Investigations of prostate epithelial stem cells and prostate cancer stem cells

Although both prostate epithelial stem cells and prostate cancer stem cells are implicated in the differentiation of the normal prostate gland and carcinogenesis of prostate cancer, there has, until recently, been little information regarding their biology. This review summarizes the recent advancements in cell biological research including various in vitro culture systems that have offered the characterization and isolation of prostate epithelial stem cells and prostate cancer stem cells. In addition, the stromal niche or microenvironment of stem cells plays an essential role in proliferation and differentiation of normal stem cells. Stroma surrounding cancer cells, which also provide another unique niche, may involve the initiation and development of cancer stem cells. Investigation of stem cells and their microenvironments in the prostate should lead to the elucidation of biological features and the development of novel treatments for prostate cancer.     

乳腺癌肿瘤干细胞体外培养体系的建立

目的建立一种适合乳腺癌肿瘤干细胞体外培养的三维培养体系。方法根据不同的培养方法分为两组:二维细胞培养组,采用传统二维细胞培养方法培养乳腺癌MCF-7细胞;三维细胞培养组,将乳腺癌MCF-7细胞接种于三维胶原支架材料上,在摇床上动态培养5d,在光学显微镜和激光共聚焦显微镜下观察乳腺癌肿瘤细胞在胶原材料上的形态学特征。采用CCK-8细胞增殖检测的方法,比较三维培养组和二维培养组细胞增殖能力,通过流式细胞术检测两组乳腺癌肿瘤干细胞标志CD44~+/CD24~-/low的表达差异。结果光学显微镜下观察结果显示:二维培养组MCF-7细胞为单层生长平展的粘附生长,呈多边形、铺路石样的上皮细胞形态;三维培养组MCF-7来源的细胞再次二维培养后,细胞展现出三角形或者长梭形及较多细胞呈不规则形,部分细胞还伸展出长浸润性伪足。激光共聚焦显微镜观察显示,MCF-7细胞均匀地生长在三维支架材料上,局部放大后清晰可见长伪足,且细胞大小不均一,细胞形态多样。三维培养组细胞增殖速度与二维培养组相似。与二维培养组相比较,三维培养组CD44~+/CD24~-细胞比例显著增加,由38.9%增加至60.3%。结论基于三维胶原支架材料的三维培养体系能够有效维持乳腺癌肿瘤干细胞干性。     

Cancer stem cells in prostate cancer

Prostate cancer (P-Ca) remains a leading cause of cancer-related death in men. Lately, increasing evidence for a hierarchically organized cancer stem cell (CSC) model emerged for different tumors entities, including P-Ca. CSCs are defined by several characteristics including self-renewal, pluripotency and tumorigenicity and are thought to be responsible for tumor recurrence, metastasis and cancer related death. In this review we discuss the recent research in the field of CSCs, its limitations and therapeutical implications in general and specifically in P-Ca.     

Effective antigen cross-presentation by prostate cancer patients' dendritic cells: implications for prostate cancer immunotherapy

Despite the potency with which dendritic cells (DCs) are able to utilize the exogenous MHC I antigen cross-presentation pathway to cross-present antigen for the activation of killer T cells in model systems, concern about defects in immune function in cancer patients has led to uncertainty regarding whether immune cells derived from patients can effectively be used to generate tumor vaccines. We have undertaken a careful analysis of the potency of using DCs obtained from prostate cancer patients to cross-present antigen derived from human prostate tumor cells for the activation of antigen-specific T cells. Such DCs can be matured ex vivo into functionally active cells and are capable of cross-presenting influenza antigen derived from internalized apoptotic prostate tumor cells. Importantly, we demonstrate effective stimulation of both CD4+ and CD8+ T cells, as evident by production of IFN-gamma, and the ability of CD8+ T cells to differentiate into effector CTLs. These results, defining conditions in which prostate cancer patient DCs can efficiently utilize the cross-presentation pathway and in which apoptotic tumor can serve as a source of antigen for DCs to activate T cells, demonstrate that this system warrants clinical study as a potential immunotherapy.     

