E-cadherin deficiency promotes prostate macrophage inflammation and bladder overactivity in aged male mice

E-cadherin in human BPH and transgenic mouse prostate

BPH and LUTS are typically associated with aging, and seldom identified in patients under the age of 51 [2, 3, 17]. In a recent study of the impact of age on the lower urogenital tract in male mice, aging mice displayed prostatic enlargement and altered urinary function compared to young animals [11], illustrating the potential relevance of an aging rodent model to identify mechanisms driving BPH/LUTS in humans. Mice reach adulthood at approximately 10 weeks of age and undergo reproductive senescence from 15–18 months of age, while post 18 months of age is considered to be the post-reproductive senescence period for mice [18]. The correlate to human adulthood is 20–51 years of age, reproductive senescence from 51–78 years of age and post-reproductive senescence period over age 78 [18]. Since BPH/LUTS incidence increases from ~ 50% in men at the age of 50 and reaches approximately 80% in men over the age of 70 [17], we chose to examine the impact of E-cadherin loss in male mice at 24 months of age – corresponding to 75–85 years of age in humans (Figure 1A). Previous studies examining the role of aging in the murine prostate have also examined mice at 24 months of age [9, 11]. To examine the impact of aging, we compared mice at 24 months of age to young adult mice at 6 months of age – corresponding to ~30 years of age in humans (Figure 1A).

E-cadherin immunostaining of the epithelial layer in normal adjacent prostate to BPH and BPH glands was uniformly distributed and most intense along the cell membrane, with decreased staining observed in BPH glands as evidenced by a reduction of clearly demarked E-cadherin positive cell borders in BPH glands (Figure 1B) [6]. This diffuse reduction in E-cadherin staining was not observed in mice with a homozygous deletion of the E-cadherin gene in luminal epithelial cells [13] (Figure 1C). While these Cdh1^-/- mice contain many prostate epithelial cells lacking E-cadherin staining, they are often found surrounded by E-cadherin positive luminal epithelial cells in a mosaic pattern [13] (Figure 1C). This is likely the result of replenishment of luminal epithelial cells from a basal progenitor that was not ablated of the E-cadherin gene [19]. In order to more closely recapitulate the uniform reduction in E-cadherin expression observed in BPH glands of patients aged 50–77 years [6], we generated a cohort of mice with heterozygous deletion of E-cadherin and examined them at age 24 months. In this way, we could determine the impact of reduced E-cadherin expression (i.e., generated by heterozygosity in the mouse model) on the aged prostate and bladder. Similar to the pattern of E-cadherin immunostaining observed in prostate specimens from BPH patients, mice at 24 months of age displayed cell membrane E-cadherin expression in the epithelial layer of both Control and Cdh1^+/- mice, with a diffuse decreased expression in prostate glands of Cdh1^+/- mice in all lobes of the prostate (blue arrows, Figure 1D, center panel, Supplementary Figure 1). Cdh1^+/- mice also displayed glands with the mosaic pattern of E-cadherin negative cells surrounded by E-cadherin positive cells (black arrows, Figure 1D, right panel), similar to the pattern observed in Cdh1^-/- mice at 6 months of age [13].

Impact of aging and E-cadherin deficiency on the murine prostate

Virgin male mice were generated with the following genotype combinations: PSA-Cre⁺; Cdh1^flox/+ (referred to herein as Cdh1^+/-) and PSA-Cre^-; Cdh1^flox/+ (Control). As an additional control for the possible effects of PSA-Cre, we also generated a cohort of three PSA-Cre⁺; Cdh1^+/+ (PSA-Cre⁺) mice for histologic analyses. At 24 months of age, no statistically significant differences were observed in mean body, organ, or prostate mass between Control and Cdh1^+/- mice (Table 1). The prostates from PSA-Cre⁺ mice also appeared histologically normal and expressed E-cadherin in a pattern similar to PSA-Cre^-; Cdh1^flox/+ controls (Supplementary Figure 2). There were no histologic defects detected in the kidney, spleen or testis and no difference in morbidity or mortality between groups at 24 months of age (data not shown). These findings suggest that the presence of PSA-Cre did not induce systemic defects.

