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1.准备盖玻片的分配数量<ol><li>清洁12毫米盖玻片低棉绒抹布。 </li><li>火焰12毫米盖玻片和14毫米盖玻片在35个毫米的玻璃底菜的微孔，通过使用镊子本生灯火焰传递它们。 </li><li>对待微孔用200μl的0.1N NaOH中进行5分钟，在室温下进行。 </li><li>吸液和空气干燥的微孔30分钟。 </li><li>对待微孔用50-100微升3-氨基丙基三甲为10分钟，在室温下于通风柜。这种化学反应，用胶;因此，使用玻璃吸管，不填微孔完全避免与菜的塑料接触。 </li><li>洗用超纯水微孔约10分钟，直到3-氨基丙基三甲变得清晰。 </li><li>冲洗用的超纯水两次用挤压瓶的微孔。 </li><li>用2毫升的超纯水洗涤微孔吨室温在摇杆定在一个中等速度（约1赫兹）10分钟。 </li><li>吸液和空气干燥的微孔30分钟。 </li><li>对待微孔用2ml 0.5％戊二醛溶液在室温下于一摇臂设置在中等速度（约1赫兹）30分钟。 </li><li>在室温下于一摇臂设置以中速（约1赫兹）10分钟洗涤用2毫升的超纯水的微孔。重复2次以上，总共30分钟。 </li><li>干微孔在一个陡峭的角度（60°或更大）30分钟。 注：盘可以存储2个月在干燥器。 </li></ol> 2.准备的PaaS的侵袭伪足测定<ol><li>为软的PAA（8％丙烯酰胺和0.05％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，25微升的2％的BIS，以及574微升的超纯水（ 表1）。 </li><li>对于硬的PAA（8％丙烯酰胺和0.35％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，175＃的181;升2％的BIS，和409微升的超纯水（ 表1）。 </li><li>为刚性的PAA（12％丙烯酰胺和0.60％BIS）的1毫升溶液，结合300微升40％的丙烯酰胺，300微升2％的BIS，以及169微升的超纯水（ 表1）。 </li><li>脱气15分钟的解决方案。在超纯水中添加200，215，和230微升的1毫克/毫升人血浆纤连蛋白的软，硬，和刚性的PAA溶液，分别。所有的溶液，加入1微升10毫克/毫升丙烯酸N-羟基琥珀酰亚胺（NHS）酯，5微升100毫克/毫升的过硫酸铵，和2微升的N，N，N&#39;，N&#39;-四甲基乙二胺（TEMED ; 表1）。混合轻轻吹打和避免解决方案产生气泡。 </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> PAA （1毫升）中</td><td> 40％ AA （微升） </td><td> 2％ （微升） </td><td>超纯 水 （微升） </td><td> 1mg / ml的 FN （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 10毫克/毫升 NHS酯 （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 100毫克/毫升 APS （微升） </td><td> TEMED （微升） </td><td>弹 系数 （PA） </td></tr><tr><td>软</td><td> 200 </td><td> 25 </td><td> 574 </td><td> 200 </td><td> 1 </td><td>五</td><td> 2 </td><td> 1023 </td></tr><tr><td>硬</td><td> 200 </td><td> 175 </td><td> 409 </td><td> 215 </td><td> 1 </td><td>五</td><td> 2 </td><td> 7307 </td></tr><tr><td>严格</td><td> 300 </td><td> 300 </td><td> 169 </td><td> 230 </td><td> 1 </td><td>五</td><td> 2 </td><td> 22692 </td></tr></tbody></table> 表1：组分VOLUMES以及由此产生的弹性模量在侵袭伪足实验中使用的软，硬，和严格的分配数量。AA =丙烯酰胺，FN =纤连蛋白，APS =过硫酸铵。 <ol start="5"><li>吸取到每个微孔的中心8.48微升的PAA解决方案。 注意：此卷理论上产生的75微米厚的PAA。 </li><li>轻轻放下上使用镊子微孔液滴的12毫米盖玻片火烧面。 </li><li>允许夹持的PAA溶液聚合15-30分钟。通过检查剩余的解决方案验证聚合。 </li><li>加2ml的1×PBS中，以每个培养皿用镊子取出12毫米盖玻片。 注：10倍PBS中是在实验室和0.011 M KH 2 PO 4，1.54 M氯化钠，和0.056，1M的Na 2 HPO 4组成。 </li><li>用2ml的1×PBS在室温下洗涤​​微孔5分钟。重复2次以上，总共15分钟。 </li></ol> 3. P部分分配的牵引力化验赔偿<ol><li>为软的PAA（8％丙烯酰胺和0.05％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，25微升的2％的BIS，以及566微升的超纯水（ 见表2）。 </li><li>对于硬的PAA（8％丙烯酰胺和0.35％BIS）的1毫升溶液，结合加入200μl40％的丙烯酰胺，175微升2％的BIS，以及401微升的超纯水（ 见表2）。 </li><li>为刚性的PAA（12％丙烯酰胺和0.60％BIS）的1毫升溶液，结合300微升40％的丙烯酰胺，300微升2％的BIS，以及161微升的超纯水（ 见表2）。 </li><li>脱气15分钟的解决方案。添加200，215，和230微升的1毫克/毫升人血浆纤连蛋白的软，硬，和刚性的PAA溶液，分别。声处理8微升200nm的红色荧光微球（的六百零五分之五百八十零nm激发/发射）下30秒。对于每种溶液，加入8微升200nm的红色荧光的微球，1微升10毫克/毫升丙烯酸NHS酯，5微升100毫克/毫升的过硫酸铵，及2微升TEMED（ 见表2）。混合轻轻吹打和避免解决方案产生气泡。 </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td style="width:40px"> PAA （1毫升）中</td><td> 40％ AA （微升） </td><td> 2％ BIS （微升） </td><td style="padding-left: 4px; padding-right: 4px;">超纯 水 （微升） </td><td> 1mg / ml的 FN （微升） </td><td> 微球 （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 10毫克/毫升 NHS酯 （微升） </td><td style="padding-left: 4px; padding-right: 4px;"> 100毫克/毫升 APS （微升） </td><td> TEMED （微升） </td><td>弹 系数 （PA） </td></tr><tr><td>软</td><td> 200 </td><td> 25 </td><td> 566 </td><td> 200 </td><td> 8 </td><td> 1 </td><td>五</td><td> 2 </td><td> 1023 </td></tr><tr><td>硬</td><td> 200 </td><td> 175 </td><td> 401 </td><td> 215 </td><td> 8 </td><td> 1 </td><td>五</td><td> 2 </td><td> 7307 </td></tr><tr><td>严格</td><td> 300 </td><td> 300 </td><td> 161 </td><td> 230 </td><td> 8 </td><td> 1 </td><td>五</td><td> 2 </td><td> 22692 </td></tr></tbody></table> 表2：成分量和产生的弹性模量在牵引力试验使用的软，硬，和僵化的分配数量 AA =丙烯酰胺，FN =纤连蛋白，APS =过硫酸铵。 <ol start="5"><li>吸取到每个微孔的中心8.48微升的PAA解决方案。 注意：此卷理论上产生的75微米厚的PAA。 </li><li>轻轻放下日的火烧面Ë12毫米盖玻片上使用镊子微孔液滴。 </li><li>允许夹持的PAA溶液聚合15-30分钟。通过检查剩余的解决方案验证聚合。 </li><li>加2ml的1×PBS中，以每个培养皿用镊子取出12毫米盖玻片。 </li><li>用2ml的1×PBS在室温下洗涤​​微孔5分钟。重复2次以上，总共15分钟。 </li></ol> 4.准备ECM的侵袭伪足测定<ol><li>加热该明胶溶液（1％明胶和1％的蔗糖），以37℃。 </li><li>从微孔的底部保持在玻璃底培养皿以45°角时，治疗用150微升的明胶溶液的部分分配为1分钟，然后小心地吸出。 </li><li>干明胶对部分分配剩余的薄层中的微孔在一个陡峭的角度（60°或更大），以优化干燥60分钟。 </li><li>治疗用2毫升冷却的0.5％戊二醛溶液微孔i上CE为15分钟，随后室温下搅拌30分钟。 </li><li>洗用2毫升的1×PBS的微孔5分钟。重复2次以上，总共15分钟。 </li><li>对待微孔用2ml的1分钟加入硼氢化钠溶液中。轻轻拍打板凳上的菜肴以去除形成明胶的表面上的泡沫。 </li><li>洗板微孔用2毫升1×PBS中5分钟。重复2次以上，总共15分钟。 </li><li>稀释FITC标记的人血浆纤连蛋白溶液用一个1×PBS中50μg/ ml的和离心175000×g离心在4℃下进行15分钟。无论是购买市售的标记纤维连接蛋白或如下标注为： <ol><li>溶解5毫克纤连蛋白在透析管10毫升硼酸盐缓冲液（170毫偏硼酸钠四水合物和40 mM氯化钠）和地点。 </li><li>通过将在200毫升含有6毫克溶解FITC的硼酸盐缓冲液的管在室温下在磁力搅拌器上标记的纤连蛋白设定在1.5小时在黑暗中的最低设置。 </li><li>用500毫升的1×PBS的磁力搅拌器上在黑暗的冷室（4℃）用2体积设定在最低设置3天透析改变了一天。 </li><li> （1：200）的基础上的稀释的等分试样的光密度计算的FITC-标记的纤连蛋白浓度在280纳米和493纳米[OD 280 - （0.36 X OD 493）] / 1.4倍的200次2稀释因子（占为甘油浓缩纤连蛋白中的下一个步骤）所得的以mg / ml单位。 </li><li>在磁力搅拌器上过夜在黑暗的冷室，在4℃下设置在最低设置为50％的甘油溶液透析过夜。） </li></ol></li><li>治疗与150微升，在室温下的FITC标记纤维连接蛋白溶液在黑暗中60分钟的微孔。 </li><li>保持在玻璃底培养皿在时小心吸从微孔底部的FITC-标记的纤连蛋白溶液一个45°角，然后填以70％的乙醇溶液中的玻璃底培养皿在室温下放置10分钟，在黑暗的细胞培养罩进行杀菌。还填充玻璃底培养皿盖或浸泡在70％的乙醇的低掉毛抹布擦拭干净。 </li><li>与1X PBS填补他们5分钟洗玻璃底菜。重复两次用2ml的1×PBS的总共15分钟。 </li></ol> 5.准备侵袭伪足化验和影像<ol><li>添加25000癌细胞在2ml侵袭伪足介质（1：1的DMEM和RPMI 1640，用5％的低蛋白质血清，10％胎牛血清，并新鲜加入表皮生长因子20纳克/毫升），以在玻璃底培养皿和孵育过夜。 </li><li>抽吸和治疗用2ml在室温下在黑暗中的3.7％多聚甲醛溶液的微孔20分钟以固定细胞。 </li><li>后两个快速洗涤用2ml的1×PBS中，治疗的微孔用2ml的0.1％的Triton X-100溶液，在室温坦佩叉涂抹在黑暗中5分钟，以透化细胞。 </li><li>后1快洗用2ml的1×PBS中，治疗用3％牛血清白蛋白阻断在室温下溶液在黑暗中进行60分钟，以阻断细胞的微孔。 </li><li>孵育150微升小鼠抗皮层蛋白抗体（1：750）的微孔阻塞，在室温下在黑暗中的溶液60分钟。 </li><li>用2ml的1×PBS中5分钟洗微孔。重复2次以上，总共15分钟。 </li><li>孵育微孔用150微升的山羊抗小鼠633抗体（1：500）和546鬼笔环肽（1：750），在封闭，在室温下在黑暗中的溶液60分钟。 </li><li>用2ml的1×PBS中5分钟洗微孔。重复2次以上，总共15分钟。 </li><li>加入6滴封固剂来填充每个微孔，盖上22×22盖玻片。 </li><li>图像皮层蛋白和肌动蛋白使用647分之632nm激发和发射滤光片，以确定侵袭伪足和五百七十〇分之五百五十六纳米，分别使用高NA，40X油浸镜头上的宽视场倒置显微镜。