一个<em>在体外</em>对雌激素敏感的乳腺癌休眠模式在骨髓中：分子机理研究与假设生成的一个工具
Summary
We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.
Abstract
The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.
Introduction
乳腺癌细胞转移到骨髓病前是可检测1，只要小的肿瘤发展的血管2,3。转移过程是快，但效率不高。细胞迅速进入新的血管，在数百万个4天，但行程到远处器官5几年生存。尽管如此，一些微生存在骨和可以发现以单细胞或在从初诊患者1骨髓抽吸小细胞团块。这些细胞抗蚀辅助化疗，其施用于消除他们6的根本目的。这种阻力被赋予的，基本上，通过存活通过与骨髓微7,8-相互作用信令发起的。微转移可以在大约三分之一的妇女局部乳腺癌的发现，代表了生存的独立指标时，单因素分析分析9。一些microme转移瘤是开始增长，但复发模式依赖于细胞类型。例三阴性乳腺癌往往1〜4年之间复发，这表明穷人控制了休眠状态。其他类型的细胞，包括ER / PR +细胞，可以保持休眠长达20年，复发10的稳定，连续速率。而在ER +和ER-乳腺癌细胞系和肿瘤之间休眠的 基因表达签名的差异反映了不同的休眠潜力11，与骨髓基质相互作用可能代表到休眠一个显著的贡献。
休眠体内的研究是非常困难的，因为微是罕见的，是由寡不敌众造血细胞超过10倍6。因此，相关模型必须产生提供体外数据可以建议机制和产生体内检验的假设。一些休眠模式，包括MATHEmatical车型12,13， 在体外模型7,8,14,15， 体内异种移植模型16， 在体外组合和异种移植模型17,18和自发肿瘤和转移模型19，已经取得了一些见解，癌细胞休眠20 。这些模型有其自身的局限性和本身用于产生有关的分子信号和支配休眠到在多种生物相关模型中进行测试的相互作用的假设主要是有用的。
界定休眠的 分子机制，以导致在周期停滞，再分化和治疗抗性和机制的微环境造成复发的ER +细胞的相互作用的总体目标，我们开发了提供选择的相关内容的体外模型基质微环境7。这种模式，而在其康波相对稀疏堂费，是足够坚固，以允许研究者得出影响休眠显著职能特定的分子机制。这些实验产生可在体内直接测试假设。该模型依赖于我们证明是有关休眠中的几个关键要素。它们包括在一个克隆的密度，其中它们的相互作用是主要与基质和介质的可溶性组分，纤连蛋白基质和碱性成纤维细胞生长因子的存在下，用雌激素 – 依赖的乳腺癌细胞中，细胞培养物（FGF-2）在介质中。
我们特征在于支配系统在体外机制，包括诱导细胞周期阻滞由FGF-2的21中 ，通过TGFβ22介导的，生存通过PI3激酶7,8和ERK 8和形态发生分化信令上皮表型，这依赖于RhoA的失活，整45;5β1上调和间质纤维连接蛋白生存7,15结扎（ 图1）。 FGF-2对MCF-7细胞的体外细胞周期的影响开始在浓度至少一个对数低于10毫微克/毫升21,23。基本原理是基于FGF-2的表达治乳腺导管形态，循环扩张和衰退在许多哺乳动物系统24-27的时间控制。我们证明了FGF-2的诱导分化，包括导管形态发生在3D培养28，而FGF-2的表达通常失去了与人类肿瘤29的恶变。 FGR1的表达仍然完好无损调查中的29和MCF-7细胞乳腺癌继续对所有4 FGF受体30。在休眠的 上下文中，FGF-2是由出口和重沉积在骨髓基质31,32在那里它在造血干细胞33的保存功能。我们DEMonstrated的FGF-2诱导ER +乳腺癌细胞纤维连接蛋白基质，还含有丰富的骨髓，它诱导分化形态培养7休眠状态。在模型中，乳腺癌细胞的生长抑制，灭活的Rho一种贯穿RhoGap GRAF，再分化到上皮表型和重新表达整联α5β1lost与恶性进展。它们结合纤连蛋白通过整合素α5β1和激活存活信号使得它们对细胞毒药物治疗7,8,15（ 图1）的抵抗力。卢的GTP酶类的抑制先前已证明诱导休眠型34。
在这里，我们将概述的具体程序，将允许调查人员建立模型，并研究规范ER +乳腺癌细胞的休眠特异的分子和细胞机制。在这里介绍的实验来说明使用该模型的我们靶向PI3K途径（ 图1B）与Akt的抑制剂和PI3K抑制剂和Rho家族（ 图1B）与泛Rho抑制剂和作为Rho激酶（ROCK）抑制剂的所有成员。
Protocol
Representative Results
Discussion
我们的模型是由休眠的骨髓中的几个关键要素。它由雌激素敏感细胞，这是可能保持休眠的 骨髓长时间10的类型，它包括纤维连接蛋白，骨髓的关键结构元件，FGF-2，生长因子大量由骨髓基质合成的和大量沉积在骨髓细胞在克隆密度，其中它们之间的相互作用主要是与底层的31,32和温育的细胞外基质。而骨髓微环境是更为复杂，因为需要问特定机械问题这个简单的系统可以建?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者什么都没有透露。
Materials
| MCF-7 cells | ATCC | HTB-22 | |
| T47D cells | ATCC | HTB-123 | |
| BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate | Corning | 354411 | |
| BD BioCoat Fibronectin 60 mm Culture Dishes | Corning | 354403 | |
| BD BioCoat Fibronectin 100 mm Culture Dishes | Corning | 354451 | |
| BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin | Corning | 354088 | |
| 6 Well tissue culture plate | CellTreat | 229106 | |
| Dulbecco Modified Eagle Medium High Glucose 10X Powder | Corning Life Sciences | 50-013-PB | |
| Heat Inactivated, Fetal Bovine Serum | Serum Source International | FB02-500HI | |
| 0.25% Trypsin/2.21 mM EDTA | Corning | 25-053-CI | |
