资助这项研究是由英国癌症研究中心。

1。逆转录病毒荧光蛋白标记的细胞<ol><li>板逆转录病毒包装细胞（ 如凤凰卫视）出0.25 × 10 6细胞，一个6孔，每孔 菜在10％胎牛血清（FBS）/ DMEM培养液。 </li><li>使用逆转录病毒DNA转染细胞48小时后细胞Effectene根据制造商的指示。 </li><li>培养24小时冲洗井两次后，再加入1.5毫升10％FBS /每口井的DMEM。 </li><li>收集包装病毒在组织培养中等48小时后吹打，并转移至2 mL离心管。 </li><li>在每分钟1600转离心5分钟沉淀的任何细胞。 </li><li>删除上清到一个干净的试管和存储于-80 ° C。 </li><li>细胞retrovirally转镀在1.5 × 10 5细胞，每孔6井的菜，在培养箱放置在37℃过夜。 </li><li>第二天，取出细胞和广告媒体D 1毫升辅以4μL聚凝胺（2毫克/毫升），每孔的病毒存货。在孵化器板更换。 </li><li> 5-6小时的潜伏期后，加入2 ml 10％胎牛血清/ DMEM培养液，每孔和板块都在37 ° C inbubator一夜之间更换。 </li><li>第二天，更换介质，并让细胞前24小时，加入适当的选择性培养基。 </li><li>当融合（这取决于细胞的生长率，逆转录病毒转导效率，但通常需要4-14天），trypsinize作为参考荧光非转导细胞的细胞和排序，收集和池细胞荧光大于非细胞和冻结在-80等分° C的将来使用。 </li></ol> 2。逆基质胶侵袭实验<ol><li>慢慢解冻一个完整的基质胶冰（图1A）（即含生长因子）等分。 </li><li>一旦解冻，淡化基质胶1:1（连同任何其他附加在冰冷的PBS在治疗前的2倍浓度稀释在PBS;图1B）。为了使更容易聚合前的基质胶处理，所有的塑料制品（如提示，管等），应该是冰冷的。 </li><li>插入许多8微米孔径6.5毫米直径的无涂层Transwells成井24孔细胞培养板所需，然后小心地移液100μL稀释基质胶入井，并在37〜30分钟离开° C到巩固（图1C）。 </li><li>在此期间，准备一个或多个荧光标记的细胞悬液，1到4 × 10 5个细胞每毫升取决于细胞系，从每个前处理条件（E.克的siRNA药物治疗）。，在其正常生长中（图1D）。 </li><li>当基质胶已固化，到过滤器（图1E）向上面临的底面反转Transwells和加入100μL的细胞悬液。 </li><li>小心地盖上24孔组织的基础Transwells培养板中，每个细胞悬液（图1F）液滴接触。 </li><li> 4小时孵育板在倒立的状态，让细胞附着（图1G）。 </li><li>这个时候，打开右侧板和每个Transwell小到2 × 1毫升血清培养基（图2A）连续浸渍清洗。 </li><li>留在一个含有任何药物或治疗所需的三分之一（图2B）Transwells。 </li><li>轻轻移液器（E. 25毫微克/毫升的克。表皮生长因子）到100μl10％FBS / DMEM培养液加趋化的凝固的基质胶/ PBS混合物顶部Transwell小，更换3-5天盖和孵化，在37 ° C的5％的CO 2（图2C）。 </li></ol> 3。染色和可视化<ol><li>细胞表达荧光蛋白可以跳过下面的步骤之前，共聚焦显微镜</li><li>对图像的非荧光的细胞入侵24以及新的菜肴和加入1毫升4微米基质胶，地方Transwells钙黄绿素AM每个基质胶插头上的污渍的解决方案，允许它波及两侧和污点从顶部和底部。