Name: molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts
Uploaded: 2014-04-29T14:00:00
Description: Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

MB是由来自MRC奖学金补助资金支持。 KP和AHM是由威塞克斯医学研究和英国癌症研究中心/ RCS（英格兰）（C28503/A10013）补助资金支持。我们感谢南安普敦组织化学研究单位大学的支持。

从结肠外植初级成纤维细胞培养1。建立<ol><li>获得正常人结肠黏膜组织样本直接从外科手术室，并暂停在7-10毫升的PBS中补充有100单位/ ml青霉素，100微克/毫升链霉素和0.25微克/毫升两性霉素。 </li><li>在实验室中，将样品在一个10厘米的组织培养皿的中心，洗3次，用PBS /笔，链球菌/两性霉素。最后一次洗涤后不要吸。 </li><li>用无菌镊子和手术刀，切割组织切成小块（约2毫米），并把每个块上绘制...

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	<li>Nystrom, M. L., Thomas, G. J., Stone, M., Mackenzie, I. C., Hart, I. R., Marshall, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J Pathol. 205 (4), 468-475 (2005).</li><li>Zhang, L., 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=miR-153+supports+colorectal+cancer+progression+via+pleiotropic+effects+that+enhance+invasion+and+chemotherapeutic+resistance.">miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance.</a> Cancer Res. , (2013).</li><li>Bullock, M. D., 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=Pleiotropic+actions+of+miR-21+highlight+the+critical+role+of+deregulated+stromal+microRNAs+during+colorectal+cancer+progression.">Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.</a> Cell Death Dis. 20 (4), (2013).</li><li>Jenei, V., Nystrom, M. L., Thomas, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+invasion+in+an+organotypic+model.">Measuring invasion in an organotypic model.</a> Methods Mol Biol. 769, 223-232 (2011).</li><li>De Wever, O., Demetter, P., Mareel, M., Bracke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+myofibroblasts+are+drivers+of+invasive+cancer+growth.">Stromal myofibroblasts are drivers of invasive cancer growth.</a> Int J Cancer. 123 (10), 2229-2238 (2008).</li><li>Fries, K. 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=Evidence+of+fibroblast+heterogeneity+and+the+role+of+fibroblast+subpopulations+in+fibrosis.">Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.</a> Clin Immunol Immunopathol. 72 (3), 283-292 (1994).</li><li>Moutasim, K. 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=Betel-derived+alkaloid+up-regulates+keratinocyte+alphavbeta6+integrin+expression+and+promotes+oral+submucous+fibrosis.">Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.</a> J Pathol. 223 (3), 366-377 (2011).</li><li>Winter, J., Jung, S., Keller, S., Gregory, R. I., Diederichs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+roads+to+maturity:+microRNA+biogenesis+pathways+and+their+regulation.">Many roads to maturity: microRNA biogenesis pathways and their regulation.</a> Nat Cell Biol. 11 (3), 228-234 (2009).</li><li>Li, J., 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=Comparison+of+miRNA+expression+patterns+using+total+RNA+extracted+from+matched+samples+of+formalin-fixed+paraffin-embedded+(FFPE)+cells+and+snap+frozen+cells.">Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.</a> BMC Biotechnol. 7 (36), (2007).</li><li>Szafranska, A. E., 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=Accurate+molecular+characterization+of+formalin-fixed,+paraffin-embedded+tissues+by+microRNA+expression+profiling.">Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.</a> J Mol Diagn. 10 (5), 415-423 (2008).</li></ol>

这里我们描述了一种方法，特别是分离和表征已购入时转移进程在上皮细胞和基质细胞的三维共培养物的最初阶段侵入癌症相关间质的能力的肿瘤细胞。 由并列含有离体人结肠癌成纤维细胞的合成基质的CRC上皮细胞共培养模型被用于研究CRC侵袭3 -维。这其中在体内条件模拟可以通过在上皮代细胞，以及使用各种解剖起源的体外成纤维细胞构成的基质适用?...

