我们感谢肯岩田（OSI制药，纽约，美国）试剂和弗雷德米勒（芭芭拉安Karmanos癌症Intitute，底特律，美国）细胞株。

1。 METHOCEL解决方案的准备（500毫升） <ol><li>高压灭菌6克甲基纤维素在500毫升瓶含有磁力搅拌器。 </li><li>甲基为20分钟，在250毫升预热的DMEM/F12无血清（60℃）溶解。 </li><li>加入250毫升的DMEM/F12含10％马血清（RT）的。混合过夜（4℃）。 </li><li>清除离心（5000G，2H，4℃）的解决方案。 </li><li>以明确的高粘度的解决方案到一个新的管（约90-95％的原液）。 </li><li>储存在4℃，直到使用C。 </li></ol> 2。球体文化<ol><li>准备了20％METHOCEL溶液（10毫升METHOCEL和40毫升的生长介质）。 </li><li>用PBS洗涤细胞两次，加入0.1％胰蛋​​白酶孵育细胞在37 ° C </li><li>重悬细胞生长介质和细胞计数。 </li><li>准备暂停10 000个细胞每毫升20％METHOCEL。 </li><li>细胞悬液加入96孔圆底小号uspension板（每孔100μL）。 </li><li>第二天，检查中悬浮板的球体形成。球体将后1-3天即可使用。 </li></ol> 3。胶原蛋白的制备<ol><li>在冰上，准备8毫升的胶原蛋白，10X PBS 1毫升，1毫升0.1 N的氢氧化钠和3滴0.1 N的盐酸溶液。检查如果pH值是7.4 pH指示剂条通过发现一些解决方案。如果超出范围，调整pH值。 </li><li>添加瓦解胶原溶液50μL，在96孔板井</li><li>在37 ° C直到胶原蛋白是凝固（90分钟）。 </li><li>准备上冰METHOCEL胶原蛋白配体的解决方案：每个配吸管1胶原蛋白溶液的一部分。新增20纳克每毫升含胶原蛋白转化生长因子-β。 METHOCEL和对不同层次的扩散稀释后，最终的TGF -β浓度将于5毫微克/毫升涡旋混合。加入1 METHOCEL解决方案的一部分。混合振荡。 </li></ol> 4。球体嵌入丁<ol><li>将200μl的针尖对针尖切断约5毫米，200μL移液器。吸用移液管的一个球体，并仔细免除到一个空板的介质。观看球体里面的提示保持。球体是一个白点可见。不释放活塞，吸100μL胶原METHOCEL混合和分配到胶原蛋白涂层96的胶原蛋白的混合物球体。 12每个条件的球体。 </li><li>胶原巩固至少30分钟，在37 ° C </li><li>取出200μL提示气泡。 </li><li>添加50μL培养基，血清和1.6％DMSO溶解抑制剂。 SB - 431542添加4μL原液（10毫米），以1毫升中等。抑制剂终浓度扩散后，将于10微米。 TGF -β和抑制剂将停留在实验的过程中。使用40X放大倍率的显微镜下的球体的图片。 </li><li>经过48H与TGF -β和/或抑制剂刺激，使用40X放大倍率的显微镜下的球体的图片。 </li><li>测量术后第2天入侵球体的面积和减去起步区。测量的面积，球体：打开图片在Adobe Photoshop Extended中（图2A）的球体，选择快速选择工具（图2b）。成圆形鼠标指针变为一个“加”号（+）（图2c）。球体的同时拖动光标，Photoshop将承认球体的边界。如果被选中球体以外的领域，这个区域可以被排除在外的选择，按Alt键或通过阴性选择的工具，这是由一个表示（ - ）在工具栏上签署。 （ - ）成圈与“减”的光标改变符号和错误选定区域拖动光标，这一方面是从选择（图2d）。如果有必要，多个区域，可以选择按了SHIFT按钮。