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Cell Growth & Differentiation Vol. 12, 631-640, December 2001
© 2001 American Association for Cancer Research

Experimental Prostate Epithelial Morphogenesis in Response to Stroma and Three-Dimensional Matrigel Culture1

Shona H. Lang2, Meg Stark, Anne Collins, Alan B. Paul, Michael J. Stower and Norman J. Maitland

YCR Cancer Research Unit [S. H. L., N. J. M.], Department of Biology [M. S.], University of York, Heslington, York YO10 5YW; Prostate Research Group, Department of Surgery, The Medical School, University of Newcastle, Newcastle Upon Tyne NE2 4HH, United Kingdom [A. C.]; Pyrah Department of Urology, St. James Hospital, Leeds LS9 7TF [A. B. P.]; and Department of Urology, York District Hospital, York YO31 8HE [M. J. S.], United Kingdom


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To reproduce the structural and functional differentiation of human prostatic acini in vivo, prostatic epithelial and stromal cells derived from human primary cultures were cocultured in Matrigel. In the absence of stroma and serum, epithelial spheroids composed of solid masses of stratified and cuboidal cells formed. Outer cells of the spheroid expressed cytokeratins 1, 5, 10, and 14, whereas the inner cells expressed cytokeratin 18. The addition of 2% serum induced formation of a lumen surrounded by a layer of one or two cuboidal and columnar epithelial cells. The further addition of stromal cultures, dihydrotestosterone, and estrogen induced polarization of the epithelium and increased spheroid-forming efficiency. Epithelium expressed either cytokeratin 18 alone or additionally cytokeratins 1, 5, 14, and 10. All spheroid epithelium expressed prostate-specific antigen and prostate-specific membrane antigen. Androgen receptor was only detected in the presence of stroma, serum, and hormones. Thus, development of a functional and morphologically correct prostate gland in vitro is dependent on extracellular matrix, steroid hormones, and factors from stromal cells and serum.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The prostate gland is composed of several zones or lobes (peripheral, central, and transitional), based on a ductal/acinar system surrounded by fibromuscular stroma. Both ducts and acini are lined by columnar/cuboidal secretory epithelium beneath, which is a continuous layer of low cuboidal basal cells upon a basement membrane (1 , 2) . It has been hypothesized that the basal layer is the proliferative compartment of the prostate, containing a stem cell population, and which can differentiate into secretory epithelium (reviewed in Ref. 3 ). The secretory cells of both ducts and acini contribute several products including PSA3 to the seminal fluid (1) .

Both human and animal studies have shown that stroma (4, 5, 6, 7) and extracellular matrix (8) are essential for functional and morphological differentiation of prostatic epithelia and the epithelia of other organs such as the urothelium (9) , skin (10) , and breast (11 , 12) . In comparison to the breast or skin, the prostate is a "quiet" gland. It is both slow to grow and renew and does not undergo any periods of remodeling during its normal lifetime. These attributes have made it difficult to study in vitro.

In men over the age of 40–50 years, the prostate represents a major medical problem because both BPH and prostatic carcinoma are becoming increasingly prevalent (13 , 14) . Both the epithelia and stroma play roles in the advancement of these diseases (15 , 16) ; therefore, good models to study the interaction of these cell types is very important. Although many animal models exist to study prostatic growth and differentiation (4 , 15 , 17) , there are no human three-dimensional models that successfully use stromal and epithelial cultures to produce morphological and functional differentiation of the prostate. Such models are essential to understand the normal physiology of the prostate and better understand disease development.

Much research has been carried out on the breast to develop such models, because postnatal remodeling has provided an ideal system with which to study differentiation. In particular, Matrigel, an artificial basement membrane, has been used to induce rat and mouse mammary three-dimensional differentiation. Early experiments by Barcellos-Hoff et al. (11) and Darcy et al. (18) cultured epithelial organoids in Matrigel and found that they reorganized into three-dimensional structures that contained lumen and underwent branching and alveolar morphogenesis. This was accompanied by milk protein and lipid synthesis, which was vectorially secreted into the lumen. Later experiments by Darcy et al. (12) found that coculturing the epithelia in Matrigel with stroma further induced the epithelial organoids to differentiate both functionally (casein secretion) and morphologically (alveoli formation). These models provide physiologically relevant models that are also growth factor and hormonally dependent.

