这项工作得到了德国研究基金会（WE 3548 / 4-1）和Gandundheitswesen（1/14）的Roland-Ernst-Stiftung的支持。

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许可，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。 1.细胞和组织的制备注意:对于每次注射，使用20μL的体积，100,000个细胞。使用基底膜基质（BMM），以防止泄漏并确保标准化注射。为了确保可重复的结果，定期进行细胞系鉴定测定（ 例如 通过 STR分析）。 <ol><li> 从新鲜组织制备原代细胞悬浮液 注意:始终在无菌条件下工作。立即转让新鲜需要从手术室将手术室切除到实验室，以确保细胞的高活力。 患者的幸福和最佳治疗必须始终是首要任务。因此，用于研究的组织样本必须以不影响随后的切除肿瘤的病理后处理和分期的方式获得。在大多数情况下，由经过培训的病理学家获得的研究样本是合理的，以确保不干扰病理诊断。 <ol><li>从被切除的标本（理想情况下〜1cm 3 ）无菌除去后，立即将组织样品置于预先填充有20-30mL磷酸盐缓冲盐水（PBS）的50mL管内。将管存放在冰上，并立即转移到实验室。 </li><li>将组织放在培养皿中，用大量PBS洗两次，以清除剩余的血液。 </li><li>用sca将组织切成小块（〜2-4mm）lpel（ 例如 ，＃20或＃36刀片）。 </li><li>根据制造商的说明使用解离器，以进一步将肿瘤组织解离成单细胞悬浮液。或者，使用酶肿瘤消化的其他方案。 </li><li>计算所得单细胞悬浮液的细胞（ 例如 ，在库尔特计数器或血细胞计数器中）。 </li><li>离心（1,500×g 5分钟），用PBS洗涤细胞悬浮液两次。 </li><li>以5×10 6个细胞/ mL的浓度将细胞重悬于BMM中，并将其保持在冰上。 </li></ol></li><li> 注射细胞系的制备 <ol><li>在标准培养条件（37℃，5％CO 2 ）下生长所有结肠直肠癌细胞系，并在手术当天进行准备。 </li><li>根据标准细胞培养方案收获细胞，计数细胞（ 例如 ，在库尔特计数器或血细胞计数器中）并计算需要量所有注射用量都取决于要注射的动物数量。 </li><li>准备3-5倍的实际需要的体积来解释注射器的移液损失和死体积。 </li><li>离心（1,500g 5分钟），用PBS洗涤细胞悬浮液两次。 </li><li>以5×10 6个细胞/ mL的浓度将细胞重悬于BMM中，并将其保持在冰上。 </li></ol></li><li> 有机体制剂 注意:所有3D有机体培养物在标准培养条件（37℃，5％CO 2 ）下生长。有机体培养的详细方案已经公布。 18，19，20，以常规的细胞系类似的，我们建议20μLBMM的每注射100,000个细胞的体积。有机体在手术当天如下: <ol><li>准备以下介质（以下简称）作为DMEM / F12 +++）:高级Dulbecco&#39;s改良的Eagle&#39;s Medium（DMEM）/ F12 + 1％HEPES（1M）+ 1％谷氨酰胺（200mM）+ 1％青霉素/链霉素。 </li><li>预先加入1 mL DMEM / F12 +++的15 mL管。 </li><li>仔细吸取培养板表面的有机物，将3至5孔的内容物转移到15 mL管中。 </li><li>通过用扩大的玻璃移液管将它们上下移动，小心地将有机体分解成更小的碎片。 </li><li>向管中加入5 mL DMEM / F12 +++，然后以1,000 xg离心5 min。 </li><li>离心后，取出上清液，将沉淀物重新悬浮在600μL酶解离缓冲液中，并将悬浮液转移到6孔板的一个孔中。 </li><li>在37°C孵育1-5分钟，然后小心地将悬浮液上下移动以溶解有机物。 注意:通过显微镜检查消化;当所有细胞簇已经解离时，它是完整的d到单细胞。 </li><li>消化完成后，加入1.4mL的DMEM / F12 +++以停止消化。然后将消化的有机体的所有孔转移到50mL管中。 </li><li>计算细胞并计算所有注射所需的量。 </li><li>准备实际需要的体积的3 - 5倍来解释注射器的移液损失和死体积</li><li>离心（1,500×g 5分钟），用PBS洗涤细胞悬浮液两次。 </li><li>将细胞重悬于BMM至5×10 6个细胞/ mL的浓度并保持在冰上。 </li></ol></li></ol>原位鼠标模型<ol><li> 接受手术的准备 注:使用6-8周龄NOD.Cg-SCID PRKDC的II2rg tm1Wjl / SZJ（NOD SCID伽马，NSG）小鼠作为收件人。 21 NSG是免疫受损最小的小鼠之一，缺乏成熟的B，T和NK细胞以及多种其他免疫原fects。 21它们可靠地生长，即使低的细胞数注射的肿瘤和非常容易发生远处转移。 NSG小鼠是优良的育种者，可以保存在常规的无特定病原体（SPF）单位中。 <ol><li>在第一次切口之前，皮下注射0.05 mg / kg的丁丙诺啡。 </li><li>使用3-3.5体积％的七氟醚进行全身麻醉。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><li>用电动剃须刀剃除腹部（脱毛膏可以替代使用），并用至少3次氯仿/己酸/碘和70％酒精消毒。 </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></ol></li><li> 中线剖腹术和盲肠暴露 <ol><li>使用剪刀（可以替代使用手术刀），使下腹部的皮肤小中线切口（3-5毫米）。用镊子拿起腹壁肌肉组织，用剪刀小心切开，打开腹腔。 </li><li>用无创伤镊子识别和仔细地将盲肠外观化。将盲肠的盲端袋定位在腹部指向头部。 </li><li>一旦外观，始终保持盲肠湿润温暖的盐水拭子。 </li></ol></li><li> 原位注射，腹部闭合和术后恢复 <ol><li>对于脑内注射，使用标准的1 mL注射器和30 G插管。将该注射器安装在显微注射泵上，显微注射泵依次安装在显微操纵器上。 </li><li>用无创伤镊子仔细抓住盲肠的尖端，轻轻抚摸它，d用第二组镊子用温盐水润湿。 </li><li>将插管直接放置在盲肠上方。 </li><li>使用双目手术显微镜在视觉控制下执行以下步骤。 </li><li>在盲肠外观部分的两端用两个无创伤镊子小心地抓住盲肠，稍微拉伸，然后缓慢地将其拉过插管，并平行并直接位于上方。至关重要的是不要穿孔整个肠壁（从而将细胞注射到内腔中），也不要穿透超出初始渗透点的浆膜，因为这将导致渗漏和腹膜传播。 <ol><li>将肠朝向插管移动，而不是将肠向肠排出。握住并拉伸两镊子之间的肠。外科医生的手必须在表面上休息，以减少震颤。 </li></ol></li><li>注射细胞之间的浆膜（看起来非常薄，半透明的衬里上面e内壁血管）和肌层。因此，套管必须在视觉上放置在血管上方和薄薄的半透膜下方。 </li><li>使用脚踏开关来开始注射，以便在插管位于肠壁内时减少震颤。 </li><li>一旦套管就位，开始注射。使用20秒的持续时间，在泵的控制单元上进行1μL/ s的设置。 注意:注入或损伤血管附近导致直接血管内传播和远处转移，因此应避免。 </li><li>注射完成后，将其向后拉，仔细取出插管。 </li><li>将干拭子放在盲肠下方，然后用蒸馏水彻底冲洗盲肠，以便裂解泄漏的细胞，从而防止人造腹膜传播。 </li></ol></li><li> 腹部闭合和术后恢复 <ol><li>之后将盲肠轻轻地倒入腹腔。 </li><li>用6-0快速吸收的运行缝合线关闭腹壁。 </li><li>用手术伤口夹闭合皮肤。 </li><li>将鼠标放置在38°C的加热图上，直到从麻醉完全恢复。 </li><li>在48小时内仔细观察小鼠的术后状况。在遇险的情况下，每12小时用0.05mg / kg丁丙诺啡治疗。 </li><li>每天至少一次监测小鼠，因肿瘤生长而引起的痛苦征兆。 </li></ol></li></ol> 3.从全血样品中分离循环肿瘤细胞注意:经麻醉小鼠经皮心脏穿刺获得血液，然后进行安乐死。理想情况下，将1000μL血液吸入预先装有100μL抗凝血剂（乙二胺四乙酸（EDTA）或肝素）的注射器）。使用抗人EpCAM（上皮细胞粘附分子）抗体以鉴定CTC。如果在小鼠模型中使用人上皮细胞系，则这很好。对于其他器具，可能需要不同的抗体。 <ol><li> 四氯化碳浓缩: <ol><li>用5 mL密度​​梯度培养基预灌装15 mL管，并小心将血液转移到15 mL管中。 </li><li>离心机（300 ug，30分钟，无刹车），小心取出上清液。 </li><li>将其余部分倒入新的15 mL管中并离心（15 min，300 xg，带制动器）。 </li><li>恢复含有单核细胞的间期（丢弃其余部分），并用PBS洗涤细胞两次。 </li><li>将沉淀重悬于200μLPBS / 1％EDTA中，加入4μLEpCAM抗体。在黑暗中在冰上孵育20分钟。 </li></ol></li><li> 筛选和拣选 <ol><li>准备以下缓冲液（以下简称拣选缓冲液）:PBS + 10％胎牛血清（FCS）+ 1％青霉素/ S雷霉霉素（1％）+ 0.8％EDTA。 </li><li>使用PAP笔在6厘米无菌培养皿中绘制〜1厘米圆（防止流体分散在培养皿中），并将700μL采摘缓冲液吸入此圆圈。 </li><li>在圆圈内的700μL采摘缓冲液中加入50μL细胞悬浮液。用显微镜检查细胞的密度。如果样品过于密集，将样品分成不同的菜肴。 </li><li>等待细胞沉降（约5分钟）。 </li><li>筛选染色（= EpCAM阳性）细胞的细胞悬浮液滴。 </li><li>一旦发现细胞，根据预期的下游分析（ 例如 ，RNA提取缓冲液或培养基），用显微操纵器挑取细胞并将其置于50μL缓冲液中。 </li><li>根据预期的下游分析，将EpCAM阴性细胞和/或培养基分离为阴性对照。 </li><li>进行下游分析（ 如细胞培养，PCR）。 </li></ol></li></ol>

