作者感谢 Rozina Hassam 和 Orsolya Cseh 的技术支持。这项研究项目部分是由加拿大创新基金会 (h. Artee Luchman 和塞缪尔？维斯) 和加拿大的干细胞网络 (也对 h. Artee Luchman 和塞缪尔. 维斯) 提供的赠款支助。

1. 从人脑胶质瘤标本中培养脑肿瘤干细胞
注: BTSC 文化是以前建立的人肾小球病患者样本6,7,8,9,10。
<ol><li>在装有70% 乙醇的烧杯中解冻一小瓶低温保存的 BTSCs, 放在37摄氏度的水浴中, 直到最后的冰解冻。在15毫升圆锥管中稀释10毫升介质中的解冻细胞, 将150相对离心力 (区域合作框架) 的细胞离心为7分钟。 注意: 在这些协议中, 完整的媒体是指用于区域性 BTSCs 的标准媒体 (以前是凯利等)。6, 典型地以成长因素存在。无生长因子的介质指的是所有组件的基本介质, 但没有任何生长因子补充。</li>
	<li>吸入介质, 并用重悬8毫升完整介质中的细胞, 将细胞和介质转移到 T25 瓶中。</li>
	<li>在不附着条件下进行1周的培养后, 每天监测烧瓶, 并根据需要补充 1-2 毫升的内容, 如果媒体开始耗尽或开始变得酸性, 如橙色黄色变化所示。通道细胞当 neurospheres 到达大约250微米的直径, 通常在 10-14 d 之间 (图 1)。 注: 不同 BTSC 文化的加倍时间差异很大。干细胞尺寸可以用眼千分尺测量。</li>
	<li>通过 BTSCs, 收集介质和 neurospheres, 吹打介质放在烧瓶的表面上, 收集所有 neurospheres 并将其转移到15毫升锥形管上, 在150区域合作框架内离心管7分钟。 注: 可在显微镜下检查烧瓶, 以确保所有 neurospheres 都已收集。可以执行额外的5毫升磷酸盐缓冲盐水 (PBS) 或媒体的清洗, 以收集任何必要的剩余 neurospheres。</li>
	<li>将 BTSC neurospheres 分离成单个细胞, 吸出培养基, 加入1毫升预热细胞脱离溶液, 孵育37摄氏度7分钟 Triturate BTSC 悬浮 40x, P1,000 微设于 800 ul。 注意: 如果 neurospheres 不离解, 细胞可以在37摄氏度的时间内孵化。</li>
	<li>添加5毫升的 PBS (用抗生素青霉素和链霉素) 到离解 BTSCs 和离心机的细胞在150区域合作框架7分钟。</li>
	<li>根据细胞颗粒的大小, 在 1-4 毫升的完整介质中吸出上清和并用重悬 BTSCs。 注: 如果浓度超出精确的细胞计数的准确检测限度, 细胞悬浮液需要进一步稀释。</li>
	<li>使用不包括死细胞的染料, 如台盼蓝, 计数的存活细胞数量和种子的 BTSCs 到瓶子或盘子的兴趣, 通常, 20万-30万细胞8毫升的完整的媒体在一个 T25 瓶。</li>
</ol>2. 脑肿瘤干细胞迁移试验
<ol><li>种子20万细胞在一个 T25 烧瓶8毫升的完整培养基 1-2 周前计划进行迁移试验。 注意: 电镀和执行化验之间的时间取决于所使用的 BTSC 文化的生长速率。<ol>
<li>为了测试药物治疗对移徙的影响, 预处理适当的车辆, 如亚砜和感兴趣的药物, 将车辆或药物储存的适用体积直接添加到含有细胞的介质的不同井中。</li>
	</ol>
</li>
	<li>用一层薄薄的胶原蛋白将其涂上, 制备趋化迁移板 (图 2A)。<ol>
<li>计算96井趋化板的井数, 需要涂上胶原蛋白。每个条件至少包括三个复制井。</li>
		<li>稀释5毫克/毫升的 i 型胶原蛋白到0.2 毫克/毫升在纯醋酸稀释到0.14% 在养殖级水。</li>
		<li>将0.2 毫克/毫升的胶原蛋白移到膜插入井和正在使用的底油层井中。 注: 底板的底部储层需要225µL, 膜插入井需要75µL 的胶原蛋白来进行适当的涂层。</li>
		<li>将迁移板放置在37摄氏度的孵化器中, 1 小时. 从水井中吸取胶原溶液, 而不划伤胶原涂层。</li>
