前列腺癌干细胞研究进展

前列腺癌是男性患者中最常见的癌症,严重危害男性的健康.目前关于前列腺癌的发病理论并不能完全解释该疾病异质性、转移性生长、耐药性和肿瘤复发的机制.前列腺干细胞是前列腺中具有自我再生和增殖功能的异质亚群,能产生构成前列腺上皮的所有细胞谱系.根据癌症干细胞假说,前列腺癌可能是一种干细胞疾病.前列腺癌干细胞具有明显的克隆形成性...     

间充质干细胞在小鼠体内可以向前列腺癌组织募集促进前列腺癌的生长

目的探索间充质干细胞在小鼠体内与前列腺癌组织生长的关系,以期为前列腺癌的治疗探索新的靶点。方法野生型C57BL/6小鼠经致死剂量照射后,接受EGFP绿色荧光转基因C57BL/6小鼠的MSCs以及野生型小鼠全骨髓细胞的混合移植。1月后在骨髓移植成功的小鼠身上建立前列腺癌RM-1细胞皮下移植瘤模型,检测前列腺癌肿瘤的生长情况。同时以荧光显微镜显像技术及流式细胞仪技术观察、检测MSCs在小鼠前列腺癌组织中的表达情况。结果皮下接种RM-1前列腺癌细胞后,给予小鼠尾静脉注射MSCs的实验组肿瘤的生长速度要快于注射生理盐水的对照组,肿瘤重量分别为(3.730±0.632)g、(2.019±0.254)g(P0.05);无论是骨髓移植后内源性的MSCs还是直接尾静脉注射的外源性MSCs都可以在前列腺癌组织中检测到。结论内源性以及外源性的MSCs都可以向前列腺癌组织趋化募集,并促进前列腺癌的生长。     

Differentiation pathways and histogenetic aspects of normal and abnormal prostatic growth: A stem cell model

A stem cell model is presented for the organization of the prostatic epithelium that may explain normal and abnormal growth in the human prostate. This model is based on recent data indicating that: 1) The three basic cell types encountered in the prostatic epithelium—i.e., secretory luminal, basal, and endocrine paracrine (EP) cells—are linked in the precursor progeny relationship. 2) The proliferative compartment of the normal and hyperplastic epithelium is located in the basal cell layer. 3) The proliferative compartment of the prostatic epithelium is androgen-independent but contains androgen-responsive target cells. 4) During the malignant transformation of the prostatic epithelium, the proliferative zone is inverted and shifts to luminal cell types. 5) Formation of neoplastic basement membrane (BM) material is crucial for the development of the invasive phenotype in prostate cancer. 6) The proliferative activities in prostate cancer are exclusively restricted to exocrine cell types, whereas endocrine differentiated tumor cells are postmitotic cells. 7) The majority of exocrine tumor cells are androgen-responsive in contrast to endocrine differentiated cell types that consistently lack the nuclear androgen receptor (AR). In this model, a small stem cell population located in the basal cell layer gives rise to all epithelial cell lineages encountered in the normal, hyperplastic, and neoplastic prostate. The differentiating process from basal cells to secretory luminal cells via intermediate phenotypes is induced by circulating androgens, and largely depends on the presence of androgen-responsive target cells in the basal cell layer. Accordingly, the abnormal growth of the secretory epithelium in benign prostate hyperplasia (BPH) may be related to an increase in the total number of androgen-responsive basal cells in the proliferative compartment. Prostate cancer derives from transformed stem cells located in the basal cell layer that acquire secretory luminal characteristics under androgenic stimulation. During tumor invasion, the malignant phenotypes adhere via specific receptors to newly formed BM-material, which, in turn, may facilitate their passage through the extracellular matrix. The occurrence of endocrine differentiation in prostate cancer reflects the pluripotency of its stem cells. The widespread absence of nuclear AR in endocrine differentiated tumor cells clearly indicates that this phenotype belongs to those cell clones in prostate cancer, that are initially androgen-independent and refractory to hormonal therapy. Accordingly, the progressive emergence of endocrine cell clones during tumor progression may represent one mechanism by which prostate cancer cells escape hormonal control. © 1996 Wiley-Liss, Inc.     