同样地，图像纤连蛋白使用的五百一十八分之四百九十二纳米的激发和发射滤光器识别ECM降解。 </li></ol> 6.制备及牵引力化验成像<ol><li>在2毫升侵袭伪足中添加15000癌细胞玻璃底菜，并培育过夜。 </li><li>用2ml的L-15培养基中，在玻璃底培养皿抽吸培养基并更换，具有相同的补充剂（5％低蛋白血清，10％胎牛血清，并新鲜加入表皮生长因子20纳克/毫升），预-warmed至37℃。 </li><li>孵育在倒置显微镜预平衡至37℃的高湿度下1小时，在玻璃底培养皿中的环境室中。 </li><li>就拿四套使用高NA，40X镜头上的宽视场倒置显微镜自动化阶段，以纪念蜂窝位置的图像： <ol><li>对图像细胞P使用相衬（“阶段”的形象）将PAAS的。 </li><li>使用六百四十五分之五百六十〇nm激发和发射滤光片由下将细胞略微聚焦直到微球在部分分配的顶部的第一层图像的荧光微球进入对焦（“紧张”的图像）。 </li><li>此外，集中于部分分配（使用微球体作为标记）的底部，并拍摄图像（“底部”的图像）。注意：此图像的目的是记录的z位置，如代表结果说明得到的实际的PAA厚度在每个位置上用于牵引力的计算。 </li><li>之后，这些图像已被收购的多个位置，轻轻在220微升的10％的Triton-X解决方案，混合，除去细胞。在部分分配的顶部在每个位置上如先前所述（“空”的图像）的图像的微球。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tense+situation:+forcing+tumour+progression.">A tense situation: forcing tumour progression.</a> Nature reviews. Cancer. 9, 108-122 (2009).</li><li>Zaman, M. 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=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 10889-10894 (2006).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+density-induced+mechanoregulation+of+breast+cell+phenotype,+signaling+and+gene+expression+through+a+FAK-ERK+linkage.">Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.</a> Oncogene. 28, 4326-4343 (2009).</li><li>Munevar, S., Wang, Y., Dembo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+of+migrating+normal+and+H-ras+transformed+3T3+fibroblasts.">Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.</a> Biophysical journal. 80, 1744-1757 (2001).</li><li>Rosel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+Rho/ROCK+signaling+in+sarcoma+cells+drives+invasion+and+increased+generation+of+protrusive+forces.">Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.</a> Mol Cancer Res. 6, 1410-1420 (2008).</li><li>Indra, I., 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=An+in+vitro+correlation+of+mechanical+forces+and+metastatic+capacity.">An in vitro correlation of mechanical forces and metastatic capacity.</a> Phys Biol. 8, 015015 (2011).</li><li>Parekh, A., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+invasiveness+by+the+physical+extracellular+matrix+environment.">Regulation of cancer invasiveness by the physical extracellular matrix environment.</a> Cell adhesion &amp; migration. 3, 288-292 (2009).</li><li>Weaver, A. M., Page, J. M., Guelcher, S. A., Parekh, A., Coutts, A. 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> Methods in Molecular Biology in Adhesion Protein Protocols. 1046, 171-189 (2013).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples).">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples).</a> Physics in medicine and biology. 52, 1565-1576 (2007).</li><li>Wang, J. H., Lin, 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=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

