Name: three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells
Uploaded: 2014-04-25T14:45:00
Description: Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

这项工作是由美国国立卫生研究院资助R01 CA107469，R01 CA125577和U01CA154224到CG Kleer的，密西根大学的癌症中心支持大学的支持。

材料1。制备及文化传媒<ol><li>解冻生长因子降低（GFR）的基底膜，在4°CO / N。 </li><li>热身完整的媒体所需的细胞系在37°C。 MCF10A-ATCC指定的介质; HME-Ham氏F-12培养基补充有5％胎牛血清，1微克/毫升氢化可的松，5微克/毫升的胰岛素，10毫微克/毫升的表皮生长因子，和100纳克/毫升的霍乱毒素; SUM149-Ham氏F-12介质补充有5％FBS，2微克/毫升氢化可的松和5微克/毫升胰岛素;和MDA-MB-231-10％FBS-DMEM中</li><li>预冷...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat. Methods. 4, 359-365 (2007).</li><li>Li, Q., Chow, A. B., Mattingly, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> J. Pharmacol. Exp. Ther. 332, 821-828 (2010).</li><li>Rosen, P. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphologies+of+breast+cancer+cell+lines+in+three-dimensional+assays+correlate+with+their+profiles+of+gene+expression.">The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression.</a> Mol. Oncol. 1, 84-96 (2007).</li><li>Pal, A., Huang, W., Li, X., Toy, K. A., Nikolovska-Coleska, Z., Kleer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCN6+modulates+BMP+signaling+via+the+Smad-independent+TAK1/p38+pathway,+acting+to+suppress+metastasis+of+breast+cancer.">CCN6 modulates BMP signaling via the Smad-independent TAK1/p38 pathway, acting to suppress metastasis of breast cancer.</a> Cancer Res. 15, 4818-4828 (2012).</li><li>Pal, A., Huang, W., Toy, K. A., Kleer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCN6+knockdown+disrupts+acinar+organization+of+breast+cells+in+three-dimensional+cultures+through+up-regulation+of+type+III+TGF-&#946;+receptor.">CCN6 knockdown disrupts acinar organization of breast cells in three-dimensional cultures through up-regulation of type III TGF-&#946; receptor.</a> Neoplasia. 14, 1067-1074 (2012).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+reversion+or+death+of+cancer+cells+by+altering+signaling+pathways+in+three-dimensional+contexts.">Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts.</a> J. Natl. Cancer Inst. 94, 1494-1503 (2002).</li><li>Weaver, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> J. Cell Biol. 137, 231-245 (1997).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+co-culture+models+to+study+prostate+cancer+growth,+progression,+and+metastasis+to+bone.">Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone.</a> Semin. Cancer Biol. 15, 353-364 (2005).</li><li>Vaughan, M. B., Ramirez, R. D., Wright, W. E., Minna, J. D., Shay, J. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+3D+microenvironment+on+phenotype,+gene+expression,+and+EGFR+inhibition+of+colorectal+cancer+cell+lines.">Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.</a> PLoS One. 8, 1-11 (2013).</li></ol>

在这里，我们已经描述了乳腺上皮细胞对重组基底膜的三维立体培养和本系统恶性与良性乳腺表现型来区分的用处。此系统还可以用于从其他腺体组织培养不同类型的细胞和已报道已被成功地用于培养的前列腺上皮细胞，支气管上皮细胞和甲状腺上皮细胞，仅举几例12-14。三维培养的重要性在于，它是生理学相关的模型。三维培养提供了深入了解，导致腺体结构破坏的恶性转化过程中的分...

三维上重建基底膜乳腺上皮细胞培养物（三维）是一种重要的模型系统来研究复杂的表型和正常乳腺和乳腺癌2,3的相应的信令。乳房的功能单元是终末导管小叶单元（TDLU），它由一个小导管，其分支成腺泡。腺泡是高度组织的结构中，两个层细胞，上皮细胞和肌上皮细胞，这是由基底膜5包围的组成。正常的腺泡的最显着特征是细胞极化，附着在底层基底膜和专门的细胞与细胞的接触。这个复杂的组织被破坏的浸润性癌。被广泛使用的二维培养，其中细胞生长为单层，不允许腺泡的形成。因此，该单层培养缺乏提供一个系统来捕捉上皮细胞之间的错综复杂的关系在正常乳腺和乳腺癌放松管制。乳腺癌细胞的功能性的单种培养上皮已经开发了几个实验室2,3。三维培养涉及乳腺癌细胞在基质胶，这是从Engelbreth-的Holm-群小鼠肉瘤细胞衍生的溶解提取物，并且是可商购的生长。我也喜欢用其他的胶原基质的3D。乳腺细胞可以通过3D嵌入式测定法，其中细胞培养嵌在基质胶2中培养在三维。此外，细胞可以通过3D顶部化验培养，也被称为3D叠加分析，这是符合成本效益，因为它需要的基质胶量较小，并促进集落形成时间推移监测相衬成像，是理想的原位成像2 -3。在上面试验中的三维已将用于定义腺泡表型和基因表达概况7之间的相关性。 三维培养可effectiv伊利用于从浸润癌区分正常和良性细胞。正常和良性乳腺细胞形成生长停止极化结构，在中心一个明确的管腔上，而基底膜浸润性癌细胞生长力强和随意，没有结算管腔。 E6/E7永生化人乳腺上皮细胞和MCF10A细胞系，这是一个自发永生化细胞系来自一个36岁的患者，纤维囊性变孤立的，已成功地用于夺回乳腺良性表型3D 3,8，并担任一个模型系统来研究几个分子8,9的致癌和抑癌功能。这些良性细胞可以被工程化以引入慢病毒/逆转录病毒操纵感兴趣的基因（次）。如果感兴趣的基因（s）是一种肿瘤抑制基因，shRNA的介导的敲除会导致，而恶性转化，如果感兴趣的基因是良性细胞癌基因的过度表达应该表现出恶性表型。在这两种情况下形成的三维腺泡结构可以捕获恶变。 良性的单细胞接种于3D开始增生并形成圆球形结构。 5-8天的细胞这个球形集合体将分化为细胞的周围偏振层，其与所述基质胶接触，并减少极化的细胞，缺乏与基质胶接触的内质。在接下来的10-15天的内细胞将开始凋亡形成一个清晰的管腔死。恶性肿瘤细胞将增长胡乱失去极性。高度恶性细胞通常形成分支结构。这些差异在表型可以通过时间的推移相衬成像和原位与相关分子标记免疫染色被捕获。最常用的标记是α-6-整，基底极性的标记，与所述增殖标志物磷酸化组蛋白3和apoptot组合IC标记物裂解的caspase-3。后者是重要的，以确定是否有管腔形成在中央的腺泡，正常和良性乳腺细胞的特征。其他极性和细胞间接触标志物包括GM130，层粘连蛋白，企业风险管理，E-cadherin的。交替的乳腺癌细胞系可被工程化来操纵感兴趣的基因。在这种情况下，复归到一个更大的增长被捕良性的表型可以被用作一个读出从癌症的表型区分。 三维培养也可以仔细用来划定参与恶性转化10-11的信号通路。使用上面提到的读数，药理抑制剂，阻断性抗体或重组蛋白可用于在分子信号传导，导致恶变被精确定位。与来自不同实验室的不断努力，可以从三维提取细胞，提取RNA，并制备裂解物用于免疫印迹和免疫noprecipitation。这些策略在协议中，并逐步详细地描述。

