Name: the colon-26 carcinoma tumor-bearing mouse as a model for the study of cancer cachexia
Uploaded: 2016-11-30T14:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia at JoVE.com

We thank Richard Lieber and Shannon Bremner for their ImageJ macro and instructions. While at Thomas Jefferson University, this work was supported by the Pennsylvania Department of Health CURE Grant TJU No. 080-37038-AI0801. Subsequently, this study was supported by a grant to AB from the National Institutes of Health (R21CA190028), and by grants to TAZ from the National Institutes of Health (R01CA122596, R01CA194593), the IU Simon Cancer Center, the Lustgarten Foundation, the Lilly Foundation, Inc., and the IUPUI Pancreas Signature Center.

伦理学声明:描述了托马斯·杰斐逊大学的机构动物护理和使用委员会和医学的印第安纳大学医学院获得批准的所有研究。 1. C26细胞生长和准备<ol><li>获得C26结肠癌细胞（俄亥俄州立大学医学中心（OSUMC）），并准备完整生长培养基（ 即，高葡萄糖的Dulbecco改良的Eagle氏培养基（DMEM）中含有10％胎牛血清（FBS），1mM丙酮酸钠，1％谷氨酰胺和1％链霉素/青霉素）。 注意:为了允许更好-实验间再现性，优选的是使用C26细胞以尽可能低的通路。 </li><li>使用市售的细胞计数器直接制版50000个细胞/厘米2在完全生长培养基的烧瓶。孵育他们在湿润气氛中，在37℃和5％的CO 2，直至亚汇合。避免在高密度培养他们上一个耳鼻喉科分化和细胞表型的漂流。 </li><li>第一通道之后，板足够的细胞在动物的计划数量被植入。对细胞植入的当天，解离通过胰蛋白酶消化细胞以每平方厘米0.02毫升0.25％胰蛋白酶-EDTA溶液中，加入每毫升胰蛋白酶的新鲜培养基至少3毫升中和胰蛋白酶，确定在细胞/ ml的细胞浓度和重悬需要在无菌PBS的实验（每只小鼠1×10 6个细胞）细胞的数量。 注:C26细胞达到汇合时，密度为约500,000个细胞/厘米2。 </li></ol> 2.小​​鼠<ol><li>随机成年CD2F1小鼠（至少8周龄）​​分组（通常控制和肿瘤承载）与至少n =每组6。 注:C26肿瘤的Balb / c小鼠生长为好。然而，根据我们的经验，CD2F1小鼠代表对于该特定肿瘤模型更通用的和经济的主机。此外，类似于临床设置，并在与癌症相关的肌肉的其它实验模型报告发现浪费14-16，在响应于C26恶病质的性别差异也可以观察到17（也见表1）。 </li><li>使用麻醉异氟烷吸入（氧气3％）鼠标。维持麻醉下的动物只要执行的肿瘤接种所需的时间。确保鼠标为了防止体热的损失位于一个加热垫。启动肿瘤注射过程之前，轻轻捏脚趾后方适当的确认麻醉。 注意:由于该过程（通常在几秒钟）的持续时间，不需要使用兽医眼膏。出于同样的原因，经受假手术对照动物预计不会减肥。 </li><li>使用无菌1毫升注射器，胰岛素用26号针头，迅速注入250微升含有T中的解决方案umor细胞在小鼠的脂肪垫皮下和intrascapularly。 注:控制动物将接受250微升无菌生理盐水intrascapularly的脂肪垫。 </li><li>将它在笼中动物回来。直接观察到鼠标至少每隔15分钟，直到它可以轻柔的操作作出反应，并收复了一个翻正反射。 注意:不要离开鼠标无人值守，并保持在一个包含固体基质温恢复笼子。另外，不要的鼠标返回到动物室，直到它完全从麻醉中恢复。通常，小鼠不需要进行单独圈养，除非研究者旨在评估个体的食物摄取。