这项工作是由美国国立卫生研究院在奖号K25CA143412美国国家癌症研究所（帕雷克）的支持。我们要感谢额外的支持是由耳鼻咽喉科提供。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

我们提出的用于制造部分分配，可以用来作为基础侵袭伪足和牵引力测定法来关联侵入性和收缩的细胞行为的方法。虽然部分分配额早已看惯了对细胞的刚性作用，并计算牵引力18,24,27，该协议是第一个开发基于分配数量与同一刚性平行检测，以响应矩阵相关侵入性和收缩的细胞行为机械性能。正确激活的玻璃底菜盖玻片确保分配数量将绑定到他们，不会脱落。虽然我们和其他人已经依靠...

肿瘤相关的ECM的刚性已被确定为在通过增加肌动球蛋白收缩1-3找到恶性行为一显著因素。虽然这种效应已主要被证明与乳腺癌细胞，基质刚性已经发现改变从多种癌症4-8表明肿瘤刚度可以以其它类型的癌症中发挥作用的来源的细胞侵入性。渗透侵入迁移过程中，交联组织，癌细胞利用肌动蛋白丰富的被称为侵袭伪足的本地化蛋白酶来灶性降低ECM 9粘合突起。侵袭伪足被认为是侵入细胞的标志，并有牵连的肿瘤细胞浸润和转移10,11。以前的工作已经表明，基质刚度可以调节侵袭伪足数目和通过肌球蛋白II活性和机械敏感蛋白12相关的ECM降解4,12。鉴于肿瘤密度和癌症侵袭性13,14之间的相关性，这些结果表明通过该癌细胞可能响应刚性肿瘤组织通过肌动球蛋白收缩驱动侵袭和转移的机制。 在体外 ECM刚性和体内组织密度已显示调节癌细胞1,15-17侵袭行为。而肌动球蛋白收缩似乎是重要的在此过程中，目前的研究冲突，以转移能力是否相关，增加或减少收缩力6,18-20。此外，目前还不清楚是否这些力量直接介导侵袭伪足的活动21。我们最近发现，肿瘤细胞收缩力是依赖于矩阵的刚度和被预测ECM降解受侵袭伪足5。这些结果表明，蜂窝力可能通过介导侵袭伪足的交流在癌症发展中起重要作用tivity响应于肿瘤微环境的机械性能。 为了关联癌细胞5侵袭和收缩性能，我们修改了协议，用于创建分配数量与以前用于调查的刚性依赖的侵袭伪足的活动4,12,22不同的僵化。通过化学整个部分分配交联的人血浆纤连蛋白，这些修改后的水凝胶可以用作基础为侵袭伪足和牵引力试验，以确保细胞经历了同样的刚性在两个实验5。在侵袭伪足测定中，纤连蛋白提供了天然的结合域为明胶以重叠的ECM链接到部分分配给检测基质降解。在牵引力测定中，纤连蛋白提供的配体的直接的细胞粘连，以检测用来计算蜂窝牵引力微球的位移。这种方法会导致我们所谓的软，硬，和僵化的分配数量绑定到玻璃底菜，并有弹性模量，E，1023，7307和22692帕5的跨度报道了正常和癌变组织23机械性能的范围内。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-Aminopropyltrimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic acid NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>A8060</td>
			<td>prepare fresh in fume hood 10 mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Life Technologies</td>
			<td>A22283</td>
			<td>can also use rhodamine</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>prepare fresh 10% solution in 1X PBS</td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>18606</td>
			<td>use six drops to fill microwells</td>
		</tr>
		<tr>
			<td>BIS (2%)</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td>make 3% for blocking solution in 1X PBS and store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9777</td>
			<td>pre-equilibrate in borate buffer for 15-30 min</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>use to make acrylic acid NHS ester solution</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PHARMCO-AAPER</td>
			<td>E200</td>
			<td>dilute with ultrapure water to 70%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30070.03</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Polysciences</td>
			<td>00639</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (35 mm coverslips)</td>
			<td>MatTek</td>
			<td>P35G-0-14-C</td>
			<td>coverslips are uncoated</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%)</td>
			<td>Polysciences</td>
			<td>01909</td>
			<td>dilute with 1X PBS to 0.5%</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa Fluor 633 antibody&#160;</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Life Technologies</td>
			<td>33016-015</td>
			<td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>PX-1565-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>mouse anti-cortactin 4F11 antibody&#160;</td>
			<td>EMD Millipore</td>
			<td>05-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>SX-0720-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>use to make 10X PBS stock and borate buffer</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>dilute with ultrapure water to 0.1 N</td>
		</tr>
		<tr>
			<td>Nu-Serum (low-protein serum)</td>
			<td>BD Biosciences</td>
			<td>355500</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros</td>
			<td>416785000</td>
			<td>typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td>
		</tr>
		<tr>
			<td>PBS (sterile)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>use for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Cellgro</td>
			<td>10-040-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>prepare fresh in fume hood 1 mg/ml in 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>sodium metaborate tetrahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0251</td>
			<td>use to make borate buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td>make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining</td>
		</tr>
	</tbody>
</table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

