Embryonic Urogenital Sinus Mesenchyme Research Articles

Human intermediate prostate cancer stem cells contribute to the initiation and development of prostate adenocarcinoma

BackgroundIntermediate cells are present in the early stages of human prostate development and adenocarcinoma. While primary cells isolated from benign human prostate tissues or tumors exhibit an intermediate phenotype in vitro, they cannot form tumors in vivo unless genetically modified. It is unclear about the stem cell properties and tumorigenicity of intermediate cells.MethodsWe developed a customized medium to culture primary human intermediate prostate cells, which were transplanted into male immunodeficient NCG mice to examine tumorigenicity in vivo. We treated the cells with different concentrations of dihydrotestosterone (DHT) and enzalutamide in vitro and surgically castrated the mice after cell transplantation in vivo. Immunostaining, qRT-PCR, RNA sequencing, and western blotting were performed to characterize the cells in tissues and 2D and 3D cultures.ResultsWe found intermediate cells expressing AR+PSA+CK8+CK5+ in the luminal compartment of human prostate adenocarcinoma by immunostaining. We cultured the primary intermediate cells in vitro, which expressed luminal (AR+PSA+CK8+CK18+), basal (CK5+P63+), intermediate (IVL+), and stem cell (CK4+CK13+PSCA+SOX2+) markers. These cells resisted castration in vitro by upregulating the expression of AR, PSA, and proliferation markers KI67 and PCNA. The intermediate cells had high tumorigenicity in vivo, forming tumors in immunodeficient NCG mice in a month without any genetic modification or co-transplantation with embryonic urogenital sinus mesenchyme (UGSM) cells. We named these cells human castration-resistant intermediate prostate cancer stem cells or CriPCSCs and defined the xenograft model as patient primary cell-derived xenograft (PrDX). Human CriPCSCs resisted castration in vitro and in vivo by upregulating AR expression. Furthermore, human CriPCSCs differentiated into amplifying adenocarcinoma cells of luminal phenotype in PrDX tumors in vivo, which can dedifferentiate into CriPCSCs in vitro.ConclusionsOur study identified and established methods for culturing human CriPCSCs, which had high tumorigenicity in vivo without any genetic modification or UGSM co-transplantation. Human CriPCSCs differentiated into amplifying adenocarcinoma cells of luminal phenotype in the fast-growing tumors in vivo, which hold the potential to dedifferentiate into intermediate stem cells. These cells resisted castration by upregulating AR expression. The human CriPCSC and PrDX methods hold significant potential for advancing prostate cancer research and precision medicine.

A developmental stage-dependent switch of the mechanisms for prostate epithelial maintenance

A developmental stage-dependent switch of the mechanisms for prostate epithelial maintenance

Dual‐label centromere and telomere FISH identifies human, rat, and mouse cell contribution to Multispecies recombinant urogenital sinus xenografts

Recombinant xenografts of human cells growing in immunocompromised rodents are widely used for studying stem cell biology, tumor biology, and epithelial to mesenchyme transitions. Of critical importance is the correct interpretation of the cellular composition of such xenografts. Here we present a rapid and robust method employing protein nucleic acid (PNA) FISH probes to dual-label centromeres and telomeres (Cen/Tel FISH). Such labeling allows unambiguous discrimination between human, mouse, and rat cells in paraffin-embedded tissue sections, providing significant advantages over current methods used to discern human versus rodent cell types. Using an in vivo prostatic developmental system where rat embryonic urogenital sinus mesenchyme is recombined with human prostate epithelial organoids and grown in an immunocompromised mouse, Cen/Tel FISH documents that all three species contribute to the development of glandular structures. The method is an indispensable tool to analyze xenograft/host interactions and prevent misinterpretation of data using tissue recombination approaches.