		<li>用无菌 1x PBS 洗涤水井2x。如果盘子没有立刻被使用, 吸入 PBS 并且存放它在4°c 2 星期。使用前, 将趋化迁移板加热至室温。</li>
	</ol>
</li>
	<li>制备趋化迁移板块的底储层。<ol>
<li>吸管225µL 的培养基 (没有生长因子), 补充10% 的血清作为趋化物, 到底部水库井的趋化迁移板块。</li>
		<li>轻轻地将膜插入到底部的储层中, 以避免产生气泡。</li>
	</ol>
</li>
	<li>将细胞板入趋化移印板的膜插入。<ol>
<li>并用重悬游离 BTSCs 在培养基中 (无生长因子), 细胞密度为5万细胞/毫升。在不同药物治疗过程中评估迁移时, 向媒体添加药物。交替, 种子相同数量的预处理细胞和控制细胞, 同时保持最终药物浓度时, 电镀。</li>
		<li>板50µL 的 BTSCs 从步2.4.1 入每个井迁移板材在期望的治疗情况下。</li>
	</ol>
</li>
	<li>图像的趋化迁移。<ol>
<li>将趋化迁移板置于活细胞成像系统中, 并设置自动成像软件, 每 1-2 小时即可获取该板的显微图像, 最多72小时. 使用商用软件 (见材料表), 右键单击时间线, 并将扫描间隔设置为每2小时24小时。 注意: 所用的 BTSC 文化之间的时间间隔和总使用时间会有所不同;开始与2小时间隔为72小时, 直到经验得到了与个体 BTSC 文化。如果自动成像系统不可用, 则可以用显微镜和连接到成像软件的照相机手动获取相同视野的图像。</li>
		<li>一旦图像采集完成, 获取整个实验的图像, 并创建一个处理定义使用分析软件, 区分细胞从背景的细胞膜和迁移毛孔 (图 2B, 红色面具).使用商业软件 (参见材料表), 选择新的处理定义并将 BTSCs 的处理定义设置设置为种子阈值52,增长阈值为 85,调整大小(像素) 1,最小面积为200微米2。将此处理定义应用于膜的底部。如果不准确区分来自背景膜的细胞, 则修改此分析。</li>
		<li>收集数据作为细胞膜底部的细胞表面积, 用于三个复制井, 这只计算通过毛孔迁移的细胞。图2中单元格的迁移 (迁移的单元格区域) 随着时间推移 (图 2C,图 3)。 注: 确保在第一时间点的膜的顶面上镀和计数的细胞相对数量在所有处理条件之间类似 (小于20% 的变化), 以避免不均匀电镀的任何影响。使用商业软件 (参见材料表) 的数据收集可以通过启动在步骤2.5.2 中创建的处理定义来执行, 单击度量值, 然后在图形/导出上, 然后再进行数据导出。.</li>
	</ol>
</li>
</ol>3. 脑肿瘤干细胞干细胞侵袭试验
<ol><li>种子20万细胞在8毫升的完整培养基在一个 T25 瓶 1-2 周前计划进行入侵检测。 注意: 从电镀到执行化验的时间取决于所使用的 BTSC 文化的增殖率。<ol>
<li>对于预处理过的细胞, 在 neurospheres 准备好进行入侵之前, 在预定的时间路线上, 将感兴趣的化合物添加到所需浓度的烧瓶中。</li>
	</ol>
</li>
	<li>等待, 直到平均干细胞大小约 150-200 微米;通常, 这需要 7-14 d, 这取决于所使用的 BTSC 区域性。 注: 观察瓶内球形尺寸分布是典型的。</li>
	<li>提示和轻轻地混合烧瓶与吸管和移动 500 ul 的 BTSC neurospheres 到1.5 毫升圆锥管为每个治疗条件;这将用于板三复制井。 注意: 步骤 3.3.1-3.3.2 是可选的, 但建议第一次入侵实验的新 BTSC 文化。<ol>
<li>要确定最终在井中的 neurospheres 的密度, 请在步骤3.3 中准备一个1.5 毫升管。轻轻地混合 neurospheres 和移动 100 ul 进入一个单独的96井板块, 并允许 neurospheres 以重力解决5分钟。</li>