如果是这种情况，并使用自动代谢笼不可用，食品消费的人工记录，必须有可能每天，每天在同一时间执行。 </li><li>每天检查小鼠并记录他们的体重。 注:录制体况评分和家笼行为可以分化程度的恶病质和病态行为，这是治疗研究18有益的。 </li></ol> 3.安乐死和采血车<ol><li>当在肿瘤中宿主体重损失比对照达到5％，10％，或15％（轻度，中度，重度恶病质，分别地）相比，其初始体重，安乐死的小鼠。 注:在一组达到体重减轻10％，在其所有的时间组处死一个典型的实验将进行。体重损失是通过测量体重包容了肿瘤的评估。 </li><li>将鼠标异氟醚全身麻醉（氧气3％）下。 （ - 1.5毫升约1）通过心脏穿刺的方法抽血。 </li><li>收集血K 2 -EDTA（18毫克），空管，将它们放在冰上。离心血液（2,000 XG在4℃15分钟），并收集血浆/血。 </li><li>用C的方式安乐死鼠标ervical错位。继续组织切除和器官集合。 </li></ol> 4.组织和器官切除注:对于使用生物化学和分子生物学实验的组织，计划权衡每个器官和组织，并立即将一个片段插入预先标记的冷冻管。快冷冻在液氮中，并储存于-80℃。 <ol><li>为了避免与毛皮组织样品的污染，喷在整个主体70％的乙醇。 </li><li>放置在解剖床鼠标在仰卧位置和由脚连接到升高的滴定管夹垂直延伸肢体。 </li><li>抓住与杜蒙钳下肢的皮肤，并使用弧形细剪刀轻轻除去皮肤和筋膜，暴露下肢下面的肌肉肚子。 </li><li>在上后股骨的外侧和内侧髁和其在插入标识腓肠肌上后下肢由其原点通过跟腱跟骨。 <ol><li>继续削减腓肠肌跟腱。抓住与杜蒙钳腓肠肌的远端拉肌腹朝它的起源。使用剪刀，切割肌肉在原点尽可能接近到股骨。将肌肉中的一道菜。立即称重并在液氮冷冻管它。 注:请不要与腓肠肌切除以及比目鱼肌。如果发生这种情况，仔细地用钳子轻轻拉动它关闭删除比目鱼。 </li></ol></li><li>继续识别和胫骨和其在通过其远端肌腱的内侧楔插入前外侧表面切除从它的起源胫骨前肌。 <ol><li>为了具有杠杆作用，通过抓住与食指和拇指数字握住脚。将杜蒙钳尖端立即胫浅表肌腱远端下，移动镊子使得BLUNT侧可用于从底层结缔组织分离胫肌腹。 </li><li>切使用细弯曲剪刀远端肌腱，然后切开肌肉在原点，尽可能靠近胫骨。 </li></ol></li><li>通过进行矢状切口在下腹区域开始和优移动到胃脘区域，停止只是隔膜上方的切口打开腹腔。 注:切口的深度应当只足以破坏腹肌壁和底层腹膜筋膜。 </li><li>小心地用钝尖镊子抓它取下肝脏，并用细弯剪刀解剖走任何血管或结缔组织附着在肝脏的腔。小心不要留下任何瓣后面。称重和捕捉冷冻肝在液氮中的管。 </li><li>使用钝头镊子，搬开肠，然后揭露和微妙删除脾脏。用钝尖镊子轻轻抓住健脾和使用细弯剪刀的背面或侧面钝解剖走任何结缔组织或血管秉承脾腔。称重和管理单元冻结它在液态氮的管。 </li><li>识别性腺脂肪垫，相邻的附睾（男性）或子宫（女性）。轻轻抓住用钝尖镊子附睾脂肪垫和拉组织了腔。使用细弯剪，切去可以连接到脂肪垫任何性腺组织。称取脂肪垫和捕捉冻结它在液氮管。 注意:通常，这种脂肪组织被认为是代表在小鼠中的总身体脂肪量的和中重量与恶病质下降。其他脂肪垫可以类似地确定，解剖，取出，称重。 </li><li>使用弧形细剪刀剪开动物的胸部。切除附着在ST的两个侧边框肋骨ernum。取出胸骨。使用两对镊子，把握胸廓的每一侧和横向拉每一侧以打开胸腔。 </li><li>娇柔与镊子拔出心脏。通过在左心房切断主动脉切除小心地用细弯剪刀心脏。确保通过微妙的印迹是对一些吸水纸空的任何残血的器官。称重和管理单元冻结它在液态氮的管。 </li></ol> 5.肌肉冻结和安装<ol><li>浸入含异戊烷入液氮的小塑料烧杯（50ml）中的下半部分。异戊烷是准备使用时稍微变粘稠并形成固体白色叠层衬烧杯内（温度:-160℃）。 注:请始终使用无火花青铜或铝制手动工具。避免吸入蒸气产品。保持通风良好。如果处理的泄漏和通风是不可能或impractic人，穿合适的呼吸器。 </li><li>由（10秒）短暂地将其浸入异戊烷冻结卡盘（软木）一些包埋剂。 </li><li>由肌腱，垂直相对于软木保持它，以维持纤维的取向，并允许截面处理肌肉（例如 ，胫骨或腓肠肌）。 </li><li>仔细定位在冷冻包埋介质顶部的新鲜肌肉的端部。 注:肌肉会坚持。不完全包围与包埋介质肌肉是很重要的。