Name: implantation and evaluation of melanoma in the murine choroid via optical coherence tomography
Uploaded: 2022-12-02T14:10:00
Description: Watch this Scientific Journal Video about Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography at JoVE.com

这项研究得到了以色列以色列科学基金会（ISF）为Arie Marcovich提供的1304/20赠款的部分支持。我们感谢来自以色列雷霍沃特卡普兰医疗中心病理学系的Shahar Ish-Shalom和Ady Yosipovich进行组织学分析。

协议中的实验已获得以色列国家动物实验委员会的批准，并符合ARVO关于在眼科和视觉研究中使用动物的声明。雌性C57BL / 6小鼠，年龄为8-10周，用于本研究，并暴露于12/12小时明暗循环。这些动物是从商业来源获得的（见 材料表）。
1. 细胞培养
<ol><li>在RPMI 1640培养基中培养B16LS9细胞，补充有10%胎牛血清，2mM L-谷氨酰胺，1mM丙酮酸钠，25mM HEPES，1%必需...

<ol>
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葡萄膜黑色素瘤是一种毁灭性的疾病，非常需要新的治疗方法。然而，葡萄膜黑色素瘤和潜在治疗方法的研究受到葡萄膜黑色素瘤动物模型1，25的技术挑战的限制。由眼内注射癌细胞诱导的眼部肿瘤在定位和大小上变化很大，可能是由于小鼠眼睛的尺寸小。这种变异性是全面评估肿瘤进展的障碍。本文描述的实验方法允许人们基于实时OCT成像<sup class="xr...

葡萄膜黑色素瘤 （UM） 是成人中最常见的眼内原发性恶性肿瘤。大约 90% 的眼部黑色素瘤起源于葡萄膜束脉络膜区域的黑色素细胞1。UM是发病和死亡的主要原因，因为据估计，近50%的患者发展为转移性疾病，肝脏是转移的主要部位2。原发性病变的早期治疗可能会减少转移的机会，但没有有效的治疗方法可以防止转移的形成3。
葡萄膜黑色素瘤的标准治疗包括放射治疗，该放射治疗与视神经病变、视网膜病变、干眼综合征和白内障导致的视力丧失有关。手术切除通常延迟到病变的生长被识别和表征。然而，这种延迟可能会使转移性疾病发展4。在某些情况下，需要徒劳的眼球摘除。当然，这种激进的程序会损害视力并导致美学急剧恶化。
已经有许多努力致力于开发研究葡萄膜黑色素瘤的实验模型。能够准确评估这种恶性肿瘤的临床前动物模型是研究葡萄膜黑色素瘤新诊断和治疗策略的关键。眼部黑色素瘤的实验动物模型主要基于小鼠、大鼠和兔5，6中肿瘤细胞的接种。小鼠模型具有成本效益，由于其快速的繁殖速度和与人类的高基因组相似性，广泛用于黑色素瘤研究。小鼠皮肤黑色素瘤细胞系B16通常用于接种C57BL6小鼠并诱导同系肿瘤。当使用该模型诱导葡萄膜黑色素瘤时，荷瘤的眼睛通常需要在接种后7-14天进行眼球摘除。此外，B16是一种高度侵入性的模型。眼睛的免疫特权性质支持转移，转移通常在肿瘤细胞接种后 3-4 周检测到。原始B16系的传代培养物显示出不同的转移特性6。例如，皇后区黑色素瘤系具有高转移率7，8。B16LS9细胞系具有树突状细胞形态，来源于注射亲本皮肤黑色素瘤系B16F19的C57BL / 6小鼠的肝转移。当注射到眼睛的后室时，这些细胞被显示形成眼内肿瘤，其组织学类似于人葡萄膜黑色素瘤，并在C57BL / 6中形成肝脏特异性转移，但不是Balb / C，小鼠10，11，12。在遗传学上，这些细胞的特征在于c-met原癌基因的更高表达，其作为肝细胞生长因子13的细胞受体。相比之下，B16F10是亲本B16的第10代，主要在眼内接种时转移到肺部14。B16F10和B16LS9都是色素12。
几个关键挑战限制了小鼠葡萄膜黑色素瘤模型的成功。首先，肿瘤细胞反流可能导致眼外或结膜下黑色素瘤。其次，黑色素瘤细胞眼内接种后的肿瘤生长通常变化很大，给评估治疗和进展带来了困难。另一个主要困难是跟踪体内肿瘤生长的能力有限。虽然生物发光成像，例如表达荧光素酶的肿瘤，通常用于监测眼部肿瘤的生长15，16，但它不能提供有关肿瘤眼内位置的信息。因此，肿瘤的评估通常在眼球摘除后进行10，17。这极大地限制了表征肿瘤进展和对治疗反应的能力。研究葡萄膜黑色素瘤的另一个主要障碍是难以监测色素小鼠的病变。需要克服这些困难的新方法来促进动物模型中葡萄膜黑色素瘤的研究。
光学相干断层扫描（OCT）提供了独特的功能，可以高分辨率深入眼睛的不同部分成像，这是其他方法（包括超声18，19）无法比拟的。OCT成像已在动物模型中用于研究各种眼部疾病20。最近，OCT成像被证明是评估眼内肿瘤生长的非侵入性手段21。这里描述的方案描述了黑色素瘤细胞在小鼠脉络膜中的植入以及利用OCT来预测细胞接种时的眼内肿瘤定位和大小。