		<li>检查显微镜下的 neurospheres 数, 以确保多个 neurospheres 在该井的区域, 将被成像而不会过度拥挤。 注意: 入侵 neurospheres 需要足够的空间, 以三重直径, 而不与相邻的 neurospheres 互动。每个井的 neurospheres 数可以通过计算每个井的 neurospheres 数来调整, 并在步骤3.3 中加入更多或更少的 neurospheres 从 T25 烧瓶到1.5 毫升圆锥管。</li>
	</ol>
</li>
	<li>将1.5 毫升锥形管与步骤3.3 中的单元格放在冰上, 并在冰上执行步骤 3.5-3.7。</li>
	<li>让 neurospheres 在1.5 毫升锥形管的底部, prechill 5 分钟, 并在冰上标上96井板。</li>
	<li>并用重悬细胞外基质蛋白中的 neurospheres。<ol>
<li>准备 500 ul 的0.4 毫克/毫升 i 型胶原蛋白溶液在冰上为每个治疗条件: 添加 459 ul 的培养基 (无生长因子), 40 ul 的5毫克/毫升胶原蛋白溶液, 0.92 ul 的无菌1N 氢氧化钠 (表 1)。 注: 500 ul 的胶原蛋白溶液是每个治疗条件要求: 100 ul 为每三个复制井和 200 ul 的过剩。药物治疗需要添加到预期的最终浓度, 从解决方案的媒体部分减去。本协议描述了 i 型胶原蛋白作为细胞外基质蛋白的应用;然而, 任何细胞外基质蛋白都可以测试。此外, 对于具有较慢的入侵率的 BTSC 文化, 可以在媒体中补充生长因子或血清, 以提高入侵率。</li>
		<li>从步骤3.5 中的 neurospheres 解决后, 尽可能多地吸入介质, 而不会失去在底部沉淀下来的干细胞颗粒。轻轻并用重悬 neurospheres 在 500 ul 的胶原溶液中制备的步骤3.6.1。</li>
	</ol>
</li>
	<li>添加 100 ul 的胶原和干细胞混合物, 三复制井的96井板在冰上。允许 neurospheres 在冰上安定5分钟。</li>
	<li>将96井板转移到37摄氏度孵化器中, 5 分钟, 以便胶原聚合。</li>
	<li>立即准备图像的96井板。<ol>
<li>将96井板放入活细胞成像系统的托盘或显微镜的舞台上, 以获取图像作为参考, 在电镀时的干细胞尺寸, 在入侵开始之前。</li>
		<li>每小时获取图像, 直至入侵率停滞不前;通常, 这在24小时内发生。 注: 成像间隔可以根据所使用的设备进行修改。对于具有不同入侵率的 BTSC 区域性, 也可以修改成像间隔和总运行时间。</li>
	</ol>
</li>
	<li>确定 BTSCs 随着时间推移侵入矩阵时的表面积增加。 注: 在电镀时的表面面积, 计算为平均三复制井, 需要类似 (小于20% 的变化, 横跨所有井) 之间的不同处理条件, 以确保在 BTSC 入侵的差异不受镀 neurospheres 的数量或大小的强烈影响。<ol>
<li>对于活细胞分析系统, 使用10X 目标进行图像采集, 并为三个复制井获取四幅图像。为了确定相对的细胞表面积, 表示为井的百分比汇流, 建立一个处理定义, 具体地强调在井的背景下的细胞。使用商用软件 (见材料表), 选择一个新的处理定义, 并将分割调整设置为 0.8, 区域最小为300微米2, 偏心最大值为0.99。如果不准确区分单元格与背景, 则修改此分析。 注: 如果使用其他方法对干细胞入侵进行图像处理, 建议在一段时间内为三个复制井获取多个视图字段。但是, 在每个时间间隔内不成像完全相同的视场将增加所收集数据的标准误差。计算机软件可以用来帮助测量每个图像中入侵细胞的表面积。</li>
		<li>在每个时间点收集数据作为每个井的总细胞表面积 (相对或绝对), 并确定三复制井的平均值。通过在一段时间内绘制增加的细胞面积来图 BTSCs 的侵袭。 注意: 使用商业软件进行数据收集 (参见材料表) 可以通过启动在步骤3.10.1 中创建的处理定义, 单击度量, 然后在图形/导出, 然后再在数据导出中执行。.</li>
	</ol>
</li>
</ol>

