Name: digital spatial profiling for characterization of the microenvironment in adult-type diffusely infiltrating glioma
Uploaded: 2022-09-13T12:50:00
Description: Watch this Scientific Journal Video about Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma at JoVE.com

作者感谢达特茅斯希区柯克卫生系统病理学和实验室医学系临床基因组学和先进技术实验室的支持。作者还承认达特茅斯癌症中心的病理学共享资源与NCI癌症中心支持补助金5P30 CA023108-37。

下面概述的协议遵循达特茅斯 - 希区柯克人类研究伦理委员会的指导方针。从本研究中包括组织样本的患者获得知情同意。有关此协议中使用的所有材料、试剂、设备和软件的详细信息，请参阅 材料表 部分。
1. 载玻片准备17
<ol><li>从人成人型弥漫性浸润性胶质瘤中检索或制备福尔马林固定的石蜡包埋组织。 注意：在该实验中，...

<ol>
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鉴于可能影响胶质瘤侵袭性的蛋白质的多样性，以及其中一些蛋白质仍未被发现的概念，高通量蛋白质定量方法是一种理想的技术方法。此外，鉴于肿瘤样品中的空间数据通常与差异表达相关18，将空间分析纳入蛋白质定量方法可以实现更有效的分析。
DSP的高通量方法还使其能够用于类似霰弹枪的方法，这是发现疾病和治疗反应的潜在新型生物标志物的理想...