刚性肿瘤组织中一直强烈牵连在调节癌细胞的迁移和侵袭。通过交联组织侵袭性迁移是由肌动蛋白富突起称为侵袭伪足该蛋白水解降解细胞外基质（ECM）提供便利。侵袭伪足的活动已被证明是依赖于ECM的刚性和癌细胞收缩力表明刚性信号可以通过肌动球蛋白收缩调节这些亚细胞结构。癌细胞的侵袭性和收缩性能可在体外使用基于不同的刚性聚丙烯酰胺凝胶（分配数量）侵袭伪足和牵引力试验相关。癌细胞的侵袭性和收缩性能可在体外使用基于不同的刚性聚丙烯酰胺凝胶（分配数量）侵袭伪足和牵引力试验相关。而在两个测定之间一些变型存在，这里介绍的协议提供了用于第创建部分分配的方法在可以在两个测定中使用，并且很容易适应用户的特定生物和技术的需求。

在侵袭伪足测定中，侵袭伪足通常标识标记像肌动蛋白和皮层蛋白在细胞体中的点状结构（ 图1）的共定位。既积极降解和非恶化侵袭伪足可以计数和由这些结构是否共定位与缺乏的FITC标记的纤连蛋白（ 图1）的荧光信号的黑色区域是有区别的。侵袭伪足手动计数，而每个单元ECM降解由细胞的轮廓内手动阈值处理这些黑色区域来确定。 在牵引力测定?...

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Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

在肿瘤微机械刚性起着通过增加侵袭伪足活性和肌动球蛋白收缩驱动恶性行为了至关重要的作用。用聚丙烯酰胺凝胶（部分分配），侵袭伪足和牵引力测定可以被用于研究癌细胞的侵入性和收缩特性响应于矩阵刚性。

聚丙烯酰胺凝胶侵袭伪足和牵引力化验的肿瘤细胞

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

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JoVE Journal

Medicine

12.0K 次观看.  Vanderbilt University Medical Center. 在肿瘤微机械刚性起着通过增加侵袭伪足活性和肌动球蛋白收缩驱动恶性行为了至关重要的作用。用聚丙烯酰胺凝胶（部分分配），侵袭伪足和牵引力测定可以被用于研究癌细胞的侵入性和收缩特性响应于矩阵刚性。 

视频: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

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

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

研究胰腺癌干细胞特性开发新的治疗策略

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

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...