这项工作得到了p53lab（BMSI、A*STAR）和JCO VIP赠款（A*STAR）的核心预算的支持。

HEK293细胞系用于协议，并在杜尔贝科的改良鹰介质中培养，补充10%胎儿牛血清，2米L-谷氨酰胺和100U/mL青霉素/链霉素。细胞在37°C的培养下，在潮湿 的环境中产生5%的二氧化碳。
1. 通过 eIF4E 对 eIF4F 复杂中断进行定量评估:eIF4G604-646 补充分析
<ol><li>eIF4E 的细胞培养和瞬态瞬变:eIF4G604-646 补充测定<ol>
<li>使用新鲜解冻的细胞，所有实验的通道少于20个。在第 1 天，使用标准细胞计数器确定细胞编号，并使用 Trypan 蓝色排除方法13计算总可行细胞。</li>
		<li>种子 6 井板与 0.9 - 1.2 x 106 的 HEK 293 细胞每井在 2 mL 的标准生长介质. 注意:为了在上述细胞系内实现最佳的转接效率，确保镀膜细胞在播种后的第二天达到70-90%的汇流。</li>
		<li>在第2天早晨，使用脂基转染试剂（见材料表）与 SubA-eIF4E 和 eIF4G604-646-SubB 质粒共同传输细胞（见下文所述）。<ol>
<li>在含有 125μL 的减少血清介质的管中稀释 9μL 脂质溶液，每次转染时不含酚红色（见 材料表），并在室温下孵育 5 分钟。</li>
			<li>通过在 125 μL 的减少血清介质中稀释每个质粒中的 3μg 来为每个输血管准备 DNA 的主混合。</li>
			<li>将 12 μL 的增强剂加入 DNA 主混合管中，混合良好，并立即添加 DNA:增强剂主混合到稀释脂体的每个管中，比例为 1:1。在室温下孵育15分钟。</li>
			<li>将DNA脂质复合物添加到每口井中，并在37°C的孵化器中孵化细胞，24小时使用5%的二氧化碳 。</li>
		</ol>
</li>
		<li>在第 3 天的早晨，用 1 mL 的 PBS 冲洗每个油井。</li>
		<li>在 37 °C 下，用 0.3 mL 的三氯苯丙辛在每口井中取出 PBS 并孵育细胞 5 分钟。</li>
		<li>通过添加2mL的减少血清介质，在每口井中不含0%FCS的酚红色，将受感染的细胞转移到15mL管中，中和三氯苯酚素。 注:酚红色会干扰路西法酶的活动:因此，细胞必须从这一步开始在中等处理，没有酚红色。</li>
		<li>在290 x g 下旋转细胞5分钟，吸气介质。在没有含有 0% FCS 的酚红色的情况下，在减少的血清介质的 2 mL 中补充细胞。按步骤 1.1 中描述的计数。</li>
		<li>种子在 96 个不透明板中传输了 HEK 293 个细胞，在 90μL 的介质中，每口油井的密度为 30，000 个细胞，没有含有 0% FCS 的酚红色。为了在同一实验中为3种不同的化合物获得3个技术复制品，60个板材的种子井，除了边缘的油井外，还有细胞。</li>
		<li>在播种受感染的细胞后，立即添加 10 μL 的 10% DMSO 复合溶液（参见步骤 1.2）。</li>
	</ol></li>
	<li>复合制备<ol>
<li>通过溶解 100% DMSO（v/v） 中的复利复利，准备 1 mM 复合库存解决方案。为了每个复合滴定有 3 个复制品，请使用 1 mM 复合库存溶液中的 8 μL。 注:所使用的1mM库存化合物的体积大于所需量。这样做是为了考虑管道错误（如果有）。</li>
		<li>执行库存复合溶液的 2 倍连续稀释，每个滴定点将 1 mM 库存中的 4μL 放入 4 μL 100% DMSO 中。 注意:对于完整的复合滴定，在 100% DMSO 中从起始库存执行 9 次连续稀释。如果需要测试多个化合物，请使用 96 井板和多通道移液器来促进这一步骤和随后的稀释。</li>