In the prostate, rat epithelial cultures have been grown within both collagen gels (19) and Matrigel (20) . In collagen, these formed acinus-like structures that secreted PSA, whereas in Matrigel, spheroids of cell masses with no normal morphological and functional differentiation were produced. Normal human prostatic epithelial cell lines cultured in Matrigel showed acinus-like structures with lumen and PSA secretion (8) ; however, primary cultures formed solid cell masses with little functional differentiation (21) .

We have established in vitro culture conditions, which are more physiological than previous studies. In the presence of serum, dihydrotestosterone, estrogen, and stromal cultures, prostatic epithelia were capable of producing three-dimensional structures in Matrigel that were similar to in vivo acini. The spheroids formed and contained lumen surrounded by one or two layers of columnar/cuboidal epithelium. The epithelium displayed characteristics of polarized secretory epithelium and showed functional differentiation (PSA, AR, and PSMA expression). Such a model will provide a relevant human model to compliment existing animal models, allowing further study of normal prostate growth and differentiation and disease formation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Optimization of Spheroid Growth.
Seven-day cultures of nonmalignant prostatic epithelia were seeded as single cells directly into Matrigel, and in the presence of KSFM, the single cells developed into spheroids that were irregular in shape (Fig. 1A)Citation . TEM demonstrated that these spheroids were solid masses of both cuboidal and stratified cells, and their appearance was consistent with a hyperplastic growth (Fig. 1B)Citation . In the center of the spheroid, there was evidence of necrosis. The central stratified/cuboidal cells had very tight cell-to cell contacts (Fig. 1C)Citation , whereas the outer cuboidal cells contacted each other much more loosely and had relatively sparse cytoplasms and elongated nuclei with prominent nucleoli. High-power magnification indicated there were multiple junctional complexes consistent with desmosomal-like and tight junction-like cell contacts, present between both cell types (examples shown in Fig. 1, C and DCitation ). The presence of estrogen, dihydrotestosterone, or medium conditioned by prostatic stromal cultures did not affect the morphology (results not shown).



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Fig. 1. Prostate epithelial spheroids grown in Matrigel and KSFM (sample C). A, phase image. Bar, 80 µm, the approximate diameter of one spheroid. B, TEM of a whole spheroid. Bar, 10 µm. C, high magnification TEM indicating both tight (TJ) and desmosomal-like (D) junctions present between the tightly associating inner cells. Bar, 0.5 µm. D, a line drawing representing C to indicate the exact location and appearance of the tight and desmosomal-like junctions.

 
Addition of 2% serum to the medium led to the spheroids appearing less dense (Fig. 2A)Citation , TEM indicated this was attributable to the spheroids developing lumen (Fig. 2B)Citation . The spheroids were 90 µm in diameter and had one or two epithelial cell layers that were cuboidal or columnar in shape. Microvilli were observed at the luminal edge of the epithelium, but other signs of polarization were not evident. Golgi bodies, secretory vesicles, and stacked rough endoplasmic reticulum were all present, consistent with a secretory function. No basal lamina was observed, and few junctional complexes were observed. Serum was included in these experiments to support stromal growth.



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Fig. 2. Prostate epithelial spheroids grown in Matrigel and KSFM plus 2% serum (sample C). A, phase image. Bar, 90 µm, the approximate diameter of one spheroid. B, TEM of a whole spheroid. Bar, 10 µm. C, high magnification TEM of a whole cell within the spheroid, showing luminal microvilli (mv), secretory vesicles (sv), and Golgi apparatus (g). Bar, 10 µm.

 
Research has shown that stroma, estrogen, and dihydrotestosterone are required to induce prostate epithelial differentiation (6 , 7 , 22, 23, 24) . The addition of these factors plus serum to epithelium growing in Matrigel led to the formation of compact spheroids that were regular in shape (Fig. 3A)Citation . TEM demonstrated that the spheroids were similar to in vivo acini because they contained lumen surrounded by one or two epithelial cell layers that were closely organized and columnar (Fig. 3B)Citation . Higher magnification (Fig. 3C)Citation indicated the cells were polarized, such that microvilli, Golgi, and secretory vesicles were organized to the luminal side, whereas nuclei were predominantly basal. Golgi were consistently large, and stacked rough endoplasmic reticulum was evident. No intact basal lamina was visible though a greater number of junctional complexes (desmosome-like and tight junction-like) were visible laterally and were predominantly toward the lumen.



