Name: four-color fluorescence immunohistochemistry of t-cell subpopulations in archival formalin-fixed, paraffin-embedded human oropharyngeal squamous cell carcinoma samples
Uploaded: 2017-07-29T14:00:00.000+00:00
Duration: 7 min 43 s
Description: Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

Simone Punt得到荷兰癌症协会授予的UL2010-4801的支持。我们要感谢原始论文的所有合作者，这份JoVE协议基于:Emilie A.Dronkers，Marij JP Welters，Renske Goedemans，SenadaKoljenović，Elisabeth Bloemena，Peter JF Snijders，Arko Gorter和Sjoerd van德伯格

患者样本按照"荷兰生物医学科学学会联合会人体组织适当二次使用行为守则"（www.federa.org）中所述的医学伦理指导进行处理。 准备幻灯片<ol><li>在获得医学伦理委员会许可使用切除材料后，从病理科获取切除的组织材料（任何种类的组织）。 注意:对于目前的实验，预处理的肿瘤样品是从1970年至2011年间在莱顿大学医学中心莱顿大学医学中心诊断的原发性口咽肿瘤获得的，如前所述（n = 162） 13选择 。组织的大小仅受到足够量的肿瘤组织的需求的限制，以可靠地在肿瘤内获得4个显微图像。 </li><li>将组织固定在4％缓冲的福尔马林中至少12小时。在ag中去除水分辐射系列乙醇。在二甲苯中洗涤，并使用自动组织处理器将其包埋在石蜡中。 </li><li> 使用切片机在玻片上切割4μm厚的福尔马林固定的石蜡包埋（FFPE）组织切片。使用标准程序将组织块放在微绒毛组织固定器中。 <ol><li>切成薄片，将其放在含有45℃去离子水的水浴表面。使用适合免疫组织化学染色的载玻片通过将载玻片放置在直接位于该部分下方的水中来切出部分。小心地将滑块向上移动一定角度，将部分从水中取出并将其粘贴在滑块上。 </li></ol></li><li>让载玻片在37℃下干燥过夜。 </li><li>将FFPE载玻片完全浸入新鲜的100％二甲苯中，使其均匀脱蜡3次，每次5分钟。在新鲜的100％乙醇，70％乙醇和50％乙醇中浸泡两次，每次5分钟，将载玻片浸没两次。 </li></ol> 2.执行抗原检索<ol><li>在去离子水中洗涤载玻片5分钟。 </li><li>使用微波将预热三乙二胺四乙酸（EDTA）缓冲液（10mM Tris + 1mM EDTA，pH9.0）加热至沸腾温度。通过将载玻片浸入预热的EDTA缓冲液中并将缓冲液在该温度下保持10分钟在微波中进行抗原恢复。 注意:可以使用功率为900 W的微波炉。 </li><li>让幻灯片在缓冲液中冷却2小时。 </li></ol> 3.组织幻灯片<ol><li>用磷酸盐缓冲盐水（PBS）洗涤载玻片两次，每次5分钟。 </li><li>用疏水笔在组织周围绘制一圈，以防止稀释的抗体从载玻片上溅出。用PBS洗一次;这是可选的。 </li><li>用1％w / v牛血清a稀释的第一抗体（抗CD3 1:50，抗FoxP3 1:200和抗IL-17 1:50）孵育组织白蛋白（BSA）在PBS中于室温过夜。 注意:每张幻灯片的体积取决于组织的大小，但通常为50 - 150μL。以与CD3，CD8和FoxP3抗体相同的稀释度（ 即，兔Ig同种型对照抗体，小鼠IgG1同种型对照抗体和山羊）以相同同种型的抗体替代对于阴性对照载玻片具有未知特异性的抗体IgG同种型对照抗体）。 </li><li>用PBS洗涤载玻片三次，每次5分钟。 </li><li> 用针对一级抗体物种和/或同种型（ 即 1:200驴抗山羊IgG Alexa 488，驴抗兔IgG A546和驴抗小鼠A647）的荧光标记的二抗的组合孵育组织，稀释于1％BSA在PBS中在室温下搅拌1小时。 