<ol>
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作者没有什么可以披露的。

尽管它们在皮下小鼠模型中具有临床前证实的活性，但大多数新型化合物在临床试验中失败，从未到达诊所。 11这明显皮下小鼠模型的功能不全，以精确地模拟肿瘤的生物学和生长模式导致基于喷射的肿瘤细胞直接导入原始器官原位小鼠模型的发展。 原位小鼠模型能够比皮下模型更准确地模拟实体瘤的生物学和传播。 15然而，主要的缺点?...

结肠直肠癌（CRC）是西方国家癌症死亡的最常见原因之一。 1原发性肿瘤经常可以切除，远处转移的发生显着恶化预后，并常常导致死亡。 2，3转移的生物学相关成分被循环肿瘤细胞（CTC），其从肿瘤分离，生存中循环，附着在上皮在靶器官，侵入器官，并最终长大到新的病灶。 4虽然CTC的已知是预后相关，5，6，7，8的，9其生物学仅部分地理解为在CRC其极端罕见的结果。 10 鼠标模型是一个强大的tool研究癌症生物学的各个方面。通过将肿瘤细胞皮下注射到受体小鼠中产生经典的皮下肿瘤模型，其可以是免疫活性的（如果使用同基因鼠肿瘤细胞）或免疫缺陷。皮下肿瘤模型价格低廉，数据量快;它们的终点肿瘤生长可以容易地和非侵入性地测量。然而，在这些模型中已经证明抗肿瘤活性的新化合物中有88％在临床试验中失败。 11这部分是由于人和小鼠之间间差异;然而，这种失败的很大一部分是由于皮下小鼠模型的预测值低。 因此，肿瘤细胞注射入原发器官并因此在其原始微环境中生长的原位小鼠模型因此越来越多地用于癌症研究。 11，12， </s达&gt; 13，14款原位不只是模拟局部肿瘤的生长条件;作为肿瘤生长的解剖学上正确的位点的结果，原位小鼠模型也允许转移的逼真的模拟，因此用于研究生物学CTC 8，15，16或它们的在CRC不同处理的响应。 13，17 原位小鼠模型的主要缺点是其技术复杂性。取决于注射细胞的器官，直到实验者能够诱导可再现肿瘤的学习曲线相当长。这尤其适用于结肠直肠癌模型，因为肿瘤细胞需要注射到肠壁中，这通常导致穿孔，肿瘤细胞渗漏或腔内肿瘤细胞损失。这是本文旨在描述从原代组织样品，2D细胞系和3D有机体培养物中注射细胞至小鼠盲肠的细胞制备方法。这里描述的技术导致高度均匀的肿瘤，并且取决于用于注射的细胞系的肿瘤生物学，在受体小鼠中可重复形成远处转移和CTC。 15

