Macrophages of multiple hematopoietic origins reside in the developing prostate.

Tissue-resident macrophages contribute to the organogenesis of many tissues. Growth of the prostate is regulated by androgens during puberty, yet androgens are considered immune suppressive. In this study, we characterized the localization, androgen receptor expression and hematopoietic origin of prostate macrophages, and transiently ablated macrophages during postnatal prostate organogenesis in the mouse. We show that myeloid cells were abundant in the prostate during puberty. However, nuclear androgen receptor expression was not detected in most macrophages. We found Cx3cr1, a marker for macrophages, monocytes and dendritic cells, expressed in interstitial macrophages surrounding the prostate and associated with nerve fibers. Furthermore, we provide evidence for the co-existence of embryonic origin, self-renewing, tissue-resident macrophages and recruited macrophages of bone-marrow monocyte origin in the prostate during puberty. Our findings suggest that prostate macrophages promote neural patterning and may shed further light on our understanding of the role of the innate immune system in prostate pathology in response to inflammation and in cancer.

Human prostate organoid generation and the identification of prostate development drivers using inductive rodent tissues.

The reactivation of developmental genes and pathways during adulthood may contribute to pathogenesis of diseases such as prostate cancer. Analysis of the mechanistic links between development and disease could be exploited to identify signalling pathways leading to disease in the prostate. However, the mechanisms underpinning prostate development require further characterisation to interrogate fully the link between development and disease. Previously, our group developed methods to produce prostate organoids using induced pluripotent stem cells (iPSCs). Here, we show that human iPSCs can be differentiated into prostate organoids using neonatal rat seminal vesicle mesenchyme in vitro. The organoids can be used to study prostate development or modified to study prostate cancer. We also elucidated molecular drivers of prostate induction through RNA-sequencing analyses of the rat urogenital sinus and neonatal seminal vesicles. We identified candidate drivers of prostate development evident in the inductive mesenchyme and epithelium involved with prostate specification. Our top candidates included Spx, Trib3, Snai1, Snai2, Nrg2 and Lrp4. This work lays the foundations for further interrogation of the reactivation of developmental genes in adulthood, leading to prostate disease.

Prostate organogenesis.

Prostate organogenesis begins during embryonic development and continues through puberty when the prostate becomes an important exocrine gland of the male reproductive system. The specification and growth of the prostate is regulated by androgens and is largely a result of cell-cell communication between the epithelium and mesenchyme. The fields of developmental and cancer biology have long been interested in prostate organogenesis because of its relevance for understanding prostate diseases, and research has expanded in recent years with the advent of novel technologies, including genetic-lineage tracing, single-cell RNA sequencing and organoid culture methods, that have provided important insights into androgen regulation, epithelial cell origins and cellular heterogeneity. We discuss these findings, putting them into context with what is currently known about prostate organogenesis.

Characterization of HOXB13 expression patterns in localized and metastatic castration-resistant prostate cancer.

HOXB13 is a key lineage homeobox transcription factor that plays a critical role in the differentiation of the prostate gland. Several studies have suggested that HOXB13 alterations may be involved in prostate cancer development and progression. Despite its potential biological relevance, little is known about the expression of HOXB13 across the disease spectrum of prostate cancer. To this end, we validated a HOXB13 antibody using genetic controls and investigated HOXB13 protein expression in murine and human developing prostates, localized prostate cancers, and metastatic castration-resistant prostate cancers. We observed that HOXB13 expression increases during later stages of murine prostate development. All localized prostate cancers showed HOXB13 protein expression. Interestingly, lower HOXB13 expression levels were observed in higher-grade tumors, although no significant association between HOXB13 expression and recurrence or disease-specific survival was found. In advanced metastatic prostate cancers, HOXB13 expression was retained in the majority of tumors. While we observed lower levels of HOXB13 protein and mRNA levels in tumors with evidence of lineage plasticity, 84% of androgen receptor-negative castration-resistant prostate cancers and neuroendocrine prostate cancers (NEPCs) retained detectable levels of HOXB13. Notably, the reduced expression observed in NEPCs was associated with a gain of HOXB13 gene body CpG methylation. In comparison to the commonly used prostate lineage marker NKX3.1, HOXB13 showed greater sensitivity in detecting advanced metastatic prostate cancers. Additionally, in a cohort of 837 patients, 383 with prostatic and 454 with non-prostatic tumors, we found that HOXB13 immunohistochemistry had a 97% sensitivity and 99% specificity for prostatic origin. Taken together, our studies provide valuable insight into the expression pattern of HOXB13 during prostate development and cancer progression. Furthermore, our findings support the utility of HOXB13 as a diagnostic biomarker for prostate cancer, particularly to confirm the prostatic origin of advanced metastatic castration-resistant tumors. © 2023 The Pathological Society of Great Britain and Ireland.

