这项工作致力于莫里茨·科赫教授的记忆。

这里提出的动物实验由机构和政府动物护理和使用委员会进行独立审查和批准，并根据实验动物科学协会联合会（FELASA）指导进行。采取一切可能的措施，尽量减少包括麻醉和镇痛在内的痛苦，或必要时提早安乐死。 局部肿瘤感染通过外科手术感染<ol><li> 手术动物的准备 注意:几乎任何条件（"floxed"）突变可以通过这里描述的方法诱导。推荐在大肠癌如Apc，Kras或Tp53相关基因中使用突变。 cre重组的效率取决于要切除的构建体的大小。大量的花粉序列的排除效率较低。所有等位基因的重组应通过PC在肿瘤中得到证实R. <ol><li>对于结肠直肠肿瘤的发展，使用以下条件等位基因的交叉进行CRC的基本模型（括号中的MGI数据库号）: Apc tm2Rak （MGI:3688435） 16 Kras tm4Tyj （MGI:2429948） 17 Tp53 tm2Tyj （MGI:3039263） 18 </li><li>如果需要荧光报告基因等位基因（ 例如 ，检测微转移），请使用以下等位基因: Gt（ROSA）26Sor tm6（CAG-ZsGreen1）Hze （MGI:3809522） 19 注意:所有上述菌株均可通过NCI Mouse Repository或Jackson Laboratory获得。不需要空腹，因为所有剩余的粪便可以在腺病毒感染之前被冲洗掉。术前禁食导致较高的围手术期死亡率，并对小型啮齿动物造成巨大压力。 </li><li>使用3〜3.5体积％的七氟烷进行全身麻醉。亏损t他的脚趾反射指示足够的麻醉。 </li><li>在第一次切开之前，皮下注射0.05 mg / kg的丁丙诺啡。 </li><li>用眼科软膏覆盖麻醉小鼠的眼睛，以避免角膜干燥。 </li><li>将小鼠放在小桌子的仰卧位置。使用非创伤性胶带来限制鼠标。 </li><li>用电动剃须刀剃除腹部（脱毛膏可以替代使用），并用酒精拭子或碘进行消毒。请使用制造商推荐的接触时间。 </li><li>用无菌盖子覆盖手术区域。 注意:围手术期抗生素的使用是可选的，并遵守机构准则。 </li><li>使用无菌单次使用或无菌仪器进行所有外科手术。 </li></ol></li><li> 中线剖腹术和结肠暴露 <ol><li>使用剪刀（可以替代使用手术刀）进行中线切口（〜15毫米）的下腹部皮肤。 </li><li>用镊子拿起腹壁肌肉组织，用剪刀仔细切开，打开腹腔。 </li><li>识别远端结肠，只用无创伤镊子触摸。用肛门近端约15毫米的精密夹具（ 例如 ，Micro Serrefine血管夹）夹住结肠。 注意:随时要特别注意冒号的脆弱性。穿孔不可避免地导致腹膜炎和败血症，需要动物的安乐死。 </li></ol></li><li> 用Adeno-cre病毒分段结肠感染 <ol><li>插入灵活的特氟隆管，经过狭窄并小心推进，直至达到预先放置在距离肛门边15mm处的夹具达到的管腔闭塞。不要使用过量的力，因为这可能会导致穿孔。 </li><li>用30G套管插管，连接标准的1 mL注射器并冲洗colon用生理盐水排出剩余的粪便物质。这可能需要几mL盐水。 </li><li>一旦远端结肠清空，就取出管子并用新鲜的特氟龙管替代，并将其直接如上所述位于夹具远端。 </li><li>使用第二个夹子将结肠置于距离近端夹具远端3 mm处（ 即 ，插入管中，距离肛门边缘约12 mm），导致3 mm的待分离的段被感染。 注意:对于远端闭塞部分，Fogarty冠状动脉夹片已被证明是最适合的，因为它们是橡胶化的，导致结肠紧闭，尽管夹子分支之间的管腔内管。 </li><li>使用第二个注射器（标准1 mL，30G插管）小心地将50 - 80μL的0.25％胰蛋白酶-EDTA注入到夹住的结肠段，并孵育10分钟。将插管和注射器连接到特氟龙管上，以防止流体泄漏。 <li>首先取下远端夹具，然后取出胰蛋白酶管。 </li><li>用约500μL生理盐水冲洗远端结肠，以除去剩余的胰蛋白酶。 </li><li>插入新的特氟隆管，将远端夹钳放回原位，并用50 - 80μL腺病毒溶液（10 11噬菌斑形成单位（PFU）/ mL，在磷酸盐缓冲盐水中）充满结肠段，并孵育30分钟图2A ）。 注意:不要溢出病毒溶液，因为与腺苷接触可能会导致有条件突变小鼠的任何组织中的肿瘤发展。 </li><li>拆下夹子和管子。 </li></ol></li><li> 腹部闭合和术后恢复 <ol><li>关闭腹壁6-0快速吸收跑步su（ 例如 ，聚二恶烷酮（PDS））。 </li><li>用手术伤口夹闭合皮肤。 </li><li>将鼠标置于38°C的加热垫上，直至从麻醉完全恢复。 </li><li>在手术后12小时再次施用0.05 mg / kg丁丙诺啡，然后每隔12 h再次使用丁丙诺啡，如有需要， </li><li>每天至少监测小鼠一次，因肿瘤生长引起的痛苦征兆。 </li></ol></li></ol>结肠镜检查注意:根据所使用的条件突变，腺病毒感染在2-4周内导致内窥镜可见的肿瘤。因此，在腺病毒诱导后2周进行第一次术后结肠镜检查，每2周重复一次。推荐用于小肠结肠镜检查的市售系统。 20 <ol><li> 用于结肠镜检查的动物的制备 注意:不需要空腹编辑。剩余的粪便通常在远端结肠中形成良好，并且可以在结肠镜检查期间被推出超过肿瘤，从而使不必要的反复禁食的紧张过程。 <ol><li>使用3〜3.5体积％的七氟烷进行全身麻醉。脚趾反射的丧失表示足够的麻醉。 </li><li>用眼科软膏覆盖麻醉小鼠的眼睛，以避免角膜干燥。 </li><li>将小鼠在仰卧位上放在小桌子上。 </li></ol></li><li> 结肠镜检查 <ol><li>将范围（直径1.9毫米，长度10厘米）通过肛门插入肠道，并在视觉控制下小心地吹入空气以扩大结肠。不要比检查要求更多的空气。 注意:对于空气吹入，可以使用结肠镜检查系统的防雾气泵。如果没有防雾气泵可用，则可以使用任何其他具有非常低压力设置的气泵柔软的空气与精致的减压阀。二氧化碳（CO 2 ）容易导致小型啮齿动物酸中毒，因此必须避免。 </li><li>仔细推动范围，直到远端结肠的粘膜病变可以被识别（ 图1A-1D ）。 </li><li>保存内窥镜图像供以后评估。之前已经描述了用于管腔内肿瘤的内窥镜评分系统。 20 </li><li>仔细取出范围，将鼠标置于38°C的加热垫上，直到其从麻醉中完全恢复。 </li></ol></li></ol>