Stem cells in prostate and prostate cancer development

Most cancers comprise a heterogenous population of cells with marked differences in their potential to proliferate as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. Dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. Cell signaling pathways shared by stem cells and cancer cells lend further evidence for a possible link between these 2 populations of cells. Study of the differentiation pathways of normal and abnormal prostate growth has led to the development of a stem cell model for prostate cancer. The basal layer of the normal prostate is believed to be populated by prostate epithelial stem cells and a population of transit-amplifying cells intermediate in differentiation to the stem and fully differentiated cells. There is recent evidence suggesting that prostate cancer occurs from malignant transformation of stem/progenitor cells, thereby resisting apoptosis and spawning proliferation. This new model for prostate cancer will have significant ramifications for the way this disease is studied and treated. Furthermore, through targeting the prostate cancer stem cell and its dysregulated self-renewal, therapies for treatment of prostate cancer are likely to improve.     

Characterization of autoimmune inflammation induced prostate stem cell expansion

BACKGROUND

The presence of inflammation in prostate cancer (PCa) and benign prostate hyperplasia (BPH) has been well described but the cellular mechanisms by which inflammation modulates the prostate are currently unclear. Prostate stem cells (PSC) not only maintain prostate homeostasis but also are considered to be the cell of origin of PCa and an important contributor to BPH. However, the impact of inflammation on PSC is not well understood. Therefore, we initiated studies to evaluate the effect of inflammation on PSC.

METHOD

Ovalbumin specific CD8⁺ T cells were intravenously delivered to intact and castrated prostate ovalbumin expressing transgenic‐3 (POET‐3) mice to induce inflammation. Lin (CD45/CD31)^?Sca1⁺CD49f⁺ cells (LSC) and progenitor cells within LSC were determined by flow cytometry. Sorted LSC were subjected to a prostate sphere forming assay to evaluate PSC clonal propagation, proliferation, immediate differentiation, and self‐renewal ability. Density of individual spheres was measured by a cantilever‐based resonator weighing system. Morphology and characterization of prostate spheres was determined by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Finally, immediate PSC differentiation in sphere formation was determined by immunofluorescence for epithelial cytokeratin markers cytokeratin (CK) 5 and CK8.

RESULT

Data presented here demonstrate a significant expansion of the proliferative (BrdU⁺) LSC population, including CK5⁺, p63⁺, CK18⁺ cells, as well as intermediate cells (CK5⁺/CK8⁺) in inflamed prostates. Histological images reveal that PSC from inflamed prostates produce significantly larger spheres, indicating that the enhanced proliferation observed in LSC is sustained in vitro in the absence of inflammatory mediators. In addition, cultures from inflamed PSC yielded increased number of tubule‐like spheres. These tube‐like spheres grown from PSCs isolated from inflamed mice exhibited stratification of a CK8⁺ luminal‐like layer and a CK5⁺ basal‐like layer. Notably, the numbers of spheres formed by inflamed and non‐inflamed PSC were equal, suggesting that even though proliferation is enhanced by inflammation, the homeostatic level of PSC is maintained.

CONCLUSION

Induction of inflammation promotes PSC expansion and immediate differentiation through highly proliferative progenitor cells while the homeostasis of PSC is maintained. Prostate 75:1620–1631, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

     

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.     

Erythropoietin stimulates growth and STAT5 phosphorylation in human prostate epithelial and prostate cancer cells

BACKGROUND: Erythropoietin (Epo), the principal regulator of erythroid progenitor survival, growth, and differentiation, initiates its action by binding to its cognate cell surface receptor (EpoR). EpoR have been identified on a variety of non-hematopoietic cells, both normal and malignant, however, little is known about the function of EpoR on malignant cells. METHODS: RT-PCR, Western blotting, and immunohistochemistry were used to demonstrate that prostate cancer cells express EpoR at both the gene and protein level. Cell proliferation assays and STAT5 phosphorylation were used to demonstrate Epo's mitogenic action and intracellular signaling, respectively. RESULTS: We have demonstrated that transformed prostate epithelial and prostate cancer cell lines, as well as primary prostate tissue, express the EpoR. Importantly, the EpoR on prostate cells are functional, as demonstrated by the observation that each of the cell lines exhibited a dose-dependent proliferative response to Epo, and that Epo triggered STAT5b phosphorylation in the cells. CONCLUSION: Human prostatic epithelial cells and prostate cancer cells express functional EpoR, and Epo serves as a growth factor for these cells. These results have implications for our understanding of normal prostatic growth and development and of the pathobiology of human prostate cancer.     