		<li>在 2 倍串行稀释的每个点添加 36μL HPLC 级无菌水，以在 10% DMSO （v/v） （即 100 μM 至 0.39 μM 10% DMSO 库存系列将导致最终解决方案为 10 μM 至 0.039 μM，添加到单元格时为 1% DMSO）。至于治疗控制，还要在HPLC级无菌水中制备10%的DMSO仅存量溶液。</li>
		<li>在 96 个不透明板中的细胞中加入 10μL 的 10 倍工作解决方案，以产生预期的最终浓度，剩余 DMSO 浓度为 1%（v/v），总容量为 100 μL，在 37 °C 时孵育 3 小时，其中 5% v/v 大气 CO2。</li>
	</ol></li>
	<li>路西法酶补充和可行性检测<ol>
<li>经过3小时的药物化，开始准备路西法酶基板试剂，将1卷基板与19卷稀释试剂相结合（见制造商的说明）。 注:路西法酶检测使用市售套件执行（参见 材料表）。</li>
		<li>使用多通道移液器立即添加 25 μL 的基板试剂。</li>
		<li>在室温下，在轨道摇床上以 350 rpm 摇动板 50 分钟。</li>
		<li>使用板阅读器评估发光。为此，将镜像读卡器设置为发光，将发射滤镜设置为 455。使用 6.5 mm 的测量高度，测量时间为 1 s。</li>
		<li>为了评估细胞的生存能力，添加 33 μL 的可生存性检测试剂，然后在室温下 15 分钟后再次测量发光，使用板阅读器。</li>
		<li>通过在发光上设置镜面读卡器，在 600 nm 上设置排放过滤器，用板阅读器评估发光。使用 6.5 mm 的测量高度，测量时间为 1 s。 注:根据制造商的说明，通过测量ATP在细胞裂解上的细胞内水平来评估可行性。在这种情况下，多路复用是可能的，因为可行性检测使用不同的路西法酶与不同的发射波长。</li>
		<li>使用数据通过将数据与 4 个参数拟合曲线方程相适应的曲线来确定每个化合物的 IC50 值: Y = （A-D）/（1+x/C）=B= D 在曲线拟合过程中必须限制数字值 D 和 A: D = Y 轴上的发光值，用于从 eIF4E:4G 补充检测中获得最小曲线无同位素或最低理论响应水平。 A = Y 轴上的发光值，用于从 eIF4E:4G 互补测定中预期的最大曲线无同位素或最大理论水平响应。 注意:最小响应的价值来自 1% DMSO （v/v） 控制处理，最大响应的价值来自高亲和力 mTOR 抑制剂的最大响应，这种抑制剂已经稳定下来，对细胞生存能力没有影响。</li>
	</ol></li>
</ol>2. 相关 eIF4E:eIF4G604-646 检测抑制与 eIF4F 在细胞中的复杂干扰
<ol><li>为了确认通过测定测量的信号与化合物 eIF4E-eIF4G 相互作用的物理中断相对应，第 1.1 步中描述的种子细胞。</li>
	<li>后天，用不含酚红色的1mL血清介质替换介质，用复合物孵育4小时。确保剩余 DMSO 浓度为 1%（v/v），总容量为 1 mL。 注意:为了轻松地允许与结果相关联，请在测量的补充测定滴定曲线的开头、中点和终点使用复合浓度。</li>
	<li>Lyse 细胞并执行 m7GTP 向下拉，以分离 eIF4F 和 eIF4E:4EBP1 复合体。关于如何执行m7GTP下拉实验的详细程序可以在其他地方找到14。</li>
	<li>通过西式污点分析检测 m7GTP 绑定 eIF4G、eIF4E 和 4EBP1 蛋白质水平，然后与 eIF4F 复杂干扰与补充检测信号抑制相关联。</li>
</ol>

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作者没有什么可透露的。

本文所述方法采用基于路西法酶的补充测定法，通过直接测量活细胞中的eIF4G-eIF4E相互作用，对eIF4F复合组件进行定量监测。我们提供了使用eIF4E-eIF4G补充系统的详细信息，也表明该系统在测量eIF4E-eIF4G相互作用16的药物诱导4EBP1介导分离方面非常准确。为了便于此检测的吞吐量，本文中描述的实验设置已设计为 96 井微板格式的使用。
为了获得最佳结果，在执?...