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Fig. 3. Prostate epithelial spheroids grown in Matrigel, KSFM plus 2% serum, 20-7 M dihydrotestosterone, 10 ng/ml estrogen, and stroma (sample C). A, phase image. Bar, 70 µm, the approximate diameter of one spheroid. B, TEM of whole spheroid. Bar, 10 µm. C, high magnification TEM of a whole cell within the spheroid. Secretory vesicles (sv) are all polarized toward the lumen, and microvilli (mv) are also visible on the luminal surface. Bar, 6 µm. D, high magnification TEM showing the luminal half of an epithelium (shown in C). The figure shows a large active golgi (g) and stacked rough endoplasmic reticulum. In addition, a tight junction (TJ) is visible at the luminal surface. Bar, 2 µm. E, a desmosomal-like junctional complex (D) present at a cell:cell interface on a luminal edge. Bar, 1 µm. F, basal edge of spheroid showing that no intact basal lamina was visible. Bar, 1 µm.

 
This experiment was carried out in parallel on three different epithelial cultures (samples B, C, and D). The results were similar for all; however, only sample C (shown in Figs. 1Citation 2Citation 3Citation ) demonstrated a high degree of polarization in the presence of stroma (Fig. 3B)Citation . Samples B and C produced lumen-containing spheroids with columnar or cuboidal epithelium but no polarization.

Table 1Citation summarizes all repeat experiments in KSFM plus 2% serum (K2), dihydrotestosterone, estrogen, and stroma. Overall, experiments in these culture conditions showed evidence of columnar polarized epithelia in two of four examined epithelial samples (C and J). In two separate experiments, spheroids did not grow in serum-free conditions (samples F and G). Growth in 2% serum consistently led to the formation of spheroids with lumen (five of seven samples), where it did not there was no growth (sample F), or there was irregular spheroid formation (sample G), as illustrated in Fig. 1Citation . The sample showing no growth produced spheroids only in the presence of stroma, and in this instance, the spheroids were irregular. The sample that produced irregular spheroids in 2% serum produced lumen in the presence of stroma, but the epithelia were cuboidal, columnar, and stratified, with no evidence of polarization. Subsequently, TEM analysis of polarization was only carried out if compact acinus-like spheroids were observed.


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Table 1 Summary of epithelium/stromal cocultures in Matrigela

 
Basal epithelium in the prostate expresses CD44 (25) and may represent a candidate epithelial population more likely to differentiate in Matrigel culture. Therefore, we selected CD44-positive epithelium from four (of seven) prostate epithelial preparations (samples F, G, I, and J). No noticeable differences were observed in spheroid formation or morphology in comparison with those produced from whole epithelial populations (samples B, C, and D). However, CD44-negative epithelial populations showed no growth within Matrigel (results not shown).

Two of the samples (B and F) formed budding and ductal structures when grown in KSFM plus 2% serum (K2), dihydrotestosterone, estrogen, and stroma, an additional two (samples D and G) also exhibited such morphologies when grown without stroma (examples shown in Fig. 4Citation ). All samples that produced budding and ductal structures were accompanied by stratified epithelia. Because epithelia are not normally stratified in prostatic duct or acini, we concentrated our studies on the acinus-like spheroids.



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Fig. 4. Examples of a budding spheroid with multiple acini and duct-like structures also with evidence of budding, in phase contrast (bars, 100 µm). Toluidene blue-stained thick sections of budding and duct-like structures, showing the presence of stratified cells (bars, 50 µm).

 
Effect of Stroma on Spheroid-forming Efficiency.
The presence of stromal cultures was found to significantly increase the spheroid-forming efficiency of epithelial samples. Fig. 5Citation indicates that approximately equal numbers of spheroids formed in KSFM or KSFM plus 2% serum (sample C). In the presence of stroma, the number of spheroids that formed approximately doubled and further increased with the addition of estrogen and dihydrotestosterone. Two other samples (B and D), examined in parallel, showed increased spheroid formation only in the presence of stroma (approximately double), but hormones had no further effects. Increased spheroid formation in the presence of stroma was reproduced on three further samples examined on separate occasions (F, G, and J). In addition, presentation of the stroma in the coculture was examined by comparing stroma within an insert to that directly mixed with epithelia in the Matrigel, or added to the top of a preset gel. Our results indicated that stroma cocultured within an insert produced maximal spheroid formation. In addition, the different ways of presenting stroma had no effect on spheroid morphology (results not shown). We also observed that the use of epithelial cells from 7-day explants rather than freshly isolated cells led to greater spheroid-forming efficiency and that the spheroids subsequently produced maintained in culture for longer periods (results not shown).



