JDM得到了INSERM /RégionAquitaine博士研究生的支持，现在得到博士后ARC研究金和西奈山医学院Tisch癌症研究所的支持。 EH得到了Ministèrede l&#39;EnseignementSupérieuret de la Recherche博士的支持。 ZE得到了国立国民大学（ANR）的博士后研究金的支持。 CM由西奈山医学院Tisch癌症研究所支持，JJBC得到NIH / NCI授权K22CA196750，TCI青年科学家癌症研究奖JJR基金和西奈山医学院Tisch癌症研究所的支持。这项工作得到ANR-13-JJC-JSV1-0005的资助。 FS由"Ligue nationale contre le cancer"支持。 VM和FS由"EquipeLabelliséeLigue Nationale contre le Cancer 2016"和Institut National du Cancer，INCA_8036和PLBio2012的资助提供支持。

注意:在固定之前，所有步骤都在无菌层流罩中完成。 二维矩阵注意:2D矩阵用于确定形成invadosomes的细胞的百分比和每个细胞的基质降解面积。 <img alt="图1" src="/files/ftp_upload/54911/54911fig1.jpg" /> 图1:协议。用于制备明胶和胶原蛋白I基质的方案总结。只能制成明胶基质或明胶和胶原蛋白I混合基质。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54911/54911fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 明胶基质 注意:荧光明胶基质用于定量基质降解，非荧光明胶用于促进胶原I纤维的粘附。荧光明胶垫嵴也用于荚膜细胞和侵袭过程研究（ 见图1 ）。 <ol><li>在无菌层流罩下，将一块石蜡膜（20厘米x 15厘米）放在工作区域，并用70％乙醇消毒。 </li><li>将一批1毫克/毫升明胶的液滴分成20毫升，间隔约1厘米;参见图1的液滴组织。 </li><li>用高压灭菌的玻璃盖玻片（直径12毫米）覆盖每个20微米的明胶液滴。 </li><li>在室温下孵育20分钟。如果明胶是荧光的，保护它免受光照。 </li><li>将等分的40μL0.5％戊二醛液滴在1x磷酸盐缓冲盐水（PBS）中，每行间隔约1厘米，低于步骤1.1.3的盖玻片线。 注意:戊二醛是一种有毒的固定剂。使用手套，不要吸入。 </li><li>使用宝石镊子，将每个明胶包被的盖玻片转移到戊二醛液滴。 注意:正常情况下，小的明胶滴留在石蜡膜上。不要将其重用于另一个实验。 </li><li>在室温下孵育40分钟。对于荧光盖玻片，保护它们免受光线影响。 </li><li>取24孔细胞培养板，每孔加入500μL无菌1x PBS。将盖玻片，明胶一面朝上放入各孔中。 </li><li>在1x PBS中洗涤两次，每次5分钟。 注意:在此步骤中，盖玻片可用于实验或在4℃下储存在1x PBS中。非荧光盖玻片可以储存一周，荧光盖玻片可以储存48小时。 </li></ol></li><li> 纤维胶原I型基质 注意:I型原纤维胶原用于研究线性侵袭体形成和相关基质降解。使用荧光胶原I和非荧光明胶盖玻片的组合将invadosome标记物与胶原纤维共定位（ 例如， F-肌动蛋白i在红色通道，红色通道中的胶原蛋白I，绿色通道中的Tks5，以及用于染色的Hoechst染料）。另一方面，使用非荧光胶原I与非荧光明胶盖玻片的组合来最大化染色的侵染体标记物的数目（ 例如，远红色通道中的F-肌动蛋白，红色通道中的cortactin，Tks5在绿色通道和Hoechst染料用于染色）。最后，使用非荧光胶原I与荧光明胶盖玻片的组合来定量明胶相关的降解（ 例如，远红色通道中的F-肌动蛋白，红色通道中的Tks5，绿色通道中的Hoechst染料为核染色）（见图1）。 Hoechst染料用于染色细胞核，并且还检查支原体污染，以及定期在所用细胞系上进行的PCR检测。 <ol><li>荧光胶原蛋白I <ol><li>使用从0.02N乙酸丙酮中的大鼠尾肌提取的商业胶原蛋白I天。取出胶原蛋白I的量，以每个盖玻片制备500μL溶液，终浓度为0.4mg / mL胶原I. </li><li>加入最终浓度为10μg/ mL的5-羧基x罗丹明琥珀酰亚胺酯，计算出胶原蛋白I溶液（nx 500μL）的最终体积。 </li><li>通过上下移液混合溶液，并在室温下孵育5分钟，防止光照。 </li><li>用含有钙和镁的1X Dulbecco的磷酸盐缓冲盐水（DPBS）稀释至最终体积，并直接使用。 </li></ol></li><li>纤维胶原I盖玻片<ol><li>使用先前在步骤1.1中制备的非荧光或荧光明胶盖玻片。 </li><li>在含有钙和镁的1X DPBS中制备终浓度为0.4mg / mL的荧光或非荧光胶原I的溶液。 </li><li>加入500μL荧光或非荧光胶原蛋白I溶液盖玻片在24孔细胞培养板中。 </li><li>在37℃下孵育4小时，进行胶原蛋白I聚合。 </li><li>孵育后，通过倾斜板和移液孔（不直接盖在盖玻片上）来移除过量的胶原蛋白I，以避免损坏基质。 注意:此矩阵无法存储。去除多余的胶原蛋白I后立即种细胞。 </li></ol></li></ol></li></ol>细胞播种，培养时间和固定<ol><li>根据细胞类型，将细胞制备在合适的培养基中，并加入细胞，每孔最终体积为500μL。 </li><li>为了表征细胞系形成线性侵袭体并降解基质的能力，将细胞在胶原I基质上铺板4小时，12小时和24小时，以确定线性侵袭体形成的动力学和相关的降解活性。 注意: 表1包含细胞系的实例在实验室测试。 <table border="1"><tbody><tr><td>细胞系</td><td>每盖玻片的细胞数</td><td>与矩阵接触线性侵袭体定量的时间</td><td>与矩阵接触的时间用于定量线性侵袭体降解活性</td></tr><tr><td> MDA MB 231 </td><td> 40000 </td><td> 4小时</td><td> 12小时</td></tr><tr><td> HUH7 </td><td> 40000 </td><td> 12小时</td><td> 24小时</td></tr><tr><td> HEP 3B </td><td> 40000 </td><td> 12小时</td><td> 24小时</td></tr><tr><td> SNU 398 </td><td> 40000 </td><td> 12小时</td><td> 24小时</td></tr><tr><td> NIH 3T3 src </td><td> 30000 </td><td> 4小时</td><td> 12小时</td></tr><tr><td> A431 </td><td> 50000 </td><td> 6小时</td><td> 24小时</td></tr><tr><td> A549 </td><td> 40000 </td><td> 12小时</td><td> 24小时＃160; </td></tr><tr><td>生的</td><td> 40000 </td><td> 12小时</td><td> 12小时</td></tr><tr><td> PAE </td><td> 40000 </td><td> 12小时</td><td> 12小时</td></tr></tbody></table> 表1:细胞接种和孵育时间参考。该表列出了用于研究线性侵袭体的细胞系列表。它表示在基质上种子的细胞数，观察线性侵袭体必需的时间以及观察相关基质降解所需的时间。线性invadosomes的寿命短。为了能够在最大数量的细胞中观察线性侵袭体，细胞必须在短时间内与胶原I基质接触。另一方面，为了能够使所报告的明胶降解可视化，细胞必须与胶原蛋白I基质接触时间比上述那些长。 </li><li>除去细胞培养基后，在500μL的4％多聚甲醛在1X PBS中将盖玻片固定10分钟。 </li><li>用1x PBS洗两次。 </li><li>储存于4°C，避光保存。 </li></ol> 3.免疫荧光<ol><li>用新鲜制备的溶液将细胞渗透10分钟 的0.2％Triton X-100在1×PBS中。不要重复使用此解决方案。 </li><li>用1x PBS洗两次盖玻片。 </li><li>将一块石板玻璃（20厘米x 15厘米）放在长凳上。 </li><li>在1×PBS中的4％牛血清白蛋白（BSA）中稀释一抗，如材料表中所述。 </li><li>在一条线上分装50μL一抗一抗溶液液滴，每个间隔约1厘米。 </li><li>将每个盖玻片放在一滴上。 </li><li>在室温下孵育40分钟，防止光照。 </li><li>孵育期后，用1x PBS洗涤两次。 </li><li>在1％的4％BSA中稀释二抗，鬼笔环肽和Hoechst染料PBS。 </li><li>如前所述，将50μL混合物的液滴分别分隔在第一行盖玻片下方约1厘米处。 </li><li>将每个盖玻片放在一滴上。 </li><li>在室温下孵育30分钟，防止光照。 </li><li>孵育后，用1x PBS洗涤两次。 </li><li>用蒸馏水洗涤一次以除去盐。 </li><li>将玻璃盖安装在玻璃载玻片上，聚合安装介质。 </li><li>在显微镜下，盖玻片在室温下干燥过夜，防止光照。 </li></ol>量化<img alt="图2" src="/files/ftp_upload/54911/54911fig2.jpg" /> 图2:线性侵袭体定量。