| Penicillin-Streptomycin Solution, 100X | Corning | 30-002-CI | |
| L-Glutamine, 100x, Liquid | Corning | 25-005-CI | |
| Recombinant Human FGF basic | R&D Systems | 234-FSE-025 | |
| Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate) | CalBiochem | 124005 | |
| LY294002 | CalBiochem | 19-142 | Chenical PI3K inhibitor |
| C3 transferase | Cytoskeleton | CTO3 | Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1 |
| Y-27632 dihydrochloride | Santa Cruz Biotechnology | 129830-28-2 | |
| BODIPY FL-Phallacidin (green) | Molecular Probes | B607 | Fluorochrome for fibrillar actin staining |
| BODIPY FL-Rhodamine phalloidin (red) | Molecular Probes | R415 | Fluorochrome for fibrillar actin staining |
| Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent | Molecular Probes | R37114 | |
| ProLong Gold Antifade Mountant with DAPI | Molecular Probes | P-36931 |
References
- Braun, S., et al. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med. 342 (8), 525-533 (2000).
- Benoy, I. H., et al. Relative microvessel area of the primary tumour, and not lymph node status, predicts the presence of bone marrow micrometastases detected by reverse transcriptase polymerase chain reaction in patients with clinically non-metastatic breast cancer. Breast Cancer Res. 7 (2), R210-R219 (1186).
- Wapnir, I. L., Barnard, N., Wartenberg, D., Greco, R. S. The inverse relationship between microvessel counts and tumor volume in breast cancer. Breast J. 7 (3), 184-188 (2001).
- Wyckoff, J. B., et al. A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer Res. 60 (9), 2504-2511 (2000).
- Phadke, P. A., et al. Kinetics of metastatic breast cancer cell trafficking in bone. Clin Cancer Res. 12 (5), 1431-1440 (2006).
- Braun, S., et al. Lack of an effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high risk breast cancer patients. J Clin Onc. 18 (1), 80-86 (2000).
- Korah, R., Boots, M., Wieder, R. Integrin α5β1 promotes survival of growth-arrested breast cancer cells: an in vitro paradigm for breast cancer dormancy in bone marrow. Cancer Res. 64 (13), 4514-4522 (2004).
- Najmi, S., Korah, R., Chandra, R., Abdellatif, M., Wieder, R. Flavopiridol blocks integrin-mediated survival in dormant breast cancer cells. Clin Cancer Res. 11 (5), 2038-2046 (2005).
- Braun, S., et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N England J Med. 353 (8), 793-802 (2005).
- Dent, R., et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 13 (15 Part 1), 4429-4434 (2007).
- Kim, R. S., et al. Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer. PLoS One. 7 (4), e35569 (2012).
- Hanin, L. Seeing the invisible: how mathematical models uncover tumor dormancy, reconstruct the natural history of cancer, and assess the effects of treatment. Adv in Exp Med & Biol. 734, 261-282 (2013).
