1 Prostate Research Group, Department of Surgery, The Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK
2 Prostate Research Group, Department of Oncology, Western General Hospital, University of Edinburgh, Edinburgh, EH4 2XU, UK
3 YCR Cancer Research Unit, Department of Biology, University of York, PO Box 373, York, YO1 5YN, UK
*Author for correspondence (e-mail: anne.collins{at}newcastle.ac.uk)
Accepted July 23, 2001
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SUMMARY |
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Key words: Stem cells, Integrins, Prostate
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INTRODUCTION |
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In self-renewing epithelial tissues, such as the gut and epidermis, the small stem cell population has the capacity for extended or unlimited growth and its progeny are either stem cells or cells with more limited proliferative capacity, termed transit amplifying cells. These daughter cells divide to maintain tissue balance but are limited to a finite number of cell divisions before they terminally differentiate (Potten, 1981; Hall and Watt, 1989). Cell types are organised whereby stem cells, transit amplifying cells and mature terminally differentiated cells occupy discrete locations within the tissue, often forming stratified layers (Potten and Morris, 1988). A similar hierarchical arrangement has been postulated for the more slowly dividing adult prostate (Isaacs, 1987; Bonkoff and Remberger, 1996). However, we have only recently begun to accumulate evidence in support of a transit amplifying compartment in the prostate (Verhagen et al., 1988; Robinson et al., 1998; van Leenders et al., 2000).
The characterisation of tissue stem cell populations remains difficult because of the lack of markers that can distinguish between stem cells and their differentiating progeny. For many tissues, panels of molecular markers have been developed to define the stem cell compartment. For example, integrins, which mediate the attachment of cells to extracellular matrix (ECM) proteins on the basement membrane, have been instrumental in the identification of stem cells in skin (Jones et al., 1995; Li et al., 1998) and testis (Shinohara et al., 1999). Epidermal stem cells express higher levels of integrins 2ß1 and
3ß1 than transit-amplifying cells and this can be used to determine the location of stem cells within the epidermis, and to isolate them directly from the tissue on the basis of rapid adherence to type IV collagen (Jones et al., 1995).
As stem cells of different tissues show certain similarities in biological behaviour, we hypothesised that they might share similar molecular properties. For instance, stem cells are usually on the basement membrane situated in a protected region, or niche, among supporting cells. In the present study we investigated whether there was a correlation between the adhesiveness of prostate cells to ECM proteins and their ability to form prostate-like glands in vivo.
The major integrins in human prostate are 2ß1,
3ß1 and
6ß4 (Bonkhoff et al., 1993; Knox et al., 1994). We focused on integrin
2ß1, which mediates adhesion to type IV collagen, type I collagen and laminin 1 and is restricted to the basal cells of the prostate (Knox et al., 1994).
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MATERIALS AND METHODS |
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Immunomagnetic positive selection of basal cells
MACS microbeads (Miltenyi Biotec Ltd, Surrey, UK), linked to anti-CD44 (Liu et al., 1997) were used to isolate basal cells from luminal cells. Epithelial cells were labelled with anti-human CD44 (clone G44-26; Pharmingen, Becton Dickinson, Oxford, UK), followed by incubation with MACS goat anti-mouse IgG microbeads. The cell suspension was then applied to a MACS column. Labelled basal cells were eluted and re-suspended in WAJC 404 complete medium (Robinson et al., 1998).
Cell adhesion to ECM proteins
CD44 positive basal cells were plated onto dishes coated with type I collagen (52 µg/ml), type IV collagen (88 µg/ml) and laminin 1 (100 µg/ml; Biocoat®, Becton Dickinson) which had previously been blocked with 0.3% bovine serum albumin (fraction V, Sigma-Aldrich, Poole, UK) in Dulbeccos phosphate buffered saline (PBS; Oxoid Ltd, Basingstoke, UK). We had determined previously that 50-100µg/ml of each protein was sufficient for maximum attachment (results not shown). After 5 minutes, dishes were washed with PBS and adherent cells were either harvested with trypsin-EDTA for grafting, or fixed in methanol at 20°C. Non-adherent cells, recovered during washing, were plated onto ECM-coated dishes and were either used for grafting or fixed in methanol at 20°C.
Basal cells were also counted and plated for the determination of colony forming efficiency (CFE). After plating, single cells were marked and examined at intervals of up to 21 days when they were subsequently fixed and colonies scored under a phase-contrast microscope. Colonies were scored if they contained >32 cells. As the number of cells selected was small, irradiated (60 Grays) STO cells were added as feeders. In control dishes the unattached cells were not removed by washing.