基因表达控制在正确执行细胞程序（如生长增殖和分化）方面起着至关重要的作用。监管控制机制可以在基因转录水平或mRNA转化水平上进行。在过去十年中，越来越明显的是，通过调节启动过程而不是后来的拉长和终止步骤进行转化控制，可以精细地调节具有广泛生物功能的蛋白质特定子集的合成。
参与生存、抗自噬和抗凋亡反应的 mRNA 的翻译增加与几种癌症有关，并且与异常激活或过度表达翻译启动因子1有因果关系。
eIF4F 综合体是翻译启动的主调节器。通过将上限结构与 mRNA 的 5' 端绑定，eIF4F 正在推动初始 mRNA-里博索姆的招聘，进而提高翻译不力的真核素 mRNA2的 mRNA 翻译效率。据报道，许多癌症模型都存在异常激活RAS/MAPK或AKT/TOR通路的癌症模型，eIF4F的中介翻译，这表明癌细胞会调节eIF4F，以增强自身的抗肿瘤活性。通过抑制eIF4F复合形成来破坏这个进气回路，因此是一个非常有希望的治疗策略3，4。
eIF4F 综合体由 （i） eIF4E 组成， eIF4F 的帽绑定子单位，与 mRNA 的 5' UTR、（ii） eIF4A、RNA 六氯酶和 （iii） eIF4G 的上限结构相互作用，后者是与 eIF4A 和 eIF4E 相互作用的脚手架蛋白，最终招募了 40S 核糖核酸亚单位5。eIF4G 与 eIF4E 关联是功能 eIF4F 复合体组装的限速步骤，由 eIF4E 结合蛋白（4EBP、成员 1、2 和3）6负调节。通过由规范和非规范 eIF4E 绑定序列7、8、9（人类 eIF4E 上的 aa 604-646 区域）组成的界面与 eIF4G 绑定竞争，4EBP 可减少积极参与翻译和防止 eIF4F 复杂形成的 eIF4E 池。这些蛋白质-蛋白质相互作用的相互作用主要受4EBP的拉帕霉素（mTOR）介质磷酸化的哺乳动物目标的调节。在偏头痛刺激下，mTOR直接磷酸化4E-BP蛋白家族的成员，减少他们与eIF4E的关联，从而促进eIF4E-eIF4G相互作用和功能eIF4F复合体10的形成。
尽管在开发针对eIF4F复合完整性的化合物方面付出了巨大努力，但缺乏测量活细胞中eIF4E-eIF4G相互作用直接中断的检测结果，限制了对细胞活性命中化合物的搜索。我们应用了基于共聚苯乙烯模拟（例如，基于纳诺鲁克的补充测定）的路西法酶检测，通过 eIF4E-eIF4G 交互实时监控 eIF4F 完整性的状态。路西法酶补充蛋白系统由18kDa蛋白片段（SubA）和11氨基酸肽片段（SubB）组成，优化为最小的自结和稳定性11。一旦表示为与人类全长eIF4E和eIF4E相互作用域从人类eiF4G1（aa 604-646）的融合产品，两种相互作用的蛋白质将把SubA和SubB片段彼此接近，并诱导形成活性荧光酶，当细胞渗透基质存在时，最终将产生明亮的发光信号（图1）。我们已在其他地方报告了eIF4E:eIF4G604-646 补充系统16的建设和验证情况。
在这里，我们描述了如何应用 eIF4E:eIF4G604-646 补充系统（可应要求提供）来准确测量活细胞中 4EBP1 介质的 eIF4E-eIF4G 中断。此外，我们通过测量目前作为潜在的癌症治疗药物12的临床试验中的几种mTOR抑制剂的效果来证明它的效用。由于目标外效应通常掩盖药物特异性活动，我们还描述了如何通过细胞生存能力正交测量来扩展 eIF4E:eIF4G604-646 系统测量的多功能性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293FT cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture microplate 96 well, F-Bottom</td>
			<td>greiner bio-one</td>
			<td>655083</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell titer Glo 2.0</td>
			<td>PROMEGA</td>
			<td>G9241</td>
			<td></td>
		</tr>
		<tr>
			<td>Envision Multilabel Reader</td>
			<td>PerkinElmer</td>
			<td>not applcable</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 Multichannel Pipettes 12-channels 30-300 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>4662070</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 Multichannel Pipettes 12-channels 5-50 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>4662050</td>
			<td></td>
		</tr>
		<tr>
			<td>FUGENE6</td>
			<td>PROMEGA</td>
			<td>E2692</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher Scientific</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/L3000015" target="_blank">L3000015</a></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT PPI Starter Systems</td>
			<td>PROMEGA</td>
			<td>N2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem I Reduced Serum Mediun, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Eppendorf</td>
			<td>not appicable</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-Aminophenyl-m7GTP (C10-spacer)-Agarose</td>
			<td>Jena Bioscience</td>
			<td>AC-155S</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring eif4f assembly by measuring eif4e-eif4g interaction in live cells

