小鼠乳腺皮下HC11和EpH4细胞的分化
Summary
我们描述了两个乳房上皮线HC11和EpH4的分化诱导技术。虽然两者都需要胎儿小牛血清、胰岛素和乳素来产生牛奶蛋白，但EpH4细胞可以在三维培养中完全分化成乳腺球。这些互补模型对于分化和肿瘤的信号转导研究非常有用。
Abstract
卡塞林在细胞分化和肿瘤的调节中起着重要的作用。在这里，我们描述了两个小鼠乳腺上皮细胞系HC11和EpH4的诱导诱导的起源和方法，以及它们用于研究乳腺发育和肿瘤转化的互补阶段。
HC11小鼠乳腺上皮细胞系源自怀孕的Balb/c小鼠的乳腺。当生长到附着在含有胎儿小牛血清和Hydrocortisone、I nsulin和Prolactin（HIP介质）的介质中附着在塑料培养皿表面时，它与众不同。 在这些条件下，HC11细胞产生乳蛋白β-酪蛋白和乳清酸性蛋白（WAP），类似于哺乳期乳腺上皮细胞，并形成称为”圆顶”的基本乳腺状结构。
EpH4细胞系来自从怀孕的Balb/c小鼠分离出来的自发不朽的小鼠乳腺上皮细胞。与HC11不同，EPH4细胞在HIP培养基的三维（3D）生长条件下培养时，可以完全分化成球体（也称为乳腺球体）。细胞被试模，悬浮在20%的基质中，由恩格尔布雷斯-霍尔姆-斯沃姆（EHS）小鼠肉瘤细胞产生的细胞外基质蛋白混合物组成，镀在一层浓缩基质层之上，涂有塑料培养皿或多孔板，并覆盖一层含有10%基质的HIP培养基。在这些条件下，EpH4细胞形成中空球体，表现出表基极性、空心流明，并产生β-酪蛋白和WAP。
利用这些技术，我们的结果表明，球红/Rac信号的强度对HC11细胞的分化至关重要。虽然 Rac1 是分化所必需的，低水平的激活 RacV12可增加分化，但高 RacV12水平会阻碍分化，同时诱发新奇。相反，EpH4细胞代表了乳腺上皮分化的早期阶段，即使低水平的RacV12也会抑制这种分化。
Introduction
在正常组织或肿瘤中，细胞在三维组织中具有大量粘附到邻居的机会，这在培养中被高密度细胞生长所模仿。细胞对细胞粘附主要通过细胞素受体进行介导，后者定义了细胞和组织结构。有趣的是，最近证明，球童在信号转导中也起着强大的作用，特别是在生存信号1中。矛盾的是，最近发现，分化和新发育不良2的细胞对细胞粘附信号中，有一些来自细胞对细胞的粘附信号。在这里，我们描述了两种具有代表性的小鼠乳腺上皮细胞系HC11和EpH4的诱导和评估方法。
HC11小鼠乳腺上皮细胞系可为研究上皮细胞分化提供有用的模型。HC11细胞是一种COMMA-1D衍生的细胞系，起源于中孕巴尔布/c小鼠3的乳腺。与其他COMMA-1D衍生克隆相比，HC11克隆不需要外生添加细胞外基质或与其他细胞类型共增，以便通过乳原激素3体外诱导内源β-酪蛋白基因。这种细胞系在分化研究中被广泛使用，因为它保留了正常乳腺上皮的重要特征：HC11细胞可以在清除的乳腺脂肪垫4中部分重组导管上皮。此外，他们可以区分在二维（2D）培养，当成长为汇合附附在塑料培养皿表面，在类固醇的存在，如Hydrocortisone或德塞马塞松，除了Insulin和Prolactin（HIP介质）缺乏表皮生长因子（EGF），分化5，6，7的抑制剂。在这些条件下，HC11细胞产生β-酪蛋白和WAP等牛奶蛋白，在诱导后4天内通过西方印迹检测。同时，HC11细胞的一部分以随机方式形成基本乳腺状结构，称为”圆顶”。诱导后4~5天可见Domes，并在第10天逐渐增大，同时增加β-酪蛋白产量8。有趣的是，HC11细胞具有突变的p539，因此代表一种前塑性状态。因此，HC11 模型非常适合研究同一细胞系统中的异化信号网络与肿瘤。
EpH4细胞是IM-2细胞的衍生物，是一种非肿瘤细胞系，最初来自自发的细胞乳腺上皮细胞，从中孕的Balb/c小鼠10中分离出来。EpH4细胞在2D培养中形成连续的上皮单层，但不分化成腺状结构10，11。然而，在由EHS小鼠肉瘤细胞12（EHS基质、基质或Matrigel，见材料表）产生的细胞外基质蛋白混合物组成的材料中，经过3D生长后，EpH4细胞还可以重述乳腺分化的初始阶段。在这些条件下，EpH4细胞形成球体（也称为乳腺球体），表现出表基极性和空心流明，能够产生乳蛋白β-酪蛋白和WAP，类似于哺乳期乳腺上皮细胞。与HC11细胞相反，它们未分化，有些表达间质标记13，EpH4细胞表现出纯发光形态14。EpH4细胞也报告通过用地塞米松、胰岛素和泌乳素15的刺激在二维培养中产生牛奶蛋白。然而，这种方法排除了对模仿3D培养中乳腺微环境的调节效应的研究。
Protocol
Representative Results
Discussion
HC11细胞非常适合与肿瘤转化一起进行分化研究。一个额外的优点是HC11细胞很容易感染基于Mo-MLV的抗逆转录病毒载体来表达各种基因。在我们手中，EpH4细胞比HC112更难感染相同的逆转录病毒载体。
细胞与细胞接触和生长抑制是HC11细胞分化的关键先决条件。因此，要在细胞层之间实现均匀的分化，在播种时实现培养皿中细胞的统一分布非常重要。如果需要区?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
HC11细胞系由D.Medina博士（德克萨斯州休斯敦）提供。作者感谢女王大学的安德鲁·克雷格博士提供许多试剂和有价值的建议。EpH4细胞是罗斯凯利博士（温哥华UBC）的礼物。科琳·希克为3D文化研究提供了出色的技术援助。