该宏需要F-actin和Tks5染色的共焦图像（格式:1,024×1,024）。 （ A ）首先打开F肌动蛋白图片并制作面膜。 （ B ）然后，打开Tks5图片和阈值。应用宏。 （ C ）分析结果将出现在两个表格中。每个细胞的线性侵袭体的数量和其他信息，如线性invadosomes在细胞中使用的百分比面积将在Summary表中。每个线性invadosome的大小将在"结果"表中。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54911/54911fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 形成线性侵袭体的细胞百分比 <ol><li>计数形成线性侵袭体的细胞，每盖玻片约100个细胞（使用技术一式三份）。每n个量化至少300个细胞，n = 3。 </li><li>通过取三次独立实验的平均值计算形成线性侵袭体的细胞的百分比，得到900个定量细胞。 </li></ol></li><li> 线性侵袭体大小和细胞数 <li>使用带有补充宏的ImageJ。 </li></ol></li><li> 每个细胞降解 <ol><li>每个条件使用三个盖玻片。 </li><li>拍摄30张荧光明胶和Hoechst染色核的盖玻片。 </li><li>使用ImageJ软件，如Martin KH 等人所述。 9 。 </li></ol></li></ol> 5. 3D胶原蛋白入侵检测<img alt="图3" src="/files/ftp_upload/54911/54911fig3.jpg" /> 图3:入侵测定方案。入侵试验方案总结。胶原蛋白I与琥珀酰亚胺酯染料交联并在37℃下聚合1小时。将细胞加入胶原蛋白I塞顶部的无血清培养基中。将插入物放置在介质wi中10％FBS。在实验条件下，将细胞接种1h后胶原Ⅰ栓塞固定在细胞接种后3天，测定细胞侵袭能力。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54911/54911fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 胶原I插头准备 <ol><li>进行第一次实验的4小时，12小时，24小时，48小时和72小时的时间过程，以确定根据细胞类型侵入凝胶的动力学。对于每个实验，添加1小时的时间点作为参考时间点，当没有入侵发生时。 </li><li>在无菌层流罩下在冰上制备胶原蛋白I溶液。为3个Boyden室插入物准备500μL溶液。 </li><li>吸出1mg市售的大鼠尾胶原I制备终浓度为2mg / mL胶原的溶液I. </li><li>加入5-羧基 - 罗丹明琥珀酰亚胺酯最终浓度为1μg/ mL，计算出胶原I溶液的最终体积（在这种情况下为500μL）。 </li><li>混合均匀，在无菌层流罩下冰上孵育5分钟，防止光照。 </li><li>加入50μL冰冷的，过滤的10X PBS和6.25μL冰冷的1M氢氧化钠（NaOH）。 </li><li>加入无菌水按照公式443.75 - xμL胶原Ⅰ。 </li><li>每Boyden腔插入物加入100μL溶液。 </li><li>在37℃孵育1小时以允许聚合。 </li><li>将富含胎牛血清（FBS）的细胞培养基加入新孔中;将插入物插入这些孔。 </li><li>在胶原蛋白I上方的种子30,000个细胞插入无FBS的培养基中。 </li><li>在37℃下培养细胞后，将胶原I栓塞在1×PBS中的4％多聚甲醛中固定30分钟。 </li><li>完全免疫荧光，如步骤3，除了以下步骤:渗透用0.2％Triton X-100在1x PBS中30分钟，而不是10分钟;分别用一次和二次抗体溶液孵育1.5小时，而不是40分钟和30分钟。染色F-肌动蛋白或细胞核定位细胞。 </li><li>免疫荧光后，使用手术刀切割插入物的膜，小心地将胶原蛋白I塞子转移到玻璃底盘。确保胶原蛋白I插头的顶部与玻璃接触。保持插入膜附着在胶原蛋白I插头的底部，以保持插头的完整性。 注意:胶原蛋白I塞在玻璃底盘中保持完整。玻璃底部和围绕玻璃底部的塑料之间的深度等于插头的厚度。 </li><li>在胶原蛋白I上插入盖玻片并用聚合安装介质安装，以防止插头变干。 </li><li>使用倒置的共聚焦显微镜来成像胶原蛋白I塞;的顶部插头与盘的玻璃底部接触。 <ol><li>从胶原蛋白I插头的顶部到底部获取一个z-stack来显现细胞侵袭。通过检查细胞侵入成像领域的深度来确定z-stack的厚度。使用Z轴步长为0.25μm的512 x 512格式进行采集。 注:作为参考，播种3天后MDA-MB-231细胞的平均z-叠层为30μm。 </li></ol></li></ol></li></ol>

