1. 2D 片上的免疫 DNA 鱼
<ol>
	<li>片制备及附着细胞播种 注: 在无菌条件下, 在层流罩下执行以下所有步骤。<ol>
		<li>将片浸入100% 乙醇消毒。 注: 我们使用 12 mm ×12 mm 硅胶支持的平方片 (所有试剂都列在材料表)。</li>
		<li>将片放在培养皿中 (直径100毫米)。干燥使用强迫空气流动5分钟。</li>
		<li>用15毫升无菌 1x Dulbecco 磷酸缓冲盐水 (PBS) 洗涤培养皿。</li>
		<li>用10毫升完全介质清洗培养皿 (参见表 1)。</li>
		<li>种子黏附细胞 (大约165万个细胞在10毫升媒介, 计数由外地资产管制系统分析) 通过轻轻地下降细胞悬浮到培养皿上获得均匀的细胞电镀 (大约3万细胞/cm2)。 注: 对于 ~ 70% 汇合, 黏附细胞应该培养在片至少 48 h。</li>
	</ol>
	</li>
	<li>固定和性 注意: 丙酮是一种易燃易怒的挥发性物质。仅使用耐丙酮的塑料或玻璃容器 (无 PVC 或 PVDF)。 注: 在化学油烟罩下执行这些步骤。<ol>
		<li>使用一次性吸气吸管去除培养基。</li>
		<li>用 1x PBS 清洗培养皿。</li>
		<li>使用镊子, 把它们浸入一个装有5毫升 1x PBS 的玻璃烧杯中, 清洗片。</li>
		<li>把片在印迹纸上晾干。</li>
		<li>在室温 (RT) 中浸泡成100% 丙酮溶液, 固定在片上的细胞。</li>
		<li>用镊子除去片。干片使用强迫气流5分钟。 注意: 在此点可以暂停协议。片应贮存在-20 ° c。</li>
	</ol>
	</li>
	<li>免疫荧光日1 注: 此阶段涉及一夜 (O/N) 孵化 (约16小时)。<ol>
		<li>两次洗 1x PBS 的片。</li>
		<li>将100µL 的抗体稀释缓冲液 (详见表 1 ) 放到片上, 在 RT 处孵育30分钟。 注: 解决方案体积应根据片表面进行调整, 以覆盖整个片, 避免溢出风险。</li>
		<li>准备抗体混合 (表 1) 使用1:20 稀释的单克隆主抗体共轭荧光和抗体稀释缓冲, 在数据表中指定的制造商提供。 注意: 强烈建议使用与耐热荧光共轭的单克隆抗体。如果使用的是 non-thermostable 荧光, 则在该协议的 DNA 鱼步骤中利用的温度可能会破坏荧光和熄灭荧光的排放。在这个协议中, 我们选择了一个 EpCAM-FITC 抗体 (1:20 稀释), 它没有显示任何显着的问题与使用的温度。</li>
		<li>在 1x PBS 中冲洗两次片。</li>
		<li>将片细胞侧放在潮湿的室内, 在片上放置100µL 的抗体混合。 注: 溶液体积应根据片表面进行调整, 以覆盖整个片, 减少溢流的风险。</li>
		<li>在4° c 的黑暗中孵育 O/N (约16小时)。</li>
	</ol>
	</li>
	<li>免疫荧光日2
	<ol>
		<li>1x PBS 片两次冲洗, 阻断抗体培养。 注: 将片浸泡在100µL 的 1x PBS 中, 在4° c 的室内, 在黑暗的潮湿环境中, 直到进行鱼化验。</li>
	</ol>
	</li>
	<li>2D DNA-鱼日2 警告: 必须特别注意仪器和潮湿室的高温。<ol>
		<li>建立杂交烤箱和干热烘箱 (在开始 DNA 鱼协议前3小时)。设置杂交烤箱在75° c, 设置干燥热烘箱在37° c 和冷却 0.4× ssc 解答 (pH 值 = 7.0 ± 0.1) (为 SSC 食谱参见表 1) 到4° c。 注: 鱼类缓冲液的 ph 值非常重要: ph 值 = 7.0 ±0.1。</li>
		<li>在 ice-cold 0.4× SSC 溶液中清洗片三次。</li>
		<li>在一个化学油烟罩下片10分钟, 在黑暗中干燥。</li>
		<li>漩涡为十年代和执行快速自旋下来 (在最大率) 探针从探针管的墙壁。</li>
		<li>将片的细胞侧下降到5µL 的鱼探针上, 然后用橡胶水泥封住片的所有边缘。 注意: 接下来的两个步骤涉及高温。戴上防护手套。</li>
		<li>将滑块放入杂交烤箱中的深色湿润腔中, 在75° c 下进行8分钟的完全 DNA 变性。</li>
		<li>将潮湿的房间移到37° c 的干热烘箱中。</li>
	</ol>
	</li>
	<li>2D DNA-鱼日3
	<ol>
		<li>设置水浴在72° c 和放置一个滑动染色罐与 0.4× SSC 缓冲入它 (表 1)。30分钟后, 检查 0.4× SSC 缓冲器的温度, 必须在72±1° c。</li>
		<li>准备两个玻璃烧杯: 一个与 2x SSC + 0.05% 补间缓冲 (pH = 7.0 ± 0.1) (表 1) 和第二个与蒸馏水。</li>
		<li>从烤箱中取出潮湿的房间, 小心地从滑梯上取下橡胶水泥, 用镊子将片分离。</li>
		<li>洗涤片通过浸泡在 0.4× SSC 2 分钟在72° c。</li>
		<li>洗片通过浸泡在 2x SSC + 0.05% 补间缓冲三十年代。</li>
		<li>用蒸馏水冲洗片。</li>
		<li>在一个化学油烟罩下片10分钟, 在黑暗中干燥。</li>
		<li>将片装在液体安装介质中, 用1.5 µg/毫升的 4´, 6-diamidino-2-吲 (DAPI) 到 counterstain 总 DNA。将片细胞侧向下放置在一张幻灯片上的5µL 安装介质上, 按片上的镊子以获得空气, 并用指甲油密封。 注: 安装介质容积应根据片表面尺寸进行调整。</li>
	</ol>
	</li>
	<li>2D 显微镜观察与分析 注: 图像可以用不同的显微镜系统获取。</li></ol>我们使用了一个传统的荧光显微镜, 赋予一个标准的商业软件的图像采集 (材料表)。<ol>
		<li>使用12位数码相机和 20×/0.50 na 和 40×/0.65 na 目标获取图像, 用适当的滤镜进行红色 (激发: 599 毫微米, 放射: 588 毫微米), 绿色 (励磁: 509, 放射: 524) 和蓝色 (励磁: 367 毫微米, 放射: 452 毫微米) 探针。</li>
		<li>使用软件选择工具分析图像。 注意: 为了评价免疫荧光染色和测向探针的定位, 我们采用了 ImageJ。幻灯片可以在-20 ° c 的黑暗中储存3周。对于长时间存储, 我们建议使用特定的安装介质进行长期存储。</li>
	</ol>
	