弥漫性浸润性胶质瘤是成人中最常见的恶性脑肿瘤类型，并且总是致命的。神经胶质瘤细胞在大脑中广泛迁移的倾向是一个主要的治疗挑战。它们的传播机制包括定向迁移和不受控制的入侵。浸润性胶质瘤细胞已被证明沿白质束1 表现出嗜性和迁移性，最近的研究表明这些束脱髓鞘是一种活跃的促肿瘤特征2。侵袭由上皮到间充质转化介导，其中胶质瘤细胞通过减少编码细胞外基质蛋白和细胞粘附分子的基因的表达来获得间充质特性，放大迁移并促进通过肿瘤微环境的繁殖3，4，5。
在分子水平上，已经证明了几种赋予细胞稳定性并与免疫原性成分界面的蛋白质的破坏6。已知浸润性胶质瘤会抑制具有抗凋亡（例如PTEN）特性的蛋白质7。它们还过表达促进逃避宿主免疫反应的蛋白质（例如，PD1/PDL1）8。这些复杂途径的失调增强了致瘤性并增加了恶性潜力。
在侵袭性胶质瘤样本中，目的是评估对细胞生长、存活和增殖以及侵袭性和非侵入性成分之间的微环境结构完整性至关重要的蛋白质的差异表达。此外，我们试图研究具有活性免疫原性作用的蛋白质的差异调节，从而深入了解受损的宿主免疫防御可能增强胶质瘤的增殖和侵袭潜力的机制。鉴于最近广泛的研究表明，恶性肿瘤中的免疫标志物和失调的驱动因素如何成为免疫治疗的靶标，这一点尤其重要。在参与免疫监视和反应性的众多蛋白质中确定可行的治疗靶点需要一种高度灵敏和全面的方法。
鉴于可以研究的候选蛋白质种类繁多，我们寻求一种类似于免疫组织化学但具有增强数据处理效率的方法。在癌症生物学领域，DSP已成为一项强大的技术，与蛋白质组学分析和定量的替代工具相比具有重要优势。DSP的特点是其高通量多重检测能力，允许同时研究样品中的几种不同蛋白质，这标志着与免疫组织化学（IHC）等标准但低重技术的重要区别9，10。DSP的多重功能不会影响其作为定量和分析工具的保真度，正如比较DSP与IHC的研究所证明的那样。例如，当用于非小细胞肺癌标本的蛋白质组学定量时，DSP已被证明具有与IHC11相似的结果。此外，DSP还提供可定制的区域规范，用户可以在其中手动定义要执行蛋白质组学分析的区域。这比全断面多重方法10，12具有优势。因此，在单轮处理中，DSP通过调查多个感兴趣区域的多个蛋白质靶标来提供多层分析。
DSP在几种不同的病理环境中都有应用。DSP在肿瘤学分析中特别有利，因为空间变化可以与细胞转化和差异蛋白表达相关。例如，DSP已被用于将乳腺癌的蛋白质组学特征与邻近的肿瘤微环境进行比较。这对于了解该肿瘤的自然史及其进展以及对治疗的潜在反应具有重要意义13。说明DSP多功能性的其他背景包括前列腺癌中蛋白质多样性的空间定量14，免疫细胞标志物表达与头颈部鳞状细胞癌疾病进展的关联15，以及蛋白质表达的上皮 - 间充质梯度的证明，以区分转移性和原发性透明细胞卵巢癌16.通过实施DSP，我们表征了可能影响胶质瘤肿瘤发生和侵袭的蛋白质的空间地形。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BOND Research Detection System</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>DS9455</td>
			<td>Open detection system containing open containers in a reagent tray</td>
		</tr>
		<tr>
			<td>BOND Wash</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>AR950</td>
			<td>10X concentrated buffer solution for washing fixed tissue</td>
		</tr>
		<tr>
			<td>Buffer W</td>
			<td>NanoString, Seattle, WA</td>
			<td>contact company</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>Cy3 conjugation kit</td>
			<td>Abcam, Cambridge, UK</td>
			<td>AB188287</td>
			<td>Cy3 fluorescent antibody conjugation kit</td>
		</tr>
		<tr>
			<td>GeoMx Digital Spatial Profiler (DSP)</td>
			<td>NanoString, Seattle, WA</td>
			<td>contact company</td>
			<td>System for imaging and characterizing protein and RNA targets</td>
		</tr>
		<tr>
			<td>GeoMx DSP Instrument BufferKit</td>
			<td>NanoString, Seattle, WA</td>
			<td>100471</td>
			<td>Buffer kit for GeoMX DSP (including buffers for sample processing and preparation)</td>
		</tr>
		<tr>
			<td>GeoMx Hyb Code Pack_Protein</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300401</td>
			<td>Controls for running GeoMX DSP experiemtns</td>
		</tr>
		<tr>
			<td>GeoMx Immune Cell Panel (Imm Cell Pro_Hs)</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300101</td>
			<td>Protein module with targets for human immune cells and immuno-oncologic targets</td>
		</tr>
		<tr>
			<td>GeoMx Pan-Tumor Panel (Pan-Tumor_Hs)</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300105</td>
			<td>Protein module with targets for multiple human tumor types and for markers of epithelial-mesenchymal transition</td>
		</tr>
		<tr>
			<td>GeoMx Protein Slide Prep FFPE</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300308</td>
			<td>Sample preparation reagents for GeoMX DSP protein analysis</td>
		</tr>
		<tr>
			<td>IDH1-R132H antibody</td>
			<td>Dianova, Hamburg, Germany</td>
			<td>DIA-H09</td>
			<td>Monoclonal antibody against human IDH1 R132H</td>
		</tr>
		<tr>
			<td>LEICA Bond RX</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>contact company</td>
			<td>Fully automated IHC stainer</td>
		</tr>
		<tr>
			<td>Master Kit--12 reactions</td>
			<td>NanoString, Seattle, WA</td>
			<td>100052</td>
			<td>Materials and reagents for use with the nCounter Analysis system</td>
		</tr>
		<tr>
			<td>nCounter Analysis System</td>
			<td>NanoString, Seattle, WA</td>
			<td>contact company</td>
			<td>Automated system for multiplex target expression quantification (to be used with GeoMx DSP)</td>
		</tr>
		<tr>
			<td>TMA Master II</td>
			<td>3DHistech Ltd., Budapest, Hungary</td>
			<td></td>
			<td>To create the tissue microarray block</td>
		</tr>
	</tbody>
</table>

digital spatial profiling for characterization of the microenvironment in adult-type diffusely infiltrating glioma