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B., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoeboid+shape+change+and+contact+guidance:+T-lymphocyte+crawling+through+fibrillar+collagen+is+independent+of+matrix+remodeling+by+MMPs+and+other+proteases.">Amoeboid shape change and contact guidance: T-lymphocyte crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases.</a> Blood. 102, 3262-3269 (2003).</li><li>Geraldo, 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=Do+cancer+cells+have+distinct+adhesions+in+3D+collagen+matrices+and+in+vivo?.">Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?.</a> Eur. J. Cell Biol. 91, 930-937 (2012).</li><li>Monteiro, P., 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=Endosomal+WASH+and+exocyst+complexes+control+exocytosis+of+MT1-MMP+at+invadopodia.">Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia.</a> J. Cell Biol. 203, 1063-1079 (2013).</li><li>Wolf, K., 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=Collagen-based+cell+migration+models+in+vitro+and+in+vivo.">Collagen-based cell migration models in vitro and in vivo.</a> Semin. Cell Dev. Biol. 20, 931-941 (2009).</li></ol>

作者没有什么可以披露的。

经典地， 在体外研究侵袭体，而不考虑微环境和细胞被电镀的基质。目前使用几种类型的基质，包括明胶，纤连蛋白，玻连蛋白或高密度纤维胶原（HDFC） 7,11 ;然而，这些通常不代表细胞存在并且不具有生理学相关性的微环境。在这里，使用由明胶和纤维I型胶原之间的关联组成的新型基质。使用I型胶原原纤维可以使我们突出出一种新颖的侵袭体，被称为线形侵袭体，其在其生理结构中特异性地形成胶原I。标记胶原蛋白我允许我们沿着纤维观察线性invadosome，并使用细胞标志物如Tks5和皮质激素量化其形成。使用mi我们已经确定了一种新的受体，即盘状结构域受体1（DDR1），参与形成线性侵袭体8 。 2D混合基质允许我们通过酶谱原位测定来定量线性侵袭体的基质降解活性。该测定通过分析荧光明胶层中黑洞的存在来报告MMPs的蛋白水解活性。有趣的是，与明胶相比，F-肌动蛋白在线性invadosomes中的组织增加了基质降解活性。该测定的一个限制是明胶是非生理学基质，并且已经证明更多的生理学基质改变细胞对微环境的反应。在明胶胶原I混合基质上定量MMP活性的方式存在另外的限制。只有明胶降解，局限在胶原蛋白I fi釉层可以量化;因此，这是量化胶原蛋白I基质降解的间接方法。可视化线性侵袭体的降解活性的另一种方法是使用针对胶原切割位点（Col1-3 / 4C，免疫球蛋白）的特异性抗体标记胶原I纤维。这允许通过免疫荧光显示切割的纤维。然而，由于细胞迁移，该抗体由于线性侵袭体形成而在2D中不是非常特异的降解。可视化和量化胶原蛋白I纤维的降解活性的另一种方法是使用多光子显微镜和二次谐波产生8 。该方法允许胶原I纤维的成像而没有任何染色。 我们的I型胶原聚合方法与文献12,13 中使用的其他方法不同。例如，Artym <em&gt; et al。离心机盖玻片涂覆有α螺旋胶原蛋白I溶液，并允许短暂聚合30分钟。这种高密度纤维状胶原蛋白（HDFC）比我们的胶原蛋白I纤维更硬，更致密，但聚合较少。矩阵结构的这种差异使得invadosome形状从线性变为点状。时间，pH，浓度和温度是必须控制ECM聚合的参数。例如，聚合温度越低，纤维越大。 为了研究线粒体侵袭细胞侵袭的情况，使用了3D胶原I塞试验。科学界已经使用3D胶原蛋白I塞来研究入侵结构或金属蛋白酶参与入侵过程14,15,16 。这些类型的3D矩阵具有关于thei的限制r刚性 - 例如，3D胶原蛋白I插塞比2D矩阵刚性低。此外，胶原蛋白I的类型和起源对于体内胶原纤维的不同组织也是重要的17 。最后，关于体内的微环境组成，仅关注细胞外基质（I型胶原）的一种元素。进一步的研究是确定线性侵袭体在体内的相关性所必需的 。 除了研究线性侵袭体外，本文所述的混合基质可以与其它类型的基质组分如纤连蛋白，玻连蛋白和其它类型的胶原（ 例如， IV型胶原）一起使用。可以根据感兴趣的矩阵元素的类型和要研究的过程来调整该协议。然而，很明显，生成复杂和生理矩阵将允许识别new途径参与细胞粘附，迁移和侵袭。