2. 电线上的免疫 DNA 鱼
<ol>
	<li>线 spike-in (3D 支持) 注: 初步设置包括基于 EpCAM 阳性的黏附细胞线的准确选择。该导线是功能化的抗 EpCAM 抗体, 使只有 EpCAM 阳性细胞染色。 注意: 请勿触摸导线的功能化部分以避免损坏。 注: 所有步骤应在无菌条件下的层流罩下进行。<ol>
		<li>用4毫升完全培养基重5毫升瓶中的 T75 烧瓶 (80% 汇合) 的全部内容;对于一个单一的 spike-in, 约200万细胞被推荐最大化细胞粘附在电线。</li>
		<li>从玻璃包装上取下电线。</li>
		<li>将金属丝的功能性黄金部分浸入小瓶中, 确保钢丝塞子是一个水密适合的瓶子。密封与实验室膜。 注: 建议使用5毫升管, 以便在铁丝瓶塞和瓶子之间进行正确的匹配。</li>
		<li>在试管转子 (0-120 角) 的 RT 中孵育30分钟。</li>
		<li>使用3不同的清洁小瓶, 在 1x PBS 溶液中冲洗三次金属丝。</li>
	</ol>
	</li>
	<li>固定和性 注意: 丙酮是一种可刺激呼吸道的易燃物质。只使用耐丙酮塑料或玻璃容器 (无 PVC 或 PVDF)。 注: 在化学油烟罩下执行这些步骤。<ol>
		<li>空气干燥的电线。</li>
		<li>通过蘸在丙酮100% 溶液中的丝在 RT 10 分钟来固定细胞. 使用5毫升管。 注意: 线塞不能与定影液接触。</li>
		<li>空气干燥的电线在 RT 5 分钟。 注意: 协议可以在此处暂停。电线必须储存在-20 ° c。当为长期储存的电线包装, 放置在功能化的尖端旁边的电线塞子, 小心地插入到存储玻璃管的电线, 注意不要损坏功能化的提示。在-20 ° c 下关闭存储玻璃和存储 (垂直或水平)。</li>
	</ol>
	</li>
	<li>免疫荧光日1 注: 此阶段涉及一个 O/N 孵化 (约16小时)。<ol>
		<li>使用5毫升管冲洗两次 1x PBS 溶液。</li>
		<li>用5毫升管在 RT 中孵育抗体稀释缓冲液30分钟。</li>
		<li>根据数据表中的规定, 在抗体稀释缓冲液中制备150µL 抗体混合物, 并将其与荧光结合的单克隆主抗体稀释。例如, EpCAM-FITC 抗体被用于1:20 稀释。</li>
		<li>使用 p200 提示进行孵化, 如下所示: 从丝塞中抽出导线, 然后通过尖端的大孔轻轻地插入导线。先插入 unfunctionalized 端, 然后慢慢将导线穿过较小的孔, 直到黄金部分刚好超出较大的孔。</li>
		<li>轻轻滴150µL 抗体混合 (例如抗 EpCAM_FITC 抗体1:20 稀释) 到提示。为了避免气泡形成的风险, 轻轻地旋转导线, 直到官能化部分完全暴跌。</li>
		<li>封住小费的洞。环绕实验室膜的孔可以帮助。</li>
		<li>在4° c 下, 在黑暗中垂直孵育 O/N (约16小时)。</li>
	</ol>
	</li>
	<li>免疫荧光日2
	<ol>
		<li>通过在 1x PBS 中洗两次丝来阻断抗体的孵化。</li>
		<li>将导线重新插入钢丝塞。 注意: 在4° c 的5毫升的小瓶中储存 1X PBS, 直到进行鱼化验。</li>
	</ol>
	</li>
	<li>3D DNA-鱼日2 警告: 必须特别注意仪器和潮湿室的高温。<ol>
		<li>建立杂交烤箱和干热烘箱 (在开始 DNA 鱼协议前3小时)。设置杂交烤箱在75° c, 设置干燥热烘箱在37° c, 并且冷却 0.4× SSC 解答 (pH 值 = 7.0 ± 0.1) 到4° c。 注: 鱼类缓冲液的 pH 值非常重要, 必须为7.0 ±0.1。</li>
		<li>在 ice-cold 0.4× SSC 溶液中清洗3次导线。 注意: 在下列过程中, 保持电线在黑暗中避免荧光漂白。</li>
		<li>在通风柜下的黑暗中干10分钟。</li>
		<li>漩涡和旋转探针 5 s。</li>
		<li>将探针的10µL 放到玻璃管内。盖上实验室胶片。</li>
		<li>旋转管内如下: 将微在干燥的吸收剂中, 把纸放入50毫升的管子中, 然后简要地旋转。</li>
		<li>小心地将干丝放入微, 并插入铁丝塞子。密封用橡胶水泥。 注意: 接下来的两个步骤涉及高温。</li></ol></li></ol>戴上防护手套。
		<li>将金属丝放入杂交烤箱中的深色湿润腔中, 在75° c 下进行8分钟的 DNA 变性。</li>
		<li>将潮湿的房间移动到37° c 的干热烘箱中。</li>
	