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作者没有什么可以披露的。

虽然它们通常易于产生和维持，但基于细胞系注射的经典CRC小鼠模型是人为的，并且不能完全重现人类疾病。因此，GEMM已经开发。第一个CRC GEMM是Apc Min小鼠，其在Apc基因中携带杂合的无效突变，因此模拟人类遗传性家族性腺瘤性息肉病（FAP）。然而，Apc Min小鼠总是发展出不限于结肠的多个肠腺瘤;此外，腺瘤形成的时间和确切位置是随机的，并且当腺瘤可以进展之前，随着小鼠死于良性肿瘤负荷，肿瘤很少发展成恶性病变。因此，Apc Min鼠标是FAP的模型，不是偶发性CRC。其他模型存在DNA错配修复基因突变。 22，23虽然其中有些小鼠是遗传性胃肠癌的优秀模型，它们不代表散发性结肠直肠癌。 除了潜在的遗传改变之外，遗传病变的位置是分开散发性和综合征性结肠直肠癌的模型。所有上述模型均具有在整个结肠或甚至整个胃肠道中组成型活性或诱导的突变。这导致多个腺瘤的形成，其不代表人类散发性CRC，几乎不能通过结肠镜检查来评估，另外通常在动物死于广泛的肿瘤负荷之前通常不会留下足够的时间来开发肿瘤。因此，散发性结肠直肠癌的小鼠模型必须纳入结肠直肠肿瘤发生的局部激活。 在本文提出的GEMM中产生的肿瘤严格限于手术夹紧和腺病毒感染的部分的结肠。这导致单一肿瘤的形成，其通过结肠镜检查易于检测和监视。此外，远端位置使肠梗阻晚期肿瘤生长并发症，允许肿瘤发展成侵入性，并且根据基因型，转移性癌在动物需要安乐死之前。节段性感染的另一个优点是不受影响的环境。在Apc Min和其他遗传性小鼠模型中，周围粘膜甚至基质细胞与肿瘤具有相同的遗传畸变，这里提出的模型中的肿瘤在正常结肠上皮和基质内发展。这使得更实际的肿瘤 - 宿主相互作用和分子研究没有限制。此外，该模型中肿瘤诱导的时间点是明确的。在Apc Min小鼠中，肿瘤在"第二次击中"后发展，其间第二个Apc等位基因是随机的在随机时间点丢失，所有所需的突变在这里描述的模型中同时被激活，从而允许以非常明确的方式研究非常早期的病变或连续的活检。 然而，这种模式也有限制。将多个等位基因交叉成一个模型需要巨大的时间和资源;此外，由于不合适的基因型，通常并不是所有的后代都可以用于实验。程序本身需要实践并且耗时。产生具有足够高滴度和大量的腺病毒悬浮液是成本密集型的​​。因此，该模型不适合替代所有其他CRC鼠标模型，并且将限于实验室，其重点在于GEM模型。 协议本身在技术上要求很高，但一些非手术人员的培训可以适当地执行程序。腺瘤过程中的关键步骤不正确的是夹紧肠段的膨胀 - 过多的腔内压力导致穿孔，压力不足降低了成功感染的发生率。这一步需要培训。 Hung et al已经出版了远端结肠腺病毒感染的方案。之前。这里介绍的协议与上述协议在几个关键方面不同。由于它经常导致未经治疗的手中的肠穿孔，所以在本方案中跳过了腺病毒孵育之前的粘膜的机械磨损。这导致肿瘤形成率降低，然而可以通过增加病毒滴度来抵消。这样，间质肿瘤的发生率也可以减少，因为没有粘膜磨损，肠壁内较少的间充质细胞暴露于腺泡。 此外，与上述协议13相反，这里描述的方案不推荐在手术或结肠镜检查前过夜禁食。相反，冲洗肠道，或者在结肠镜检查的情况下，使用简单的机械操作来去除剩余的粪便物质。协议的这种改进大大提高了动物福利。隔夜禁食是对小型啮齿动物的高度压力的程序，已知其对鼠代谢有很强的影响，因此也影响实验结果。 24,25在这里提供的手稿中可以避免过夜禁食的这些不必要的影响。 Hung等人提出的方案和协议之间的另一个重要区别是被夹紧（从而被感染）段的长度。虽然这里描述的方案建议短的长度（〜3毫米），Hung et al。推荐20毫米的远端结肠被感染。已经选择较短的段长度来减少数量的重组隐窝，从而导致肿瘤。由于绝大多数患有散发性CRC的患者发生单个肿瘤而不是多发性肿瘤，因此该方法增加了该模型的临床相关性。肿瘤形成率似乎不受感染结肠表面减少的影响。 该技术的未来应用包括在结肠粘膜内需要局部复发的所有方案。癌症研究中的大多数应用将包括条件致癌基因，导致结肠直肠肿瘤的形成;然而，也可以使用本文提出的方案来实现所有其它应用，例如在结肠粘膜内诱导报道构建体。 总之，这里提出了高度复杂的基因工程小肠结肠直肠癌模型的结合以及用于检测和监测发展中的肿瘤的高分辨率结肠镜检查的选择，是研究CRC的生物学和治疗的一个很好的环境。