细胞外基质（ECM）重塑发生在生理和病理过程中，如血管生成和肿瘤细胞侵袭。在生理条件下，许多细胞类型，主要来自造血系，能够降解ECM元件。例如，巨噬细胞能够跨越解剖障碍以达到组织，并且破骨细胞降解骨基质以确保钙稳态。在全球范围内，身体中的所有基质更新以维持其物理和化学性质。在癌组织中，肿瘤微环境组成被改变。例如，在乳腺癌和肺癌中，I型胶原蛋白被过度表达并在肿瘤周围积聚。此外，这种积累与发生转移的风险增加相关1,2 。癌转移依赖于癌细胞降解ECM并侵入邻近组织的能力。 该细胞的侵袭活性归因于被称为invadosome的专门的肌动蛋白丰富的结构。该术语包括分别存在于正常和癌细胞（ 例如，巨噬细胞，内皮细胞和癌细胞如MDA-MB-231乳腺癌细胞系）中的囊体和隐球菌。 体外 ，invadosomes可以组织成不同的形状:点，聚集体或花环3 。经典地，侵袭体由含有若干蛋白质的F-肌动蛋白核心组成，如骨架蛋白Tks5和皮质激素，被粘附斑块分子包围，如整联蛋白和连锁蛋白4 。最近，显示Tks5和RhoGTPase Cdc42可以用作功能性侵袭体5的最小分子标记。这些结构能够通过募集和激活特异性金属蛋白酶蛋白（如MT1-MMP和MMP-2 6）来降解ECM。在之前的研究中，我们报道了I型胶原原纤维与I型胶原原纤维的特异性受体的盘状蛋白结构域受体1（DDR1）之间的相互作用导致形成一类称为线性侵袭体的新型invadosome。沿着I型胶原纤维7形成线性侵袭体。这些结构能够通过募集和激活MT1-MMP / MMP2来降解I型胶原原纤维。此外，它们的形成取决于Tks5和Cdc42 5 。 在体外 ，我们证明线性侵袭体的存在和DDR1参与其形成和活动8使用由各种基质元素的组合组成的不同策略，包括:i）荧光明胶包被的盖玻片，ii）荧光I型胶原原纤维涂层盖玻片，和iii）3D I型胶原蛋白塞。由于使用不同的矩阵，我们能够学习和品格ize线性侵袭体形成及其降解活性7,8 。 最后，为了更好地了解入侵细胞的生物学，使用2D和3D培养系统中的ECM组分的组合，模拟肿瘤微环境组成的复杂性。以下是在2D和3D培养系统中用明胶/ I型胶原涂覆盖玻片的方案。

