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℃。添加到细胞中的含FBS的DMEM / F12培养基的10％以淬灭0.25％胰蛋白酶。 </li><li>离心在80×g下将细胞3分钟。洗在无血清培养基中的细胞两次以除去残留的血清，用血球计数细胞并在Hank氏平衡盐溶液（HBSS）在1重新悬浮- 5×10 6个细胞/ ml，取决于细胞系。保持冰直到注射时间。 注:MDA-MB-231乳腺癌细胞和K1735黑素瘤细胞在该研究中使用，并在2×10 6个细胞/ ml的浓度被用于这两种细胞系。保持胰蛋白酶消化持续时间尽可能地短的过度治疗可能影响转移测定法的结果。 </li></ol> 2.准备小鼠肿瘤细胞注射<ol><li>麻醉通过腹膜内（IP）小鼠氯胺酮注射/甲苯噻嗪混合物（氯胺酮100毫克/千克，甲苯噻嗪10mg / kg的）。 </li><li>按捏脚动物的确认完全麻醉，观察毫无反应。 </li><li>放置在玻璃板上的鼠标（用于铸造蛋白凝胶），并用橡皮筋固定。 </li><li>如果需要的话，通过刮或施加脱毛产品少量并用纸巾擦拭头发断除去颈部的头发。注意:如果使用裸鼠，跳过这一步，因为他们没有体毛。 </li><li>通过应用清洁颈部皮肤聚维酮碘和70％乙醇。 </li><li>将鼠标放在解剖显微镜的阶段</li><li>使在皮肤约1cm的切口长用外科手术刀。 </li><li>说白了解剖用有齿镊肌肉底下露出颈动脉。 </li><li>分开用外科钳子相邻的迷走神经的颈动脉。 </li><li>采用手术镊子，从棉花球分离一些棉花，滋润，并塑造成一个较小的棉花球。把这个缓冲区滋润棉球注射期望的站点的颈动脉下方。 </li><li>放置缝合线远端和近端的棉球，使松节。收紧近端结阻断血液流进注射部位。 </li><li>继续癌细胞注入如果在棉花球颈动脉出现新鲜的红血全压。 </li></ol> 3.肿瘤细胞注射到颈动脉<ol><li>涡和借鉴100微升Ø˚F癌细胞进入注射器。 </li><li>下的解剖显微镜，慢慢插入31克针（带斜面向上）插入颈动脉坐在棉球的顶端的内腔中。 </li><li>缓缓注入从注射器到颈动脉中的细胞。成功注射可以在显微镜下通过附近的血管和肌肉的变色的观察时缓冲癌细胞推入循环。 </li><li>当整个体积的注射完成后，轻轻提起远端颈内动脉，以防止反流。 </li><li>很快收紧远端结完成注射过程。 </li><li>在完成注入后，拧紧连字和解剖显微镜下把过多的丝线用微型剪刀。移回分离的肌肉覆盖伤口部位。与两主食合皮肤。 </li></ol> 4.手术恢复<ol><li>移动运营动物到一个加热垫进行恢复。这可能需要30 - 60分钟为动物恢复知觉。在此期间， 通过皮下注射作为手术后的镇痛施用丁丙诺啡（0.05毫克/千克）。注:镇痛应根据当地IACUC批准的协议来提供。 </li><li>一旦动物唤醒，恢复正常的行为，返回老鼠他们的住房位置。数天手术后提供凝胶食品的小鼠。保持手术记录，并保持它在房间里。 </li></ol> 5.非侵入性的体内成像<ol><li>为标记有荧光素酶报道构建体的细胞， 使用体内成像系统14测量癌细胞注入效率。 未在注射当天进行成像，以避免过大的应力，以研究动物:注。 </li></ol>

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The authors have nothing to disclose.

