Name: intracarotid cancer cell injection to produce mouse models of brain metastasis
Uploaded: 2017-02-08T14:00:00
Description: Watch this Scientific Journal Video about Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis at JoVE.com

The authors have no acknowledgements.

伦理学声明：所有的动物研究由得克萨斯大学MD安德森癌症中心的大学机构动物护理和使用委员会（IACUC）的批准。 1.准备注射癌细胞<ol><li>注射前种子癌细胞一天或两天。使用补充有10％FBS，除非特殊介质在文献表明对于特定的细胞系的DMEM / F12培养基。 </li><li>外科手术当天，收获细胞，当他们到达70 - 通过用无血清培养基一旦第一洗涤80％汇合胰蛋白酶（0.25％）1前 - 2分钟，在37℃。添加...

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颈动脉注射癌细胞成功的最重要的步骤是：1）小鼠中为外科手术制备的深度麻醉; 2）对棉载体的顶部的颈动脉的稳定放置; 3）成功后注入颈总动脉结扎紧。 中〜30分钟，深麻醉通常是在解剖显微镜下手术稳定性能必要的。我们用鼠标麻醉商用现成使用氯胺酮/甲苯噻嗪鸡尾酒。在生理盐水中在调整用于麻醉的体积提供了更大的灵活性2：以避免潜在的过量，氯胺酮/赛拉嗪鸡尾?...

癌症中枢神经系统（CNS）的转移是一种破坏性的疾病，并且可涉及任何脑实质或（“脑转移”指的是既在本文中）的软脑膜。它是主要的颅内恶性肿瘤，由&gt; 10数量上超过初级胶质瘤：1 1，2。肺癌，乳腺癌和黑色素瘤是产生脑转移3,4的高发生率前三位主要肿瘤疾病。近年来，以新颖的癌症疗法跨出去已大幅延长生存期，改善生活质量为众多癌症患者。然而，在复发，脑转移瘤的发病率迅速上升。例如，所述抗HER2抗体曲妥单抗（赫赛汀）展示了在患者的HER2 +乳腺癌显著临床疗效;但令人不安的趋势已经出现在这些患者中：最多那些颅外全身性疾病最初从赫赛汀治疗获益以后开发的脑转移5，6，7的1/3。不幸的是，患者的脑转移是难治几乎所有目前的治疗，通常遇到的生活质量的创伤恶化，以及他们的一年存活率诊断后仅为〜20％8。对于脑转移瘤目前的疗法（包括类固醇，颅放疗，手术切除的患者选择）仅仅是治标不治本，不能治愈9。因此，脑转移正在成为这个时代新的癌症治疗方法的下一个宏伟的挑战。为了解决患者每天面对的诊所未得到满足的挑战，我们迫切需要更好地了解脑转移的基本机制，并利用这些知识来开发新疗法。 <p类=“jove_content”&gt;脑转移性癌细胞的成功定居需要一系列的由多个生物球员，如血脑屏障（BBB）的旁通错综复杂的相互作用介导的功能的性能，并从大脑的固有逃脱免疫防御机制10，其中没有在离体 或体外系统概括。因此，适当的和忠实的体内模型对于脑转移瘤的研究是至关重要的。 在体内转移法传统的经尾静脉注射引入癌细胞，导致大部分细胞变成肺提出的。脑转移灶在这些模型中很少产生前的11肺部引起的肿瘤负荷的动物死亡。癌细胞的直接脑内注射产生一致的肿瘤生长在CNS和广泛应用于初级胶质瘤的研究。然而，SUCH注入损害血脑屏障并引起外伤性损伤在注射部位，关注的两个主点作为这种模式的生理相关性。另一种常用癌细胞引进路线，心内注射，易于管理，并确实会产生实验性转移到中枢神经系统。然而，并行转移到除中枢神经系统其它器官部位总是产生，并可能导致动物的死亡11;因此，高度该模型的变异性使得它不适合的生物学机制或用动物的数量有限的治疗剂的定量评估。 这里，我们描述的程序通过注射癌细胞进入颈总动脉，以产生实验脑转移。我们已经用这种方法来单个基因的贡献解剖脑转移的转移级联，并评估治疗干预的有效性第12条 ，13。这种方法的主要优点是高度重现性和低度变性的;的主要缺点是执行显微所需的复杂性和灵巧。

