Am J Physiol Renal Physiol 308: F1421–F1430, 2015. First published April 29, 2015; doi:10.1152/ajprenal.00488.2014.

Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate Liang Wang,1,6 Marloes Zoetemelk,1 Brahmananda R. Chitteti,2 Timothy L. Ratliff,3,6 Jason D. Myers,1 Edward F. Srour,2,4,5,6 Hal Broxmeyer,5,6 and Travis J. Jerde1,6 1

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana; 2Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana; 3Department of Comparative Pathobiology, Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana; 4Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana; 5Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana; and 6Melvin and Bren Simon Cancer Center-Indiana Basic Urological Research Working Group, Indiana University, Indianapolis, Indiana Submitted 29 August 2014; accepted in final form 23 April 2015

Wang L, Zoetemelk M, Chitteti BR, Ratliff TL, Myers JD, Srour EF, Broxmeyer H, Jerde TJ. Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate. Am J Physiol Renal Physiol 308: F1421–F1430, 2015. First published April 29, 2015; doi:10.1152/ajprenal.00488.2014.—Prostatic inflammation is a nearly ubiquitous pathological feature observed in specimens from benign prostate hyperplasia and prostate cancer patients. The microenvironment of the inflamed prostate is highly reactive, and epithelial hyperplasia is a hallmark feature of inflamed prostates. How inflammation orchestrates epithelial proliferation as part of its repair and recovery action is not well understood. Here, we report that a novel epithelial progenitor cell population is induced to expand during inflammation. We used sphere culture assays, immunofluorescence, and flow cytometry to show that this population is increased in bacterially induced inflamed mouse prostates relative to naïve control prostates. We confirmed from previous reports that this population exclusively possesses the ability to regrow entire prostatic structures from single cell culture using renal grafts. In addition, putative progenitor cells harvested from inflamed animals have greater aggregation capacity than those isolated from naïve control prostates. Expansion of this critical cell population requires IL-1 signaling, as IL-1 receptor 1-null mice exhibit inflammation similar to wild-type inflamed animals but exhibit significantly reduced progenitor cell proliferation and hyperplasia. These data demonstrate that inflammation promotes hyperplasia in the mouse prostatic epithelium by inducing the expansion of a selected epithelial progenitor cell population in an IL-1 receptor-dependent manner. These findings may have significant impact on our understanding of how inflammation promotes proliferative diseases such as benign prostatic hyperplasia and prostate cancer, both of which depend on expansion of cells that exhibit a progenitor-like nature. hyperplasia; inflammation; interleukin-1; progenitor; prostate PROSTATIC INFLAMMATION is a critical area of urological research due to its common colocalization with prostate cancer and benign prostatic hyperplasia (BPH) (3, 9). Inflammation in the human prostate is associated with dysplastic changes including focal disruption of the epithelium, polymorphisms of epithelial cell nuclei, and increased epithelial proliferation (3, 6, 9). Inflammation in the prostate is manifested by leukocytic infiltration and the release of pro-inflammatory cytokines, chemokines, prostanoids, and growth factors (7). Initial causes of

Address for reprint requests and other correspondence: T. J. Jerde, IU Simon Cancer Center, A417 VanNuys Medical Sciences Bldg., 635 Barnhill Drive, Indianapolis, IN 46202 (e-mail: [email protected]). http://www.ajprenal.org

inflammation in the prostate remain unclear and are likely multifactorial, and proposed initiating factors have included infection from culturable and nonculturable organisms (18, 26) as well as numerous nonbacterial potential causes, including viruses, environmental and dietary components, systemic steroid action (especially estrogens), oxidative stress, systemic inflammation associated with metabolic syndrome, and urinary reflux of noxious stimuli into the prostatic ducts (12). A number of potential chemicals in urine may represent a major inflammatory stimulus in the prostate. However inflammation is induced, the mechanistic understanding of how prostatic inflammation promotes the survival and growth of prostate cancer and benign prostatic hyperplasia is a major gap that impedes the development of superior therapies to BPH and prostate cancer. BPH is commonly defined as a benign enlargement or growth of the prostate gland (35), but the clinical manifestation of BPH is a characterized symptom profile known as “lower urinary tract symptoms.” As a hyperplastic disease, BPH also includes induced benign proliferation of both epithelial and stromal compartments of the tissue (35). BPH tissues exhibit histologically evident severe and widespread inflammation; the overall prevalence of inflammation in BPH specimens published in the existing literature ranges from 75% to 100% (10, 27, 29, 37). One recent study (10) of autopsy specimens found widespread chronic inflammation in 75% of prostates with histological evidence of BPH, whereas only 50% of prostates not affected by BPH exhibited inflammation. A supporting study (29) found substantial inflammation in all tissues obtained from 80 men undergoing prostatectomy as a treatment for symptomatic BPH. In addition, 78% of 8,224 prostate biopsies from men enrolled in the REDUCE trial exhibited inflammation. BPH-associated inflammation exhibits an abundance of T cells and macrophages and a high abundance of inflammatory cytokines including IL-1, IL-6, and IL-8, which are known to induce prostatic proliferation and growth (27, 37). Notably, the most tightly correlated histological finding to prostate symptomology and prostate volume is histologically evident prostatic inflammation (28). These reports demonstrate a clear association of inflammation with BPH, suggest a significant role for inflammation in BPH, and demonstrate the need for a mechanistic understanding. We have previously shown that activation of signaling pathways shared by inflammation and organ development is necessary for inflammation-induced prostate epithelial hyper-