	
	<li>3D DNA-鱼日3
	<ol>
		<li>将 0.4× SSC 溶液的滑动染色罐放入水浴中, 设置在72° c。</li>
		<li>检查 0.4× SSC 溶液温度直到达到72±1° c。</li>
		<li>准备两个玻璃烧杯, 一个与 2x SSC + 0.05% 吐温 (pH = 7.0 ± 0.1) 解决方案和第二个与蒸馏水。</li>
		<li>从干燥的烘箱中取出潮湿的房间, 用镊子小心地将橡皮水泥从钢丝塞子上取出, 取出铁丝。 注意: 小心地把未涂上的铁丝端通过塞子, 注意不要损坏功能性的尖端。</li>
		<li>将导线浸入 0.4× SSC 溶液中, 在72° c 时为2分钟。</li>
		<li>在 RT 的三十年代 2x SSC + 0.05% 补间解决方案中清洗电线。</li>
		<li>在 RT 中用蒸馏水冲洗铁丝。</li>
		<li>在黑烟罩下烘干电线10分钟。</li>
		<li>准备一个 DAPI 1.43 µM 在2毫升的 1x PBS 的股票解决方案。</li>
		<li>用 DAPI 溶液在2毫升的小瓶中孵育电线的功能性尖端, 在室温下为1小时。</li>
		<li>1x PBS 和风干两次冲洗铁丝。</li>
	</ol>
	</li>
	<li>3D 显微镜观察与分析
	<ol>
		<li>将导线放置在导线架上。小心地将功能性的笔尖插入到特殊刀柄的入口点, 直到笔尖与耦合点匹配为止。注意不要损坏功能化提示 (图 1a)。</li>
		<li>将特殊支架放在显微镜台上。用20×透镜调整显微镜的焦距。首先粗略地专注于特殊的支持, 然后在 DAPI 的细胞上进行精细的调整。</li>
		<li>只把焦点放在透镜中央的细胞上。 注: 图像可以用不同的显微镜系统获取。在这个设置中, 我们使用一个传统的荧光显微镜赋予一个标准的商业软件的图像采集。</li>
		<li>使用12位数码相机和 20×/0.50 na 和 40×/0.65 na 目标获取图像, 用适当的滤镜进行红色 (激发: 599 毫微米, 放射: 588 毫微米), 绿色 (励磁: 509, 放射: 524) 和蓝色 (励磁: 367 毫微米, 放射: 452 毫微米) 探针。 注意: 图像可以使用不同的工具和软件进行可视化。我们使用 ImageJ 评价免疫荧光染色和测向探针定位。 注意: 协议可以在此处暂停。电线必须储存在-20 ° c。当为长期储存的电线包装, 放置在功能化的尖端旁边的电线塞子, 小心地插入到存储玻璃管的电线, 注意不要损坏功能化的提示。在-20 ° c 下关闭存储玻璃和存储 (垂直或水平)。</li>
	</ol>
	</li>