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C., Park, A. Y., Guan, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nature Protocols. 2 (2), 329-333 (2007).</li><li>Naber, H. P., Wiercinska, E., Ten Dijke, P., van Laar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+Assay+to+Measure+TGF-&#946;-induced+Invasion.">Spheroid Assay to Measure TGF-&#946;-induced Invasion.</a> Journal of Visualized Experiments. (57), (2011).</li></ol>

作者没有什么可透露的。

鉴于癌细胞正在积极增殖, 这对研究细胞迁移构成了潜在的技术挑战。在这里描述的迁移试验中, BTSCs 被镀没有生长因子, 这限制了增殖, 而趋化板的趋性梯度不完全平衡超过72小时。此外, 随着时间的推移, 细胞被监测使用活细胞成像, 细胞可以进行视觉监测, 以确保广泛的细胞分裂没有发生。为了成功的迁移化验, 重要的是把 neurospheres 完全分离到单个细胞和准确计数的细胞数在电镀之前。电镀球或电镀太多的细胞, 导致结块, 可以防止细胞通过小毛孔迁移 (图 4A和4B)。同样重要的是, 避免制造气泡时, 电镀的细胞在趋化迁移板块或当放置膜插入到底部水库。气泡可以改变焦点平面, 防止精确量化 (图 4C)。整个板块需要涂上薄薄的胶原层, 因为这给细胞提供了一个表面来导航到盘子里的毛孔, 因为这种化验要求细胞坚持, 然后在生物相关的表面上迁移到趋化因子。
此处描述的 BTSC 迁移协议允许对单个细胞迁移进行动态评估, 而与现有协议相比, 测试不同治疗条件所需的细胞较少。这对于生长非常缓慢的文化很重要, 因为它很难收集大量的细胞进行实验。趋化迁移板块的低孔隙密度, 使趋化因子梯度的平衡速度减慢, 大于72小时, 更有效地模拟了更长时间内趋化迁移的生物过程。该协议的优化, 以在趋化移印板上有一个非常薄层的胶原涂层, 以确保检测措施的迁移, 而不是入侵。它还有助于成像和定量的单细胞, 因为他们坚持胶原基质涂层的膜插入。然而, 这项技术的一个限制是, 利用商业软件大大简化和加快了跨多口井的迁移量化 (见材料表)。这里所描述的迁移分析的一个显著优势是该协议对其他单元类型的广泛适应性。这将是特别有用的量化的趋化细胞系的迁移, 无论是缓慢增长, 难以坚持, 或迁移缓慢。
为了确保 BTSC 入侵检测的成功, 必须在 neurospheres 超过200µm 之前收集它们. 较大的球体有一个焦点的平面, 它太大, 无法始终如一地进行图像和量化 (图 5C)。此外, neurospheres 可以开始与其他 neurospheres 丛和聚合。这将影响获取准确和一致的数据。此外, 重要的是, neurospheres 被允许定居的底部的井, 而胶原蛋白仍然在冰上, 以允许 neurospheres 在一个单一的平面图像的焦点 (图 5D)。同样, 重要的是, 让胶原完全设置, 而不移动板块, 因为这可能会扰乱甚至矩阵的形成, 这可能会对图像质量产生负面影响 (图 5D)。在这里描述的 BTSC 入侵化验, 其中一个主要优点是, BTSCs 养殖 neurospheres 可以直接评估入侵, 而不需要种植 adherently。此外, BTSC neurospheres 可以嵌入任何细胞外基质, 无论是使用细胞外基质的个别成分从一个特定的组织或使用一个商业上可用的基底膜混合物。指定细胞外基质可以帮助识别或测试特定的入侵机制。例如, 有了这个协议, 就有可能评估针对矩阵 metallopeptidase 家族的单个成员的效果, 这些基因是已知的降解特定的矩阵成分。另外, 虽然此协议特定于 BTSC neurospheres, 但任何以球体形式生长的细胞都可以用类似的方式使用。例如, 以 mammospheres 或原发结直肠癌细胞培养为 tumorspheres 的乳癌细胞;但是, 这将技术限制为可以在区域性中作为球体增长的单元格类型。迁移和入侵检测的其他优点是它们易于设置, 它们的工作方式是96井格式, 数据随着时间的推移可量化。
总的来说, 为了防止治疗后的肾小球复发, 必须制定方法, 以促进调查 BTSC 迁移和入侵。这里描述的迁移协议比以前建立的迁移分析具有许多优势, 特别是对 BTSCs 的研究。这种改进的迁移协议包括在干细胞条件下培养的游离 BTSCs, 以及在96层胶原蛋白涂层趋化迁移板上镀单细胞。使用活细胞成像系统, BTSCs 的迁移可以随时监测和量化。这里描述的入侵检测允许监测 BTSC 入侵从 neurospheres 到一个矩阵。这种检测包括将 neurospheres 嵌入胶原基质, 或在96井板中植入另一种富含蛋白质的细胞外基质。用活细胞时间推移成像系统对钢板进行成像, 可以在不同的治疗条件下对 BTSC 入侵进行分析。因此, 这些化验为科学家们提供了一个宝贵的工具, 希望能更好地了解 BTSC 迁移和入侵的机制。

