Name: characterization of tumor cells using a medical wire for capturing circulating tumor cells: a 3d approach based on immunofluorescence and dna fish
Uploaded: 2017-12-21T15:00:00.000+00:00
Duration: 10 min 56 s
Description: 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

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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+the+enumeration+and+phenotyping+of+CTC.">Challenges in the enumeration and phenotyping of CTC.</a> Clin. Cancer Res. 18 (20), 5711-5718 (2012).</li><li>Allard, W. J., Terstappen, L. W. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR+20th+Anniversary+Commentary:+Paving+the+Way+for+Circulating+Tumor+Cells.">CCR 20th Anniversary Commentary: Paving the Way for Circulating Tumor Cells.</a> Clin. 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. M., 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=Enumeration+and+Molecular+Characterization+of+Tumor+Cells+in+Lung+Cancer+Patients+Using+a+Novel+In+Vivo+Device+for+Capturing+Circulating+Tumor+Cells.">Enumeration and Molecular Characterization of Tumor Cells in Lung Cancer Patients Using a Novel In Vivo Device for Capturing Circulating Tumor Cells.</a> Clin. Cancer Res. 22 (9), 2197-2206 (2016).</li></ol>

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。

<table><tbody><tr><td>Ethanol</td><td>Carlo Erba</td><td># 414605</td><td></td></tr><tr><td>Tween 20</td><td>BIO RAD</td><td># 1706531</td><td></td></tr><tr><td>PBS (Phosphate Buffered Saline)&amp;nbsp;</td><td>Medicago</td><td># 09-9400-100</td><td></td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td># 534064-500ML</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td># A7906</td><td></td></tr><tr><td>Triton X-100 Detergent</td><td>BIO RAD</td><td># 1610407</td><td></td></tr><tr><td>RUBBERCEMENT</td><td>Royal Talens</td><td># 95306500</td><td></td></tr><tr><td>Vysis 20X SSC (66g)</td><td>Abbott Molecular Inc.&amp;nbsp;</td><td># 30-804850</td><td>powder to be resuspended in distilled H&lt;sub&gt;2&lt;/sub&gt;O, as recommended</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td># 31870-025</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Gibco</td><td># 10270-098</td><td></td></tr><tr><td>L-Glutamine (200mM)</td><td>Gibco</td><td># 25030-024</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td># 15140-122</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;0</td><td>MilliPore</td><td></td><td></td></tr><tr><td>XT ALK BA</td><td>MetaSystems Probes</td><td># D-6001-100-OG</td><td></td></tr><tr><td>DAPI, FluoroPure grade</td><td>Invitrogen</td><td># D21490</td><td></td></tr><tr><td>SlowFade Diamond Antifade Mountant with DAPI</td><td>Life Technologies</td><td># S36973</td><td></td></tr><tr><td>EpCAM-FITC (clone: HEA-125)</td><td>Mitenyi</td><td># 130-080-301</td><td></td></tr><tr><td>Micro tubes M-Tube18</td><td>Gilupi Nanomedizin</td><td></td><td></td></tr><tr><td>Detektor CANCER01 EpCAM</td><td>Gilupi Nanomedizin</td><td></td><td>Functionalized wire</td></tr><tr><td>STAR FROST Microscope Slides</td><td>Knittel GL&amp;Auml;SER</td><td>VS1117# 076FKB</td><td></td></tr><tr><td>SecureSlip Silicone Supported Coverglass</td><td>GRACE BIO-LABS</td><td># 104112</td><td></td></tr><tr><td>ZEISS Fluorescent Microscope Axioskop&amp;nbsp;</td><td>ZEISS</td><td></td><td></td></tr><tr><td>Nikon NIS-Elements BR 4.11.00 64 bit software</td><td>Nikon</td><td>BR 4.11.00</td><td></td></tr><tr><td>Nikon DS-QiMc 12 bit digital camera</td><td>Nikon</td><td>DS-QiMc</td><td></td></tr></tbody></table>

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

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

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 ...

科研

癌症研究

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

医学

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

在3D揭示侵入性癌症细胞骨架组织

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, where success depends on the distribution of specific immune cell subpopulations. Over the past decade, multiple sophisticated technologies have been introduced to achieve detailed resolution of the TME using two-dimensional sections from either formalin-fixed, paraffin-embedded (FFPE) or fixed-frozen tissues. While these thin sections are easier to procure and analyze, they lack the three-dimensional architecture needed to reliably and comprehensively characterize a tumor. To address this limitation, a tissue mounting and imaging technique was developed to enable the three-dimensional analysis of tumor lesions in their native in vivo state. This protocol outlines the procurement of human tumor tissue, the mounting of samples on custom-printed platforms, and staining procedures for post-fixation samples. The multiplexed immunofluorescence technique, IBEX (Iterative Bleaching Extends Multiplexity), was adapted to characterize the three-dimensional TME with up to 15 markers for tumor, immune, and stromal cells using commercially available antibodies. Imaging depths of up to 100 &#181;m were achieved using an inverted white-light laser confocal microscope with a custom-printed imaging adaptor and commercial glass-bottom dishes to ensure optimal tissue orientation. This protocol highlights the potential of the IBEX method to expand multiplexed immunofluorescence studies, providing a more comprehensive understanding of TME composition.

This article describes a protocol for multiplex immunofluorescence optimized to characterize the three-dimensional architecture of peritoneal metastases.

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

肿瘤微环境下免疫细胞种群的可视化、定量和映射

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

免疫与感染

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

生物学

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

冷冻组织部分多光谱荧光成像的快速方法

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Immunofluorescence Assay: A Method to Identify Tumor Cells Captured on a Medical Wire

Source: Gallerani, G. et.al., <a href="https://www.jove.com/v/56936/characterization-tumor-cells-using-medical-wire-for-capturing">Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH.</a> J. Vis. Exp. (2017).
This video describes the technique of immunofluorescence staining of circulating tumor cells captured on a functionalized medical wire. The functionalized wire allows isolation of CTCs in vivo, directly from patient&#8217;s blood stream.&#160;

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

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