Isolation, Propagation and Characterization of Breast Stem Cells

乳腺干细胞的分离、增殖和表征

基本信息

项目摘要

Normal stem cells: The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma. Alterations in this communication can lead to the progression or expansion of malignant growth. The human mammary gland contains two distinctive types of fibroblasts within the stroma. The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts. The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. We compared the gene expression profiles between the two stromal subtypes. Fresh normal breast tissue was collected from reduction mammoplasty patients and immediately placed into embedding medium and frozen on dry ice. Tissue sections were subjected to laser capture microscopy to isolate the interlobular from the intralobular fibroblasts. RNA was prepared and subjected to microarray analysis using the Affymetrix Human Genome U133 GeneChip. Data was analyzed using the Affy and Limma packages available from Bioconductor. Findings from the microarray analysis were validated by RT-PCR and immunohistochemistry. No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR. However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different. This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland. While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested. These data also highlight the importance of studying gene regulation at both the transcriptional and post-translational level. We are continuing to advance the recapitulation of human breast epithelial morphogenesis by humanizing the mammary fat pad of NOD/ SCID mice as described by Kuperwasser et al. 2004. The stromal cells fill over 50% of the mouse mammary glands but normal epithelial cell outgrowths are very rare. The humanized gland are also used to influence the tumorigenicity of breast cancer cells. We are investigating alternative approaches to enhance epithelial cell growth such as the addition of primary human macrophages. Preliminary results show a significantly higher number of epithelial outgrowths in the presence of macrophages. Additionally, we have successfully isolated human mammary extracellular matrix. This will be useful: 1. In vivo to replace Matrigel with a more physiological matrix; 2. to study the epithelial/tumor cells in response to ECM proteins derived from specific subsets of the population (for example: age, race, parity); 3. to study matrix composition/interactions from a specific tumor subset (for example: Her2 or BRCA). Cancer stem cells: Recent data suggest that solid tumors are initiated and propagated by a rare population of cells that are unique from the tumor bulk in their ability to self-renew indefinitely, give rise to heterogeneous lineages and form tumors. In the breast, this population of cancer initiating cells has been prospectively identified as CD44+ and CD24-/dim while CD24+ cells are considered to be of a terminally differentiated luminal subtype. We have definitively illustrated, contrary to data in the literature, that (1) the presence of CD44+CD24- cells in a pleural effusion (PE) does not ensure that the PE can give rise to tumors in NOD/SCID mice, (2) in both PE and xenograft tumors, CD44+CD24- and CD44+CD24+ are equally tumorigenic and can give rise to each other and (3) CD44+CD24- cells do not faithfully recapitulate the CD44/CD24 profile of the PE in the resulting xenograft. Of 8 clinical samples, only 1 PE behaved as predicted based on the data of Al-Hajj et al. As few as 200 CD44+CD24- cells gave rise to tumors whereas 5,000 CD44+CD24+ cells failed to give rise to tumors. Contrary to these observations, CD44+CD24+ cells from several PEs were capable of initiating tumors. Furthermore, CD44+CD24- cells from 3 additional PEs failed to give rise to tumors even after 11 months. In xenografts derived from PEs and a primary tumor fragment, CD44+CD24- and CD44+CD24+ cells were consistently equally tumorigenic. CD44- cells are non-tumorigenic regardless of their source (i.e. PE vs. xenografts). With one exception that was ER+PR+HER2-, all tumors examined were of a basal phenotype (ER-, PR-, HER2+/-). In all cases, the receptor status of the primary tumor/PE was identical to that of its resulting xenograft. However, PE cells and their xenografts had markedly disparate CD24 profiles. Pleural effusions that had substantial C24- populations