我们特别感谢范德比尔特大学医学中心的患者，他们为分子神经外科组织库提供了宝贵的研究材料。我们感谢那些建立和维护组织银行的人，里德·汤普森MD（首席调查员），樱桃金纳德RN（研究护士）和拉里A.皮尔斯MS（经理）。组织学服务部分由范德比尔特大学医学中心 （VUMC） 转化病理共享资源（由 5P30 CA068485 奖支持范德比尔特-英格拉姆癌症中心）执行。这项工作得到了国家发展局（1R21NS070139）、伯劳斯威康基金和VMC发展基金向MKC提供的赠款的支持。MKC 由退伍军人事务部、退伍军人健康管理局、研究和发展办公室、生物医学实验室研究和发展部通过赠款 1 I01 BX000744-01 获得支持。内容不代表退伍军人事务部或美国政府的意见。

1. 肿瘤细胞悬架的准备
注： 需要为使用患者材料和动物提供适当的机构批准，以建立和维持原发性正畸胶质瘤异种移植物。在范德比尔特大学医学中心，在患者同意下收集超过诊断所需量的肿瘤材料，用于研究组织库。标本标本标注带有随机的 5 位 REDcap 数据库编号，并删除所有患者特定标识符。REDcap 数据库包含组织存储库中每个标本的分离临床数据，包括性别、年龄（年）、种族、生存状态、病理学、癌症治疗、切除年份和进展时间。为了保持无菌性和优化主要异种移植方法的成功，所有试剂、蒸汽自动切割手术器械和手术部位应提前组装或准备。
注： 胶质瘤标本通常含有大量的骨髓和碎片。根据标本大小，可能需要使用超过 4 个不连续梯度管来充分清除骨髓和碎屑。
注： 当没有或很少清晰可见的未分离组织剩余时，这个过程就会完成。避免在三次加工过程中引入气泡。
注： 分离后的细胞生存能力通常为 90%>%。较低的通过率可能反映许多变量，包括从手术室的次优组织处理或运输时间、冷冻保存方法或木瓜分离技术。然而，只要以适当的体积移植足够数量的可行细胞，较低的细胞通过率可能仍然足够。每只小鼠的体积为2μl，可植入50，000-200，000个可行的细胞。由于注射器针的斜尖，应在最后音量中加入额外的 5 μl 细胞悬浮，以确保每次注射时都能将足够的材料抽入注射器中。例如，为 5 只小鼠注射 2μl/鼠标，最终体积应为 15 μl （15 μl = （5 x 2 μl） = 5 μl）。
<ol><li>将刚被重新解剖的、身份不明的患者材料放在冰上无菌的封顶容器中，以便运往实验室。将标本直接加工为移植或冷冻保存在液氮中的标本（见下文），以储存在液氮中，然后使用。</li>
	<li>准备在无菌罩与制造商提供的套件组件和说明书包解决方案（帕潘溶液，洗涤/蛋白酶抑制剂溶液和不连续梯度溶液）。标记无菌 15 毫升聚苯乙烯锥形管，并分发如下解决方案：<ul>
<li>管1：5毫升的帕金溶液</li>
			<li>管 2： 3 毫升洗涤/蛋白酶抑制剂溶液</li>
			<li>管 3-6： 5 毫升每个不连续梯度溶液</li>
		</ul>
</li>
	<li>将胶质瘤标本放在管 1 中，配有 5 毫升木瓜溶液，并在 10-20 分钟内用 5 毫升移液器进行三重奏。</li>
	<li>将材料上下吹10次，然后在室温下孵化材料2-3分钟，然后启动下一个三轮。</li>
	<li>将 5 毫升移液器的尖端放置在圆锥管底部，每个三轮车循环，使尖端在最后 2 个周期中接触管的底部。</li>
	<li>离心机在室温下300 x g 5分钟。在 3 毫升的洗涤/蛋白酶抑制剂溶液中上下 10 倍地取出超自然剂和移液器。</li>
	<li>小心地将等量的悬架分层到每个 5 毫升不连续梯度溶液（管 3-6），以"重力"分配速度设置管道。</li>
	<li>将管子放在装有摆动桶转子的离心机中，加速速度降低到最低设置，制动器关闭。离心机在室温下76 x g 12分钟。随着制动器的关闭，离心工作一般在20分钟后完成。</li>
	<li>取出超自然物，再喷出并结合每个颗粒在5毫升的无菌平衡盐溶液中，并将细胞放在冰上。</li>
	<li>取出细胞悬架的一部分（10-100 μl），并通过用血细胞计排除盘蓝色来确定可行细胞的密度。</li>
	<li>离心机在300 x g 5分钟。去除超自然物质，以每μl25，000-125，000个可行细胞的密度重新消耗细胞颗粒。</li>
	<li>将足够容量的细胞悬架转移到 200μl 微中微管 （PCR 管） 中，并放置在冰上。</li>
</ol>2. 准备手术场地和仪器
注： 手术过程中佩戴无菌手术手套。根据每个机构的要求，可能需要手术服、帽子和护脸器。
<ol><li>准备一个消毒的台面啮齿动物手术与分开的相邻区域，动物准备，手术场和动物恢复。</li>
	<li>在蒸汽高压灭菌器械中消毒。随后，在从一种动物过渡到另一种动物时，使用热珠灭菌器对仪器进行闪光消毒。</li>
</ol>3. 感应、麻醉和镇痛
注： 从小鼠麻醉后超过15分钟的手术需要接触热源。为了防止体温过低，在术前和术后期间，小鼠会获得热源（循环热水毯）。 
<ol><li>准备氯胺酮/西拉津鸡尾酒进行感应麻醉，使每只小鼠每克体重注射10微克/千克的鸡尾酒，每克体重可获得氯胺酮100毫克/千克和10毫克/千克的西拉津。</li>