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Fig. 5. Stromal cultures increased spheroid-forming efficiency. Epithelial sample C was mixed into Matrigel and grown for 2 weeks in either KSFM, KSFM plus 2% serum (K2), K2 and primary stroma (S) or K2, S, and 20-7 M dihydrotestosterone (D) and 10 ng/ml estrogen (O). Bars, SE.

 
Effect of Stroma on Spheroid Size.
The size and type (irregular or acinus-like) of epithelial spheroids forming within Matrigel varied between samples (summarized in Table 1Citation ). However, we consistently observed that stromal cocultures predominantly produced smaller sized spheroids. Fig. 6Citation shows that, after 1 week in Matrigel and KSFM plus 2% serum, equivalent numbers of 0.1- and 0.2-mm diameter epithelial spheroids had grown (22 cells/field did not form spheroids but remained as single cells). In total, an average of 30 spheroids/field formed. In the presence of primary and cell line stroma (STO), 36 and 43 total average spheroids/field, formed, respectively, but were predominantly 0.1 mm in diameter. Notably, coculture with STO cells (a fibroblast cell line) led to greater numbers of spheroids forming.



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Fig. 6. Stromal cultures affect spheroid size. Epithelial sample J was mixed into Matrigel and grown for 1 week in either KSFM plus 2% serum (K2) or K2 plus primary stroma (S), 20-7 M dihydrotestosterone (D), and 10 ng/ml estrogen (O) or K2 plus STO cells, D and O. Spheroid size was measured using a graticule. Bars, SD.

 
Effect of Stroma on Spheroid Phenotype.
Spheroids were sectioned and stained by fluorescence immunohistochemistry to compare the phenotypic profiles of those grown in serum-free conditions to those grown with sera, stroma, and hormones. The spheroids were phenotyped by investigating a variety of differentiation markers. Luminal prostatic epithelium were identified using cytokeratin 18 and PSA (26 , 27) , whereas basal epithelial cells were identified using basal cytokeratin (1 , 5 , 10 , 14) , CD44, and ß1 integrin (25 , 27 , 28) . Vimentin was analyzed because it can reflect differentiation (29) . Androgen receptor, PSA, and PSMA served as functional differentiation markers (30 , 31) . Finally, the cell adhesion molecules, E-cadherin and desmoglein, were also analyzed. The results are summarized in Table 2Citation , and examples of each stain are shown in Figs. 7Citation , 8Citation , and 9Citation . Intermediate filaments stained at similar intensities between the different spheroid types (Table 2)Citation . However, localization of cytokeratins 18 and 1, 5, 10, and 14 varied between the different spheroids (Fig. 7)Citation . Spheroids grown in the presence of KSFM showed expression of cytokeratins 1, 5, 10, and 14 in the epithelia at the outer edge of the spheroid, whereas cytokeratin 18 was expressed independently by the cells in the middle of the spheroid. Spheroids grown in the presence of serum and/or stroma were predominantly cytokeratin 18 positive, but also colocalization of cytokeratins 18 and 1, 5, 10, and 14 was observed. PSA was strongly expressed in all of the spheroids, but expression was polarized (toward the lumen) in spheroids grown in the presence of stroma (Fig. 8)Citation . PSMA was strongly expressed by all spheroid types, but expression was stronger in the outer cells of spheroids grown in serum-free conditions (Fig. 9)Citation . Androgen receptor was only weakly detected in spheroids grown with stroma (Fig. 9)Citation . E cadherin and desmoglein were expressed by all spheroids at cell to cell contacts. CD44 and ß1 integrin were likewise strongly expressed by all spheroids at the cell membrane, but noticeably both markers were only expressed by the outer cells of spheroids grown in serum-free conditions. In addition, ß1 integrin expression was strongly polarized (basally) in the presence of stroma (Fig. 8)Citation .


