in carcinomas may also be associated with PDGF (9). PDGF, GM-CSF and TGF-ß can stimulate {alpha}-SMA expression and can, thus, modulate fibroblast/myofibroblast phenotype (9,19,21,33). Since differentiation often accompanies a decrease in growth, the induction of myofibroblastic differentiation and growth inhibition by TGF-ß, that we and others have observed (19,20), are in agreement.

On the basis of these observations, we suggest that stromal proliferation in BPH and stromal reaction in invading prostate cancer may mimic changes associated with inflamation, wound healing and tissue remodeling. A loss of balance between the proliferation-inducing and -inhibiting factors would result in prostatic stromal hyperplasia seen in BPH and a stromal reaction seen in cancer. The myofibroblast cell line WPMY-1 and the non-tumorigenic and tumorigenic epithelial cell lines, derived from the same prostate and maintained in serum-free media, provide novel and useful models to study stromal–epithelial interactions. It is hoped that further studies, using these cell models, will provide new insights into cancer prevention and treatment.


    Acknowledgments
 
We thank Gillian Bice for karyotype analysis, and Nestor Deocampo and Salmaan Quader for their valuable advice in the performance of zymographic analysis.


    Notes
 
5 To whom correspondence should be addressed Email: mwebber{at}pilot.msu.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 14, 1998; revised February 19, 1999; accepted March 17, 1999.