Name: culture and imaging of ex vivo organotypic pseudomyxoma peritonei tumor slices from resected human tumor specimens
Uploaded: 2022-12-09T13:55:00
Description: Watch this Scientific Journal Video about Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens at JoVE.com

作者要感谢摩尔斯癌症中心成像核心设施的Kersi Pestonjamasp对显微镜的帮助UCSD专业癌症支持中心P30资助2P30CA023100。这项工作还得到了JoVE出版补助金（JRW）的支持，以及伊丽莎白和Ad Creemers遗产，Euske家庭基金会，胃肠道癌症研究基金和腹膜转移研究基金（AML）的慷慨捐赠。

所有组织的去识别和采集均在加州大学圣地亚哥分校根据IRB批准的协议进行。
1. 制备用于组织处理和培养的人PMP组织
<ol><li>肿瘤组织的运输和显微切割<ol><li>准备运输和培养基：完成 10% （v/v） Dulbecco 改良鹰培养基 （DMEM）、10% FBS、2 mM L-谷氨酰胺、1% 青霉素/链霉素（笔链球菌）。</li>
		<li>组织到达后，根据机构IRB批准的方案，将PMP肿瘤组织转移到35个直径为6厘...

<ol>
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本手稿描述了一种可用于培养、询问和分析人腹膜假黏液瘤 （PMP） 肿瘤标本的技术。我们利用了许多下游功能检测来询问肿瘤免疫微环境和从工作台到床边测试的平台。
虽然该方法在我们手中非常有效，但使用振动切片机切割肿瘤标本需要一些练习。也就是说，我们遇到了由于高度粘液的样品以及不正确地显微解剖以去除不可切割支架（腹壁，腹水，各种ECM蛋白）而导致的...

腹膜假黏液瘤 （PMP） 是一种罕见综合征，发病率为每年每百万人 1 例1。大多数PMP病例是由阑尾肿瘤转移引起的。鉴于小鼠没有类似人类的阑尾，对这种类型的癌症进行建模仍然极具挑战性。虽然原发性疾病通常可以通过手术切除治愈，但转移性疾病的治疗选择有限。因此，开发这种新型器官型切片模型的基本原理是研究PMP的病理生物学。迄今为止，还没有可以永久培养的阑尾类器官模型;然而，最近的模型被证明可用于治疗剂和免疫疗法的药理学测试2。因此，我们采用了一种器官型切片培养系统，该系统已用于其他类型的人类癌症，例如脑癌、乳腺癌、胰腺癌、肺癌、卵巢癌等3，4，5，6。
除阑尾肿瘤外，PMP偶尔还由其他肿瘤类型引起，包括卵巢癌7，在极少数情况下，导管内状粘液肿瘤8 和结肠癌9。此外，这些肿瘤往往生长缓慢，在患者来源的异种移植（PDX）模型中移植率低10，11。鉴于这些挑战，开发模型来研究这种疾病的需求尚未得到满足，以开始了解PMP的病理生物学，以及这些癌细胞如何被招募到腹膜表面，增殖并逃避免疫监视。
虽然从体循环中切割出来，但肿瘤切片确实含有细胞和无细胞成分，包括细胞外基质、基质细胞、免疫细胞、癌细胞、内皮细胞和神经。这种半完整的微环境允许对这些细胞类型进行功能研究，与仅由癌细胞组成的3D类器官培养物相比，这是独特的优势12。虽然器官型切片培养在某些方面是有利的，但与可以扩增的3D类器官相比，它们本质上也是一种基于低通量的方法，适用于多重研究性治疗药物筛选13，14，15。就PMP而言，没有报告记录PMP衍生类器官的可靠建立和永久传代16。这可能是由于PMP衍生的肿瘤细胞生长缓慢，以及在这些粘液肿瘤中发现的恶性上皮细胞数量很少。鉴于需要开发模型来研究PMP，器官型切片特别适合研究这种疾病。我们提出了一种用于制备，成像和分析人类标本PMP的方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M CaCl2 solution</td>
			<td>Sigma</td>
			<td>21115</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES solution</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2 solution&#160;</td>
			<td>Sigma</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>100 micron filter</td>
			<td>ThermoFisher</td>
			<td>22-363-549</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 40 glass coverslips</td>
			<td>Daiggerbrand</td>
			<td>G15972H</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M KCl solution</td>
			<td>Sigma</td>
			<td>60135</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl solution</td>
			<td>Sigma</td>
			<td>S5150</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP&#947;S&#160;</td>
			<td>Tocris&#160;</td>
			<td>4080</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM&#160;</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>CD206&#160;</td>
			<td>Biolegend</td>
			<td>321140</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Biolegend</td>
			<td>555333</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4&#160;</td>
			<td>Biolegend</td>
			<td>357410</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45&#160;</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#160;</td>
			<td>Biolegend</td>
			<td>344721</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo&#160;</td>
			<td>Promega</td>
			<td>G9681</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Thermo Fisher</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, heat inactivated</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block&#160;</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4</td>
			<td>Thermo Fisher</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Collagenase/Hyaluronidase&#160;</td>
			<td>Stem Cell</td>
			<td>7912</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Chamber Platform</td>
			<td>Warner Instruments</td>
			<td>PH-1</td>
			<td></td>
		</tr>
		<tr>
			<td>LD-Blue&#160;</td>
			<td>Biolegend</td>
			<td>L23105</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM</td>
			<td>ThermoFisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD imaging dyes</td>
			<td>Thermofisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti microscope&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Includes: A1R hybrid confocal scanner including a high-resolution (4096x4096) scanner, LU4 four-laser AOTF unit with 405, 488, 561, and 647 lasers, Plan Apo 10 (NA 0.8), 20X (NA 0.9) dry objectives.&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump&#160;</td>
			<td>Isamtec</td>
			<td>ISM832C</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum silicone grease</td>
			<td>Sigma</td>
			<td>Z273554-1EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