1931-857X/15 Copyright © 2015 the American Physiological Society

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plasia (16). The similarities between tissue development and adult tissue regeneration suggested a critical role of pluripotent cells in inflammation-induced tissue repair. Pluripotent prostate epithelial progenitor cells have been identified in normal prostate, BPH, and prostate cancer tissues (25, 33). They were capable of regenerating other prostate cells, such as basal cell and luminal cells, in vitro or in vivo (19, 21, 24). However, their responses to inflammation and their roles in the pathogenesis of prostate diseases are still unknown. Therefore, the role of epithelial progenitor cells in prostate inflammation needs to be further investigated. We propose that a unique population of progenitor cells can respond to inflammation by proliferation and eventual differentiation to compensate for cell loss during inflammation. To investigate this hypothesis, we examined the progenitor characteristics of isolated “four-marker” epithelial cells that have been shown to be capable of regenerating entire functioning prostatic structures in grafts in vivo (21). These four-marker cells express Sca-1, CD44, CD133, and c-Kit (CD117). Since we have already shown that IL-1 receptor 1 (IL-1R1) signaling is essential for both prostate development and inflammationinduced epithelial cells (16, 11), we also postulated that progenitor cell expansion by inflammation is IL-1 signaling dependent. MATERIALS AND METHODS

In vivo induction of inflammation, proliferation, and assessment of reactive hyperplasia. All animal experiments were conducted under the approval and supervision of the Animal Care and Use Committee of the Indiana University School of Medicine and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Esherichia coli strain 1677 (2 ⫻ 106 bacteria/ml, 100 ␮l/mouse) was instilled through catheters into the urinary tract of C57BL/6J wild-type (WT) and IL-1R1⫺/⫺ mice (The Jackson Laboratory, Bar Harbor, ME; verified by genotyping) at 8 wk of age as previously described (2, 16). Mice were inoculated with 100 ␮g of bromodeoxyuridine (BrdU; Roche) 2 h before euthanization, and groups were euthanized daily 1–7 days after bacterial induction. PBS-instilled animals were used as naïve controls. Prostates were harvested for prostate epithelial cell preparation or fixed (4% paraformaldehyde at 4°C for 24 h) for immunofluorescences assay. Prostate epithelial cell preparation. Mouse prostates were washed with PBS and cut into ⬃1-mm3 segments in collagenase (1% collagenase in DMEM supplied with 5% serum, 1% antibiotics, and 1% HEPES). Tissues were then subjected to three steps of 1% collagenase digestion of 30 min each followed by three steps of 1% trypsin digestion, again for 30 min each. Cell suspensions were washed three times with PBS with centrifugation to collect cells. The collected slurry was then filtered through a 40-␮m filter (BD, San Jose, CA) to collect single cell suspensions for further experiments. All cells were then plated on polypropylene tissue culture dishes for 12 h, time for stromal cells to attach but sufficiently short for epithelial cells to remain unattached. The collected supernatant was then used for experimentation as described below. Flow cytometry analysis/sorting of four-marker progenitor cells. Single prostate cell suspension was washed with stain wash buffer (PBS supplemented with 1% serum and 1% antibiotics) twice. Cell concentrations were counted, and cells were treated with excess (2 ␮l/107 cells) of the following conjugated antibodies for the isolation of four-marker cells (20): lineage markers (phycoerythrin-conjugated CD45R, CD31, Ter119, CD5, Ly-6G, Ly-6C, CD11b, PerCp-Cy5.5conjugated Sca-1, allophycocyanin-conjugated CD117, FITC-conjugated CD133, and allophycocyanin-Cy7 conjugated-CD44, all Becton-Dickinson, BD Biosciences) on ice for 15 min. Cells were washed

