Name: three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation
Uploaded: 2016-08-18T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

注:没有道德的考虑，因为动物和人类癌细胞购买或慷慨地提供给我们。 1.使胶原蛋白插头（伪原发灶） <ol><li>制备胶原分散液。 I型从鼠尾腱（RTT）既可以提取胶原消毒如先前报道17，或购买。分散用混合机冻干RTT胶原（3.25-3.50毫克/毫升0.02N的乙酸）;对于均匀的混合（高速设置5 2分钟运行）。 </li><li>收获（胰蛋白酶-EDTA，通常情况下），并使用台盼蓝排除了使用血球计数活细胞。调整到所需的细胞密度（每插头5×10 4细胞）。 </li><li>制备在无菌条件下的所有解决方案（氢氧化钠，胎牛血清，DMEM 5倍， 碳酸氢钠 ）分别（ 表1），并保持在冰上冷却。注:加入各种解决方案的顺序是重要的，以防止渗透或酸性震荡细胞。 </li><li>进行细胞分散液（1.25×10 6个细胞）到最终的胶原溶液（5毫升）尽快。拌匀（通过上下吹打），同时避免气泡，然后在96孔板的每个孔中迅速分布200微升准备使用的溶液。轻轻敲击细胞培养罩的工作区表面上的多孔板，以去除气泡，并均匀地散布在孔内的溶液。 </li><li>填充所有孔之后（此步骤需要每96孔板约15-20分钟），将其存储到孵化器。 </li><li>从2小时到过夜孵育，在37℃下的板。胶原凝胶化（ 即 ，原纤维）在30分钟内发生。培养基（100μl/孔）添加到培养以进行过夜孵育。 </li></ol> 2.纤维蛋白胶的第一层<ol><li>纤维蛋白原溶液制备方法。 注:同一批次的纤维蛋白胶的理想情况下应该使用更重现resulTS，如血纤维蛋白凝胶形成可以商业冻干纤维蛋白原的不同批次之间有所不同。 <ol><li>始终使用新鲜配制的纤维蛋白原溶液。在打开的小瓶，以避免水合物晶体形成之前使冻干的纤维蛋白原至室温。 </li><li>逐渐溶解在预热（37℃）的Hank氏平衡盐溶液（HBSS）以钙 /镁离子以3毫克/毫升的工作浓度（考虑制备15％过量的最小最终体积的所需的纤维蛋白原: 例如 ，17.25毫克5.75毫升为5ml的溶液）。 <ol><li>添加预热的HBSS滴加起初以溶解纤维蛋白原片段。分解较大的片段与在烧杯刮刀。搅动烧杯不时以促进混合。在手术过程中不要使用搅拌器板。通过上下吹打悬浮液溶解剩余的粉末。 </li></ol></li><li>保持纤维蛋白原溶液不冷不热，而免缝使其通过一个0.22微米的过滤器LIZING溶液。注意:如果HBSS是不够暖或血纤维蛋白原不完全溶解，该溶液可能堵塞过滤器。如果适用的话，更换过滤器一次或两次，这可以降低纤维蛋白原浓度，从而纤维蛋白凝块的刚度。 </li><li>暂停的利益（ 例如 ，内皮细胞）的细胞分化为准备使用的纤维蛋白原溶液中，同时调整其最终体积，作为替代程序。 </li></ol></li><li>准备凝血酶解决方案。 <ol><li>准备在DDH 2 O（50 NIH单位/ ml）的储备液，然后用0.22微米的过滤消毒的。 </li><li>使用的纤维蛋白原/凝血酶比率≥1:0.0075（体积/体积），以生成血纤维蛋白凝胶。 </li></ol></li><li>生成纤维蛋白胶。 <ol><li>保持无菌纤维蛋白原和在所有的后续步骤凝血酶在冰上原液。血纤维蛋白凝胶允许形成在24孔板中。 </li><li>及时覆盖日每个电子表面很好地与纤维蛋白原溶液（200μl/孔），同时避免气泡形成。一次处理6个孔。 </li><li>一旦纤维蛋白原溶液完全覆盖孔的表面，倾斜板以45°角和通过丢弃凝血酶入井的中心添加1.5微升的凝血酶溶液到第一井，然后轻轻水平摇动板1-2秒。 <ol><li>离开板在层流罩（5-10分钟），直至凝胶化/凝固过程已经完成下一个稳定的位置（注:在聚合过程中必须不被干扰， 例如 ，通过输送板的孵化器）。 </li></ol></li><li>一旦第一个六口井已经聚合，重复相同的序列（ 即 3之前的步骤），在接下来的6口井，直到所有的水井都被处理。 </li></ol></li></ol> 3.纤维蛋白胶并夹塞胶原第二层<ol><li>选项答:（使用胶原蛋白立即插件）。 <ol><li>确保纤维蛋白胶的第一层已在所有孔中通过微妙地倾斜板聚合。放置装有由侧的胶原凝胶插头侧与24孔板（包含纤维蛋白凝胶）的96孔板来缓解胶原插头传送。 </li><li>添加HBSS一滴到含有胶原塞板的各孔中。 </li><li>除去从井各胶原插头与安装在一注射器（用作把手）细针或使用微勺（见视频）。每个胶原插头传送到使用一个或两个微匙第一纤维蛋白凝胶层，同时确保胶原塞孔中心到井和无菌状态保持良好。 </li><li>用的纤维蛋白原溶液的第二层覆盖在预先形成的纤维蛋白胶（300微升/孔），并在2.3中描述介绍凝血酶，保持一个最小的1:在一个时间0.0075比率和六个孔中的序列&lt;/ LI&gt; </ol></li><li>选项​​B（涂敷胶原插头与生长因子减少基底膜（GFRBM）的薄层）。 <ol><li>酷派所有准备的解决方案和手段事先并自GFRBM冷冻等分处理过程中让他们在4℃或冰上（ 例如 ，移液器，吸头，试管）的解冻过程中过度加热速度非常敏感（按照制造商的说明） 。 </li><li>从板孔中以下的去除，浸泡各胶原插头2分钟在1.5毫升离心管中含有100μl的纯GRFBM溶液的冰上。 </li><li>每个涂覆插头转印到第一纤维层，同时确保它是良好居中，如前面所述。在37°C孵育含插件板5分钟，以允许GRFBM以形成凝胶。添加第二个纤维层在步骤3.1.4。 </li></ol></li></ol> 4.细胞培养基条件<ol><li>用培养基填充每口井（400微升）。培养基和补充剂将基于所述细胞系和实验条件中选择。 </li><li>在100激肽释放酶抑制剂单位（KIU）/ ml的终浓度添加抑肽酶，一个抗纤维蛋白溶解剂，培养基中。 注:在用于测试的细胞系的条件下的细胞培养孵化器存放板。 </li><li>补充用新鲜培养基培养隔日或根据实验时间表，并添加抑肽酶。加入新鲜培养基之前，稍有倾斜板（以30-35°角）和倾斜吸管针对井的侧，同时小心抽吸下恒定观测条件培养基。 </li></ol>