At 6 months of age, the prostates of control and Cdh1^+/- mice appeared histologically normal and did not display any epithelial pathologic changes (Table 2). Aging in control mice induced a nonsignificant increase in prostate hyperplasia and mPIN (murine prostatic intraepithelial neoplasia). The prostates of Cdh1^+/- mice at 24 months of age displayed a statistically significant increase in epithelial changes including epithelial hyperplasia and mPIN lesions compared to both age-matched controls and Cdh1^+/- mice at 6 months of age (Table 2, Supplementary Figure 3). One Cdh1^+/- mouse at 24 months of age (1/7 Cdh1^+/- vs. 0/9 Cdh1^+/+, p = 0.44) developed prostate cancer.

The proliferative marker Ki-67 was used to detect dividing cells in the prostates of Cdh1^+/- and control mice. There was no significant difference in the number of Ki-67-positive epithelial cells in all lobes of the Cdh1^+/- mice compared to controls (Figure 2A). In contrast, there was a statistically significant increase in Ki-67-positive stromal cells in the ventral and lateral lobes of Cdh1^+/- mice, indicative of stromal hyperplasia (Figure 2B, 2C). Masson’s trichrome staining showed an increase in extracellular matrix (ECM) deposition in the ventral lobes of Cdh1^+/- mice compared to control prostates (Figure 2D, 2E). In a post hoc comparison of control mice at 24 months of age to mice at 6 months of age, there was a significant increase in both epithelial and stromal proliferation in aged mice (Figure 3). We did not observe a significant increase in ECM deposition in aged mice compared to 6 months old controls (Supplementary Figure 4). These results suggest that aging is associated with an increase in epithelial and stromal proliferation, and that E-cadherin deficiency further increased stromal proliferation in the aged murine prostate.

We recently showed that in aging human prostate, increased inflammation is correlated with down-regulation of epithelial barrier proteins E-cadherin [6], which was previously found to be down-regulated in BPH [14–16]. Previous studies have also demonstrated an association between macrophage infiltration and fibrosis in the murine prostate [20]. Thus, we analyzed the number of CD19-positive B-cells, CD3-positive T-cells, and CD68-positive macrophages in aged mice. Lymphomas composed of both CD19-positive B cells and CD3-positive T-cells were observed and identified by a board-certified animal pathologist (LHR, V.M.D.) in the prostates of both controls (4/9) and Cdh1^+/- mice (3/7) at 24 months of age, while neither cohort displayed lymphoma at 6 months of age (0/7 in controls, 0/3 in Cdh1^+/- mice). Although the age-related increased incidence was not statistically significant (p = 0.07 in controls, p = 0.48 in Cdh1^+/- mice), the spontaneous development of lymphoma in aged C57BL/6J mice is common [21]. The incidence of lymphoma was similar in both cohorts (p = 1.00), and this made it difficult to detect an increase in B-cells or T-cells in the prostates of Cdh1^+/- mice compared to age-matched controls at 24 months of age. However, there was a significant increase in the number of CD68-positive macrophages in all lobes of the prostates of Cdh1^+/- mice at 24 months of age (Figure 4), and a significant increase in macrophages, T-cells, and B-cells in the prostates of control mice at 24 months of age compared to controls at 6 months of age (Figure 5). These results suggest that heterozygous deletion of E-cadherin in aged animals was associated with a further increased number of macrophages in the prostate of aged animals.

