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>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-induced+cancer+cell+fusion:+a+mechanism+of+breast+cancer+metastasis.">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li><li>McArdle, T. J., Ogle, B. M., Noubissi, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types.">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li><li>Noubissi, F. K., Ogle, B. 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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?...

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><tbody><tr><td>MatTek P35G-1.5-10C</td><td>MatTek Corporation, Ashland, MA</td><td>P35G-1.5-10-C</td><td>mattek glass bottom dishes</td></tr><tr><td>Rat tail collagen I</td><td>Corning, Corning, NY, USA</td><td>354236</td><td>Collagen gel</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>H1758</td><td>HCl</td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>10010023</td><td>PBS</td></tr><tr><td>10X Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>D2429</td><td>10X DMEM</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Confocal Fluorescent microscope</td><td>Olympus America, Center Valley, PA, USA</td><td>Olympus IX81</td><td>Confocal&amp;nbsp; microscope</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td><td>SH30070.03</td><td>FBS</td></tr><tr><td>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><td>Imaris software</td><td>Bitplane Inc. Zurich, CH</td><td>Imaris 7.5.2</td><td>Imaris software</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>D9542</td><td>DAPI</td></tr><tr><td>Paraformaldehyde</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>50980487</td><td>PFA</td></tr><tr><td>&amp;alpha;-minimum essential medium</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>M0894</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC; Manassas, VA, USA</td><td>HBT-22</td><td></td></tr><tr><td>MSC</td><td>Trivedi and Hematti, 2007</td><td></td><td></td></tr><tr><td>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。?...

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

Universidad de Cartagena UDEC

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

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

科研

医学

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 ...

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 ...

癌症研究

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

生物学

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

发育生物学

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

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

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

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

癌症生物学中的 3D 模型:异球体中的侵袭动力学

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

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

作者聚焦:破译头颈癌进展过程中癌症和免疫细胞之间的串扰

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

分析 3D 培养物体外细胞侵袭的替代策略

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

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

向上移动: 一个简单而灵活的体外三维入侵检测协议

摘要

探索更多视频

此视频中的章节

球体的检测，以测量TGF -β诱导的入侵

3维建模和成像集体细胞的侵袭

三维流状入侵检测中的全人肿瘤基基质

一个 FluoroBlok肿瘤侵袭实验

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

一种改进

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

三维（3D）肿瘤球体侵袭试验