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B., 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=Clinical+Experience+With+Crizotinib+in+Patients+With+Advanced+ALK+-Rearranged+Non-Small-Cell+Lung+Cancer+and+Brain+Metastases.">Clinical Experience With Crizotinib in Patients With Advanced ALK -Rearranged Non-Small-Cell Lung Cancer and Brain Metastases.</a> J. Clin. Oncol. 33 (17), 1881-1888 (2015).</li><li>Fischer, J. C., 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=Diagnostic+leukapheresis+enables+reliable+detection+of+circulating+tumor+cells+of+nonmetastatic+cancer+patients.">Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients.</a> Proc. Natl. Acad. Sci. U. S. A. 110 (41), 16580-16585 (2013).</li><li>Coumans, F. A. W., Ligthart, S. T., Uhr, J. W., Terstappen, L. W. M. 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Cancer Res. 21 (13), 2883-2885 (2015).</li><li>Theil, G., 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=The+use+of+a+new+CellCollector+to+isolate+circulating+tumor+cells+from+the+blood+of+patients+with+different+stages+of+prostate+cancer+and+clinical+outcomes+-+A+proof-of-concept+study.">The use of a new CellCollector to isolate circulating tumor cells from the blood of patients with different stages of prostate cancer and clinical outcomes - A proof-of-concept study.</a> PLoS One. 11 (8), 1-14 (2016).</li><li>Gorges, T. 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作者声明他们没有竞争的金融利益。

