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
• 负责人: Barbara Vonderhaar
• 金额: $ 72.84万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965627
• 关键词: African; African American; Age; BMI1 gene; Biological Assay; Biological Models; Breast; Breast Cancer Cell; Breast Cancer Model; CD44 gene; Cancer Patient; Cancer cell line; Cell Line; Cells; Chest; Clinical; Development; Early Diagnosis; Environment; Epithelial; Epithelial Cells; Estradiol; Estrogens; Etoposide; Event; Extracellular Matrix; Fatty acid glycerol esters; Fibroblasts; Fibrous capsule of kidney; Future; Gene Expression; Genes; Gland; Goals; Growth; Heterogeneity; High Risk Woman; Hormone Receptor; Human; Image; Immune; Implant; Incidence; Injection of therapeutic agent; Invaded; Knowledge; Lobular; MCF7 cell; Malignant Neoplasms; Mammary gland; Mesenchymal; Messenger RNA; Methodology; Methods; Methylation; MicroRNAs; Molecular; Morphogenesis; Mus; NOD/SCID mouse; Paracrine Communication; Patients; Phenotype; Play; Pleural effusion disorder; Population; Premenopause; Primary Neoplasm; Production; Proteomics; Protocols documentation; Race; Regulation; Role; Sampling; Site; Skin; Small Interfering RNA; Sorting - Cell Movement; Source; Stem cells; Structure; Supporting Cell; Surgical incisions; T47D; Time; Transcript; Tumor Volume; Tumorigenicity; Woman; Work; Xenograft Model; Xenograft procedure; cancer cell; cancer stem cell; caucasian American; cell growth; chemotherapy; cytokine; extracellular; in vivo; in vivo Model; insight; macrophage; malignant breast neoplasm; matrigel; mouse model; novel; promoter; self-renewal; stem; stem cell fate; stem cell niche; subcutaneous; tumor; tumorigenesis; tumorigenic

Normal stem cells: 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). We have been successful in using alternative approaches to enhance epithelial cell growth in the humanized model system. We have optimized a protocol for the isolation and differentiation of human macrophages. The addition of primary human macrophages has significantly increased the formation of human epithelial lobular structures. The paracrine signaling interactions remain to be determined; however, it is clear that the addition of human macrophages provides a more permissive niche for the growth of human cells, and potentially human mammary stem cells, in the in vivo model. To begin to understand the paracrine signaling between the macrophages and epithelial cells, we have identified the cytokines secreted by the differentially activated macrophages via quantitative real time PCR array analysis. To gain insight into the role of the microenvironment of breast stem cell growth and tumorigenicity, we have investigated the role of the stromal fibroblasts on the tumorigenicity of primary metastatic breast cancer cells. We chose to examine the effect of fibroblasts derived from pre-menopausal African- or Caucasian-American patients given the high incidence of aggressive (i.e. triple negative), metastatic breast cancer development in pre-menopausal African-American women. Results show that when the gland is humanized with Caucasian-American fibroblasts then injected with pleural effusion cells derived from metastatic breast cancer patients, the resultant tumors are significantly larger compared to the glands humanized with African-American fibroblasts. These results were also repeated using the well-characterized breast cancer T47D cell line. Quantitative real-time PCR array analysis has indicated that the Caucasian-American fibroblasts have increased cytokine production, potentially leading to the more permissive environment for tumorigenesis. Using our newly optimized protocol for isolating human breast extracellular matrix, we have shown that human breast ECM can alter the tumorigenicity of breast cancer cells, depending on the source of the ECM (i.e. age and race of the patient) and the hormone receptor status of the breast cancer cells. We have used proteomics to begin to identify the components of the breast ECM and will use this knowledge for future