结肠直肠癌（CRC）仍然是西方国家与癌症相关死亡的主要原因之一。 1尽管早期疾病患者的预后良好，但是在后期阶段诊断出许多肿瘤，尽管有许多治疗方案，预后有限。 2，3，4，5 目前大多数CRC的小鼠模型都是基于将衍生自细胞系或患者肿瘤的肿瘤细胞植入免疫缺陷小鼠。 6,7,8这导致局部，并且取决于注射部位和用于注射的肿瘤细胞，有时是转移性肿瘤。 9，10然而，所得到的异种移植模型具有巨大的作用r限制。它们必须建立在免疫缺陷小鼠中，从而消除肿瘤与宿主免疫系统之间的复杂相互作用。另外，由于肿瘤基质来自宿主细胞，所以人类肿瘤实质与鼠基质之间的相互作用是有缺陷的，因此不能代表疾病。这些缺陷可以通过使用注射用鼠细胞系来避免。然而，只有少数鼠CRC细胞系可用，并且与大多数可用的人类CRC细胞系相似，是单克隆和高度间变性的。总之，目前大多数目前可用的CRC小鼠模型是高度人为的，并不完全代表人类疾病。 CRC的遗传工程小鼠模型（GEMM）可以避免这些缺点，因为它们具有通过诱导结肠中CRC的关键突变而产生的真正的小鼠肿瘤。 12，13 ，这可以通过在结肠直肠粘膜中通过cre重组酶激活条件（floxed）种系突变来实现。在许多其他肿瘤实体的GEMM中，使用由组织特异性启动子驱动的种系（诱导型）cre表达，种系cre不能用于结肠，因为这导致整个结肠中的大量腺瘤通过良性肿瘤负荷导致死亡很年轻因此，在这里描述的模型中，使用表达cre的腺病毒载体来感染短的结肠段。这导致在研究者定义的时间点诱导该粘膜部分的肿瘤发生，导致腺瘤最终进展到侵袭性和转移性癌。肿瘤是真正的小鼠肿瘤，在完整的微环境中生长，因此能够模拟包括肿瘤 - 宿主相互作用和转移性级联在内的整体结直肠癌发生。这个模型是因此是癌症生物学和临床前治疗试验研究的有吸引力的平台。 CRC遗传工程小鼠模型的一个主要缺点是其技术复杂性。以前已经描述了使用直肠腺苷灌注剂的小鼠携带Floced Apc等位基因的局部妊娠然而，这种技术，肠肿瘤的发生率，多样性和位置可能是高度可变的。因此，开发了通过手术夹紧待诱导的片段限制腺病毒感染的技术。 13我们修改了这一程序，以改善动物福利，并减少死亡率和肿瘤数量。有了这个协议，所有具有小型啮齿动物手术经验的实验室都应该能够复制模型并产生高度可重复性且容易接近结肠镜检查的肿瘤。取决于条件m用于肿瘤发生的术语，腺瘤的全谱，侵袭性癌和转移灶可以观察到。由于肿瘤位于远端结肠，因此在该模型中可以很容易进行连续内镜评估。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents / consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Life Technologies GmbH</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%, Phenol-Red)</td>
			<td>Life Technologies GmbH</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline 0.9% (E154)</td>
			<td>Serumwerk Bernburg AG</td>
			<td>10013</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua ad injectabilia</td>
			<td>B. Braun Melsungen AG</td>
			<td>235144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ad5CMV-Cre (adenovirus, c = 2E+11 PFU/mL)</td>
			<td>Gene Transfer Vector Core 
			University of Iowa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL, 50 mL centrifuge tubes</td>
			<td>Greiner Bio-One GmbH</td>
			<td>188271/227270</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes 1.5 mL/ 2 mL</td>
			<td>Sarstedt AG &#38; Co.</td>
			<td>72,695,400</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish PS 100/15 mm (sterile, Nuclon)</td>
			<td>Fisher Scientific GmbH</td>
			<td>10508921/ NUNC150350</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe (without dead volume) - Injekt-F SOLO</td>
			<td>Braun/neoLab</td>
			<td>194291661</td>
			<td></td>
		</tr>
		<tr>
			<td>30G injection needle</td>
			<td>BECTON DICKINSON</td>
			<td>304000</td>
			<td></td>
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			<td>Name</td>
			<td>Company</td>
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			<td>Analgesia / anesthesia</td>
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			<td>Sevoflurane (Sevoflurane AbbVie)</td>
			<td>AbbVie Germany GmbH &#38; Co. KG</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical oxygen</td>