	<li>
表1。麻醉鸡尾酒。
		<table><tbody>
<tr>
<td rowspan="2"></td>
					<td rowspan="2">库存集中度</td>
					<td colspan="3">要准备的音量 </td>
				</tr>
<tr>
<td>一只鼠标</td>
					<td>五只老鼠</td>
					<td>十只老鼠</td>
				</tr>
<tr>
<td>氯胺酮</td>
					<td>100毫克/毫升</td>
					<td>25 微升</td>
					<td>125 微升</td>
					<td>250微升</td>
				</tr>
<tr>
<td>西拉津</td>
					<td>100毫克/毫升</td>
					<td>2.5 微升</td>
					<td>12.5 微升</td>
					<td>25 微升</td>
				</tr>
<tr>
<td>伊索托尼盐水</td>
					<td></td>
					<td>223 微升</td>
					<td>1，113 微升</td>
					<td>2，225 微升</td>
				</tr>
<tr>
<td colspan="2">总</td>
					<td>250 ul</td>
					<td>1，250微克</td>
					<td>2，500微克</td>
				</tr>
</tbody></table>
</li>
	<li>通过稀释库存溶液（10毫克/毫升）1：20无菌、无阳激素的水，为镇痛准备酮蛋白溶液，使每只小鼠通过每克体重注射10微克溶液，获得5毫克/千克的剂量。</li>
	<li>称重并记录术前体重。单个鼠标重量各不相同，但一般范围为 18-22 克。</li>
	<li>用胰岛素注射器将每克小鼠体重10微克氯胺酮/西拉津鸡尾酒注射到腹膜内空间。例如，为 20 克鼠标注射 200 μl。</li>
	<li>确定鼠标是否通过后腿的脚趾捏完全麻醉。鼠标通常需要 3-5 分钟才能不再从捏中退出。</li>
	<li>用胰岛素注射器在侧翼松弛的皮肤中每克小鼠体重皮下注射10μl的酮蛋白溶液。例如，为 20 克鼠标注射 200 μl。</li>
	<li>用剪子在头骨上剃头发，用棉尖施用器擦洗裸露的头皮，然后擦拭酒精垫。重复这些步骤，多擦两次， 波维酮碘擦洗和酒精擦拭。</li>
	<li>将眼药膏涂抹在鼠标的眼睛上。</li>
	<li>用无菌手术刀刀片将1厘米的中线切口从耳朵后面延伸到眼睛前部。</li>
	<li>将鼠标置于解剖范围下，并露出头骨以识别缝合线。使用带钻头的圆形运动，将一个2.5毫米横向的毛刺孔中心到胸前，将1毫米的后孔集中到日冕缝合线。</li>
</ol>4. 颅内移植
注： 注射量超过2.5微克对小鼠可能致命。
<ol><li>将鼠标钩在切口杆上的上切口并应用鼻夹，使头骨牢牢地保持在中性位置，从而将鼠标置于立体定形框架上。</li>
	<li>将微中线管中5-10μl的细胞上下多次抽取到粘在立体定向操纵器探针支架中的10μl汉密尔顿注射器中，以混合悬架并消除气泡。压低柱塞，使注射器装载2μl（足够的体积注入一种动物）。</li>
	<li>拉皮切口的横向边缘，露出毛刺孔，并使用微脉冲器将针头引入毛刺孔，使斜面部分就在头骨表面以下。<ol>
<li>将针头推进 3 mm，然后取出针头 0.5 mm 以创建注射空间。</li>
			<li>将柱塞缓慢推进超过 30 秒，然后让针头在大脑中再停留 2 分钟。通过提升 0.5 mm 并等待额外的 30 秒，然后从大脑中取出针头，逐渐取出针头。</li>
		</ol>
</li>
	<li>将鼠标从立体定型框架中取出，用自动剪辑关闭伤口。</li>
	<li>将鼠标放在加热垫上以保持体温，并持续监测粘膜和裸露组织（脚底）的呼吸模式和颜色。</li>
	<li>一旦老鼠从麻醉（麻醉和流动）完全恢复，将鼠标放在无菌微分离器笼中，并将其送回动物收容所。</li>
</ol>5. 植入后动物护理和观察
注： 肿瘤移植和渗透生长的最可靠迹象是渐进的，持续的体重减轻，伴随着驼背姿势和粗糙的发衣的发展。在极少数情况下，神经缺陷被注意到，包括厌食症，帕雷西斯和癫痫发作。正交原发性异种移植物具有很强的侵入性，并长期发展。小鼠通常在移植后3-6个月出现肿瘤生长症状。
<ol><li>每天观察有正位异种草的小鼠，在切口部位观察食物和水的摄入、行为、梳妆和感染迹象。</li>
	<li>如果术后观察到疼痛或痛苦，则施用酮丙酮（皮下5毫克/千克，每天1-2次）。</li>
	<li>手术后 8-10 天取出自动剪辑。</li>
	<li>每周称量小鼠。</li>
	<li>监测肿瘤移植的体征和症状。</li>
</ol>6. 肿瘤收获
注： 通过这种方法，第一个日冕切片（#1）包含嗅球的后部方面和前叶的前侧。受精肿瘤在随后的3个冠状切片（切片#2、#3和#4）的右半球最为丰富，注射视线通常包含在冠状切片#3中。根据实验需要，该材料可用于肿瘤通道、冷冻组织部分、形式素固定石蜡嵌入组织部分、分离组织流动细胞测量、细胞培养或用于DNA、RNA或蛋白质分析的溶解酶。肿瘤的一部分可以低温保存，以满足未来的需要，细胞存活率在80-95%不等。
