Name: a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion
Uploaded: 2015-04-24T14:00:00.000+00:00
Duration: 6 min 36 s
Description: Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1.准备不同的媒体和其他组件<ol><li>事先进行实验，制备介质选自由DMEM或其他指定的介质中，并添加任一正常胎牛血清，木炭剥离的FBS，或木炭剥离的FBS加组件进行测试。需要注意的是多个组件可以在每个实验进行测试。 </li><li>称出并稀释的激素，生长因子，细胞因子或适当地在生理浓度溶解在木炭脱色的血清。 </li></ol> 2.准备胶原基质上的冰<ol><li>制备2毫升胶原I基质中2.2毫克/毫升通过在冰上加入以下无菌过滤组件：加入200μl10倍的PBS（pH为7.4），5.4微升的1N NaOH，600微升双蒸H 2 O为和1.2毫升胶原I （在3.63毫克/毫升）。 </li><li>保持胶原蛋白I溶液在冰上，直到准备板。 </li></ol> 3.准备迁移/侵袭板用于分析&lt;/ P&gt; <ol><li>对于要测试的每个细胞系，使用一种24孔室板，其中12孔含有插入。使用额外的12个孔不含有插入用于将趋化介质和转印刀片为实验的设置。 注：在平板上用聚乙烯对苯二甲酸盐（PET）膜和8微米孔径胶原基质是最适合的细胞系用在这里。然而，在预涂板一个基质胶基质也可以取代的孔径根据细胞系被调查下降。 </li><li>清楚地标注板，采用每条件3口井被分析（FBS迁移，CS-FBS迁移，FBS侵袭，而CS-FBS侵袭以及FBS迁移的控制，非侵入性的细胞系）。通过移动通孔运动中的PET膜，并测定侵袭通过胶原或基质胶基质，然后通过在所述膜孔测定迁移。使用不同的颜色标记为每个小区条件到标识在电镀过程。 </li></ol> 4.分装的入侵矩阵<ol><li>仔细吸取用于入侵检测75微升胶原基质溶液进入插入物。请小心，以避免气泡。通过将一个倒枪头表面驱散气泡。 </li><li>该板用胶原涂覆的刀片转移到37℃和5％CO 2的培养箱中30分钟，使凝胶固化。 </li></ol> 5.板上的细胞上膜或入侵矩阵<ol><li>同时，trypsinize细胞和有10％FBS补充培养基。自旋细胞在200×g离心5分钟，在台式离心机和在无血清培养基冲洗3次。 </li><li>重悬在无血清培养基。计数细胞用血细胞计数器或自动幻灯片计数器。添加无血清培养基以5×10 4个细胞/ ml的终浓度。 </li><li>当胶原基质凝固（30分钟后），加入700μl的媒体无论是2％FBS的定义或活性炭处理的FBS到每个孔中。每盘12个刀片： <ul><li> 3插入有胶原蛋白和井媒体+ 2％FBS </li><li> 3插入有胶原蛋白和井媒体+ 2％CS-FBS </li><li> 3插入没有胶原蛋白和井媒体+ 2％FBS </li><li> 3插入没有胶原蛋白和井媒体+ 2％CS-FBS </li></ul><ol><li>使用取决于被测试的因子的数目和与对应于每个状态的无胶原控制附加平板上。 </li></ol></li><li>添加细胞悬浮液的刀片以5×10 4个细胞/ ml，在所有的迁移和侵袭插入电镀每个刀片500微升的细胞。 </li><li>孵育细胞22小时，在37℃。 </li></ol> 6.量化迁移和侵入细胞的数量<ol><li>使用甲醇固定液，曙红，和苏木，在分开的井孔中的设置染色。 </li><li>用棉签从每孔除去细胞和基质。Rrepeat为每个井第二拭子应用。 </li><li>用钳子，沾每次插入1秒5次到各3解决方案连续。 </li><li>允许插入干O / N。 </li><li>任我）去除过滤器，用手术刀，小心地切割周围的边缘，并安装在盖玻片和浸油的幻灯片，或ii）允许插入干O / N反转，直接使用插入显微镜。 </li><li>第二天，查看幻灯片或插入与20X物镜的显微镜下，并采取5张从过滤器的不同区域。为了提高一致性，需要4外场和一个中心。 </li><li>计数细胞用于使用ImageJ的软件的所有条件，并适用于下面的公式。 </li><li>确定百分入侵如下： 平均＃细胞通过胶原侵入我插入=一 平均＃细胞通过控制插入的迁移= B ％入侵=（A / B）×100 </li><li>确定入侵指数在2％FBS如下： ％侵袭细胞被测定（在2％FBS）= C ％侵袭控制非侵入细胞（2％FBS）= D 浸润指数（FBS）=（C / D） </li><li>确定入侵指数在2％CS-FBS如下： ％侵袭的细胞被检测（在2％CS-FBS）= E ％入侵控制非侵入性的细胞（2％CS-FBS）的= F 入侵指数（CS-FBS）=（E / F） </li></ol> 7.重复实验协议比较活性炭处理FBS为活性炭处理FBS + X n，其中多因素综合<ol><li>如使用不同的组件为“X”或组分的组合需要重复该过程多次。 </li><li>应用计算，以确定各因素“X”的迁移和侵袭影响的贡献。 </li></ol>