Voiding behavior and conscious cystometry in Cdh1^+/- mice at 24 months of age

Previously, we and others have reported an increased urinary frequency [22] and bladder overactivity in mice and rat models of bacterial- or chemical-induced prostatic inflammation [23, 24]. We also reported that mice with homozygous deletion of Cdh1 developed prostatic inflammation and displayed an increase in the number of voids, non-voiding contractions and a decrease in the average single voided volume compared to controls at 6 months of age [13]. Post-hoc comparison of control mice at 6 months of age vs. 24 months of age revealed that aged mice displayed a significant decreased average single voided volume in voiding assays during the four-hour observation period despite larger bladder mass at necropsy and a trend increase of ICI during cystometry in 24 months-old control mice (Table 3). Because bladder voiding efficiency calculated as a percent ratio of voided volume against bladder capacity was lowered to 66% in aged control mice compared to control mice at 6 months of age (Table 3), it is assumed that aged mice exhibited an underactive bladder condition resulting in inefficient voiding, in line with a previous study in which voiding dysfunction due to reduced bladder contractility was observed with aging in male mice [12]. In addition, there was a trend decrease in total urine output volume (1101.2 vs. 567.7, p = 0.07) during four hours in control mice at 24 months of age compared to control mice at 6 months of age (Table 3).

Although the difference was not statistically significant, the measured bladder volume at necropsy of Cdh1^+/- mice at 24 months of age was much larger than that of age-matched control mice (3.31 vs. 0.931 cm³, p = 0.068) (Table 3). In accordance, aged Cdh1^+/- mice displayed trend increases in ICI (p = 0.090) and bladder capacity (p = 0.053) in cystometry compared to age-matched controls (Table 3) although enlarged bladders did not display obvious histological defects compared to age-matched controls (Figure 6). However, because bladder voiding efficiency was not different between aged Cdh1^+/- mice and age-matched control mice (56 vs. 66%, p = 0.478) (Table 3), the aging-related underactive bladder condition does not seem to be significantly worsened by E-cadherin downregulation. In contrast, cystometric tracing of awake Cdh1^+/- mice demonstrated a significantly increased mean number of non-voiding contractions (NVC) per minute compared to controls (Figure 7) in aged mice, suggesting that prostatic defects induced by E-cadherin heterozygosity contributed to the development of bladder overactivity during the storage phase in aged mice. Cystometric analysis was not performed on Cdh1^+/- mice at 6 months of age since they did not display any prostatic defects. Taken together, these changes suggest that aging contributes to bladder underactivity during the voiding phase function while decreased E-cadherin in the aging prostate can additionally induce bladder overactivity during the storage phase in aged mice.

E-cadherin immunostaining in human prostate specimens

E-cadherin immunostaining was performed on sections from a previously described experimental cohort of prostate tissue specimens [6, 31], which included 14 patients who received simple prostatectomy for symptomatic BPH, and six young healthy organ donors without any history of chemo-, radio-, or hormone therapy [31]. BPH specimens were composed of mixed hyperplastic nodules with both glandular and stromal expansion. The “deidentified” specimens were retrieved from the clinical files of UPMC by the University of Pittsburgh Biospecimen Core (PBC) with approval from the University of Pittsburgh Institutional Review Board for this research project under protocol #17010177. PBC also provided deidentified pathology reports for the patients constituting the study cohort through their IRB-approved Honest Broker System, HB015, to ensure research integrity. All participating patients or their next of kin provided informed consent for the banking protocol.

Samples were fixed in 10% formalin for at least 24 hours, then embedded in paraffin, sectioned at 5 μm. Immunohistochemical staining was performed on five-micron sections of paraffin-embedded prostate tissue specimens. Briefly, sections were deparaffinized and rehydrated through a graded series of ethanol. Heat-induced epitope retrieval was performed using a BioCare Decloaking Chamber (BioCare Medical, Pacheco, CA, USA), followed by 5 minutes rinsing in TBS buffer. Sections were stained with rabbit monoclonal anti-E-cadherin (ab76319, EP913(2)Y, Abcam, Cambridge, MA, USA), then counterstained in hematoxylin and cover-slipped. Stained sections were imaged with a Leica DM LB microscope (Leica Microsystems Inc., Bannockburn, IL, USA) equipped with an Imaging Source NII 770 camera (The Imaging Source Europe GmbH, Bremen, Germany) and NIS-Elements Documentation v 4.6 software (Nikon Instruments, Inc., Mellville, NY, USA). All tissues were examined by a board-certified genitourinary pathologist (R.D.) using light microscopy.