<ol><li>通过长期接触二氧化碳，然后进行宫颈错位，使小鼠安乐死。</li>
	<li>用一把较大的手术剪刀在大脑底部（前兆巨无霸水平）将安乐死的小鼠斩首，并将皮肤从切口向前拉，以完全暴露头骨。</li>
	<li>移除大脑。<ol>
<li>将一把细手术剪刀插入前体巨无霸的横向侧面，以达到左外听觉肉的水平，沿头骨左侧切割骨骼。在右侧执行相同的切割。</li>
			<li>沿着冠状平面将冠状骨从一个外部听觉肉切到另一个，并取出骨骼以暴露小脑。</li>
			<li>切开眼睛之间的鼻骨板。</li>
			<li>切下垂缝合线，沿下垂缝合线从鼻骨板切割到胸骨。通过从羊肉到布雷格玛的切口，沿着下垂缝合线完成中线切口。</li>
			<li>小心地将一对细钳的尖端沿两侧的下垂开口插入，将头骨的任何粘附物撕裂到大脑，然后横向剥去两个头骨的两半，以横向暴露大脑和嗅觉球茎。</li>
			<li>在嗅球的腹腔方面和头骨之间滑动钳子以释放任何粘附。</li>
			<li>将头骨向后倾斜，使大脑被缩回，使视神经和其他颅骨神经暴露在外。切断颅骨神经，将大脑释放到含有无菌PBS的盘子中。</li>
		</ol>
</li>
	<li>用称重铲将大脑转移到矩阵切片器中，用剃须刀叶片生成 2 毫米日冕切片。</li>
	<li>将日冕片放在含有无菌PBS的盘子中，以进行进一步的目视检查和进一步的组织处理。</li>
</ol>7. 肿瘤冷冻保存
<ol><li>准备冷冻保存介质的库存解决方案。<ol>
<li>添加 50 毫升增殖补充剂、5 毫升 100 倍青霉素和链霉素溶液，以及 450 μl 的 0.2% 肝素至 450 毫升基底介质。</li>
			<li>在 4 °C 下存储库存解决方案，防止光线照射。</li>
		</ol>
</li>
	<li>准备冷冻保存介质的工作解决方案。<ol>
<li>将 47.5 毫升低温保存介质和 2.5 毫升无菌 DMSO 混合在 50 毫升聚丙烯锥管中，混合均好。</li>
			<li>Aliquot 1.0 毫升的工作解决方案，以每个低温和存储在 4 °C 保护免受光。</li>
		</ol>
</li>
	<li>将一片2毫米的日冕状粒状大脑放入冰上含有冷冻保存介质的低温内。</li>
	<li>将小瓶紧紧盖住，放在-80°C的冷冻容器中。 第二天将低温气流转移到液氮罐中，以进行更长的储存。</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=50+years+of+preclinical+anticancer+drug+screening:+empirical+to+target-driven+approaches.">50 years of preclinical anticancer drug screening: empirical to target-driven approaches.</a> Clin. Cancer Res. 11, 971-981 (2005).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor+xenografts+as+predictive+preclinical+models+for+anticancer+drug+activity+in+humans:+better+than+commonly+perceived-but+they+can+be+improved.">Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved.</a> Cancer Biol. Ther. 2, 134-139 (2003).</li><li>Park, C. Y., Tseng, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell-directed+therapies:+recent+data+from+the+laboratory+and+clinic.">Cancer stem cell-directed therapies: recent data from the laboratory and clinic.</a> Mol. Ther. 17, 219-230 (2009).</li><li>Carlson, B. L., Pokorny, J. L., Schroeder, M. A., Sarkaria, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment,+maintenance+and+in+vitro+and+in+vivo+applications+of+primary+human+glioblastoma+multiforme+(GBM)+xenograft+models+for+translational+biology+studies+and+drug+discovery.">Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.