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	<tbody>
		<tr>
			<td>10 &#956;L glass syringe (Hamilton Co., Bonaduz, Switzerland)</td>
			<td>Hamilton</td>
			<td>721711</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needles</td>
			<td>BD Microbalance</td>
			<td>2025-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezole hydrochloride</td>
			<td>Orion Phrma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B16LS9 cells</td>
			<td>from Hans Grossniklaus USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine&#160;</td>
			<td>richter pharma</td>
			<td>102047</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 female mice</td>
			<td>Envigo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Essential vitamin mixture</td>
			<td>satorius</td>
			<td>01-025-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>rhenium</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>satorius</td>
			<td>03-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyethylcellulose 1.4% eye drops</td>
			<td>Fisher Pharmaceutical</td>
			<td>390862</td>
			<td></td>
		</tr>
		<tr>
			<td>InSight OCT segmentation software&#160;</td>
			<td>Phoenix Micron, Inc&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>bremer pharma GMBH (medimarket)</td>
			<td>17889</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>satorius</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine&#160;</td>
			<td>zoetis (vetmarket)</td>
			<td>102532</td>
			<td></td>
		</tr>
		<tr>
			<td>Ofloxacin 0.3% eye drops</td>
			<td>allergan</td>
			<td>E92170</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical coherence tomography&#160;</td>
			<td>Phoenix Micron, Inc&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxybuprocaine 0.4%</td>
			<td>Fisher Pharmaceutical</td>
			<td>393050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin-amphoteracin</td>
			<td>satorius</td>
			<td>03-033-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)&#160;</td>
			<td>satorius</td>
			<td>02-023-1a</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI cell media</td>
			<td>satorius</td>
			<td>01-104-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>satorius</td>
			<td>03-042-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Zeiss</td>
			<td>OPMI-6 CFC</td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide 0.5%</td>
			<td>Fisher Pharmaceutical</td>
			<td>390723</td>
			<td></td>
		</tr>
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</table>

implantation and evaluation of melanoma in the murine choroid via optical coherence tomography

建立实验性脉络膜黑色素瘤模型在正确定位诱导肿瘤的能力方面具有挑战性。此外， 在体内 观察后脉络膜黑色素瘤的困难限制了肿瘤的实时定位和生长评估。这里描述的方法优化了通过多步骤亚脉络膜B16LS9细胞注射 程序在 小鼠中建立脉络膜黑色素瘤的技术。为了能够精确地注射到小鼠葡萄膜的小尺寸中，在显微镜下进行完整的程序。首先，在眼睛的背颞区形成结膜围膜。然后，通过将针插入暴露的巩膜来创建进入脉络膜下空间的束。随后将钝针插入尿道并将黑色素瘤细胞注射到脉络膜中。注射后立即利用无创光学相干断层扫描（OCT）成像来确定肿瘤的位置和进展。视网膜脱离被评估为肿瘤部位和大小的预测指标。所提出的方法能够可重复地诱导小鼠脉络膜局部黑色素瘤和肿瘤生长评估的实时成像。因此，它为研究眼内肿瘤提供了有价值的工具。

注射B16LS9细胞后立即通过OCT检查眼睛。注射后观察到局部视网膜脱离。小鼠表现出三种RD模式：局灶性（图2，上图），玻璃体渗漏（图2，中图）和扩展RD（图2，下图）。延长的RD可能是由注射损伤引起的。注射后立即的RD模式与注射后5-7天的肿瘤定位之间存在关联。如图2所示，局灶性RD与仅限于脉络?...