studies to identify the role of the breast microenvironment and the stem cell niche. Current studies are being conducted to determine if ECM molecules have the ability to re-direct stem cell fate and tumorigenesis. Development of a standard human xenograft breast cancer model: Breast cancer studies often utilize xenograft mouse models by implanting human cancer cells under the renal capsule, subcutaneously, or orthotopically into the mammary gland. However, these procedural differences and the discretionary use of estrogen and immune suppressants (etoposide) can make seemingly relatable results incomparable. To standardize the in vivo breast cancer model, we directly compared the effects of orthotopic, transdermal, and subcutaneous sites of injection on tumorigenicity, in the presence or absence of estrogen and/or etoposide pretreatment. Cells derived from a human breast cancer pleural effusion were injected into the inguinal or thoracic mammary gland of NOD/SCID mice either after surgical incision (orthotopic) or through the skin (transdermal). Tumors in inguinal glands had significantly decreased latency and increased volume and incidence compared to those in thoracic glands, independent of method of injection. Within the inguinal gland, no statistically significant difference in tumor volume was found between groups treated with and without estrogen or etoposide. In addition, athymic NCr-nu/nu mice treated estradiol were injected orthotopically or subcutaneously with well-characterized human breast cancer cell lines (MCF7, ZR-75-1, MDA MB231, or MCF10A1h). Increased tumor volume was observed with all four cell lines injected orthotopically. These studies strongly suggests that the inguinal gland is the preferred site for a xenograft model of breast cancer and illustrates the influence of the local microenvironment on in vivo models. Cancer stem cells: Our work has been focused on developing ways to enrich and/or identify tumor initiating cells (TIC) in both established breast cancer cell lines and clinical samples. We demonstrated that in ER negative clinical samples and resulting xenografts, cells lacking CD44 were largely non-tumorigenic while CD44+CD24+ and CD44+CD24- cells were equally tumorigenic. Subsequently, we have focused on segregating CD44+ cells into two distinct fractions: one tumorigenic and one non-tumorigenic. We have identified a novel extracellular marker profile that effectively enrichs TIC from human ER- breast cancer. FACS sorted TIC cells from primary tumors and their resulting xenografts give rise to tumors in NOD/SCID mice with as few as 50 cells. Contrary to this, 2,500 cells lacking the TIC marker profile fail to yield tumors. Furthermore, the novel TIC cells faithfully recapitulate tumor molecular sub-type and immunophenotypic heterogeneity through multiple passages. At the transcript level, our novel TIC profile enriches a population of cells with elevated expression genes known to play a role in stem cell self-renewal including BMI1, nanog, and sox2. We currently are planning to evaluate global mRNA, miRNA, and promoter methylation status of the TIC and non-tumor initiating cells. Additionally, we demonstrated that in the MCF10 Ca1a cell line, CD44+CD24- cells were more invasive than CD44+CD24+ cells. Furthermore, we demonstrated that both expression of CD24 and the epithelial-like phenotype associated with its expression was under dynamic regulation. Specifically, epithelial-like CD44+CD24+ cells readily give rise to invasive mesenchymal-like CD44+CD24- cells and vice versa. We demonstrated similar events with MCF7, SUM159, and MDA MB231 cells. Also, we demonstrated that CD24 promoter CpG methylation was similar between CD44+CD24+ and CD44+CD24- cells, supporting the observation that this gene is susceptible to dynamic regulation. Using siRNA methodologies, we found that CD24 depletion increased a cells ability to invade through Matrigel. However, CD24 knockdown failed to yield the mesenchymal phenotype observed in cells where CD24 is down-regulated endogenously.