注意:每张幻灯片的体积取决于组织的大小，但通常为50 - 150μL。 <ol><li>后添加二次抗体，通过将它们存放在暗盒中或通过将盒子包裹在铝箔中，尽可能地保持滑片免受光的影响。 </li></ol></li></ol>装载和复制组织幻灯片<ol><li>在PBS中洗涤载玻片三次，每次5分钟。 </li><li>使用含有核反复染料（ 例如 DAPI）的水性防褪色装载介质安装载玻片。尽快（两周内）使用荧光显微镜分析载玻片。将载玻片在4°C的黑暗中保存，直到分析。 </li></ol> 5.分析染色组织幻灯片<ol><li> 使用装备有20 - 25X物镜的荧光显微镜获取每张幻灯片的四张图像。在包含在幻灯片中的整个肿瘤区域的随机位置拍摄图像，以捕获肿瘤的代表性部分。 注意:应使用250X的标准放大倍率。任何荧光o可以使用r激光扫描显微镜，只要它具有可视化所用荧光染料所需的滤光片和激光器即可。 <ol><li>为了可视化Alexa 488，使用最大激发490，最大发射525。对于Alexa 546的可视化，使用最大激发最大值556，排放最大值为573;为了可视化Alexa 647，使用最大激发650，最大发射665;并且为了可视化DAPI，使用激发最大值为358，发射最大值为461。 </li><li>将图像作为图像文件（ 即 jpeg和tiff）保存在用户可用的软件中。 </li></ol></li><li> 在咨询病理学家之后，基于肿瘤上皮与基质细胞的核和细胞形状，区分肿瘤上皮和肿瘤基质区域。 <ol><li>为了确定总基质表面积，在基质细胞（而不是肿瘤细胞）占据的所有区域周围绘制一条线d使用由显微镜制造商提供的图像分析软件的表面积。 <ol><li>点击图片下的"覆盖"标签;选择"封闭多线"形状并点击它。通过点击该区域直到达到第一个点，划分肿瘤上皮和间质区域之间的边界。这提供了该领域的措施。 注意:一旦形状是封闭的，一个数字将旁边的形状包围所选择的图像的表面面积（A = X微米2）出现。 </li></ol></li><li>从总图像表面积（以图像属性提供）减去该表面积，以计算上皮表面积。通过在其周围绘制线排除血管，坏死和自发荧光区域，记录所提供区域的大小并从上皮表面区域减去。 </li><li>在电子表格文件中保存所有表面积号，以计算平均表面积每张幻灯片的细胞数</li><li>使用图像分析软件，通过检查所有四个通道的荧光来确定细胞是单次，双次还是三次阳性。 <ol><li>使用ImageJ计算每个图像中的单，双和三阳性细胞。 <ol><li>下载ImageJ。点击"文件"和"打开"打开图像并选择要分析的图像。右键单击"点工具"（四行中间的一个红色方块），然后选择"多点工具"。双击"多点工具"，通过为每个单元格类型选择不同的计数器编号来计算图像中不同单元格的数量。 注意:计算每种不同细胞类型的所有细胞，如通过存在一种，两种或所有三种荧光颜色所鉴定的。排除DAPI，因为这种核复染素存在于所有细胞中，并用于区分肿瘤细胞与免疫细胞。每个单元格类型都被计数使用不同的计数器号。通过每次选择不同的计数器，在肿瘤上皮和基质区域中分别计数每种细胞类型的细胞数。不要在血管和大部分自身荧光区域内计数细胞。 </li><li>记录电子表格文件中每个图像在每个计数器仓中分析的单元格数。 </li><li>将肿瘤上皮和肿瘤基质的总表面积划分为每种细胞类型的细胞总数。 </li></ol></li></ol></li></ol></li></ol>统计分析注意:所有统计分析应与统计学家讨论，以确保数据质量。对于一般统计，请使用任何医疗统计指南14 。 <ol><li>使用Spearman等级相关rho测试来测试小区频率之间的相关性。 </li><li>测试阳性细胞数量的统计学差异在使用Wilcoxon Mann-Whitney测试的患者组之间。 </li><li>通过使用Kaplan-Meier曲线生成和对数进行基于细胞数的细胞分组，测试细胞数与无病生存之间的相关性（ 即从诊断到局部或远程复发死亡或疾病的时间）排名分析15 。 </li><li>使用Cox比例风险回归分析进行多变量分析。 </li></ol>

