作者感谢Drs。 Fahad Bafakih和Jim Mandell收购和鉴定患者样本，Garrett F. Beeghly为免疫组化提供帮助，生物制剂和组织研究设施，心血管研究中心组织学核心和弗吉尼亚大学生物分子分析设备协助样本采集，免疫组织化学和成像。

该方案鉴定了福尔马林固定石蜡包埋（FFPE）样品中的细胞组分，如同样的临床患者样本。石蜡包埋允许细胞和组织形态的最佳维持以及具有更好的切片寿命。用于该分析的样品通过弗吉尼亚大学生物原理和组织研究设施进行访问。根据2010年至2013年在弗吉尼亚大学完成肿瘤切除术的胶质母细胞瘤（星形细胞瘤，WHO IV级）的确定性诊断，由神经病理学家选择患者样本，并在该分析之前被去除11 。 FFPE样品脱蜡和再水化注意:该协议的这一部分是针对FFPE样本的。而石蜡包埋的样品由于保留细胞和t而可能对这一分析更有用问题形态，这个分析也可以用冻结部分完成。如果使用冷冻切片，该部分可以省略，直接进行显色免疫组织化学。 <ol><li>进行以下洗涤5分钟:二甲苯，二甲苯，100％乙醇，100％乙醇，95％乙醇，95％乙醇，70％乙醇，去离子水，去离子水</li></ol> 2.抗原检索注意:该方案的这一部分是破坏FFPE样品福尔马林固定期间形成的亚甲基桥所必需的，并暴露抗原位点以进行结合。 <ol><li>稀释Tris型高pH抗原揭示溶液，制造商推荐在蒸馏水中。 </li><li>使用微波进行热介导的抗原检索。其他形式的热介导回收（如压力锅，蔬菜蒸笼或沸水）也足够了。 <ol><li>将稀释后的未曝光溶液加入非密封微波炉中船只。将幻灯片放入血管。将幻灯片放入微波炉。 </li><li>在大功率下煮20分钟。监测液体的蒸发量，并根据需要用蒸馏水补充。 </li></ol></li><li>使样品在室温下在溶液中冷却1小时。 </li></ol> 3.显色免疫组织化学<ol><li>用疏水笔勾画组织样本，以尽量减少覆盖样品所需的试剂体积。 注意:确保保持组织样本的水分，不要让它们变干，因为这会影响染色效果。 </li><li>吸管足够的透化溶液（Tris缓冲盐水（TBS）+ 0.01％Triton-X）以覆盖组织样品（通常约100-200μL）。 </li><li>取出并丢弃溶液并重复步骤3.2。 </li><li>在室温下用封闭溶液（2.5％马血清+透化溶液）孵育样品。 </li><li>在4℃孵育样品过夜，用一级抗体稀释在阻塞解决方案。 注意:此处使用的所有初级抗体均以1:200稀释，但最佳稀释度可用制造商推荐的连续稀释度确定。此协议中使用的抗体的详细信息先前已发布11 。 </li><li>使用与一抗体宿主动物相对应的辣根过氧化物酶聚合物试剂检测一抗，按照制造商方案。 </li><li>吸取足够的透化溶液以覆盖组织样品并孵育5分钟。 </li><li>删除并丢弃解决方案并重复步骤3.7。 </li><li>在1×TBS中的0.3％H 2 O 2中孵育15分钟。 </li><li>用过氧化物酶二氨基联苯胺（DAB）底物开发样品2-10分钟，直到达到所需的染色强度。 </li><li>复制样品以鉴定细胞核，如用苏木精，按照制造商协议。 </li><li>用100％脱水样品乙醇和二甲苯。 </li><li>使用安装介质永久安装样品。 </li></ol> 4.利益区域识别<ol><li>图像幻灯片在明场显微镜下能够进行高分辨率图像。 注意:使用成像细胞组分至少20x分辨率。 </li><li>将相机移动到整个组织样本的特定感兴趣区域。 </li><li>将图像保存为TIFF文件进行定量。 </li></ol>图像分析<ol><li>在ImageJ中打开图像以量化百分比覆盖率。 </li><li>使用阈值颜色插件从苏木精染色的核中去除紫色。 </li><li>将图像转换为8位。 </li><li>添加阈值，不带深色背景。 </li><li>使用17个预加载的ImageJ阈值滤波器（ 即 MaxEntropy）中的一个处理图像，因此只有DAB染色部分被包括在阈值中。 注意:选择最小化的最佳预加载阈值过滤器包括背景染色。这可以取决于抗体的染色质量和特异性。对每个患者样本中的所有技术重复使用相同的阈值。 </li><li>应用阈值。 </li><li>测量阈值图像的百分比面积。 </li><li>每个样本中多个区域的平均面积百分比覆盖率。 </li></ol>

