Research in Zhang lab was supported by X. H.-F. Z.'s NCI CA151293, CA183878, Breast Cancer Research Foundation, U.S. Department of Defense DAMD W81XWH-13-1-0195, a Pilot Award of CA149196-04, McNair Medical Institute and by H.W.'s U.S. Department of Defense DAMD W81XWH-13-1-0296.

所有的动物工作是按照药品的贝勒医学院的动物护理准则进行。 1.细胞的制备注意:不同的癌细胞系可以用于IIA注射取决于研究目的。我们已经使用了乳腺癌细胞系MCF7，4T1，4T07，MDA-MB-361，MDA-MB-231，MDA-MB-436和前列腺癌细胞系C4-2在我们的研究。我们通常使用两种GFP-和萤火虫荧光素酶标记的肿瘤细胞对我们的研究，并从GFP荧光素酶标记的MCF7细胞系在这里显示的一些数据。 <ol><li>保持在5％CO 2含有10％FBS和0.1mg / ml的青霉素-链霉素，在37℃的细胞在DMEM中。 </li><li> IIA注射前，trypsinize在80细胞 - 90％汇合，用0.25％胰蛋白酶/ EDTA溶液。用冷PBS洗细胞两次，并重新悬浮细胞在10ml PBS中的细胞计数。以除去PBS，离心细胞，在800×g离心5分钟。 </li><li>重新悬浮细胞在冰冷的PBS一个的5×10 6个细胞/ ml的浓度。 </li><li>用100微升GFP-和萤火虫荧光素酶标记的肿瘤细胞注射IIA一个鼠标的。 注意:因此，每个动物接收的细胞数为5×10 5个该数目可能需要基于对细胞系的侵袭性被改变。 </li><li>置于冰上细胞悬液。 可能需要的振动，以防止细胞聚集每隔几分钟，直到它准备好用于注射:音符。对于某些细胞系（如MDA-MB-231），可能需要更严格的程序（ 例如 ，通过45微米的细胞滤网），以防止聚集。请注意，血管的堵塞可导致组织坏死，并且因此混杂的实验。 </li></ol> 2.动物的制备<ol><li>动物疼痛管理，得到5毫克/公斤/天卡洛芬（或其它止痛药）与膳食补充剂给小鼠手术前不小于24小时。管理剂量的手术前0.1毫克/毫升丁丙诺啡皮下注射60分钟。注意:任选的步骤是植入雌二醇托盘到动物的背部，以提供额外的雌二醇。这将加速ER +肿瘤细胞的增长（ 如 MCF-7细胞）9。 </li><li>麻醉4 - 第6周龄小鼠（约20克）用氯胺酮（80 - 100毫克/千克）和赛拉嗪（10 -12.5毫克/毫升）腹腔注射。 </li><li>通过确认脚趾捏20 G鼠标和呼吸频率减少50％的观察镇静的适当水平和粉红色粘膜（如鼠耳皮肤和皮肤爪子）。保持手术过程中监测这些生命体征，每15分钟。注:动物的无动作表明，该动物是充分麻醉，准备手术。 </li><li>使用于眼部兽医药膏，以防止干燥。 </li><li>剃须鼠标放在下腹部，尤其是右腹股沟区域，手术和注射会发生。 注意:如果使用无胸腺裸鼠这是没有必要的。 </li><li>把鼠标在它的后面，传播腿在它们的自然位置，坚持脚趾用胶带剥离移动纸板。 </li><li>请用70％乙醇异丙醇浸泡消毒棉签右腹股沟区，随后与优碘手术擦洗几次。 </li></ol> 3.髂总静脉和动脉位置，并分离<ol><li>使1.0皮肤切口- 1.2厘米长的第4 次 ，并使用在3号手柄保持无菌，＃10碳钢手术刀片5 个乳头在右下腹部之间。然后用无菌手术盖布来覆盖动物体除外切口部位。 </li><li>移动动物标准台式解剖显微镜。使用4X的放大率注射。 </li><li>根据解剖显微镜的放大4倍，将脂肪组织和peritoneu之间钝性分离钳m和向外推两侧组织以暴露髂血管和神经（参见图1）。 注意:从中线主动脉和动脉分支的下部之间的动脉被称为髂总动脉。这是其中注入发生。 </li><li>使用一对直线细镊子通过血管和神经之间的结缔组织（如膜）打破，接近船只。 </li><li>切换直细镊子左手。抓住一对用右手角度的细镊子的。将右手镊子到同一位置，结缔组织已经被打破。然后通过粘右手镊子，容器的下方。 </li><li>同时，插入左手镊子插入血管的左侧的结缔组织，以指导右手镊子经历。 </li><li>一旦右手镊子得到船底下，试图将船从周围的TI分开ssue多一点点，然后让他们在右手镊子的顶部。 </li><li>使用左手钳提供一个4-0丝线缝合的尖端右手镊子，然后通过血管下方轻柔缓慢地拉出缝合。注:现在无论是常见的静脉和动脉血管被缝合举起来（ 见图2）。 </li></ol> 4.注射后注射护理<ol><li>准备用于注射的细胞在31克胰岛素注射器。使用100μl的细胞悬浮液在PBS中每次注射。确保吸取上下数次以分离细胞并避免聚合，使得注射的细胞不会堵塞和阻塞血液流动。 </li><li>与在左手倾斜钳，把钳子沿着缝线的引导容器的下方，然后打开钳子的两个臂，具有对镊子轻轻保持的血管。 注意:钳子的两个臂之间的间隔将是第injection网站。 </li><li>持用100μl细胞悬浮液的31克针在右侧，与针的斜面向上，准备用于注射。 </li><li>针插入动脉管腔（血液会进入针头）。然后推细胞悬浮液缓慢地注入细胞。将细胞悬浮液将推开红细胞中的容器中。尽量不要打破船只或泄漏细胞悬液出来。 </li><li>当注射完毕后，轻轻地拉了回来针，并继续保持船只在左边手钳止血。话扯远了缝合。 </li><li>拉开左手钳，赶紧用棉签按压切开动脉区止血。 注意:通常5 - 10分钟的持续压力足以止血。 </li><li>当出血停止，用皮胶，关闭手术区域的皮肤边缘。使耳标签，并检查生物发光信号来检查注射的细胞的分布。&lt;BR /&gt;注:一个成功的注入将导致类似的图像中图3。 <ol><li>以100微升15毫克/毫升的荧光素在PBS注射75毫克/经由帧内眶窦千克小鼠浓度检查的生物发光信号。 <ol><li>对于帧内入轨，把鼠标两侧眼球的手指，使用压力，使眼球从眼睛框稍微弹出，插入眼球和鼻子之间的28克胰岛素注射器针头，然后慢慢推注射器注入荧光素。 注意:在正确的位置，针应能达到2的深度 - 打硬组织（骨）前3毫米。 </li><li>鼠标放置到成像系统用于体内整个动物成像10 - 5分钟内60秒（ 见图3）。 注意:可选的步骤是植入雌二醇粒料到动物的背部，以提供额外的雌二醇。这将加速ER +肿瘤细胞的生长（ 例如 ，MCF-7细胞）15。 </li></ol></li></ol></li><li>离开鼠标放在一个温暖的加热垫，直到醒来。监控出血，肿胀，在手术后的一段开裂和疼痛的迹象。把老鼠回用镇痛药覆盖率（carprofin膳食补充剂的建议）疼痛管理给定的，直到没有痛苦的迹象笼。在手术后7天密切关注和照顾动物。 </li></ol> 5.监测转移性生长<ol><li>组织形态测定: <ol><li>收获肿瘤携带骨组织，固定在4％低聚甲醛的骨头24小时，然后脱钙它们在pH 7.0的3 14％的EDTA溶液-第5天，并使其受到石蜡包埋如前所述14。 </li><li>部以3微米的厚度的石蜡嵌入骨组织和如先前所述14进行苏木精/曙红染色的标准的组织学方法。 </li></ol></li><li> Immunohistochemis尝试: <ol><li>受试者石蜡包埋的肿瘤轴承骨滑动如前所述14免疫组织化学染色用于各种目的。简言之，将免疫染色的MCF7肿瘤细胞通过抗GFP抗体，和免疫染色的成骨细胞（预成骨细胞和成骨细胞）分别由抗Osterix的抗体和抗碱性磷酸酶（ALP）的抗体。 </li></ol></li></ol>