同样重要的是要保持的截面积的连续测定的垂直方向。 </li><li>蘸用所附肌肉入异戊烷浴夹头（通常冷冻时间为7 - 15秒，这取决于样品的大小和组合物）。 注:浸没在冷冻溶液不应持续超过需要完全冻结标本。冻结时间过长会压裂TURE的组织块，而时间太短会造成冰晶形成。一个良好的冰冻标本会变得更白。 </li><li>冷冻样品后，将其放入一个小塑料袋或样品管（50毫升）中，并立即在-80℃或在液氮中保存在冰柜。 </li></ol> 6.原始数据的分析<ol><li>为了控制在小鼠的大小变化，正常化的最终体重（FBW）;无瘤体重;和胎体，器官，并根据该初始体重（IBW）组织重量（以克表示），以及表达它们为"重量/ 100毫克IBW"。 注意:可替换地，一些研究者正常化肌肉量对于胫骨的长度。 </li><li>使用数据分析软件。找到的归一化权重的平均值和SD。执行与控制和承载C26小鼠之间不成对学生t检验进行统计分析。 注:结果可以相对于身体IBW呈现（和FBW）无肿瘤，肿瘤，器官和组织重量。 </li></ol> 7.肌肉切片<ol><li>低温恒温器内室的工作温度设置为围绕-23℃。 </li><li>允许标本（以前保存在-80°C）的工作温度（一两个小时应该足够）适应新环境。 </li><li>切的多个检体（优选胫前或腓肠肌），并收集它们在载玻片上的8微米厚的切片。 注:以肌肉的中腹区切段。 另外，为了准确起见，切段垂直于所述安装轴线。 </li><li>保持载玻片低温恒温器腔室内部。不要让如果目的是执行IF / IHC研究或酶染色它们解冻。 </li><li>在-80℃下用于进一步分析存储的幻灯片。 </li></ol> 8.评估肌纤维大小的层粘连蛋白免疫（方案1） 19。虽然肌肉切片的H＆E染色代表形态分析有价值的，方便的方法，使用一个IF方法14是显著更快，谦虚比基于E-H＆方法更准确。 H＆E方法描述如下。 <ol><li>无论从低温恒温器或-80℃储存空间中移除的组织切片，并允许他们平衡至室温（约5 - 10分钟）。 注意:为了避免与在小鼠中引发初级抗体相关的非特异性相互作用，最好是在另一个物种要么凸起使用抗体或利用市售的"小鼠 - 上 - 小鼠"免疫检测试剂盒的。 </li><li>浸入预冷的章节编甲醇（-20℃）10分钟。 </li><li>洗在室温下1×PBS中的部分5分钟。 </li><li>除去PBS中并用于免疫组织化学应用的笔以环绕在幻灯片的节。不要让部分在任何时候完全干燥。 </li><li>应用一个合适的封闭缓冲液（5 - 8％FBS的PBS或PBS中的5％山羊血清）到部分在RT 1小时。 </li><li>除去封闭缓冲液。 </li><li>在潮湿的腔室申请任一个的兔抗层粘连蛋白（1:30）或小鼠抗肌营养不良蛋白（1:30）的第一抗体在室温封闭缓冲液的章节2小时（或在4℃过夜）。 </li><li>洗用1×PBS切片5分钟。 </li><li>除去洗涤缓冲液，并在1×PBS中再次洗涤切片5分钟。 </li><li>除去洗涤缓冲液，并应用适当的荧光第二抗体（1:在封闭缓冲液1,000）在潮湿的腔室在RT 1小时至部分。 </li><li>去除二级抗体并用1×PBS洗涤切片5分钟。 </li><li>除去洗涤缓冲液并重复用1×PBS洗涤5分钟。除去洗涤缓冲液并覆盖使用的水基介质与玻璃盖的部分。 </li><li>通过使用染色的部分的数字图像识别肌肉的形态学特征。为了做到这一点，使用倒置荧光显微镜光IF部分（10倍或20倍放大倍率的目标是合适的），以及用数码相机和图像采集软件。将每个数字图像中的比例尺（20 - 30随机字段被推荐用于各肌肉部分），方便的CSA的量化。图像应保存在.TIF格式为与使用ImageJ的程序的图像分析软件兼容。 </li><li>使用ImageJ的宏通过荧光第二抗体在20肌内膜存在区分收缩组织（肌纤维大小）结缔组织。 注:宏和说明可从AUTHORS，理查德·利伯和Shannon布雷姆纳。 </li><li>通过双击桌面上的图标，打开ImageJ的程序。 </li><li>通过一次单击"插件"选项卡中加载CSA宏。向下移动光标通过单击一次选择"安装"选项。 </li><li>从已保存在文件夹中选择IT负载截宏，然后单击"打开"。 </li><li>一旦宏已被打开，点击"文件"，然后"打开"，从工具栏中的ImageJ打开包含比例尺的组织形象。 </li><li>一旦图像被打开，通过点击与直线的图标选择在ImageJ的程序中的"直线工具"。 </li><li>一旦"直线工具"时，上规模栏最左边的结束单击一次，然后在比例尺的最右边的结束单击一次。当正确完成，应该有覆盖在比例尺上的黄线。 </li><li>在确保黄釉W线性仍然覆盖规模栏上，单击一次选择从ImageJ的工具栏中的"分析"选项卡。 </li><li>在"分析"选项卡，将光标向下移动，并通过点击一次选择"设置规模"。 </li><li>在"设置规模"的窗口，不改变"以像素为单位距离"框中的值。在"已知距离"栏中输入的比例尺的已知距离。更改单位的"长度单位"框与比例尺单位（ 即微米=微米）相对应。最后，选择通过单击框"环球"一旦左边的"全球性"。 </li><li>关闭"设置规模"的窗口。 </li><li>打开第一图像通过按键盘上的"O"按钮来测量。观察一个窗口弹出窗口，允许用户查找并选择图像从已保存的文件夹进行测量。通过点击一次，然后点击选择图像"打开"。 </li> &lt;LI&gt;一旦打开，一个窗口应该出现在可接受范围CSA测量和圆的选择。 注意:这些设置通常被保留为默认值。但是，如果希望更改这些设置，一定要保持的值相同的每个连续图像中的实验来衡量。 </li><li>设置CSA范围和圆后，观察覆盖红色像素的图像与"门槛"窗口。调整图像的使用酒吧的门槛中的"阈值"窗口，直到肌肉纤维被准确地红色像素填充。 注:尝试以获得不会触碰彼此的纤维的最大数量;如果纤维是接触，它们会作为一个大的纤维，这将需要下游编辑来测量。 </li><li>设置阈值后，按键盘上的"1"按钮来衡量的肌肉纤维。 </li><li>圆圈黄线个别肌肉纤维。通过图像移动和移除伪像或已经由程序选择双纤维。要做到这一点，选择不点击他们一次纤维结构，然后按键盘上的"D"按钮。这将从测量删除它们。 </li><li>选择并从复制三围"测量值窗口"，并将其粘贴到进行进一步分析表格。 注意:得到的值也可以用于评估纤维分布分析，其通常表示一个有用的参数，以确定肿瘤生长是否促进向更小的肌纤维的转变。 </li><li>通过点击"X"，在窗口的左上角关闭所有在ImageJ的打开的窗口。 </li><li> 8.29 - 打开一个新的形象，按"O"，并重复步骤8.24来衡量。 </li></ol> 9.从H＆E染色切片肌纤维大小的评估（方案2） <ol><li>放置玻璃含有过滤哈里斯苏木8分钟滑入科普林罐子。 注:或者，如果可以不那么激烈的污渍需要使用迈耶苏木。 </li><li>冲洗在去离子水中的幻灯片。 </li><li>浸在自来水中的幻灯片长达5分钟。 </li><li>快速冲洗他们在去离子水中，然后孵育它们在过滤的1％伊红溶液为小于2分钟。 注意:如果一个更强烈的染色是期望的，加入1 - 乙酸2滴（0.01％或更少）的曙红溶液。 </li><li>开始脱水步骤。冲洗70％的乙醇的幻灯片不到1分钟。 </li><li>浸在90％乙醇的幻灯片1分钟。 </li><li>浸在95％乙醇的幻灯片1分钟。 </li><li>蘸100％乙醇的幻灯片2分钟。 </li><li>浸在二甲苯幻灯片2分钟。移动幻灯片含有新鲜二甲苯2分钟以上另外科普利罐子。 注意:保持在二甲苯的幻灯片（不超过1小时），直到它们被盖上盖玻片。 </li><li>放置coversliPS使用Permount或选择的基础二甲苯安装介质中的肌肉部分。 </li><li>观察H＆E染色载玻片用放大20倍的显微镜下，并获得整个肌肉部分区域的照片。通过使用一个明亮的光显微镜，数码相机和图像捕捉软件获取图像。 注意:在相同的放大倍数下拍摄一个微米的比例尺或图像的存在将要求确定的CSA。如上所述，至少20 - 30每块肌肉部分随机领域。 </li><li>评估使用ImageJ软件肌纤维的大小。打开所有的数字图像。设置如上述的比例。测量每使用手绘选择工具的肌肉至少1,500-2,000肌纤维，跟踪用鼠标纤维周围或更快的输入，与平板输入设备和一支笔。 </li></ol> 10.数据收集和分析<ol><li>为了建立肌肉纤维大小，评估的平均纤维"区域。"计算为每个肌肉，包括的面积和的Feret直径获得的测量装置和SD。另外，为了评价是否实验设置与朝任一较小或较大的肌纤维的移位相关联，分析了"纤维分布"。 </li><li>通过为两个组进行不成对t-检验评价结果的意义。确定用于C26小鼠组和对照组所获得的值之间的比率。上绘制报告手段SD和频率分布/柱状图旨在展示在纤维浸润转移的图表中的值。 </li></ol>