胶质瘤 (肾小球) 是最常见和最具攻击性的脑癌形式, 尽管目前的治疗方法包括手术切除术, 其次是放疗和化疗, 但患者平均存活率仅为15月1。肾小球是侵袭性, 高度耐药性, 最终是一种不治之症, 需要继续研究其固有的生物学, 以确定新的治疗途径。肾小球病患者的高死亡率主要是由于细胞侵袭邻近正常脑组织和沿血管迁移导致复发的疾病后治疗2,3.一个细胞的子集, 被称为脑肿瘤干细胞 (BTSCs), 是缓慢的循环和治疗的抗性, 已被推测导致疾病复发。肾小球 BTSCs 显示克隆自我更新, 多向分化潜能和肿瘤启动电位4,5,6的性质。BTSCs 还保留其父母肾小球瘤7、8、9、10的遗传、分子和表型异质性。这些特性使 BTSCs 成为一个非常有用的模型系统的研究的肾小球和探索新的治疗策略。在体内, 这些茎样细胞在植入新生大鼠11和非肥胖的糖尿病/严重联合免疫机能丧失 (点头/有缺陷) 小鼠4的大脑时, 能够形成肿瘤、生长和侵入。BTSCs 在体内反复形成侵袭性肿瘤的能力, 重述其原发肿瘤的细胞和组织学特征, 有力地支持它们作为肿瘤启动细胞的作用12。
新的治疗方法正在开发的目标 BTSCs 和帮助改善长期结果的肾小球病患者。目前, 对于与治疗耐药性和复发有关的细胞迁移和侵袭过程的理解有限。迁徙与入侵是截然不同的现象;迁移是细胞定向运动所依据的过程, 它涉及到骨架、黏附分子的重组以及焦点接触点的组装/拆卸, 而入侵也需要物理上的屏障破坏。如胞外基质13。一个广泛接受的研究细胞迁移和入侵的方法是博伊登室化验14,15。对于这种技术, 一个双腔充满了媒体, 与媒体在底部室补充了趋化。趋化因子是通过特定的生长因素或细胞因子, 或胎牛血清作为非特异趋性因子触发的。随着趋化因子的梯度, 细胞通过多孔膜迁移。在端点上, 通过细胞学或荧光染料染色, 可以在膜的底部进行可视化和量化。博伊登迁移试验可以通过涂敷具有细胞外基质成分如 i 型胶原或基底膜样基质的多孔过滤器来进行侵袭性检测。侵入细胞会降解基质, 进入多孔过滤器的底部, 并且可以以类似的方式对迁移试验进行量化。其他常用的迁移方法包括划伤和细胞排除区化验13。划伤的方法是通过在细胞单层中抓一个缝隙, 观察细胞从伤口边缘的迁移, 并定期进行成像, 直到抓痕闭合。在细胞排斥试验中, 用物理屏障在细胞播种之前创建一个无细胞区。当细胞汇合后, 屏障被移除, 细胞迁移到无细胞区的过程通常是16。
这些以前建立的化验经常被用来研究迁移或入侵的黏附和均质细胞种群, 但在研究肾小球 BTSCs 或其他非均质癌细胞群体时造成相当大的不利因素。博伊登室化验只能产生端点测量与细胞运动的动力学评估。随着时间的推移, 观察对 BTSC 迁移的测量具有很高的相关性, 因为不同文化的细胞通常以不同的速率迁移。因此, 必须为每种文化类型优化化验的条件和时间, 并需要耗费大量时间的劳动力进行适当的抽样和量化。划痕和细胞排斥检测不太适合 BTSC 文化, 即使在 BTSCs 是在层粘连蛋白涂层板上培养的, 我们观察到, BTSCs 似乎抵制运动进入开放空间, 并希望保持密切接近其他细胞。此外, 这些已建立的迁移化验不允许在整个实验中对单个细胞进行可视化和监测。随着时间的推移, 对单个细胞的监测对于评估非均质细胞种群 (如 BTSCs) 的迁移具有重要价值. 博伊登室、划痕和细胞排斥区化验 BTSC 文化的其他缺点是, 他们要求较高的细胞数, 可以很耗时地设置, 或者快速平衡或没有趋化因子梯度。因此, 这些化验并不理想, 用于罕见或缓慢增长的细胞群体或药物筛选。此外, 这些化验不适合测量入侵的三维 (3D) 格式, 这是特别重要的 BTSCs 生长在干细胞条件下。
在这里, 我们描述专门修改的观察和量化的个体 BTSCs 的迁移, 以及入侵的肾小球 BTSCs 培养为 neurospheres。第一种分析描述了用活细胞的时间推移成像和趋化移印板来测定趋性细胞迁移13的博伊登室的适应性。多井格式的活细胞成像允许在多种治疗条件下进行细胞迁移的可视化和量化。本文所描述的第二种检测方法是13、17的球体入侵检测, 该方法测定了干细胞条件下培养的 BTSCs 的侵入性, 并将其嵌入3D 胞外基质中进行各种处理。条件。总的来说, 这些化验比以前描述的方法更符合研究异种 BTSC 文化的迁移和侵入性特性。它们还提供了更好的机会, 以调查新的治疗策略, 以移徙和入侵为目标, 这大大有助于疾病复发和致死。