碱性成纤维细胞生长因子在富集结直肠癌肿瘤干细胞中的作用

目的 研究不同浓度碱性成纤维细胞生长因子（bFGF）作用于结直肠癌细胞不同时间,富集肿 瘤干细胞的效果。方法? 结直肠癌DLD-1细胞分别在含5 ng/mL、10 ng/mL、20 ng/mL bFGF的无血清培 养基中悬浮培养,分为G1、G2、G3组,设置培养时间梯度为10 d、20 d、30 d,获得球细胞。采用流式 细胞术（FCM）检测细胞球中的CD44+、CD133+及CD44+CD133+双阳性的细胞表达比例,Real-time PCR 检测球细胞中的干细胞基因（KLF4、Nanog）;上皮间质转化基因（E-cadherin、Snail）;Wnt/β-catenine通路 基因（Wnt-3a）的 mRNA表达情况,成球实验检测细胞球的自我更新能力。结果 FCM检测结果：CD44+ 阳性表达的细胞表达以G2组20 d最高,CD133+及CD44+CD133+双阳性的细胞表达均以G2组20 d及G3 组10 d最高,差异有统计学意义（F=98.895、147.641、13.321,P<0.05）。 Real-time PCR检测结果：各组中 KLF4、Nanog、Snail以及Wnt-3a mRNA的表达均以G2组20 d表达最高（F=2.424、7.694、2.951、3.771, 均P<0.05）, E-cadherin基因G2组20 d mRNA表达最低（G2组30 d、G1组10 d除外）（F=10.620,P<0.05）。 成球实验结果：各组比较G1组20 d和G2组20 d成球数量明显多于其他组,差异具有统计学意义（F=14.279, P<0.01）;但 G1组20 d和G2组20 d比较,无统计学差异（t=0.605,P=0.578）。 结论 碱性成纤维细胞生长 因子无血清悬浮培养能够有效富集肿瘤干细胞,其中G2组培养20 d较其余组能更有效地富集结直肠癌肿 瘤干细胞。     

Human prostate cancer stem cells: new features unveiled

Cancer stem cells (CSCs) are a rare sub-population of phenotypically distinct cancer cells exhibiting stem cell characteristics.They are tumourigenic, meanwhil...     

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)。     

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。     

Bi-directional interactions of prostate cancer cells and bone marrow endothelial cells in three-dimensional culture

BACKGROUND: Prostate cancer preferentially metastasizes to bone, yet little is known about the cellular and molecular factors that support this growth. Endothelial cells are likely the initial contact for circulating prostate cells entering the bone microenvironment. METHODS: Using co-culture and conditioned media experiments, we studied cellular and molecular interactions of prostate cancer cells of varying aggressiveness (PC-3 and LNCaP) with bone marrow endothelial (HBME-1) cells in collagen gels. RESULTS: In co-culture, HBME-1 cells stimulated proliferation ( approximately 90% increase) and migration of the more aggressive PC-3 cell line, while having little effect on LNCaP cell proliferation or migration. Concomitantly, HBME-1 cell growth was inhibited by both PC-3 and LNCaP cells and their conditioned media. Additionally, HBME-1 cells underwent significant morphological changes in co-culture, forming large, branching, cord-like structures, which mimic angiogenesis. Prostate cancer cell conditioned media induced a similar effect on HBME-1 cells. In comparison, conditioned media from PC-3 cells also inhibited growth of non-bone marrow-derived endothelial cells, but did not affect their morphology. CONCLUSIONS: Significant bi-directional interactions, including secreted factors and direct cellular interactions, exist between bone marrow endothelial cells and highly metastatic prostate cancer cells, and may underlie the propensity for prostate cancer to metastasize to the bone.     

In vitro propagated dendritic cells from prostate cancer patients as a component of prostate cancer immunotherapy

T cell-mediated cancer immunotherapy requires efficient antigen-presenting cells. Dendritic cells (DCs) are arguably the most efficient antigen-presenting cells studied to date. Individuals with prostate cancer often undergo various therapies which may compromise their immune system, including the state of their DC precursors. We report the in vitro propagation of DCs from peripheral blood of patients with prostate cancer, most of whom are in clinical stages D₁ or D₂ and have undergone radiation therapy. After 7 days in culture, the number of DCs recovered were 20-50-fold higher than those isolated directly from peripheral blood. This number is comparable to findings of previous studies with healthy individuals. Cultured patients' DCs were capable of presenting tetanus toxoid to autologous T cells in vitro. Furthermore, T cells from 2 of 4 patients proliferated when cultured with their DCs and the lysate of a human prostate cancer cell line (LNCaP), demonstrating the potential role of autologous DCs in prostate cancer immunotherapy studies. © 1995 Wiley-Liss, Inc.     