弥漫性浸润性胶质瘤由于肿瘤扩散的浸润性而与高发病率和死亡率相关。它们是形态复杂的肿瘤，在肿瘤本身及其异质微环境中具有高度的蛋白质组学变异性。这些肿瘤的恶性潜力通过参与几个关键途径的蛋白质失调而增强，包括维持细胞稳定性和保持微环境结构完整性的过程。尽管已经进行了大量和单细胞胶质瘤分析，但这些蛋白质组学数据的空间分层相对缺乏。了解内在肿瘤、侵袭边缘和微环境之间致瘤因子和免疫细胞群的空间分布差异，为肿瘤增殖和增殖的机制提供了有价值的见解。数字空间剖析 （DSP） 代表了一种强大的技术，可以为这些重要的多层分析奠定基础。
DSP是一种有效定量组织标本中用户指定空间区域内蛋白质表达的方法。DSP是研究区分区域内和跨区分区域多种蛋白质差异表达的理想选择，可实现多层次的定量和定性分析。DSP协议是系统且用户友好的，允许对蛋白质组学数据进行定制的空间分析。在该实验中，组织微阵列由存档的胶质母细胞瘤核心活检构建。接下来，选择一组抗体，靶向样品中感兴趣的蛋白质。然后将预偶联到紫外光切割DNA寡核苷酸的抗体与组织样品一起孵育过夜。在抗体的荧光显微镜可视化下，用样品定义用于定量蛋白质表达的感兴趣区域（ROI）。然后将紫外线照射到每个ROI，切割DNA寡核苷酸。微吸寡核苷酸在每个ROI内计数，在空间基础上量化相应的蛋白质。

图4 显示了对胶质母细胞瘤样本进行的DSP实验的代表性结果。展示了一个热图，说明了使用DSP软件直观地捕获数据的方法之一。行代表蛋白质靶标，每列对应于一个感兴趣区域。蓝色到红色的颜色范围分别表示低到高表达。行内颜色的变异性反映了区域蛋白质的异质性，并表明可能与差异蛋白质表达的空间关联。例如，在本实验中，S100和CD56普遍较高，因为它们是神经标记。...

Watch this Scientific Journal Video about Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma at JoVE.com

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

蛋白质组学失调在弥漫性浸润性胶质瘤的扩散中起重要作用，但几种相关蛋白质仍未鉴定。数字空间处理（DSP）提供了一种高效、高通量的方法，用于表征可能导致浸润性胶质瘤侵袭和迁移的候选蛋白的差异表达。

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 ...