In vitro induction of prostate buds from murine urogenital epithelium in the absence of mesenchymal cells.

The prostate is a male reproductive gland which secretes prostatic fluid that enhances male fertility. During development and instigated by fetal testosterone, prostate cells arise caudal to the bladder at the urogenital sinus (UGS), when the urogenital mesenchyme (UGM) secretes signals to the urogenital epithelium (UGE). These initial mesenchymal signals induce prostate-specific gene expression in the UGE, after which epithelial progenitor cells form prostatic buds. Although many important factors for prostate development have been described using UGS organ cultures, those necessary and sufficient for prostate budding have not been clearly identified. This has been in part due to the difficulty to dissect the intricate signaling and feedback between epithelial and mesenchymal UGS cells. In this study, we separated the UGM from the UGE and tested candidate growth factors to show that when FGF10 is present, testosterone is not required for initiating prostate budding from the UGE. Moreover, in the presence of low levels of FGF10, canonical WNT signaling enhances the expression of several prostate progenitor markers in the UGE before budding of the prostate occurs. At the later budding stage, higher levels of FGF10 are required to increase budding and retinoic acid is indispensable for the upregulation of prostate-specific genes. Lastly, we show that under optimized conditions, female UGE can be instructed towards a prostatic fate, and in vitro generated prostate buds from male UGE can differentiate into a mature prostate epithelium after in vivo transplantation. Taken together, our results clarify the signals that can induce fetal prostate buds in the urogenital epithelium in the absence of the surrounding, instructive mesenchyme.

Androgen action in cell fate and communication during prostate development at single-cell resolution.

Androgens/androgen receptor (AR)-mediated signaling pathways are essential for prostate development, morphogenesis and regeneration. Specifically, stromal AR signaling has been shown to be essential for prostatic initiation. However, the molecular mechanisms underlying AR-initiated mesenchymal-epithelial interactions in prostate development remain unclear. Here, using a newly generated mouse model, we have directly addressed the fate and role of genetically marked AR-expressing cells during embryonic prostate development. Androgen signaling-initiated signaling pathways were identified in mesenchymal niche populations at single-cell transcriptomic resolution. The dynamic cell-signaling networks regulated by stromal AR were additionally characterized in relation to prostatic epithelial bud formation. Pseudotime analyses further revealed the differentiation trajectory and fate of AR-expressing cells in both prostatic mesenchymal and epithelial cell populations. Specifically, the cellular properties of Zeb1-expressing progenitors were assessed. Selective deletion of AR signaling in a subpopulation of mesenchymal rather than epithelial cells dysregulated the expression of the master regulators and significantly impaired prostatic bud formation. These data provide novel, high-resolution evidence demonstrating the important role of mesenchymal androgen signaling in the cellular niche controlling prostate early development by initiating dynamic mesenchyme-epithelia cell interactions.

Stromal androgen and hedgehog signaling regulates stem cell niches in pubertal prostate development.

Stromal androgen-receptor (AR) action is essential for prostate development, morphogenesis and regeneration. However, mechanisms underlying how stromal AR maintains the cell niche in support of pubertal prostatic epithelial growth are unknown. Here, using advanced mouse genetic tools, we demonstrate that selective deletion of stromal AR expression in prepubescent Shh-responsive Gli1-expressing cells significantly impedes pubertal prostate epithelial growth and development. Single-cell transcriptomic analyses showed that AR loss in these prepubescent Gli1-expressing cells dysregulates androgen signaling-initiated stromal-epithelial paracrine interactions, leading to growth retardation of pubertal prostate epithelia and significant development defects. Specifically, AR loss elevates Shh-signaling activation in both prostatic stromal and adjacent epithelial cells, directly inhibiting prostatic epithelial growth. Single-cell trajectory analyses further identified aberrant differentiation fates of prostatic epithelial cells directly altered by stromal AR deletion. In vivo recombination of AR-deficient stromal Gli1-lineage cells with wild-type prostatic epithelial cells failed to develop normal prostatic epithelia. These data demonstrate previously unidentified mechanisms underlying how stromal AR-signaling facilitates Shh-mediated cell niches in pubertal prostatic epithelial growth and development.