加拿大自然科学和工程研究理事会、加拿大卫生研究院、加拿腺癌基金会（加拿腺癌基金会，安大略省分会）、加拿腺癌研究联盟）的财政援助，安大略省英才中心、乳腺癌行动金斯敦（BCAK）和克莱尔·纳尔逊遗赠基金通过向LR提供赠款，对此表示感谢。BE获得CIHR、CBCF、BCAK和癌症研究协会的资助。PTG由加拿大研究主席、加拿大创新基金会、CIHR、NSERC和加拿大癌症协会提供支持。MN得到了NCIC的特里·福克斯跨学科癌症研究培训项目、研究生奖（QGA）和皇后大学院长奖的支持。MG得到了美国陆军乳腺癌项目、安大略省研究与创新部以及皇后大学咨询研究委员会博士后奖学金的支持。VH得到了CBCF博士奖学金和特里·福克斯基金会跨学科癌症研究培训计划的博士后奖学金的支持，并与CIHR合作。哈纳德·阿丹是NSERC暑期学生班的获得者。BS获得女王大学研究生奖的支持。
Materials
| 30% Acrylamide/0.8% Bis Solution | Bio Rad | 1610154 | Western Blotting |
| Anti-Cyclin D1 antibody | rabbit, Santa Cruz | sc-717 | Western Blotting |
| Anti-p120 antibody | mouse, Santa Cruz | sc-373751 | Western Blotting |
| Anti-β actin antibody | mouse, Cell Signalling technology | 3700 | Western Blotting |
| Anti-β casein antibody | goat, Santa Cruz Biotechnology | sc-17971 | Western Blotting |
| Aprotinin | Bio Shop | APR600 | Lysis Buffer |
| Bicinchoninic Acid Solution | Sigma | B9643-1L-KC | Protein Determination |
| Bovine serum albumin | Bio Shop | ALB007.500 | Protein Determination |
| Clarity Western ECL Substrate | Bio Rad | 170-5061 | Western Blotting |
| Copper(II) sulphate | Sigma | C2284-25ML | Protein Determination |
| DAPI | Thermofisher Scientific | D1306 | Staining |
| Digitonin | Calbiochem, Cedarlane Laboratories Ltd | 14952-500 | Staining |
| EDTA | Bio Shop | EDT001.500 | |
| Epidermal Growth Factor | Sigma | E9644 | Medium for HC11 Cells |
| Fetal calf serum | PAA | A15-751 | Cell Culture Medium |
| Goat- Anti Rabbit-HRP | Santa Cruz | SC-2004 | Western Blotting |
| Hepatocyte Growth Factor recombinant | Gibco | PHG0321 | |
| Hepes | Sigma | 7365-45-9 | Cell Culture |
| Horse Anti-Mouse HRP | Cell Signalling Technology | 7076 | Western Blotting |
| Hydrocortisone | Sigma | H0888 | HIP Medium |
| insulin | Sigma | I6634 | HIP Medium |
| Laminar-flow hood | BioGard Hood | Cell Culture | |
| Leupeptin | Bio Shop | LEU001.10 | Lysis Buffer |
| Matrix (Engelbreth-Holm-Swarm matrix, Matrigel) | Corning | CACB 354230 | |
| Mouse Anti-Goat HRP | Santa Cruz | sc-8360 | Western Blotting |
| Mowiol 4-88 Reagent | Calbiochem, Cedarlane Laboratories Ltd | 475904-100GM | Staining |
| Multi-photon confocal microscope | Leica TCS SP2 | ||
| Na3VO4 | Bio Shop | SOV850 | Lysis Buffer |