</a> Curr. Protoc. Pharmacol. Chapter. 14, (2011).</li><li>Hambardzumyan, D., Parada, L. F., Holland, E. C., Charest, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modeling+of+gliomas+in+mice:+new+tools+to+tackle+old+problems.">Genetic modeling of gliomas in mice: new tools to tackle old problems.</a> Glia. 59, 1155-1168 (2011).</li><li>Sarangi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+inhibition+of+the+Hedgehog+pathway+in+established+malignant+glioma+xenografts+enhances+survival.">Targeted inhibition of the Hedgehog pathway in established malignant glioma xenografts enhances survival.</a> Oncogene. 28, 3468-3476 (2009).</li><li>Valadez, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Hedgehog+pathway+responsive+glioblastomas+by+isocitrate+dehydrogenase+mutation.">Identification of Hedgehog pathway responsive glioblastomas by isocitrate dehydrogenase mutation.</a> Cancer Lett. 328, 297-306 (2013).</li><li>Bar, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclopamine-mediated+hedgehog+pathway+inhibition+depletes+stem-like+cancer+cells+in+glioblastoma.">Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma.</a> Stem Cells. 25, 2524-2533 (2007).</li><li>Ehtesham, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-dependent+activation+of+the+hedgehog+pathway+in+glioma+progenitor+cells.">Ligand-dependent activation of the hedgehog pathway in glioma progenitor cells.</a> Oncogene. 26, 5752-5761 (2007).</li><li>Kelly, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglioma+cell+lines+containing+t(1;19)(q10;p10.">Oligodendroglioma cell lines containing t(1;19)(q10;p10.</a> Neuro-oncology. 12, 745-755 (2010).</li><li>Quintana, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+tumour+formation+by+single+human+melanoma+cells.">Efficient tumour formation by single human melanoma cells.</a> Nature. 456, 593-598 (2008).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R+gamma+null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.</a> J. Immunol. 174, 6477-6489 (2005).</li><li>Singh, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li></ol>