Developmental stage and infection status may affect drug distribution in the prostate of rats.

Prostate inflammation is often treated with drugs which are ineffective. Antibacterial agents fail to reach the prostate epithelium, and the blood-prostate barrier (BPB) may affect the drug transport process. Factors affecting drug efficacy remain unclear.Rats were categorised into groups A and B, corresponding to adulthood and puberty, respectively. Group C included the model of chronic prostate infection. Dialysates of levofloxacin and cefradine were collected from the prostate gland and jugular vein and evaluated. Pharmacokinetic analysis was conducted.The free concentrations of antimicrobials in the prostate and plasma samples of all groups peaked at 20 min, then gradually decreased. The mean AUC0-tprostate/AUC0-tplasma ratio in the levofloxacin group were 0.86, 0.53, and 0.95, and the mean values of AUC0-∞prostate/AUC0-∞plasma ratio were 0.85, 0.63, and 0.97. The corresponding values in the cefradine group were 0.67, 0.30 and 0.84, and 0.66, 0.31, and 0.85, respectively. The mean values in group B were lower than those in group A, and those in group C were higher than those in group B.The maturity of the prostate may affect the ability of the drug to cross the BPB. Infection may disrupt the BPB, affecting drug permeability.

Spatiotemporal regulation of multipotency during prostate development.

The prostate is formed by a branched glandular epithelium composed of basal cells (BCs) and luminal cells (LCs). Multipotent and unipotent stem cells (SCs) mediate the initial steps of prostate development whereas BCs and LCs are self-sustained in adult mice by unipotent lineage-restricted SCs. The spatiotemporal regulation of SC fate and the switch from multipotency to unipotency remain poorly characterised. Here, by combining lineage tracing, whole-tissue imaging, clonal analysis and proliferation kinetics, we uncover the cellular dynamics that orchestrate prostate postnatal development in mouse. We found that at an early stage of development multipotent basal SCs are located throughout the epithelium and are progressively restricted at the distal tip of the ducts, where, together with their progeny, they establish the different branches and the final structure of prostate. In contrast, pubertal development is mediated by unipotent lineage-restricted SCs. Our results uncover the spatiotemporal regulation of the switch from multipotency to unipotency during prostate development.

Androgen Signaling in the Tumor Microenvironment.

The key function of mesenchymal/stromal androgen receptor (AR) signaling for prostate development has been well documented by tissue recombination experiments. Some studies have addressed the expression and function of AR in stromal cells in prostate cancer, yet our understanding of the role of stromal AR in other tissues beyond prostate is still insufficient.Genomic analysis has revealed that cellular responses to androgens differ between epithelial and stromal cells. AR in stromal cells seems not to act via classical AR transcription factors such as FOXA1 but rather depends on the JUN/AP1 complex. Stromal AR appears to have tumor-promoting and tumor-protective functions depending on tumor stage. Loss of AR signaling in fibroblasts has been detected already in premalignant lesions in the skin and prostate and has been associated with tumor induction in xenografts of skin cancer and aggressive disease features and poor patient prognosis in prostate cancer. Moreover, AR expression is found on virtually all tissue-infiltrating immune cells and plays critical roles in immune cell function. These findings suggest a potential deleterious impact of current androgen deprivation therapies which inhibit both epithelial and stromal AR, highlighting the need to develop tissue-specific AR inhibitors.

RET-mediated glial cell line-derived neurotrophic factor signaling inhibits mouse prostate development.