Urothelial transdifferentiation to prostate epithelia is mediated by paracrine TGF- β signaling

The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF- β signaling directed the prostatic induction. UGM from TGF- β type II receptor stromal conditional knockout mice (Tgfbr2 fspKO) or control mice (Tgfbr2 floxE2/floxE2) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2 floxE2/floxE2 UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2 fspKO UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2 fspKO UGM compared with Tgfbr2 floxE2/floxE2 UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF- β activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF- β signaling to mediate epithelial Wnt activity.

Anchorage-independent culture maintains prostate stem cells

Freshly isolated mouse prostate epithelial cells regenerate fully differentiated prostate tissue when combined with embryonic urogenital sinus mesenchyme and grafted in vivo. We show here that this regenerative capacity, which has been attributed to a small population of pleuripotential progenitor epithelial cells, is rapidly lost when the cells are placed in monolayer culture but can be maintained by culture in anchorage-independent conditions. Epithelial cells placed in anchorage-independent culture formed proliferating spheres that could be serially passaged and exhibited increased expression of putative stem cell markers as compared to cells grown in monolayer culture. Epithelial cells isolated from the fetal urogenital sinus, the newborn, and adult prostate formed spheres with similar efficiency, while cells isolated from the post-castration prostate exhibited significantly higher sphere-forming abilities. When passaged spheres were recombined with E17 rat urogenital sinus mesenchyme and grafted in vivo, they generated fully differentiated mouse prostate glandular epithelium containing both p63+ basal cells and p63− luminal cells and expressing a variety of prostate-specific and terminal differentiation markers.

In vivo regeneration of murine prostate from dissociated cell populations of postnatal epithelia and urogenital sinus mesenchyme.

The existence of a postnatal prostate stem cell is supported by several types of evidence. Withdrawal of androgen leads to involution of the gland, but readdition can rapidly stimulate regeneration. Tissue fragments derived from mouse or rat prostatic epithelia from midgestation embryos or adult mice, when combined with tissue fragments from urogenital sinus mesenchyme and grafted under the kidney capsule, can regenerate prostatic structures. Indirect evidence supports that the stem cell population is contained within the basal layer. Purified prostatic stem cell preparations would be useful to define the physical and functional properties required for regeneration and to compare with cells that accumulate during abnormal growth states, like prostate cancer. We have developed a regeneration system using dissociated cell populations of postnatal prostate epithelia and embryonic urogenital sinus mesenchyme. Efficient in vivo regeneration of prostatic structures in the subcapsular space of the kidney was observed within 4-8 wk with as few as 103 epithelial cells from prostates derived from donors 10 d to 6 wk of age. The regenerated structures show a branching tubular epithelial morphology, with expression of a panel of markers consistent with prostate development. Donor epithelial populations can be readily infected with GFP expressing lentiviral vectors to provide integration markers and easy visualization. The cell preparations of urogenital sinus mesenchyme can be expanded in short-term in vitro culture while their inductive capabilities are retained. Further definition of the subpopulation of prostate epithelial cells containing the regeneration activity should be possible with such technologies.

The influence of mesenchyme of neonatal seminal vesicle and embryonic urogenital sinus on the morphologic and functional cytodifferentiation of dunning prostatic adenocarcinoma: Roles of growth factors and proto-oncogenes