and resuspended in stain wash buffer for flow cytometry analysis (BD LSRII) or sorting (BD FACS ARIA). Prostasphere formation assay. Sphere-forming prostatic epithelial cells were collected and cultured as previously described (36). A single prostate cell suspension isolated as above was cultured in growth medium (DMEM supplemented with 10% serum, 1% antibiotics, and 1% HEPES) for 6 h (37°C/5% CO2) to attach stromal cells. Unattached epithelial cells were collected, washed with PBS, and resuspended in sphere growth medium (DMEM supplied with 20 ng/ml EGF, 10 ng/FGF, 1% HEPES, 1% antibiotics, and 2% B27 supplement, GIBCO). Cells were cultured in 60 mm low-attachment culture plates (Corning) at a concentration of 10,000 cells/ml and 3 ml/dish for 21 days (37°C/5% CO2). Pictures were taken on day 21 of culture; sphere diameters were measured by Photoshop CS and normalized by single cell diameter. Four-marker and nonfour-marker prostate epithelial cells were sorted by flow cytometry into lowattachment 96-well plates containing sphere growth medium at a concentration of 100 cells/100 ␮l per well. Pictures were taken on day 7 of culture. Sphere diameters were measured by Photoshop CS. Sphere volumes were calculated and normalized by single cell volume. For dual color sphere formation, prostate epithelial cells were collected and cultured as previously described. Prostate epithelial cells from noninflamed green fluorescent protein (GFP)-expressing mice and noninflamed dtTomato-red fluorescent protein (RFP)-expressing mice or cells from inflamed GFP-expressing mice and inflamed dtTomato-RFP-expressing mice were mixed at a 1:1 ratio to make a solution with a total cell concentration of 5,000 GFP-expressing cells/ml (1.5 ml) and 5,000 dtTomato-RFP-expressing cells/ml (1.5 ml) and cultured in a 3 ml total volume in low-attachment dishes (3 ml/dish) for 21 days. Pictures were taken on day 21 of culture using fluorescence microscopy (Zeiss), and sphere diameters were measured by Photoshop CS. Single four-marker progenitor cell-derived spheres in renal capsule implantation. Renal grafts from single prostatic four-marker progenitor and nonprogenitor prostate epithelial cells were performed as previously published (21). Single four-marker cells from inflamed and control mice were sorted by flow cytometry into low-attachment 96-well plates containing implantation medium (50 ␮l/well, DMEM supplied with 20 ng/ml EGF, 10 ng/ml FGF, 1% HEPES, and 1% antibiotics). Single spheres were mixed with 8,000 urogenital mesenchymal cells in transplant medium (50 ␮l/well) and cultured for 3 days in 50 ␮l/well volumes in DMEM supplied with 20 ng/ml EGF, 10 ng/ml FGF, 1% HEPES, 1% antibiotics, and 4 ␮g/ml Matrigel. The formed Matrigel plugs were then implanted surgically under the renal capsules of nude mice (CD-1 background) such that one plug was inserted into each kidney. Mice were euthanized on day 60 after transplantation. Kidneys were harvested and fixed in 4% paraformaldehyde (Fisher Scientific) at 4°C for 72 h followed by routine histological processing. Immunofluorescence. Fixed tissues were processed by dehydration through an ethanol gradient to xylene and embedded into paraffin blocks. Five-micrometer sections were cut via a microtome and fixed to glass slides. Sections were rehydrated through xylene and methanol to an aqueous phase in water. Antigen retrieval was performed in boiled citrate buffer for 20 min and washed in PBS ⫹ 0.025% Tween (PBST). Sections were blocked with blocking buffer (10% donkey serum ⫹ 1% BSA in PBST) for 2 h at room temperature. Sections were then stained with one of the following primary antibodies: anti-probasin (Cell Signaling, 1:100) or anti-RFP (Abcam, 1:50) overnight in a humidified chamber at 4°C. After sections had been washed with PBST, secondary antibodies (1:200) were applied for 1 h at room temperature. After a serial wash with PBT, sections were incubated with 20 ␮g/ml Hoechst 33342 in PBST for 10 min. Sections were washed and mounted by Aqua mounting medium and glass coverslips. Pictures were taken by fluorescence microscope (Zeiss). Statistical considerations. One-way ANOVA was performed to determine progenitor cell analysis. Student t-test was performed to

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determine sphere number and sphere size change. Two-way ANOVA was performed to determine the effect of IL-1R1 knockout and inflammation on epithelial progenitor cell populations. When appropriate (two-sided P ⬍ 0.05), pairwise comparisons applying Fisher’s exact test were calculated. Ethical approval declarations. All animal experiments were approved by the appropriate Institutional Review Board and conducted in strict adherence to protocols approved by the Indiana University School of Medicine and University of Wisconsin-Madison Animal Care and Use Committees and in accordance with National Institutes of Health guidelines for animal research. RESULTS

Epithelial cells cultured from inflamed prostates exhibit greater sphere-growing capacity than those from control prostates. Acute inflammation of 3 days increased the proportion of epithelial cells capable of forming large spheres in the mouse prostate but not total sphere number per 10,000 epithelial cells (Fig. 1). Isolated epithelial cells from inflamed and control mice were grown in anchorage-independent conditions for 21 days at a density of 10,000 cells/10 ml culture. After growth, cell groups meeting the criteria for counting as a “sphere” (⬎3 cells in diameter) were counted by microscopy, and their size (as measured by the number of cells in diameter in a two-dimentional plane) was determined. The total number of spheres per 10,000 cells was not significantly different between epithelial cells cultured from inflamed and control prostates (Fig. 1A). However, the number of spheres of intermediate size (5–20 cells in diameter) was increased 3-fold from 10 spheres formed from 10,000 cells to 31, and the number of very large (⬎20 cells in diameter) spheres increased 10-fold from 1 cell per 10,000 to 10 (Fig. 1, B and C). The average diameter of spheres formed by noninflamed prostate cells was

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4.3 cells, whereas the average diameter of inflamed spheres was 6.8 cells (Fig. 1D). A representative sphere image from control mice is shown in Fig. 1E, whereas Fig. 1F shows a typical very large sphere cultured from an inflamed mouse, representative of those quantified in Fig. 1C. To determine if there were any cell size differences between individual cells growing in these spheres, we assessed the size of the cells within the growing spheres and found that cells from 20 to 30 spheres cultured from 3 mice from both inflamed and control mice exhibited no difference in cell size between inflamed and naïve mice (n ⫽ 3, P ⫽ 0.86). These data demonstrate that total sphere number was not increased in cells cultured from inflamed prostates but suggest that epithelial cells cultured from inflamed prostates had the capacity to form larger spheres than those from control prostates. Interestingly, BrdU labeling of spheres 1 h before fixing followed by embedding, sectioning, and immunofluorescence for BrdU labeling did not reveal an increase in proliferation within these spheres (data not shown), but the total number of cells cultured from all spheres was increased in spheres cultured from inflamed mice relative to control mice. Larger spheres exhibit adhesive qualities in vitro. To determine if the larger spheres cultured from inflamed prostates exhibit adhesive qualities in vitro, we isolated cells from control or inflamed prostates from GFP- and dtTomato-RFPexpressing mice and cocultured both under anchorage-independent conditions mixing 7,500 cells (5,000 cells/ml, 1.5 ml) from GFP-expressing mice with 7,500 cells (5,000 cells/ml, 1.5 ml) from dtTomato-RFP-expressing mice for 7 days (Fig. 2, A–D). This was performed for both inflamed and control mice. Fluorescent images from these spheres showed that the majority of spheres contained cells from both mice, indicating that