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Table 2 Immunofluorescent phenotype of primary prostate epithelial spheroids grown in Matrigel

 


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Fig. 7. Dual immunostaining of cytokeratin 18 (green) and cytokeratins 1, 5, 10, and 14 (red) of prostatic epithelia grown in Matrigel. Epithelia (sample C) were grown in the presence of KSFM, KSFM plus 2% serum (K2) or K2 plus primary stroma (S), 20-7 M dihydrotestosterone (D) and 10 ng/ml estrogen (O) for 2 weeks. Cell nuclei in spheroids were counterstained with DAPI (blue). Bar, 80 µm.

 


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Fig. 8. Polarization of PSA and ß1-integrin in Matrigel epithelial spheroids when cocultured with stroma. Using confocal analysis, the expression of PSA and ß1-integrin was compared between epithelial spheroids (sample C) grown in KSFM plus 2% serum (K2) or K2 plus primary stroma (S), 20-7 M dihydrotestosterone (D) and 10 ng/ml estrogen (O) for 2 weeks. Bar, 70 µm.

 


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Fig. 9. Examples of immunohistochemical staining of prostatic epithelial Matrigel spheroids. All spheroids shown were grown in KSFM, except for that illustrating AR expression, which was cultured in the presence of KSFM plus 2% serum, 20-7 M dihydrotestosterone, 10 ng/ml estrogen, and primary stroma. Spheroid nuclei were counterstained with DAPI (blue). The epithelial sample C was cultured for 2 weeks. Bar, 80 µm.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The present study demonstrates for the first time that human primary prostate epithelium seeded into Matrigel can form acinus-like structures in the presence of stroma, androgen, estrogen, and serum. These acini show a high degree of functional (PSA+/PSMA+/AR+) and morphological differentiation consistent with human prostatic acini in vivo. This represents a very useful model with which to study prostatic biology and will complement existing animal models (4 , 15 , 17) by reducing the complexity inherent in such systems.

PSA expression can be induced by Matrigel alone (shown here) or stroma alone (6 , 7) . Induction of PSA expression by epithelium without stroma indicates that epithelial differentiation is partly inherent. In our model, both Matrigel and stroma were clearly required to induce architectural organization, AR expression, and polarized secretion of PSA. Previously, AR expression in human primary prostate has been observed in both epithelia and stroma when cocultured together but not in isolation (7) , emphasizing the importance of both cell types for terminal epithelial differentiation. The requirement for stroma to induce the correct architectural organization has been demonstrated previously in mouse models (32) . Our results also found that stromal coculture produced greater numbers of small spheroids, which may indicate that the stroma and hormones either reduced growth or increased adhesion (thereby compacting the cells into a smaller spheroid). Stroma was clearly important for increasing spheroid-forming efficiency. The ability of stroma to double spheroid-forming efficiency suggests that stroma can recruit more epithelia to form spheroids. It is possible that epithelium in isolation can form spheroids if they have already received signals to differentiate but are then unable to undergo proper differentiation. The factors governing these differentiation pathways are unknown. One stroma-derived factor, hepatocyte growth factor, was found to increase the growth of primary lung epithelium and also increase the numbers of spheroids formed in Matrigel 2-fold (33) . Hepatocyte growth factor is clearly worth further investigation in our own model system.

The addition of unknown serum factors to Matrigel went some way to producing the correct morphological organization of spheroids into acinus-like structures, but stromal coculture was required for greater differentiation. Experiments examining the behavior of primary mammary epithelium cultured in Matrigel alone found that breast-specific proteins can also be expressed (34 , 35) . However, breast epithelial spheroids can be grown in Matrigel and demonstrate both functional and morphological differentiation in the presence of serum and hormones alone (11) . Stromal coculture systems increased differentiation and, in agreement with our own results, produced alveolar morphogenesis rather that ductal morphogenesis (12) . Breast studies have shown that differences in growth factor/kinase receptor activation can account for alveolar or ductal morphologies (36) . Recent studies indicate that a complex mix of growth factors and hormones can override the need for serum when breast epithelium is grown in Matrigel with stromal coculture (12) , and such studies are clearly now required to understand the important factors in prostatic differentiation. Stromal coculture is also required for the induction of functional and morphological differentiation in other organ models, such as ovarian epithelium (37) . The differentiation of urothelium in collagen matrix is also dependent on the formation of a basement matrix specifically driven by stromal interactions and not by soluble stromal factors (38) . The breast and ovarian models discussed above all found evidence of a complete basal lamina forming beneath the epithelium, whereas our results found only an incomplete basal lamina, suggesting that Matrigel alone was sufficient to induce differentiation, a phenomenon also reported with rat prostate (19) .

