Name: application of aldesense to stratify ovarian cancer cells based on aldehyde dehydrogenase 1a1 activity
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这项工作得到了美国国立卫生研究院（R35GM133581至JC）和伊利诺伊州癌症中心研究生奖学金（授予SG）的支持。JC感谢Camille和Henry Dreyfus基金会的支持。作者感谢Thomas E. Bearrood博士为准备AlDeSense和AlDeSense AM股票所做的初步贡献。我们感谢奥利弗·皮查多·佩盖罗先生和约瑟夫·福尔扎诺先生协助制备各种合成前体。我们感谢Erik Nelson教授（UIUC分子与综合生理学系）的Caov-3，IGROV-1和PEO4细胞。我们感谢Paul Hergenrother教授（UIUC化学系）的BG-1细胞。我们感谢Carl R. Woese基因组生物学研究所的核心设施使用蔡司LSM 700共聚焦显微镜和相应的软件。我们感谢流式细胞术设施使用BD LSR II CMtO分析仪。我们感谢Sandra McMasters博士和细胞培养基设施在制备细胞培养基方面的帮助。

1. 通过荧光法 测量 卵巢癌细胞匀浆中的总 ALDH1A1 活性
<ol><li>在 5 mL 以下细胞培养基中的 T25 细胞培养瓶中解冻 1 ×10 6 个细胞：<ol><li>IGROV-1 和 PEO4：罗斯威尔公园纪念研究所 （RPMI） 1640 培养基，含 10% 胎牛血清 （FBS） 和 1% 青霉素/链霉素 （P/S）。</li>
		<li>BG-1和Caov-3：Dulbecco的改良鹰培养基（DMEM），具有10%FBS和1%P / S。</li>
		<li>OVCAR-3：RPMI 1640，含 20% FBS、1% P/S 和 0.01 mg...

<ol>
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泛选择性是许多 ALDH 探头的主要限制;然而，最近报道了几种亚型选择性实例32，33，34，35，36，37，38，39，40，41。本研究中使用的亚型选择性?...