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+exercise+training+attenuates+wasting+of+the+extensor+digitorum+longus+muscle+in+mice+bearing+the+colon-26+adenocarcinoma.">Resistance exercise training attenuates wasting of the extensor digitorum longus muscle in mice bearing the colon-26 adenocarcinoma.</a> Biol Res Nurs. 2, 155-166 (2001).</li><li>Bonetto, 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=JAK/STAT3+pathway+inhibition+blocks+skeletal+muscle+wasting+downstream+of+IL-6+and+in+experimental+cancer+cachexia.">JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia.</a> American journal of physiology. Endocrinology and metabolism 303. 303, E410-E421 (2012).</li><li>Samuels, S. 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=Liver+protein+synthesis+stays+elevated+after+chemotherapy+in+tumour-bearing+mice.">Liver protein synthesis stays elevated after chemotherapy in tumour-bearing mice.</a> Cancer Lett. 239, 78-83 (2006).</li><li>Cornwell, E. W., Mirbod, A., Wu, C. L., Kandarian, S. C., Jackman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C26+cancer-induced+muscle+wasting+is+IKKbeta-dependent+and+NF-kappaB-independent.">C26 cancer-induced muscle wasting is IKKbeta-dependent and NF-kappaB-independent.</a> PloS One. 9, e87776 (2014).</li><li>Penna, 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=Muscle+wasting+and+impaired+myogenesis+in+tumor+bearing+mice+are+prevented+by+ERK+inhibition.">Muscle wasting and impaired myogenesis in tumor bearing mice are prevented by ERK inhibition.</a> PloS One. 5, e13604 (2010).</li></ol>