| Na4P207 | Sigma | 125F-0262 | Lysis Buffer |
| NaCl | Bio Shop | SOD001.1 | Western Blotting |
| NaF | Fisher Scientific | 7681-49-4 | Lysis Buffer |
| Nikon digital Camera | Coolpix 995 | ||
| Nitrocellulose | Bio Rad | 1620112 | Western Blotting |
| NP-40 | Sigma | 9016-45-9 | |
| Paraformaldehyde | Fisher Scientific | 30525-89-4 | Staining |
| Phase Contrast Microscope | Olympus IX70 | Cell Culture | |
| Phenylmethylsuphonyl fluoride | Sigma | 329-98-6 | Lysis Buffer |
| pMX GFP Rac G12V | Addgene | 14567 | |
| Prolactin | Sigma | L6520 | HIP Medium |
| RPMI-1640 | Sigma | R8758 | Cell Culture Medium |
| Tissue Culture Dish 35 | Sarstedt | 83.3900. | Cell Culture |
| Tissue Culture Plate-24 well | Sarstedt | 83.1836.300 | Cell Culture |
| Transfer Apparatus | CBS Scientific Co | EBU-302 | Western Blotting |
| Tris Acetate | Bio Shop | TRA222.500 | Western Blotting |
| Trypsin | Sigma | 9002-07-7. | Cell Culture |
| Tween-20 | Bio Shop | TWN510.500 | Western Blotting |
| Veritical Gel Electrophoresis System | CBS Scientific Co | MGV-202-33 | Western Blotting |
References
- Geletu, M., Guy, S., Arulanandam, R., Feracci, H., Raptis, L. Engaged for survival; from cadherin ligation to Stat3 activation. Jaks-Stat. 2, 27363-27368 (2013).
- Niit, M., et al. Regulation of HC11 mouse breast epithelial cell differentiation by the E-cadherin/Rac axis. Experimental Cell Research. 361 (1), 112-125 (2017).
- Ball, R. K., Friis, R. R., Schoenenberger, C. A., Doppler, W., Groner, B. Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. The EMBO Journal. 7 (7), 2089-2095 (1988).
- Humphreys, R. C., Rosen, J. M. Stably transfected HC11 cells provide an in vitro and in vivo model system for studying Wnt gene function. Cell growth & differentiation. 8 (8), 839-849 (1997).
- Merlo, G. R., et al. differentiation and survival of HC11 mammary epithelial cells: diverse effects of receptor tyrosine kinase-activating peptide growth factors. European Journal of Cell Biology. 70 (2), 97-105 (1996).
- Wirl, G., Hermann, M., Ekblom, P., Fassler, R. Mammary epithelial cell differentiation in vitro is regulated by an interplay of EGF action and tenascin-C downregulation. Journal of Cell Science. 108, 2445-2456 (1995).