钙黄绿素AM（钙黄绿素acetoxymethyl酯）是一个活细胞染料，绿色，污渍整个细胞，无需固定。 </li><li>孵育1小时，在37℃，5％CO2饱和湿度，此时细胞完全染色，共聚焦显微镜成像。 </li><li>另外，可能是固定的，非荧光细胞侵入基质胶染色描述在3.5以下3.9。 </li><li>每个Transwell小转移到一个新的24孔板。覆盖1 4％para-formaldehyde/0.2％的Triton X - 100毫升。 </li><li>室温孵育0.5小时。 </li><li>取出固定液，用1 ml PBS洗3次。 </li><li>删除30分钟用100微克/毫升核糖核酸的核糖核酸酶处理的细胞质RNA。用PBS洗2次。 </li><li>用于可视化添加0.01 mg / ml的碘化丙啶（PI）的PBS稀释，并在室温下离开中DARK为0.5小时。用PBS洗3次。在这个阶段，PI染色Transwell小，可在保持室温在黑暗中至少有1个月。 </li><li>共聚焦显微镜的精确成像方法取决于可用资源。使用与残余PBS的少量非浸泡20 ×目标和捕捉光的部分从底部的基质胶插头每10-15微米（确保无气泡存在）到大盖玻片倒置共聚焦显微镜，Transwell小的地方。个人光片可用于量化入侵的程度（图3A），或建立相应的软件使用，如Volocity（图3B）到3维对象。 </li></ol> 4。代表性的成果如图3A所示的光学切片Z系列的一个例子。在这种情况下，细胞与钙黄绿素上午染色和细胞入侵的过滤器的数量可以看出，随着距离的减少。量化在vasion可以通过分析钙黄绿素的比例，我在每个时间间隔，或使用上面详细的固定/染色法和计数在每个位置PI阳性细胞核的负面像素的积极像素。钙黄绿素的优势之一AM染色细胞浸润，三维重建，可装配使用，如Volocity软件，给人一种视觉描绘的入侵模式（图3B）。如果细胞是由荧光蛋白的表达标记，然后每种颜色的单元格的位置可以进行可视化三维重建，无论是从侧面（图3C），或通过重建（图3D）片观看。 <img alt="图1" src="/files/ftp_upload/3525/3525fig1.jpg" /> 图1。设立反入侵检测所涉及的步骤示意图。一）基质胶流脑在冰上解冻。 B）基质胶含有任何药物治疗，终浓度的两倍，用PBS稀释为1:1。三）<html> Transwell小插入多孔板放入，基质胶吸管到每个。 D）细胞悬液制成所需浓度。 E）的基质胶一旦成立，该板块倒置和删除，细胞被镀上Transwell小插入底部过滤器。 F）在倒转位置，中空板是经过精心放置在Transwell小插入，使细胞悬液接触。 G）的细胞是可以坚持4个小时的过滤器。 <img alt="图2" src="/files/ftp_upload/3525/3525fig2.jpg" /> 图2。继续反入侵检测的原理图。 a）一旦细胞坚持到无血清培养基，浸每个Transwell小两次，以去除松散的细胞。 b）将洗到最后的媒体以及含有加治疗Transwell小。 c）媒体趋化因子（如10％胎牛血清）的治疗需要时，仔细分层到基质胶。 耳鼻喉科“&gt; <img alt="图3" src="/files/ftp_upload/3525/3525fig3.jpg" /> 图3。逆侵袭实验结果的代表性的图像。一）与钙黄绿素染色细胞的光学部分AM入侵基质胶，共聚焦显微镜在15微米间隔。二）重建三维重建一叠焦Z系列图像，从侧面看一个细胞浸润。转载自参考文献10。三）GFP和RFP标记的入侵，从侧面观看的基质胶细胞的三维重建。 D）光片通过GFP和RFP标记的细胞三维重建。转载自参考文献10。