The authors have nothing to disclose.

特别是在其最新的阶段，大肠癌与恶病质的发展，这是负责不良预后和患者的生活质量降低有关。许多研究都集中在继发于癌症疾病的治疗;然而，尽管在这个方向上许多努力，但仍是癌症恶病质21没有批准的治疗。因此，当务之急是动物模型，以便最大限度地发现的翻译尽可能类似于人体病理学。 C26荷瘤小鼠是癌症恶病质22-24的一种常用的实验模型。这种模?...

肌肉消瘦是各种临床病症，如癌症，败血症，肝，肝硬化，心脏和肾功能衰竭，慢性阻塞性肺疾病，与爱滋病的严重并发症。特别是，肌肉消瘦在癌症患者1的至少50％是显而易见的。在从增加的蛋白质降解由于骨骼肌蛋白水解系统和/或从的过度活化癌症结果骨骼肌损失蛋白质合成6下降。脂解也是明显的，导致脂肪组织的消耗。在临床上，恶病质与减少质量和生活的长度相关联，并且估计为死亡的20的原因-癌症患者7的30％。实验模型，尽可能地类似于人类疾病的使用将是有益的。最佳动物模型的特点是高的再现性，以及由来自不同疗法有限干扰和不可预知的因素饮食，性别，遗传背景，通常是与临床病症8相关联。到目前为止，癌症恶病质已经主要研究了在动物模型特征在于癌细胞或致癌物注射的移植，虽然新的方法是使用转基因小鼠易患癌症的发展。 荷的C26癌（也被称为结肠-26和腺癌）代表癌症恶病质2,5的充分表征的和广泛使用的模型。的C26肿瘤导致的身体和肌肉重量损失的生长，主要是通过加强脂肪和蛋白质分解代谢9。一般地，10％的肿瘤重量与总体重与减少的骨骼肌重量20-25％和脂肪3,10更大消耗相关联。肝脾肿大也与肿瘤生长观察，与急性期反应的活化和促infla的高度沿mmatory细胞因子水平3,11。在这些中，它是众所周知的，IL-6起着在C26模型中介肌肉消瘦举足轻重的作用，即使该细胞因子可能不是恶病质12的唯一诱导剂。升高的IL-6引起肌肉萎缩通过JAK / STAT3通路的活化，并且抑制该转录因子能够防止肌肉萎缩3,4。 期间C26诱导肌肉消瘦，如肌肉萎缩的许多条件，肌肉质量在很大程度上是通过横跨肌纤维在肌肉蛋白质含量减少丢失，不通过细胞死亡或纤维13的损失。在C26恶病质，向更小的截面积的移位在两个糖酵解和氧化纤维2被观察到。这也具有降低的肌力5一致。全球许多团体，以便及时发现癌症CAC肌肉萎缩的新的调解员或临床相关的药品采取了C26型号的优势河虾。然而，对于使用这种模式的许多不同的程序已经报道，提高对所获得的数据的一致性的担忧，并在不同的实验条件下构成障碍重复性。这里，我们报告的产生标准化的和可重复的数据癌症恶病质的研究中通常会使用此模式。