To determine the optimum time to select the stem cell population, basal cells were plated onto ECM-coated dishes at 37°C for 5, 20, 60 and 3 hours and the percentage of adherent, CK18-expressing cells was determined.
Xenografts
To determine the ability of rapidly-adherent basal cells to induce ductal branching morphogenesis, growth and functional cytodifferentiation in vivo, epithelial cells (1x105 to 1x106), obtained from adult prostate after immunomagnetic selection of the basal cell fraction, were directly plated onto petri dishes coated with type I collagen. Nonadherent cells were removed after 5 minutes. The adherent cells were trypsinised and combined with cultured stromal cells (passage 1-4), at a ratio of 1:1 epithelium to stroma and injected subcutaneously into 6-8 weeks old male, nude mice (strain ICRF-nu). The mice were sacrificed 6 weeks after grafting. Grafts were removed, fixed in phosphate-buffered formalin, embedded in paraffin, and sectioned. Serial sections (4 µm) were stained and examined for the development of organised prostatic glandular tissue. The capacity to differentiate in vivo to the secretory phenotype (expression of androgen receptor, prostate specific antigen (PSA) and prostatic acid phosphatase (PAP)) was taken as evidence for an epithelial stem cell population. Cells that had not adhered after 20 minutes to type I collagen were used as controls.
Immunofluorescent staining of tissue sections
Prostate cells expressing the 2-integrin subunit were identified by direct immunofluorescent staining using anti-
2 mouse monoclonal antibody (clone AK-7; TCS Biologicals Ltd, Buckingham, UK) directly conjugated with fluorescein isothiocyanate (FITC). Confocal microscopy was used to quantify fluorescence by recording the intensity of pixels along a transect through lateral cell borders. A series of 1 µm optical sections through the entire thickness of the tissue was obtained using the 60x objective of the confocal microscope, and a Z series was constructed from these sections. In order to provide a clear definition of integrin-bright cells, and account for variation in intensity as a consequence of bleaching, bright cells were defined as those in which the fluorescence intensity across cell borders was at least twice the average of other cells in the transect.
Immunocytochemical staining and FACS of isolated cells and grafts
Tissue sections taken from xenografts were fixed in formalin and paraffin-embedded. Isolated cells and colonies were fixed in methanol at 20°C. After incubating with the primary antibody, a biotinylated antibody was applied to the specimens followed by incubation with avidin-biotin complex reagents (Dako Ltd, UK). The staining was developed with diaminobenzidine tetrahydrochloride (DAB; Sigma).
The anti-cytokeratin antibodies used were 34ßE12 (which identifies cytokeratins 1, 5, 10 and 14 of the basal cell compartment in prostate; Dako Ltd), CY-90, designated CK18, which reacts with cytokeratin 18 and identifies differentiating epithelium in prostate (Sigma) (Robinson et al., 1998) and RCK 108 (designated CK19) which binds to basal and luminal epithelial cells and recognises cytokeratin 19. Anti PSA (clone ER-PR8) and anti PAP (clone PASE/4LJ) antibodies are markers of secretory luminal cells (Dako Ltd). Anti androgen receptor (clone AR27) antibody was obtained from Novocastra Laboratories Ltd, Newcastle upon Tyne, UK). The mesenchymal markers vimentin and smooth muscle actin was detected by antibodies V9 and 1A4 respectively (Sigma). Basal cells were analysed on a Becton-Dickinson FACScan. At least 10,000 events were acquired for each sample. Cells positive for propidium iodide were gated out. The antibodies used for FACS analysis were anti-
2 mouse monoclonal antibody (clone AK-7; TCS Biologicals Ltd), anti-
3 mouse monoclonal antibody (clone Mikd2; TCS Biologicals Ltd) and anti-
6 mouse monoclonal antibody (clone 4F10; TCS Biologicals Ltd). The antibodies were directly conjugated with FITC.
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RESULTS |
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Rapidly adherent cells grow more slowly in culture than total basal cells
We compared the types of colony produced by the total basal population and by basal cells selected by rapid adherence to type I collagen (Fig. 6A,B). Colonies were scored if they contained more than 32 cells after 21 days growth. We identified three types of colony. Type I colonies were founded by cells that did not divide in culture for at least 7 days. After this initial lag period they formed large colonies by 28 days (>15 cell doublings). The perimeter of these colonies was nearly circular and many small cells were observed throughout the colony and in some colonies they had concentrated around the perimeter. The interior was often stratified; the upper differentiating layers consisting of larger, flattened cells. By contrast, type II colonies grew more rapidly and were often larger than type I colonies at 21 days. The perimeter was irregular, as the colonies were heterogeneous; small cells were intermingled with differentiating, larger, flattened cells. Type III colonies were small, irregular and terminal. Such colonies contained <32 cells, and all the cells were large, squamous and terminally differentiated after a few rounds of cell division (data not shown). Although we were able to select proliferative basal cells by their rapid attachment to type collagen I, we were unable to select for type I or type II colonies. That is, cells that adhered to type I collagen, within 5 minutes formed either type I or type II colonies. By contrast, cells that attached to type I collagen after 20 minutes founded type III colonies.