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我们在这里提出的方法是一种定量的方法来分析使用传统的显色免疫组织化学染色的组织学样品。此类分析的现行方法包括相似的染色方案，随后由独立病理学家进行分级。该方法是可靠的，但是对于许多应用，需要更精确地了解细胞构成，例如更好地理解与肿瘤相关的异质性和用于体外研究的肿瘤的准确重现。 该方案证明了用于定量分析脑肿瘤微环境内胶质细胞群体的直?...

成胶质细胞瘤（GBM），最常见的恶性脑肿瘤，从初级肿瘤主体，其特征在于高度扩散侵入周围健康脑实质1,2。此漫入侵使得肿瘤尤其难以完全切除，并且保持治疗后入侵癌细胞是不可避免的复发2，3，4中的最常见的原因。以前，我们发现抑制弥漫性胶质瘤细胞侵袭是治疗有益的5 ，但是对于有助于GBM入侵的复杂机制知之甚少。肿瘤微环境中，或组织中癌症周围，已牵涉于肿瘤的进展在多种癌症6,7。胶质母细胞瘤肿瘤微环境，关节炎，相对低于特征，是独特复杂的，由多种胶质细胞组成，如星形胶质细胞，小神经胶质细胞和少突胶质细胞，以及细胞外基质，可溶性因子和生物物理因素。在实验上，星形胶质细胞和小胶质细胞已被证明能增加神经胶质瘤进展和侵袭8，9，10，但所有的神经胶质细胞中的天然人脑微环境中的组合物是未知的。 我们以前显示微环境成分可以通过定量分析胶质母细胞瘤微环境的细胞成分并将我们的分析纳入比例风险模型来预测患者存活11 。在这里，我们描述了定量分析方法，用于鉴定胶质母细胞瘤患者肿瘤切除肿瘤体积和相邻区域内胶质细胞的数量秒。我们使用显色免疫组织化学来鉴定胶质细胞群和ImageJ，以分析每个胶质细胞群体的染色百分比。评估百分比覆盖率创建一个简单的测量来确定细胞的形态学差异，特别是那些受癌细胞相互作用影响的细胞。以前的定量组织病理学染色的研究使用标准染色，如苏木精和曙红12或Masson的三色染色体13 ，其不利用基于抗体的免疫组织化学染色的特异性。我们的方法被开发直接量化胶质母细胞瘤患者肿瘤切除术中的胶质细胞群，我们旨在阐明复杂胶质母细胞瘤微环境。