癌症干细胞（CSC）是具有干细胞样特性的肿瘤细胞亚群1。与非癌性对应物类似，CSC具有非凡的自我更新和增殖能力。与其他内置机制（例如ATP结合盒转运蛋白的上调）一起，CSC通常免于初始手术减瘤工作以及随后的辅助治疗2。由于CSCs在治疗耐药性3，复发4和转移5中的关键作用，已成为癌症研究的优先事项。尽管有多种细胞表面抗原（例如CD133）可用于鉴定CSC6，但利用细胞质中发现的醛脱氢酶（ALDH）的酶活性已成为一种有吸引力的替代方法7。ALDHs 是 19 种酶的超家族，负责催化反应性内源性和异生素醛氧化为相应的羧酸产物8。
一般来说，醛解毒对于保护细胞免受可能损害干细胞完整性的不良交联事件和氧化应激至关重要9。此外，1A1亚型控制视黄酸代谢，而视黄酸代谢又通过视黄醛信号传导影响干性10。AlDeSense 11，12 是一种基于活性的小分子传感 （ABS） 探针，用于选择性检测 ALDH1A1 活性，最近被开发出来。ABS设计通过化学变化而不是结合事件实现分析物检测，从而实现高选择性和减少脱靶响应13，14，15，16。亚型选择性荧光探针的设计原理依赖于供体光诱导电子转移（d-PeT）猝灭机制17，该机制源自醛官能团，用于抑制探针18的荧光特征。在 ALDH1A1 介导的转化为羧酸后，辐射松弛被解锁以产生高荧光产物。由于d-PeT猝灭从来都不是100%有效的，因此在通过开发Ctrl-AlDeSense（一种具有匹配的光物理特性（例如，量子产率）和细胞中相同细胞质染色模式的非响应试剂）建立该测定时，考虑了可能导致假阳性结果的残留荧光。当同时使用时，这种独特的配对可以通过荧光、分子成像和流式细胞术可靠地区分具有高 ALDH1A1 活性的细胞和低水平细胞。与传统的免疫组织化学方法相比，使用亚型选择性可活化染料有几个关键优势。例如，假设CSCs深埋在肿瘤中，因此相对于大抗体更容易被小分子接近19。此外，翻转的荧光产物不会共价修饰任何细胞成分，这意味着它可以通过洗涤循环轻松去除，使CSC处于未修饰状态。最后，开启反应仅识别活细胞和功能，与MTT测定非常相似，因为它依赖于NAD +辅因子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64713/64713fig01.jpg"> 图 1：演示 AlDeSense 荧光开启的示意图。 这种亚型选择性染料由 ALDH1A1 激活，可用于通过荧光、分子成像和流式细胞术 鉴定 卵巢癌细胞中升高的 ALDH1A1 活性。 <a href="https://www.jove.com/files/ftp_upload/64713/64713fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
在过去的工作中，亚型选择性荧光探针测定成功地从K562人慢性白血病细胞、MDA-MB-231人乳腺癌细胞和B16F0小鼠黑色素瘤细胞中的ALDH低（ALDH-）细胞中分层了ALDH高（ALDH+）细胞。这一点很重要，因为对于许多癌症类型，高 ALDH1A1 蛋白表达意味着临床预后较差20。这假设 ALDH1A1 水平升高表明 CSC 可以逃避治疗、产生耐药性并传播到全身。然而，在卵巢癌的情况下，有研究报告了相反的发现（高 ALDH1A1 表达与改善患者生存率有关）21，22，23，24。虽然乍一看这似乎是矛盾的，但表达不一定与酶活性相关，酶活性可能受到肿瘤微环境变化（例如pH通量、氧梯度）、NAD+辅因子或醛底物的可用性、羧酸水平（产物抑制）以及可能改变酶活性的翻译后修饰的影响25.此外，卵巢癌分为五种主要的组织学类型（高级别浆液性、低级别浆液性、子宫内膜样、透明细胞和粘液），我们假设其 ALDH1A1 活性水平不同26。为了研究卵巢肿瘤中的 ALDH1A1 活性，采用亚型选择性荧光探针测定法鉴定属于上述不同组织学类型的五种卵巢癌细胞系中的 ALDH1A1+ 群体。本研究中测试的细胞系包括BG-1，Caov-3，IGROV-1，OVCAR-3和PEO4细胞，涵盖透明细胞和浆液组织型。本文强调了探针的多功能性和可推广性，以鉴定CSC，以便寻求在其他永生化癌细胞系以及患者样本中进行类似研究的研究人员使用CSC。AlDeSense的使用将揭示复杂组织微环境中CSC维持所涉及的生化途径，并可能作为确定预后和测量癌症侵袭性的临床工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate</td>
			<td>Corning</td>
			<td>&#160;&#160;25-050-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffer Saline</td>
			<td>Corning</td>
			<td>21-040-CMX12</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuSpin Micro 17R</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>AlDeSense</td>
			<td></td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>BG-1</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Paul Hergenrother Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>BioLite 25cm2 Flask</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>130189</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet 1300 series A2</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caov-3</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>Cell homogenizer</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5180R</td>
			<td>Eppendorf</td>
			<td>22627040</td>
			<td></td>
		</tr>
		<tr>
			<td>Contrl-AlDeSense</td>
			<td></td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>DMEM, 10% FBS, 1% P/S</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5mL</td>
			<td>Corning</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS Express 6</td>
			<td></td>
			<td></td>
			<td>Provided by UIUC CMtO</td>
		</tr>
		<tr>
			<td>FL microscope</td>
			<td>EVOS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer</td>
			<td>Photon Technology International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Series II Water-Jacketed CO2&#160;Incubator</td>
			<td>Fisher Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr>
			<td>IGROV-1</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Innova 42R Incubated Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 700</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSR II</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered #1.0 Borosicilate Coverglass System</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr>
			<td>OVCAR-3</td>
			<td>ATCC</td>
			<td>HTB-161</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO4</td>
			<td></td>
			<td></td>
			<td>A gift provided by the Prof. Erik Nelson Lab, University of Illinois Urbana-Champaign</td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor Tablets</td>
			<td>Thermo Scientific</td>
			<td>A32963</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI, 10% FBS, 1% P/S</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
		<tr>
			<td>RPMI, 20% FBS, 1% P/S, 0.01 mg/mL Insulin</td>
			<td></td>
			<td></td>
			<td>Prepared by UIUC cell media facility</td>
		</tr>
	</tbody>
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application of aldesense to stratify ovarian cancer cells based on aldehyde dehydrogenase 1a1 activity