eIF4F复合物的形成已被证明是信号通路收敛的关键下游节点，这些信号通路经常在人类中经历致癌活化。eIF4F 是一个上限绑定复合体，涉及翻译启动的 mRNA - 相关招聘阶段。在许多癌症的细胞和临床前模型中，eIF4F 的放松管制导致参与癌症增殖和生存的特定 mRNA 子集的翻译增加。eIF4F 是一个由上限绑定子单位 eIF4E、六角酶 eIF4A 和脚手架子单元 eIF4G 构建的异质三元复合体。活性eIF4F复合物组装的关键在于eIF4E和eIF4G蛋白质之间的蛋白质-蛋白质相互作用。在本文中，我们描述了一个测量 eIF4F 组件的协议，该程序可监控活细胞中的 eIF4E-eIF4G 交互状态。eIF4e:4G 基于细胞的蛋白质-蛋白质相互作用检测还允许准确可靠地评估 eIF4F 复杂完整性的药物诱导变化。我们设想，这种方法可以应用于验证市售化合物的活性，或进一步筛选有效破坏eIF4F复合物形成的新化合物或方式。

为了验证eIF4E:eIF4G604-646 补充系统的灵敏度，使用mTOR抑制剂对eIF4F复合组件进行了4EBP1介停抑制评估。通过抑制4EBP蛋白家族的mTORC1激酶依赖磷酸化，mTOR抑制增强4EBP1与eIF4E的关联，因此，eIF4F拆解15。测试了两类机械上不同的mTOR激酶抑制剂:拉帕洛格（如拉帕霉素），它们是mTORC1的同位素抑制剂，但不是mTORC2和ATP基于竞争的抑制剂（例如PP242），它们...

Watch this Scientific Journal Video about Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells at JoVE.com

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

在这里，我们提出了一个协议，以测量eIF4E-eIF4G在活细胞中的相互作用，使用户能够评估药物引起的扰动eIF4F复杂动态的筛选格式。

通过测量活细胞中的 eIF4E-eIF4G 交互来监控 eIF4F 装配

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

monitoring-eif4f-assembly-measuring-eif4e-eif4g-interaction-live

Research

JoVE Journal

Cancer Research

3.0K 次观看.  A*STAR (Agency for Science, Technology and Research). 在这里，我们提出了一个协议，以测量eIF4E-eIF4G在活细胞中的相互作用，使用户能够评估药物引起的扰动eIF4F复杂动态的筛选格式。

视频: Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

科研

癌症研究

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

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 ...

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 ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

通过颈内动脉注射癌细胞模拟脑转移

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...