<ol>
	<li>Olson, M. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

作者什么都没有透露。

基质胶侵袭实验历来设立细胞到细胞外基质蛋白与趋化因子诱导的蠕动层对通过底部的过滤器放在。侵袭得分多少细胞可以在过滤器的底面计算的函数。虽然实际上有以上所述的“逆”入侵检测的差别不大，有相当多的入侵过程中，可以通过可视化入侵的细胞，因为他们通过基质胶和确定的信息。例如，除了能够看到不同的标记细胞研究领导与集体入侵细胞（图3C和3D），使用这种方法使细胞固定...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> DMEM（贝科改良Eagle培养基） </td><td> GIBCO公司 </td><td> 21969 </td></tr><tr><td>胎牛血清</td><td> 临时机场管理局 </td><td> A15 - 101 </td></tr><tr><td>青霉素链霉素</td><td> GIBCO公司 </td><td> 15140 </td></tr><tr><td> 200毫米L -谷氨酰胺（100X） </td><td> GIBCO公司 </td><td> 25050-032 </td></tr><tr><td>嘌呤霉素</td><td> Sigma - Aldrich公司 </td><td> P8833 </td></tr><tr><td> 0.05％胰蛋白酶EDTA </td><td> GIBCO公司 </td><td> 25300 </td></tr><tr><td>凝聚胺</td><td> Sigma - Aldrich公司 </td><td> AL - 118 </td></tr><tr><td>脂质体2000试剂</td><td> Invitrogen公司 </td><td> 11668019 </td></tr><tr><td> 6.5毫米Transwells，8.0微米孔径</td><td> 康宁 </td><td> 3422 </td></tr><tr><td>完整的基质胶</td><td> BD公司 </td><td> 354234 </td></tr> &lt;TR&gt; <td>钙黄绿素AM </td><td> Invitrogen公司 </td><td> C1430 </td></tr><tr><td>核糖核酸酶</td><td> Qiagen公司 </td><td> 19101 </td></tr><tr><td>碘化丙啶</td><td> Sigma - Aldrich公司 </td><td> P4864 </td></tr><tr><td>共焦microcope </td><td> 徕卡 </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

一个定义特征是癌症的恶性肿瘤侵袭和转移1。在某些癌症（E. 克 。胶质瘤2），局部浸润到周围的健康组织的疾病和死亡的根源。其他癌症（E. 克 。乳腺癌，肺癌等 ），它是在转移过程中，肿瘤细胞从原发肿瘤的质量，殖民远端站点，并最终有助于器官功能衰竭，最终导致发病率和死亡率3。据估计，侵袭和转移是负责90％的癌症死亡 4 。因此，有浓厚的兴趣，提高诊断和治疗5的目的确定的分子过程，侵袭和转移的关键蛋白质调解员。 癌症科学家面临的一个挑战是要发展入侵检测， 充分类似于在体内的情况启用准确的疾病建模 6 。二维细胞活力检测信息约一个入侵方面，并没有考虑到帐户外基质（ECM）蛋白的重塑，这也是一个关键因素。最近，研究完善我们了解肿瘤细胞的侵袭，并透露，单个细胞的移动拉长或圆形模式 7 。此外，有更大的集体入侵的贡献表示赞赏，特别是在高度分化的肿瘤，维持上皮细胞的特点，使细胞股，床单和集群入侵 ，8癌症的蔓延。 我们目前的候选蛋白的研究贡献的集体入侵10改进后的方法9。特别是在单独池工程细胞表达不同的荧光蛋白，它是分子剖析的活动和proteiNS需要在领导与那些需要在以下细胞的细胞。使用的RNAi实验拆卸单个细胞的侵袭，以及在不同岗位的集体入侵所涉及的过程提供了分子工具。在此过程中，荧光标记的细胞的混合物镀以前与基质胶流脑蛋白填补了Transwell小插入底部，然后让其通过过滤器进入基质胶入侵的“向上”。 Z系列的形象栈，通过共聚焦成像到三维表示，重建允许集体入侵链分析和荧光标记的细胞，在领导与以下职位的任职可视化。

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

成三维的细胞外基质肿瘤细胞的侵袭模型更好地反映<em&gt;在体内</em&gt;比二维的蠕动检测情况。可使用矩阵入侵检测结合共焦成像与荧光标记的细胞，对领导与下面的细胞入侵模式和独特的贡献的详细信息。

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 ...

modeling-and-imaging-3-dimensional-collective-cell-invasion

Research

JoVE Journal

Medicine

16.6K 次观看.  University of Strathclyde. 成三维的细胞外基质肿瘤细胞的侵袭模型更好地反映在体内比二维的蠕动检测情况。可使用矩阵入侵检测结合共焦成像与荧光标记的细胞，对领导与下面的细胞入侵模式和独特的贡献的详细信息。

视频: Modeling and Imaging 3-Dimensional Collective Cell Invasion

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

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

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

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

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

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 ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...