Mice

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh and were conducted in strict accordance with the standards for humane animal care and use as set by the Animal Welfare Act and the National Institutes of Health guidelines for the use of laboratory animals under Animal Welfare Assurance number A3187-01. Animals were provided water and irradiated chow (Prolab^® IsoPro^® RMH 3000 5P75, LabDiet, St. Louis, MO, USA) ad libitum. Male mice were generated by cross-breeding the B6.129-Cdh1^tm2Kem/J mouse [32] (JAX stock #005319, The Jackson Laboratory, Bar Harbor, ME, USA) with the inducible Cre line PSA-CreER^T2 (generously provided by Dr. Pierre Chambon and Dr. Daniel Metzger, IGBMC, Illkirch, France) on a C57BL/6J background [33]. Experimental cohorts were virgin male mice that were either hemizygous or negative for the Cre allele (PSA-Cre⁺ or PSA-Cre^-) and had heterozygous or wild-type expression of Cdh1 floxed alleles (Cdh1^flox/+ and Cdh1^+/+) resulting in genotype combinations: PSA-Cre⁺; Cdh1^flox/+, PSA-Cre^-; Cdh1^flox/+, or PSA-Cre⁺; Cdh1^+/+. Mice herein were referred to as Cdh1^+/-, control and PSA-Cre+, respectively. Genotyping was performed using PCR analysis of mouse tail genomic DNA at age 21 days and muscle genomic DNA after euthanization as previously [13].

Tamoxifen induction of Cre activity in male mice containing the PSA-CreER^T2 allele was performed as previously for 5 consecutive days at 7 weeks of age [13, 34]. Tamoxifen (Sigma Chemical Co., St. Louis, MO, USA) suspended in Kolliphor EL (Sigma) 3 mg/40 g of body weight was injected i.p. daily. All animals in the study were treated with the same regimens of tamoxifen injections and were examined at 6 months or 24 months of age. Body mass was measured using a triple beam balance immediately prior to euthanization. Length was determined by measuring the animal from the nose to the anus [35]. Bladder was measured in situ in three dimensions with a precision caliper as described [36] and bladder volume was estimated by calculating the volume of an ellipsoid in cubic millimeters [37]. The heart, spleen, left kidney, left testis and lower urinary tract were removed and fixed in 10% formalin. Fixed tissues were cleaned of excess fat and membrane with phosphate-buffered saline; mass of each organ and each prostate lobe was determined after blotting with filtration paper to remove excess liquid.

Histopathologic analysis of murine specimens

Murine tissue samples were fixed in 10% formalin for at least 24 hours, then embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. All tissues were examined by a board-certified animal pathologist in a blinded fashion (LHR, V.M.D.). Prostatic defects were identified as epithelial hyperplasia, stromal hyperplasia, inflammation and mPIN and adenocarcinoma per the criteria published by Shappell, et al., commonly used to score prostate lesions in transgenic mouse models [38].

