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​​类型和研究人员的偏好，可以对二次抗体和使用的安装介质进行修改。如果没有达到预期的结果，我们建议您查看使用的玻片的类型，因为已知一些已知导致背景信号的问题<html>对于特定种类的染色溶液（ 例如， Perma Blue）。其次，通过使用热诱导的表位检索可能会破坏某些抗原。在这种情况下，研究人员可以用其他技术取代缓冲液的加热步骤，例如酶诱导的表位检索。第三，如果使用疏水笔，标记不应该太靠近组织，因为这将防止染色溶液正常工作。关于每个样本的部分数量和每个部分所需的图像数量，由研究人员和具体研究问题决定用足够的统计学能力来回答研究问题所需的数量。对于我们的分析，我们每个样本使用一个部分，每个部分使用四个图像。最后，当使用免疫荧光染色时，使用指甲油时要注意密封盖子，因为指甲油中存在的醇会影响荧光信号。 ve_content"&gt;这里描述的协议不涉及高通量分析，我们已经尝试自动化这一步，但是细胞大小和形态的主观解释使得当时可用的软件包的分析自动化变得复杂，尽管组织制备并且染色可以在很大程度上自动化，手动计数细胞。 我们能够使用所述方案研究所研究的浸润性T细胞亚群与临床结果之间的相关性。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细胞可能与在OPSCC不良的免疫反应，这与在癌症患者中16 IL-17和低存活率之间描述的相关协议。 [曝光]已经使用的软件程序已经成为商业上可用的自动化步骤，目前正在取代手动细胞观察和计数，这将导致更可靠，客观，可重现和快速的结果。 此外，允许荧光免疫组织化学标记多路复用的商业套件的可用性目前正在上升17 。这将允许多达7种不同的免疫标记物/生物标志物与核复染剂组合。然而，我们认为，这里报告的方案仍将更加有效和轻松地应用于缺乏复杂而昂贵的基础设施，显微镜和多光谱染色分析软件包的实验室。