本文首次提出了一种结合免疫荧光染色和 DNA 鱼的方法, 用于功能化丝的应用。这种方法被称为免疫 DNA 鱼。这项技术是为了允许在3D 导线上同时识别假定 ctc 的基础上的型参数, 如积极性到 EpCAM (事件 1), 并促进检测分子的变化, 如与基因状态 (事件 2)。同时标识两个事件, 可以检测其共存。因此, 这种方法可以定制, 以确定和表征 ctc 从血液中提取的癌症患者。haemo 成分和白细胞对染色过程的潜在影响通常不会干扰手术。特别是, 文献报道的数据表明, haemo 成分和白细胞不影响免疫荧光染色的细胞连接到电线, 关键步骤, 以确定实际 ctc1,15。
免疫 DNA 鱼的荧光亮度略高于传统的 immunophototyping 检测, 是鱼类测定所需高温的结果。然而, 免疫荧光染色仍然是可观的。例如, 微弱的信号仍然是可探测的在低 EpCAM 表达的细胞线, NCIH1975。鱼类检测所需的高温是选择与抗体相关的右荧光的主要限制因素。在本协议中, 抗体必须与耐热荧光相连接, 以耐受 DNA 变性温度。例如, 藻, 一个热敏荧光, 不建议在这个设置, 因为荧光退化造成的高温。荧光素异硫氰酸酯是一种很好的选择, 通常荧光用于鱼探针。在标准2D 支架上, 免疫 DNA 鱼自发荧光信号非常差。在导线支撑上, 聚合物层可能会诱发轻微的自发荧光信号, 特别是在红色通道上。与2D 背景不同的是, 具体的背景信号在导线上是可辨识的, 但不影响细胞核识别或细胞特征。
由于在读取圆柱形3D 结构的表面上的光学显微镜的局限性, 对金属丝的显微评估比2D 的支撑更累人。然而, 这一困难可以克服通过旋转导线持有人和获取的图像, 主要是沿中线线。导线持有者允许导线的360°旋转。一旦聚焦在细胞核上, 改变荧光通道并不一定会把焦点放在目标区域上。因此, 必须保持对内细胞靶区的关注。
这种技术可以用来检测和表征不同种类的3D 支持的细胞。也可能使用不同的抗体和鱼探针。当选择使用不同的抗体时, 荧光热稳定性和靶抗原在细胞膜上的表达水平是重要的考虑因素。cytoplasmatic 抗原也可以染色。当使用不同的鱼探针时, 重要的是要检查数据表规格的变性过程和准备细胞的鱼化验相应。