In humans and rodents, the prostate gland develops from the embryonic urogenital sinus (UGS). The androgen receptor (AR) is thought to control the expression of morphogenetic genes in inductive UGS mesenchyme, which promotes proliferation and cytodifferentiation of the prostatic epithelium. However, the nature of the AR-regulated morphogenetic genes and the mechanisms whereby AR controls prostate development are not understood. Glial cell line-derived neurotrophic factor (GDNF) binds GDNF family receptor α1 (GFRα1) and signals through activation of RET tyrosine kinase. Gene disruption studies in mice have revealed essential roles for GDNF signaling in development; however, its role in prostate development is unexplored. Here, we establish novel roles of GDNF signaling in mouse prostate development. Using an organ culture system for prostate development and Ret mutant mice, we demonstrate that RET-mediated GDNF signaling in UGS increases proliferation of mesenchyme cells and suppresses androgen-induced proliferation and differentiation of prostate epithelial cells, inhibiting prostate development. We also identify Ar as a GDNF-repressed gene and Gdnf and Gfrα1 as androgen-repressed genes in UGS, thus establishing reciprocal regulatory crosstalk between AR and GDNF signaling in prostate development.

An Indispensable Role of Androgen Receptor in Wnt Responsive Cells During Prostate Development, Maturation, and Regeneration.

Androgen signaling is essential for prostate development, morphogenesis, and regeneration. Emerging evidence indicates that Wnt/ß-catenin signaling also contributes to prostate development specifically through regulation of cell fate determination. Prostatic Axin2-expressing cells are able to respond to Wnt signals and possess the progenitor properties to regenerate prostatic epithelium. Despite critical roles of both signaling pathways, the biological significance of androgen receptor (AR) in Axin2-expressing/Wnt-responsive cells remains largely unexplored. In this study, we investigated this important question using a series of newly generated mouse models. Deletion of Ar in embryonic Axin2-expressing cells impaired early prostate development in both ex vivo and tissue implantation experiments. When Ar expression was deleted in prostatic Axin2-expressing cells at pre-puberty stages, it results in smaller and underdeveloped prostates. A subpopulation of Axin2 expressing cells in prostate epithelium is resistant to castration and, following androgen supplementation, is capable to expand to prostatic luminal cells. Deletion of Ar in these Axin2-expressing cells reduces their regenerative ability. These lines of evidence demonstrate an indispensable role for the Ar in Wnt-responsive cells during the course of prostate development, morphogenesis, and regeneration, which also imply an underlying interaction between the androgen and Wnt signaling pathways in the mouse prostate. Stem Cells 2018;36:891-902.

Dual regulation of decorin by androgen and Hedgehog signaling during prostate morphogenesis.

BACKGROUND: Prostate ductal branching morphogenesis involves a complex spatiotemporal regulation of cellular proliferation and remodeling of the extracellular matrix (ECM) around the developing ducts. Decorin (Dcn) is a small leucine-rich proteoglycan known to sequester several growth factors and to act as a tumor suppressor in prostate cancer. RESULTS: Dcn expression in the developing prostate paralleled branching morphogenesis and was dynamically regulated by androgen and Hedgehog (Hh) signaling. DCN colocalized with collagen in the periductal stroma and acellular interstitium. Exogenous DCN decreased epithelial proliferation in ex vivo organ cultures of developing prostate, whereas genetic ablation of Dcn resulted in increased epithelial proliferation in the developing prostate. CONCLUSIONS: Dcn expression and localization in the developing prostate is consistent with a primary role in organizing collagen around the developing ducts. Regulation of Dcn expression appears to be complex, involving both androgen and Hh signaling. The growth inhibitory effect of Dcn suggests a unique linkage between a structural proteoglycan and epithelial growth regulation. This may serve to coordinate two elements of the morphogenetic process: ductal growth and organization of the collagen matrix around the nascent duct. Developmental Dynamics 247:679-685, 2018. © 2018 Wiley Periodicals, Inc.

Beta-catenin and estrogen signaling collaborate to drive cyclin D1 expression in developing mouse prostate.