<table border="0" cellpadding="0" cellspacing="0" width="1156">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Ketamine hydrochloride/xylazine hydrochloride solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:151px;">K113</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Routine Stereomicroscope Leica M50</td>
			<td style="width:151px;">Leica</td>
			<td style="width:151px;">M50</td>
			<td>The microscope is modular and highly configurable to fit particular space requirements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Surgical disposable scapel</td>
			<td style="width:151px;">Integra Miltex</td>
			<td style="width:151px;">4-410</td>
			<td>to make skin incisions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Tissue Forceps - 1x2 Teeth</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11021-12</td>
			<td>to bluntly dissect mucles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Dumont #5 - Mirror Finish Forceps</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11252-23</td>
			<td>to separate and prepare the carotid artery for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Spring Scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">15025-10</td>
			<td>to cut sutures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">EZ Clip Kit</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:151px;">59020</td>
			<td>for wound cloure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BD Insulin Syringe</td>
			<td style="width:151px;">Becton Dickinson</td>
			<td style="width:151px;">328438</td>
			<td>for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">IVIS Spectrum In Vivo Imaging System</td>
			<td style="width:151px;">PerkinElmer</td>
			<td style="width:151px;">124262</td>
			<td></td>
		</tr>
	</tbody>
</table>

intracarotid cancer cell injection to produce mouse models of brain metastasis

转移，扩散和恶性细胞的生长在一个患者体内的辅助站点，占癌症相关死亡率的&gt; 90％。近日，在新的治疗跨出去已大幅延长生存期，改善生活质量为众多癌症患者。可悲的是，脑转移复发的发病率正在快速上升，目前所有的疗法只是治标不治本。因此，迫切需要良好的实验动物模型，以促进在深入疾病生物学的研究和评估新的治疗方案临床前评估。然而， 在 经由癌细胞尾静脉注射的体内转移测定法的标准主要是产生肺转移灶;动物脑转移任何有意义的产物，通常前屈从于肺部肿瘤负荷。肿瘤细胞心内注射产生转移灶多器官部位包括大脑;然而，variabi这种模式产生的肿瘤生长lity较大，抑制其效用评价治疗效果。为了产生脑转移瘤的研究可靠和一致的动物模型，在这里我们描述了通过肿瘤细胞的颈动脉注射产生的家鼠（ 小家鼠 ）实验性脑转移的过程。这种方法允许一个产生大量具有相似的生长和死亡特性脑转移瘤轴承小鼠，从而方便研究工作，以学习基本的生物学机制，并评估新的治疗药物。

有在该注射液的质量进行评估的两点。第一次机会是通过注射期间改变血管的颜色操作者的观察。来自贫穷注射泄漏解剖显微镜下很容易观察到。鼠标主体的稳定和安全的放置（ 图1A）和在支撑棉球其颈动脉（ 图1B）是用于顺利成功注射到颈动脉的关键因素。第二点来评价喷射质量是手术后的非侵入性成像。为了这个目的，癌细胞通常标记有荧光...

Watch this Scientific Journal Video about Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis at JoVE.com

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

脑转移已成为迫切满足医疗需要，因为其发病率有增加，而治疗方法仍然治标不治本。 经由颈动脉动脉注射癌细胞创建脑转移的实验动物模型有利于新型干预方案的疾病生物学和评价的机理研究。