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Fig. 1. Inflammation increases the proportion of epithelial cells capable of forming large spheres in the mouse prostate but not the total sphere number. Isolated epithelial cells from inflamed and control mice were grown in anchorageindependent conditions for 21 days at a density of 10,000 cells/10 ml culture. After 21 days, the number of cell groups that met the criteria for being called a sphere (⬎3 cells in diameter) were counted and their size was determined by microscopy (n ⫽ 6). A: the total number of spheres per 10,000 cells did not significantly increase with inflammation. B: the number of spheres 5–20 cells in diameter was increased 3-fold. C: the number of very large (⬎20 cells in diameter) spheres increased 10-fold. D: the average diameter of spheres cultured from inflamed prostates increased significantly compared with controls. E: example spheres from control prostates. F: example of a very large sphere (⬎10 cells in diameter) from an inflamed prostate as quantified in Fig. 1C. *P ⬍ 0.05 inflamed vs. control (n ⫽ 6, ANOVA).

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Sphere Size (Number of cells in diameter) Fig. 2. Larger spheres exhibit adhesive qualities in vitro. A–D: fluorescent images of spheres isolated from control and inflamed prostates from green fluorescent protein (GFP)- and dtTomato-red fluorescent protein (RFP)-expressing mice and cultured in anchorage-independent conditions for 7 days. A: spheres cultured from control GFP-expressing mice. B and C: spheres cultured from inflamed GFP-expressing (B) and dtTomato-RFP-expressing (C) mice. D: spheres cultured from a 1:1 mix of cells from GFP- and dtTomato-RFP-expressing mice, illustrating spheres with cells from multiple mice. E: the majority of spheres cultured contained cells from both mice, indicating cell-cell aggregation, and all spheres with a diameter ⱖ8 cells had both GFP and RFP components.

epithelial cells cultured in sphere culture are aggregating cells. In particular, all spheres of a size of eight cells in diameter or larger had both GFP and RFP components, indicating that the larger spheres were made up of multiple potential sphereforming cells (Fig. 2E) and suggesting that cell aggregation contributes to larger sphere formation. Flow cytometry analysis of the four-marker putative progenitor population demonstrates expanded progenitor cells during prostate inflammation. Since the sphere data suggested an increase in cells capable of forming spheres and increased aggregation of spheres, we sought to determine if inflammation increases the percentage of epithelial cells expressing the bona fide progenitor four-marker phenotype (Lin⫺, Sca-1⫹, CD44⫹, CD133⫹, c-Kit⫹) panel (putative progenitor cells) in the prostate. These cells have previously been shown to be capable of forming prostatic structure from a single cell (21). We isolated epithelial cells from control and inflamed mouse prostates each day during the 5-day inflammation induction process and analyzed them by flow cytometry for cells positive for the critical four-marker panel (Fig. 3). The number of four-marker cells was significantly increased 3 days after the induction of inflammation (Fig. 3A). As several other publications have used the integrin CD49F as a stem cell marker, we analyzed this marker as a percentage of the four-marker population and found that 100% of the four-marker population also expressed CD49F (Fig. 3B). Correspondingly, every four-marker cell was positive for both CD49F and Sca-1, whereas only 29% of the

total epithelial population is positive for these two markers. This demonstrates that a Sca-1/CD49F panel does include the four-marker cell population but does not exclusively contain four-marker cells. When using this model of inflammation, we typically use histology and hematoxylin and eosin staining to confirm that we adequately inflamed each prostate to allow adequate analysis of prostatic inflammation. This is usually accomplished by scoring the inflammatory cell number to criteria met in our previously published study (2). In the case of isolation of four-marker cells, the entire prostate needed to be used to isolate an adequate number of cells for analysis. As a surrogate for histological confirmation of inflammation criteria, we calculated the percentage of hematopoietic Lin⫹ cells in inflamed prostates, as inflammatory cells express at least one of the Lin⫹ markers. Preliminary experiments indicated that prostates reaching inflamed criteria histologically described by Boehm et al. (2) had at least 50% of the total live cell population being of the Lin⫹ fraction. Control prostates typically contained ⬍20% of the total live cell population that were Lin⫹. We then sought to determine if the four-marker cell population correlated to the severity of inflammation using Lin⫹ cell percentage as the measure of inflammatory severity and found that four-marker cells correlated with Lin⫹ cell percentage, with a Pearson correlation coefficient of 0.957 (Fig. 3C). In this large analysis of 10 inflamed and 13 control prostates, one of the control prostates contained 32% of its live cells as Lin⫹,