<table>
	<tbody>
		<tr>
			<td>Rat Collagen I</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>VWR</td>
			<td>BDH3247-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Millipore Sigma</td>
			<td>AX0073-6</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Eppendorf</td>
			<td>30730119</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 Nunc EasYFlask</td>
			<td>ThermoFisher</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15ml conical tube</td>
			<td>ThermoFisher</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte ClearView 96-Well Chemotaxis plate</td>
			<td>Essen Bioscience</td>
			<td>4582</td>
			<td></td>
		</tr>
		<tr>
			<td>IncuCyte Zoom Live-Cell Analysis System</td>
			<td>Essen Bioscience</td>
			<td>4545</td>
			<td></td>
		</tr>
		<tr>
			<td>IncuCyte Zoom Live-Cell Analysis Software</td>
			<td>Essen Bioscience</td>
			<td>2016A Rev1</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax cell detachment solution</td>
			<td>Innovative Cell Technologies</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin, streptomycin&#160;</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture grade water</td>
			<td>Lonza BioWhittaker</td>
			<td>17-724Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>12483-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

live-cell imaging assays to study glioblastoma brain tumor stem cell migration and invasion

胶质瘤 (肾小球) 是一种侵袭性脑肿瘤, 它的控制不善, 目前可用的治疗方案。GBMs 的主要特点包括快速增殖和普遍侵入正常的大脑。复发被认为是由于存在的射频和抗化疗的脑肿瘤干细胞 (BTSCs) 入侵远离最初的癌症肿块, 从而逃避手术切除。因此, 靶向 BTSCs 的治疗方法和他们的侵袭能力可以改善这种疾病的预后不佳。我们的小组和其他人已经成功地建立和特点 BTSC 文化从肾小球患者样本。这些 BTSC 文化显示了基本的癌症干细胞的性质, 如克隆自我更新, 多血统分化, 和肿瘤启动的免疫缺陷小鼠。为了改善目前治疗方法的肾小球, 更好地了解 BTSC 迁移和入侵的机制是必要的。在肾小球中, 迁移和入侵的研究受到限制, 部分原因是由于现有技术的局限性, 这并不充分说明 BTSCs 的体外生长特性 neurospheres。在这里, 我们描述了快速和定量的活细胞成像检测, 以研究 BTSCs 的迁移和入侵性质。所描述的第一种方法是 BTSC 迁移法, 测量向趋化梯度的迁移。第二种方法是 BTSC 入侵检测, 图像和量化的细胞入侵从 neurospheres 到一个矩阵。这里描述的化验是用于定量的 BTSC 迁移和入侵的时间和在不同的处理条件。