culture and imaging of ex vivo organotypic pseudomyxoma peritonei tumor slices from resected human tumor specimens

腹膜假黏液瘤 （PMP） 是一种罕见的疾病，由粘液原发性肿瘤的播散和分泌粘蛋白的肿瘤细胞在腹膜腔中积累引起。PMP可由各种类型的癌症引起，包括阑尾癌，卵巢癌和结直肠癌，尽管阑尾肿瘤是迄今为止最常见的病因。PMP由于其（1）罕见性，（2）有限的小鼠模型和（3）粘液，无细胞组织学，研究具有挑战性。这里介绍的方法允许在肿瘤微环境（TME）保持完整的制剂中使用患者来源的 离体 器官型切片对这些肿瘤类型进行实时可视化和询问。在该协议中，我们首先描述使用振动切片机制备肿瘤切片并随后进行长期培养。其次，我们描述了肿瘤切片的共聚焦成像以及如何监测活力、钙成像和局部增殖的功能读数。简而言之，切片装有成像染料，并放置在可以安装在共聚焦显微镜上的成像室中。延时视频和共聚焦图像用于评估初始活力和细胞功能。该程序还探索TME中的平移细胞运动和旁分泌信号传导相互作用。最后，我们描述了用于流式细胞术分析的肿瘤切片的解离方案。定量流式细胞术分析可用于从实验室到床边的治疗测试，以确定免疫景观和上皮细胞含量内发生的变化。

简而言之，来自PMP的人肿瘤标本是根据IRB批准的方案获得的。制备组织，显微解剖并在琼脂糖模具中固化，以使用振动切片机切割（图1A;视频 1）。切割后，将组织切片放置在可渗透的插入膜上并培养（图1B），可用于原位成像测定，以及使用流式细胞术，共聚焦成像分析和细胞毒性测定进行细胞和功能检查。人PMP组织切片培养和处?...

Watch this Scientific Journal Video about Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens at JoVE.com

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

我们描述了一种用于人类癌症的生产，培养和可视化的方案，这些癌症已经转移到腹膜表面。使用振动切片机切割切除肿瘤标本，并在可渗透的插入物上培养以增加氧合和活力，然后使用共聚焦显微镜和流式细胞术进行成像和下游分析。

