We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

道德守则 所有的动物研究批准了纽约市立大学亨特学院的机构动物护理和使用委员会（IACUC）。 注意：八只BALB / c小鼠在本实验过程中使用。五只BALB / c小鼠植入5×10 6，BNL 1ME A.7R.1小鼠肝癌细胞产生的原发肿瘤。三只BALB / c小鼠，没有植入用作对照。 BNL 1ME A.7R.1是从鼠非致瘤性肝细胞细胞系，BNL CL.2衍生由化学转变该鼠肝癌细胞株。 BNL 1ME A.7R.1从美国典型培养物保藏中心（ATCC）获得。 1.预手术<ol><li>确保小鼠全部存活手术是健康的。为了确保小鼠是健康的，由皮肤的隆起检查脱水的迹象。不要用小鼠疾病或D的任何证据istress在这些实验中。 </li><li>称取小鼠以确保它们不减持。确保重量至少16-20克。 </li><li>准备5×10 6 BNL 1ME A.7R.1小鼠肝癌细胞每只小鼠植入： <ol><li>抽吸媒体从10ml的60％ - BNL 1ME A.7R.1小鼠肝癌细胞70％汇合细胞培养皿。用5ml PBS洗涤两次。加2ml胰蛋白酶的菜和在培养箱与空气和5％的CO 2 5分钟在37℃。 </li><li>接着，移液管加入10μl胰蛋白酶消化细胞分化成在显微镜下在10倍的放大倍率的手动血细胞计数用于细胞计数。算在四个大角落正方形中发现的细胞的总数和由细胞的总数乘以稀释因子。然后，通过平方计数的数分裂并通过10 6乘以为细胞浓度（每毫升的细胞）。 </li><li>使用的计算，收集每只小鼠在灭菌的5百万个细胞1.5ml微量管。标签的1.5 ml离心管中。 </li></ol></li><li>重悬在100μl磷酸盐缓冲盐水（PBS）中的5×10 6，BNL 1ME A.7R.1小鼠肝癌细胞。保持这些细胞在冰上，直到植入手​​术。 </li><li>进行必要的准备小鼠外科手术如麻醉诱导和头发修剪远离消毒设备和材料，以避免污染的活动。 </li><li>使用与氧一起的异氟烷蒸发器以1L每分钟（约2.1％）的恒定浓度流速诱导小鼠通过吸入异氟烷麻醉。注意：该系统是自立，和包括一个精密汽化器，氧的供应和链接到所需要的气/麻醉剂清除呼吸回路一活性炭罐。 </li><li>确保麻醉足以防止动物从手术过程中经历的疼痛。执行脚趾捏，观察动物的非responsiveness确认麻醉。 </li><li>此时，切换阀管线，密封的框和开放到鼻锥。 </li><li>只要鼠标被麻醉，敷眼软膏的一个微小的量，以角膜为从干燥和损坏防患于未然。 </li><li>接着，将动物以​​制备区域，用鼻锥体正确地放置和连接的（用于维持麻醉）。准备手术切口进行手术通过脱毛的部位，在这个准备区。 </li><li>确保手术的区域是在不含所有其他活动的位置。 </li><li>确保工作表面坚固，并适当消毒，未弄脏的，易用的手术前。放置一个无菌的一块亚麻或手术托盘在它的。 </li><li>确保手术视野仅可用于进行外科手术的人员。确保手术室进行清洗和清扫用合适的清洁剂，并用适当的disinfectant。 </li><li>确保它也位于远离门窗作为气流可以传播粉尘导致现场的污染。 </li><li>执行生存手术无菌器械。或者，根据制造商的建议，或高压釜浸泡仪器消毒剂。使用基本无菌物品是可以使用了。 </li><li>根据功能的不同工具，以帮助防止无菌区域受到污染。 </li><li>剃周围的切口（正中切口）站点约1英寸访问动物的肝脏。从动物手术的部位取下的头发了。 </li><li>剃须后，擦洗肥皂消毒剂（优碘外科洗手）的区域，并用无菌PBS液清洗多余的消毒液了。执行这几次，与整个磨砂工艺处理大约持续5分钟。卸下并丢弃用过的手套。 </li><li>后来，改为防护服，如surgic人长衫，磨砂顶和面罩。接着，把手术手套前，双手消毒用适当的溶液擦洗。注：这是一个必要的步骤，以防止脱落，污染手术部位与潜在的污染物。 </li></ol> 2.手术<ol><li>提供一个外部热源温暖整个麻醉的整个时期。动物可能会遇到一些低体温是因为麻醉导致体温调节障碍;因此，维持体温。 </li><li>莱鼠标在整个手术的背面。 </li><li>修复前肢，后肢和鼠标尾部的手术台与手术磁带件。 </li><li>做一个直线切口立即逊于xyphisternum。 </li><li>使用无菌的自固手术用牵引器具，以暴露肝脏。 </li><li>与具有注射器20G针，缓慢而小心地注入含有5×10 6，BNL 1ME A.7R.1 100微升PBS小鼠肝癌细胞到肝脏的右叶。 </li><li>除去针后，应用消毒棉签至少1分钟，以停止任何潜在的出血由于肝脏高血管。 </li><li>关闭皮下组织与可吸收缝合线之后皮肤缝合手术用夹子。随后，优碘擦洗一次清理伤口。注意：这将确保手术伤口闭合。 </li><li>皮下给予100微升生理盐水。 </li></ol> 3.后手术<ol><li>立即手术后，鼠标转移到一个干净的笼子与手术后的身份证，以通知的鼠标状态照料。提供食物和水。注：根据我们的经验，刚刚动过手术的境遇最好的，如果单独住用湿的食物和水的最初24小时内术后鼠标。 </li><li>保持动物温暖，热灯产生的热量不超过38ºC。 ü瑟温度计来测量温度，直到鼠标完全恢复。 </li><li>众议院的动物关在笼子里最好用纸张的床上用品，以确保舒适。 </li><li>初步恢复后，监测动物的不适，出血，疼痛和痛苦的迹象。给予丁丙诺啡（0.05 -0.1毫克/千克），以减轻疼痛。密切监控活动，行为和外观，以及水和食物消耗量为约2小时。如果动态，将动物回区域老鼠经常正常的住房，并持续监控在6-12小时，间隔24小时后手术。如果需要执行重量检查。 </li><li>但是，如果动物是有问题休养，采取适当的支持治疗措施。如果小鼠无法恢复，人道安乐死通过CO 2窒息和颈椎脱位鼠标。按照安乐死认可机构的指导方针。 </li><li>手术后，检查肝癌的临床证据通常是observab乐为显著减少，体况评分。注意：在这一点上，初级肿瘤可能已经开发了在肝脏和转移性沉积物在肺部有可能开发的。 </li><li>此时，牺牲参与本实验由人道的安乐死所有小鼠（实验和对照）。若使小鼠二氧化碳窒息：非常缓慢地释放二氧化碳进入室在一段5 -10分钟。这将导致呼吸骤停。 </li></ol>