在这里, 我们描述了数据的例子, 可以获得使用活细胞成像检测 BTSC 迁移和入侵研究。BTSCs 被镀成单个细胞, 可以作为自由浮动的 neurospheres 生长 (图 1)。我们表明, BTSCs 可以被分离和镀在膜插入的水井中的一个趋化移印板, 涂一层薄薄的 i 型胶原蛋白 (图 2A)。个体 BTSCs 均匀地分散在膜插入物的顶部在试验初。在 24-72 小时的时间内, 细胞黏附在膜上, 沿着胶原涂层的表面移动, 并通过96个小毛孔中的一个进入膜的底部, 向油藏中的趋化物迁移。在突出的细胞在膜的顶部黄色, 在蓝色的毛孔, 和细胞膜底部的细胞在红色, 细胞可以看到进入在上面的毛孔 (黄细胞接近蓝孔) 和退出毛孔在底部 (红色细胞出口蓝色毛孔) (图 3)。重要的是, 细胞可以被药物处理和镀在同一趋化移印板作为车辆处理的控制细胞, 以研究治疗干预对 BTSC 迁移的影响 (图 2B)。趋化迁移板允许成像的细胞在顶部和底部的膜插入。因此, 通过计算迁移到膜的底部的细胞 (图 2C), 可以在一段时间内量化细胞迁移。
我们发现, 如果 BTSCs 没有完全分离, 那么细胞就不会通过毛孔迁移, 保持小 neurospheres (图 4A)。它也重要的是不板超过 2500 BTSCs/好, 因为这导致细胞聚集, 而不是在迁移到膜的底部 (图 4B)。最后, 在镀膜单元和将膜插入到底部储层时, 避免在薄膜插入井中制造气泡, 因为这样可以防止精确的成像和定量 (图 4C)。
接下来, 我们表明, BTSC 入侵, 从 neurospheres 到周围的矩阵, 可以成像和量化随着时间的96井板格式 (图 5A)。入侵可以通过量化细胞的表面积来测量, 因为它们会随着时间的推移入侵矩阵。使用图像分析软件, 应用了一个处理定义, 将掩码覆盖到单元格表面区域, 从而允许随着时间的推移自动量化 (图 5B)。还可以创建 BTSC 入侵的时间推移视频, 以方便地观察 BTSC 入侵的整个过程 (图 6)。此外, 荧光显微术可用于图像 BTSCs 的表达荧光蛋白, 如绿色荧光蛋白 (GFP) 或红色荧光蛋白 (RFP)。在这里, 我们创建了一个时间推移视频 BTSCs 表达一个胞浆形式的 GFP, 以帮助跟踪个别 BTSCs, 因为他们侵入基底膜样基质 (图 7)。这种 BTSC 干细胞入侵检测还允许在复制井中添加药物治疗, 以直接评估其对 BTSC 从 neurospheres 侵入母体的影响 (图 8A)。在复制井中多 neurospheres 的量化可以随着时间的推移进行计算和绘制, 以评估药物在多种浓度下的作用 (图 8B)。
如果不遵守上文所述的协议, 可能导致无法获得可靠和准确的数据。嵌入的 BTSC neurospheres 太大, 直径大于200µm, 导致焦点的平面太大, 无法精确地量化视野中的所有球体 (图 8C)。同样, 如果在嵌入 neurospheres 时不保持矩阵在4摄氏度, 也会导致不在同一平面上的球体和没有均匀凝固的胞外基质, 从而阻止了准确数据的收集 (图8D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58152/58152fig1.jpg"> 图 1: 培养 BTSCs.这些面板是一个典型的例子, BTSCs 生长从单一细胞到 neurospheres 在14维的文化时期。在这里显示的 neurospheres 在14天是一个适当的大小为传代。<a href="https://www.jove.com/files/ftp_upload/58152/58152fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58152/58152fig2.jpg"> 图 2: BTSC 迁移的活细胞成像.(a) 这是一个96良好的趋化移印板, 涂上一层薄薄的 i 型胶原蛋白, 然后再用 BTSCs 进行迁移试验。图像显示 i) 一个板块, 所有三部分 (盖子, 膜插入, 和储层) 放置在一起, ii) 全部三个部分分离, iii) 一个特写图像的顶部视图的膜插入, iv) 的特写图像的底部视图的膜插入和 v) 底部储层的特写图像。(B) 这些是实验开始 (0 小时) 和24小时 BTSC 迁移的代表性图像。图像聚焦在膜插入的顶端, 细胞最初是被镀的。迁移到趋化迁移板块底部的单元格以红色突出显示。在0小时, 几个细胞迁移到膜的底部。24小时后, 迁移的细胞数量急剧增加。用车辆与药物 X 治疗细胞的24小时图像比较表明, 药物治疗减少了 BTSC 迁移。刻度条代表600µm. (C) 本小组显示了在使用车辆或药物 x 预处理后进行 BTSC 迁移的量化。该图表明, 药物治疗对 BTSCs 的迁移有很强的影响。数据点是三技术复制井的平均值, 误差条表示标准偏差 (SD)。<a href="https://www.jove.com/files/ftp_upload/58152/58152fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58152/58152fig3.jpg"> 动画图 3: BTSC 迁移延时视频.该视频显示 BTSCs 的迁移使用 i 型胶原涂层趋化迁移板。膜插入顶部的细胞以黄色突出显示, 迁移到膜插入底部的细胞以红色突出显示, 膜中的孔隙以蓝色突出显示。焦点的平面被设置为膜底部的图像细胞。该视频是使用每小时 51 h 拍摄的时间间隔图像创建的, 每秒4帧编译。刻度条代表200µm.<a href="https://www.jove.com/files/ftp_upload/58152/JOVE_Fig_3.mov" target="_blank">请点击这里查看此视频。(右键单击可下载.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58152/58152fig4.jpg"> 图 4: 迁移分析设置中的错误示例.(A) 本小组展示了在趋化移印板块顶膜插入之前未完全分离的 BTSCs 的例子。由于球体的体积大, 膜孔体积小, 细胞无法迁移到底层油藏。(B) 本小组展示了一种迁移化验的例子, 其中有太多的细胞被电镀。细胞的聚集干扰细胞的迁移, 损害量化。(C) 本小组展示了在镀 BTSCs 的膜上插入气泡形成的例子。气泡导致图像质量较差, 并防止了精确的迁移量化。刻度条代表600µm.