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Fig. 3. Inflammation increases the number of four-marker (Lin⫺, Sca-1⫹, CD44⫹, CD133⫹, c-Kit⫹) putative progenitor cells in the prostate. A: isolated epithelial cells from control and inflamed mouse prostates from days 1 to 5 of inflammation were analyzed by flow cytometry for cells positive for the four-marker panel (n ⫽ 6). The number of four-marker cells was significantly increased 3 days postinflammation. *P ⬍ 0.05 vs. control (n ⫽ 6, one-way ANOVA). B: flow cytometry data comparing the expression of CD49F (an alternate progenitor cell marker) in the four-marker population relative to that in total epithelial cells. C: correlation data representing the number of four-marker cells plotted with the number of hematopoietic Lin⫹ cells in the same prostate, as an indicator of inflammation severity. Four-marker cells correlated with Lin⫹ cell number, suggesting that four-marker cell abundance correlates with inflammation severity. Pearson correlation coefficient ⫽ 0.957, n ⫽ 10 inflamed prostates and 13 control prostate. #Four-marker cells from a control mouse that did not meet the criteria of being noninflamed (⬍20% Lin⫹ cells/total viable cells). D: sorted four-marker cells have greater sphere-forming capacity than total epithelial cells do, but those isolated from inflamed prostates have no greater sphere-forming capacity than those from control prostates (n ⫽ 4). PEPC, primary epithelial prostate cells.

putting it outside the criteria for “control.” As an internal control to verify the induction of inflammation, we analyzed the Lin⫹ cell population as a percentage of total living cells and found that the percentage of Lin⫹ cells peaked on day 3 after inflammation, when an average of 60% of the living cells were Lin⫹, indicating substantial inflammation (data not shown). By 5 days after inflammation, the Lin⫹ fraction had reduced to ⬍20% of living cells. To determine if sorted four-marker cells from inflamed prostates had greater sphere-forming capacity compared with those sorted from control prostates, we compared the number of isolated epithelial cells and number of four-marker cells (sorted by flow cytometry) that had the capacity to form spheres of at least five cells in diameter. As expected, fourmarker cells had much greater sphere-forming capacity than the cell population not expressing all four markers; however, we found no difference between those isolated from inflamed and control prostates (Fig. 3D). Taken in total, the results shown in Figs. 2 and 3 demonstrate that inflammation increases the number of four-marker cells in the mouse prostate and that isolated four-marker cells from inflamed prostates have a greater tendency to aggregate compared with those isolated from control prostates. No difference was found in the ability of four-marker cells to form spheres between inflamed and control groups. Four-marker cells have the capacity to form prostatic structures in a renal graft from a single cell. To verify that four-marker cells were enriched for stem/progenitor capability,

we isolated single four-marker cells by flow sorting, grew them into spheres in anchorage-independent conditions, combined them in reduced Matrigel with urogenital mesenchymal cells from dtTomato-RFP-expressing mice, and implanted the resulting pellet under the renal capsule for 60 days (Fig. 4). We implanted 10 spheres from four-marker cells cultured from inflamed and control mouse prostates in this method and compared them with nonfour-marker cells (epithelial cells that do not express all four markers). In spheres grown from cultured four-marker cells, 7 of 10 implants grew prostatic structures, as shown in Fig. 4, whereas no implants from the nonfour-marker population were able to grow prostatic structures. There was no difference in the graft growing ability between implants from control or inflamed prostates. We verified these epithelial structures as prostatic by probasin immunofluorescence. As others have previously published (17), probasin is localized on the luminal secretory side of the epithelial structures (Fig. 4D). To demonstrate that the epithelium did not grow from UGM support cells in the graft, we stained for dt-Tomato and found that no epithelial cells expressed this RFP protein, whereas fibroblasts in the resulting stroma did, as expected. c-Kit⫹ cells expand from the basal to luminal compartment during inflammation. While the total percentage of isolated cells concurrently expressing all four markers in the normal mouse prostate was 0.2%, marker expression individually was as follows: Sca-1 was expressed in 32% (⫾8.6%) of isolated cells, CD44 in 40% (⫾12.2%), CD133 in 23% (⫾7.8%), and

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mice. IL-1R1 is the receptor for IL-1␣, IL-1␤, IL-1F10, and possibly IL-1F8. It is also the receptor that is antagonized by IL-1F3, also known as IL-1RA. Given that 3 days was the time point of inflammation exhibiting maximal induction of progenitor cells, we inflamed WT and IL-1R1⫺/⫺ mice for 3 days and analyzed them for c-Kit⫹ cell expansion by immunofluorescence or four-marker cell expansion by flow analysis (Fig. 6). The inflammation-induced c-Kit⫹ population, as measured by immunofluorescence, and the total four-marker population, as measured by flow cytometry, were partially attenuated by genetic loss of IL-1R1, indicating that IL-1R signaling is at least partially involved in prostate progenitor cell expansion. DISCUSSION

Fig. 4. Four-marker cells have the capacity to form prostatic structures in a renal graft from a single cell. Four-marker cells were isolated into single cells by flow sorting and grown into spheres in anchorage-independent conditions. Spheres were then combined with urogenital mesenchymal cells from dtTomato-expressing mice and implanted under the renal capsule. A–C: hematoxylin and eosin-stained sections of an example graft that grew into a prostatic structure at ⫻10 (A), ⫻20 (B), and ⫻40 (C) magnification. D: immunofluorescence imaging demonstrating prostatic structure-expressing probasin (red) off the luminal epithelium and dtTomato (green) in a fibroblast population.