			<td>Air Liquide Medical GmbH</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>Indivior Eu Ltd.</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthen - ophthalmic ointment</td>
			<td>Bayer Vital GmbH</td>
			<td>10047757</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Research Anesthesia Machine x/O2 Flush w/ Sevoflurane Vaporizer</td>
			<td>Parkland Scientific</td>
			<td>V3000PS/PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>Surgical Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose swabs</td>
			<td>Lohmann &#38; Rauscher Deutschland</td>
			<td>13356</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe EMG 1 mL (with 30G cannula)</td>
			<td>B. Braun Melsungen AG</td>
			<td>9161627S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Bore Tubing (bore: 0.28 mm/ diameter: 0.61mm)</td>
			<td>Smiths Medical Deutschland</td>
			<td>800/100/100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissor - ToughCut</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoClip Kit</td>
			<td>Fine Science Tools</td>
			<td>12020-00</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS Z1012H 6/0 C1 (surgical suture)</td>
			<td>Johnson &#38; Johnson Medical GmbH</td>
			<td>Z1012H</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Micro Serrefine Vascular Clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Fogarty Spring Clips</td>
			<td>Edwards</td>
			<td>CDSAFE 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot Plate 062</td>
			<td>Labotect</td>
			<td>13854</td>
			<td></td>
		</tr>
		<tr>
			<td>Isis - Hair shaver</td>
			<td>Aesculap - Braun</td>
			<td>-</td>
			<td></td>
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		<tr>
			<td>Name</td>
			<td>Company</td>
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		<tr>
			<td>Colonoscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold Light Fountain XENON 175 SCB</td>
			<td>Karl Storz</td>
			<td>20132101-1</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>Fiber Optic Light Cable</td>
			<td>Karl Storz</td>
			<td>69495NL</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>TRICAM Three-Chip Camera Head</td>
			<td>Karl Storz</td>
			<td>20221030</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>TRICAM SLII Camera Control Unit</td>
			<td>Karl Storz</td>
			<td>20223011-1</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>15&#34; Flat Screen Monitor EndoVue</td>
			<td>Karl Storz</td>
			<td>9415NN</td>
			<td>Karl Storz Coloview System Mainz</td>
		</tr>
		<tr>
			<td>HOPKINS Straight Forward Telescope 
			diameter 1.9 mm; length 10 cm 
			autoclavable 
			fiber optic light transmission incorporated</td>
			<td>Karl Storz</td>
			<td>64301AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Protection and Examination Sheath</td>
			<td>Karl Storz</td>
			<td>61029C</td>
			<td></td>
		</tr>
	</tbody>
</table>

