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），并把每个块上绘制在一个12孔板的手术刀刀片交叉的中心。轻轻摇动手术刀在组织，使其能够坚持。 </li><li>培养在750微升的试样初级成纤维细胞生长培养基（DMEM补充有20％胎牛血清（FCS），100 U / ml青霉素，100微克/毫升链霉素和292微克/毫升L-谷氨酰胺），这应该次亚RGE组织而不使其浮动。避免打扰板块未来5天，惟重新饲料每2天。 </li><li>在未来5天的成纤维细胞会慢慢开始从组织的边缘长出来。在大约7天，这将更加明显。经过约3-4周，如果有足够的成纤维细胞生长出来，加750μL胰蛋白酶，每孔。经过细胞脱离（这可能需要5-10分钟），游泳池和转移到T-25组织培养瓶。 </li></ol> 2，器官准备编制成纤维浸渍胶器官的： <ol><li>制备含每器官型5×10 5细胞在DMEM补充有10％FCS和292微克/毫升L-谷氨酰胺成纤维细胞悬浮液。 </li><li>弥补在冰上的器官型凝胶，以以下比率： <ul><li> 7卷的鼠尾胶原蛋白：基质胶混合（1:1） </li><li> 1体积的过滤DMEM 10倍</li><li> 1体积的FCS </li><li> 1体积的含有5×10 5的成纤维细胞的成纤维细胞的细胞悬浮液 9凝胶（凝胶每1ml）的准备好10毫升，轻轻混匀，避免气泡。 </li></ul></li><li>加入1 ml至24孔板的各孔中并孵育1小时，在37 °C在一个潮湿气氛中，使凝胶进行设置。 1小时后，加入1 ml DMEM（10％FCS）/过剩的凝胶的顶部，并返回到培养箱中过夜。 </li><li>翌日，从凝胶板和含有5×10 5个CRC上皮细胞介质1毫升的顶部吸出培养基。 </li></ol>制备凝胶涂层尼龙片： <ol start="5"><li>准备灭菌，高压灭菌尼龙布测量1.5厘米×1.5厘米，在前进。 </li><li>用无菌镊子，将尼龙片材的所需数量在10厘米的培养皿（通常4尼龙片可以每皿容纳）。 </li><li>弥补冰凝胶下列顺序（250μl总音量每尼龙板必填项）： <ul><li> 7卷的鼠尾胶原蛋白</li><li> 1体积的过滤DMEM 10倍</li><li> 1体积的FCS </li><li> 1体积的DMEM 10％FCS /过剩</li></ul></li><li>如果溶液是黄色的，通过添加0.1M的NaOH在50微升等分试样，直至溶液变成粉红色中和。 </li><li>加入250μl该解决方案的每个尼龙板，并在37 ℃，5％CO 2中30分钟，使凝胶来设置的潮湿气氛。 </li><li>补1％戊二醛的PBS溶液中。加入10 ml每10 6cm培养皿一旦凝胶已经设置并孵育在4℃下1小时。 </li><li>洗涤尼龙片3倍的PBS中和1x的DMEM（10％FCS）/谷氨酸和在DMEM /谷氨酸孵育过夜，在4°C。 </li></ol>养器官凝胶上钢格栅的入侵： <ol start="12"><li>通过折叠不锈钢板成三脚架形成预做准备脚手架网格。高压灭菌后才能使用。</li><li>使用镊子灭菌的乙醇放置一个钢格到6孔板的各孔中。 </li><li>将凝胶涂层尼龙板与胶原侧至上，到每个网格。 </li><li>小心地从24孔板转移的器官型凝胶用无菌乙醇刮刀凸起尼龙片。 </li><li>填充井用DMEM 10％FCS /过剩补充有10％FCS，直到尼龙片是在与介质接触，但不被水淹没。确保介质不接触的器官凝胶。 </li><li>孵育14天，在37℃下在5％CO 2的潮湿气氛中，更换媒体，每2天。 </li></ol> 3。器官枷<ol><li>从很好地去除整个器官，包括尼龙板，并放置在保鲜膜。 </li><li>平分器官并用洁净的一次性手术刀尼龙板和甲醛固定两半24小时在室温下。 </li><li>用70％乙代替甲醛醇后24小时，石蜡包埋，切片，染色和前离开过夜。 </li></ol> 4，激光的入侵保证金显微切割<ol><li>第10微米厚的膜上安装滑轨。 </li><li> Deparaffinize部分通过应用二甲苯，持续1分钟，然后除去二甲苯和75％乙醇固定进行1分钟。 </li><li>除去乙醇并用染色1分钟0.125％甲酚紫溶液。甲酚紫突出上皮细胞，并允许从基质容易受到歧视。 </li><li>除去甲酚紫和用100％乙醇漂洗之前申请的100％乙醇进行1分钟。让载玻片在空气中干燥30分钟。 </li><li>使用选择的平台（ 如徕卡的AS系统）进行激光显微切割。 </li><li>将载玻片与染色部分进行显微切割面朝下放在显微镜载物台。 </li><li>安装0.5 ml离心管放入回收盒，并加入50微升细胞裂解缓冲液（ 例如，DNA，RNA或蛋白质的裂解缓冲液）到帽成显微组织将收集。 </li><li>在直视下，用摇杆来确定感兴趣的组织，并使用该软件的界面，注释染色的有机部分，突出细胞在肿瘤侵犯前进行显微切割。 </li><li>指示激光射击，这都应该切出的突出部分和推动显微组织到微量离心管的盖子。 </li><li>一旦有足够的材料被收集，弹出收集盒，关闭离心管中，轻轻旋转绘制裂解缓冲液到管的底部。 </li><li>将样品置于冰上，直到显微切割完成。过程中样品通过适当的方法来提取目标分析物。 </li></ol>

<ol>
	<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>

作者没有披露潜在的利益冲突。

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 ...

molecular-profiling-invasive-tumor-microenvironment-3-dimensional

Research

JoVE Journal

Medicine

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

11.0K 次观看.  University of Southampton School of Medicine. 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。 

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

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 ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

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 studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

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 ...

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

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat 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

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 cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

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

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

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

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

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

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

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

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

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

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

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 ...