gave rise to xenografts that were largely CD24+. In all xenografts examined, the xenograft immunophenotype remained static after numerous passages. We have recently begun efforts to employ lentiviral mediated, doxycycline inducible shRNA production to knockdown expression of CD44. We have achieved a 4-fold reduction in CD44 protein abundance and a concomitant 6.8 fold increase in the percentage of CD44- cells in the MCF10 Ca1a.c11 cell line. While statistically significant, the biological relevance (i.e. in vivo tumorigenicity) of this knockdown is questionable given that even after shRNA induction, the fraction of CD44+ cells remains approximately 3.5%. Established Breast Cancer Cell Lines: Similarly, in breast cancer cell lines of three subtypes (ER+PR+HER2-, ER+PR+HER2+, and ER-PR-HER2-) CD44+CD24- and CD44+CD24+ cells are equally tumorigeniic at the orthotopic site with very low numbers. In vitro, however, CD24- cells possess markers characteristic of epithelial-mesenchymal transition. CD24- cells have elevated levels of Vimentin and Slug, reduced levels of E-cadherin, and increased invasiveness relative to isogenic CD24+ cells. Strikingly, CD24- cells give rise to less invasive CD24+ progeny as effectively as CD24+ cells give rise to more invasive CD24- progeny. These observations demonstrate that acquisition of CD24 expression is not permanent and not all progeny of CD24+ cells express the marker. While functionally different in vitro, in vivo tumorigenicity is similar between CD24- and CD24+ populations. Additionally, the ability to recapitulate functional heterogeneity, considered a key hallmark of the cancer stem cell, is shared by both CD44+CD24+ and CD44+CD24- cells. In summary, our observations suggest that in the majority of clinical samples and established breast cancer cell lines, cells lacking CD44 are largely non-tumorigenic, but CD24+ cells and CD24- cells are equally tumorigenic. We have also observed that cells enriched for tumorsphere forming ability are not enriched for CIC. Efforts to identify novel extracellular markers capable of enriching CIC are currently underway.
正常干细胞:乳腺上皮细胞的正常生长和功能依赖于与支持基质的相互作用。这种交流的改变可导致恶性生长的进展或扩大。人乳腺间质中含有两种不同类型的成纤维细胞。上皮细胞被连接松散的小叶内成纤维细胞包围,随后被更紧密的小叶间成纤维细胞包围。这些成纤维细胞与上皮细胞的不同接近程度表明这两种亚型具有不同的功能。我们比较了两种基质亚型之间的基因表达谱。从缩乳术患者身上收集新鲜的正常乳腺组织,立即放入包埋介质中,在干冰上冷冻。组织切片采用激光捕获显微镜分离小叶间和小叶内成纤维细胞。制备RNA,并使用Affymetrix Human Genome U133基因芯片进行微阵列分析。使用Bioconductor提供的Affy和Limma软件包对数据进行分析。微阵列分析结果经RT-PCR和免疫组织化学验证。微阵列分析和RT-PCR均未发现小叶间和小叶内成纤维细胞基因表达谱差异有统计学意义。然而,对于某些测试基因,两种亚型成纤维细胞之间的蛋白质表达模式显着不同。这项研究首次报道了人类乳腺中两种不同成纤维细胞群体的基因表达谱。虽然基因表达谱在两组之间没有显著差异,但几种被测蛋白的表达模式存在明显差异。这些数据也强调了在转录和翻译后水平研究基因调控的重要性。根据Kuperwasser等人2004年的描述,我们正在通过人源化NOD/ SCID小鼠的乳腺脂肪垫,继续推进人类乳腺上皮形态发生的再现。间质细胞占小鼠乳腺的50%以上,但正常上皮细胞的生长非常罕见。人源化腺体也被用于影响乳腺癌细胞的致瘤性。我们正在研究促进上皮细胞生长的替代方法,如添加原代人巨噬细胞。初步结果显示,巨噬细胞存在时,上皮增生的数量明显增加。此外,我们还成功分离了人乳腺细胞外基质。这将是有用的:在体内用更生理的矩阵代替matrix;2. 研究上皮/肿瘤细胞对来自人群特定亚群(例如:年龄、种族、胎次)的ECM蛋白的反应;3. 研究特定肿瘤亚群(例如:Her2或BRCA)的基质组成/相互作用。癌症干细胞:最近的数据表明,实体瘤是由一种罕见的细胞群发起和繁殖的,这种细胞群在肿瘤群体中是独一无二的,它们具有无限自我更新的能力,产生异质谱系并形成肿瘤。在乳腺中,这群癌症起始细胞已被前瞻性地鉴定为CD44+和CD24-/dim,而CD24+细胞被认为是终末分化的管腔亚型。我们已经明确地说明,与文献数据相反,(1)胸膜积液(PE)中CD44+CD24-细胞的存在并不能确保PE在NOD/SCID小鼠中引起肿瘤,(2)在PE和异种移植肿瘤中,CD44+CD24-和CD44+CD24+具有相同的致瘤性,可以相互引起,(3)CD44+CD24-细胞不能忠实地再现所产生的异种移植中PE的CD44/CD24特征。在8个临床样本中,只有1个PE表现符合al - hajj等人基于数据的预测。只有200个CD44+CD24-细胞产生肿瘤,而5000个CD44+CD24+细胞不能产生肿瘤。与这些观察结果相反,来自几种pe的CD44+CD24+细胞能够引发肿瘤。此外,来自另外3个pe的CD44+CD24-细胞即使在11个月后也没有产生肿瘤。在来自pe和原发肿瘤片段的异种移植物中,CD44+CD24-和CD44+CD24+细胞具有相同的致瘤性。无论来源如何(即PE与异种移植),CD44细胞都是非致瘤性的。除ER+PR+HER2-外,所有检查的肿瘤均为基础表型(ER-, PR-, HER2+/-)。在所有病例中,原发肿瘤/PE的受体状态与由此产生的异种移植物的受体状态相同。然而,PE细胞及其异种移植物具有明显不同的CD24谱。含有大量C24-的胸腔积液会产生大量CD24+的异种移植物。在所有检查的异种移植物中,经过多次传代后,异种移植物的免疫表型保持不变。我们最近开始尝试使用慢病毒介导的强力霉素诱导的shRNA生产来敲低CD44的表达。我们已经实现了CD44蛋白丰度降低了4倍,同时MCF10 Ca1a中CD44细胞的百分比增加了6.8倍。C11细胞系。虽然具有统计学意义,但这种敲除的生物学相关性(即体内致瘤性)值得怀疑,因为即使在shRNA诱导后,CD44+细胞的比例仍约为3.5%。建立的乳腺癌细胞系:同样,在三种亚型乳腺癌细胞系(ER+PR+HER2-、ER+PR+HER2+和ER-PR-HER2-)中,CD44+CD24-和CD44+CD24+细胞在原位位置具有相同的致瘤性,但数量很少。然而,在体外,CD24-细胞具有上皮-间质转化的特征标记。与等基因CD24+细胞相比,CD24-细胞的Vimentin和Slug水平升高,E-cadherin水平降低,侵袭性增加。引人注目的是,CD24-细胞产生侵袭性较低的CD24+子代与CD24+细胞产生侵袭性较强的CD24-子代一样有效。这些观察结果表明,CD24表达的获得不是永久性的,并且并非所有CD24+细胞的后代都表达该标记物。虽然体外功能不同,但CD24-和CD24+群体的体内致瘤性相似。此外,CD44+CD24+和CD44+CD24-细胞都具有重现功能异质性的能力,这被认为是癌症干细胞的一个关键标志。总之,我们的观察结果表明,在大多数临床样本和已建立的乳腺癌细胞系中,缺乏CD44的细胞在很大程度上是非致瘤性的,但CD24+细胞和CD24-细胞同样具有致瘤性。我们还观察到,增强肿瘤球形成能力的细胞不增强CIC。目前正在努力鉴定能够富集CIC的新型细胞外标记物。

