基底样乳腺癌和管腔A 乳腺癌干祖细胞的生物学行为比较*

目的:从人基底样和管腔A 乳腺癌分离肿瘤干/祖细胞,并比较其增殖、自我更新和分化能力等生物学特点。方法:应用乳腺微球无血清悬浮培养法富集乳腺癌干/祖细胞,并通过细胞生长曲线测定和连续克隆形成实验检测了干/祖细胞在体外的增殖能力和自我更新能力,建立裸鼠移植瘤检测在体内的成瘤情况,流式细胞术检测CD44和CD24的表达水平。结果:基底样和管腔A 乳腺癌均能在无血清培养基中形成乳腺微球,基底样癌的肿瘤干细胞含量较高,并形成细胞数量多、直径大的微球,球内细胞在体内、外有更强的扩增能力。基底样癌传代过程中,细胞容易贴壁分化,传代多不超过15代;而管腔A 癌,虽然丧失克隆形成能力的悬浮单细胞明显增多,但均可传代20次以上;两者CD44和CD24的表达也有明显差异。结论:基底样乳腺癌和管腔A 乳腺癌的干/祖细胞表现出差别明显的生物学行为,乳腺癌的肿瘤干细胞可能是异质性的。      

乙醛脱氢酶阳性乳腺癌干细胞的分离培养及其生物学特性分析

 目的　证实人类乳腺癌MCF-7细胞系中存在ALDH+乳腺癌干细胞,并研究ALDH+乳腺癌干细胞体外增殖、侵袭能力等生物学特性。方法　应用流式细胞术从MCF-7细胞中分离并培养ALDH+ 乳腺癌干／祖细胞,通过划痕试验、MTT法生长曲线测定、以及Transwell法等检测ALDH+乳腺癌干细胞的生物学特性。结果　MCF-7细胞系中,CD-／low24 CD+44 细胞比例约为1.4 %,ALDH+ CD-／low24 CD+44 细胞比例约为1.2 %; ALDH+乳腺癌干细胞与CD-／low24 CD+44 细胞两者的生长曲线基本一致;CD-／low24 CD+44 ,ALDH+ CD-／low24 CD+44 组细胞划痕区细胞间距离明显缩短,其迁移能力明显强于对照组,且两群干细胞之间存在差异;Transwell实验结果,与对照组相比,CD-／low24 CD+44 、ALDH+ CD-／low24 CD+44 两组细胞有大量细胞过膜,三组MTT检测吸光度值分别为1.05±0.098、1.56±0.075、1.67±0.032。结论　MCF-7细胞系中存在CD-／low24 CD+44 和ALDH+ CD-／low24 CD+44 乳腺癌干细胞,ALDH可以作为鉴定乳腺癌干细胞的分子标志之一。     

ALDH+乳腺癌干细胞的原代分离培养及生物学特性分析

目的 证实乳腺癌组织中存在ALDH+乳腺癌干细胞,并研究ALDH+乳腺癌干细胞体外增殖、侵袭能力等生物学特性.方法 应用流式细胞法从人乳腺癌组织细胞中分离并培养ALDH+乳腺癌干/祖细胞,通过克隆形成实验,生长曲线测定,以及transwell法等实验方法检测ALDH+乳腺癌干细胞的生物学特性.结果 在乳腺癌组织内,我们检测到ALDH+ CD24-/lowCD44+约占总细胞量的16％,无血清培养72 h后逐渐出现细胞球,2～3周时细胞球数量达到高峰.Transwell法检测ALDH+乳腺癌干细胞侵袭性较ALDH-乳腺癌干细胞侵袭性强.结论 乳腺癌组织中存在ALDH+ CD24-/low CD44+乳腺癌干细胞,ALDH可以作为鉴定乳腺癌干细胞的分子标志之一.     

乳腺干细胞和乳腺癌

乳腺干细胞是一种未分化的细胞，具有永远增殖和自我更新能力。乳腺癌中的癌症干细胞可能起源于正常干细胞，以细胞表面标志CD44^＋/CD24^-/low识别。癌症干细胞可能为肿瘤转移和复发的原因。识别乳腺癌干细胞和普通癌症细胞之间的差异，可以发展更有效的诊断和治疗方法。     