来自切除人肿瘤标本的 离体 器官型腹膜假粘液瘤肿瘤切片的培养和成像

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

这项研究展示了一种在临床前环境中研究阑尾癌治疗和病理生物学的新方法。这些方法可能更广泛地适用于其他恶性肿瘤的研究。阑尾癌很难研究，因为在小鼠中没有用于疾病建模的阑尾的情况下，阑尾癌的发病率很低。该技术允许研究肿瘤微环境中的细胞相互作用。该方法可用于研究具有转移到网膜倾向的多种癌症的生物学，例如结直肠癌和卵巢癌。首先准备运输和培养基。组织到达后，将腹膜假粘液瘤肿瘤组织转移到35个完整的DMEM中，其中包含6厘米直径的培养皿。除了去除任何液化粘蛋白或高度粘液的组织区域外，还可以去除任何难以用手术刀切割的组织。将肿瘤组织结节大致切成一立方厘米的小块。对于琼脂糖制备和组织包埋，如果组织到达，则在同一天在PBS中制备5%的低熔点琼脂糖溶液，不含胎牛血清或FBS。将液体5%琼脂糖溶液加入包含两到三个较小肿瘤标本的六厘米培养皿中，直到覆盖标本。将嵌入的组织放入四摄氏度的冰箱中 30 分钟。拆卸振动切片机上的组织标本，从冰箱中取出凝固的螺旋钻，然后用手术刀切割略大于组织宽度的琼脂糖立方体。将强力胶涂在振动切片机载物台上，然后将琼脂糖立方体轻轻放在强力胶上一到两分钟进行固定。将刀片固定在振动切片机上，并将组织切片的厚度设置为大约 150 至 250 微米，以获得最佳下游成像。为了培养组织切片，通过在渗透膜上方添加两毫升完整的DMEM培养基和在渗透膜下方添加三毫升来制备可渗透的插入板。接下来，使用细画笔轻轻地将组织切片从振动切片机的切割室中取出，并将两到四个切片放入含有完整DMEM的渗透性培养皿中。将切片在 37 摄氏度的 5% 二氧化碳中培养长达 7 天。在培养基更换期间，每 24 小时加入硼替佐米作为细胞杀伤对照，并将可测试的化疗 5-氟尿嘧啶或 5-FU 添加到培养基中以进行药物干预。将肿瘤切片用于共聚焦成像分析，置于含有 1%FBS 的 PBS 的 12 孔培养皿的单孔中。对于钙成像实验，代替PBS，将切片在额外的细胞钙溶液中孵育。用成像染料对样品进行染色，以确定组织切片的活力。加入活性染料后孵育10分钟至1小时。孵育后，将切片转移到含有具有1%FBS的PBS的光学透明玻璃底培养皿中，用于共聚焦成像设备上的成像。从一毫升移液器吸头的末端切下一小块以扩大开口，通过移液进行剧烈的机械解离。在 37 摄氏度下在 1 毫升消化缓冲液中旋转孵育切片 5 至 15 分钟，在使用机械解离孵育期间剧烈破坏组织 2 至 3 次。一旦组织被消化，用消化培养基将破碎的组织转移到放置在50毫升锥形管顶部的70微米过滤器上。使用无菌镊子或移液管挑选较大的碎片。使用塑料五毫升注射器的钝后端，捣碎任何较大的未解离切片，并用含有2%FBS的四毫升PBS清洗。取解离的细胞上清液，以300G离心5至10分钟。除去上清液，用一毫升含有2%FBS的PBS洗涤沉淀。为了制备用于流式细胞术的样品，加入含有50微升人FC封闭的封闭缓冲液，并在室温下孵育15分钟。使用含有2%FBS的50微升PBS中的相应抗体进行额外的细胞染色。等分五毫升用于控制和处理条件的完整培养基。在铺在渗透性培养皿上的两到四片上，加入来自对照处理等分试样的培养基和用于下游活性、活死分析的其他联合疗法。将组织切片留在含有完整DMEM的培养物中两到五天，每隔一天更换培养基以及硼替佐米和5-FU。对于发光活性分析，将500微升PBS加入12孔培养皿中，并使用画笔或1毫升移液器将顺序匹配的组织切片转移到PBS溶液中。从切片中取出 PBS。根据条件加入500微升发光活性溶液，并在室温下在摇床上缓慢旋转孵育30分钟，然后使用发光板读数器读取发光。使用周期性酸移染色和粘蛋白特异性抗体可视化组织切片中的粘蛋白染色。在钙黄绿素AM和碘化丙啶中孵育决定了健康和活力。细胞核与培养过程中增殖的细胞重叠。对这些结果进行定量以确定肿瘤切片内增殖细胞的数量。在CD11b PE偶联抗体和Fluo-4AM中孵育的切片，标记CTU中的局部免疫细胞。显示细胞内钙水平的假彩色缩放图像允许可视化细胞内钙的不同水平。此处显示了突出显示的CD11b阳性免疫细胞内细胞间钙反应之前，期间和之后的自发活动跟踪和时间流逝。定量了CD11b免疫细胞以及其他反应和无反应细胞中钙反应的原始痕迹。此处显示了腹膜假黏液瘤患者肿瘤样本免疫分型的代表性结果。肿瘤免疫谱的定量表明，供体标本337具有高水平的M2巨噬细胞，表明利用假粘液瘤腹膜衍生的组织切片可以询问患者肿瘤样本的独特供体细胞景观。来自腹膜假粘液瘤人体组织的药物分型和下游分析显示，细胞毒性分析和隧道共聚焦成像证实了肿瘤切片中细胞的杀伤。响应细胞毒性药物硼替佐米和5-氟尿嘧啶。使用顺序匹配的切片进行发光活性分析。成功生产粘液肿瘤切片需要细致的解剖和去除细胞和无细胞组织成分，如脂肪和致密的腹壁组织。该技术可以模拟癌细胞与肿瘤微环境中多种细胞类型之间的相互作用，例如脂肪，血管和免疫细胞相互作用。

Research

JoVE Journal

Cancer Research

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

科研

癌症研究

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

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 ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

小鼠膀胱肿瘤器官的培养、操作和正交移植

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

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 ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

来自切除人肿瘤标本的 离体 器官型腹膜假粘液瘤肿瘤切片的培养和成像

来自切除人肿瘤标本的离体器官型腹膜假粘液瘤肿瘤切片的培养和成像