作者宣称他们没有相互竞争的经济利益。

培养细胞系、异种移植和转基因小鼠是模拟胶质瘤的最常见方法，每个模型系统3，13，14都有明显的益处和局限性。原位正交胶质瘤异种移植的相关好处包括渗透性生长，这些生长是弥漫性胶质瘤的典型代表，以及保留遗传改变和重要的信号机制，这些机制在培养的胶质瘤细胞中可能极其难以维持。例如，isocitrate脱氢酶突变和声波刺猪配体生产可以维持在原位矫形异种格拉夫特...

恶性胶质瘤是发生在大脑和脊髓的中枢神经系统的原发性胶质肿瘤。胶质瘤由世界卫生组织（世卫组织）根据组织学上与星形细胞、寡头细胞或子细胞的相似性进行分类，然后根据恶性病理特征进行数值分级（I至IV）。最常见的组织学亚型是天体细胞瘤、寡头性腺胶质瘤和混合寡头肌细胞瘤。包括世卫组织二至四级在内的恶性胶质瘤的特点是侵入性生长和对现有疗法的顽固不化。在美国，每年约有15，750人被诊断为恶性胶质瘤，估计有12，740名患者死于这种疾病。这些统计数字突出了恶性胶质瘤的特别致命性，以及提高疗效的重要需要。
癌症模型对于研究肿瘤生物学和疗法至关重要。人类癌细胞系是体外操纵和体外异种移植研究（继发性异种移植）1的重要第一步。然而，标准癌细胞培养经历表型和基因型转换2-4，可能无法恢复在继发性异种移植5。此外，基因改变，如异辛酸盐脱氢酶（IDH）突变6，独特的干细胞种群7和对关键信号通路8的依赖，可能会在癌细胞培养中消失。基因组特征可以在癌症领域培养中更好地保持，但仍不能完全反映原肿瘤的基因型2，3。直接正交移植否定了体外培养的影响，提供了适当的微环境，并保留了肿瘤启动细胞9，10的完整性。因此，原生异种移植物是一种强大而相关的临床前模型，用于严格测试靶向剂，以帮助合理设计未来的临床试验5，11，12。

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:211px;">
			Phosphate buffered saline
			</td>
			<td style="width:142px;">
			Life Technologies
			</td>
			<td style="width:140px;">
			14040-133
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Papain dissociation system
			</td>
			<td style="width:142px;">
			Worthington Biochemical Corp.
			</td>
			<td style="width:140px;">
			LK003150
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Trypan blue solution 0.4%
			</td>
			<td style="width:142px;">
			Life Technologies
			</td>
			<td style="width:140px;">
			15250061
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Ketamine HCl
			</td>
			<td rowspan="4" style="width:142px;">
			Obtained from institutional pharmacy or local veterinary supply company
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Xylazine HCl
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Ketoprofen
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Ophthalmic ointment
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Povidone-iodine
			</td>
			<td style="width:142px;">
			Fisher Scientific
			</td>
			<td style="width:140px;">
			190061617
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Cryopreservation medium and proliferation supplement
			</td>
			<td style="width:142px;">
			StemCell Technologies
			</td>
			<td style="width:140px;">
			05751
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			0.2% Heparin sodium salt in PBS
			</td>
			<td style="width:142px;">
			StemCell Technologies
			</td>
			<td style="width:140px;">
			07980
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Penicillin-streptomycin
			</td>
			<td style="width:142px;">
			Life Technologies
			</td>
			<td style="width:140px;">
			15140-122
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Dimethyl sulfoxide
			</td>
			<td style="width:142px;">
			Sigma-Aldrich
			</td>
			<td style="width:140px;">
			D6250-5X10ML
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			NOD.Cg-Prkdcscid I/2rgtm1Wjl/SzJ mice
			</td>
			<td style="width:142px;">
			The Jackson Laboratory
			</td>
			<td style="width:140px;">
			005557
			</td>
			<td style="width:97px;">
			NSG mice
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Anti-human vimentin antibody
			</td>
			<td style="width:142px;">
			Dako
			</td>
			<td style="width:140px;">
			M7020
			</td>
			<td style="width:97px;">
			Use 1:200 to 1:800
			</td>
		</tr>
		<tr>
			<td style="width:211px;">
			Anti-human IDH1 R132H antibody
			</td>
			<td style="width:142px;">
			Dianova
			</td>
			<td style="width:140px;">
			DIA-H09
			</td>
			<td style="width:97px;">
			Use 1:100 to 1:400
			</td>
		</tr>
	</tbody>
</table>




<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:210px;">
			Material 
			</td>
			<td style="width:140px;">
			Company
			</td>
			<td style="width:143px;">
			Catalogue Number
			</td>
			<td style="width:97px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Centrifuge with swinging bucket rotor
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:143px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Pipetter with dispensing speed control
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:143px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Disposable hemocytometer
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			22-600-100
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Sterile surgical gloves
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			11-388128
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Disposable gown
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			18-567
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Surgical mask
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			19-120-1256
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Tuberculin syringe
			</td>
			<td style="width:140px;">
			BD
			</td>
			<td style="width:143px;">
			305620
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Alcohol pads
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			22-246-073
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Portable electronic scale
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			01-919-33
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Zoom stereomicroscope
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:143px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Surgical clipper
			</td>
			<td style="width:140px;">
			Stoelting
			</td>
			<td style="width:143px;">
			51465
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Scalpel handle
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			10003-12
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Scalpel blades, #10
			</td>
			<td style="width:140px;">
			