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 border="0" cellpadding="0" cellspacing="0" width="1379">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Pathos Delta Ultra Rapid Tissue Processor</td>
			<td style="width:167px;">Milestone and Histostar</td>
			<td style="width:143px;"></td>
			<td style="width:696px;">Automated tissue processor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Histostar</td>
			<td style="width:167px;">Thermo Scientific&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:696px;">Tissue block embedding machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Formaldehyde</td>
			<td style="width:167px;">Baker</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Xylol</td>
			<td style="width:167px;">Merck</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ethanol</td>
			<td style="width:167px;">Merck</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">milliQ water</td>
			<td style="width:167px;">Elaga Purelab Chorus</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Paraffin wax/Paraclean</td>
			<td style="width:167px;">Klinipath</td>
			<td style="width:143px;">5079A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Microtome tissue holder&#160;</td>
			<td style="width:167px;">Leica</td>
			<td style="width:143px;">RM225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flex IHC side</td>
			<td style="width:167px;">Dako</td>
			<td style="width:143px;"></td>
			<td>Tissue slide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris</td>
			<td style="width:167px;">Merk-Milipore</td>
			<td style="width:143px;">1,083,821,000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">EDTA</td>
			<td style="width:167px;">Baker</td>
			<td style="width:143px;">1073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">PBS</td>
			<td style="width:167px;">Bio-Rad</td>
			<td>BUF036A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BSA</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:143px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rabbit anti-CD3</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:143px;">ab828</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Mouse IgG1 anti-FoxP3</td>
			<td style="width:167px;">Abcam</td>
			<td style="width:143px;">ab20034</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Goat IgG anti-IL-17</td>
			<td style="width:167px;">R&#38;D Systems</td>
			<td style="width:143px;">AF-317-NA</td>
			<td>Titrate required antibody dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rabbit Ig isotype control antibody</td>
			<td style="width:167px;">Abcam</td>
			<td>ab27472</td>
			<td>Use at same final concentration as anti-CD3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Mouse IgG1 isotype control antibody</td>
			<td style="width:167px;">Abcam</td>
			<td>ab91353</td>
			<td>Use at same final concentration as anti-FoxP3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Goat IgG isotype control antibody</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">02-6202</td>
			<td>Use at same final concentration as anti-IL-17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-rabbit IgG A546</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A10040</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-mouse-A647</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A31571</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Donkey anti-goat IgG A488</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:143px;">A11055</td>
			<td>Dilute 1:200 in 1% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">VectaShield containing DAPI</td>
			<td style="width:167px;">Vector Laboratories</td>
			<td style="width:143px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM700 confocal laser scanning microscope&#160;</td>
			<td style="width:167px;">Zeiss</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective</td>
			<td style="width:167px;">Zeiss</td>
			<td>420852-9972-720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM Zen Software</td>
			<td style="width:167px;">Zeiss</td>
			<td>version 2009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LSM Image Browser</td>
			<td style="width:167px;">Zeiss</td>
			<td style="width:143px;">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 height="21">
			<td height="21" style="height:21px;width:375px;">SPSS</td>
			<td style="width:167px;">IBM Corp.</td>
			<td style="width:143px;">version 20.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ImageJ</td>
			<td style="width:167px;"></td>
			<td style="width:143px;">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性仅在特定抗体对预期定位（在细胞膜或细胞核）处染色阳性。阴性对照载玻片在背景组织自体荧光以上没有显示任何特异性染色（ 图2和图4 ），证实所有信号对于主要识别的靶标是特异性的抗体。发现所有肿瘤样品都被不同程度的CD3 + T细胞浸润。 FoxP3 + Tregs总是表达CD3，是主要的浸润性T细胞群体之一。表达IL-17（Th17细胞）的CD3细胞是浸润性T细胞的次要群体。通过CD3表达IL-17 -细胞是另一个丰富浸润细胞群。 IL-17 + FoxP3 +细胞不超过所有FoxP3 +细胞的0.01％。 所有统计检验均为双侧，p值低于0.05被认为是显着的14 。提出了上皮内或总细胞数与存活之间的相关性，因为基质或总细胞数与患者存活之间的相关性相似。大量浸润的总CD3 + T细胞呈现与无病生存改善相关的趋势（0.086， 图5A ）与低数量的T细胞相比（ 即，最低四分位数）。当专门研究IL-17 +细胞数少的患者时，大量的总浸润性T细胞与改善的无病生存率相关（p = 0.012， 图5B ）。在肿瘤浸润性IL-17 +细胞数量较多的患者组中，肿瘤浸润性T细胞的预后效应丧失（数据未显示）。因此，肿瘤浸润性T细胞在OPSCC中的作用可能与存在的低IL-17 +细胞数有关。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55589/55589fig1.jpg" /> 图1:通过四色荧光IHC染色的FFPE口咽癌组织。 CD3（ A ，红），IL-17免疫荧光染色的代表性图像（ B </str<html> ong&gt;，绿色）和FoxP3（ C ，蓝色），以及与DAPI（灰色）（ D ）组合的合并图像。两个自体荧光细胞用箭头表示。 <a href="http://ecsource.jove.com/files/ftp_upload/55589/55589fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55589/55589fig2.jpg" /> 图2:CD3阴性对照。 FFPE口咽癌组织的代表性图像，如方案中所述染色，用具有未知特异性的兔Ig同种型对照抗体取代抗CD3抗体。结合IL-17（ B ，绿色），FoxP3（ C ，蓝色）和DAPI（ D ，灰色）染色的兔Ig（ A ，红色）没有染色。2large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55589/55589fig3.jpg" /> 图3:IL-17的阴性对照。 FFPE口咽癌组织的代表性图像，如方案中所述染色，用具有未知特异性的山羊Ig同种型对照抗体取代抗IL-17抗体。结合染色CD3（ A ，红色），FoxP3（ C ，蓝色）和DAPI（ D ，灰色）的山羊Ig（ B ，绿色）无染色。 <a href="http://ecsource.jove.com/files/ftp_upload/55589/55589fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55589/55589fig4.jpg" /> F图4:FoxP3的阴性对照。 FFPE口咽癌组织的代表性图像，如方案中所述染色，用具有未知特异性的小鼠IgG1同种型对照抗体取代抗FoxP3抗体。显示与CD3（ A ，红色），IL-17（ B ，绿色）和DAPI（ D ，灰色）染色结合的小鼠IgG1（ C ，蓝色）没有染色。 <a href="http://ecsource.jove.com/files/ftp_upload/55589/55589fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55589/55589fig5.jpg" /> 图5:Kaplan-Meier生存曲线。对于IL-17 +细胞/ mm 2低于中位数的患者，无病生存曲线显示非常低（ 即最低四分位数）与高数目的total T细胞（ A ）和低（ 即低于中位数）相对于总肿瘤区域中的总T细胞数量（ B ）。转载癌症免疫学免疫杂志许可13 。 <a href="http://ecsource.jove.com/files/ftp_upload/55589/55589fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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

Nanyang Technological University

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

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

在头颈部鳞状细胞癌的神经浸润模型

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

科研

癌症研究

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌细胞外基质的制备与舌鳞癌体外重建

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

石蜡嵌段细胞免疫细胞化学法测定培养体细胞蛋白质表达水平

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

pT3N0M0 胃癌组织石蜡嵌入幻灯片上的弹性染色

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

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 ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

多维共同培养系统模拟肺鳞癌进展

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

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 ...