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The authors have no disclosure.

作为一个重要的技术脚注，至关重要的是，没有间隙存在于中央和周边的凝胶之间的接口。否则，可能会降低细胞迁移/侵袭纤维蛋白凝胶的能力。如果凝血酶未被适当地稀释的胶原蛋白和纤维蛋白凝胶之间的空间可以培养的第24小时的过程中可形成。它也可能是测试可能导致的胶原蛋白凝胶的细胞系培养过程中收缩，从而导致相当大的空间都凝胶之间形成。当基质细胞中的胶原凝胶与癌细胞由于?...

调查癌细胞侵袭/迁移和随后的转移机构的基本和生物医学特征是强烈的研究1,2的主题。转移是癌症的最终阶段，临床管理仍然遥遥无期。在细胞和分子水平上更好地了解转移将使更有效的疗法3的发展。 转移细胞的几个属性可以在体外 4包括它们的干性和潜在的获得的过渡状态（ 例如 ，上皮-间质转化）迁移和内部以及从原发肿瘤5侵入进行探讨。然而，侵袭/转移过程的体外评估一直是一个挑战，因为它几乎排除了血液/淋巴循环的贡献。在胶原凝胶中嵌入肿瘤碎片器官文化有页上一页狡猾被用于监测癌症的侵袭性。虽然肿瘤的复杂性被保留（ 例如 ，非癌细胞的存在），肿瘤片段暴露于有限介质扩散，以采样变化，并且对基质细胞6的过度生长。另一种方法包括在细胞外基质（ECM），它模仿了三维（3D）细胞环境的组分中生长的癌细胞。的乳腺癌细胞系中胶原凝胶和/或基底膜衍生基质的扩散是之中三维细胞培养物的最佳表征的例子。通过使用特定的三维细胞培养物的环境中，在标准条件下生长的乳腺癌细胞中观察到的紊乱组件可被反向以自发形成乳腺腺泡和管状结构7-10的。此外，来自腺癌细胞的多细胞肿瘤球状体的形成使用不同的技术（聚集例如，悬滴，浮球体，琼脂嵌入）现在构成了最常用的三维细胞培养物测定法11-13。然而，该测定是由限制集合的癌细胞系可以形成球状体，并通过提供研究细胞在这些条件下，短周期的限制。 在这种可视化技术，我们这里所介绍其中感兴趣癌细胞嵌入在胶原凝胶，以允许可替代地涂覆有基底膜衍生矩阵的伪原发肿瘤的体外形成复杂的三维细胞培养物测定法。一旦形成，所述伪原发性肿瘤，然后夹在无细胞基质（在本例中纤维蛋白胶），它允许癌细胞跨越两个矩阵隔间之间的界面（参见图1）。有趣的是，从伪原发性肿瘤始发侵袭性癌细胞沿继发性肿瘤样结构出现在纤维蛋白胶。这样的三维培养系统提供调查所需的灵活性，例如，抗癌药物，基因表达和细胞-细胞和/或细胞-ECM相互作用14-16。 <img alt="图1" src="/files/ftp_upload/54322/54322fig1.jpg" /> 图 1: 该方法的概述 ，生成三维细胞培养系统作为癌症研究的模型方法的原理总结<a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Freeze-dried collagen</td><td>Sigma-Aldrich</td><td>C7661</td><td>from rat tail tendon (soluble dispersion) or home-made (see Rajan &lt;i&gt;et al&lt;/i&gt;., ref.#14)</td></tr><tr><td>Fibrinogen (freeze-dried)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F8630</td><td>Type I-S, 65 - 85% protein with &amp;ge; 75% of protein is clottable</td></tr><tr><td>Thrombin</td><td>EMD Chemicals Inc.</td><td>605157</td><td>&amp;nbsp;Gibbstown, NJ; NIH units/mg dry weight&amp;nbsp;</td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</td><td>Corning</td><td>356234</td><td>Previously from BD Biosciences</td></tr><tr><td>Aprotinin</td><td>Sigma-Aldrich</td><td>A6279&amp;nbsp;</td><td>&amp;nbsp;solution at 5 - 10T IU/ml (Trypsin Inhibitor Unit)&amp;nbsp;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Micro-spoons</td><td>Fisher Scientific</td><td>2140115</td><td>Fisherbrand Handi-Hold Microspatula</td></tr><tr><td>96 well plate, round base</td><td>Sarstedt</td><td>3925500</td><td></td></tr><tr><td>24 well plate</td><td>Sarstedt</td><td>3922</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s Medium</td><td>Sigma Chemical, Co.</td><td>D5546</td><td>DMEM</td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>CAA15-701</td><td>FBS, Canadian origin.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma Chemical, Co.</td><td>T4049</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>Sigma Chemical, Co.</td><td>H8264</td><td>HBSS</td></tr></tbody></table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