体外琼脂克隆培养对胎肝干细胞分化的影响

目的 探讨和建立体外扩增胚胎肝干细胞并抑制其分化的培养条件及方法.方法 胶原酶结合机械消化法制备14 d胎龄大鼠胎肝干细胞悬液.将细胞随机分为两组.一组接种至Ⅰ型胶原包被的培养板中,常规贴壁培养.另一组细胞则接种至软琼脂培养基中进行悬浮培养.倒置显微镜下观察两组细胞的生长.培养2周后收集两组细胞,电镜观察两组细胞超微结构的差异,流式细胞仪检测其CD90.1+、CD49F+等干细胞标志物表达特征,碱性磷酸酶染色检测其分化状态的差异.结果 悬浮培养的胎肝干细胞具有高核浆比、胞浆内细胞器不发达等超微结构特征,并高表达CD90.1、CD49F等干细胞标志物,碱性磷酸酶染色阳性;而常规贴壁培养的胎肝干细胞则在培养过程中显示出显著的分化特征.结论 琼脂克隆培养的胎肝干细胞较有血清贴壁培养分化程度低,在琼脂克隆培养条件下能增殖形成具有显著干细胞特征的克隆球.     

Identification,characterization, and biological relevance of prostate cancer stem cells from clinical specimens

Cancer stem cells (CSCs) are a reservoir of tumor cells that exhibit the properties of self-renewal and the ability to re-establish the heterogeneous cell population of a tumor. They appear therapy-resistant and may be the underlying cause of recurrent disease. Using prostate as a model, this review presents the CSC hypothesis and discusses the role of the androgen receptor in CSCs, the methods used for isolating CSCs, and the therapeutic challenges CSCs have for cancer therapy.     

骨髓间充质干细胞向前列腺癌趋向转移的研究

目的探讨人骨髓间充质干细胞（hMSCs）在体内向前列腺癌微环境趋向转移的特性。方法应用人前列腺癌细胞株PC-3异种皮下种植建立前列腺癌严重联合免疫缺陷小鼠（SCID）模型，通过密度梯度离心和贴壁法分离、培养hMSCs，将体外4,6-联脒-2-苯基吲哚（DAPI）标记的4～6代hMSCs经尾静脉和肿瘤周围注射入荷瘤SCID鼠体内。分别在经尾静脉注射后17d和肿瘤周围注射后7、10、14d处死小鼠，收集肿瘤和肝、肺、脾、肾等脏器作冰冻切片和石蜡切片。在荧光显微镜下观察肿瘤及各脏器中hMSCs的分布情况。结果前列腺癌SCID鼠模型成瘤率83．3％，经尾静脉注射DAPI标记的hMSCs后17d和肿瘤周围注射DAPI标记的hMSCs后7、10、14d，荧光显微镜下显示，前列腺癌组织中可见DAPI标记的hMSCs的核呈蓝色荧光。而肝、肺、脾、肾等脏器中均未见hMSCs的存在。结论hMSCs在体内具有向前列腺癌微环境趋向转移的特性。     

Selective growth of epithelial basal cells from human prostate in a three-dimensional organ culture

BACKGROUND: A three-dimensional organotypic culture method has been developed for selectively growing epithelial basal cells from human benign prostate. METHODS: Tissue fragments were cultured on sponges for several weeks and the viability of luminal and basal epithelium and cellular responses to 4,5alpha-dihydrotesterone (DHT) stimulation were studied. RESULTS: The gland tissue could be successfully maintained showing preservation of ducts and lobules as in vivo. Without DHT, a progressive hyperplasia of basal cells was observed: these cells proliferated with retention of the lumen or forming nests with squamous differentiation, lining the surface of the fragment and migrating to the underlying sponge. In contrast, secretory cells disappeared. Epithelial cells isolated from long-term cultures showed a typical basal cell-immunophenotype. DHT-treated tissues maintained a much higher percentage of luminal cells than untreated tissues. CONCLUSIONS: These systems allow the study of proliferation and differentiation of basal cells within their natural microenvironment as well as prostate pathobiology.