LoVo细胞系中结肠癌干细胞样细胞的分离、培养及鉴定

摘 要　目的：从结肠癌LoVo细胞系中分离、鉴定具有CD44+/EPCAM high特异表型的结肠癌干细胞样细胞,观察其生物学行为,证实该细胞系中结肠癌干细胞样细胞的存在。方法：从普通血清培养的LoVo细胞系中以流式细胞仪分选具有CD44+/EPCAM high表型的细胞,接种于添加生长因子的无血清培养基中,观察其增殖过程,继而诱导分化。MTT法、流式细胞术检测CD44+/EPCAM high、EPCAM low和未分选LoVo细胞的增殖能力及细胞周期分布。3种细胞接种裸鼠,比较不同细胞的成瘤率;免疫荧光技术检测小鼠次代CD44+/EPCAM high细胞中 CD44/EPCAM的表达。结果：LoVo细胞中有17.4%的CD44+/EPCAM high细胞,并能在添加生长因子的无血清培养基中呈细胞球样生长,且可连续传代;在血清的诱导下,呈贴壁分化生长,其形态与未分选LoVo细胞无差别。CD44+/EPCAM high细胞增殖能力高于EPCAM low细胞及未分选LoVo细胞,且细胞周期多集中在G0/G1期。以500个CD44+/EPCAM high细胞接种裸鼠成瘤率为90%（9/10）,而1×104个EPCAM low细胞成瘤率为0（0/10)。小鼠移植瘤中次代CD44+/EPCAM high细胞仍能少量表达CD44和EPCAM。结论：LoVo细胞中存在CD44+/EPCAM high结肠癌干细胞样细胞,CD44+/EPCAM high可用于结肠癌肿瘤干细胞的深入研究。     

无血清悬浮法培养MCF-7细胞系并筛选该细胞系中肿瘤干细胞相关亚群的初步研究

目的：研究无血清培养基悬浮培养MCF-7乳腺癌细胞系,筛选并鉴定MCF-7乳腺癌细胞系中的肿瘤干细胞相关亚群。方法：应用乳腺癌培养基在无血清的条件下悬浮培养MCF-7乳腺癌细胞系。通过无血清培养筛选乳腺癌细胞系肿瘤干细胞相关亚群,将其接种于含血清培养基,观察分化。应用单克隆形成实验、表面标志检测、HOECHST33342染色检测来确定培养出的细胞中肿瘤干细胞的比例及其培养后肿瘤干细胞含量的变化。结果：乳腺癌MCF-7细胞系中有约2.12%的肿瘤细胞在无血清培养基中能够存活、增殖,形成自由漂浮的细胞球。细胞球可连续传代,若重新接种于含血清培养基中可重新贴壁分化,贴壁分化后细胞形态与直接在含血清培养基中培养的MCF-7无明显差别。流式细胞仪表面标志检测,细胞球中约含83.13%表达CD24^-CD44^＋的细胞,HOECHST33342染色提示细胞球中约含10.06%的侧亚群（sidepopulation）细胞。结论：MCF-7细胞系可在含生长因子的无血清培养基中悬浮生长,并维持细胞系。该细胞系中含有比例较低的具有增殖和分化能力的乳腺癌干细胞相关亚群。表面标志以及HOECHST33342检测差异提示细胞球中仅约1/8细胞具有干细胞的功能。     

乳腺癌干细胞临床与基础研究的进展

乳腺癌是一类高度异质性的肿瘤,包括18种组织病理学类型和5 种分子亚型。乳腺癌干细胞（CSCs）是乳腺癌组织中极少数具有自我更新能力和多向分化潜能的细胞,具有肿瘤启动作用。CD44+/CD24-/low表型为乳腺CSCs的标志,大约30% 乳腺癌组织中存在CD44+/CD 24-/low表型细胞,CD44+/CD 24-/low表型细胞与病理组织学类型有关,在呈现CD44+/CD 24-/low表型的肿瘤中,浸润性导管癌所占比例最多（78%）,其次为髓样癌（11%）和浸润性小叶癌（7%）。 CD44+/CD 24-/low表型细胞与乳腺癌的分子亚型有关,在依据基因表达谱得到的5 种分子亚型中,CD44+/CD 24-/low表型在基底样癌中最常见。乳腺癌干细胞通常为ER- 和PR- 的表型,但ER- 和PR- 的多潜能干细胞能产生ER+ ,PR+ 的肿瘤细胞。HER2 过表达能够增加CSCs的数量并促进乳腺癌的发生、进展和浸润,但乳腺癌组织中CD44+/CD 24-/low表型通常与HER2 低表达或阴性表达呈正相关。迄今有关干细胞与乳腺癌的临床基础研究提出了更多新的问题,引发了更多的思考。      