Previous attempts to produce the prostatic model described here have failed to produce morphological differentiation, most likely because of the absence of stromal cultures. Early attempts using primary rat epithelia (20) and more recently human primary epithelia (21) both successfully grew spheroids in serum-free medium, and in both instances spheroids of solid cells exhibiting a phenotype of hyperplastic growth were produced. Such morphologies were evident even in the presence of soluble stromal factors and the expression of AR (21) . In contradiction of this, prostatic cell lines can undergo morphological and functional differentiation in Matrigel when plated without stroma (8) , suggesting that the immortalization process can override the requirement for stromal cells to induce full differentiation as described here. The importance of mesenchyme for epithelial differentiation is fundamental and has been demonstrated by numerous animal studies (4 , 15 , 17) . Mesenchyme from different origins can induce epithelia to differentiate along different pathways. For example, urogenital mesenchyme can induce bladder epithelium to undergo prostatic differentiation, indicating the potential existence of a urogenital stem cell (39) . More recently, a greater contribution of stroma toward the development of disease is being considered. Studies have shown that stroma from different reproductive states of the of the breast (40) or prostate tumors (41) can modulate invasion and motility of the epithelium, characteristics clearly important for cancer progression. Our model will provide a useful tool for studying how epithelial/stromal interactions contribute toward cancer progression.

The formation of spheroids in Matrigel in the presence of serum had distinct effects on the phenotypic profile of the epithelium. Spheroids grown in serum-free medium showed two distinct cellular compartments. The outer cells of the spheroid were basal in morphology and phenotype (cytokeratins 1, 5, 10, and 14+/cytokeratin 18-/CD44+/ß1-integrin+), whereas the central cells were intermediate (cytokeratins 1, 5, 10, 14+/cytokeratin 18+/CD44-/ß1-integrin-) or luminal in phenotype (cytokeratins 1, 5, 10, and 14-/cytokeratin 18+/CD44-/ß1-integrin-). The presence of PSA in all of the cell populations indicates that the basal-like cells are more likely early intermediate in phenotype. These serum-free spheroids are similar to those produced previously by Hudson et al. (21) and also to the budding structures produced in monolayer culture by van Leenders et al. (42) . Phenotypically, the spheroids produced in serum-free conditions are more similar to in vivo acini because they contain separate cellular compartments (basal and luminal-like layers). The lack of lumen and columnar, luminal epithelium means they are morphologically dissimilar. Spheroids grown in the presence of serum, hormones, and stroma produced spheroids that are morphologically very similar to in vivo acini. Phenotypically, they show intermediate (cytokeratins 1, 5, 10, and 14+/cytokeratin 18+/PSA+/AR+) or luminal-like (cytokeratins 1, 5, 10, and 14-/cytokeratin 18+/PSA+/AR+) epithelial profiles, but the presence of a distinct basal layer is lost. The presence of AR and a more complete morphology indicates that these spheroids are more differentiated than those grown in serum-free conditions. It is possible that the majority of spheroids (grown under any conditions) are derived from early basal cells, and only a few are derived from stem cell populations. Those derived from early basal cells would have the capacity to differentiate but not replace a basal cell population. Those elusive spheroids derived from stem cells may therefore contain basal and luminal cells in a morphologically differentiated spheroid. It is highly likely that a proportion of the primary epithelium used in these studies were stem cell like (or early basal cells), because they were proliferative and pluripotent (capable of producing both basal and luminal cells, stratified, columnar or cuboidal cells, and also acinus-like or duct-like structures). Spheroids derived from CD44+ (basal) cells certainly gave rise to PSA+/cytokeratin 18+/CD44- (luminal) cells. This study provides further evidence for the hierarchical relationship in which basal and luminal cells are linked in a precursor progeny relationship. The heterogeneous expression of several markers of basal and luminal cells suggest that the putative stem cell population, reside within the basal layer, and give rise to intermediate cells (cytokeratins 1, 5, 10, and 14+/cytokeratin 18+/PSA+) and terminally differentiated cells (cytokeratin 18+/PSA+/AR+).