			</td>
			<td style="width:143px;">
			
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Stereotaxic instrument
			</td>
			<td style="width:140px;">
			Stoelting
			</td>
			<td style="width:143px;">
			51730
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			High-speed drill
			</td>
			<td style="width:140px;">
			Stoelting
			</td>
			<td style="width:143px;">
			51449
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">Drill bit, 0.6 mm</td>
			<td style="width:140px;">Stoelting</td>
			<td style="width:143px;">514552</td>
			<td style="width:97px;"></td>
		</tr>
		<tr>
			<td style="width:210px;">
			Hamilton syringe
			</td>
			<td style="width:140px;">
			Hamilton
			</td>
			<td style="width:143px;">
			80336
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Autoclip, 9 mm
			</td>
			<td style="width:140px;">
			BD
			</td>
			<td style="width:143px;">
			427630
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Circulating water warming pad
			</td>
			<td style="width:140px;">
			Kent Scientific
			</td>
			<td style="width:143px;">
			TP-700
			TP-1215EA
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Hot bead dry sterilizer
			</td>
			<td style="width:140px;">
			Kent Scientific
			</td>
			<td style="width:143px;">
			INS300850
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Surgical scissors
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			14101-14
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Fine scissors
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			14094-11
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Spring scissors
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			15018-10
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Dumont forceps
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			11251-30
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Semimicro spatulas
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			14374
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Mouse brain slicer matrix
			</td>
			<td style="width:140px;">
			Zivic Instruments
			</td>
			<td style="width:143px;">
			BSMAS002-1
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			Fisher Scientific
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			12-567-501
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primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase i mutation

恶性胶质瘤是一组具有明显临床和分子特征的高度渗透性胶质肿瘤的异质性群体。原位矫形异种格拉夫特在前科动物模型中重新概括恶性胶质瘤亚型的组织病理学和分子特征。为了模拟世卫组织在移植检测中的I级和IV级恶性胶质瘤，人类肿瘤细胞被异种移植到免疫功能低下小鼠的正交部位——大脑中。与利用培养肿瘤细胞的继发性异种移植物相比，人类胶质瘤细胞与被切除的标本分离，在组织培养中无需事先通过移植即可产生原发性异种移植。本报告中的程序详细说明了肿瘤样本准备、颅内移植到免疫功能低下的小鼠、肿瘤移植监测以及肿瘤收获，供随后进入受体动物或进行分析。肿瘤细胞准备需要2小时，外科手术需要20分钟/动物。

分离的胶质瘤细胞被直接移植到免疫功能低下的小鼠的大脑中，以获得原位正畸异种移植线。每个肿瘤标本在移植前被分配一个随机数字，作为去除受保护的健康信息的分离过程的一部分。为此，我们使用 5 位 REDcap 数据库编号。 图1 说明了从胶质母细胞瘤（GBM 17182）与同心酸脱氢酶1（IDH1）突变精氨酸132到组织丁（R132H）建立异种移植线的过程和命名。移植后，肿瘤识别号中添加"X"?...

Watch this Scientific Journal Video about Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation at JoVE.com

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

恶性胶质瘤是一组具有明显临床和分子特征的高度渗透性胶质肿瘤的异质性群体。原位矫形异种格拉夫特在前科动物模型中重新概括恶性胶质瘤亚型的组织病理学和分子特征。

原正畸胶质血异草回顾渗透生长和同源脱氢酶 I 突变

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

primary-orthotopic-glioma-xenografts-recapitulate-infiltrative-growth

Research

JoVE Journal

Medicine

7.9K 次观看.  Vanderbilt University Medical Center. 恶性胶质瘤是一组具有明显临床和分子特征的高度渗透性胶质肿瘤的异质性群体。原位矫形异种格拉夫特在前科动物模型中重新概括恶性胶质瘤亚型的组织病理学和分子特征。

视频: Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

科研

医学

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

大鼠原位小肠移植

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

大鼠原位肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

成像神经胶质瘤启动<em在体内</em&gt;通过抛光和增强的薄膜头骨的颅窗

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.