癌症治疗后的复发通常归因于称为癌症干细胞（CSC）的肿瘤细胞亚群的持续存在，其特征在于其显着的肿瘤起始和自我更新能力。根据肿瘤的起源（例如卵巢），CSC表面生物标志物谱可能会有很大差异，这使得通过免疫组织化学染色 鉴定 此类细胞是一项具有挑战性的工作。相反，醛脱氢酶 1A1 （ALDH1A1） 因其在几乎所有祖细胞（包括 CSC）中的保守表达谱而成为鉴定 CSC 的极好标志物。ALDH1A1 亚型属于 19 种酶的超家族，负责将各种内源性和异生素醛氧化成相应的羧酸产物。Chan等人最近开发了AlDeSense，这是一种用于检测ALDH1A1活性的亚型选择性“开启”探针，以及一种非反应性匹配对照试剂（Ctrl-AlDeSense），用于解决脱靶染色。这种亚型选择工具已被证明是一种多功能化学工具，可检测 K562 骨髓性白血病细胞、乳腺球和黑色素瘤衍生的 CSC 异种移植物中的 ALDH1A1 活性。在本文中，通过额外的荧光法、共聚焦显微镜和流式细胞术实验展示了探针的实用性，其中在一组五种卵巢癌细胞系中测定了相对 ALDH1A1 活性。

卵巢癌细胞匀浆的总 ALDH1A1 活性 从该测定中获得的每个细胞系的平均折叠开启为：BG-1（1.12±0.01）;IGROV-1（1.30 ± 0.03）;Caov-3（1.72 ± 0.06）;PEO4 （2.51 ± 0.29）;和OVCAR-3（10.25±1.46）（图2）。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64713/64713fig02.jpg"> 图 2：通过荧光法测量的每?...

Watch this Scientific Journal Video about Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity at JoVE.com

Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity

测量活细胞中 ALDH1A1 活性的方法在癌症研究中至关重要，因为它是干性的生物标志物。在这项研究中，我们采用了亚型选择性荧光探针来确定一组五种卵巢癌细胞系中 ALDH1A1 活性的相对水平。

AlDeSense在基于醛脱氢酶1A1活性的卵巢癌细胞分层中的应用

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are ...