Our experiments showed a variation between tissue samples. This is not unexpected, given the diversity of tissue samples and the heterogeneous nature of prostatic disease. Indeed, this type of analysis will bring us closer to the phenotype of prostate tumors than the analysis of a few cell lines. The age of the patient from which the tissue was obtained also plays a role in culture. In our experiments (Table 1)Citation , cell culture from the tissue of 70–90-year-old patients was less successful than that from younger patients (54–57 years of age). This may indicate that the model requires a viable stem cell population, because stem cells will be more predominant in younger tissue. Thus, our future studies will concentrate on the use of younger tissue while trying to analyze the contribution of age and stem cell populations to the formation of acinus-like structures.

In summary, using primary human epithelial and stromal cultures, we have produce an in vitro model of a three-dimensional prostatic acinus. The acinus-like spheroids were grown in Matrigel and show both functional and morphological differentiation. Importantly, this model will allow the study of mesenchymal/epithelial interactions in the differentiation and growth of the normal prostate. In addition, the contribution of androgens, other hormones, or growth factors to prostatic differentiation can be studied and as well as how such control pathways break down during disease progression. Ultimately, such models will allow a reassessment of standard therapies, perhaps on an individual patient basis to tailor treatments from a primary biopsy.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
General chemicals were purchased from Sigma Chemical Co. (Poole, United Kingdom), tissue culture medium from Life Technologies, Inc. (Paisley, United Kingdom), and tissue culture plastic from Corning Costar Ltd. (High Wycombe, United Kingdom) unless otherwise stated. Antibodies were purchased from Dako (High Wycombe, United Kingdom) unless stated.

Cell Line Culture.
STO cells (mouse embryonic fibroblast cell line) were obtained from the European Collection of Animal Cell Cultures (Porton Down, United Kingdom) and were routinely cultured in DMEM culture medium (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 10% FCS (PAA Laboratories, Linz, Austria) and 2 mM glutamine (Life Technologies, Inc.). Cells were routinely cultured without antibiotics in a humidified atmosphere at 37°C and 5% CO2.

Prostate Tissue Collection.
Nonmalignant tissue was obtained from consenting patients undergoing transurethral resection for BPH or cystoprostatectomy for bladder cancer. Seven samples were collected for epithelial culture (age range, 54–86 years) and five for stromal cultures (age range, 57–89 years), summarized in Table 1Citation .

Prostate Primary Cell Culture.
Epithelial and stromal cultures were prepared and characterized as described before (43) ; these methods were based on those by Chaproniere and McKeehan (44) . Briefly, prostatic tissue was digested by collagenase and trypsin, and differential centrifugation was used to enrich for epithelial and stromal fractions. The enriched stromal fraction was resuspended in stromal cell growth medium (RPMI 1640 supplemented with 10% FCS and 1% antibiotic/antimycotic solution) and cultured routinely in 75-ml tissue culture flasks. Stromal cultures were used between passages 2–5. The epithelial fraction was resuspended in keratinocyte serum-free medium supplemented with 5 ng/ml epidermal growth factor, 50 µg/ml bovine pituitary extract, and 1% antibiotic/antimycotic solution (medium subsequently referred to as KSFM) and passed through a cell sieve (40 µm) to obtain single cells. Single cells were used immediately for additional experiments, frozen for storage, or plated into 25-ml flasks in 8 ml of KSFM and grown for 1 week.

Medium conditioned by stroma was collected from confluent cultures of stromal cells by incubating the cultures for 48 h in 15 ml of serum-free medium (DMEM/F12 supplemented with 10 µg/ml insulin, 5 µg/ml transferrin, and 1 ng/ml selenium). Conditioned medium was removed, filtered (0.2 µm pore), and frozen at -20°C until required.