a genetically engineered mouse model of sporadic colorectal cancer

尽管具有易于适用性和成本效益的优点，但是基于肿瘤细胞注射的结直肠癌小鼠模型具有严重的局限性，并且不能准确模拟肿瘤生物学和肿瘤细胞传播。引入基因工程小鼠模型来克服这些局限性;然而，这种模型在技术上是苛刻的，特别是在大器官如结肠中仅需要单个肿瘤。 结果，开发了免疫活性的基因工程的结肠直肠癌小鼠模型，其发展高度均匀的肿瘤，并且可以用于肿瘤生物学研究以及治疗试验。肿瘤发展是通过复合条件突变小鼠中腺瘤病毒的远端结肠的手术，节段性感染开始的。可以通过结肠镜检查容易地检测和监测肿瘤。我们这里描述了节段性腺病毒感染的外科技术结肠， 通过高分辨率结肠镜检查监测肿瘤，并呈现所得的结肠直肠肿瘤。

如果充分进行，&gt; 85％的动物会发展成肿瘤。这里介绍的外科手术的死亡率&lt;5％，结肠镜检查的死亡率几乎不存在。在大多数小鼠中，检测到单个病变;在大约30％的2〜3个小腺瘤中，可以检测到肿瘤诱导后2-3周内通常融合到单个肿瘤。 所得肿瘤的表型和生物学行为高度依赖于动物的条件突变。 Apc的局部cre介导的敲除足以诱导肿瘤发生，最初导致腺瘤，其可以在腺病毒感染后2-4周检测，并且在12-16周内进展到侵袭性腺癌。可以通过添加条件致癌性Kras突变（Kras G12D）来加速该过程，所得到的肿瘤迅速进入侵袭性腺癌。在加入致癌Tp53 R172H后，肿瘤迅速进入侵袭性和转移性癌。生存强烈依赖于肿瘤的基因型;中位生存通常在肿瘤诱导后约80-200天。绝大多数病例的死亡原因是继发于肿瘤生长的大肠梗阻。 可以通过结肠镜检查（ 图1 ）容易地和反复地监测肿瘤，而不会对动物造成重大压力。 疾病表现与人类CRC相似。动物发展为远端结肠腺瘤和最终腺癌（ 图2B，2C，2F ）。根据小鼠的条件基因型，肿瘤也转移到腹膜（ 图2D ），肝脏（ 图2E ）和罕见<html>肺部（未显示）。如果使用cre记者等位基因（ 例如 ，ZSGreen 19 ），则可以容易地鉴定所有的肿瘤表现。 ZSGreen记者等位基因具有荧光蛋白表达，其明亮度足以在日光下可见（ 图1B ， 图2C ）。 组织学上，95％的发展中的肿瘤是腺癌。大约5％的肿瘤是间质来源的， 例如 ，纤维肉瘤或平滑肌肉瘤，可能是由已被意外感染腺病毒的基质细胞发育出来的。腺癌的组织形态学非常类似于人类CRC，其特征在于从非侵入性腺瘤到腺癌侵入周围结构的整个谱图（ 图3A-3C ）。 <html> files / ftp_upload / 55952 / 55952fig1.jpg"/&gt; 图1.结肠直肠肿瘤的结肠镜图像（括号中给定动物的条件等位基因）。 正常远端结肠。 B.腺病毒感染后2周早期腺瘤。注意由于这个小鼠中GFP报告基因等位基因导致的绿色。 （Apc tm2Rak ，Kras tm4Tyj ，Gt（ROSA） 26Sortm6（CAG-ZsGreen1）Hze ）。晚期腺瘤（Apc tm2Rak ，Kras tm4Tyj ）。 D.结肠直肠腺癌（通过结肠镜检查图像后的病理诊断; Apc tm2Rak ，Kras tm4Tyj ，Tp53 tm2Tyj ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55952/55952fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> /&gt; 图2. A.术中情况。注意底部有大橡胶的Fogarty夹子和用于腺泡注射的经导管插管（红色箭头）。 B.腺病毒感染后8周连续大肠梗阻的结肠直肠肿瘤（白色箭头）（Apc tm2Rak ，Kras tm4Tyj ，Tp53 tm2Tyj ）。 C.腺病毒感染后10周的结肠直肠肿瘤（白色箭头）（Apc tm2Rak ，Kras tm4Tyj ，Gt（ROSA） 26SorTm6（CAG-ZsGreen1）Hze ）。注意由于这种小鼠中绿色荧光蛋白（GFP）报告基因等位基因而导致的绿色。 D. GEMM中的腹膜癌（黑色箭头）（Apc tm2Rak ，Kras tm4Tyj ，Tp53 tm2Tyj ）。肝和肠已经被去除以暴露肾脏和隔膜。 E.腺体中12周后GEMM中肝转移灶感染（Apc tm2Rak ，Kras tm4Tyj ）。 F.从图2C所示的动物去除后，结肠肿瘤。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55952/55952fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55952/55952fig3.jpg" /> 图3.来自GEMM的结肠直肠肿瘤的苏木精/曙红（H / E）染色部分。 A.粘膜，粘膜下层和肌层粘膜浸润的正常粘膜到腺癌的过渡区（Apc tm2Rak ，Kras tm4Tyj ）。 B.正常结肠粘膜到侵袭性腺癌的过渡区（Apc tm2Rak ，Kras tm4Tyj ）。 C.高级腺癌与周围组织浸润（Apc tm2Rak，Kras tm4Tyj ，Tp53 tm2Tyj ）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55952/55952fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer at JoVE.com

