这项工作得到了国家卫生研究院 (NIMHD-G12MD007581) 的支持, 通过 RCMI 环境健康中心在杰克逊州立大学和美国国防部, 想法奖 11-1-0205。

注意: 此协议在麦卡德尔et中描述。11. 在图 1中提供了时间线。
1. 培养板的制备
<ol><li>用1毫升1米盐酸 (HCl) 洗涤35毫米培养皿, 含10毫米的玻璃底井, 以降低玻璃底部的疏水性。</li>
	<li>用1毫升磷酸盐缓冲盐水 (PBS) 洗两次培养板, 除去所有的 HCl。</li>
	<li>用1毫升70% 乙醇洗两次培养板。</li>
	<li>用1毫升的细胞特定培养基冲洗培养基 (在这里, 使用α-记忆补充10% 热灭活血清和1% 非必需氨基酸)。 注意:包含培养基培养基的经处理的培养板可存储在 CO2孵化器中, 其加湿器为37摄氏度, 至第二天。</li>
	<li>生长细胞 (MSCs 和 MCF-7 共培养) 在被处理的玻璃底部井的35毫米培养皿到100% 融合。共培养3.5万的 MSC 细胞和 7.5万 MCF7 细胞为 24 h。</li>
</ol>2. 胶原凝胶的制备
<ol><li>存储用于吹打胶原凝胶的微提示, 防止胶原蛋白在接触时聚合。 注意:这是最关键的一步。</li>
	<li>根据胶原蛋白的浓度, 确定大鼠尾胶原的体积。制备100µL 胶原凝胶。 注意:高浓度的大鼠尾胶原我的股票工作更好。确定 10x Dulbecco 的改性鹰培养基的体积, 以相应稀释胶原蛋白。</li>
	<li>结合冰鼠尾胶原蛋白 I (3.8 µg/毫升), 10x Dulbecco 的改良鹰的培养基和1x 细胞培养培养基补充2% 胎牛血清和适当的趋化蛋白, 以最终浓度的胶原2.4 毫克/毫升。</li>
	<li>加入4.5% 碳酸氢钠溶液的混合物中和胶原凝胶。 注意:所添加的碳酸氢钠溶液的体积根据其他试剂的确切 pH 值可能会有所不同。最好是用 ph 条来测试混合物, 以确保中性溶液, 或者在培养基中使用 ph 指示剂。</li>
	<li>放置80µL 的胶原蛋白混合物在细胞内的玻璃底部井的文化菜。</li>
	<li>允许胶原凝胶在 CO2孵化器中聚合30分钟, 加湿器为37摄氏度。</li>
	<li>用2毫升的细胞培养基与减少的血清 (2%) 覆盖胶原凝胶, 并在 CO2孵化器中孵育37摄氏度的细胞, 其中有24、48或 72 h 的加湿器。 警告:该钢板应小心处理, 以防止胶原凝胶从玻璃底部分离。</li>
</ol>3. 用3.8 µg/毫升的大鼠尾胶原蛋白制备胶原凝胶的实例
<ol><li>在冰上, 结合114.5 µL 的大鼠尾胶原蛋白, 16.6 µL 10x DMEM, 33.1 µL 的培养基中含有趋化因子。</li>
	<li>用14.0 µL 碳酸氢钠中和胶原蛋白混合物。</li>
	<li>继续执行步骤2.5。</li>
</ol>4. 细胞的成像
<ol><li>轻轻地从盘子中取出介质。</li>
	<li>用1毫升的 PBS 轻轻冲洗凝胶。</li>
	<li>用1毫升4% 多聚甲醛的细胞固定15分钟。</li>
	<li>用1毫升的 PBS 冲洗两次细胞。</li>
	<li>在室温下用 DAPI 溶液 (100 ng/毫升) 将细胞染色20分钟。</li>
	<li>在不同位置使用共聚焦显微镜对细胞进行图像处理, 并在16µm 步骤中拍摄每个位置的400µm z 叠加图像。使用的显微镜有一个磁盘扫描单元, 使共焦成像使用白光, 弧励磁源。使用20X 透镜, 数值孔径为0.75。</li>
	<li>重建图像。<ol>
<li>打开一个给定的凝胶 (大约25图像) 的图像, 并通过点击图像→堆栈→图像堆叠创建图像栈。第一个图像对应于凝胶的底部 (z = 0 µm), 第二个图像对应于第一步在 z 方向 (z = 16 µm), 第三个图象对应了到第二个步在 z 方向 (z = 32 µm)。评估每个核在每个图像的深度和指定的深度, 核是最尖锐的焦点作为入侵深度的特定细胞。</li>
	</ol>
</li>
</ol>