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The authors have nothing to disclose.

虽然只有髂动脉被注射癌细胞的目标，我们建议既髂静脉和动脉从周围组织中分离，并把它们一起抬起作为包。这是因为静脉和动脉广泛相互接触，与静脉血管壁薄，容易断裂。因此，对于一个成功的注射，这样可以节省时间和精力，将两个容器保持在一起，虽然癌细胞仅注射到动脉。 4-0丝线来帮助这个过程如图2所示，缝合线还可以帮助它应该出现止血。 需要?...

转移占引起实体瘤死亡的90％以上。骨是受各种癌症类型，特别是乳腺癌和前列腺癌的转移中最常见的器官。当诊断在临床上，骨转移一般都已经进入高级阶段，在骨无论是溶骨性或成骨细胞的改变，常伴有神经症状。 以往的研究主要集中在明显溶骨骨转移1-3，但是我们目前有溶骨过程来临前限制骨骼微的理解。这至少部分是由于缺乏合适的实验模型和方法。乳腺癌的基因工程小鼠模型常转移至肺部，但更高效地骨4。同样，原位移植瘤很少自发的骨转移，一些骨热带4T1乳腺carcinom子的克隆和MSP过表达PyMT转基因小鼠模型例外5-7。帧内胫钻孔可以提供癌细胞的骨8-10，但它也导致损伤和炎症到局部组织。目前的乳腺癌细胞系心内注射已经调查骨定植11-13的主要方法。然而，在癌症细胞导入左心室只有有限的比例将最终达到骨和骨髓，使得它难以跟踪在一个量化的方式微观转移。 在这项研究中，我们建立了一种技术，即帧内髂动脉（ⅡA）注射14，癌细胞选择性地递送到后肢组织中，从而丰富癌细胞在骨和骨髓而不导致破坏局部组织。由于骨特异性，该方法还允许有足够的时间好逸恶劳癌细胞的一个前殖民最终imals屈从于原发肿瘤或转移的其他重要器官。当与各种其他技术，如生物发光成像，免疫荧光染色和骨形态计量学结合，ⅡA注射是相关的骨转移研究目的宽范围可能有用的，尤其是跟踪从单个癌细胞多小区进展微转移。特别是，我们表明，IIA族注射使我们能够可视化癌细胞和各种类型的在骨微环境周围细胞之间的相互作用。