CD44+CD24-/low乳腺癌干细胞分选鉴定及其多药耐药性研究

目的:观察MACS免疫磁珠法分选CD44+CD24-/low乳腺癌干细胞活性,并检测其与多药耐药的关系。方法:运用MACS免疫磁珠法从多药耐药乳腺癌细胞株MCF-7/ADR中分选CD44+CD24-/low乳腺癌干细胞,流式细胞术测定分选前后CD44+CD24-/low细胞比例,微球体培养法检测分选细胞自我更新能力,流式检测CD44+CD24-/low细胞表面P-糖蛋白(P-gp)表达水平,Real-time PCR检测多药耐药相关基因MDR1表达水平。结果:MACS免疫磁珠法分选后,CD44+CD24-/low细胞比例为93.85%,其成球能力明显强于non-CD44+CD24-/low细胞亚群。MCF-7/ADR细胞株和CD44+CD24-/low乳腺癌干细胞P-gp表达强度分别为101 177.10±2 171.86和114 906.70±2 560.19,P<0.05。CD44+CD24-/low乳腺癌干细胞MDR1基因表达水平为MCF-7/ADR细胞株的(1.07±0.02)倍,P<0.05。结论:经MACS免疫磁珠法分选所得CD44+CD24-/low细胞亚群有更强的自我更新能力,高表达P-gp蛋白和MDR1基因可能是引起乳腺癌多药耐药的重要原因。     

人卵巢癌细胞系A2780肿瘤干细胞分离培养与鉴定

目的:从人卵巢癌细胞系A2780中分离培养卵巢癌干细胞,并对其特性进行鉴定.方法:将A2780细胞消化,离心制成单细胞悬液,悬浮于含有生长因子的无血清培养基(SFM)中获得肿瘤细胞微球体,流式细胞仪检测细胞表面分子标志CD44和CD133的表达;Transwell小室侵袭实验检测肿瘤微球体的体外侵袭能力;CCK-8检测微球体细胞的增殖能力,比较两种细胞的倍增时间;将肿瘤微球体置于含有血清的培养基使其诱导分化,并观察其形态学变化.结果:A2780可在SFM中形成可悬浮生长、稳定传代的肿瘤细胞微球体.与贴壁的肿瘤细胞相比,微球体高表达干细胞标志CD44和CD133(P＜0.05),具有更强的体外侵袭力(t=9.354,P＜0.05)和增殖能力(t=13.682,P＜0.05),及更短的倍增时间(t=3.773,P＜0.05),在血清环境下可分化为普通的肿瘤细胞.结论:用含有生长因子的SFM悬浮卵巢癌细胞系A2780可获得卵巢癌肿瘤细胞微球体,此细胞球体中富集有肿瘤干细胞.     

乳腺浸润性导管癌干细胞标志物ALDH1、CD133与肿瘤血管生成相关性

目的 探讨乳腺浸润性导管癌干细胞标志物乙醛脱氢酶1(aldehyde dehydrogenase1,ALDH1)、细胞分化标志133(cluster of differentiation 133,CD133)与肿瘤血管生成的相关性.方法 收集颈胸外科肿瘤手术切除乳腺癌标本95例,所选标本均经病理确诊为乳腺浸润性导管癌,采用免疫组织化学双染法检测所选标本中ALDH1、CD133干细胞样细胞,并应用免疫组织化学单染法检测其肿瘤血管性标志CD4、CD105及血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)的表达情况,分析ALDH1、CD133与肿瘤血管生成的相关性.结果 95例乳腺浸润性导管癌组织中存在8例(8.3％) ALDH1 +/CD133+表型,ALDH1 +/CD133+表型与ER、VEGF表达以及肿瘤血管生成指标CD34 MVD、CD105 MVD均有相关性(均P<0.05).结论 ALDH1、CD133可作为乳腺肿瘤细胞的特异性标志物,其中ALDH1 +/CD133+表型干细胞可以促进肿瘤新生血管的生成.     