c-Kit (CD-117) in 1.6% (⫾0.6%) of isolated cells. As such, the most selective of the four markers used to isolate four-marker cells was c-Kit. To follow the localization of c-Kit⫹ cells during inflammation-induced hyperplasia, we stained sections from our inflammation time course by immunofluorescence (Fig. 5A). c-Kit⫹ cells are a rare population of cells exclusively found in the basal layer of epithelium in the control prostate. These cells expand beyond the basal layer during 2 and 3 days of inflammation into the luminal and hyperplastic “intermediate” layers (defined loosely as cells in between basal and luminal layers in hyperplastic prostate glands). Expansion occurs in “nests” where upon proliferation promoted by hyperplasia, the c-Kit⫹ population exists as multiple positive cells grouped together (Fig. 5, B and C, arrows)., Mitotic c-Kit⫹ cells are particularly demonstrated in Fig. 5C. The total number of c-Kit⫹ cells maximized at 3 days postinflammation and, like four-marker cells, decreased thereafter. The inflammationinduced increase of the c-Kit⫹ population by immunofluorescence well replicated the expansion calculated by flow analysis (Fig. 5D). We stained prostates from BrdU-labeled control (Fig. 5E) and 2-day inflamed (Fig. 5F) mice for c-Kit and BrdU to determine if c-Kit⫹ cells were selectively proliferative in the prostate. We found that 46% of c-Kit⫹ cells from inflamed prostates was also BrdU positive, indicating that c-Kit⫹ cells are proliferating. In contrast, no c-Kit⫹ cells were proliferating in control mice (n ⫽ 4, P ⫽ 0.03). The progenitor cell increase is IL-1R signaling dependent. To determine if increases in prostate progenitor cells as a result of inflammation are mechanistically similar to the IL-driven epithelial expansion observed in prostate development and reactive hyperplasia (11, 16), we compared c-Kit⫹ cell increases in mice lacking the IL-1R with those in C57BL/6J WT

Our data indicate that the number of functional prostate epithelial progenitor cells is induced substantially by inflammation in mouse prostates. This is indicated by the increased growth capacity of sphere-forming cells isolated from inflamed prostates, by the increased number of previously verified (21) four-marker cells, and by our demonstration that these specialized cells have the capacity to generate functional and secreting prostatic structures when grown in graft. Inflammation increased the number of four-marker cells from 0.2% of isolated prostatic epithelial cells sixfold to 1.2% at day 3 after inflammation and returned to basal levels by day 5. This decline of four-marker cells suggests that four-marker cells may be differentiating to other epithelial cells (most likely transit amplifying cells), losing their stem cell markers, and thus no longer are recognized as four-marker cells by flow cytometry. The current experiments do not evaluate this, nor provide a mechanism for this proposed differentiation. To determine this, future studies of lineage tracing differentiated cells that repopulate the tissue secondary to inflammation would have to be performed, and future work will be directed to this question. We further found that the most rigorous of the four-marker panel, c-Kit, was found exclusively in the basal epithelial layer (the layer proposed to harbor tissue stem cells in the normal prostate) and that c-Kit positivity increased laterally upon inflammation from niche regions of the basal layer, upward toward the luminal surface of the epithelium. This proliferation is likely to be a significant part of the repair and recovery process that the damaged inflamed tissue undergoes to repopulate the tissue. In addition, inflammation increased the BrdU⫹ positivity rate of c-Kit⫹ cells from 0% of identified cells to 46% by the second day of inflammation, indicating a proliferation of these cells. However, given that we also show increased aggregation capacity in vitro for these cells, the possibility that the “nests” of c-Kit⫹ cells in the basal layer of the epithelium might be due to an aggregation of positive cells cannot be ruled out with the present study. Future work will include linage-tracing studies aimed at determining if these collections of c-Kit⫹ cells represent an expansion of progenitor cells or if aggregation of positive cells is responsible for these collections of cells. Our first experiments of this work involved sphere-forming culture in which the isolated epithelium was grown in anchorage-independent conditions. The ability to grow spheres in serum-free conditions is a hallmark feature of cells with progenitor cell characteristics (20), and this has been described for four-marker cells (36, 38, 39). Surprisingly, our results initially

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14

% of Epithelial Cells C-Kit-Positive

Calculated by Immunofluorescence 12 Calculated by Flow Cytometry 10

8

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Fig. 5. c-Kit⫹ cells expand from the basal to luminal compartment during inflammation. A: immunofluorescence image (c-Kit, green; pan-cytokeratin, red; yellow arrows denote dual-stained cells) demonstrating a rare c-Kit⫹ cell in the basal layer of a control prostatic duct. B and C: c-Kit⫹ cells expand beyond the basal layer during 2 and 3 days of inflammation into the intermediate and luminal layers. D: calculations for expanded c-Kit⫹ cells, as measured by immunofluorescence and flow cytometry, demonstrating the consistency of the results. Four ⫻20 fields were counted per specimen for each single data point, and there were 6 prostates in each group. *P ⬍ 0.05 vs. control prostates (by ANOVA). n ⫽ 4 for immunofluorescence and 6 for flow cytometry. E and F: costaining of c-Kit⫹ (green) and bromodeoxyuridine (BrdU)⫹ (red) in control (E) and 2-day inflamed (F) prostates indicating that half of the c-Kit⫹ cells were also proliferative (BrdU⫹), whereas we did not observe BrdU⫹/c-Kit⫹ cells in control prostates. Green arrows indicate c-Kit⫹ BrdU⫺ cells; magenta arrows indicate BrdU⫹/c-Kit⫹ cells.