<a href="https://www.jove.com/files/ftp_upload/58152/58152fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58152/58152fig5.jpg"> 图 5: BTSC 干细胞入侵检测.这些面板显示了一个例子, BTSCs 入侵从干细胞成0.4 毫克/毫升 i 型胶原蛋白基质在6小时内。在这里, (A) 每个时间点的相位对比图像显示 (B) 与具有代表性的定量掩码覆盖, 如协议步骤3中详细描述的那样。<a href="https://www.jove.com/files/ftp_upload/58152/58152fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58152/58152fig6.jpg"> 图 6: BTSC 干细胞在 i 型胶原蛋白中侵入的延时视频.该视频显示从干细胞侵入 BTSCs 0.4 毫克/毫升 i 型胶原基质 (也显示为图 5A中静止图像)。这些图像是用10X 的目标每小时获取12小时, 每秒编译4帧。刻度条代表300µm.<a href="https://www.jove.com/files/ftp_upload/58152/JOVE_Fig_6.mov" target="_blank">请点击这里查看此视频。(右键单击可下载.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58152/58152fig7.jpg"> 图 7: BTSC 干细胞在基底膜状基质中侵入的时间推移视频.这个视频显示入侵 BTSCs, 即表达胞浆 GFP, 从干细胞成一个基底膜状基质。这些图像是使用4X 目标每30分钟48小时获得的, 每秒编译8帧。刻度条代表800µm.<a href="https://www.jove.com/files/ftp_upload/58152/JOVE_Fig_7.mov" target="_blank">请点击这里查看此视频。(右键单击可下载.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58152/58152fig8.jpg"> 图 8: BTSC 干细胞入侵检测与治疗.(A) BTSC neurospheres 嵌入在与车辆一起制备的基底膜状基质中, 或每小时采集到的药物 x 图像浓度;这里显示的是在电镀时和16小时后拍摄的图像。刻度条代表200µm. (B) 按照议定书步骤3的规定, 每小时对 BTSCs 从 neurospheres 侵入周围基质的情况进行量化。数据点是三技术复制井的平均值, 误差条表示平均值 (SEM) 的标准误差。(C) 此面板显示嵌入在 i 型胶原蛋白中的 BTSC neurospheres, 其球体太大, 无法量化。刻度条代表300µm. (D) 此面板显示 BTSC neurospheres 嵌入在未完全设置的 i 型胶原蛋白中。刻度条代表300µm.<a href="https://www.jove.com/files/ftp_upload/58152/58152fig8large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">胶原蛋白溶液制备</td>
		</tr>
<tr>
<td>库存 [胶原蛋白] (镁/毫升)</td>
			<td>5</td>
		</tr>
<tr>
<td>最终 [胶原蛋白] (镁/毫升)</td>
			<td>0。4</td>
		</tr>
<tr>
<td>所需水井数:</td>
			<td>5</td>
		</tr>
<tr>
<td>总容积 (ul)</td>
			<td>500</td>
		</tr>
<tr>
<td>库存胶原量 (ul)</td>
			<td>40</td>
		</tr>
<tr>
<td>1N 氢氧化钠 (ul) 容积</td>
			<td>0.92</td>
		</tr>
<tr>
<td>媒体量 (ul)</td>
			<td>459</td>
		</tr>
</tbody></table>表 1: 制备0.4 毫克/毫升型胶原蛋白溶液.列出的是已知的开始和期望的最终浓度的 i 型胶原蛋白溶液。列出的卷足以将 neurospheres 板成三个复制井, 再加两个用于超额, 并且可以包括单个处理条件, 如协议步骤3中详细描述的那样。

Watch this Scientific Journal Video about Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion at JoVE.com

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

在这里, 我们描述了活体细胞成像技术, 以定量测量的迁移和入侵胶质瘤脑肿瘤干细胞的时间和在多种治疗条件下。

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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JoVE Journal

Cancer Research

10.1K 次观看.  University of Calgary. 在这里, 我们描述了活体细胞成像技术, 以定量测量的迁移和入侵胶质瘤脑肿瘤干细胞的时间和在多种治疗条件下。

视频: Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

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. ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

脱细胞和细胞肺模型研究肿瘤转移

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

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 ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...