Previous studies have demonstrated that male genital tract mesenchymes can induce morphogenetic changes in the rat prostatic Dunning tumor (DT). However, the mechanism of these cellular interactions remains unknown. The aims of this paper are: (I) to examine the influence of neonatal seminal vesicle mesenchyme (SVM) and embryonic urogenital sinus mesenchyme (UGM) on the growth, and morphologic and functional cytodifferentiation of DT; (2) to investigate the possible role of growth factors and receptors including epidermal growth factor and its receptor, basic fibroblast growth factor and transforming growth factor-beta, and two proto-oncogenes, c-fos and c-jun, in these tumor-mesenchymal interactions. Combination of mesenchymes (SVM or UGM) with DT enhanced the growth and induced an apparently more normal morphologic cytodifferentiation in vivo with formation of large tubules lined by highly differentiated columnar epithelial cells and reappearance of fibromuscular stroma. The SDS-PAGE analysis has shown that the DT + UGM enlarged and small tubules secreted proteins different from those of parental DT, demonstrating that mesenchymes can also modulate the functional expression of DT. Interestingly, our immunohistochemical data demonstrate that all the selected growth factors, receptors, and proto-oncogenes are upregulated in the mesenchyme-induced DT epithelial cells, suggesting that these cellular regulators may be closely associated with the mesenchymal induction on the DT phenotypic changes in vivo. This finding implicates the potential role of these growth factors, receptors, and proto-oncogenes in the epigenetic pathway of prostate carcinogenesis via tumor-stromal interactions.

Epithelial-mesenchymal interactions in prostatic development. I. morphological observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder.

Tissue recombinants of embryonic urogenital sinus mesenchyme (UGM) and epithelium of the urinary bladder (urothelium, BLE) of adult rats and mice were grown for 3-30 d in male syngeneic hosts. Short-term in vivo growth indicated that prostatic morphogenesis is initiated as focal outgrowths from the basal aspect of the adult urothelium. The solid epithelial buds elongate, branch, and subsequently canalize, forming prostatic acini. After 30 d of growth in the male hosts, prostatic acini exhibit secretory activity. The marked changes in urothelial morphology induced by the UGM are accompanied by the expression of fine-structural features indicative of secretory function (rough endoplasmic reticulum, Golgi apparatus, and secretory granules). During this process, urothelial cells express prostatic histochemical markers (alkaline phosphatase, nonspecific esterase, glycosaminoglycans) and prostate-specific antigens. The expression within BLE of prostatic characteristics is associated with the loss of urothelial characteristics. These data indicate that adult urothelial cells retain a responsiveness to embryonic mesenchymal inductors. Furthermore, mesenchyme-induced changes in urothelial cytodifferentiation appear to be coupled to changes in functional activity.

Stromal-epithelial interactions: II. Regulation of prostatic growth by embryonic urogenital sinus mesenchyme.

Embryonic urogenital sinus mesenchyme (UGM) has been demonstrated previously to be a potent inductor of prostatic morphogenesis and functional differentiation when associated with either embryonic urogenital sinus epithelium (UGE) or urothelium derived from adult urinary bladder (ABLE) and grown in male hosts. To determine the role of mesenchyme in prostatic acinar growth, homotypic tissue recombinants of 16-day-old embryonic mouse urogenital sinus mesenchyme and epithelium (UGM mouse + UGE mouse) were prepared in which the relative amounts of UGM to UGE were varied from approximately 0.1:1 to 100:1. Recombinants were grown for 1 month in intact male hosts after which the amount of acinar growth was assessed by histological analysis and by determination of wet weight and DNA content. The latter criteria are valid measures of acinar content of histologically normal prostatic tissue because the rodent prostate is composed of greater than 80% ductal-acinar tissue. From this analysis the amount of acinar growth was found to be determined by the amount of mesenchymal tissue and was independent of the amount of epithelium utilized to prepare the tissue recombinants. Similarly, the magnitude of growth in hetero-specific rat-mouse tissue recombinants prepared with urogenital sinus epithelium or adult bladder epithelium (ABLE) and urogenital sinus mesenchyme (UGM mouse + UGE rat, UGM mouse + ABLE rat, UGM rat + UGE mouse, or UGM rat + ABLE mouse) is determined by the source of UGM. Although the initial DNA content per UGM is comparable between mouse and rat, the overall acinar growth induced by UGM rat is about 10-fold greater than that induced by UGM mouse when recombined reciprocally with UGE mouse and UGE rat, respectively. These results suggest that UGM is of fundamental importance as a regulator of prostatic epithelial growth. The relevance of this finding to the development of human benign prostatic hyperplasia is discussed.