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c-Kit+ cells (Percent of Epithelium)

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INFLAMMATION INDUCES PROSTATIC EPITHELIAL PROGENITOR CELLS

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Strain of Mouse Fig. 6. Mice lacking intact IL-1 receptor (IL-1R) exhibit decreased expansion of putative progenitor cells. A: the inflammation-induced c-Kit⫹ population was attenuated by loss of IL-1Rs, as a percentage of total epithelial cells. *P ⬍ 0.05, wild-type vs. IL-1R1⫺/⫺ mice. n ⫽ 6. B: the 3-day inflamed four-marker cell population was attenuated in IL-1R1⫺/⫺ mice, as a percentage of epithelial cells. *P ⬍ 0.05, inflamed vs. control tissues; #P ⬍ 0.05, wild-type vs. IL-1R1⫺/⫺ inflamed tissues. n ⫽ 5– 8.

did not show an increase in the total sphere-forming isolates from inflamed prostates, but the spheres that did grow demonstrated what appeared to be greatly enhanced growth capacity. Paradoxically, BrdU labeling of these spheres and subsequent immunofluorescence did not demonstrate a significant increase of proliferation within these spheres compared with control prostates. Furthermore, the significant increase in the fourmarker population was clear evidence of increased cells that had previously been shown to have sphere-forming capacity. This disconnect led us to consider that the increase in sphere size could be due to aggregation of the spheres in culture, resulting in larger spheres and an underestimation of sphereforming isolated cells. To investigate this, we cultured spheres in a 1:1 ratio of epithelial cells from GFP-expressing mice and dtTomato-RFP-expressing mice and found that all of the large spheres (⬎8 cells in diameter) contained both green- and red-fluorescing regions, some with multiple elements of each color. This indicated to us that aggregation was occurring and was more frequent in isolates from inflamed prostates. The mechanism for why aggregation of growing spheres is increased in cells isolated from inflamed prostates is unclear, and a detailed investigation of this is beyond the scope of this study. Our sphere cell recovery experiment suggested a higher number of sphere-forming cells in the inflamed group, and this may increase the chance of cellular aggregation. However, it is certainly possible that more mechanisms may be involved in sphere formation regulation, including modified integrin signaling or matrix protein secretion. This will be an active area of investigation for future studies.

The existence of prostate progenitor cells was postulated after the findings that normal prostate regeneration can occur after repeated cycles of androgen ablation and readdition (14). Several cell surface marker panels have been identified as expressed on prostate cells with stem characteristics, including Sca-1, CD133, CD44, and CD-117/c-Kit (4, 5, 19, 21). In particular, Gao and associates (21) determined that the c-Kit stem cell factor receptor was identified as a rare adult mouse progenitor cell marker, and the addition of this marker to the isolation panel has been shown to be critical in isolating cells that have the capacity to generate a prostate from a single cell after transplantation in vivo. As such, c-Kit⫹ cells could generate functional secretion-producing prostates when transplanted in vivo, and these cells have long-term self-renewal capacity, as evidenced by serial isolation and transplantation in vivo. In that large study, 25% of isolated four-marker cells had the capacity to generate functional prostate from a single cell, whereas no cells that did not express the full panel, most notably c-Kit, had this capacity. To verify in our study that four-marker cells were indeed functional prostate progenitor cells, we performed a smaller confirmatory study of implants from single cell isolates, including the full four-marker panel, and compared them with isolates missing the critical c-Kit marker. In our study, 7 of 10 four marker-positive isolates grew functional prostate grafts, whereas no grafts arose from c-Kit⫺ isolates. No difference was observed in graft growing capacity between inflamed and control prostates. This confirms the four-marker panel as capable of identifying epithelial progenitor cells. Other surface marker panels have been used in the isolation of cells with progenitor cell characteristics, most notably the Sca-1 marker combined with CD49F, as previously described by Lukacs et al. (23). However, this combination was not shown to have the capacity to grow complete functional prostatic prostates from single cell isolates but does produce significant enrichment of the cells that have progenitor characteristics, including prostate graft growth from large cell populations. To compare the panels, we analyzed by flow cytometry our four-marker isolates for the concurrent expression of CD49F and Sca-1 and found that 100% of our four-marker cells also expressed these markers. This indicates that there is significant overlap between these two widely used marker panels, and it suggests that those cells with progenitor characteristics, within the CD49F/Sca-1 panel, may also concurrently express c-Kit. Epithelial progenitor cells have been postulated to play a role in prostate disease, including prostate cancer. Primary tumor cells and established cell lines and xenografts in immunodeficient mice demonstrate high proliferative potential in colony-forming assays as well as the ability to differentiate into luminal and basal cell types (5). Various approaches have been used to identify putative cancer stem cells from prostate tumor xenografts that have been established from primary tumors, either by passaging through cell culture or through serial passaging in immunodeficient mice (30, 31, 32). In addition, putative prostate cancer stem cells from established human prostate cancer cell lines, including PC3 (22), DU145 (40), and LNCaP cell lines (13), have been isolated. Most strikingly, these cells can initiate serially transplantable tumors after subcutaneous injection. While the above studies do not conclusively prove that prostate cancer arises from prostatic pro-