颈动脉肿瘤细胞注射，产生脑转移瘤小鼠模型

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

The overall goal of this microsurgical injection of cancer cells through the carotid artery is to produce consistent in vivo experimental brain metastasis models in mice. So this method can help answer key questions in the brain metastasis field, such as whether a new therapeutic may show efficacy in treating brain metastasis. So the main advantage of this technique is that it allows the production of in vivo experimental brain metastasis with high reproducibility and low variability.Generally individuals new to this technique will struggle initially because a fair amount of practice is needed to maintain a steady hand to inject cancer cells into the carotid artery of these mice under the microscope. Begin this procedure by seeding the cancer cells with DMEM F12 media supplemented with 10%FPS and culturing them for one to two days before injection. On the day of surgical operation, remove the cells from the incubator and harvest them when they reach 70%to 80%confluence by washing with serum-free media.Next, incubate the cells with 0.25%trypsin for one to two minutes at 37 degrees Celsius. Afterward, remove them from the incubator and add 10%FBS containing DMEM F12 media to the cells to quench chipsenation. Centrifuge the cells at 80 times G for three minutes.Then, remove the medium and wash the cells in serum-free twice to remove residual serum. Next, count the cells with a hemocytometer and resuspend them in HBSS. Then, keep the cells on ice until the time of injection.In this procedure, anesthetize the mouse with ketamine/xylazine cocktail by intraperitoneal injection. Confirm complete anesthesia by pinching the animal's feet and observe the lack of response. Next, remove the neck hair by shaving or using a small amount of hair removal product.Then, place the mouse on a glass plate and secure it with rubber bands. Clean the skin by applying 70%alcohol in povidone iodine. Now, make an incision in the skin about one centimeter long with a surgical scalpel.Then, bluntly dissect the muscle with surgical forceps to expose the carotid artery underneath. Separate the carotid artery from the adjacent vagus nerve with surgical forceps. Afterward, place two sutures.One distal and the proximal to the cotton ball and make loose knots. Tighten the proximal knot to block the blood flow into the injection site. After that, moisten a small cotton ball with buffer and place it underneath the carotid artery of the intended site of injection.Then, proceed to cancer cell injection if the carotid artery on the cotton ball appears fully pressurized with fresh red blood. In this step, vortex and draw 100 microliters of cancer cells into the syringe. Under the dissecting microscope, slowly insert the 31 gauge needle with bevel up into the lumen of the carotid artery, sitting on top of the cotton ball.Slowly inject the cells from the syringe into the carotid artery. Successful injection can be observed by the changing color of nearby blood vessels and musculature when the buffered cancer cells are pushed into the circulation. When the injection is complete, lift the distal ligature gently to prevent regurgitation.Then, quickly tighten the distal knot to complete the injection process. After completing the injection, tighten the ligatures and trim the excess silk suture with micro scissors. Move back the separated muscle to cover the wound site and close the skin with two staples.For recovery, transfer the animal onto a heating pad. Then, administer buprenorphine via subcutaneous injection as post-surgical analgesia. Once it regains consciousness and resumes normal behavior, return the mouse to its housing location.Provide the animal with gel food for several days post-surgery. These images show that the modified MDA MB231 breast cancer cells were labeled with loose foraise reporter gene ex-vivo. Starting from 24 hours post-carotid artery injection, brain metastasis development was monitored non-invasively by repeated in vivo imaging system imaging.Quantification of imaging data indicates an initial decline of tumor burden post-carotid artery injection, followed by an exponential outgrowth of brain metastasis until animal's more abundity. So what is mastered this technique can be done in less than 10 minutes for each mouse injection if it's performed properly. So while performming this procedure, the single most important thing to remember is you have to ensure, study and secure placement of the carotid artery on top of the cotton ball prior to proceeding to the injection of the cancer cells.Following this procedure, other methods can be performed such as therapeutic treatments to answer other questions. For example whether a new drug would be effective inhibiting brain metastasis outgrows. After its development, this technique paved the way for researchers in the brain metastasis field to explore basic or translational questions in vivo experimental model in mice.So after watching this video you should have a good understanding of how to inject the cancer cells into the carotid artery of mice to produce a in vivo experimental brain metastasis model.

Research

JoVE Journal

Cancer Research

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

科研

癌症研究

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

患者衍生的中枢神经系统转移异种移植模型的建立与利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

门静脉注射结直肠癌类器官研究肝转移基质

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...