<ol>
	<li>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> The Biology of Cancer. , (2013).</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=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (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 (1), 57-70 (2000).</li><li>Valastyan, S., 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=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Dang, Z. C., Lowik, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+serum+factors+by+charcoal+treatment+promotes+adipogenesis+via+a+MAPK-dependent+pathway.">Removal of serum factors by charcoal treatment promotes adipogenesis via a MAPK-dependent pathway.</a> Mol Cell Biochem. 268 (1-2), 159-167 (2005).</li><li>Yohay, D. A., Zhang, J., Thrailkill, K. M., Arthur, J. M., Quarles, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+serum+in+the+developmental+expression+of+alkaline+phosphatase+in+MC3T3-E1+osteoblasts.">Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts.</a> J Cell Physiol. 158 (3), 467-475 (1994).</li><li>Zucker, S. N., Bancroft, T. A., Place, D. E., Des Soye, ., Bagati, B., Berezney, A., R, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dominant+negative+Cx43+mutant+differentially+affects+tumorigenic+and+invasive+properties+in+human+metastatic+melanoma+cells.">A dominant negative Cx43 mutant differentially affects tumorigenic and invasive properties in human metastatic melanoma cells.</a> J Cell Physiol. 228 (4), 853-859 (2013).</li><li>Fox, E. M., Andrade, J., Shupnik, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+actions+of+estrogen+to+promote+proliferation:+integration+of+cytoplasmic+and+nuclear+pathways.">Novel actions of estrogen to promote proliferation: integration of cytoplasmic and nuclear pathways.</a> Steroids. 74 (7), 622-627 (2009).</li><li>Meierjohann, S., 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=MMP13+mediates+cell+cycle+progression+in+melanocytes+and+melanoma+cells:+in+vitro+studies+of+migration+and+proliferation.">MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation.</a> Molecular Cancer. 9, 201 (2010).</li><li>Smalley, K. S., Haass, N. K., Brafford, P. A., Lioni, M., Flaherty, K. T., 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=Multiple+signaling+pathways+must+be+targeted+to+overcome+drug+resistance+in+cell+lines+derived+from+melanoma+metastases.">Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases.</a> Mol Cancer Ther. 5 (5), 1136-1144 (2006).</li><li>Both, N. J., Vermey, M., Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+evaluation+of+various+invasion+assays+testing+colon+carcinoma+cell+lines.">A comparative evaluation of various invasion assays testing colon carcinoma cell lines.</a> Br J Cancer. 81 (6), 934-941 (1999).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+cancer+metastasis:+the+'seed+and+soil'+hypothesis+revisited.">The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited.</a> Nat. Rev. Cancer. 3, 453-458 (2003).</li><li>Huang, Y., 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=Epithelial+to+mesenchymal+transition+in+human+breast+epithelial+cells+transformed+by+17beta-estradiol.">Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol.</a> Cancer Res. 67 (23), 11147-11157 (2007).</li><li>Huang, Y. H., 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=Thyroid+hormone+regulation+of+miR-21+enhances+migration+and+invasion+of+hepatoma.">Thyroid hormone regulation of miR-21 enhances migration and invasion of hepatoma.</a> Cancer Res. 73 (8), 2505-2517 (2013).</li><li>Scheel, C., 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=Cancer+stem+cells+and+epithelial-mesenchymal+transition:+concepts+and+molecular+links.">Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.</a> Semin. Cancer Biol. 22 (5-6), 396-403 (2012).</li><li>Kojima, Y., 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=Autocrine+TGF-beta+and+stromal+cell-derived+factor-1+(SDF-1)+signaling+drives+the+evolution+of+tumor-promoting+mammary+stromal+myofibroblasts.Proc.">Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts.Proc.</a> Natl Acad Sci U S A. 107 (46), 20009-20014 (2010).</li><li>Maehara, Y., 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=Role+of+transforming+growth+factor-&#946;1+in+invasion+and+metastasis+in+gastric+carcinoma.">Role of transforming growth factor-&#946;1 in invasion and metastasis in gastric carcinoma.</a> J. Clin. Oncology. 17 (2), 607-614 (1999).</li><li>Lu, Z., Jiang, G., Blume-Jensen, P., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor-induced+tumor+cell+invasion+and+metastasis+initiated+by+dephosphorylation+and+downregulation+of+focal+adhesion+kinase.">Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase.</a> Mol. Cell Biol. 21 (12), 4016-4031 (2001).</li><li>Korkaya, H., Liu, S., Wicha, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+stem+cells,+cytokine+networks,+and+the+tumor+microenvironment.">Breast Cancer stem cells, cytokine networks, and the tumor microenvironment.</a> J. Clin. Invest. 121 (10), 3804-3809 (2011).</li><li>Rosen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interrogation+of+HGF-SF+with+other+cytokines+in+tumor+invasion+and+metastasis.">The interrogation of HGF-SF with other cytokines in tumor invasion and metastasis.</a> EXS. 65, 301-310 (1993).</li><li>Sanz-Moreno, V., 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=ROCK+and+JAK1+signaling+cooperate+to+control+actomyosin+contractility+in+tumor+cells+and+stroma.">ROCK and JAK1 signaling cooperate to control actomyosin contractility in tumor cells and stroma.</a> Cancer Cell. 20 (2), 229-245 (2011).</li><li>Fleming, J. 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=Paracrine+interactions+between+primary+human+human+fibroblasts+enhance+murine+mammary+gland+humanization+in+vivo.">Paracrine interactions between primary human human fibroblasts enhance murine mammary gland humanization in vivo.</a> Breast Cancer Res. 14 (3), (2012).</li><li>Sikora, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.J.,+V.,+M.E.,+M.D.,+J.M.">M.J., V., M.E., M.D., J.M.</a> Mechanisms of estrogen-independent breast cancer growth driven by low estrogen concentrations are unique versus complete estrogen deprivation.Breast Cancer Res Treat. 134 (3), 1027-1039 (2012).</li></ol>