<table><tbody><tr><td>Xylene</td><td>Fisher Chemical</td><td>X3P</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>High pH antigen unmasking solution</td><td>Vector Labs</td><td>H-3301</td><td></td></tr><tr><td>TBS</td><td></td><td></td><td></td></tr><tr><td>Triton-X</td><td>Amresco</td><td>9002-93-1</td><td></td></tr><tr><td>Horse serum</td><td></td><td></td><td></td></tr><tr><td>Anti-ALDH1L1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab56777</td><td></td></tr><tr><td>Anti-Iba1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab5076</td><td></td></tr><tr><td>Anti-Oligodendrocyte Specific Protein1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab53041</td><td></td></tr><tr><td>ImmPRESS anti-goat</td><td>Vector Labs</td><td>MP-7405</td><td></td></tr><tr><td>ImmPRESS Universal (anti-mouse/rabbit)</td><td>Vector Labs</td><td>MP-7500</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich</td><td>216763</td><td></td></tr><tr><td>ImmPACT DAB substrate</td><td>Vector Labs</td><td>SK-4105</td><td></td></tr><tr><td>Hematoxylin counterstain</td><td>ThermoScientific</td><td>72404</td><td></td></tr><tr><td>Histochoice Mounting Media</td><td>Amresco</td><td>H157-475</td><td></td></tr><tr><td>Aperio Scanscope</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Image Scanscope</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Super HT PAP Pen</td><td>Research Products International</td><td>195506</td><td></td></tr></tbody></table>

quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections

随着对肿瘤微环境的兴趣越来越大，我们着手开发一种方法，专门确定患者胶质母细胞瘤，最致命和最具侵袭性脑癌患者样本中的微环境成分。不仅定量方法有利于准确描述患病组织，而且还可能有助于更精确的预后，诊断和组织工程系统和替代品的发展。在胶质母细胞瘤中，胶质细胞，如小神经胶质细胞和星形胶质细胞，与病理学家分级的不良预后独立相关。然而，这些细胞和其他神经胶质细胞成分的状态在定量上尚未得到很好的描述。由于标记这些胶质细胞的大过程，这可能是困难的。此外，大多数组织学分析集中在整体组织样本或仅在大部分肿瘤内，而不是基于区域的划分量化在高度异质的组织。在这里，我们描述了一种用于鉴定和定量分析来自成胶质细胞瘤患者的肿瘤切除肿瘤体积和相邻区域内的神经胶质细胞群体的方法。我们使用显色免疫组织化学来鉴定患者肿瘤切除术和ImageJ中的胶质细胞群，以分析每个胶质细胞群体的染色百分比。通过这些技术，我们能够更好地描述胶质瘤肿瘤微环境区域的神经胶质细胞。

对于这一分析，我们的肿瘤切除术中的两个兴趣区域 - 主要肿瘤体积和相邻区域，主要由具有扩散性侵袭性癌细胞的健康组织组成（ 图1A ， 1B ） - 通过合成神经病理学家在苏木精和伊红染色患者样本。在每个患者样品中，通过显色免疫组织化学鉴定了星形胶质细胞（ 图1C ），小胶质细胞（ 图1D</str...

Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

患者胶质母细胞瘤切除中细胞微环境的定量免疫组织化学

该方案被开发用于使用显色免疫组织化学和ImageJ定量鉴定成胶质细胞瘤患者切除中的肿瘤微环境成分。

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

quantitative-immunohistochemistry-cellular-microenvironment-patient

Research

JoVE Journal

Medicine

9.6K 次观看.  University of Virginia. 该方案被开发用于使用显色免疫组织化学和ImageJ定量鉴定成胶质细胞瘤患者切除中的肿瘤微环境成分。 

视频: Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

科研

癌症研究

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 ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

医学

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

用于表征成人型弥漫性浸润性胶质瘤微环境的数字空间剖析

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

肿瘤微环境的多重免疫荧光和空间图像分析

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

作者聚焦：多重免疫荧光结合空间图像分析用于肿瘤微环境的临床和生物学评估  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

多重免疫荧光结合空间图像分析，用于肿瘤微环境的临床和生物学评估

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

绘制肿瘤免疫景观图谱：深入的多重 IHC 分析方案

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

作者聚焦：解锁对肿瘤免疫细胞景观的见解

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

患者胶质母细胞瘤切除中细胞微环境的定量免疫组织化学

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