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INFLAMMATION INDUCES PROSTATIC EPITHELIAL PROGENITOR CELLS

genitor cells, they are suggestive of an expansion of cells with progenitor-like qualities. Given the strong localization of prostate cancer with inflammation, the concept shown in our study that inflammation drives expansion of cells with progenitor cell characteristics could be critical toward our understanding of how inflammation and inflammatory signals promote cancer initiation in the prostate and at sites of metastases. One recent report (19) has demonstrated a role for inflammation in prostate progenitor cell biology, an effect of transdifferentiation from a basal to luminal phenotype within these cells. Kwon et al. conducted a lineage tracing study of basal cells using a K14-CreER;mTmG genetic construct in a mouse model of prostatic inflammation induced by uropathogenic bacterium CP9, a similar strain to the E. coli 1677 strain used in our work. Interestingly, they showed that the inflamed tissue microenvironment induced a differentiation of basal cells into luminal cells, an event that rarely occurs under normal physiological conditions. This event significantly accelerated the initiation of prostatic intraepithelial neoplasia relative to a naïve prostatic microenvironment and that tumor initiation occurred within this transdifferentiated phenotype. Our data supplement this previous report by demonstrating that the inflammatory microenvironment also expands the population of progenitor cells, and, together, these two studies suggest that inflammation may promote prostate tumorigenesis by both expansion and transdifferentiation of the protected basal progenitor cell population in the prostate. BPH is a slow progressive enlargement of the prostate gland characterized histologically by hyperproliferation of epithelial and stromal cells in the transition zone of the prostate gland. Little is known in terms of biological processes that contribute to the development of BPH, but it is noteworthy that inflammation is the single most associative histological feature within BPH specimens correlating to symptom severity (28). The hypothesis that progenitor cell expansion occurs during benign prostatic growth has been proposed. This hypothesis states that a progenitor cell population residing in the prostate gland is increased due to abnormal proliferation and apoptosis of progenitor cells, which may eventually contribute to BPH pathogenesis (1, 15). Cells isolated from the BPH samples have been shown to both possess sphere-forming capacity and the capacity to undergo clonal proliferation and the generation of branching ductal structures (41). These studies, demonstrating the presence of these high proliferative cells with progenitor characteristics in BPH tissue samples, suggest that BPH could occur as a result of expansion of cells with progenitor properties that could ultimately give rise to a clonal expansion of cell populations. Given the extremely high prevalence of inflammation in sections cut from BPH specimens, it is quite feasible that inflammation may be postulated to be a driver of these events. Previous work from our laboratory has demonstrated that development of the prostate requires activation of IL-1-driven IGF signaling and that this event is recapitulated during the induction of prostatic inflammation (11, 16). Inflammation is associated with many developmental-like features as part of the repair and recovery process, and the present study demonstrates that expansion of tissue progenitor cells is yet another process of developmental biology that is reactivated by inflammation. Since IL-1R-mediated signaling is critical for prostate growth during development and reactive hyperplasia, we

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sought to determine if its activity is also critical for inflammation-driven progenitor cell expansion. The finding that progenitor cell expansion is reduced by ⬎50% in IL-1R1⫺/⫺ mice relative to C57BL/6J control mice indicates that IL-1R signaling plays a role in inflammation-induced progenitor cell expansion. Of note, IL-1R-null mice still mount an effective inflammatory reaction complete with neutrophils, lymphocytes, and macrophages (16), so the effect of IL-1R on hyperplasia resulting from inflammation appears to be at the epithelial-stromal interaction level rather than in reducing inflammation. It must also be noted, however, that this was not a complete abrogation; therefore, other cytokines and growth factors are likely involved in this process. These may include IGF-1, as a target of IL-1R signaling, as well as pathways induced by IL-6, IL-8, IL-12, and other cytokines, in addition to FGFs, platelet-derived growth factor, sonic hedgehog, and other developmental mediators known to be induced by inflammation and known to play a role in the promotion of proliferative prostate diseases. Future studies arising from this work will the investigation into further mechanisms of inflammation-induced progenitor cell expansion and cell survival. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Kai-Ming Chou for fluorescent microscopy expertise and use and Dr. Xu Dong Shi and Dr. Wade Bushman (University of Wisconsin, Madison, WI) for expertise and experimental design for anchorage-independent prostasphere protocols. The IU Simon Cancer Center provided core services, including the Flow Cytometry Core and the Biological Imaging Core for confocal imaging. GRANTS This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-092366-01A1. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: L.W., M.Z., B.R.C., T.L.R., J.D.M., E.F.S., H.B., and T.J.J. conception and design of research; L.W., M.Z., B.R.C., J.D.M., and T.J.J. performed experiments; L.W., M.Z., B.R.C., J.D.M., E.F.S., H.B., and T.J.J. analyzed data; L.W., M.Z., B.R.C., T.L.R., J.D.M., E.F.S., H.B., and T.J.J. interpreted results of experiments; L.W., J.D.M., and T.J.J. prepared figures; L.W., M.Z., and T.J.J. drafted manuscript; L.W., M.Z., B.R.C., T.L.R., J.D.M., E.F.S., H.B., and T.J.J. approved final version of manuscript; T.L.R., E.F.S., H.B., and T.J.J. edited and revised manuscript. REFERENCES 1. Bajek A, Pokrywka L, Wolski Z, De˛bski R, Drewa T. Prostate epithelial stem cells are resistant to apoptosis after ␣1-antagonist treatment. The impact for BPH patients. Cent European J Urol 64: 256 –257, 2011. 2. Boehm BJ, Colopy SA, Jerde TJ, Loftus CJ, Bushman W. Acute bacterial inflammation of the mouse prostate. Prostate 72: 307–317, 2012. 3. Bostanci Y, Kazzazi A, Momtahen S, Laze J, Djavan B. Correlation between benign prostatic hyperplasia and inflammation. Curr Opin Urol 23: 5–10, 2013. 4. Burger PE, Xiong X, Coetzee S, Salm SN, Moscatelli DN, Goto K, Wilson EL. Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue. Proc Natl Acad Sci USA 102: 7180 –7185, 2005. 5. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65: 10946 –10951, 2005. 6. Cotran RS, Kumar V, Robbins SL. Prostatitis. In: Pathologic Basis of Disease (6th ed.), edited by Robbins SL. Philadelphia, PA: Saunders, 1999, p. 1025–1027.

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