为了更准确地工作，鼠标指针的大小可以调整，通过改变工具栏上的画笔直径（图2E）。当整个球体被选中，选择面积计算，通过菜单栏中的的分析测量，或按CTRL + SHIFT + M（图2F）。测量记录“窗口中显示的文件名和以像素为单位的选择方面，以及其他数据（图2G）。如果选择多个测量，第一行显示所有地区的总和。个人选择的测量是在下面的线条。所有的测量数据可以导出到一个文本文件，并在Excel中打开。 </li></ol> 5。代表性的成果： MCF10A1供应量（M1）正常乳腺上皮细胞的球体检测的一个例子，H - RAS转化MCF10AneoT供应量（M2）细胞和M2衍生MCF10CA1a（M4）是在图3中给出。 M1细胞表明，只有疲软的入侵无刺激，但显著入侵后，由TGF -β刺激。相比之下，在RAS转化的货币供应量M1衍生M2的入侵已经没有刺激除了高效，这是进一步提高TGF -β治疗后的4-5倍。 M4的细胞表现出最强的入侵，与TGF -β此外。 下一步，我们检查我们是否可以在这个球体模型使用化学抑制剂与TGF -β-诱导的入侵干扰。为此，我们使用了SB - 431542，ATP模拟和TβRI激酶活性的选择性抑制剂，以及Ib型受体激活素（ActRIB）和激活素样受体激酶（ALK）7月12日。正如所料，SB - 431542有效抑制TGF -β诱导的入侵货币供应量M1，M2和M4的细胞（图4），表明TGF -β诱导的入侵是TβRI激酶依赖。此外，基底入侵的球体也强烈抑制，表明基底入侵也是德对转化生长因子-β下垂。 <img alt="图1" src="/files/ftp_upload/3337/3337fig1.jpg" /> 图1。3D球体检测流程图。首先，细胞非贴壁条件下在U形悬浮板镀金。细胞aggregrate multcellular球体和可嵌入成胶原基质。进入这个胶原基质，它们能侵入。 <img alt="图2" src="/files/ftp_upload/3337/3337fig2.jpg" /> 图2：使用Adobe Photoshop的球体面积的量化扩展。 （一）球体的图片打开ADOBE PHOTOSHOP Extendend（二）选择“快速选择”工具拖动球体光标选择球体（四）去除一个wongly包括使用面积面积（三）负面的快速选择工具（E）快速选择工具的笔刷大小的调整，以便更准确地选择面积（F）的选择测量命令记录（G）的测量记录的例子。 <img alt="图3" src="/files/ftp_upload/3337/3337fig3.jpg" /> 图3。TGF -β诱导自发永生MCF10A1供应量（M1）（一）球体的侵袭，RAS转化MCF10AneoT供应量（M2（B）和转移性衍生MCF10CA1a（M4）（C）货币供应量M1，M2和M4的球状体嵌入胶原5ng/ml的TGF -β处理48h后表示，AC代表图片嵌入一天，两天后，D相对入侵第2天减0天的区域差异的量化结果表示以平均值± SD 13日改编。 <img alt="图4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="图4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> 图4。基底和TGF -β诱导的球体入侵抑制bŸ SB - 431542。货币供应量M1，M2和M4的球状体嵌入到胶原蛋白和5ng/ml TGF -β和/或10微米的SB - 431542作为治疗。术后第2天（一）采取的代表图片。第2天减第0天（二）面积差异，相对的入侵进行了量化。结果表示为平均值± SD 13日改编。