我们的实验方案允许用户测量 ALDH1A1 活性，这是一种干细胞生物标志物，使用从流式细胞术到活细胞分子成像的各种方式。这种方法的主要优点是探针激活涉及以高信差比为特征的周转响应，以及选择性 ALDH1A1 亚型检测。演示这一程序的将是我实验室的Michael Lee，Sarah Gardner和Rodrigo Tapia Hernandez研究生。首先，从八孔室载玻片中感兴趣的培养细胞中吸出生长培养基。每孔加入500微升无血清培养基，补充有两微摩尔亚型选择性荧光探针或非反应性对照探针。将载玻片在室温下孵育30分钟。孵育后，立即进行共聚焦显微镜成像。以 10 倍放大倍率定位细胞。本实验需要FITC通道和透射光通道。使用透射光通道定位并聚焦细胞，以在整个实验中聚焦在同一 Z 平面上以消除偏差。将激光功率和FITC增益调整到适当的设置，在该设置中，来自控制AlDeSense AM样本的信号可检测性最小，同时在AlDeSense AM样本中仍能看到信号。通过滑动相应的条来调整每个参数。可能需要调整几次设置才能识别正确的参数。优化后，使用相同的参数完成该细胞系内的其余实验。每个孔拍摄三个图像，每个处理条件总共三个孔并保存图像。确保仅在平面和孔之间切换时使用透射光通道进行聚焦，以避免偏置。接下来，使用图像处理软件将CZI文件拆分为不同的通道，并计数细胞和荧光细胞的总数。要确定醛脱氢酶1A1阳性细胞的百分比，请将荧光细胞的数量除以每个图像中的细胞总数。对每个图像进行相同的计数，以避免混淆变量。取出含有培养箱中维持的细胞的T25细胞培养瓶。添加胰蛋白酶进行细胞分离，并使用自动细胞计数器对细胞进行计数。然后在15毫升离心管中沉淀细胞，在180G下在25摄氏度下离心5分钟。将细胞重新悬浮在PBS中的一毫升两微摩尔探针或对照探针溶液中。在室温下摇动细胞60分钟，以确保均匀暴露于染料中。孵育期后，通过在25摄氏度下以180G离心5分钟来沉淀细胞。然后将细胞重新悬浮在0.5毫升PBS中。将细胞通过35微米尼龙网状细胞过滤器运行到放置在冰上的管子上，以去除可能堵塞流式细胞仪的细胞团块。打开流式细胞仪仪器并运行启动方案。检查鞘液和空腰。用10%漂白剂和水运行生产线，每次五分钟。然后运行质量控制珠以确保功能正常。在设置选项卡中，为荧光滤光片选择前向散射侧散射和FITC进行。为图形中心附近的主细胞群绘制前向散点区域与侧向散点区域图。接下来，绘制一个前向散点区域，而不是指示单线的窄水平带的前向散点宽度图。然后绘制FITC面积与前向散射面积图，以观察由亚型选择性荧光开启探针分类的细胞分布。接下来，绘制FITC区域直方图以观察基于FITC的群体变化并确定醛脱氢酶1A1阳性细胞的百分比。为了优化FITC激光功率，请使用探头运行样品，使直方图曲线的右尾接近最大FITC面积信号。激光功率优化步骤可能必须重复多次，但一旦为实验指定了设置，就不应在样品之间改变激光功率。随后，将样品添加到筛子中，并用对照探针运行它。应该可以观察到种群偏移以显示最大动态范围。运行每个样品进行 10， 000 次计数，一式三份。完成样品收集后，用10%漂白剂和水运行管线各五分钟，然后关闭仪器。使用流式细胞术软件处理数据并步态所需的细胞群。使用矩形栅极选择，设置醛脱氢酶1A1负步态，使对照探针样品中99.5%以上的事件发生在该栅极内。其余细胞将被视为醛脱氢酶1A1阳性。对探针样品应用相同的门。量化醛脱氢酶1A1阴性和1A1阳性的事件数。测定每个细胞系的平均折叠开启次数，以量化总醛脱氢酶1A1活性。通过调整激光功率和增益来确定每个细胞系中醛脱氢酶1A1阳性细胞的百分比。为了最小化对照AlDeSense AM处理样品中的信号，在AlDeSense AM处理的细胞中优化荧光信号。通过计数醛脱氢酶A1A阳性细胞的数量，测定醛脱氢酶A1A阳性细胞的百分比。通过应用亚型选择性荧光探针，通过对对照AlDeSense AM处理群体中最亮的细胞的前0.5%进行门控，在每个细胞系内对醛脱氢酶1A1阳性细胞群进行定量。对卵巢癌细胞小组的分析揭示了醛脱氢酶1A1阳性细胞的百分比。从所测试的癌细胞系获得的结果可以得出结论，BG-1具有最低的醛脱氢酶1A1活性和最低的醛脱氢酶1A1阳性群体。此外，共聚焦成像和流式细胞术显示Caov-3细胞中醛脱氢酶1A1阳性细胞的百分比最大。但细胞系的整体活性仅居第三高。或者，OVCAR-3细胞含有第三高的醛脱氢酶1A1阳性群体，但表现出最高的总活性。AlDeSense是一种多功能且可推广的探针，用于鉴定永生化细胞系和患者样品中的CSC。我们希望将其视为临床环境中的预后工具。

Research

JoVE Journal

Cancer Research

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

科研

癌症研究

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

卵巢癌组织线粒体的制备与控制卵巢组织进行定量蛋白质组学分析

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

使用单链DNA免疫荧光量化卵巢癌细胞中的复制应激

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

使用非放射性化学发光检测定量端粒长度

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

TAGGG端粒长度测定性能参数优化

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...