Isolation of CD44+ Epithelial Cells.
Single cell suspensions of primary prostatic epithelia (106 cells) were labeled with 2.5 µg of anti-CD44 (PharMingen, Becton Dickinson UK Ltd., Oxford, United Kingdom) for 5 min at 4°C and then washed extensively using PBS supplemented with 2 mM EDTA and 0.5% (w/v) BSA. Antibody was then linked to 20 µl of goat antimouse MACS microbeads (Miltenyi Biotec, Ltd., Bisley, United Kingdom) at 4°C for 15 min, the cells were again washed extensively, after which they were added to a MACS column, and the labeled basal cells were eluted and resuspended in appropriate culture medium (basal cells formed 10–43% of the total epithelial cell population).

Cell Culture in Matrigel.
Epithelial cells were prepared at a concentration of 60,000 cells/ml in KSFM. On ice, they were mixed 1:1 (v/v) with Matrigel (Becton Dickinson, Oxford, United Kingdom), and 0.25-ml aliquots were subsequently plated into 24-well plates. The Matrigel was set by incubating at 37°C for 30 min. For experiments requiring stromal coculture, stroma was pregrown onto cell culture inserts; these were then placed on top of the Matrigel/epithelial cell mix (illustrated in Fig. 10Citation ). One ml of required growth medium was added to each well; thereafter, medium for cells was changed every 3 days, by the removal of 0.5 ml of spent medium and the addition of 0.5 ml of fresh medium. Equivalent batches of Matrigel were used throughout. Phase images were observed with a Nikon TE300 inverted microscope and captured with a JVC 3-CCD video camera. Images were subsequently prepared using Adobe Photoshop 4.



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Fig. 10. Illustration of prostatic epithelial and stromal cell coculture in Matrigel.

 
TEM.
Cells growing in Matrigel were washed twice with PBS and then fixed for 1 h at room temperature in 100 mM phosphate buffer, 4% paraformaldehyde (TAAB, Aldermaston, United Kingdom), and 2.5% Ultrapure glutaraldehyde. Cells were further processed for electron microscopy as described by Allen and de Wynter (45) . Thick sections were cut at 1 µm and stained with 0.6% toluidene blue in 0.3% sodium bicarbonate. Seventy-nm sections were cut and stained with saturated uranyl acetate in 50% ethanol, followed by Reynolds lead citrate and observed with a Jeol JEM 1200 Ex transmission electron microscope.

Fluorescent Immunostaining.
Cells grown in Matrigel were snap frozen in liquid nitrogen after embedding the gel in OCT Compound (BDH, Poole, United Kingdom). Embedded gels were stored at -20°C. Seven-µm sections were cut on a Leica cryostat and mounted onto Super frost microscope slides (BDH).

Immunostaining was carried out according to Table 3Citation . Antibodies were prepared in PBS supplemented with 1% BSA. Each step was followed by three washes in PBS. Primary antibodies were incubated at room temperature for 1 h and secondary antibodies for 30 min. Spheroids were counterstained with 1 µg/ml DAPI. Coverslips were mounted to slides using Cityfluor (Agar Scientific Limited, Stansted, United Kingdom). Immunostained cultures were observed and photographed using a Nikon Eclipse TE300 fluorescent microscope. Digital images were subsequently prepared using Adobe Photoshop 4.


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Table 3 Antibodies and procedures for fluorescent immunostaining

 
Confocal Microscopy.
Samples embedded in OCT were sectioned at 20 µm and immunostained as described above. Sections were then observed at 1-µm layers using a MRC1000 Bio-Rad Confocal Microscope (Hemel, Hempstead, United Kingdom).


    Acknowledgments
 
We thank Dr. I. N. Reid for assistance in examining the pathologies of the spheroids and T. D. Allen for analysis of the electron microscopy. In addition, our thanks to Kath Ramsay, Catherine Hyde, and June Hall for technical assistance.


    Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Yorkshire Cancer Research. Back

2 To whom requests for reprints should be addressed, at YCR Cancer Research Unit, Department of Biology, University of York, Heslington, York YO10 5YW, United Kingdom. Phone: 01904-432932; Fax: 01904-432615; E-mail: SHL2{at}york.ac.uk. Back

3 The abbreviations used are: PSA, prostate-specific antigen; PSMA, prostate-specific membrane antigen; KSFM, keratinocyte serum-free medium; BPH, benign prostatic hyperplasia; TEM, transmission electron microscopy; AR, androgen receptor; DAPI, 4',6-diamidino-2-phenylindole. Back

Received for publication 8/10/01. Revision received 10/23/01. Accepted for publication 10/25/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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