<ol>
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提交人宣布, 没有任何竞争利益存在。

在这里, 我们描述了一个简单的倒置垂直入侵检测, 这是方便的各种细胞类型, 包括细胞是罕见的和/或依赖于其微环境。在这3D 入侵检测中, 细胞保持在原微环境中, 并被含有趋化蛋白的胶原凝胶所覆盖。细胞然后迁移和侵入胶原蛋白。在这种化验中, 细胞可以被染色和容易地监测在凝胶内。该方法的简便、重现性使其成为研究3D 系统中癌细胞迁移和侵袭的方便工具。然而, 强烈的趋化因子似乎需要打破细胞亲和的文化菜的底部。
对35毫米培养皿中的倒置垂直侵入试验进行了优化。这种文化菜有一个10毫米的玻璃底部井, 这是适合成像。这种培养皿的一个限制是需要大量的胶原凝胶来进行化验。寻找更小的文化菜肴与图像兼容的设计将更经济。适合 35 mm 菜肴设计的凝胶层由于其强度很细腻, 如果不小心处理, 可以很容易地从底板中分离出来。不同的细胞培养设计可以提高胶原凝胶层的稳定性, 但要优化胶原凝胶强度与细胞活力的相容性。
尽管有这些局限性, 反向垂直入侵检测相比其他3D 入侵检测格式具有许多优点。主要的好处是细胞留在其原始的文化环境中, 这不是在博伊登室化验的情况。该协议保留细胞微环境, 有利于严格控制的细胞微环境, 如干细胞15。此检测还可防止细胞损伤或细胞丢失, 特别是对于微妙或罕见的10,11的细胞。此外, 通过这种检测, 细胞形态学和相关的动力学可以评估在凝胶, 这是不可能的博伊登室化验。此外, 非生理聚碳酸酯或聚丙烯过滤器的博伊登室化验系统不使用的倒置垂直侵入试验。倒置垂直入侵检测是一种静态设计, 它不模拟微流控入侵检测设计提供的体内动态环境, 但它提供了一个更简单、更低挑战性和成本效益高的3D 环境来量化在保存和控制的微环境中迁移和侵入多种细胞类型。
倒置垂直入侵检测在病理生理学研究中有许多潜在的应用。这种方法可以作为一个体外平台, 用于研究胚胎发育、创面愈合和/或炎症反应中细胞的运动情况3。在迁移和入侵过程中, 与 ECM 和其他细胞类型的细胞相互作用也可以使用该协议进行研究。这种化验也可作为初步药物筛选抗转移药物的有用工具。采用倒置垂直侵袭试验, 建立了器官特异性肿瘤转移模型体系。
为了规避某些细胞从原微环境中的位移在博伊登室检测系统中的迁移, 提出了倒置垂直入侵检测方法。本试验简便、可靠、重现性好;它是灵活的, 可用于量化各种细胞类型的侵入电位, 包括敏感和稀有细胞类型。该方法可用于检测与基质降解相关的迁移活动, 也可用于研究不同基质基质的选择性降解活性。