<table><tbody><tr><td>Gelatin solution</td><td>Sigma</td><td>G1393</td><td>Stock 20 mg/ml used at 1 mg/ml</td></tr><tr><td>Gelatin from pig skin Oregon green 488 conjugate</td><td>Molecular probe life technologies</td><td>G13186</td><td>5mg dilute at 1 mg/ml in sterile water</td></tr><tr><td>Microscope cover glasses</td><td>Marlenfeld GmbH &amp;amp; Co</td><td>111520</td><td>&amp;Oslash;12mm No.1</td></tr><tr><td>2.5% Glutaraldehyd in 0.1M sodium cacodylate buffer pH 7.4</td><td>Electron microscopy science</td><td>15960</td><td>Dilute at 0.5 % in sterile water</td></tr><tr><td>1x PBS pH 7.4</td><td>Gibco by life technologies</td><td>10010-015</td><td>Use this PBS in all steps before fixation</td></tr><tr><td>Collagen I Rat tail</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>5 carboxy X rhodamin siccinimidyl ester</td><td>Life technologies</td><td>C-6125</td><td></td></tr><tr><td>DPBS 1x + calcium + magnesium</td><td>Gibco by life technologies</td><td>14040-091</td><td></td></tr><tr><td>Paraformaldehyde 16% solution</td><td>Electron microscopy science</td><td>15710</td><td>Dilute at 4% in 1X PBS</td></tr><tr><td>Triton X 100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>10x PBS buffer pH 7.4</td><td>Ambion</td><td>AM9625</td><td>Dilute at 1X in water Use in steps after fixation</td></tr><tr><td>Tks5 antibody</td><td>Santa Cruz</td><td>sc-30122</td><td>Invadosome markers Dilution : 1/100</td></tr><tr><td>Cortactin 4F11 antibody</td><td>Millipore</td><td>5180</td><td>Invadosome markers Dilution :1/100</td></tr><tr><td>DDR1 antibody</td><td>Cell signaling</td><td>5583</td><td>Linear invadosome receptor</td></tr><tr><td>Dilution :1/100</td><td></td><td></td><td></td></tr><tr><td>Phalloidin FluoProbes</td><td>Interchim</td><td>FT-AZ0330</td><td>Fibrillary actin marker Dilution :1/200</td></tr><tr><td>Hoechst</td><td>Sigma</td><td>33258</td><td>Nucleus marker Dilution :1/200</td></tr><tr><td>Secondary antibodies FluoProbes</td><td>Interchim</td><td>FP-488 FP-547H or FP-647H</td><td></td></tr><tr><td>Albumin from bovine serum</td><td>Sigma</td><td>A2153</td><td>Dilute at 4% in 1X PBS</td></tr><tr><td>Fluoromount G mounting medium</td><td>Interchim</td><td>FP 483331</td><td></td></tr><tr><td>ImageJ software</td><td>Public domain</td><td></td><td>http://www.macbiophotonics.ca/imagej/</td></tr><tr><td>Cell culture insert</td><td>Corning</td><td>353097</td><td>8.0&amp;micro;m pore size / 24 wells</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma</td><td>221465</td><td></td></tr></tbody></table>

2d and 3d matrices to study linear invadosome formation and activity

细胞粘附，迁移和侵袭参与许多生理和病理过程。例如，在转移形成期间，肿瘤细胞必须跨越解剖障碍以侵入和迁移穿过周围组织以便达到血液或淋巴管。这需要细胞和细胞外基质（ECM）之间的相互作用。在细胞水平上，包括大多数癌细胞在内的许多细胞能够形成invadosomes，这是能够降解ECM的基于F-肌动蛋白的结构。侵袭体是募集和激活基质金属蛋白酶（MMP）的突出肌动蛋白结构。 ECM的分子组成，密度，组织和刚度在调节invadosome形成和激活中是至关重要的。 在体外 ，明胶测定是用于观察和定量侵袭体降解活性的标准测定法。然而，变性胶原I的明胶不是生理基质e字元素。开发了使用I型胶原原纤维的新型测定法，并用于证明该生理基质是侵入体的有效诱导剂。由于它们在纤维上的线性组织，沿着胶原纤维形成的侵袭体被称为线性侵袭体。此外，线性invadosomes的分子分析表明，盘状结构域受体1（DDR1）是参与其形成的受体。这些数据清楚地表明了使用生理相关基质以了解细胞与ECM之间的复杂相互作用的重要性。