<table border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Materials</td>
			<td style="width:303px;"></td>
			<td style="width:123px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:303px;">HyClone</td>
			<td style="width:123px;">SH30022.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:303px;">Gibco</td>
			<td style="width:123px;">16000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pen/Strep Amphatericin B</td>
			<td style="width:303px;">Lonza Biowhittaker</td>
			<td style="width:123px;">17-745E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:303px;">Lonza Biowhittaker</td>
			<td style="width:123px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin/EDTA solution</td>
			<td style="width:303px;">HyClone</td>
			<td style="width:123px;">SH30042.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">45uM cell strainer</td>
			<td style="width:303px;">VWR International Laboratory</td>
			<td style="width:123px;">195-2545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MediGel CPF with carprofen&#160;</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td>For pain management</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine&#160;</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td>For pain management</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Estradiol pellet</td>
			<td style="width:303px;">Innovative Research of America</td>
			<td style="width:123px;">SE-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine and xylazine</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vet ointment</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td>Avoid eye dryness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaver</td>
			<td style="width:303px;">Oster</td>
			<td style="width:123px;">78005-050</td>
			<td>For furred mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropyl ethanol</td>
			<td style="width:303px;">ACROS</td>
			<td style="width:123px;">67-63-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine surgical scrub</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#10 scalpel blades</td>
			<td style="width:303px;">Ted Pella, Inc</td>
			<td style="width:123px;">549-3CS-10</td>
			<td>Multiple</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">No. 3 handle</td>
			<td style="width:303px;">Ted Pella, Inc</td>
			<td style="width:123px;">541-31</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical drape</td>
			<td style="width:303px;">Sai Infusion Technology</td>
			<td style="width:123px;">PSS-SD1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight forceps&#160;</td>
			<td style="width:303px;">Roboz Surgical Instrument</td>
			<td style="width:123px;">RS-5132</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight fine forceps</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">11253-20</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Edged fine forceps</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">11253-25</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 Vicryl silk suture</td>
			<td style="width:303px;">Johnson &#38; Johnson Health Care</td>
			<td style="width:123px;">J214H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">31G insuline syringes</td>
			<td style="width:303px;">BD</td>
			<td style="width:123px;">328418</td>
			<td>Multiple</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Q-tips cotton swabs (Sterile)</td>
			<td style="width:303px;">VWR International Laboratory</td>
			<td style="width:123px;">89031-272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Skin glue</td>
			<td style="width:303px;">Henry Schein Animal Health</td>
			<td style="width:123px;">31477</td>
			<td>For surgery site skin closure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ear Tag Applicator</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">24220-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ear tags</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">24220-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-luciferin</td>
			<td style="width:303px;">Gold Biotechnology</td>
			<td style="width:123px;">LUCK</td>
			<td>Avoid light and put on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">28G insulin syringes</td>
			<td style="width:303px;">BD</td>
			<td style="width:123px;">329410</td>
			<td>For intra-orbital injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformadehyde</td>
			<td style="width:303px;">Alfa Aesar</td>
			<td style="width:123px;">30525-89-4</td>
			<td>For tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:303px;">OmniPur</td>
			<td style="width:123px;">4050</td>
			<td>For bone tissue decalficication</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:303px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:303px;">Leica</td>
			<td style="width:123px;">Leica S6E stereo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS Lumina II imaging system</td>
			<td>Advanced Molecular Vision</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibodies</td>
			<td></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-GFP antibodies (JL-8)</td>
			<td>Clontech</td>
			<td>632381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-ALP antibodies</td>
			<td>Abcam</td>
			<td>ab108337</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Osterix antibodies</td>
			<td>Abcam</td>
			<td>ab22552</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

图1示出的解剖位置和髂总动脉（红色）和静脉（蓝色）的关系。 图2显示了在解剖镜下髂血管和神经的相对位置。 如图2A所示，血管和神经的右腹膜壁下方和皮肤切口由与腹膜推开后，可以发现。髂总静脉是在左侧，并与动脉是更大和更暗。动脉是在中间，并期待粉红色。它比静脉薄，?...

Watch this Scientific Journal Video about Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis at JoVE.com

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

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 ...

intra-iliac-artery-injection-for-efficient-selective-modeling

Research

JoVE Journal

Cancer Research

16.1K 次观看.  Baylor College of Medicine. 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. 

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

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 ...

科研

癌症研究

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

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. ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

通过将表达的腺病毒注射到小鼠乳腺腺中,模拟乳腺癌

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

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 Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

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 ...