Watch this Scientific Journal Video about Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography at JoVE.com

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

本协议描述了利用光学相干断层扫描在小鼠脉络膜中植入和评估黑色素瘤。

通过光学相干断层扫描 在 小鼠脉络膜中植入和评估黑色素瘤

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

将肿瘤确定为眼睛的后部具有挑战性。该方法能够在小鼠中诱导可重复的脉络膜黑色素瘤肿瘤，并对肿瘤生长进行实时成像以进行评估。该协议利用OCT实时成像来评估肿瘤细胞接种时已经存在的肿瘤位置，并跟踪活体动物的肿瘤生长。葡萄膜黑色素瘤是一种毁灭性的疾病，转移率接近 50%。开发实验模型是推进旨在实现新治疗可能性的研究的关键。这种方法的一个优点是，对注射成功的即时评估允许到达具有同质肿瘤的研究组，并通过实时成像连续跟踪它们。检查肿瘤进展。注射到小老鼠的眼睛需要练习。可视化注射细胞的能力使您可以立即评估您的成功，并在必要时对更多小鼠重复。演示该程序的将是来自卡普兰医学中心眼科研究实验室的Dimitri Gaber博士。首先，将0.4%局部眼科麻醉剂Oxybuprocaine应用于麻醉小鼠的双眼。然后局部涂抹0.5%托吡卡胺双眼以扩大瞳孔，然后用1.4%羟乙基纤维素作为润滑剂，以避免眼睛干燥。在手术显微镜下观察鼠标的一只眼睛。用无菌眼内钳保持眼睑张开。然后握住颞上缘结膜，拉向鼻下位置。通过在整个手术过程中保持边缘结膜来确保该位置。使用 30 号针尖在颞背区进行小结膜周围切开，距角膜缘后方约 1-2 毫米。从围膜开口处取出多余的肌腱囊。插入针尖以穿透巩膜，在脉络膜下空间形成一条轨迹，直到脉络膜的棕色通过巩膜暴露的白质出现。将细胞装入无菌10微升玻璃汉密尔顿注射器中，该注射器装有以45度角扭曲的32号钝针。将装载的注射器的针头插入创建道约两毫米，并注射两微升细胞悬液。注射后将针头固定到位两到三秒钟，直到所有液体清除。轻轻拉出针头，以免从尿道中泄漏。黑色素瘤细胞接种后，立即通过OCT扫描观察动物的注射眼睛。根据扫描结果，根据局部 RD、玻璃体渗漏或扩展 RD 三类对视网膜脱离或 RD 模式进行分类。打开OCT分割分析软件，根据RD高度预测肿瘤大小。在OCT扫描中测量本地RD的高度，并将其分为三组。在术后阶段，局部施用0.3%的氧氟沙星。该图描述了基于OCT的脉络膜下细胞注射后肿瘤生长预测。小鼠表现出RD，局灶性，玻璃体泄漏和扩展RD三种模式。注射后立即的RD模式与注射后5天的肿瘤定位之间存在关联。局灶性RD与仅限于脉络膜的肿瘤细胞生长有关。然而，在显示局灶性RD的动物注射后玻璃体细胞的观察表明玻璃体腔和脉络膜中的肿瘤生长。当注射后观察扩展RD时，肿瘤在5天后分散在整个脉络膜和玻璃体。该模型中诱导的肿瘤大小范围可分为小、中、大。这里显示的是注射后RD高度的OCT测量的代表性图像，相应的眼底图像，注射后第五天肿瘤高度和宽度的水平OCT扫描，及其相应的眼底图像，以及相应的H&E染色眼切片，显示可比的肿瘤测量。本文介绍了细胞注射后立即呈现小，中或大RD的小鼠的肿瘤体积测量。调整注射力会影响肿瘤的大小和位置。重要的是要校准这一点，并评估注射后OCT诱导视网膜脱离的模式。脉络膜肿瘤的实时成像是一种重要的实验工具，可以检查不同因素或潜在疗法对肿瘤进展的影响。

Research

JoVE Journal

Cancer Research

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

A Mouse Model of Fatigue Induced by Peripheral Irradiation

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

科研

癌症研究

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

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

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 ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

初级毛质素黑色素细胞和纤维细胞培养的快速生成

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

在免疫缺陷小鼠中生成肝脏正交人子宫黑色素瘤异种移植平台

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

毛尿胰腺腺腺腺癌超声引导正射植入

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

通过炎症性乳腺癌细胞的尾静脉注射模拟脑转移

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

通过光学相干断层扫描 在 小鼠脉络膜中植入和评估黑色素瘤

通过光学相干断层扫描在小鼠脉络膜中植入和评估黑色素瘤