<ol>
	<li>Akhurst, R. J., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling+in+cancer--a+double-edged+sword.">TGF-&#946; signaling in cancer--a double-edged sword.</a> Trends. Cell. Biol. 11, S44-S51 (2001).</li><li>Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+in+Cancer.">TGF-&#946; in Cancer.</a> Cell. 134, 215-215 (2008).</li><li>Ghellal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+TGF-&#946;1+and+TGF-&#946;+in+human+breast+carcinoma.">Prognostic significance of TGF-&#946;1 and TGF-&#946; in human breast carcinoma.</a> Anticancer. Res. 20, 4413-4413 (2000).</li><li>Sheen-Chen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+levels+of+transforming+growth+factor-&#946;1+in+patients+with+breast+cancer.">Serum levels of transforming growth factor-&#946;1 in patients with breast cancer.</a> Arch. Surg. 136, 937-937 (2001).</li><li>ten Dijke, P., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+TGF-&#946;-Smad+signaling.">New insights into TGF-&#946;-Smad signaling.</a> Trends Biochem. Sci. 29, 265-265 (2004).</li><li>Moustakas, A., Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Smad+TGF-&#946;+signals.">Non-Smad TGF-&#946; signals.</a> J. Cell Sci. 118, 3573-3573 (2005).</li><li>Soule, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+spontaneously+immortalized+human+breast+epithelial+cell+line,+MCF-10.">Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.</a> Cancer Res. 50, 6075-6075 (1990).</li><li>Strickland, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+premalignant+MCF10AT+generates+heterogeneous+malignant+variants+with+characteristic+histologic+types+and+immunohistochemical+markers.">Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers.</a> Breast Cancer Res. Treat. 64, 235-235 (2000).</li><li>Santner, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+MCF10CA1+cell+lines+derived+from+premalignant+human+breast+epithelial+MCF10AT+cells.">Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells.</a> Breast Cancer Res. Treat. 65, 101-101 (2001).</li><li>Smalley, K. S., Lioni, M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+isn't+flat:+taking+cancer+biology+to+the+next+dimension.">Life isn't flat: taking cancer biology to the next dimension.</a> In Vitro Cell Dev. Biol. Anim. 42, 242-242 (2006).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+multicellular-mediated+resistance+to+alkylating+agents+in+cancer.">Acquired multicellular-mediated resistance to alkylating agents in cancer.</a> Proc. Natl. Acad. Sci. U. S. A. 90, 3294-3294 (1993).</li><li>Inman, 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=SB-431542+is+a+potent+and+specific+inhibitor+of+transforming+growth+factor-b+superfamily+type+I+activin+receptor-like+kinase+(ALK)+receptors+ALK4,+ALK5,+and+ALK7.">SB-431542 is a potent and specific inhibitor of transforming growth factor-b superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.</a> Mol. Pharmacol. 62, 65-65 (2002).</li><li>Wiercinska, 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+TGF-&#946;/Smad+pathway+induces+breast+cancer+cell+invasion+through+the+up-regulation+of+matrix+metalloproteinase+2+and+9+in+a+spheroid+invasion+model+system.">The TGF-&#946;/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system.</a> Breast Cancer Res. Treat. 128, 657-657 (2011).</li><li>Tang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+switches+from+tumor+suppressor+to+prometastatic+factor+in+a+model+of+breast+cancer+progression.">TGF-&#946; switches from tumor suppressor to prometastatic factor in a model of breast cancer progression.</a> J. Clin. Invest. 112, 1116-1116 (2003).</li><li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC. Med. 6, 11-11 (2008).</li><li>Ramaswamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+signature+of+metastasis+in+primary+solid+tumors.">A molecular signature of metastasis in primary solid tumors.</a> Nat. Genet. 33, 49-49 (2003).</li><li>Debnath, J., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+glandular+epithelial+cancers+in+three-dimensional+cultures.">Modelling glandular epithelial cancers in three-dimensional cultures.</a> Nat. Rev. Cancer. 5, 675-675 (2005).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat. Rev. Mol. Cell. Biol. 8, 839-839 (2007).</li><li>Lee, G. Y. <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, 359-359 (2007).</li><li>Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells.">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 9064-9064 (1992).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> 30, 256-256 (2011).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-Activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-Activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> 10, 314-314 (2011).</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, 821-821 (2010).</li></ol>