ctc 代表癌细胞传播的一个关键步骤1。他们的存在在患者的外周血与 (转移) 复发和疾病进展相关2,3。CTC 从血液中分离和鉴定肿瘤患者是一种非侵入性液体活检。近年来, 越来越明显的是, 使用这种分析方法监测肿瘤对不同治疗的进展和反应, 提供了重要的临床信息4,5。液体活检是更有用的, 当手术是不可行的, 或当原发肿瘤组织不可用,即, 为 non-biopsiable 病变。因此, 这种方法是有希望在特定的癌症的设置, 如转移性 NSCLC, 其中存在的 ctc 已经证明有一个负面的预后的作用6。NSCLC 是一种肿瘤, 特别是受益于靶向治疗方法7,8,9 , 旨在对特定分子 (分子靶) 采取行动, 已知参与生长, 进展, 和疾病的传播。因此, 需要在疾病进展过程中检测特定的靶点。反恐委员会的调查是一个非常有趣的诊断方法, 以检测和监测药物的目标, 而无需主要或转移组织。例如, 在 NSCLC 细胞中检测易位基因, 与 crizotinib 的敏感性有关, 这是一种特定的靶向治疗10。然而, 目前, 易位的检测只在细针物或小活检上执行;因此, 没有肿瘤组织的分析是不可能的。ctc 是一个潜在的替代肿瘤组织调查, 并代表了一个非常有希望的同伴诊断方法。
尽管他们的潜在重要性, ctc 仍然是一个大争论的主题在研究之中, 主要由于他们的稀有 (1-10 细胞/毫升的外周血11)。目前的液体活检方法使用有限的血量 (即, 1-30 毫升)12,13, 但这会造成 ctc 检测的次优灵敏度的情况. 因此, 有必要研究寻找方法和开发用于在更大的外周血量上执行 CTC 靶向液体活检的装置。
另一种装置, 一种功能化的医用金属丝 (参见材料表), 已经开发来克服血液取样限制, 并获得更具代表性的 ctc 分析。这种功能化的电线是一个 CE 认证的医疗设备, 捕获 ctc 直接从血液中的癌症患者1。它是由一个16厘米长的不锈钢线 (图 1a) 组成, 2 厘米长的功能性尖端涂有 0.2-µm 厚的金层。该层依次由1至5µm-厚羧酸水凝胶地层共价结合, 抗体针对 EpCAM, 其中最广泛表达的抗原的表面 ctc14。导线的官能化的末端被介绍入患者的胳膊的静脉并且保持在位置至少30分钟。这种方法允许隔离 ctc在体内,直接在外周血, 并筛选约 1.5-3.0 升的血液 (大约300倍以上的其他方法使用的体积)1.
、 et al。证明了这种方法的效果, 将 ctc 直接分离到肺癌患者的臂静脉中15。他们进行了线免疫荧光染色识别 ctc 使用常规抗体针对 EpCAM 和泛角, CD45 白细胞检测。导线在光学荧光显微镜下被审查了15。作者证明, 该装置能够隔离 ctc, 但他们没有调查任何与治疗有关的目标, 如易位。
所提出的方法旨在根据型参数,例如, EpCAM 阳性和分子生物标志物的存在, 例如 ctc 的状态 (图 1b), 来识别 NSCLC 细胞系中的假定的不确定因素。这3天长的过程结合了功能化线和免疫荧光染色与 dna 荧光-原位杂交 (dna 鱼), 命名为免疫 dna 鱼。鉴于 ctc 是稀有的实体, 该协议的优点是, ctc 可以在同一条线上的型特征和 DNA 重的特点。

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characterization of tumor cells using a medical wire for capturing circulating tumor cells: a 3d approach based on immunofluorescence and dna fish