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DISCUSSION |
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One important property attributed to stem cells is the ability to regenerate the different cell types that constitute the tissue in which they exist. Thus, transplanted cells should be capable of self-renewal and to produce progeny that differentiate into a fully functional epithelium. By definition, only stem cells could produce this result. The most adhesive cells in our experiments fulfilled these requirements, as they were able to regenerate a fully differentiated prostate epithelium when grafted into nude mice. By contrast, the slowly adherent cells were incapable of forming prostate epithelium in vivo. These results provide the strongest evidence yet that stem cells are present in the slowly cycling prostate epithelium and that they can be selected by high surface expression of integrin 2ß1.
As prostatic development and maintenance of function in the adult is dependent upon stromal interactions (Cunha et al., 1983), epithelial cells were grafted together with stroma to induce epithelial morphogenesis and cytodifferentiation. Although we cannot comment on the nature of this interaction without further study, it was interesting to note that the stromal cells closely associated with the grafts were smooth muscle cells, which begs the question of whether this phenotype is induced by the epithelium (Hayward et al., 1992).
In classical self-renewing tissue, stem cells reside in an optimal microenvironment, or niche. The dividing cells are located in one place and the stem cells lie elsewhere. The histological structure of most epithelia is clearly composed of structural units, and the number of stem cells is directly related to tissue architecture (Slack, 2000). In the epidermis, for example, stem cells reside at the tips of the deep rete ridges in palm and can be located by their high surface expression of integrins 2ß1 and
3ß1. Integrins hold cells in the right place in a tissue, and loss or alteration of integrin expression ensures departure from the stem cell niche through differentiation or apoptosis (Zhu et al., 1999). In our study we examined the position and number of integrin-bright cells within prostatic acini. However, it should be noted that we used tissue from patients that would have had some degree of BPH and that the number and position of stem cells within acini may not reflect the picture in normal prostate. Integrin-bright cells were not confined to tips of acini, for example, but were randomly located throughout each acinus unlike the epidermis, where the integrin-bright cells are arranged in clusters (Jones et al., 1995); no more than one bright cell was found together in acini examined. Approximately 1% of cells were classed as integrin bright by confocal microscopy, but this number was nearer 15% by flow cytometry; however, it is difficult to compare the two systems. From the confocal images we defined
2-integrin-bright cells as those in which the fluorescence intensity across cell borders was at least twice the average of other cells in the transect, whereas all the cells that bound to type I collagen within 5 minutes were classed as
2-integrin bright. However, it is clear that we cannot determine the number of stem cells by quantifying expression of a single cell surface marker. For example, 40% of cells in the basal layer of the epidermis are integrin
2 bright, yet kinetic analysis would predict that the percentage of stem cells is closer to 10% (Potten and Morris, 1988). Kaur and Li recently reported that epidermal cells that had begun to differentiate within the basal layer retain high levels of activated ß1-integrin, but downregulate
6ß4 expression (Kaur and Li, 2000). These cells retain their adhesive capacity, indicating that induction of differentiation does not correlate with decreased ß1 expression or function. By contrast, however, we found that in prostate epithelia the rapidly adherent population also express high levels of integrins
3 and
6. Mutagenesis studies in which a visible cell label can be produced will be required to resolve the clonal makeup of prostate epithelia as well as the spatial relationship between stem cells and their progeny.
In conclusion we have shown that cells that express high levels of the 2-integrin subunit have properties that indicate that they are equivalent to the stem cells of the prostate as they have the potential to establish and maintain a prostate epithelium similar to that found in vivo, with associated secretory activity. These findings are in agreement with several reports that the stem cell populations of other self-renewing tissues express similar ß1-integrin molecules (Jones et al., 1995; Hirsch et al., 1996; Jacques et al., 1998). The degree of enrichment of stem cells attainable by this method will allow further fractionation and analysis of the stem cell population to identify a set of additional markers unique for prostate epithelial stem cells. A systematic evaluation of surface molecules on the stem cells will facilitate identification and purification of these cells and greatly contribute to our understanding of pathways governing proliferative regulation and differentiation. Most importantly, stem cell research will provide a foundation for therapeutic advancement in the treatment of prostate cancer.
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ACKNOWLEDGMENTS |
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