<table border="0" cellpadding="0" cellspacing="0" width="1294">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Cell culture Media and Components</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td style="width:457px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Advanced DMEM F12</td>
			<td style="width:297px;">Invitrogen</td>
			<td style="width:119px;">12634010</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HEPES (1 M)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15630056</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Glutamax-I Supplement (200 mM)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">35050038</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">DMEM</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">61965026</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TrypLE Express enzymatic dissociation buffer</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">12604021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Matrigel basement membrane matrix (BMM, phenol red free)</td>
			<td style="width:297px;">CORNING B.V. Life Sciences</td>
			<td style="width:119px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Trypsin-EDTA (0,25%, Phenol-Red)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">6-/48-well plates with lid</td>
			<td style="width:297px;">CORNING</td>
			<td style="width:119px;">3516/3548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 75cm&#178;, 250 mL</td>
			<td style="width:297px;">VWR International GmbH</td>
			<td style="width:119px;">734-2066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 150cm&#178;, 600 mL</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td style="width:119px;">355001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Eppendorf tubes 1,5 mL / 2 mL</td>
			<td style="width:297px;">Sarstedt AG &#38; Co.</td>
			<td style="width:119px;">72.706.400/ 72.695.400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">15 ml, 50 ml centrifuge tubes</td>
			<td style="width:297px;">Greiner-Bio-One GmbH</td>
			<td style="width:119px;">188271/227270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TC10 Counting Slides (for TC20 Counting Machine)</td>
			<td style="width:297px;">Bio-Rad Laboratories GmbH</td>
			<td style="width:119px;">1450016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Pasteur pipettes (glass, 150 mm)</td>
			<td style="width:297px;">Fisher Scientific GmbH</td>
			<td style="width:119px;">11546963/ FB50251</td>
			<td>thinly pulled by using a bunsen burner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS Dissociator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSmix Tube Rotator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C Tubes</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Tumor Dissociation Kit</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-095-929</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 70&#181;m Cell Strainer</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td>352350</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Surgical Equipment</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Sevoflurane</td>
			<td style="width:297px;">AbbVie Germany GmbH &#38; Co. KG</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Medical oxygen</td>
			<td style="width:297px;">Air Liquide Medical GmbH</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Buprenorphine</td>
			<td style="width:297px;">Temgesic</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Bepanthen - opthalmic ointment</td>
			<td style="width:297px;">Bayer Vital GmbH</td>
			<td style="width:119px;">10047757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Normal saline 0.9% (E154)</td>
			<td style="width:297px;">Serumwerk Bernburg AG</td>
			<td style="width:119px;">10013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Aqua ad injectabilia</td>
			<td style="width:297px;">Braun</td>
			<td style="width:119px;">235144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">1 mL Syringe (without dead volume) - Injekt-F SOLO</td>
			<td style="width:297px;">Braun/neoLab</td>
			<td style="width:119px;">194291661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">30G injection needle</td>
			<td style="width:297px;">BECTON DICKINSON</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cellulose swabs</td>
			<td style="width:297px;">Lohmann &#38; Rauscher Deutschland</td>
			<td style="width:119px;">13356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Micro-Adson Forceps</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">11018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Iris Scissor - ToughCut</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Olsen-Hegar Needle Holder</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">AutoClip Kit</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12020-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PDS Z1012H 6/0 C1 (surgical suture)</td>
			<td style="width:297px;">Johnson &#38; Johnson Medical GmbH</td>
			<td style="width:119px;">Z1012H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">V3000PS/PK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">UltraMicro Pump with Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">UMP3-4</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Footswitch for SYS-Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">15867</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Three-axis Manual Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M325</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Magnetic Stand for Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M10</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Steel Base Plate for M10 Magnetic Stand</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">5479</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Hot Plate 062</td>
			<td style="width:297px;">Labotect</td>
			<td style="width:119px;">13854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Isis - Hair shaver</td>
			<td style="width:297px;">AESCULAP - Braun</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Binocular Surgical Microscope</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">VS-2Z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">CTC isolation</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Density gradient medium &#8211; Ficoll</td>
			<td>StemCell - Lymphoprep</td>
			<td>7801</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4</td>
			<td>BioLegend</td>
			<td>324210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8</td>
			<td>BioLegend</td>
			<td style="width:119px;">118210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish, &#248; 60 x 15 mm, 21 cm&#178;, Vent</td>
			<td>Greiner bio-one</td>
			<td>628102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Cell Culture Microscope</td>
			<td style="width:297px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferman 4r Micromanipulator</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTram Air</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:457px;">aspiration pump connected to the micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dmz Universal Microelectrode Puller</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td>required for the manufacturing of micro capillaries for single cell aspiration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism Glass Capillaries</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PAP pen</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab2601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Immunohistochemistry</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Purified anti-human CD326 (EpCAM) antibody clone 9C4</td>
			<td>BioLegend</td>
			<td style="width:119px;">324201</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HRP rabbit anti-mouse IgG</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab97046</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
	</tbody>
</table>