使用两种类型的基质，明胶和I型胶原的组合（ 图1和图4 ），我们突出了一种称为线性侵袭体的新型invadosome。这些基质的标记允许观察沿胶原I纤维的线性侵袭体形成及其降解能力（ 图5 ）。然后可以通过前面描述的宏来量化侵入性结构的数量（步骤4.2），还可以使用ImageJ软件来确定降解活性，如Martin 等人所述。和Diaz 等 9，10 。混合基质允许通过将DDR1定义为线性侵袭体形成和功能所必需的受体来表征线性侵袭体（ 图6 ）。 图3和7 ）。该测定允许我们通过使用z-stack重建来确定入侵到胶原I插塞中的细胞数以及行进的距离。 <img alt="图4" src="/files/ftp_upload/54911/54911fig4.jpg" /> 图4:使用共焦显微镜比较明胶和明胶胶原I混合基质。显示在顶板上组织荧光明胶盖玻片的示意图。 （ A ）共焦z叠层重建表明，荧光明胶基质形成覆盖盖玻片整个表面的薄均匀层。 （ B ）在混合基质中，在盖玻片上沉积荧光明胶后，I型胶原原纤维聚合在上面。胶原蛋白I原纤维染成红色。胶原蛋白I基质在盖玻片上是稠密和异质的。胶原I纤维的分布取决于胶原Iα链的聚合。刻度棒=10μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54911/54911fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/54911/54911fig5.jpg" /> 图5:明胶和明胶胶原I基质对侵袭体形成和活性的影响。用于这些测定的细胞是MDA-MB-231乳腺癌细胞。 （ A ）示意图表示在顶板上荧光明胶盖玻片上接种的细胞。由于荧光的降低，降解区域呈黑色可视化。 Tks5染色用作invadosome标记。在明胶上，形成的adosome被组织成点。 （ B ）示意图表示接种在明胶和胶原I混合基质上的细胞。胶原蛋白I以红色标记; I型胶原纤维的添加增加了细胞降解明胶的能力。有趣的是，invadosome标记Tks5被重组，并且点被代表线性invadosome的线性结构取代。由于荧光的降低，降解区域呈黑色可视化。刻度棒=5μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54911/54911fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/54911/54911fig6.jpg" /> 图6:在明胶和混合基质条件下共聚焦显微镜分析侵袭体的分子组成和组织。 （ A ）在gelatin条件，invadosome以点组织，F-肌动蛋白（红色）与Tks5（绿色，底部面板）共同定位，但不与DDR1（绿色，顶部面板）共定位。这些是经典的invadosome。刻度棒=5μm。 （ B ）在明胶胶原I混合基质条件下，DDR1（绿色，顶部）与胶原蛋白I原纤维（红色，顶部面板），Tks5（绿色，底部）和F-肌动蛋白（红色，底部）共定位面板）。 I型胶原纤维诱导线形侵袭体的形成。刻度棒=10μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54911/54911fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图7" src="/files/ftp_upload/54911/54911fig7.jpg" /> 图7:胶原I插塞中MDA-MB-231细胞的细胞侵袭测定。接种细胞1小时后，对照插入物固定染色。 （ A ）z-stack a使用共焦显微镜检查胶原蛋白I插塞。在这个时间点细胞不会侵入胶原蛋白I塞;相反，它们仍然保留在胶原蛋白I插头的顶部。因此，这被用作没有入侵发生的控制时间点。 MMP激活对于细胞以此密度侵入胶原蛋白I栓塞是必需的。 （ B ）播种三天后，有些细胞侵入胶原蛋白I塞。该测定允许观察和定量细胞侵袭。另外，可以用来研究各种药物或siRNA的影响。刻度棒=20μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54911/54911fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about 2D and 3D Matrices to Study Linear Invadosome Formation and Activity at JoVE.com

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

该协议描述了如何制备由明胶和胶原蛋白I组成的2D混合基质，以及3D胶原I插塞来研究线性侵袭体。这些方案允许研究线性侵袭体形成，基质降解活性和原代细胞和癌细胞系的侵袭能力。

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

2d-and-3d-matrices-to-study-linear-invadosome-formation-and-activity

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.9K 次观看.  INSERM U1053. 该协议描述了如何制备由明胶和胶原蛋白I组成的2D混合基质，以及3D胶原I插塞来研究线性侵袭体。这些方案允许研究线性侵袭体形成，基质降解活性和原代细胞和癌细胞系的侵袭能力。 

视频: 2D and 3D Matrices to Study Linear Invadosome Formation and Activity

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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

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

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

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

医学

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

三维流状入侵检测中的全人肿瘤基基质

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

在3D揭示侵入性癌症细胞骨架组织

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

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) ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

生物工程

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

2D和3D矩阵研究线性侵袭体形成和活性

摘要

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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

3维建模和成像集体细胞的侵袭

使用自动活单元成像仪的简单迁移/入侵工作流

三维流状入侵检测中的全人肿瘤基基质

在3D揭示侵入性癌症细胞骨架组织

分析 3D 培养物体外细胞侵袭的替代策略

分析 3D 培养物体外细胞侵袭的替代策略

分析 3D 培养物体外细胞侵袭的替代策略

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下