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/赛拉嗪鸡尾酒可以稀释1:1或1。可替代地，非受控药物三溴也可以用作麻醉剂;然而，我们已发现，三溴降解的倾向，使作为有效麻醉剂比氯胺酮/赛拉嗪鸡尾酒该物质不可靠。 一个稳步放置并充分加压颈动脉大大方便癌细胞的注射液;因此，我们强烈建议花时间仔细准备在显微镜下动脉的位置，而不是次优条件下注射进行。 多种癌细胞将颈动脉注射侧的脉管系统中提出。有时使两个半球出现承受在垂死的动物转移灶的显著量的转移细胞的显著部分可能会迁移到脑的另一侧。 转移灶中可能会出现的实质或CNS的软脑膜，或在这两个位置。似乎有独特的肿瘤微环境的相互作用所依据这些表型生物15。因此，当一个新的细胞系是用于其生产的实验脑转移能力被测试，它是necessARY要特别注意这些表型。 内部和外部颈动脉分别供给血液进入脑实质和软脑膜。因此，肿瘤细胞沉积到实质或软脑膜可通过该协议的轻微的修改来实现。允许细胞仅进入软脑膜，颈内动脉可由前肿瘤细胞注射的缝合线被连接，从而引导细胞进入颈外动脉;另一方面，在注射之前，颈外的结扎将允许细胞条目只到内部颈动脉和脑实质。 总体来说，相比于尾静脉腔内或注射的颈动脉注射方法产生重复性更高和更低的可变性的数据;相比于直接脑内注射法还避免了注射诱导的中枢神经系统的炎症。因此，该方法是目前最脑转移的定量研究旨在使用动物的数量有限，产生显著统计结论的价值。同时，所有的实验模型有局限性。类似于血肿瘤细胞导入的其他方法，在动脉注射仅概括在癌细胞传播到循环后的转移级联后续步骤。因此，它不是一个合适的模型，研究，例如，该确定原发癌细胞的传播进入血液的生物因素。

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素酶报道体外注射前。为了尽量减少对动物应力， 体内成像，通常进行后24小时注射。一个成功的注射导致颅内强肿瘤细胞负荷，鼠标头部阳性是唯一区域ignals（ 图2A）。 活体成像系统中使用跟踪荧光素标记的肿瘤细胞，我们观察到天注射后内信号强度的初始下降;然而，能够在CNS生长的癌细胞最终管理分裂生成的体内成像系统的信号增加为脑转移发展（ 图2B）。动脉注射通常会产生具有相对低的变异，这是使用这种方法，以产生实验脑转移的一大优势在体内成像系统的信号相一致。作为生长的肿瘤病灶置换脑组织，健康的动物的质量具有快速进展脑转移可以迅速恶化，镜像人脑转移的患者的临床表现。表明脑转移的显著存在的典型症状的行为包括损失重量，弯腰驼背的姿势，并不断盘旋。在实验结束点，动物的大脑可以提取用于生化指标的检查或组织学检查。除了黑色素转移灶（ 图3A），它通常是难以从周围之前组织学评价（ 图3B）的脑组织区分肿瘤病灶。在这样的情况下，建议以标记肿瘤细胞与荧光报告（ 如 GFP）的分离转移性细胞（ 图3C）的便利性。或者，整个大脑可以解离成单细胞和肿瘤细胞通过磁珠标记的抗体分离。 <img alt="图1" src="/files/ftp_upload/55085/55085fig1.jpg" /> 图1:用于颈动脉注射制备。一）麻醉小鼠被放置在玻璃板上，并与る固定BBER带。站点和切口的长度标有在颈部有一条黑线。 B）毕竟准备手术步骤完成后，露出颈动脉被放置在支持一个企业棉球，准备对癌症细胞注射。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55085/55085fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55085/55085fig2.jpg" /> 图2:脑转移瘤生长无创监测。 A）的改性的MDA-MB-231乳腺癌细胞用萤光素酶报道基因体外 。从后24小时颈动脉注射开始，脑转移发展中监测非侵入通过体内成像系统成像（比例尺= 50毫米）重复。 B）的 体内成像系统伊马量化晋数据表明肿瘤负荷，颈动脉后注入的初始下降，其次是脑转移瘤的生长指数直到动物濒死。误差棒反映总发光信号的平均（SEM）的标准误差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55085/55085fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55085/55085fig3.jpg" /> 图3:收获脑转移病灶。 A）K1735黑色素细胞产生的同基因C3H小鼠大脑，极大地方便了从周围间质脑转移灶的分离，但仅限于某些机型黑色素黑色素脑转移病灶。 B）游离的乳腺肿瘤细胞转基因MMTV-NEU / PTEN的 -null小鼠产生明显的脑转移AF经颈动脉注射植入之三。肿瘤/间质利润率很难尽管血管转移灶的存在明显区别。 C）的 MDA-MB-231乳腺癌细胞进行预标记有绿色荧光蛋白（GFP）记者体外 ，以促进通过手术切除或细胞分选脑转移病灶提取。注意，大多数转移性病灶都集中在何处颈动脉注入发生在脑的相同侧。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55085/55085fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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. ...

intracarotid-cancer-cell-injection-to-produce-mouse-models-brain

Nanyang Technological University

Research

JoVE Journal

Cancer Research

26.2K 次观看.  The University of Texas MD Anderson Cancer Center. 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。 

视频: Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

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

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

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

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

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

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 cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

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

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

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

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

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

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 colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

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

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

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

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 diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

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

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

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

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

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 ...