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

遗传工程小鼠模型的散发性结肠直肠癌

提出了通过节段性腺病毒感染建立结肠直肠癌基因工程小鼠模型的方法及其通过高分辨率结肠镜检查进行的监测。

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

a-genetically-engineered-mouse-model-of-sporadic-colorectal-cancer

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.4K 次观看.  Technische Universität Dresden. 提出了通过节段性腺病毒感染建立结肠直肠癌基因工程小鼠模型的方法及其通过高分辨率结肠镜检查进行的监测。 

视频: A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

科研

癌症研究

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Establishing a Competing Risk Regression Nomogram Model for Survival Data

The Kaplan&#8211;Meier method and Cox proportional hazards regression model are the most common analyses in the survival framework. These are relatively easy to apply and interpret and can be depicted visually. However, when competing events (e.g., cardiovascular and cerebrovascular accidents, treatment-related deaths, traffic accidents) are present, the standard survival methods should be applied with caution, and real-world data cannot be correctly interpreted. It may be desirable to distinguish different kinds of events that may lead to the failure and treat them differently in the analysis. Here, the methods focus on using the competing regression model to identify significant prognostic factors or risk factors when competing events are present. Additionally, nomograms based on a proportional hazard regression model and a competing regression model are established to help clinicians make individual assessments and risk stratifications in order to explain the impact of controversial factors on prognosis.

Presented here is a protocol to build nomograms based on the Cox proportional hazards regression model and competing risk regression model. The competing method is a more rational method to apply when competing events are present in the survival analysis.

为生存数据建立竞争风险回归 Nomogram 模型

The Kaplan&#8211;Meier method and Cox proportional hazards regression model are the most common analyses in the survival framework. These are relatively ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

超高频超声成像分析黑色素瘤Braf/Pten基因工程小鼠模型中的淋巴结体积

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

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 ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

分子和免疫技术在胃肠道间质瘤基因工程小鼠模型中

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

An R-Based Landscape Validation of a Competing Risk Model

The Cox proportional hazard model is widely applied for survival analyses in clinical settings, but it is not able to cope with multiple survival outcomes. Different from the traditional Cox proportional hazard model, competing risk models consider the presence of competing events and their combination with a nomogram, a graphical calculating device, which is a useful tool for clinicians to conduct a precise prognostic prediction. In this study, we report a method for establishing the competing risk nomogram, that is, the evaluation of its discrimination (i.e., concordance index and area under the curve) and calibration (i.e., calibration curves) abilities, as well as the net benefit (i.e., decision curve analysis). In addition, internal validation using bootstrap resamples of the original dataset and external validation using an external dataset of the established competing risk nomogram were also performed to demonstrate its extrapolation ability. The competing risk nomogram should serve as a useful tool for clinicians to predict prognosis with the consideration of competing risks.

The present protocol describes codes in R for evaluating the discrimination and calibration abilities of a competing risk model, as well as codes for the internal and external validation of it.