循环肿瘤细胞 (ctc) 与转移癌的生存率低下有关。它们的鉴定、分和基因分型可以使人们更好地了解肿瘤的异质性, 从而便于选择病人进行个性化治疗。然而, 由于稀有的 ctc, 这是受阻的。我们提出了一种创新的方法来取样高量的患者血液和获得有关的存在, 表型和基因易位的 ctc。该方法结合免疫荧光染色和 dna 荧光-原位杂交 (dna 鱼), 并以功能化的医疗导线为基础。这根导线是一种创新的设备, 允许在体内的 ctc 隔离大量的外周血。由30分钟的导线管理的血液容量是大约 1.5-3 L。为了证明这一方法的可行性, 在非小细胞肺癌 (NSCLC) 细胞系中, 用功能性金属丝捕捉上皮细胞黏附分子 (EpCAM) 的表达和染色体易位。用免疫 DNA 鱼的方法染色我们的主要挑战是在3D 结构, 功能化的金属丝上进行化验, 并使用传统的荧光显微镜来确定免疫表型和鱼类信号。结果表明, 捕捉 ctc 和分析其表型和染色体重排可能代表一种新的伴侣诊断方法, 并提供一个创新的策略, 以改善个性化的癌症治疗。

使用上述步骤, 可以对 ctc (或其他等效细胞) 进行免疫-DNA 鱼的检测, 这是由官能化的金属丝所丰富的。在建立这个协议之前, 两种技术的相容性被确定 (免疫荧光与鱼) 在标准2D 支持。两个不同的 NSCLC 细胞线表达 EpCAM (需要坚持与抗体结合抗 EpCAM) 被选中。它们具有不同的状态, 这对测试不同的初始条件很有用。第一种, NCI-H1975, 有野生型 (重量) 与基因, 而第二, NCI-H3122, 是以易位为特征的。
采用了一种用于鱼检测的基因分离检测系统。该系统由两个探针, 一个 (橙色) 杂交到近端区域内的2p23 和一个 (绿色) 杂交远端到。在2D 支持, 免疫 DNA 鱼为抗体和探针提供了明确的信号 (图 2)。特别是, 两个细胞线显示明确的 EpCAM 膜定位。此外, 探针信号出现明亮和尖锐: NCI-H1975 细胞显示重叠的橙色和绿色探针, 确认野生的地位的基因 (图 2a, b)。相反, NCI-H3122 细胞显示重叠的信号和单一的绿点, 反映了一个删除的表达基因 (图 2c, d)。当免疫 DNA 鱼检测在3D 支持功能化线上进行时, 抗体和探针信号的定义一般少于2D 支持。EpCAM 染色可见。测向探测信号的定义较少, 但仍反映了预期的通信状态 (图 3)。结果表明, 免疫荧光染色不干扰 DNA 鱼的信号。探针专门与他们的目标杂交。NCI-H3122 细胞系显示出一个异常的, 与 NCI-H1975, 与野生类型的 "与" (图 3a) 的基因状态 (图 3b)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56936/56936fig1.jpg"> 
图 1: 功能化导线、与分离探头的示意图排列、对重排的预期模式和特殊持有者的方案.(a) 红色方框突出显示线的三主要部分: 功能化的尖端、线塞和 un-functionalized 尖端。功能性的尖端是最微妙的部分, 在处理过程中必须不接触, 以避免细胞脱落或损害。(b) 绿色和红色探针分别与序列的上游和下游基因 (在左边) 的顺序绑定。在右边, 显示了 exemplificative 模式的安排。正常细胞上的红色和绿色共存信号;分离的绿色和红色信号表明, 一个与之有关的基因染色体断裂 (易位基因) 和一个绿色橙色的定位信号 (异常 cell-1)。一个红色信号和两个绿色信号表明一个红色信号的丢失, 提示染色体易位和删除 (异常 cell-2)。(c) 导线架;在显微镜分析过程中, 红色方框强调在处理导线时需要注意的部位。通过转动转子, 导线可以经受360°分析。<a href="//ecsource.jove.com/files/ftp_upload/56936/56936fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56936/56936fig2.jpg"> 
图 2: 基于2D 支持 (片) 的免疫 DNA 鱼检测.(a, b)NCI-H1975 细胞弱阳性的 EpCAM。EpCAM FITC 信号是可观的。在 NCIH1975 细胞系中, 与之分离的橙色和绿色探针重叠, 证实了该基因的重度状态。显示两个以上配对信号的细胞由于其倍而存在。(c, d)在高 EpCAM 表达的 NCIH3122 细胞系中, 免疫荧光信号是明亮的, 在细胞间的连接中更加突出。鱼分析证实了该基因的缺失, 这是典型的细胞系。红色箭头表示单绿色探针 (没有相应的橙色探针), 反映了对基因的删除。<a href="//ecsource.jove.com/files/ftp_upload/56936/56936fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56936/56936fig3.jpg"> 
图 3:免疫-DNA 鱼检测使用功能化金属丝作为3D 支持.(a) 线上 NCI-H1975 细胞的代表性图像。虽然在导线上的细胞的3D 形状很难获得充分对焦图像, EpCAM 信号是很好的可见的所有细胞。(b) 线上 NCI-H3122 细胞的代表性图像。与先前在2D 支持上看到的一样, 测向探针显示了基因缺失 (红色箭头)。可见的背景信号很可能是由于聚合物层的存在。然而, 它没有显着影响细胞识别或荧光分析。<a href="//ecsource.jove.com/files/ftp_upload/56936/56936fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>缓冲区</td>
			<td>组成</td>
			<td>注意</td>
			<td>股票</td>
		</tr>
		<tr>
			<td>细胞完整培养基</td>
			<td>RPMI 1640 + 2 毫米谷氨酰胺 + 5-10% 胎儿牛血清 (FBS)。</td>
			<td></td>
			<td>RPMI: 4 ° c;谷氨酰胺, FBS:-20 ° c</td>
		</tr>
		<tr>
			<td>抗体 Diluition 缓冲</td>
			<td>1% BSA, 0.3% 海卫 X-100 在 1x PBS</td>
			<td></td>
			<td>实验室胶片密封。</td>
		</tr>
		<tr>
			<td>20x SSC 解决方案</td>
			<td>3 M 氯化钠, 300 毫米柠檬酸三钠溶于 ddH20</td>
			<td>过滤器溶液和 pH 值 = 7.0 +/-0.1 与 HCl</td>
			<td>实验室胶片密封。</td>
		</tr>
		<tr>
			<td>0.4x SSC 解决方案</td>
			<td>稀释库存 20x SSC 在蒸馏 H2O</td>
			<td>过滤器溶液和 pH 值 = 7.0 +/-0.1 与 HCl</td>
			<td>rt.</td></tr></tbody></table>密封与实验室膜。
		