细胞运动是细胞的正常发育和生理过程。然而, 细胞迁移和侵袭的模式和能力的变化与病理状况有关, 包括炎症性疾病和肿瘤转移1,2,3。转移可以说是癌症中最不了解的方面。对于转移, 癌细胞必须降解额外的细胞基质 (ECM), 侵入局部组织和 intravasate。一旦循环, 癌细胞就会传播到远处的部位, extravasate 并形成次生肿瘤。因此, 细胞降解 ECM、迁移和入侵的能力与它们的转移电位有关。对细胞的迁移和入侵能力进行了分析和量化, 并提出了不同的方法。最常用的方法包括伤口愈合试验、时间推移显微镜和博伊登室化验。创伤愈合试验4和时间失效显微镜是简单和方便的化验, 将初步了解细胞迁移。但是, 这两种检测都是2D 检测, 不反映单元格存在体内的3D 环境。研究表明, 与2D 一个5、6相比, 单元格对3D 环境的响应不同。此外, 伤口愈合化验难以重现, 并产生定量结果7,8,9。细胞排斥区的检测, 这是一个3D 修改的伤口愈合化验, 是一个更生理学相关的化验, 但它不适合的细胞数低, 如混合细胞产生的细胞融合事件10,11.
博伊登室化验是一项3维的化验, 为细胞研究提供相关的微环境。然而, 它要求细胞从原来的微环境中移除, 并存放到博伊登化验系统的上腔中, 以便向含有培养基的趋化因子迁移并向下侵入。此3D 方法不适用于分析易受其微环境影响的细胞的迁移和入侵能力, 并且/或限制在数字10、11、12中。一个新的方法来分析细胞迁移和入侵是微流控梯度室化验13。虽然在迁移和入侵研究中, 这种方法是适当和可靠的, 但这种检测方法更昂贵, 在技术上对一个典型的实验室具有挑战性。这里我们描述了一个简单的倒置垂直入侵检测, 它与许多细胞类型兼容, 包括对其微环境敏感的细胞和/或数量限制。这项化验是根据霍普. et . 提出的协议改编的。14. 在本实验中, 细胞保持在原微环境中, 当它们在含有趋化因子11的胶原凝胶中向上移动时, 它们的迁移和侵入受到监测。本试验重现性好, 在35毫米培养皿中进行了优化和验证。它可以适应不同的检测条件, 包括不同的细胞培养大小, 不同的基质或黏附力, 以及不同的趋化因子。这种检测可以作为一种体外平台, 用于药物发现过程的初步筛选。

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">MatTek P35G-1.5-10C</td>
			<td style="width:269px;">MatTek Corporation, Ashland, MA</td>
			<td style="width:151px;">P35G-1.5-10-C</td>
			<td style="width:232px;">mattek glass bottom dishes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I</td>
			<td>Corning, Corning, NY, USA</td>
			<td align="right">354236</td>
			<td>Collagen gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>H1758</td>
			<td>HCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">10010023</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>D2429</td>
			<td>10X DMEM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Fluorescent microscope</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>Olympus IX81</td>
			<td>Confocal&#160; microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defined Fetal Bovine Serum</td>
			<td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td>
			<td>SH30070.03</td>
			<td>FBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ti:Sapphire multi-photon laser scanning microscope</td>
			<td>Prarie Technologies, Sioux Falls, SD, USA</td>
			<td>40x NA 0.8 objective</td>
			<td>MPLSM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris software</td>
			<td>Bitplane Inc. Zurich, CH</td>
			<td>Imaris 7.5.2</td>
			<td>Imaris software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td style="width:269px;">Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>D9542</td>
			<td>DAPI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">50980487</td>
			<td>PFA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;-minimum essential medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MDA-MB-231</td>
			<td>ATCC; Manassas, VA, USA</td>
			<td>HBT-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MSC</td>
			<td>Trivedi and Hematti, 2007</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20X lens UPLAPO</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>36-064</td>
			<td>Olympus UPLSAPO 20X Objective</td>
		</tr>
	</tbody>
</table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

尽管已经开发了3D 种入侵检测, 但在不影响其微环境完整性的情况下研究细胞仍然是一个挑战。传统的3D 检测 (如博伊登室) 要求细胞从原始的文化位置迁移到新的环境中。这不仅扰乱了微环境固有的细胞过程, 而且常常导致细胞的丧失。这些问题在处理罕见的细胞, 或对其微环境极其敏感时尤其具有挑战性。在这里, 我们描述了一个3D 入侵检测的发展, 避免了这两个问题。在这个实验中, 细胞被镀在一个小井里, 一个含有趋化因子的 ECM 矩阵被放置在细胞之上。这不需要细胞移位, 并且允许细胞向上侵入母体。在这种检测中, 细胞侵袭以及细胞形态学可以在胶原凝胶内进行评估。利用这一方法, 我们对稀有和敏感细胞的侵袭能力进行了表征;由乳腺癌细胞 MCF7 和间充质/多向干细胞 (MSCs) 融合而成的混合细胞。

