我们要感谢杰弗里·格林L.博士（芝加哥大学）的Luc2-tdTomato质粒和HCT116细胞系，安仁索兰奇先生（动物资源中心）的小鼠管理，拉拉莱尼博士的协助与DLIT。荧光灯和发光强度Quantifications在综合小动物成像研究资源在芝加哥上IVIS频谱大学（珀金埃尔默，霍普金顿，MA）进行。这项工作是由弗吉尼亚和DK路德维希基金会为癌症研究的支持，肺癌症研究基金会（LCRF），前列腺癌基金会（PCF）和癌症中心支援津贴（P30CA014599）。该资助者在研究设计，数据收集和分析，决定发表或准备手稿中没有作用。

所有动物的程序是由机构动物护理和使用委员会在芝加哥大学（协议＃72213-09）批准，并在无菌条件下进行的。 1.准备<ol><li>使500毫升对HCT116肿瘤细胞的培养基:Dulbecco氏补充有10％胎牛血清（FBS），100U / mL青霉素，和100毫克/毫升链霉素改良的Eagle培养基（DMEM）。 </li><li>高压釜所用的仪器为脾注射模型，包括3 - 4手术巾，纱布，两个小斜角肌挤压拾音器，针驱动器，两双剪刀，小钳，和3 microclips，在251°F下20分钟。 </li><li>术后准备麻醉小鼠，待脾注射后皮下给药。稀2 - 4微克丁丙诺啡以每只小鼠0.9生理盐水500微升。 </li><li>准备萤火虫荧光素150微克/ 150微升等份腹腔注射DURING生物发光检测。在-20°C存储和避光。 </li></ol> 2.生成荧光素酶/ tdTomato标记的单克隆细胞系10,11的<ol><li>解冻并保持HCT116人结肠直肠癌细胞和293FT人胚肾细胞在DMEM含10％FBS，100U / mL青霉素，和100毫克/毫升链霉素。在5％CO 2和37℃下维持培养。 </li><li>产生高滴度慢病毒。 <ol><li>第D 1，板10 - 12×10 6 293FT在18毫升完全DMEM的（cDMEM）通道3和10 15个1cm池培养皿之间1％非必需氨基酸的细胞有10％FBS，1％青霉素/链霉素和。在37℃和5％的CO 2。 </li><li>上D 2，使用以下的转染溶液转染细胞: <ol><li>对于每道菜，请使用以下内容:pFUG慢病毒载体的37.5微克插入荧光素酶（Luc2）和tdTomato一个DNA混合物构建11（从杰弗里·格林博士在芝加哥大学的礼品），pCMVΔ8.74的25微克和12.5微克pMD2.G的悬浮在总共无菌H 2 O. 1062微升加入2M的氯化钙2 188μL。 </li><li>添加1250 2×HEPES-缓冲盐水（HBS，50mM的HEPES，1.5毫摩尔的 Na 2 HPO 4，和180 mM氯化钠，pH值7.12）的DNA / 氯化钙溶液中，在一个时间一滴微升，同时通过吹泡泡溶液用移液管。 </li><li>孵育在室温20分钟，然后添加15.5毫升RT cDMEM和氯喹，以使25μm的终浓度。 </li></ol></li><li>吸媒体在293FT细胞每个培养皿的转染溶液替换。添加16毫升慢慢转染溶液，作为电镀的细胞容易地移去。 </li><li>第D 3,8后 - 16小时，除去转染溶液，并用Dulbecco磷酸盐缓冲盐水（DPBS）一次洗涤细胞，再塔基NG不在乎打跑细胞。用18毫升新鲜cDMEM用10毫摩尔的Hepes更换介质。 注:缓慢移动的菜，吸出的转染溶液，并通过板的侧壁添加介质，以便不去除细胞。 </li><li>第D 4，在从转染时48小时，通过用移液管慢慢吸以免撞出细胞收集从碗碟介质。离心机在300×g离心5分钟收集的媒体以沉淀大细胞碎片。 </li><li>使用过滤病毒上清用0.45μm低蛋白结合以50,000 xg离心4℃使用SW-28转子2小时过滤烧瓶中并离心。立即倒出上清液离心结束后，干燥与雨刷所述管的内侧。 </li><li>有1％FBS重悬在低血清培养基（RSM）的50微升的病毒颗粒。等分试样可以储存在-80℃以供将来使用（病毒股票）。 </li></ol></li><li>转导的慢病毒ES进入HCT116细胞和产生tdTomato阳性克隆的面板。 <ol><li>板的HCT116细胞在病毒感染前1 10 5×24孔板1天的密度。 </li><li> （:100稀释1）与1％青霉素/链霉素和8微克/毫升聚凝胺（CRSM）在4mL的RSM的稀重悬病毒的40微升（从2.2.7）。 </li><li>感染的当天，用DPBS洗涤细胞一次，然后从步骤2.3.2添加稀释的病毒的250μL到每个孔中。孵育在37℃下4小时和5％的CO 2，然后添加cDMEM 1毫升到各孔中;孵育在37℃和5％CO 2的48 - 72小时。 </li><li>悬浮细胞用1mL 0.05％胰蛋白酶和0.53毫摩尔EDTA，然后添加1mL的cDMEM以中和胰蛋白酶。转移细胞到一个离心管中，并沉淀在300×g离心4分钟。在1mL的Hanks&#39;平衡盐溶液（HBSS）中加2％FBS的洗涤沉淀3次。 </li><li>从步骤2.3.4悬浮颗粒用FACS缓冲液（2％FBCS和2mM EDTA的PBS中）成单细胞悬浮液以1×10 6个细胞/ mL。 </li><li>排序使用细胞分选仪，门控PE阳性细胞的tdTomato阳性HCT116细胞（激发/发射:五百八十五分之五百五十六纳米）。生长于10-cm培养皿中收集的细胞在5％CO 2和37℃，以产生亲tdTomato阳性HCT116细胞（ 图1A）。 </li><li>悬浮亲tdTomato阳性HCT116细胞从2.3.6用8mL 0.05％胰蛋白酶和0.53毫摩尔EDTA为3 - 5分钟，然后添加cDMEM 8毫升，以中和胰蛋白酶。 <ol><li>转移细胞到一个50毫升管中并沉淀在300×g离心4分钟。除去上清液和稀释亲tdTomato阳性HCT116细胞在cDMEM（ 图1B）每200微升1细胞。 </li></ol></li><li>在5％CO 2和37℃（ 图1C）板在96孔平板每孔的单个小区（稀释的200微升）。 </li><li>在5％的CO 2和37℃，生长在瓶中个别单克隆。 （ 图1D）。 </li></ol></li></ol> 3.荧光信号强度校正为细胞数的函数<ol><li>板转染HCT116细胞，现在被称为HCT116-L2T，在0密度，10 3，2×10 4，3×10 5，5×10 4，7×10 4，9×10 4个 ，10个5细胞/孔在一式三份的每个密度的96孔板。 </li><li>孵育5小时后，量化使用IVIS 10 tdTomato荧光强度。 <ol><li>点击桌面上的图像软件图标启动软件。点击"初始化"按钮初始化成像系统。初始化完成后（大约需要几分钟）后，选择"荧光"，"4秒"的曝光时间，"中"分档，"2"F /停止，"535"激发滤光片，和"580"的发射滤波器。 </li><li>将成像站上96孔板，然后点击"获取"开始成像。图像被获取后，点击"投资回报工具"，在工具选项板，并选择12×8从狭缝图标。 </li><li>创建12×8的狭缝，以覆盖96孔板的图像上的信号的区域，并点击测量标签。观察每球面度为每平方厘米每一台S光子单位投资回报测量的表的一个窗口（光子/秒/ SR /厘米2）。 </li></ol></li><li>构造用参数（X轴）等于细胞的数目和代表信号强度的函数（Y轴）的散点图。计算使用数据分析软件来计算对应于荧光强度的单元电池的数量（ 图2）10回归曲线。 </li></ol> 4.肝转移的动物模型<ol><li>一旦HCT116-L2T细胞达到70- 80％汇合，前约1小时，他们准备脾注射。分离使用0.05％胰蛋白酶将细胞在0.53毫摩尔EDTA为3 - 5分钟，然后与等量cDMEM的中和。请务必仔细吸取避免结块。 </li><li>伯爵用自动细胞计数细胞。 </li><li>重悬在1×DPBS细胞以1.2的浓度- 2×10 6个细胞/ 100微升。确保吸取，彻底悬浮细胞，以避免结块。 </li><li>保持冰细胞注射前，偶尔它们再悬浮在管。 </li><li>此前麻醉小鼠，提供术前麻醉和流体通过静推注射皮下步骤1.3中所述的丁丙诺啡混合500微升。辖额外丁丙诺啡混合相同剂量每天两次，直到疼痛（行动不便，驼背的身材，没有新郎，处理时发声）的迹象已经解决。 </li><li>麻醉6至8周-old雌性无胸腺裸小鼠中氧气2％异氟烷在麻醉室中。确认老鼠完全麻醉下按捏尾巴。使用对眼睛兽医药膏，以防止干燥时的麻醉下。每个麻醉鼠标转移到一个单独的鼻锥健脾注入。前切口，将每只小鼠应该用无菌手术盖布覆盖。 </li><li>确定脾通过对裸鼠的左翼皮肤外看到一个紫色区域。采用显微剪刀，使皮肤上的只是脾脏上方8mm的左翼切口。 <ol><li>接下来，抬起腹壁作一小切口。允许空气进入腹腔，使内脏从切口部位移开。放大腹壁切口大约5毫米。 </li><li>轻轻用棉签将切口周围的压力暴露脾脏。如果需要的话，该胰脂肪可轻轻人ipulated以帮助曝光，以免伤害脾胃。 </li></ol></li><li>缓缓注入100μL用1毫升注射器，用27克针插入暴露脾根尖细胞的。缓缓注入，历时至少30 - 60秒，以避免额外的肝肿瘤。 </li><li>放置在脾脏一个microclip在注射结束时除去针，以防止注入细胞悬液泄漏之前。 </li><li>离开代替microclip 5分钟。 </li><li> 5分钟postinjection，使用手持式烧灼装置止血执行脾切除。解剖脾门，距前身边做起。当解剖大血管，预凝固与相邻的脂肪组织的血管的近端侧，利用烧灼避免过度出血。 注:止血方法:采用烧灼直接凝固出血点，施加压力，出血点为3 - 5分钟，或缝合结扎出血点用5-0丝质领带。 </li&gt; <li>关闭每个小鼠的侧面切口在两层。首先，关闭腹壁在单个水平针5-0可吸收缝线编织（如薇乔）。接着，用一个单一的水平缝关闭用4-0不可吸收的单纤维缝合线（ 例如，prolene线）皮肤，一次。 </li><li>喷洒70％的异丙醇，以保持每道工序之间的无菌条件下清洗所有仪器。 注意:确保当他们从麻醉中醒来的动物温暖。监视所有小鼠手术后，直到他们成为日间及恢复正常活动。通常情况下，恢复的时间大约为3 - 5分钟。直到他们重新获得足够的意识，以保持胸骨斜卧不要让动物无人值守。不要返回已动过手术，以公司的其他动物，直到它已经完全康复的动物。 </li></ol> 5. 在体内生物光学成像<ol><li>进行成像上每周测量和随时间推移定量生物发光变化。 </li><li>放置小鼠麻醉室，并与在氧气2％异氟烷麻醉它们。确认老鼠完全麻醉下按捏尾巴。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 </li><li>其次，从腹腔1.4步注入每只小鼠用的预制的150微升萤火虫萤光素。 </li><li>将带有个人的鼻锥管理异氟醚在IVIS小鼠。放置在仰卧位的小鼠中之间的间隔的信号，以相邻的小鼠最小化。最多5只小鼠一可以在一个时间被成像。 </li><li>测量和分析生物发光强度荧光素注射后3分钟。 <ol><li>如在步骤3.2.1描述初始化IVIS后，选择"发光"，"1秒"的曝光时间，和"中"分箱。 </li><li>点击"获取"按钮，开始IMAGING。确保在荧光素注射后一致的时间（3分钟）执行每个成像。 </li><li>图像获取后，将图像窗口和工具调色板将出现。点击"的ROI工具"，选择"1"，"2"，"3"，"4"，或"5"，从圆圈图标，对应于成像的小鼠的数目。 </li><li>要定义利息（投资回报）的地区，圈在小鼠腹腔发光信号的信号区域，然后单击投资回报率的"三围"为辐射效率的任意单位（（光子/秒/厘米2 /球面度）/（μW/厘米2））。确保有背景的额外投资回报循环。 </li></ol></li><li>四周后喷射和前动物牺牲，使用IVIS评价使用个别肿瘤集落的生物发光强度的实时3D重建肿瘤负荷和分配执行漫射发光成像断层扫描（DLIT）。 <o升&gt; <li>麻醉小鼠和注射萤光素，如在步骤5.2.4说明。 DLIT是在时间一个鼠标完成。 </li><li>初始化IVIS后，按步骤3.2.1所述，选择"映像向导"，"生物发光"，然后单击"下一步"。选择"DLIT"，然后单击"下一步"。选择"萤火虫"的探头和点击"下一步"。选择"鼠标"的形象主题，"自动"曝光参数，视"C-13 CM"领域，"0.5 CM"主题的高度，然后单击"下一步"。点击"采集"按钮，开始成像。 </li><li>图像被获取后，选择"DLIT三维重建"选项卡和"分析"选项卡，然后单击"重建"来生成三维重建图像。 </li><li>点击"工具"和"三维动画"。在3D动画窗口中选择"Y轴旋转CCW"。点击"记录"和"保存"成.MOV格式文件。 </li></ol></li></ol> 6. 离体荧光成像<ol><li>通过颈椎脱位牺牲小鼠而麻醉，或根据在4机构指南 - 第6周后的脾注射。 </li><li>收获肝脏。建立从左边到右侧水平切口。抓住剑突与皮卡，解剖镰状，肝静脉和下腔静脉。 <ol><li>解剖右肝肾韧带，肝十二指肠韧带，和左韧带肝肾，小心不要弄伤尾状叶，而解剖肝十二指肠韧带。收获肝脏后，取出胆囊，因为这将显示自体荧光。记存在的任何附加额外的肝肿瘤。 </li></ol></li><li>记下号码和肝肿瘤的大小目前宏观，然后将收获的肝脏在DPBS。 注意:肿瘤肉眼可见的宏观为白色的肿瘤（为一个代表性的例子，参见图4）。 </li><li>之前的成象片上的位置，采取各肝脏和轻轻印迹在纸巾上除去过量的液体。 </li><li>测量从使用IVIS每个肿瘤集落的荧光强度。 <ol><li>选择"荧光"，"4秒」的曝光时间，"中"分箱，"2"的F /停止，"535"激发滤光片，和"580"发射滤光片。点击"采集"按钮，开始成像。 </li><li>获取图像后，找到图像窗口和工具调色板。点击"工具的投资回报率"，并从圆形图标选择"1"。要定义的ROI，圈出映像中的单个肿瘤殖民地的信号区域，然后单击投资回报率的"三围"为辐射效率的任意单位（（光子/秒/厘米2 /球面度）/（μW/厘米2） ）。确保有背景的额外投资回报循环。 </li></o升&gt; </li><li>通过假设它是一个球体计算总的肿瘤体积。肿瘤体积= 4×πXR 3/3（R =半径）。计算肿瘤集落形成细胞（Fc）的作为肿瘤的肝脏由splenically注射细胞的数目除以数的分数。 </li><li>从线性近似从步骤3.3计算每个菌落的细胞总数（= X）。计算细胞分裂（= Y）的数量为Y =登录2×和TD作为Td的（天）= 28 / Y. </li></ol>

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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=Survival+after+hepatic+resection+for+metastatic+colorectal+cancer:+trends+in+outcomes+for+1,600+patients+during+two+decades+at+a+single+institution.">Survival after hepatic resection for metastatic colorectal cancer: trends in outcomes for 1,600 patients during two decades at a single institution.</a> J. Am. Coll. Surg. 210 (5), 744-752 (2010).</li><li>Smakman, N., Martens, A., Kranenburg, O., Borel Rinkes, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+bioluminescence+imaging+of+colorectal+liver+metastases+in+the+mouse.">Validation of bioluminescence imaging of colorectal liver metastases in the mouse.</a> J. Surg. 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Rep. 5 (10946), (2015).</li><li>Liu, H., 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=Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models.">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc. Natl. Acad. Sci. U. S. A. 107 (42), 18115-18120 (2010).</li><li>Wang, X. 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=Integrative+analyses+identify+osteopontin,+LAMB3+and+ITGB1+as+critical+pro-metastatic+genes+for+lung+cancer.">Integrative analyses identify osteopontin, LAMB3 and ITGB1 as critical pro-metastatic genes for lung cancer.</a> PLoS One. 8 (2), e55714 (2013).</li><li>Fidler, I. J., Kripke, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+results+from+preexisting+variant+cells+within+a+malignant+tumor.">Metastasis results from preexisting variant cells within a malignant tumor.</a> Science. 197 (4306), 893-895 (1977).</li><li>Yachida, 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=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467 (7319), 1114-1117 (2010).</li><li>Khodarev, 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=STAT1+pathway+mediates+amplification+of+metastatic+potential+and+resistance+to+therapy.">STAT1 pathway mediates amplification of metastatic potential and resistance to therapy.</a> PLoS One. 4 (6), e5821 (2009).</li><li>Langley, R. R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell-organ+microenvironment+interactions+in+the+pathogenesis+of+cancer+metastasis.">Tumor cell-organ microenvironment interactions in the pathogenesis of cancer metastasis.</a> Endocr. Rev. 28 (3), 297-321 (2007).</li><li>Lussier, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligo-+and+polymetastatic+progression+in+lung+metastasis(es)+patients+is+associated+with+specific+microRNAs.">Oligo- and polymetastatic progression in lung metastasis(es) patients is associated with specific microRNAs.</a> PLoS One. 7 (12), e50141 (2012).</li><li>Lussier, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+characterizes+oligometastasis(es).">MicroRNA expression characterizes oligometastasis(es).</a> PLoS One. 6 (12), e28650 (2011).</li><li>Calon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependency+of+colorectal+cancer+on+a+TGF-beta-driven+program+in+stromal+cells+for+metastasis+initiation.">Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation.</a> Cancer Cell. 22 (5), 571-584 (2012).</li><li>Vanharanta, S., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+metastatic+traits.">Origins of metastatic traits.</a> Cancer Cell. 24 (4), 410-421 (2013).</li><li>Khodarev, N. N., Roizman, B., Weichselbaum, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathways:+Interferon/Stat1+Pathway:+Role+in+the+tumor+resistance+to+genotoxic+stress+and+aggressive+growth.">Molecular pathways: Interferon/Stat1 Pathway: Role in the tumor resistance to genotoxic stress and aggressive growth.</a> Clin. Cancer Res. 18 (11), 3015-3021 (2012).</li><li>Li, C., 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=Interferon-stimulated+gene+15+(ISG15)+is+a+trigger+for+tumorigenesis+and+metastasis+of+hepatocellular+carcinoma.">Interferon-stimulated gene 15 (ISG15) is a trigger for tumorigenesis and metastasis of hepatocellular carcinoma.</a> Oncotarget. 5 (18), 8429-8441 (2014).</li><li>Cespedes, M. 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=Orthotopic+microinjection+of+human+colon+cancer+cells+in+nude+mice+induces+tumor+foci+in+all+clinically+relevant+metastatic+sites.">Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.</a> Am. J. Pathol. 170 (3), 1077-1085 (2007).</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), (2007).</li><li>Soares, K. 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The authors have nothing to disclose.

在目前的报告中提出的动物模型是基于两种主要方法。首先，为了确保观察具有不同倾向转移克隆定殖并在肝脏增殖的能力，高度异质的单克隆细胞系的组建立了，而不是建立普通癌细胞系12,13。单克隆办法转移发展受到最近的基因组数据14合理的，以前被成功地用于转移过程10,15,16建模。第二，通过萤光素酶和tdTomato标记的亲代细胞系的双标记，以便提供转移性生长在?...

从初级结直肠癌（CRC）肝转移患者被预后不良表征。初级非转移性的CRC的5年生存率（阶段I - III）中被估计为75 - 88％的3,4-，而肝转移患者（IV期）具有的只有8 5年生存率- 12％5 6。然而，转移性患者是一组异质性，不同数量的转移和复发的不同时代的呈现。临床观察表明，转移（其可以是成比例的定植能力或定植频率）和任何单转移（正比于本地生长速率）的大小的数目是独立的预后因素1,7。换句话说，转移克隆定植肝脏的成功取决于两个主要的性能:它们生长的能力和它们的传播，并在肝微生存能力。 该设计成功的临床模型与捕获和定量转移克隆的性能可显着提高我们的肝转移生物学的理解，并提供的潜在的治疗方法的设计的有效工具的能力。实验肝转移模型已经先前报道8,9，但是他们都没有提供定量捕获，并描述在体内和离体个体转移性克隆的性质的能力。 在这里，我们提出肝转移，其中包括肿瘤克隆具有不同的肝脏定植效率和生长特性的产生一个新的，先进的模式。我们采用与荧光素酶和tdTomato荧光蛋白与具有在转移能力固有差异单克隆细胞系的产生癌细胞双标记的组合。在此实验模型中，数据表明，开发肝转移可以在定植频率方面和倍增时间（Td），这是与临床观察结果一致进行说明。该模型的定量性质使得它易于采用用于药物发现和诊断的目的。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td>124262</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>LivingImage 4.0 Software</td>
			<td>Caliper Life Sciences</td>
			<td>128165</td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>VAD-MGX Research Anesthetic Machine</td>
			<td>Vetamac</td>
			<td>VAD-MGX</td>
			<td>Inhalation anesthesia machine</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid (10,000 units penicillin;10,000 &#956;g streptomycin)</td>
			<td>Invitrogen</td>
			<td>15140163</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>24020117</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>Buprenex Injection (0.3mg/mL)</td>
			<td>Reckitt Benckiser Healthcare Ltd.</td>
			<td>12496-0757-5</td>
			<td>Buprenorphine hydrochloride</td>
		</tr>
		<tr>
			<td>Gemini Cautery System</td>
			<td>Braintree Scientific</td>
			<td>GEM 5917</td>
			<td>Hand-held cautery for splenectomy</td>
		</tr>
		<tr>
			<td>Micro Clip; Straight; 70 Grams Pressure; 1.5mm Clip Width; 10mm Jaw Length</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5426</td>
			<td>Hemoclip: Hemostasis instruments after spleen injection</td>
		</tr>
		<tr>
			<td>D-luciferin, potassium salt</td>
			<td>Goldbio Technology</td>
			<td>LUCK-1G</td>
			<td>Luciferin potassium salt</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td>Reduced Serum Medium</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>BIO-RAD</td>
			<td>1450102</td>
			<td>Automatic cell counter</td>
		</tr>
		<tr>
			<td>JMP10 software&#160;</td>
			<td>SAS Institute</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>BD FACSAria II cell sorter</td>
			<td>BD Biocsiences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
	</tbody>
</table>

advanced animal model of colorectal metastasis in liver: imaging techniques and properties of metastatic clones

肝转移和进展的速度缓慢的有限数量的患者可以用局部治疗成功治疗方法1,2。然而，知之甚少肝转移的异质性，并且需要能够评估个体转移性集落的发育的动物模型。这里，我们提出，提供定量可视个体肿瘤克隆的发展，在肝脏和估计它们的生长动力学和移效率的能力肝转移的一种先进的模型。我们产生与荧光素酶和tdTomato和拥有不同生长特性稳定标记HCT116人结肠癌细胞的单克隆衍生物的面板。用脾注射后跟脾切除，大多数这些克隆能够产生肝转移，但与定植的不同的频率和不同的生长率。使用体内成像SYSTE米（IVIS），它是可能的可视化和量化转移发展与体内的发光和离体荧光成像。此外，漫射发光成像断层扫描（DLIT）提供肝转移体内三维分布。 体外收获肝脏荧光成像提供个别肝脏转移菌落定量测量，从而允许肝定植的频率的评估和生长动力学的转移。由于该模型类似于临床上观察到的肝脏转移，它可以作为用于检测与肝脏转移相关基因和用于肝脏转移性疾病测试潜在烧蚀或辅助治疗的一种模式。

该实验的目的是建立与潜在一致和容易复制动物模型的体内转移性肿瘤负荷的串行量化和对定植频率和显影肝转移生长动力学的估计。 图2-6，有传说，是从我们以前的出版物经Creative Commons CC-BY许可10提供。 双标肿瘤细胞单克隆生成首先，用不同的转移能?...

Watch this Scientific Journal Video about Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones at JoVE.com

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

肝大肠癌转移的高级动物模型:成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

advanced-animal-model-colorectal-metastasis-liver-imaging-techniques

Research

JoVE Journal

Cancer Research

12.4K 次观看.  The University of Chicago. The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

视频: Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

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 ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

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 ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

缺血再灌注损伤中转移性肝肿瘤的模样

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

大肠癌HT29衍生癌症干细胞类肿瘤球中的驱动基因发现

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...