正常干细胞：根据Kuperwasser等人（2004）的描述，我们通过人源化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。我们已经成功地使用替代方法来增强人源化模型系统中的上皮细胞生长。我们优化了一种分离和分化人巨噬细胞的方案。原代人巨噬细胞的加入显著增加了人上皮小叶结构的形成。旁分泌信号相互作用仍有待确定；然而，很明显，在体内模型中，人巨噬细胞的加入为人类细胞和潜在的人类乳腺干细胞的生长提供了一个更宽松的生态位。为了开始了解巨噬细胞和上皮细胞之间的旁分泌信号，我们通过定量实时PCR阵列分析鉴定了差异活化巨噬细胞分泌的细胞因子。为了深入了解乳腺干细胞生长和致瘤性的微环境的作用，我们研究了基质成纤维细胞在原发性转移性乳腺癌细胞的致瘤性中的作用。考虑到绝经前非洲裔美国妇女侵袭性（即三阴性）转移性乳腺癌的高发病率，我们选择检查来自绝经前非洲裔或高加索裔美国患者的成纤维细胞的影响。结果表明，将白种美国人成纤维细胞人源化腺体，然后注射转移性乳腺癌患者的胸腔积液细胞，所得肿瘤明显大于非裔美国人成纤维细胞人源化腺体。使用具有良好特征的乳腺癌T47D细胞系也重复了这些结果。实时荧光定量PCR阵列分析表明，高加索裔美国人成纤维细胞增加了细胞因子的产生，可能导致更宽松的肿瘤发生环境。使用我们最新优化的分离人乳腺细胞外基质的方案，我们已经表明，人乳腺ECM可以改变乳腺癌细胞的致瘤性，这取决于ECM的来源（即患者的年龄和种族）和乳腺癌细胞的激素受体状态。我们已经使用蛋白质组学开始识别乳腺ECM的组成部分，并将在未来的研究中使用这些知识来确定乳房微环境和干细胞生态位的作用。目前正在进行研究，以确定ECM分子是否具有重新引导干细胞命运和肿瘤发生的能力。标准人类异种移植乳腺癌模型的建立：乳腺癌研究通常利用异种移植小鼠模型，将人类癌细胞植入肾包膜下、皮下或原位植入乳腺。然而，这些程序上的差异和随意使用雌激素和免疫抑制剂（依托泊苷）可以使看似相关的结果无法比较。为了规范体内乳腺癌模型，我们直接比较了在存在或不存在雌激素和/或依托泊苷预处理的情况下，原位、透皮和皮下注射部位对致瘤性的影响。从人乳腺癌胸腔积液中提取的细胞在手术切口（原位）或通过皮肤（透皮）注射到NOD/SCID小鼠的腹股沟或胸部乳腺。与注射方式无关，腹股沟腺肿瘤潜伏期明显降低，体积和发生率明显高于胸腺肿瘤。在腹股沟腺内，使用雌激素或依托泊苷组与未使用雌激素或依托泊苷组的肿瘤体积无统计学差异。此外，将经雌二醇处理的胸腺NCr-nu/nu小鼠原位或皮下注射具有良好特征的人乳腺癌细胞系（MCF7、ZR-75-1、MDA MB231或MCF10A1h）。原位注射四种细胞系后，肿瘤体积均增加。这些研究强烈提示腹股沟腺是乳腺癌异种移植模型的首选部位，并说明了局部微环境对体内模型的影响。癌症干细胞：我们的工作一直集中在开发方法来丰富和/或鉴定肿瘤起始细胞（TIC）在已建立的乳腺癌细胞系和临床样本。我们证明，在ER阴性的临床样本和由此产生的异种移植物中，缺乏CD44的细胞在很大程度上是非致瘤性的，而CD44+CD24+和CD44+CD24-细胞同样具有致瘤性。随后，我们专注于将CD44+细胞分离成两个不同的部分：一个致瘤性和一个非致瘤性。我们已经确定了一种新的细胞外标记谱，可以有效地从人ER-乳腺癌中富集TIC。在NOD/SCID小鼠中，从原发肿瘤中提取的经FACS分类的TIC细胞及其产生的异种移植物可产生肿瘤，只有50个细胞。与此相反，2500个缺乏TIC标记谱的细胞不能产生肿瘤。此外，新的TIC细胞通过多次传代忠实地概括了肿瘤的分子亚型和免疫表型异质性。在转录水平上，我们的新TIC谱丰富了已知在干细胞自我更新中起作用的表达基因升高的细胞群，包括BMI1、nanog和sox2。我们目前正计划评估TIC和非肿瘤起始细胞的mRNA、miRNA和启动子甲基化状态。此外，我们证明了在MCF10 Ca1a细胞系中，CD44+CD24-细胞比CD44+CD24+细胞更具侵袭性。此外，我们证明CD24的表达和与其表达相关的上皮样表型都受到动态调控。具体来说，上皮样CD44+CD24+细胞很容易产生侵袭性间充质样CD44+CD24-细胞，反之亦然。我们在MCF7、SUM159和MDA MB231细胞中证实了类似的事件。此外，我们证明CD24启动子CpG甲基化在CD44+CD24+和CD44+CD24-细胞之间相似，支持该基因易受动态调控的观察。使用siRNA方法，我们发现CD24缺失增加了细胞通过Matrigel入侵的能力。然而，CD24敲低不能产生在CD24内源性下调的细胞中观察到的间充质表型。