Immunohistochemistry and Masson’s trichrome staining of murine specimens

Immunohistochemical stains were performed on five-micron sections of paraffin-embedded murine tissue specimens as described previously [39]. Briefly, sections were de-paraffinized and rehydrated through a graded series of ethanol. Heat-induced epitope retrieval was performed using a BioCare Decloaking Chamber (BioCare Medical, Pacheco, CA, USA), followed by 5 minutes rinsing in TBS buffer. Primary antibodies for immunostaining of murine tissue sections were mouse monoclonal anti-E-cadherin (Clone 36, #610181, BD Biosciences, San Jose, CA, USA), rabbit monoclonal anti-Ki-67 (D3B5, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-CD3 (P, CP215, BioCare Medical), rabbit monoclonal anti-CD3 (D4V8L, #99940, Cell Signaling Technology), CD19 (D4V4B, #90176, Cell Signaling Technology), and mouse monoclonal anti-CD68 (KP1, CM033, BioCare Medical). Slides were then counterstained in hematoxylin and coverslipped. Masson’s Trichrome histochemical staining was performed using HT15 (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Stained sections were imaged with a Leica DM LB microscope (Leica Microsystems Inc., Bannockburn, IL, USA) equipped with an Imaging Source NII 770 camera (The Imaging Source Europe GmbH, Bremen, Germany) and NIS-Elements Documentation v 4.6 software (Nikon Instruments, Inc., Mellville, NY, USA). All tissues were examined by a board-certified veterinary pathologist (L.H.R.) and a board-certified genitourinary pathologist (R.D.) using light microscopy.

Mean Ki-67-positive cell density was determined by analysis of sections from at least six independent mice from each genotype and from all lobes of the prostate. The average number of Ki-67-positive luminal epithelial cells or stromal cells in each prostate lobe for each mouse was determined from at least four nonoverlapping fields imaged at 40× magnification. The average number of CD19-positive B cells, CD68-positive macrophages and CD3-positive T cells was also determined similarly for each prostate lobe in each mouse.

Extracellular matrix (ECM) was quantified by calculating the average intensity of blue staining in Masson’s trichrome stain using Adobe PhotoShop 2021, as described [40]. Seven fields from each section were analyzed and an average score was determined for each mouse.

Void spot assay

Mice were placed in metabolic cages and voiding behavior was evaluated for 4 hours. Filter paper was placed under the wire mesh bottom of each metabolic cage and urine stains on the paper were analyzed to determine voiding behavior and voided volumes using the Voided Stain on Paper (VSOP) method [41]. Before each analysis, mice were treated with subcutaneous injections of sterile water (20 μl/g body weight) to enhance urine production. The voided volume for each urine spot was calculated using ImageJ (NIH, Bethesda, MD, USA).

Cystometry

Mice were examined by cystometric investigation under a conscious condition as previously [23]. Under isoflurane anesthesia, following a lower midline abdominal incision, the bladder was exposed and catheterized with PE-50 tubing (Clay Adams Division of Becton Dickinson, Parsippany, NJ, USA) inserted through the bladder dome. After recovery from anesthesia, conscious mice were placed in a restraining cage (Economy holder 15 to 30 g, Kent Scientific, Torrington, CT, USA). After 1–2 hours of acclimation, saline was infused into the bladder at 0.01 ml/min to induce micturition. At least three reproducible micturition cycles were recorded after an initial stabilization period (60 min). After several micturition cycles, the saline infusion was stopped to evaluate intercontraction intervals (ICI), and non-voiding contractions (NVCs), bladder capacity and bladder voiding efficiency using Chart software (AD Instruments, Colorado Springs, CO, USA). NVCs were defined as rises in intravesical pressure that exceeded 20% of voiding pressure over the baseline without fluid elimination from the urethral orifice during bladder filling.

Post-hoc comparison of data from control mice at 24 months of age (PSA-Cre-; Cdh1^flox/+) to previously generated control mice at 6 months of age (PSA-Cre-; Cdh1^flox/flox) [13] was performed to determine whether prostate inflammation and bladder function were altered with aging. Control mice from both 6 months and 24 months of age cohorts were treated with tamoxifen at 7 weeks of age.

Statistical analysis

Comparisons between groups were calculated using the student’s t-test or two-tailed Fisher exact test using the method of summing small p-values as appropriate. The Pearson correlation coefficient was used to determine the correlation between extracellular matrix density, proliferation, and infiltration of inflammatory cells in the combined samples from aged mouse prostate (control and Cdh1^+/-). A p-value of p < 0.05 was considered significant. GraphPad Prism version 9 was used for graphics (GraphPad Software, San Diego, CA, USA). Values are expressed as mean ± S.D.