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作者声明没有商业或金融利益冲突。

对于所述的方案，最关键的步骤之一是确定所使用的一抗的正确稀释度。标记的二次抗体的稀释度为1:200，如这些特异性Alexa标记的抗体的制造商所推荐的。然后通过连续稀释来确定一抗的稀释度，优选使用两种不同样本的预期组织类型（在这种情况下，OPSCC）。最佳工作稀释度是在没有背景噪声的情况下获得清除信号并且在相同浓度下没有阴性对照的信号的稀释。根据可用的显微镜​​类型和研?...

口咽鳞状细胞癌（OPSCC）是起源于口咽部的鳞状细胞癌的异质组。危险因素包括OPSCC人乳头瘤病毒（HPV）感染和使用酒精和烟草1,2。免疫反应的作用以及如何在临床环境中使用这种作用刚刚开始探索。肿瘤微环境被吸引到癌症部位的免疫细胞渗透。尽管高CD8 +细胞毒性T细胞频率已经与OPSCC患​​者3的改善生存率相关，但其他T细胞亚群（包括Treg和Th17细胞）的作用尚不清楚4 。而Th1和Th17细胞被认为有助于靶向肿瘤细胞的免疫应答，Treg因其抑制其他T细胞活性的能力而众所周知5 。但是，T的存在暂存器已经发现，在不同的肿瘤类型6有利和不利的响应相关。由于并非所有存在于血液中的免疫细胞以相同的程度渗透到肿瘤中，所以研究局部肿瘤微环境提供了针对肿瘤的免疫应答的最可靠的测量。本研究的目的是确定免疫细胞数量和类型与临床结果之间的相关性。我们使用四色荧光IHC成像分析人类OPSCC中各种T细胞亚群的数量和定位。 我们专注于总T淋巴细胞（CD3 + ），Th17细胞和免疫抑制性FoxP3 + Treg，其分化途径与Th17细胞密切相关。 Th17细胞的特征在于CD3和IL-17的组合。细胞因子IL-17也可以由非T细胞产生7 。我们确定了intra-epit的分布在一系列OPSCC病例中的Helic和基质T细胞，Treg，Th17和IL-17 +非T细胞，并分析与患者生存的相关性。多色荧光IHC用于鉴定CD3，Foxp3和IL-17的表达，与DAPI复染色结合。该测定允许容易且清楚地鉴定肿瘤细胞（使用DAPI核染色）和浸润性T细胞群体（使用不同标记物的组合）。样品制备和染色后，使用荧光显微镜和成像软件分离所使用的不同荧光颜色，并确定存在于肿瘤上皮细胞和肿瘤相关基质中的细胞的数量和类型。 用于量化和表型免疫细胞群体的替代测定法是通过肿瘤或外周样品（ 即血液或腹水）的流式细胞术分析或通过飞行时间（CyTOF）分析的细胞计数。使用这个技术，丢失了关于不同细胞类型的定位和相对分布的所有信息。外周样品的使用和分析也不提供关于哪些细胞能够渗透肿瘤微环境的信息。血液和腹水免疫细胞的分析已经示出不以反映肿瘤组织8,9的免疫细胞浸润的表型和频率。 另一个选择是使用明视野显微镜。这种技术优于荧光成像的优点是没有组织自体荧光。虽然一些样品含有更多的自发荧光 - 特别是红细胞，但也包括其他细胞类型，包括嗜中性粒细胞 - 这些区域可以在几乎所有样品的分析中容易地去除。免疫荧光提供了通过使用一组靶向荧光分析一个样品中的多个标记物的优点nt波长。由于缺乏足够数量的标记物和用于特定抗原的商业上可获得的抗体同种型，目前对于明视野显微术来说，这是不可能的。 这里所描述的多色荧光IHC技术已在许多不同的癌症类型和抗体组合被用于研究不同的免疫细胞群，以及肿瘤细胞表达的分子，如人白细胞抗原（HLA）和PD-L1 10，11， 12 。该方案已经使用许多不同类型的样品和抗体建立和验证。

<table><tbody><tr><td>Pathos Delta Ultra Rapid Tissue Processor</td><td>Milestone and Histostar</td><td></td><td>Automated tissue processor</td></tr><tr><td>Histostar</td><td>Thermo Scientific</td><td></td><td>Tissue block embedding machine</td></tr><tr><td>Formaldehyde</td><td>Baker</td><td></td><td></td></tr><tr><td>Xylol</td><td>Merck</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td></td><td></td></tr><tr><td>milliQ water</td><td>Elaga Purelab Chorus</td><td></td><td></td></tr><tr><td>Paraffin wax/Paraclean</td><td>Klinipath</td><td>5079A</td><td></td></tr><tr><td>Microtome tissue holder</td><td>Leica</td><td>RM225</td><td></td></tr><tr><td>Flex IHC side</td><td>Dako</td><td></td><td>Tissue slide</td></tr><tr><td>Tris</td><td>Merk-Milipore</td><td>1,083,821,000</td><td></td></tr><tr><td>EDTA</td><td>Baker</td><td>1073</td><td></td></tr><tr><td>PBS</td><td>Bio-Rad</td><td>BUF036A</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Rabbit anti-CD3</td><td>Abcam</td><td>ab828</td><td>Titrate required antibody dilution</td></tr><tr><td>Mouse IgG1 anti-FoxP3</td><td>Abcam</td><td>ab20034</td><td>Titrate required antibody dilution</td></tr><tr><td>Goat IgG anti-IL-17</td><td>R&amp;amp;D Systems</td><td>AF-317-NA</td><td>Titrate required antibody dilution</td></tr><tr><td>Rabbit Ig isotype control antibody</td><td>Abcam</td><td>ab27472</td><td>Use at same final concentration as anti-CD3</td></tr><tr><td>Mouse IgG1 isotype control antibody</td><td>Abcam</td><td>ab91353</td><td>Use at same final concentration as anti-FoxP3</td></tr><tr><td>Goat IgG isotype control antibody</td><td>ThermoFisher Scientific</td><td>02-6202</td><td>Use at same final concentration as anti-IL-17</td></tr><tr><td>Donkey anti-rabbit IgG A546</td><td>ThermoFisher Scientific</td><td>A10040</td><td>Dilute 1:200 in 1% BSA/PBS</td></tr><tr><td>Donkey anti-mouse-A647</td><td>ThermoFisher Scientific</td><td>A31571</td><td>Dilute 1:200 in 1% BSA/PBS</td></tr><tr><td>Donkey anti-goat IgG A488</td><td>ThermoFisher Scientific</td><td>A11055</td><td>Dilute 1:200 in 1% BSA/PBS</td></tr><tr><td>VectaShield containing DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>LSM700 confocal laser scanning microscope&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr><tr><td>LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective</td><td>Zeiss</td><td>420852-9972-720</td><td></td></tr><tr><td>LSM Zen Software</td><td>Zeiss</td><td>version 2009</td><td></td></tr><tr><td>LSM Image Browser</td><td>Zeiss</td><td>version 4.2.0.121</td><td>Available to download at www.zeiss.com/microscopy/int/website/downloads/lsm-image-browser.html.</td></tr><tr><td>SPSS</td><td>IBM Corp.</td><td>version 20.0</td><td></td></tr><tr><td>ImageJ</td><td></td><td>version 1.50i</td><td>Available to download at http://rsb.info.nih.gov/ij.</td></tr></tbody></table>