在单层哺乳动物细胞培养被广泛用于研究各种生理和分子过程。然而，这种方法来研究生长的细胞往往会产生不希望的痕迹。因此，在一个三维（3D）环境的细胞培养，通常使用细胞外基质组分，成为一个有趣的选择，因为它接近相似的体内组织或器官中的原生。我们开发了使用两个室，即（i）一个三维细胞培养系统包含嵌入在胶原凝胶充当伪主macrospherical肿瘤和（ii）的周边无细胞隔室制成的纤维蛋白凝胶的癌细胞的中心隔室， 即细胞外基质组分从在中心使用的不同，其中，癌症细胞可以迁移（侵入前）和/或形成代表仲或卫星肿瘤微球肿瘤。卫星肿瘤的外围隔室的形成是显着相关与天然肿瘤细胞的已知攻击性或转移性原点，这使得这种三维培养系统是唯一的。这种细胞培养物的方法可以考虑以评估癌细胞侵袭和运动性，细胞 - 细胞外基质相互作用并作为评价抗癌药物性能的方法。

如前面提到的，这个三维细胞培养物测定法的一个有趣的特征是肿瘤细胞不仅可以从胶原插头向相邻的血纤维蛋白凝胶迁移，而且还建立继发性肿瘤（ 例如 ，卫星瘤样结构）。这可以在用凝胶厚度低和高放大倍数的倒置相差显微镜直接观察到，尤其是具有长工作距离聚光镜（ 图2）。采用这种三维细胞培养的方法，公知的转移性细胞的行为可以容易地?...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

Research

JoVE Journal

Medicine

10.3K 次观看.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

视频: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

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

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

癌症研究

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

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 ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

Source: Cvetkovi&#263;, D., et al. <a href="https://www.jove.com/v/51341/quantification-breast-cancer-cell-invasiveness-using-three">Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model.</a> J. Vis. Exp. (2014).
This video provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment.

3D 培养基底膜测定:在 3D 基底膜蛋白基质上培养癌细胞以研究细胞侵袭

Source: Cvetkovi&#263;, D., et al. Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model. J. Vis. Exp. (2014).
This video ...

JoVE Encyclopedia of Experiments

乳腺癌

In vitro studies

3-Dimensional Culture of Lung Carcinoma Cells: A Method To Study Cell-Matrix Interactions

Source: Chen, S. et. al. <a href="https://www.jove.com/v/60644/multidimensional-coculture-system-to-model-lung-squamous-carcinoma">Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression</a>. J. Vis. Exp. (2020).
This video describes the technique of 3-D culture of lung carcinoma cells to study the cell-matrix interactions. In the protocol, we will perform the 3D culture of TUM622 lung carcinoma cells to explore its potential of forming organoids in vitro.

肺癌细胞的 3 维培养:一种研究细胞-基质相互作用的方法

Source: Chen, S. et. al. Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression. J. Vis. Exp. (2020).
This video describes the ...