Intermediate basal cells of the prostate: in vitro and in vivo characterization

BACKGROUND: Progenitor cells within the prostate basal layer may play important roles in differentiation and carcinogenesis; however, prostate stem cell populations remain uncharacterized. METHODS: Immunohistochemical and immunoblot analyses were used to characterize prostate epithelial cells (PrEC), a commercially available prostate basal cell isolate. RESULTS: Proliferating PrECs exhibited immunophenotypic characteristics most consistent with basal cells, but during senescence PrECs up-regulated androgen receptor (AR) mRNA, p27, and low-molecular-weight cytokeratin (LMWCK) expression, suggestive of partial differentiation. PrECs also stained strongly for involucrin, which marked a subset of intermediate prostate basal cells in vivo. Basal hyperplasia consisting of involucrin-positive cells was prevalent in prostate tissue from androgen-ablated patients, and formed epithelial clusters flanked by involucrin-negative basal and luminal monolayers. Cultivation of PrECs on matrigel together with androgen-treated stromal conditioned media resulted in dense aggregates, with a peripheral rim of basal-like cells expressing p63 and basal cytokeratins. CONCLUSIONS: PrEC represents an epithelial population whose basal characteristics are modified in response to matrigel, stromal factors, and senescence, consistent with a transient amplifying population. These cells may derive from a previously unrecognized, involucrin-positive subset present in vivo.     

Delta-like 1 (Dlk-1), a novel marker of prostate basal and candidate epithelial stem cells, is downregulated by notch signalling in intermediate/transit amplifying cells of the human prostate

Background

There is a lack of understanding of the processes that regulate differentiation in the prostate.

Objective

To determine localisation, activity, and regulation of cytodifferentiation-modulatory proteins in the human adult prostate.

Design, Settings, and Participants

Eighteen volunteering patients with organ-confined prostate cancer were prospectively enrolled at a single university hospital.

Intervention

All patients underwent radical prostatectomy, and normal/benign tissue was excised and obtained from the transition zone.

Measurements

Expression and activity of Notch-protein family members, including the Notch-homologous protein Delta-like 1 (Dlk-1/Pref1), were investigated immunohistochemically in normal/benign tissue and explant cultures. The effect of the Notch inhibitor L-685,458 on Dlk-1 expression and cell number was investigated in primary cell cultures, and data were analysed with Student t test.

Results and Limitations

Mature luminal cells were found to co-express Notch-1 and its ligand Jagged1, but epithelia in normal/benign tissue showed no active Notch signalling. The basal cell layer, rare candidate epithelial stem cells, and a subpopulation of neuroendocrine cells expressed the differentiation protein Dlk-1. In explant cultures, luminal cells and Jagged1 expression were lost, whereas intermediate cells downregulated Dlk-1 concomitant with Notch-1 upregulation and activation. Notch inhibition in primary cell cultures led to lower cell densities (p < 0.001) and suppressed downregulation of Dlk-1. This is a small study; current results need to be confirmed in larger investigations.

Conclusions

We demonstrate that Notch-1 is upregulated in differentiation of prostate epithelia, and that the novel prostate progenitor marker Dlk-1 is downregulated by Notch signalling in intermediate cells. The identification of Dlk-1–expressing candidate stem and neuroendocrine cells suggests a hierarchical relationship.     

Renal differentiation from adult spermatogonial stem cells

Abstract

There is considerable interest in the use of multi-potent stem cells in kidney tissue regeneration. We studied if spermatogonial stem cells have the ability to undergo kidney differentiation. Spermatogonial stem cell differentiation was induced using in vitro and ex vivo co-culture techniques. Conditioned media from human kidney fibroblasts induced the expression of epithelial and endothelial lineages in spermatogonial stem cells, consistent with nephrogenesis. Furthermore, we showed that these cells up-regulated renal tubular-specific markers alkaline phosphatase, mineralocorticoid receptor, renal epithelial sodium channel and sodium-glucose transporter-2 (p?<?0.05). GFP-labeled spermatogonial stem cells were engrafted into metanephric kidney organ cultures harvested from E12.5 mouse embryos. After 5 days of organ culture, focal anti-GFP staining was detectable in all inoculated kidneys demonstrating integration of spermatogonial stem cells into the developing kidney (p?<?0.01). Histological assessment showed early nephron-like architecture. In summary, we show that spermatogonial stem cells have the potential to generate renal tissue and lay the foundations for further investigations into a novel therapeutic approach for renal insufficiency.     