<table><tbody><tr><td>Cell culture Flasks</td><td>Falcon - Becton Dickinson</td><td>35-5001</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>10-017-CV</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>26140</td><td></td></tr><tr><td>Streptomycin-Penicillin&amp;nbsp;</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>CD2F1 mice</td><td>Harlan</td><td>060</td><td></td></tr><tr><td>Anesthesia apparatus</td><td>EZ-Anesthesia</td><td>EZ-7000</td><td></td></tr><tr><td>2-Methyl Butane</td><td>Sigma-Aldrich</td><td>M32631</td><td></td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1850</td><td></td></tr><tr><td>Cork disks</td><td>Electron Microscopy Sciences</td><td>63305</td><td></td></tr><tr><td>Superfrost plus glass slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Anti-Laminin Rabbit polyclonal antibody</td><td>Sigma-Aldrich</td><td>L9393</td><td></td></tr><tr><td>Anti-Dystrophin Mouse Monoclonal antibody</td><td>Vector Laboratories</td><td>VP-D508</td><td></td></tr><tr><td>Alexa Flour 594 anti-mouse IgG</td><td>Life Technologies</td><td>A11062</td><td></td></tr><tr><td>Alexa Flour 594 anti-rabbit IgG</td><td>Life Technologies</td><td>A21211</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>GHS216</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich</td><td>HT110332</td><td></td></tr><tr><td>Xylene</td><td>Acros Organics</td><td>422680025</td><td></td></tr><tr><td>Cytoseal-XYL</td><td>Thermo</td><td>8312-4</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Observer.Z1&amp;nbsp;</td><td></td></tr><tr><td>Bamboo Tablet</td><td>Wacom</td><td>CTH-661</td><td></td></tr><tr><td>Prism 7.0 for Mac OS X</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr><tr><td>Excel for Mac 2011</td><td>Microsoft Corp.</td><td></td><td></td></tr><tr><td>ImageJ</td><td>US National Institutes of Health</td><td>IJ1.46</td><td>http://rsbweb.nih.gov/ij/download.html</td></tr><tr><td>Microtainer</td><td>BD</td><td>365873</td><td></td></tr></tbody></table>

the colon-26 carcinoma tumor-bearing mouse as a model for the study of cancer cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

C26肿瘤生长动力学显示第一个7滞后相位 - 注射后8天，随后指数细胞生长（4 - 5 D）。肿瘤块最终到达〜体重的10％（约2克; 图 1A-B）。在第一阶段中，肿瘤可以仅触诊被定位和显示为皮肤的一小突出部。在第二阶段中，肿瘤被观察为在皮肤下质量。很少，肿瘤变得溃烂，导致一个开放的伤口;在这种情况下，鼠标从实验组中排除，并且安乐死。 </p...

Watch this Scientific Journal Video about The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia at JoVE.com

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

the-colon-26-carcinoma-tumor-bearing-mouse-as-model-for-study-cancer

Research

JoVE Journal

Cancer Research

16.1K 次观看.  Indiana University School of Medicine. Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting. 

视频: The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

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

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

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. ...

科研

癌症研究

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 ...

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

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

医学

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 ...

多细胞球体的肺内接种以模拟原位 NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

作者聚焦：建立小鼠非小细胞肺癌模型以开发抗癌药物纳米制剂

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

摘要

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此视频中的章节

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

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

遗传工程小鼠模型的散发性结肠直肠癌

结直肠癌原位小鼠模型

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

门静脉注射结直肠癌类器官研究肝转移基质

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

摘要

探索更多视频

此视频中的章节

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

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

遗传工程小鼠模型的散发性结肠直肠癌

结直肠癌原位小鼠模型

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

门静脉注射结直肠癌类器官研究肝转移基质

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

模拟疾病进展和预测药物活性的非小细胞肺癌原位异种移植模型的构建

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）