The authors have nothing to disclose.

肿瘤转移是一个多步骤的过程。细胞必须通过基底膜断裂，intravasate成要么淋巴系统或血液微血管，它们被运到远处位点。肿瘤细胞然后外渗和移成macrometastasis 12。通过上皮间质转化（EMT），肿瘤侵袭和转移的进展已增强了类固醇激素13,14，生长因子15-18和细胞因子19-21。这些分子是如此关键，解决肿瘤进展的信号通路，即化验的发展，以解决他们在肿瘤侵入的潜在...

一种新颖的侵袭实验研制了具有确定具体激素，细胞因子和/或生长因子如癌细胞侵袭化学引诱物的参与的目标。肿瘤细胞侵入通过细胞外基质（ECM）的能力，是在转移表型1-3的一个标志。入侵室已被广泛用作体外的工具来研究癌细胞的侵袭和转移，并可以提供知识， 体内肿瘤的侵袭和转移的机制。 腔室包括嵌套细胞培养板的孔中的圆柱形的细胞培养插入物。插入件的底部是限定孔径的半透聚碳酸酯或聚苯乙烯膜。 在标准侵袭实验，将细胞接种到在插入膜用无血清培养基，并置于细胞培养孔被填充有血清或血清样化学引诱物送等了一个趋化的力量。移动至该力驱动细胞通过的半透膜（迁移）或可替代地，通过细胞外基质（侵入），然后可以使用常规的染料检测和定量细胞数染色的涂层。细胞数，然后根据迁移和侵袭的非侵入性的细胞系的范围内归一化。此方法允许研究者评估在各种条件，包括遗传操作不同类型的细胞的侵袭性潜力。 激素，细胞因子和生长因子是血清的关键组成部分，并越来越多地被示出为与驱动侵袭性表型4相关联。然而，在自然界，角色，和特异性这些趋化血清分子仍然介导入侵仍然无法实现。我们面临的挑战仍然是确定哪些具体因素血清或血清样趋化因子负责驾驶癌细胞侵袭以及什么分子可以抑制入侵过程。在这份报告中，我们描述了一种方法来评估潜在激素，细胞因子和/或血清中存在的生长因子的参与，如分子找到癌细胞的趋化和侵袭。 在此提出的改性协议，木炭剥离用于去除激素，细胞因子和生长因子，由2％胎牛血清（FBS），以产生2％的活性炭脱色的FBS（CS-FBS）。两个2％FBS和2％CS-FBS的被用作试剂来建立趋化力来驱动癌细胞侵袭。用2％CS-FBS的作为相比于正常的2％FBS的趋化剂能提供若干好处，包括第一，最突出的是，以研究在减少/相对缺乏激素，细胞因子和生长因子的癌症侵袭的表型的能力。它还使研究者评估是否集体去除激素，生长因子，以及CYtokines引起侵袭性指数的增加或减少。该测定然后设计，以确定是否增加一个单独的组件，被指定为“X”，可以抑制，减少，增加，或侵入索引恢复到原来的值（参见图2和3）。虽然这种方法是具体用于实现生理水平是从血清中木炭提除去的组件，还应当注意的是，木炭汽提也可以影响信号转导途径。例如，已经报道，木炭剥离能降低骨祖细胞的碱性磷酸酶活性，并通过减少MAPK活化剂5的诱导脂肪生成。木炭剥离介质是可商购，并且基于协议概述，结合木炭与葡聚糖和孵育胎牛血清O / N 6。 该测定是在respo开发NSE到报告的Zucker 等人 7的结果的作者证明朝向媒体与正常的2％FBS的迁移时的突变体的表型影响间隙连接通讯证明增加的侵袭索引。这一增长与活性炭处理血清替代被淘汰。从该结果中，作者得出结论认为，这种突变影响朝向亲脂性分子迁移（更具体地，激素，生长因子，细胞因子或）7。它公知的是激素，生长因子和细胞因子介导的信号参与肿瘤促进和侵入4通路。因此，对于科学调查，以确定哪些因子（S）的驱动的肿瘤侵袭其特定的癌细胞或突变体的研究之中是重要的。该试验的目的是为解决在其生理浓度各组分的作用，因为，根据该成分的浓度之间的差来确定与在正常的FBS和CS-FBS的X“;＃8220。通过此法的发展，研究人员可以更深入地了解具体的侵入途径管理他们的系统。 激素，生长因子，和细胞因子经常被归类为促癌8。其中的一些因素，如EGF被用作化学引诱物中浸润室9直接来源。因此，它很可能是这些代表了血清肿瘤直接侵犯的主要组成部分。该协议提出了一种简单的，但显著，修改现有的体外侵袭测定法，其允许研究者评估在介导癌细胞的侵袭性潜力激素，生长因子和细胞因子的参与。但是，在测定的目的是提供一个答案的研究者以过程是否将是有效的对他们的研究在分析的早期步骤，以免使用宝贵的时间而r物资跟不上，如果认为没有必要。此方法使用的CS-FBS和依赖于研究者酌情决定哪个分子追求铅候选潜在介导癌细胞侵袭。从这些分析的结果应该被证明在确定其中血清组分充当被研究的化学引诱物或抑制剂对特定细胞系或突变体是有用的。此外，这种方法可以帮助研究者确定主要的信号通路或者促进或抑制癌细胞侵袭;从而引导未来的药物设计。