Androgen, beta-catenin (CTNNB1), and estrogen pathways stimulate proliferative growth of developing mouse prostate but how these pathways interact is not fully understood. We previously found that androgens induce CTNNB1 signaling in mouse urogenital sinus (UGS) epithelium from which prostatic ductal epithelium derives. Others have shown that low estradiol concentrations induce UGS epithelial proliferative growth. Here, we found that CTNNB1 signaling overlaps cyclin D1 (CCND1) expression in prostatic buds and we used a genetic approach to test whether CTNNB1 signaling induces CCND1 expression. We observed an unexpected sexually dimorphic response to hyperactive CCNTB1 signaling: in male mouse UGS it increased Ccnd1 mRNA abundance without increasing its protein abundance but in female UGS it increased Ccnd1 mRNA and protein abundance, suggesting a potential role for estrogens in stabilizing CCND1 protein. Treating wild type male UGS explants with androgen and either 17ß-estradiol or a proteasome inhibitor increased CCND1 protein and KI67 labeling in prostatic bud epithelium. Together, our results are consistent with an epithelial proliferative growth mechanism linking CTNNB1-driven Ccnd1 transcription and estrogen-mediated CCND1 protein stabilization.

Morphoregulatory pathways in prostate ductal development.

The mouse prostate is a male sex-accessory gland comprised of a branched ductal network arranged into three separate bilateral lobes: the anterior, dorsolateral, and ventral lobes. Prostate ductal development is the primary morphogenetic event in prostate development and requires a complex regulation of spatiotemporal factors. This review provides an overview of prostate development and the major genetic regulators and signaling pathways involved. To identify new areas for further study, we briefly highlight the likely important, but relatively understudied, role of the extracellular matrix (ECM). Finally, we point out the potential importance of the ECM in influencing the behavior and prognosis of prostate cancer. Developmental Dynamics 246:89-99, 2017. © 2016 Wiley Periodicals, Inc.

Intrauterine exposure to oestradiol promotes sex-specific differential effects on the prostatic development of neonate gerbils.

The effects of intrauterine exposure to 17ß-oestradiol (E2) are well studied for the male prostate and there are accumulating evidences that the exposure to high dosages leads to a hypomorphic development. However, there is a lack of information about the effects of intrauterine exposure to E2 in the prostate of rodent females, and such research becomes relevant in view of the presence of functional prostate in a proportion of women, and the morphophysiological similarities between the prostate of female rodents and the prostate of women. This study uses histochemical, immunohistochemical, immunofluorescence and three-dimensional (3D) reconstruction techniques to evaluate the effects of intrauterine exposure to E2 (500 BW/d) on neonatal prostate development in both male and female gerbils. It was verified that intrauterine exposure to E2 promotes epithelial proliferation and growth of prostatic budding in females, whereas in males the prostatic budding shows hypomorphic growth in the VMP (Ventral Mesenchymal Pad) as well as reduced epithelial proliferation. Together, the data demonstrate that intrauterine exposure to E2 causes different effects on male and female prostates of the gerbil even at the early postnatal development of the gland.

Impact of maternal and postnatal zinc dietary status on the prostate of pubescent and adult rats.

Zinc is important for cell physiology and alteration of its levels during development can modulate a series of biological events. The aim of this study was to investigate whether dietary zinc deficiency or supplementation during morphogenesis and early postnatal development could interfere in prostate maturation. Pregnant rats were exposed to a standard diet (NZ:35 mg Zn/kg chow), low-zinc diet (LZ:3 mg of Zn/kg chow) and zinc-supplemented diet (HZ:180 mg/Kg chow) from gestational day 10 (GD10) through postnatal day 21 (PND21). After weaning, male offspring were divided into three groups that were submitted to the same food conditions as their mothers until PND53. The animals were euthanized at PND53 and PND115. The ventral prostate was removed, weighed and its fragments were subjected to histological, western blot and zymography analysis. PND53: body and prostate weight were lower in LZ compared to NZ; the epithelial compartment was reduced while the stromal compartment was increased in LZ compared to NZ; there was an increase in the amount of collagen and reduction in AR and SIRT1 expression in LZ compared to NZ. PND115: body weight was lower in LZ compared to NZ and prostate weight was similar among the groups; peripheral physiological hyperplasia was observed, as well as an increased epithelial proliferation index and reduced PAR4 expression in LZ and HZ compared to NZ. Zinc deficiency during prostate morphogenesis and differentiation is potentially harmful to its morphology, however, by restoring the standard dietary environment, the gland responds to the new microenvironment independent of the previous dietary condition.

Neonatal exposure to ethinylestradiol increases ventral prostate growth and promotes epithelial hyperplasia and inflammation in adult male gerbils.