TGFBR3 loss and consequences in prostate cancer

BACKGROUND: Resistance to transforming growth factor-beta (TGF-beta) is important in tumorigenesis. TGF-beta resistance mechanisms in prostate cancer are not well understood. METHODS: We have conducted a systematic analysis of TGF-beta pathway components with a meta-analysis of seven microarray studies using Oncomine and evaluated the results of TGFBR3 expression in prostate cell lines. Furthermore, we knocked down TGFBR3 in prostate epithelial cells and analyzed the consequences of TGFBR3 knockdown. RESULTS: We found that TGFBR3 is the TGF-beta component most commonly downregulated among localized human prostate cancer studies. TGFBR3 knockdown led to focus formation and enhanced expression of CD133, a marker found on prostate cancer stem cells. DNA microarray analysis of TGFBR3 knockdown cells identified 101 genes regulated by TGFBR3. Seven of these genes show a corresponding decrease in clinical prostate cancer specimens, which include genes involved in prostate mass and vasculature. CONCLUSIONS: TGFBR3 downregulation is an important step in prostate tumorigenesis.     

A tumour stem cell hypothesis for the origins of prostate cancer

Cancer stem cells undoubtedly exist in many tumour types, including the prostate. This hypothesis can explain both the heterogeneity of prostate tumours and their variable responses to several conventional therapies. In the longer term, therapies directed against tumour stem cells should offer a real possibility of long-term cure, rather than current palliative therapy. Identifying specific tumour stem-cell markers will enhance this process, but the scarcity of these cells within the mass of more differentiated amplifying progeny that comprise >99.9% of most cancers makes this a severe technical challenge. In addition, many tumour stem-cell markers are probably shared with normal stem cells, both in prostate and in stem cells from other tissues, but tumour-specific patterns of gene expression, probably designed to allow the tumour stem cell to survive outside its protective 'niche' in normal tissues, will be the best initial targets for new therapeutic agents.     

细胞株源人前列腺肿瘤干细胞的分选与鉴定

目的:论证人前列腺癌(prostate cancer,PCa)细胞株中是否存在干细胞亚群。方法:分别用免疫表型法和侧群(side population,SP)细胞法从5种人PCa细胞株(Du145、IA8、LNCaP、TSU-PrL和PC-3)中富集类干细胞,再应用软琼脂克隆形成试验初步验证类干细胞亚群的体外生长方式及成瘤能力。选择LNCaP源SP细胞(LNCaP/SP),依次采用免疫细胞化学技术、Transwell、MTT以及裸鼠致瘤试验,分别检测其干细胞标记物的表达情况、鉴定其体外增殖和侵袭能力以及动物体内的致瘤和转移潜能。结果:5种细胞株中均难以分选出免疫表型为CD133+CD44+的细胞亚群。除PC-3外,其余4株细胞可分选出呈现典型克隆性生长特点的SP细胞。体外克隆形成率在IA8、LNCaP和TSU-PrL源SP细胞与非侧群(non-side population,NSP)细胞间有显著性差异(P<0.05)。与LNCaP/NSP相比,LNCaP/SP的体外增殖和侵袭能力显著增强,同时阳性表达整合素α2、Nanog、CD44、OCT4以及ABCG2等5种干细胞标记物。而且,LNCaP/SP的皮下成瘤率、骨转移率及瘤体体积亦显著高于LNCaP/NSP(P<0.01)。结论:SP分选法更适合富集人PCa细胞株中类干细胞,LNCaP/SP细胞是PCa细胞株LNCaP中的肿瘤干细胞(cancer stem cell,CSC)。     

Interferon-gamma induces neuroendocrine-like differentiation of human prostate basal-epithelial cells