The anti-diabetic drug metformin suppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells

We here demonstrate that the anti-diabetic drug metformin interacts synergistically with the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin?) to eliminate stem/progenitor cell populations in HER2-gene-amplified breast carcinoma cells. When using the mammosphere culture technique, graded concentrations of single-agent metformin (range 50–1,000 μmol/l) were found to dose-dependently reduce the number of mammospheres formed by SKBR3 (a Tzb-naïve model), SKBR3 TzbR (a model of acquired auto-resistance to Tzb) and JIMT-1 (a model of refractoriness to Tzb and other HER2-targeted therapies ab initio) HER2-overexpressing breast cancer cells. Single-agent Tzb likewise reduced mammosphere-forming efficiency (MSFE) in Tzb-naïve SKBR3 cells, but it failed to significantly decrease MSFE in Tzb-resistant SKBR3 TzbR and JIMT-1 cells. Of note, CD44-overexpressing Tzb-refractory SKBR3 TzbR and JIMT-1 cells retained an exquisite sensitivity to single-agent metformin. Concurrent combination of metformin with Tzb synergistically reduced MSFE as well as the size of mammospheres in Tzb-refractory SKBR3 TzbR and JIMT-1 cells. Flow cytometry analyses confirmed that metformin and Tzb functioned synergistically to down-regulate the percentage of Tzb-refractory JIMT-1 cells displaying the CD44^pos/CD24^neg/low stem/progenitor immunophenotype. Given that MSFE and mammosphere size are indicators of stem self-renewal and progenitor cell proliferation, respectively, our current findings reveal for the first time that: (a) Tzb refractoriness in HER2 overexpressors can be explained in terms of Tzb-resistant/CD44-overexpressing/tumor-initiating stem cells; (b) metformin synergistically interacts with Tzb to suppress self-renewal and proliferation of cancer stem/progenitor cells in HER2-positive carcinomas.     

Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells

The epithelial components of the mammary gland are thought to arise from stem cells with a capacity for self-renewal and multilineage differentiation. Furthermore, these cells and/or their immediate progeny may be targets for transformation. We have used both in vitro cultivation and a xenograft mouse model to examine the role of hedgehog signaling and Bmi-1 in regulating self-renewal of normal and malignant human mammary stem cells. We show that hedgehog signaling components PTCH1, Gli1, and Gli2 are highly expressed in normal human mammary stem/progenitor cells cultured as mammospheres and that these genes are down-regulated when cells are induced to differentiate. Activation of hedgehog signaling increases mammosphere-initiating cell number and mammosphere size, whereas inhibition of the pathway results in a reduction of these effects. These effects are mediated by the polycomb gene Bmi-1. Overexpression of Gli2 in mammosphere-initiating cells results in the production of ductal hyperplasia, and modulation of Bmi-1 expression in mammosphere-initiating cells alters mammary development in a humanized nonobese diabetic-severe combined immunodeficient mouse model. Furthermore, we show that the hedgehog signaling pathway is activated in human breast "cancer stem cells" characterized as CD44+CD24-/lowLin-. These studies support a cancer stem cell model in which the hedgehog pathway and Bmi-1 play important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells.     

克隆柱法提取MCF-7乳腺癌细胞系中癌干细胞

目的通过克隆柱法提取MCF-7乳腺癌细胞中的乳腺癌干细胞,并进行鉴定。方法采用单细胞克隆加低密度干细胞培养基筛选获得呈“球样”生长的乳腺癌干细胞,行CD44、CD24免疫荧光细胞化学染色及诱导分化实验。结果CD44＋CD24^-/Low细胞的比例为12．1％,且在全克隆中富含CD44＋CD24^-/Low细胞。在形成的细胞球中富含CD44＋CD24^-/Low细胞,并且在诱导分化时可见明显类管腔样结构形成,并表达CK18及CK14。结论克隆柱法是提取细胞系中癌干细胞的简单有效方法。     

Treatment of breast cancer stem cells with oncolytic herpes simplex virus

Cancer stem cells have recently been isolated from several different solid tumors. In breast cancer, the CD44(+)CD24(-/low) population is considered to comprise stem-like cells. The identification of cancer stem cells has provided new targets for the development of therapeutics. Oncolytic herpes simplex viruses (oHSVs) are an effective strategy for killing breast cancer cells and treating breast tumors in preclinical models. Here, we examined the efficacy of the oHSV G47Δ in killing breast cancer stem cells. Human breast cancer cell line SK-BR-3 and human primary breast cancer cells were cultured in suspension under conditions conducive to the growth of stem cells. They generated mammospheres, which had cancer stem cell properties. The proportion of CD44(+)CD24(-/low) cells in these mammospheres exceeded 95%, as determined by flow cytometry. The mammospheres were found to be highly tumorigenic when implanted subcutaneously in nude BALB/c mice. G47Δ contains the LacZ gene, and X-gal staining of infected cells in vitro and in vivo showed the replication and spread of the virus. G47Δ was found to be highly cytotoxic to the CD44(+)CD24(-/low) population in vitro, even when injected at low multiplicities of infection, and G47Δ treatment in vivo significantly inhibited tumor growth compared with mock treatment. This study demonstrates that oHSV is effective against breast cancer stem cells and could be a beneficial strategy for treating breast cancer patients.     

Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties

Breast cancer-initiating cells have been recently identified in breast carcinoma as CD44+/CD24(-/low) cells, which exclusively retain tumorigenic activity and display stem cell-like properties. However, at present, direct evidence that breast cancer-initiating cells can be propagated in vitro is still lacking. We report here the isolation and in vitro propagation of breast cancer-initiating cells from three breast cancer lesions and from an established breast carcinoma cell line. Our breast carcinoma-derived cultures encompassed undifferentiated cells capable of self-renewal, extensive proliferation as clonal nonadherent spherical clusters, and differentiation along different mammary epithelial lineages (ductal and myoepithelial). Interestingly, cultured cells were CD44+/CD24- and Cx43-, overexpressed neoangiogenic and cytoprotective factors, expressed the putative stem cell marker Oct-4, and gave rise to new tumors when as few as 10(3) cells were injected into the mammary fat pad of SCID mice. Long-term cultures of breast tumorigenic cells with stem/progenitor cell properties represent a suitable in vitro model to study breast cancer-initiating cells and to develop therapeutic strategies aimed at eradicating the tumorigenic subpopulation within breast cancer.     

Progesterone stimulates progenitor cells in normal human breast and breast cancer cells

The epithelium of the human breast is made up of a branching ductal–lobular system, which is lined by a single layer of luminal cells surrounded by a contractile basal cell layer. The co-ordinated development of stem/progenitor cells into these luminal and basal cells is fundamentally important for breast morphogenesis. The ovarian steroid hormones, progesterone (P) and 17β-estradiol, are critical in driving this normal breast development, yet ovarian activity has also been shown to be a major driver of breast cancer risk. We previously demonstrated that P treatment increases proliferation and augments the number of progenitor-like cells, and that the progesterone receptor (PR) is also expressed in the bipotent progenitor-enriched subfraction. Here we demonstrate that PR is expressed in a subset of CD10+ basal cells and that P stimulates this CD10+ cell compartment, which is enriched for bipotent progenitor activity. In addition, we have shown that P stimulates progenitor cells in human breast cancer cell lines and expands the cancer stem cell population via increasing the stem-like CD44+ population. As changes in cell type composition are one of the hallmark features of breast cancer progression, the demonstration that progenitor cells are stimulated by P in both normal breast and in breast cancer cells has critical implications in discerning the mechanisms of how P increases breast cancer risk.     

原代乳腺癌干细胞富集及其与临床病理特征的相关性分析

目的:采用微球体培养法富集原代乳腺癌干细胞,并探讨微球体形成与乳腺癌临床病理特征之间的相关性.方法:采用细胞悬浮培养法对45例乳腺癌组织来源的原代乳腺癌细胞进行微球体培养.采用FCM分析CD44 +/CD24low/-表型细胞所占百分率,采用实时荧光定量PCR (real-time fluorogenic quantitative-PCR,RFQPCR)检测干细胞相关基因Nanog、KLF4、OCT-4、SOX2和MDR1在微球体细胞中的表达,分析微球体形成与乳腺癌临床病理特征之间的关系.结果:细胞悬浮培养14 d时,原代乳腺癌细胞微球体形成.乳腺癌组织来源的原代乳腺癌细胞微球体细胞中CD44 +/CD24 low/-表型细胞所占百分率高于原代乳腺癌细胞(分别为24.71％和1.30％,P＜0.05).原代乳腺癌细胞微球体形成与发病年龄、初潮年龄、绝经情况、组织学分级、孕激素受体(progestogen receptor,PR)状态、人表皮生长因子受体2(human epidermal growth factor receptor 2,HER-2)状态和肿瘤复发明显相关(P＜0.05).微球体形成与否与无病生存期(disease-free survival,DFS)和总生存期(overall survival,OS)无明显相关性(P＞0.05).结论:细胞悬浮培养法可有效富集原代乳腺癌干细胞,微球体形成与部分临床病理特征存在相关性,可能是影响预后的因素之一.     

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the 'mammosphere' system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44(+)/CD24(-) fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations-all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes.