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>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2016.">Cancer statistics, 2016.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li><li>Weitz, J., Koch, M., Debus, J., H&#246;hler, T., Galle, P. R., B&#252;chler, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer.">Colorectal cancer.</a> Lancet. 365 (9454), 153-165 (2005).</li><li>Sch&#246;lch, S., 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=Circulating+tumor+cells+of+colorectal+cancer.">Circulating tumor cells of colorectal cancer.</a> Cancer Cell Microenviron. 1 (5), (2014).</li><li>Steinert, G., Sch&#246;lch, S., Koch, M., Weitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+significance+of+circulating+and+disseminated+tumour+cells+in+colorectal+cancer.">Biology and significance of circulating and disseminated tumour cells in colorectal cancer.</a> Langenbecks Arch Surg. 397 (4), 535-542 (2012).</li><li>Bork, U., 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=Prognostic+relevance+of+minimal+residual+disease+in+colorectal+cancer.">Prognostic relevance of minimal residual disease in colorectal cancer.</a> World J Gastroenterol. 20 (30), 10296-10304 (2014).</li><li>Bork, U., 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=Circulating+tumour+cells+and+outcome+in+non-metastatic+colorectal+cancer:+a+prospective+study.">Circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study.</a> Br J Cancer. 112 (8), 1306-1313 (2015).</li><li>Rahbari, N. N., 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=Compartmental+differences+of+circulating+tumor+cells+in+colorectal+cancer.">Compartmental differences of circulating tumor cells in colorectal cancer.</a> Ann Surg Oncol. 19 (7), 2195-2202 (2012).</li><li>Rahbari, N. N., 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=Metastatic+Spread+Emerging+From+Liver+Metastases+of+Colorectal+Cancer:+Does+the+Seed+Leave+the+Soil+Again?.">Metastatic Spread Emerging From Liver Metastases of Colorectal Cancer: Does the Seed Leave the Soil Again?.</a> Ann Surg. 263 (2), 345-352 (2016).</li><li>Rahbari, N. N., 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=Meta-analysis+shows+that+detection+of+circulating+tumor+cells+indicates+poor+prognosis+in+patients+with+colorectal+cancer.">Meta-analysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer.</a> Gastroenterology. 138 (5), 1714-1726 (2010).</li><li>Steinert, 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=Immune+Escape+and+Survival+Mechanisms+in+Circulating+Tumor+Cells+of+Colorectal+Cancer.">Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer.</a> Cancer Res. 74 (6), 1694-1704 (2014).</li><li>Sharpless, N. E., Depinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mighty+mouse:+genetically+engineered+mouse+models+in+cancer+drug+development.">The mighty mouse: genetically engineered mouse models in cancer drug development.</a> Nat Rev Drug Discov. 5 (9), 741-754 (2006).</li><li>Roper, J., Hung, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priceless+GEMMs:+genetically+engineered+mouse+models+for+colorectal+cancer+drug+development.">Priceless GEMMs: genetically engineered mouse models for colorectal cancer drug development.</a> Trends Pharmacol Sci. 33 (8), 449-455 (2012).</li><li>Sch&#246;lch, S., 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=Radiotherapy+combined+with+TLR7/8+activation+induces+strong+immune+responses+against+gastrointestinal+tumors.">Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors.</a> Oncotarget. 6 (7), 4663-4676 (2015).</li><li>Sch&#246;lch, S., Rauber, C., Weitz, J., Koch, M., Huber, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+activation+and+ionizing+radiation+induce+strong+immune+responses+against+multiple+tumor+entities.">TLR activation and ionizing radiation induce strong immune responses against multiple tumor entities.</a> Oncoimmunology. 4 (11), e1042201 (2015).</li><li>Sch&#246;lch, S., 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=Circulating+tumor+cells+exhibit+stem+cell+characteristics+in+an+orthotopic+mouse+model+of+colorectal+cancer.">Circulating tumor cells exhibit stem cell characteristics in an orthotopic mouse model of colorectal cancer.</a> Oncotarget. 7 (19), 27232-27242 (2016).</li><li>Nanduri, L. K., Garc&#237;a, S., Weitz, J., Sch&#246;lch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Colorectal+Cancer-Derived+Circulating+Tumor+Cells.">Mouse Models of Colorectal Cancer-Derived Circulating Tumor Cells.</a> Med Chem (Los Angeles). 6 (7), 497-499 (2016).</li><li>van Noort, V., 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=Novel+Drug+Candidates+for+the+Treatment+of+Metastatic+Colorectal+Cancer+through+Global+Inverse+Gene-Expression+Profiling.">Novel Drug Candidates for the Treatment of Metastatic Colorectal Cancer through Global Inverse Gene-Expression Profiling.</a> Cancer Res. 74 (20), 5690-5699 (2014).</li><li>Sato, T., 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Boj, S. F., 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Gao, 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=Organoid+cultures+derived+from+patients+with+advanced+prostate+cancer.">Organoid cultures derived from patients with advanced prostate cancer.</a> Cell. 159 (1), 176-187 (2014).</li><li>Ito, 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=NOD/SCID/gamma(c)(null)+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Simon, M. 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=A+comparative+phenotypic+and+genomic+analysis+of+C57BL/6J+and+C57BL/6N+mouse+strains.">A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains.</a> Genome Biol. 14 (7), R82 (2013).</li><li>Kalish, S., 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=C57BL/6N+Mice+Are+More+Resistant+to+Ehrlich+Ascites+Tumors+Than+C57BL/6J+Mice:+The+Role+of+Macrophage+Nitric+Oxide.">C57BL/6N Mice Are More Resistant to Ehrlich Ascites Tumors Than C57BL/6J Mice: The Role of Macrophage Nitric Oxide.</a> Med Sci Monit Basic Res. 21, 235-240 (2015).</li><li>Tseng, W., Leong, X., Engleman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+model+of+colorectal+cancer.">Orthotopic mouse model of colorectal cancer.</a> J Vis Exp. (10), e484 (2007).</li><li>Roper, 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=Combination+PI3K/MEK+inhibition+promotes+tumor+apoptosis+and+regression+in+PIK3CA+wild-type,+KRAS+mutant+colorectal+cancer.">Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer.</a> Cancer Lett. 347 (2), 204-211 (2014).</li><li>Coffee, E. 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=Concomitant+BRAF+and+PI3K/mTOR+blockade+is+required+for+effective+treatment+of+BRAF(V600E)+colorectal+cancer.">Concomitant BRAF and PI3K/mTOR blockade is required for effective treatment of BRAF(V600E) colorectal cancer.</a> Clin Cancer Res. 19 (10), 2688-2698 (2013).</li><li>Belmont, P. 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=Resistance+to+dual+blockade+of+the+kinases+PI3K+and+mTOR+in+KRAS-mutant+colorectal+cancer+models+results+in+combined+sensitivity+to+inhibition+of+the+receptor+tyrosine+kinase+EGFR.">Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.</a> Sci Signal. 7 (351), ra107 (2014).</li><li>Hung, K. 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=Development+of+a+mouse+model+for+sporadic+and+metastatic+colon+tumors+and+its+use+in+assessing+drug+treatment.">Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.</a> Proc Natl Acad Sci USA. 107 (4), 1565-1570 (2010).</li><li>Wang, F., Johnson, R. L., Snyder, P. W., DeSmet, M. L., Fleet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Inducible,+Large-Intestine-Specific+Transgenic+Mouse+Model+for+Colitis+and+Colitis-Induced+Colon+Cancer+Research.">An Inducible, Large-Intestine-Specific Transgenic Mouse Model for Colitis and Colitis-Induced Colon Cancer Research.</a> Dig Dis Sci. 61 (4), 1069-1079 (2016).</li><li>Xue, Y., Johnson, R., Desmet, M., Snyder, P. W., Fleet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+transgenic+mouse+for+colorectal+cancer+research+with+intestinal+cre+expression+limited+to+the+large+intestine.">Generation of a transgenic mouse for colorectal cancer research with intestinal cre expression limited to the large intestine.</a> Mol Cancer Res. 8 (8), 1095-1104 (2010).</li><li>Tetteh, P. W., 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=Generation+of+an+inducible+colon-specific+Cre+enzyme+mouse+line+for+colon+cancer+research.">Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.</a> Proc Natl Acad Sci USA. 113 (42), 11859-11864 (2016).</li></ol>

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><tbody><tr><td>&lt;strong&gt;Cell culture Media and Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advanced DMEM F12</td><td>Invitrogen</td><td>12634010</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>HEPES (1 M)</td><td>Life Technologies GmbH</td><td>15630056</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>Glutamax-I Supplement (200 mM)</td><td>Life Technologies GmbH</td><td>35050038</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>Penicillin/Streptomycin (PenStrep)</td><td>Life Technologies GmbH</td><td>15140122</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>DMEM</td><td>Life Technologies GmbH</td><td>61965026</td><td>basic medium of 2D cell lines (DMEM/10% FCS)</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>BIOCHROM AG</td><td>S 0115</td><td>basic medium of 2D cell lines (DMEM/10% FCS)</td></tr><tr><td>TrypLE Express enzymatic dissociation buffer</td><td>Life Technologies GmbH</td><td>12604021</td><td></td></tr><tr><td>Matrigel basement membrane matrix (BMM, phenol red free)</td><td>CORNING B.V. Life Sciences</td><td>356231</td><td></td></tr><tr><td>Dulbecco&#039;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>6-/48-well plates with lid</td><td>CORNING</td><td>3516/3548</td><td></td></tr><tr><td>cell culture flask 75 cm&amp;sup2;, 250 mL</td><td>VWR International GmbH</td><td>734-2066</td><td></td></tr><tr><td>cell culture flask 150 cm&amp;sup2;, 600 mL</td><td>Corning B.V. Life Sciences</td><td>355001</td><td></td></tr><tr><td>Eppendorf tubes 1,5 mL / 2 mL</td><td>Sarstedt AG &amp;amp; Co.</td><td>72.706.400/ 72.695.400</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>TC10 Counting Slides (for TC20 Counting Machine)</td><td>Bio-Rad Laboratories GmbH</td><td>1450016</td><td></td></tr><tr><td>Pasteur pipettes (glass, 150 mm)</td><td>Fisher Scientific GmbH</td><td>11546963/ FB50251</td><td>thinly pulled by using a bunsen burner</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>for primary tumor tissue preparation</td></tr><tr><td>MACSmix Tube Rotator</td><td>Miltenyi Biotec</td><td>130-090-753</td><td>for primary tumor tissue preparation</td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td>for primary tumor tissue preparation</td></tr><tr><td>Human Tumor Dissociation Kit</td><td>Miltenyi Biotec</td><td>130-095-929</td><td>for primary tumor tissue preparation</td></tr><tr><td>Falcon 70 &amp;micro;m Cell Strainer</td><td>Corning B.V. Life Sciences</td><td>352350</td><td>for primary tumor tissue preparation</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sevoflurane</td><td>AbbVie Germany GmbH &amp;amp; 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</td><td>Temgesic</td><td>-</td><td></td></tr><tr><td>Bepanthen - opthalmic ointment</td><td>Bayer Vital GmbH</td><td>10047757</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>Braun</td><td>235144</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></tr><tr><td>cellulose swabs</td><td>Lohmann &amp;amp; Rauscher Deutschland</td><td>13356</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>FST - Fine Science Tools</td><td>11018-12</td><td></td></tr><tr><td>Iris Scissor - ToughCut</td><td>FST - Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Olsen-Hegar Needle Holder</td><td>FST - Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>AutoClip Kit</td><td>FST - Fine Science Tools</td><td>12020-00</td><td></td></tr><tr><td>PDS Z1012H 6/0 C1 (surgical suture)</td><td>Johnson &amp;amp; Johnson Medical GmbH</td><td>Z1012H</td><td></td></tr><tr><td>Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer</td><td>Parkland Scientific</td><td>V3000PS/PK</td><td></td></tr><tr><td>UltraMicro Pump with Micro4 Controller</td><td>World Precision Instruments</td><td>UMP3-4</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Footswitch for SYS-Micro4 Controller</td><td>World Precision Instruments</td><td>15867</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Three-axis Manual Micromanipulator</td><td>World Precision Instruments</td><td>M325</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Magnetic Stand for Micromanipulator</td><td>World Precision Instruments</td><td>M10</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Steel Base Plate for M10 Magnetic Stand</td><td>World Precision Instruments</td><td>5479</td><td>equipment for highly controlled orthotopic injection</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></tr><tr><td>Binocular Surgical Microscope</td><td>Parkland Scientific</td><td>VS-2Z</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;CTC isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EDTA</td><td>Roth</td><td>8040.1</td><td></td></tr><tr><td>Density gradient medium &amp;ndash; Ficoll</td><td>StemCell - Lymphoprep</td><td>7801</td><td></td></tr><tr><td>Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4</td><td>BioLegend</td><td>324210</td><td></td></tr><tr><td>Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8</td><td>BioLegend</td><td>118210</td><td></td></tr><tr><td>Petri Dish, &amp;oslash; 60 x 15 mm, 21 cm&amp;sup2;, Vent</td><td>Greiner bio-one</td><td>628102</td><td></td></tr><tr><td>Fluorescence Cell Culture Microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Transferman 4r Micromanipulator</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>CellTram Air</td><td>Eppendorf</td><td></td><td>aspiration pump connected to the micromanipulator</td></tr><tr><td>Dmz Universal Microelectrode Puller</td><td>Dagan Corporation</td><td></td><td>required for the manufacturing of micro capillaries for single cell aspiration</td></tr><tr><td>Prism Glass Capillaries</td><td>Dagan Corporation</td><td></td><td></td></tr><tr><td>PAP pen</td><td>Abcam</td><td>ab2601</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Life Technologies GmbH</td><td>14190169</td><td>picking buffer</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>BIOCHROM AG</td><td>S 0115</td><td>picking buffer</td></tr><tr><td>Penicillin/Streptomycin (PenStrep)</td><td>Life Technologies GmbH</td><td>15140122</td><td>picking buffer</td></tr><tr><td>EDTA</td><td>Roth</td><td>8040.1</td><td>picking buffer</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunohistochemistry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Purified anti-human CD326 (EpCAM) antibody clone 9C4</td><td>BioLegend</td><td>324201</td><td>EpCAM immunohistochemistry (cf, fig 2C)</td></tr><tr><td>HRP rabbit anti-mouse IgG</td><td>Abcam</td><td>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

Universidad de Cartagena UDEC

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

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

科研

癌症研究

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

结直肠癌原位小鼠模型

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

生物学

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

循环肿瘤细胞系:基础和转化研究的创新工具

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

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 ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

医学

Intracecal Tumor Cell Injection: A Technique to Deliver Cancer Cells for Establishing Orthotopic Colorectal Cancer Mouse Model

Source: Kochall, S., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/55357"> Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer </a> J. Vis. Exp. (2017)
The video describes the technique for intracecal injection of cancerous cells to develop an orthotopic mouse model with highly uniform tumors. Therefore, the orthotopic mouse model allows realistic simulation of metastasis, providing a valuable tool to study the phenotype changes during tumor growth and dissemination.

肿瘤细胞注射液:一种递送癌细胞以建立原位结直肠癌小鼠模型的技术

Source: Kochall, S., et al.  Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer  J. Vis. Exp. (2017)
The video ...

JoVE Encyclopedia of Experiments

Colorectal Cancer

Animal model studies

Establishing Experimental Metastases Mouse Model: Implanting GFP Expressing CRC Organoid Cells in PDX Model to Detect Micrometastases

Source: Okazawa, Y. et al.&#160; <a href="https://jove.unicartagenaproxy.elogim.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a> J. Vis. Exp. (2018).
This video describes the protocol for generating metastases by the PDX derived CRC organoids labeled with GFP lentivirus into the spleen of an immunodeficient mouse. Cancer micrometastases colonizing other organs can be detected using a fluorescence microscope to assess the GFP expression.

建立实验转移小鼠模型:在 PDX 模型中植入表达 GFP 的 CRC 类器官细胞以检测微转移

Source: Okazawa, Y. et al.&#160;  High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a ...

Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

Source: Okazawa, Y., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.&#160;