		<tr>
			<td>2x SSC + 0.05% 吐温20解决方案</td>
			<td>稀释的库存 20x SSC 在蒸馏的 H2O + 0.05% 吐温20</td>
			<td>过滤器溶液和 pH 值 = 7.0 +/-0.1 与 HCl</td>
			<td>实验室胶片密封。</td>
		</tr>
		<tr>
			<td>DAPI 解决方案 30 nM</td>
			<td>1x PBS 稀释库存1.43 µM</td>
			<td></td>
			<td>-20 ° c 在黑暗的情况准备好使用 aliquote</td>
		</tr>
	

表 1: 解决方案食谱。

Watch this Scientific Journal Video about Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH at JoVE.com

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

我们提出了一种新的方法来描述肿瘤细胞。我们结合免疫荧光与 DNA 荧光-原位杂交, 以评估细胞捕获的功能性医用电线, 能够在体内的,丰富 ctc 直接从病人的血液。

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

characterization-tumor-cells-using-medical-wire-for-capturing

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

6.9K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 我们提出了一种新的方法来描述肿瘤细胞。我们结合免疫荧光与 DNA 荧光-原位杂交, 以评估细胞捕获的功能性医用电线, 能够在体内的,丰富 ctc 直接从病人的血液。

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

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

捕获和血液中的活循环肿瘤细胞释放

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

科研

癌症研究

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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 ...

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

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 patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

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 ...