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		<col />
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		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced matrigel</td>
			<td style="width:83px;">BD biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Avoid repeat freeze thaw&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass hamber slides 8-well</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177402</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass chamber slides 2- well&#160;</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177380</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">goat anti mouse F(ab&#180;)2 fragment</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">115-006-006</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Normal goat serum</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent conjugated Alexa antibodies</td>
			<td style="width:83px;">Molecular probes</td>
			<td style="width:119px;"></td>
			<td>Please look at molecular probes website</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Prolong gold antifade reagent with DAPI</td>
			<td style="width:83px;">Molecular Probes</td>
			<td style="width:119px;">P36935</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">3D Culture Cell Harvesting kit</td>
			<td style="width:83px;">Trevigen</td>
			<td style="width:119px;">3448-020-k</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Apha-6-integrin</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">MAB1378</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-GM130</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610822</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-phospho-histone 3 (Ser10)FITC Conjugated</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">16-222</td>
			<td>1:100 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Cleaved caspase 3 (Asp 175)</td>
			<td style="width:83px;">Cell Signaling</td>
			<td style="width:119px;">9661s</td>
			<td>1:50 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-E-cadherin</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610181</td>
			<td>1:200 dilution for IF</td>
		</tr>
	</tbody>
</table>

three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

乳腺浸润性癌是一组恶性上皮性​​肿瘤特征的侵袭邻近组织，并倾向转移的。信号癌细胞和微环境之间的相互作用发挥对乳腺癌的生长和生物学行为1强大的影响力。然而，大多数来自细胞外基质，这些信号的丢失或当细胞生长在二维培养（2D）作为单层的相关性被充分研究。近年来，三维（3D）上的重组基底膜培养已成为首选的方法扼要重述良性和恶性乳腺细胞的组织结构。在三维培养的细胞保留来自细胞外基质的重要线索，并提供一个生理相关的体外系统2,3。值得注意的是，有越来越多的证据表明细胞有不同的行为时，在3D种植相比，2D 4。三维培养可以有效地使用，以从良性乳腺表型分化的恶性表型的装置和用于支撑的细胞和分子信号传导参与3。一种良性上皮细胞的显着特点是它们被极化，使得根尖细胞质是朝向管腔和基底细胞质搁置在基底膜。这apico - 基底极性消失在乳腺浸润性癌，其特点是细胞解体和形成网状和分支小管是随意渗入周围的基质。良性腺体和浸润癌之间的这些病理的差异可以被复制在3D 6,7。使用了适当的读奏像单轮腺泡结构的定量，或差的验证分子标记物的细胞增殖，极性和凋亡与其它分子和细胞生物学技术，三维文化视角组合表达E能提供更好的理解过程中的恶性转化，并划定了负责信号的细胞变化的重要工具。

三维培养物可被有效地用于区分人类乳腺上皮细胞的恶性表型的良性乳腺。镀在基质胶单良性细胞成长为有组织的球形结构采用X射线相衬成像，可以很容易地成像为单轮平腺泡在一个平面上。镀在基质胶的单一的恶性细胞生长随意，显示出非常高的折射率，而且难以像在一个平面上。 如图1，良性HME和MCF10A细胞形成球形寻找腺泡而高转移性乳腺癌细胞系SUM149和MDA-MB-231成长为分支的管?...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

对重组基底膜乳腺上皮细胞的三维培养是扼要重述在体内的体系结构的良性乳房，并从良性乳腺表型分化的恶性表型的有效方法。重要的是，该系统可以应用到研究浸润性癌中的其他组织中。

三维文化：一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

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Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

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乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...