数字空间分析（DSP）提供了一种有效的空间分层蛋白质定量方法。它的实施使我们能够表征肿瘤和微环境不同区域的蛋白质表达。DSP的一个关键特性是其多路复用能力，可实现高吞吐量数据处理。定义自定义感兴趣区域的能力还为数据分析提供了空间维度。DSP 可应用于任何在空间上定量蛋白质或 RNA 表达可能有助于阐明病理或生理过程的样品。这在肿瘤学中尤其重要，因为区域靶标变异性可能与恶性生长或侵袭相关。展示该程序的将是组织技术专家Scott Palisoul和基因组技术专家Rachael Barney。为了准备载玻片，首先从每个活检中取出几个两毫米的核心，并将它们放入单个组织微阵列块中。从块上切割四微米的部分，并将它们安装在载玻片上。将每张载玻片放在载玻片支架垫圈内，并在 60 摄氏度下孵育 30 分钟。在半自动IHC系统中，每张载玻片用150微升洗涤缓冲液加上5毫升死体积填充位置一的容器，并保持盖子打开。在位置二的容器中填充相同量的封闭溶液。将试剂容器托盘装载到机器上。选择“处理 IHC”，然后在“标记”字段的下拉框中选择阻止解决方案名称。完成所有幻灯片后，单击“关闭”。然后单击“打印标签”，并选中“所有尚未打印的幻灯片标签”，然后单击“打印”。将标签粘贴到幻灯片的顶部。将载玻片装载到载玻片托盘上，确保样品和标签朝上。按下 LED 按钮降低托盘，让仪器开始扫描和识别载玻片。单击“开始”按钮开始实验。运行完成后，按下 LED 按钮，使其呈绿色闪烁。从仪器上取下托盘，然后小心地从每张幻灯片上提起盖板。将载玻片放入1X磷酸盐缓冲盐水中。去除多余的缓冲液，并用疏水笔勾勒出每个组织切片的轮廓，以形成疏水屏障。通过每张载玻片添加 8 微升每种抗体并使用缓冲液 W 达到 200 微升的最终体积来制备抗体 PC 寡核苷酸溶液。然后将抗体PC寡核苷酸溶液涂在载玻片上，并将载玻片在4摄氏度下孵育过夜。孵育后，将载玻片放入加湿的托盘中，并在1X TBST中洗涤三次10分钟。在室温下在4%多聚甲醛中后固定30分钟，然后在1X TBST中洗涤两次5分钟。在室温下加入 SYTO 13 核染色剂 15 分钟，然后用 1X TBST 洗涤两次。将鼠标悬停在控制中心的数据收集上，然后选择新建或继续运行。将载玻片放在载玻片支架中，标签朝向用户。降低滑动托盘夹，确保组织在细长的窗口中可见。加入六毫升缓冲液S.按照控制中心的提示操作。使用 x 和 y 滑块在不同轴之间缩放，以描绘要扫描的区域。选择“扫描”，并允许扫描继续，直到对整个定义的目标区域进行成像。生成 20 倍图像。通过为每个组织核心选择三个直径为 250 微米的相同大小的圆形 ROI 来定义 ROI。通过单击退出扫描工作区按钮批准投资回报率。然后等待紫外线将寡核苷酸从抗体中切割出来。完成清洁仪器过程后，选择“新建数据收集”以继续处理当前载玻片或孔板。双击板图标区域打开最终的板窗口。输入杂交码包批号，然后单击更新。然后点击完成。按照提示拆下载玻片支架和收集板。将载玻片储存在TBST中，或用水性介质和盖玻片覆盖它们以长期储存。使用渗透密封密封板，并在顶部打开的热循环仪中以65摄氏度的温度干燥吸气物。加入七微升焦碳酸二乙酯处理的水并混合。在室温下孵育 10 分钟，然后快速旋转。通过选择适当的探针R和探针U方程来制备探针缓冲液混合物，应用文本协议中所述的公式。向每个杂交代码包中加入 84 微升探针缓冲液混合物。在新鲜的 96 孔杂交板中，将 8 微升每种杂交代码预混液加入指定行的所有 12 孔中。将7微升从DSP收集板转移到杂交板中的相应孔中。热封，旋转板，并在67摄氏度下孵育过夜。孵育后，将板在冰上冷却，然后进行快速旋转。将每个孔的杂交产物汇集到带管中，轻轻移液每个孔五次。向下旋转，并将带管装入分析系统。在DSP仪器上，将CDF文件保存到USB驱动器上，然后将数据传输到数字分析仪。在屏幕上，按开始处理。选择高灵敏度，然后选择下一步。按全选以获取含样品的孔数，然后按完成，然后在电子邮件通知上按下一步。然后点击开始。制备站完成后，密封小柱并将其转移到数字分析仪中。按开始计数。选择舞台位置。按加载现有 CDF 文件，然后选择以前上传的文件。按完成。再次选择舞台位置，按完成，然后启动以运行程序。将报告器计数转换文件的 ZIP 文件从分析系统保存到 USB 驱动器。将驱动器插入 DSP 计算机。在 DSP 控制中心中，将鼠标悬停在数据收集上，然后单击上传帐户。选择相关的 ZIP 文件。单击DSP控制中心中的记录。要查看队列中的扫描，请选择“将所选扫描添加到队列”，然后选择“我的分析队列”。通过将鼠标悬停在控制中心的数据分析选项上，从队列中选择新算例。对胶质母细胞瘤样本进行DSP实验，结果可视化为热图。行代表蛋白质靶标，每列对应于一个感兴趣区域。表达级别由颜色范围表示，从蓝色（表示低表达）到红色（表示高表达）。一行内颜色的变异性反映了区域蛋白质的异质性，并表明可能与差异蛋白质表达的空间关联。在这个实验中，S100和CD-56普遍很高，因为它们是神经标记。变异性最大的标志物包括 B7-H3、KI-67、CD-44 和纤连蛋白，已知它们与肿瘤增殖、迁移和转移有关。从大量的候选目标中，DSP可以识别出那些表现出显着区域差异的目标。这为研究与变异性相关的因素及其与疾病的相关性奠定了基础。

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

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

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 ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Analysis of Side Population in Solid Tumor Cell Lines

固体肿瘤细胞系侧面人口分析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

组织中蛋白质和RNA表达的空间分析：一种微调虚拟显微切割的方法

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

小儿弥漫性中线胶质瘤的活-3D细胞免疫细胞化学测定

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

工业化、人工智能引导的激光显微切割，用于肿瘤微环境的微尺度蛋白质组学分析

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...