没有利益冲突。

我们建立了一个球体的模型，其中MCF10A1细胞和其恶性衍生物成胶原基质中的TGF -β-依赖性入侵。首先，球体形成96井圆底暂停存在甲基板。当然，也U形聚甲基丙烯酸羟乙酯涂层板，可用于这一目的。甲基纤维素是绝对必要的，在这一步，，因为细胞就会死亡中的甲基纤维素的情况下。 TGF -β是直接添加到胶原METHOCEL混合，因为我们发现，细胞的反应较差的TGF -β时，这个因素是添加胶原蛋白的中型?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>甲基纤维素</td><td> Sigma - Aldrich公司</td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td>高级Biomatrix </td><td> 5005 - B </td><td></td></tr><tr><td> pH指示剂条</td><td>默克公司</td><td> 9533 </td><td></td></tr><tr><td> 96，以及悬挂 板</td><td>格雷纳生物一</td><td> 650 185 </td><td></td></tr><tr><td> 96孔粘连训练班 板</td><td>格雷纳生物一</td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF -β有反对乳腺癌进展中的角色，作为一个在初始阶段的抑癌， 但在稍后阶段1,2刺激的侵袭和转移。此外，TGF -β是经常过度表达在乳腺癌中，其表达与预后差， 转移 3,4有关。其中TGF -β诱导的入侵是不能很好地理解的机制。 TGF -β诱发细胞通过TGF -β型II（TβRII）和I型（TβRI）受体反应。当TGF -β诱导的异聚复合物的形成，TβRII磷酸化TβRI。激活TβRI启动Smads的磷酸化受体（R - Smads的），即Smad2的和Smad3的细胞内的规范信号转导通路。这些活化的R - Smads的形式与Smad4的异聚复合物，积聚在细胞核内调节靶基因的转录 5 。除了以前ð旁切Smad通路，在其他几个非Smad信号通路的激活受体激活的结果，例如丝裂原活化蛋白激酶（MAPK）途径6。 要研究的TGF -β在乳腺癌的不同​​阶段中的作用，我们使用的MCF10A电池系统。该系统由自发永生MCF10A1供应量（M1）乳房上皮细胞的H - RAS转化M1衍生MCF10AneoT供应量（M2），在小鼠8癌前病变，生产和M2衍生MCF10CA1a（M4），成立M2的异种移植和形式与能力转移到肺9高年级癌。这MCF10A系列提供了可能性，研究与恶性肿瘤的不同等级是由不同的遗传背景不偏颇细胞的反应。 对于TGF -β诱导的入侵的分析，我们产生的同型MCF10A球体细胞培养EMBEdded在三维胶原基质在体外 （图1）。这种模型非常类似于人类肿瘤在体内建立一个氧气和营养物质的梯度， 造成 10球体内部的外部和静态的，甚至坏死细胞活跃，侵入细胞。球体为基础的检测也已证明，以更好地复述比单层培养 11的耐药性。这MCF10三维模型系统允许我们调查的乳腺细胞的侵入特性，在不同阶段的恶性肿瘤的TGF -β信号的影响。

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

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

描述一个分析，定量分析转化生长因子（TGF）-β诱导的三维胶原凝胶侵袭。这个实验需要的细胞株MCF10A系列，代表不同阶段乳腺癌的发展优势。通过这种方法可以用于其他细胞系，并可能被用来调查其他潜在的激活或入侵抑制剂。

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 ...

spheroid-assay-to-measure-tgf-induced-invasion

Research

JoVE Journal

Medicine

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

21.7K 次观看.  Leiden University Medical Centre. 描述一个分析，定量分析转化生长因子（TGF）-β诱导的三维胶原凝胶侵袭。这个实验需要的细胞株MCF10A系列，代表不同阶段乳腺癌的发展优势。通过这种方法可以用于其他细胞系，并可能被用来调查其他潜在的激活或入侵抑制剂。

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

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

一个实时的基于电阻抗技术测量癌细胞入侵内皮细胞单层

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

科研

医学

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

测量方法轴向和近端肌肉音

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

精度在牙科医学，一种新的方法来测量正确度与精密度

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

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 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

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 ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

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 ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Vivo模型中的小鸡胆膜模型,用于评估头颈部癌症的围神经入侵

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...