- Wilkie, K. P. A review of mathematical models of cancer-immune interactions in the context of tumor dormancy. Adv in Exp Med & Biol. 734, 201-234 (2013).
- Barkan, D., Green, J. E. An in vitro system to study tumor dormancy and the switch to metastatic growth. J Vis Exp. (54), e2914 (2011).
- Barrios, J., Wieder, R. Dual FGF-2 and intergrin α5β1 signaling mediate GRAF-induced RhoA inactivation in a model of breast cancer dormancy. Cancer Microenvironment. 2 (1), 33-47 (2009).
- Ganapathy, V., et al. Luminal breast cancer metastasis is dependent on estrogen signaling. Clin & Exp Metastasis. 29 (5), 493-509 (2012).
- Barkan, D., et al. Inhibition of metastatic outgrowth from single dormant tumor cells by targeting the cytoskeleton. Cancer Res. 68 (15), 6241-6250 (2008).
- Ghajar, C. M., et al. The perivascular niche regulates breast tumour dormancy. Nat Cell Biology. 15 (7), 807-817 (2013).
- Marshall, J. C., et al. Effect of inhibition of the lysophosphatidic acid receptor 1 on metastasis and metastatic dormancy in breast cancer. J Nat Cancer Inst. 104 (17), 1306-1319 (2012).
- Almog, N. Genes and regulatory pathways involved in persistence of dormant micro-tumors. Adv in Exp Med & Biol. 734, 3-17 (2013).
- Wang, H., et al. Basic FGF causes growth arrest in MCF-7 human breast cancer cells while inducing both mitogenic and inhibitory G1 events. Cancer Res. 57 (9), 1750-1757 (1997).
- Fenig, E., et al. Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor. Breast Cancer Res Treat. 70 (1), 27-37 (2001).
- Fenig, E., et al. Basic fibroblast growth factor confers growth inhibition and Mitogen-activated Protein Kinase activation in human breast cancer cells. Clinical Cancer Research. 3 (1), 135-142 (1997).
- Coleman-Krnacik, S., Rosen, J. M. Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development. Molecular Endocrinology. 8 (2), 218-229 (1994).
- Plath, A., et al. Expression and localization of members of the fibroblast growth factor family in the bovine mammary gland. J Dairy Science. 81 (10), 2604-2613 (1998).
- Lavandero, S., Chappuzeau, A., Sapag-Hagar, M., Oka, T. In vivo and in vitro evidence of basic fibroblast growth factor action in mouse mammary gland development. FEBS letters. 439 (3), 351-356 (1998).
- Sinowatz, F., Schams, D., Plath, A., Kolle, S. Expression and localization of growth factors during mammary gland development. Adv in Exp Med & Biol. 480, 19-27 (2000).
- Korah, R., Sysounthone, V., Scheff, E., Wieder, R. Intracellular FGF-2 promotes differentiation in T47-D breast cancer cells. Biochem Biophys Res Comm. 277 (1), 255-260 (2000).
- Korah, R., Das, K., Lindy, M. E., Hameed, M., Wieder, R. Co-ordinate loss of FGF-2 and laminin 5 expression during neoplastic progression of mammary duct epithelium. Human Pathology. 38 (1), 154-160 (2007).
- Wieder, R., et al. Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation. J. Cellular Physiology. 177, 411-425 (1998).
- Brunner, G., Gabrilove, J., Rifkin, D. B., Wilson, E. L. Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan. J Cell Biol. 114 (6), 1275-1283 (1991).
- Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., Gabrilove, J. L. Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures. Blood. 77 (5), 954-960 (1991).
- Gupta, P., McCarthy, J. B., Verfaillie, C. M. Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells. Blood. 87 (8), 3229-3236 (1996).
- Chatterjee, M., van Golen, K. L. Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype. Int J Cancer. 129 (1), 61-69 (2011).
- Korah, R., Sysounthone, V., Golowa, Y., Wieder, R. Basic fibroblast growth factor confers a more differentiated phenotype in MDA-MB-231 human breast cancer cells. Cancer Res. 60 (3), 733-740 (2000).
- Dontu, G., et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes & Dev. 17 (10), 1253-1270 (2003).
- Tivari, S., Lu, H., Dasgupta, T., Wieder, R. Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy. Proceedings of the 105th Annual Meeting of the American Association for Cancer Research. , (2014).