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The authors declare that they have no competing financial interests.

在这篇文章中进行了深入描述的方法的是考虑到最近报道Ogunwobi和成功地建立了肝癌转移的原位同基因小鼠模型的同事（图2）3。肿瘤的接受率在此过程中普遍偏高。我们先前观察到的100％3肿瘤接受率。然而，接受率可以根据研究者的能力是可变的。在由下一个训练新研究生完成的最新的实验中，将肿瘤接受率较低，为40％。尽管大多数实验终止于4 - 由于在体况评分?...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table border="0">
	<tbody>
		<tr>
			<td>Micro Dissecting Tweezer&#160;</td>
			<td>Roboz</td>
			<td>(RS-5040)</td>
			<td>Tip .20 x .12mm</td>
		</tr>
		<tr>
			<td>Graefe Micro Dissecting Forceps, serrated curved tip</td>
			<td>Roboz</td>
			<td>(RS-5111)</td>
			<td>1 X 2 teeth, curved tip width 0.6mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6501)</td>
			<td>Blunt 3 X 3 prongs, depth 4mm, spread 25mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6503)</td>
			<td>Blunt, 3 X 3 prongs, depth 4mm, spread 19mm</td>
		</tr>
		<tr>
			<td>Jameson Caliper (Measures tumors)</td>
			<td>Roboz</td>
			<td>(RS-6466)</td>
			<td>80mm/3 inch scale, chrome plated&#160;</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td>
			<td>Roboz</td>
			<td>(RS-5852)</td>
			<td>23mm blades, length 4 inches, flat shanks and large rings</td>
		</tr>
		<tr>
			<td>Scalpel with blades, for delicate dissecting procedures</td>
			<td>Roboz</td>
			<td>(RS-9861-36)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle&#160;</td>
			<td>Roboz</td>
			<td>(RS-9884)</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>Littauer Stitch Sissors</td>
			<td>Roboz</td>
			<td>(RS-7074)</td>
			<td>Length 4.5 inches</td>
		</tr>
		<tr>
			<td>Brown needle holder, for easy suture tying</td>
			<td>Roboz</td>
			<td>(RS-7960)</td>
			<td>Convex jaw, fine serrations</td>
		</tr>
		<tr>
			<td>Reflex 7MM wound clips with reflex 7 clip applier</td>
			<td>Roboz</td>
			<td>(RS-9262)</td>
			<td>safe, secure alternative method of wound closure</td>
		</tr>
		<tr>
			<td>Instrument tray and lid</td>
			<td>Roboz</td>
			<td>RT-1350S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-clipper with detachable blade</td>
			<td>Roboz</td>
			<td>(RS-5903)</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td>
			<td>Roboz</td>
			<td>Ds-400, Ds-401, DS-501</td>
			<td>For fast decontamination of micro-dissecting instruments.&#160;&#160; Instruments decontaminate within 15 seconds.&#160;</td>
		</tr>
		<tr>
			<td>Microinjection needles</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
	</tbody>
</table>

a "patient-like" orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

Balb / c小鼠的肝脏植入5×10 6，BNL 1ME A.7R.1小鼠肝癌细胞。肝癌发展的临床证据是观察到63天后手术。因此，将小鼠安乐死。尸体剖检在安乐死的小鼠进行的。肺和肝脏切除和仔细肉眼检查以鉴定宏观肿瘤。在一个鼠标，浅表肿瘤观察到一些附着到腹壁（图1A）的肝脏的表面上。在第二个鼠标，异常好看肝脏进行了观察。异常非常强烈地看起来像一个浅表肿瘤（图1B?...

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A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

癌症的转移扩散是癌症相关死亡的主要原因。我们提供了一个深入的描述，我们赖以生存的手术方法，为建立“以病人为像”原位同基因小鼠模型系统研究的实质器官肿瘤转移的机制。

“以病人为像”肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

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A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

12.1K 次观看.  Hunter College of the City University of New York. 癌症的转移扩散是癌症相关死亡的主要原因。我们提供了一个深入的描述，我们赖以生存的手术方法，为建立“以病人为像”原位同基因小鼠模型系统研究的实质器官肿瘤转移的机制。 