four-color fluorescence immunohistochemistry of t-cell subpopulations in archival formalin-fixed, paraffin-embedded human oropharyngeal squamous cell carcinoma samples

四色荧光免疫组织化学（IHC）技术是一种量化目标细胞群的方法，同时考虑到其相对分布及其在组织中的定位。该技术已被广泛应用于研究各种肿瘤类型的免疫浸润。肿瘤微环境被吸引到肿瘤部位的免疫细胞浸润。已经发现不同的免疫细胞群体在肿瘤微环境中发挥不同的作用，并且对疾病的结果具有不同的影响。本手册以口咽鳞状细胞癌（OPSCC）为例介绍了多参数荧光IHC的应用。该技术可以扩展到其他组织样本和感兴趣的细胞类型。在本研究中，我们分析了大型OPSCC队列的上皮内和间质隔室（n = 162）。我们专注于总T淋巴细胞（CD3 + ），免疫抑制调节（Tregs， 即 FoxP3 + ）和T辅助细胞17（Th17）细胞（ 即 IL-17 + CD3 + ），使用核复染色体将肿瘤上皮细胞与基质区分开来。发现大量的T细胞与肿瘤内IL-17 +非T细胞数量较少的患者的无病生存率相关。这表明IL-17 +非T细胞可能与OPSCC中的不良免疫反应相关，这与IL-17和癌症患者的存活率差相关。目前，正在开发使用多达7种不同荧光染料的新型多参数荧光IHC技术，并且能够使肿瘤微环境中的免疫细胞更精确地表征和定位。

从1970年至2011年间，莱顿大学医学中心莱顿大学医学中心（Leiden，荷兰莱顿大学医学中心）诊断的一系列FFPE预处理肿瘤样本，如前所述（n = 162）选择，使用所述方案13进行染色。分析每张幻灯片的1到4个随机图像（ 图1 ）。指出一些自体荧光细胞，其可以通过其完整的黄色外观而明确区分。在这种情况下，细胞真正的双重或三重阳?...

Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

多参数荧光免疫组织化学可用于评估肿瘤微环境中免疫细胞群体的数量，相对分布和定位。该手稿描述了使用这种技术来分析口咽癌中的T细胞亚群。

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

four-color-fluorescence-immunohistochemistry-t-cell-subpopulations

Research

JoVE Journal

Cancer Research

9.6K 次观看.  Leiden University Medical Center. 多参数荧光免疫组织化学可用于评估肿瘤微环境中免疫细胞群体的数量，相对分布和定位。该手稿描述了使用这种技术来分析口咽癌中的T细胞亚群。 

视频: Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

癌症研究

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since &#34;low&#34; or &#34;high&#34; HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

原位杂交的色原性杂交是HPV相关头颈部癌症诊断的工具

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...

遗传学

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 ...

使用 mIHC 破译肺鳞状癌中的肿瘤微环境

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

作者聚焦：增强肺癌样本中的多色荧光定位

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

石蜡包埋肺癌组织的多重免疫组化染色

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...

癌症组织中免疫细胞标志物的原位免疫组织化学检测

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

作者聚焦：使用荧光免疫组织化学高效检测癌症组织中的免疫细胞浸润

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

摘要

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此视频中的章节

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

在使用定量免疫肿瘤的异质性蛋白表达的映射

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

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