BACKGROUND: Prostatic neuroendocrine (NE) cells are intraglandular hybrid epithelial-neural-endocrine cells that express and secrete numerous hormones and neuropeptides, which presumably regulate growth, differentiation, and secretory activity of the prostatic epithelium. This specialized cell type appears to differentiate from a common basal/precursor/stem cell that also gives rise to the secretory epithelium. In order to elucidate mechanisms of NE-differentiation the effects of type 1 (alpha, beta) and type 2 (gamma) interferons (IFNs) on human prostate basal cells (PrECs) were evaluated. METHODS AND RESULTS: Application of alpha/beta IFN increased the expression of the cell-cycle inhibitor p21(CIP1) and inhibited DNA synthesis, while only IFN-gamma led to increased apoptosis, cell-cycle inhibitor p27(KIP1) upregulation, and differentiation of PrECs into NE-like cells. In vitro differentiated NE-like cells expressed the glycolytic enzyme neuron-specific enolase (NSE) and chromogranin A (CgA), known markers of NE-cells in vivo in the prostate. These NE-like cells also changed cytokeratin (CK) expression patterns by upregulating CK 8/18, predominantly found in terminally-differentiated secretory luminal/NE epithelial cells. CONCLUSIONS: IFN-gamma produced locally in the prostate by basal cells and, under proinflammatory conditions, by infiltrating lymphocytes could support NE cell differentiation and play a role in NE differentiation processes of tumor cells in hormone-refractory prostate cancer.     

前列腺神经内分泌细胞与前列腺疾病的关系

前列腺神经内分泌细胞(NECs)是一种非常独特的细胞,它不仅具有神经细胞的特性,同时还具有内分泌细胞和上皮细胞的特性。前列腺组织中存在的少量NECs能分泌神经内分泌肽,参与组成神经内分泌调节系统,通过内分泌、旁分泌、自分泌等多种形式,对前列腺的发育和生长分化及内分泌起着调节作用。在前列腺疾病,如慢性前列腺炎、前列腺增生及前列腺癌的发生、发展及转归中,NECs均起到重要作用。尤其是NECs缺乏雄激素受体,在去势治疗时仍可继续生长,可能通过旁分泌的方式促进肿瘤雄激素非依赖性增值,引起肿瘤复发。本文就近年来对NECs分泌的各种细胞因子及受体进行鉴定、深入研究的进展做一综述。     

Prostate stem cells: The niche and cell markers

Abstract:   Prostate cancer and benign prostatic hyperplasia are common diseases in elderly men worldwide. Identifying the prostate stem cell is an important tool to investigate the mechanism of these prostatic diseases. Although the prostate stem cell has not yet been detected, progress has been made. The 'niche' or place in which the prostate stem cell resides is thought to be located in the proximal region of the murine prostate, near the urethra. Several candidate prostate stem cell markers are currently under investigation. In this review, we summarize the historical approaches and recent evidence regarding the niche and prostate-specific stem cell markers.     

Prostatic stromal cells derived from benign prostatic hyperplasia specimens possess stem cell like property

INTRODUCTION: The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. METHODS: Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. RESULTS: Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. CONCLUSIONS: Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia.     

Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model

INTRODUCTION: The phenotypic plasticity of the human prostate stem cell within human prostate tissue was examined to determine the response of the stem cell to changes in the androgenic environment. METHODS: Prostate xenografts were transplanted into athymic nu/nu mice implanted with testosterone pellets, allowed to establish for 1 month time point, the hosts were castrated and pellets removed, and following 1 month of androgen deprivation, the hosts were stimulated with androgen for 2 days to induce proliferation of the residual population of stem cells (2-month time point). RESULTS: Glands in benign xenografts harvested at the 1- and 2-month time points contained basal cell layers that expressed p63 and high molecular weight cytokeratin, and in which essentially all of the cellular proliferation was localized, consistent with the proposed localization of the prostate stem cell. Benign glandular structures in the xenografts were populated by basal, secretory epithelial, neuroendocrine (NE), or squamous cells overlaying the basal cell layer, whereas, adenocarcinoma glands in the xenografts resembled the original prostate cancer (CaP) tissue. CONCLUSIONS: In this human prostate primary xenograft model, the residual stem cell population that survives transplantation, or androgen deprivation, maintains significant pluripotentiality as demonstrated by the capacity to generate progeny that differentiate along multiple lineages in response to microenvironmental signals, particularly along the secretory epithelial lineage in response to androgen, and along the NE cell lineage in response to androgen deprivation.