<table><tbody><tr><td>BioCoat Control Inserts with 8.0 &amp;micro;m PET membrane</td><td>Corning</td><td>354578</td><td>12 in each of two 24-well plates. PET = polyethylene teraphthalate</td></tr><tr><td>Collagen I, rat tail</td><td>Corning</td><td>354236</td><td>Source = rat tail tendon, purity = 90%</td></tr><tr><td>Fixative; Diff-Quick</td><td>Thermo Fisher Scientific</td><td>NC9844047</td><td>Methanol-based fixative, hematoxylin/eosin</td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>16000</td><td>Designated as FBS</td></tr><tr><td>Charcoal stripped fetal bovine serum</td><td>Life Technologies</td><td>12676</td><td>Designated as CS-FBS</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific Hyclone</td><td>SH30070</td><td>Designated as FBS</td></tr><tr><td>Charcoal stripped fetal bovine serum</td><td>Thermo Scientific Hyclone</td><td>SH30068</td><td>Designated as CS-FBS</td></tr></tbody></table>

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

血清作为趋化对其中的癌细胞迁移和侵袭，促进他们的血管内进入微血管。然而，实际的分子朝向该细胞迁移仍然无法实现。本变形侵袭实验已经开发，以确定哪一个驱动器细胞迁移和侵袭的目标。这种技术比较下三个条件的侵袭索引，以确定是否一个特定的激素，生长因子，细胞因子或在介导癌细胞的侵袭性潜力的作用。这些条件包括：i）正常牛胎儿血清（FBS），ⅱ）活性炭处理的FBS（CS-FBS）的功能，从而激素，生长因子，以及细胞因子和iii）的CS-FBS +分子（表示为“X”）。相比FBS细胞侵袭与CS-FBS的显著的变化，表明激素，细胞因子和生长因子介导的变化的参与。各个分子然后可以重新添加到CS-FBS以测定他们的重新能力诗句或抢救入侵表型。此外，两个或更多的因素可以结合，以评估该添加剂或多种分子在驾驶或抑制侵袭协同效应。总体而言，该方法能够使研究者确定是否激素，细胞因子，和/或生长因子通过作为化学引诱物或侵入的抑制剂为特定类型的癌细胞的或特定的突变体在细胞侵袭的作用。通过识别特定的趋化因子和抑制剂，这个修改后的入侵检测可能有助于阐明信号通路，直接癌细胞侵袭。

入侵指数是根据归一化，以一种非侵入性的细胞系计算每个条件。对于我们的实验中，我们使用1205LU黑素瘤细胞系，建立了变体的稳定细胞株作为我们的侵入线以及癌前无创变型中，WM793从中1205LU细胞衍生10用作一个逻辑控制。我们还利用I型胶原作为基质侵入，因为这是真皮的主要成分。这是根据先前的研究，从而最优侵袭基质变化的基础上的细胞系和一致性的与体内的程度的结果...

Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

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 ...

a-modified-vitro-invasion-assay-to-determine-potential-role-hormones

Research

JoVE Journal

Medicine

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

10.7K 次观看.  Roswell Park Cancer Institute. 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.

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

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

科研

癌症研究

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.

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 ...

医学

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

遗传学

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

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 ...

癌症生物学中的 3D 模型:异球体中的侵袭动力学

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

作者聚焦:破译头颈癌进展过程中癌症和免疫细胞之间的串扰

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

分析 3D 培养物体外细胞侵袭的替代策略

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

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 ...

一种改进

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此视频中的章节

向上移动: 一个简单而灵活的体外三维入侵检测协议

球体的检测，以测量TGF -β诱导的入侵

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

监测三D培养中的癌细胞入侵和T细胞毒性

在实时的间皮细胞卵巢癌球体附件和入侵评估

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分析 3D 培养物体外细胞侵袭的替代策略

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