项目成果

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Barbara Vonderhaar其他文献

Barbara Vonderhaar的其他文献

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{{ truncateString('Barbara Vonderhaar', 18)}}的其他基金

Prolactin Interactions in Mammary Gland Development and Tumorigenesis
催乳素在乳腺发育和肿瘤发生中的相互作用
  • 批准号:
    7965103
  • 财政年份:
  • 资助金额:
    $ 25.03万
  • 项目类别:
Prolactin Interactions in Mammary Gland Development and Tumorigenesis
催乳素在乳腺发育和肿瘤发生中的相互作用
  • 批准号:
    8175288
  • 财政年份:
  • 资助金额:
    $ 25.03万
  • 项目类别:
Isolation, Propagation and Characterization of Breast Stem Cells
乳腺干细胞的分离、增殖和表征
  • 批准号:
    7965627
  • 财政年份:
  • 资助金额:
    $ 25.03万
  • 项目类别:
Isolation, Propagation and Characterization of Breast Stem Cells
乳腺干细胞的分离、增殖和表征
  • 批准号:
    7592917
  • 财政年份:
  • 资助金额:
    $ 25.03万
  • 项目类别:
Isolation, Propagation and Characterization of Breast Stem Cells
乳腺干细胞的分离、增殖和表征
  • 批准号:
    8175341
  • 财政年份:
  • 资助金额:
    $ 25.03万
  • 项目类别:

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