isolation of circulating tumor cells in an orthotopic mouse model of colorectal cancer

尽管皮下小鼠模型具有易于适用性和成本效益的优点，但其严重限制并不能准确模拟肿瘤生物学和肿瘤细胞传播。引入原位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，特别是在诸如大肠的中空器官中。为了产生可靠地生长和转移的均匀的肿瘤，肿瘤细胞制备和注射的标准化技术至关重要。 我们已经开发了一种大肠癌（CRC）的原位小鼠模型，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。将来自原发性肿瘤，二维（2D）细胞系或3维（3D）有机体的肿瘤细胞注射到盲肠中，并且根据注射的肿瘤细胞的转移潜能形成高度转移性肿瘤。此外，定期发现反恐委员会。我们这里描述来自2D细胞系和3D组织以及原发性肿瘤组织的肿瘤细胞制备技术，手术和注射技术以及来自荷瘤小鼠的CTC的分离以及目前的故障提示。

在该模型中成功和可重复地产生结肠直肠肿瘤严重依赖于细胞的精确注射而没有溢出或渗漏。如果这样做，这种模式是非常可靠的，很少造成人造腹膜传播。肿瘤的生长动力学及其传播模式取决于所用组织和细胞的生物学特性。 15在本模型中，HCT116细胞可靠地转移到肝脏，SW620细胞形成原位肿瘤，但不转移。 15 <p class="jove_content" fo:kee...

Watch this Scientific Journal Video about Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer at JoVE.com

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

我们描述了通过将肿瘤细胞或有机体注入小鼠盲肠并随后从该模型中分离循环肿瘤细胞（CTC）来建立原位结肠直肠肿瘤。

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 ...

isolation-circulating-tumor-cells-an-orthotopic-mouse-model

Research

JoVE Journal

Cancer Research

9.9K 次观看.  Technische Universität Dresden. 我们描述了通过将肿瘤细胞或有机体注入小鼠盲肠并随后从该模型中分离循环肿瘤细胞（CTC）来建立原位结肠直肠肿瘤。 

视频: Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of 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

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

捕获和血液中的活循环肿瘤细胞释放

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

遗传工程小鼠模型的散发性结肠直肠癌

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

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

转移性结直肠癌患者循环微 rna 自定义面板的检测

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

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 ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

小鼠膀胱肿瘤器官的培养、操作和正交移植

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

毛尿胰腺腺腺腺癌超声引导正射植入

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...