The aim of this study was to analyse morphologically the ventral prostate of adult Mongolian gerbils exposed to ethinylestradiol (EE) during the first week of postnatal development. Lactating females received daily, by gavage, doses of 10 µg/kg of EE diluted in 100 µl of mineral oil from the 1st to 10th postnatal day of the pups (EE group). In the control group (C), the lactating females received only the vehicle. Upon completing 120 days of age, the male offspring were euthanized and the prostates collected for analyses. We employed morphological, stereological-morphometrical, immunohistochemical and ultrastructural methods. The results showed that the postnatal exposure to EE doubled the prostatic complex weight, increasing the epithelial and stromal compartments, in addition to the secretory activity of the ventral lobe of the prostate. All glands exposed to EE showed strong stromal remodelling, and some foci of epithelial hyperplasia and inflammatory infiltrate in both luminal and epithelial or stromal compartments. Cells positive for anti-AR and anti-PCNA reactions increased into the epithelial and stromal tissues. ERα-positive cells, which are normally found in the stromal compartment of intact prostates, were frequently observed in the prostatic epithelium of treated animals. This study demonstrated that the exposure to EE during postnatal development causes histophysiological alterations in this gland, predisposing to the development of prostatic lesions during life. These results are important for public health, considering that women worldwide have commonly used EE. Moreover, the bioaccumulation of this chemical has increased in different ecosystems.

Paracrine Signaling in the Prostatic Stroma: A Novel Role for the Telocytes Revealed in Rodents' Ventral Prostate.

The telocytes have recently been described in the prostate gland. In mature gland, they exist in close association with the acini and their telopodes form networks whose functions remain unclear. In this chapter, our group gives a brief introduction to telocytes and explores the history that led to such a concept and then discusses hypotheses and presents new evidences about the roles exerted by telocytes in the prostate. First is given emphasis on the role that these cells possibly play in paracrine signaling employed in the differentiation of smooth muscle periacinar are then discussed other roles potentially performed by telocytes in the prostate, such as the organizational, where these cells would act in order to delimit stromal microenvironments, thereby assisting the differentiation of the prostatic anatomical components. In addition, the pacemaker function of smooth muscle cells contraction, as evidenced by the presence of caveolae and gap-type junction and, finally, the role of telocytes in prostate remodeling and the possible action as adult progenitor cells. Generally speaking, the chapter reaffirms the existence of telocytes as distinct cells of other stromal cells and the importance of this new cell type for normal metabolism and prostate development.

Intrauterine exposure to bisphenol A promotes different effects in both neonatal and adult prostate of male and female gerbils (Meriones unguiculatus).

Substances that mimic endogenous hormones may alter the cell signaling that govern prostate development and predispose it to developing lesions in adult and senile life. Bisphenol A is able to mimic estrogens, and studies have demonstrated that low levels of exposure to this compound have caused alterations during prostate development. The aim of this study was to describe the prostate development in both male and female neonatal gerbils in normal conditions and under exposure to BPA during intrauterine life, and also to analyze whether the effects of intrauterine exposure to BPA remain in adulthood. Morphological, stereological, three-dimensional reconstruction, and immunohistochemical methods were employed. The results demonstrated that in 1-day-old normal gerbils, the female paraurethral glands and the male ventral lobe are morphologically similar, although its tissue components-epithelial buds (EB), periurethral mesenchyme (PeM), paraurethral mesenchyme (PaM) or ventral mesenchymal pad (VMP), and smooth muscle (SM)-have presented different immunolabeling pattern for androgen receptor (AR), and for proliferating cell nuclear antigen (PCNA). Moreover, we observed a differential response of male and female prostate to intrauterine BPA exposure. In 1-day-old males, the intrauterine exposure to BPA caused a decrease of AR-positive cells in the PeM and SM, and a decrease of the proliferative status in the EB. In contrast, no morphological alterations were observed in ventral prostate of adult males. In 1-day-old females, BPA exposure promoted an increase of estrogen receptor alpha (ERα) positive cells in PeM and PaM, a decrease of AR-positive cells in EB and PeM, besides a reduction of cell proliferation in EB. Additionally, the adult female prostate of BPA-exposed animals presented an increase of AR- and PCNA-positive cells. These results suggest that the prostate of female gerbils were more susceptible to the intrauterine BPA effects, since they became more proliferative in adult life. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1740-1750, 2016.