器官型共培养物是组织工程化的模型，其帮助在含有必需的细胞外基质组分1-3的胶原凝胶并列恶性上皮细胞和基质细胞重建体内肿瘤微环境中3维。主要设想为测量肿瘤侵袭的方法，organotypics减少依赖于体内动物模型，并避免其它的体外技术如Transwell小室测定法，其中侵入细胞是人工强制进入单分散状态2-4的缺点。包含基质细胞在这些模型中，如成纤维细胞，反映了肿瘤微环境中调节恶性肿瘤的侵袭和转移3-4中的重要作用，然而基质的表型异质性是这样的，为了最大限度地提高器官的生理相关性模型，器官特异性，准确的解剖体外成纤维细胞应该包括尽可能3,6。 Organotypics是一个通用平台，研究肿瘤和基质细胞之间的相互作用，并越来越多地以新颖的方式来检查对肿瘤侵袭化学抑制剂的影响，以及靶基因的改变7,8使用。研究侵袭性肿瘤边缘是一个特别令人兴奋的前景。在器官模型，建立结肠直肠癌（CRC）细胞系中通常会产生一个很好的分层上皮细胞层，并在截面中，已经获得侵入细胞外基质（ECM）的能力的细胞是容易辨别的。鉴于肿瘤和肿瘤相关间质细胞在体内的既定微地形异质性，采用激光显微切割和学习他们孤立地提取这些细胞，可以揭示有关大肠癌转移的来历重要的生物学见解以及它会如何更有效地有针对性的。我n以下的方法，谁捐赠签署知情同意书的组织样本，并研究的所有患者被批准为机构区域研究伦理委员会。

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colorectal cancer cell lines - example shown SW480</td>
			<td style="width:132px;">ATCC</td>
			<td style="width:119px;">ATCC CCL-228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matrigel</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon membrane</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">VVLP01300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Metal grid</td>
			<td style="width:132px;">The Mesh Company</td>
			<td>WSS20-A4</td>
			<td>themeshcompany.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser microdissection platform</td>
			<td style="width:132px;">Leica Microsystems</td>
			<td style="width:119px;">Leica AS LMD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Membrane mounted slides</td>
			<td style="width:132px;">Molecular devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cresyl Violet</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">1052350025</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

入侵结直肠癌（CRC）细胞已获得挣脱他们的姐姐细胞，浸润间质，和重塑细胞外基质（ECM）的能力。这个特征明显的表型组细胞的生物学转移级联过程中可以大大提高我们的早期事件的理解。 肿瘤侵袭是一个动态过程由​​恶性上皮和相关肿瘤间质之间的双向相互作用促进。为了检查细胞特异性应答在我们结合器官共培养和激光显微解剖技术在肿瘤基质界面。 器官模型，其中关键的基质成分，如成纤维细胞是3 - dimentioanally共培养与癌症上皮细胞，是高度可操作的实验工具，使浸润和肿瘤 - 基质的相互作用在近pH值加以研究ysiological条件。 激光显微切割（LMD）是这需要手术剥离和提取肿瘤组织内的各阶层，以微米级精度的技术。 通过结合这些技术与基因组，转录组和表观遗传分析，我们的目标是发展的入侵肿瘤细胞和周围间质组织的分子特性有更深的了解，并在这样做可能揭示新的生物标志物，并在CRC的药物的发展机遇。

本公司已申请上述方法的CRC细胞系和间质细胞的多种组合。其中一个例子这里介绍的是SW480上皮CRC细胞系与原代人类体外结肠成纤维细胞。三维共培养物对细胞之间差异表达的miRNA在侵袭性肿瘤前比较，构造（ 图1），进行显微镜和激光捕获显微切割（ 图2），并通过微RNA（miRNA）的谱（ 图3）分析以及在该分层（非侵入）的肿瘤区域。 <p class="jove_conte...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

激光显微切割细胞和基质由合成的，组织工程肠癌模型的分子分析代表了一种新的和可操作的方法在肿瘤细胞之间的界面来表征和解剖的独特生物学在浸润前沿和癌症相关的基质细胞。

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Research

JoVE Journal

Medicine

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Modeling and Imaging 3-Dimensional Collective Cell Invasion

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

科研

医学

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

An Ex vivo Model to Study Hormone Action in the Human Breast

An Ex vivo Model to Study Hormone Action in the Human Breast

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

三维（3D）肿瘤球体侵袭试验

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

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...