我们使用了一个35毫米培养皿与玻璃底部和鼠尾胶原 i 开发和优化体外3D 入侵检测协议。利用这一方法, 我们表明, 乳腺癌细胞 MCF7 和 MSC 细胞可能侵入胶原凝胶到平均距离 0.04 1.8 µm, 41.3 IGF1 76.0 µm 分别在 48 h 时 (18.6 ng/毫升) 被用作趋化因子 (图 2)。更重要的是, 我们表明, 从 MSC 和 MCF7 细胞的融合产品, 这是罕见的细胞, 可能侵入胶原凝胶以及。他们的平均入侵距离在48小时是 10.7 12.4 µm 当胰岛素样生长因子 I 在 18.6 ng/毫升被使用作为趋化因子 (图 2)。MCF7 与骨髓间充质干细胞入侵能力差异有统计学意义 (P < 0.01)11。融合产物与 MCF7 或 MCSs 的入侵能力差异无统计学意义。然而, 这种检测一致表明, 癌细胞融合产品具有更大的迁移和侵入能力比父母的癌细胞, MCF7。
细胞在整个实验中保持健康, 如其形态学和数量 48 h 后凝胶聚合 (图 3a) 所示。采用倒置垂直侵入试验设计, 可以分析细胞迁移和入侵的动力学 (图 3b, 4) 和细胞形态学可以评估 (图 4)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56568/56568fig1.jpg"> 图 1: 反向垂直入侵检测设计的时间线  <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56568/56568fig1large.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/56568/56568fig2.jpg"> 图 2: 肿瘤细胞融合产品的侵袭能力.采用倒置垂直侵入法测定肿瘤细胞融合产物的侵袭能力。数字代表四独立融合产品的平均入侵能力从三个分离的联合文化和他们的父母细胞线分析从八个不同的领域在每个试验之内。本实验以三项进行。方差分析与 Tukey 的 HSD 后临时测试。** < 0.01。误差线表示标准偏差 (SD)。此数字已从11中进行了调整。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56568/56568fig2large.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/56568/56568fig3.jpg"> 图 3: 倒置垂直入侵检测的细胞活力和 Z 系列. a) 20X 亮场图像, 显示两天后胶原凝胶下细胞的形态。从左到右 MCF7 和理学硕士, MCF7 和理学硕士;b) 表示 z 系列显示两个位置 (i 和 ii) 在一个菜从底部到顶部。连续的图像是16µm 分开。3.8% 的血清被用作趋化因子。两个面板上的绿色圆圈表示入侵细胞;注意, 在底部的凝胶和焦点在顶部凝胶, 侵入到凝胶。两个面板上的红色圆圈表示 z 系列 (非侵入单元格) 底部的单元格。刻度条 (a):15 µm;刻度条 (b):10 µm。此数字已从11中进行了调整。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56568/56568fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56568/56568fig4.jpg"> 图 4: 倒置垂直侵入检测中的细胞形态学.从底部 (z = 0 µm) 到 Z = 20 µm 的胶原凝胶中的 MSCs 的形态学. 3.8% 的血清被用作趋化因子。长箭指的是 MSC 侵入凝胶的附属物。箭头表示一个 MSC, 它沿着凝胶的底部对齐, 而不是侵入。蓝色显示核 DAPI 染色, 而红色显示罗丹明染色肌动蛋白丝。此数字已从11中进行了调整。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56568/56568fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

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

该协议描述了一种倒置垂直入侵检测方法, 可用于量化三维环境中细胞的迁移和入侵能力, 同时保持细胞微环境。本试验适用于稀有和/或敏感细胞。

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. ...

moving-upwards-simple-flexible-vitro-three-dimensional-invasion-assay

Nanyang Technological University

Research

JoVE Journal

Cancer Research

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

6.0K 次观看.  University of Minnesota - Twin Cities. 该协议描述了一种倒置垂直入侵检测方法, 可用于量化三维环境中细胞的迁移和入侵能力, 同时保持细胞微环境。本试验适用于稀有和/或敏感细胞。

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

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

科研

癌症研究

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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 ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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 ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

研究结肠癌干细胞的球体三维模型

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...