- Arbeithuber, B., et al. Aquaporin 5 expression in mouse mammary gland cells is not driven by promoter methylation. BioMed Research International. 2015, 460598 (2015).
- Morrison, B., Cutler, M. L. Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation. Journal of Visualized Experiments. (32), e1265 (2009).
- Merlo, G. R., et al. Growth suppression of normal mammary epithelial cells by wild-type p53. Oncogene. 9, 443-453 (1994).
- Reichmann, E., Ball, R., Groner, B., Friis, R. R. New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally. Journal of Cell Biology. 108 (3), 1127-1138 (1989).
- Somasiri, A., Wu, C., Ellchuk, T., Turley, S., Roskelley, C. D. Phosphatidylinositol 3-kinase is required for adherens junction-dependent mammary epithelial cell spheroid formation. Differentiation. 66 (2-3), 116-125 (2000).
- Mroue, R., Bissell, M. J. Three-dimensional cultures of mouse mammary epithelial cells. Methods in Molecular Biology. 945, 221-250 (2013).
- Williams, C., Helguero, L., Edvardsson, K., Haldosen, L. A., Gustafsson, J. A. Gene expression in murine mammary epithelial stem cell-like cells shows similarities to human breast cancer gene expression. Breast Cancer Research. 11 (3), 26 (2009).
- Montesano, R., Soriano, J. V., Fialka, I., Orci, L. Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties. In Vitro Cellular & Developmental Biology – Animal. 34 (6), 468-477 (1998).
- Nagaoka, K., Zhang, H., Watanabe, G., Taya, K. Epithelial cell differentiation regulated by MicroRNA-200a in mammary glands. PLoS One. 8 (6), 65127 (2013).
- Arulanandam, R., et al. Cadherin-cadherin engagement promotes survival via Rac/Cdc42 and Stat3. Molecular Cancer Research. 17, 1310-1327 (2009).
- Geletu, M., et al. Classical cadherins control survival through the gp130/Stat3 axis. BBA-Molecular Cell Research. 1833, 1947-1959 (2013).
- Raptis, L., Arulanandam, R., Geletu, M., Turkson, J. The R(h)oads to Stat3: Stat3 activation by the Rho GTPases. Experimental Cell Research. 317 (13), 1787-1795 (2011).
- Raptis, L., et al. Beyond structure, to survival: Stat3 activation by cadherin engagement. Biochemistry and Cell Biology. 87, 835-843 (2009).
- Niit, M., et al. Regulation of Differentiation of HC11 Mouse Breast Epithelial Cells by the Signal Transducer and Activator of Transcription-3. Anticancer Research. 39 (6), 2749-2756 (2019).
- Brinkmann, V., Foroutan, H., Sachs, M., Weidner, K. M., Birchmeier, W. Hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells. Journal of Cell Biology. 131, 1573-1586 (1995).
- Starova, B. . Role of cell adhesion microevironment and the Src/Stat3 axis in autocrine HGF signaling during breast tumourigenesis. , (2008).
- Raptis, L., et al. Rasleu61 blocks differentiation of transformable 3T3 L1 and C3HT1/2-derived preadipocytes in a dose- and time-dependent manner. Cell Growth & Differentiation. 8, 11-21 (1997).
- Greer, S., Honeywell, R., Geletu, M., Arulanandam, R., Raptis, L. Housekeeping gene products; levels may change with confluence of cultured cells. Journal of Immunological Methods. 355, 76-79 (2010).
- Vultur, A., et al. Cell to cell adhesion modulates Stat3 activity in normal and breast carcinoma cells. Oncogene. 23, 2600-2616 (2004).
- Debnath, J., Muthuswamy, S. K., Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods. 30 (3), 256-268 (2003).
- Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nature Methods. 4 (4), 359-365 (2007).
- Hoskin, V. C. . A novel regulatory role of Ezrin in promoting breast cancer cell invasion and metastasis. , (2015).
- Su, H. W., et al. Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression. Journal of Biological Chemistry. 282 (13), 9883-9894 (2007).
- Ostrovidov, S., et al. Skeletal muscle tissue engineering: methods to form skeletal myotubes and their applications. Tissue Engineering Part B: Reviews. 20 (5), 403-436 (2014).
- Cass, J. . Novel Stat3 and Stat5 pathways in epithelial cell differentiation. , (2013).
