This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.

坚持上新加坡国立大学动物使用和处理（协议号084/12）的机构动物护理和使用委员会（IACUC）的指导方针所有动物的工作。 1.成长MB49-PSA细胞在体外和测量PSA分泌<ol><li>保持小鼠膀胱癌细胞MB49 - PSA 15在完整的Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），2毫摩尔/升L-谷氨酰胺，和0.05毫克/毫升青霉素-链霉素在培养箱在37 ℃和5％的CO 2。加入200微克/毫升潮霉素B至保持的PSA分泌细胞的选择压力。 </li><li>以确定的PSA分泌，板1×10 6个细胞在6孔培养板中，在5％的CO 2存在下48小时，在37°C孵育它。 </li><li>两天后，收集的PSA测量上清。 <ol><li>使用巴斯德吸管，转移SUPernatant到15毫升离心管中。 </li><li>在250 xg离心离心5分钟，除去漂浮细胞和碎片。如果PSA测定是在不同的一天进行，存储在上清 - 30℃下，在1.5毫升微量离心管中。如果不是，则进入立即进行下一步。 </li><li>确定使用无人类- PSA ELISA试剂盒7所述PSA浓度。 </li></ol></li><li>枚举铺的细胞。 <ol><li>使用巴斯德吸管，洗涤细胞轻轻地用1毫升的DMEM。吸并完全取出介质。 </li><li>添加1毫升的新鲜培养基中，用细胞刮刀轻轻刮去，以从井中移走细胞。 </li><li>转移细胞到一个离心管中并彻底重新悬浮它们确保单细胞悬浮液。 </li><li>混合细胞悬液和0.4％台盼蓝溶液的等体积。计数使用血球活细胞（不占用染料）。 </li></ol></li><li>计算PSA表达为NG的PSA /毫升培养基/ 10 6个细胞。 MB49 - PSA细胞用于在小鼠中植入的PSA表达为80 - 120毫微克/毫升/ 10 6个细胞。 </li></ol> 2.确定PSA测量用ELISA和实时荧光定量PCR分析灵敏度<ol><li>板MB49 - PSA细胞在完全DMEM培养基中以不同量的亲MB49细胞的6孔培养板上，使得有一个最大每孔1×10 6个细胞（ 即 10 0，10 1，10 2，10是3，10 4，10 5或10 6个 MB49 - PSA细胞与10 6个培养，分别为10 5，10 4，10 3，10 2，10 1，或10 0 MB49细胞）。 </li><li>一天后，收集PSA测量上清液，如步骤1.3所述。确定使用无人类- PSA ELISA试剂盒7所述PSA浓度。 </li><li>提取从细胞中的RNA。 <ol><li>裂解细胞直接在板中加入1毫升的RNA提取试剂。 </li><li>孵育在室温下5分钟，细胞裂解物转移到一个离心管中。添加0.2毫升氯仿中并剧烈涡旋振荡样品15秒。孵育它们在室温下3分钟。 </li><li>离心样品以12,000 xg离心在4℃下15分钟。小心转移上层水相（0.5mL）中，以一个新的试管中，而不会干扰相间。 </li><li>加入0.5毫升的异丙醇中。孵育在室温下10分钟。离心样品以12,000 xg离心在4℃下10分钟。完全除去上清液，而不会在该管的底部干扰RNA的沉淀物。 </li><li>用1mL的75％乙醇洗涤沉淀一次。离心机7500 xg离心在4℃下5分钟。去除所有的乙醇并允许沉淀风干10分钟。 </li><li>溶解在不含核酸酶的水30微升的RNA。孵育5分钟，在60℃，以增加这样lubility。 </li><li>通过使用紫外（UV）光谱法在260nm（A260）测定的吸光度定量测定RNA。为了评估RNA的纯度，测量在280nm（A280）的吸光度，并计算其应该高于1.6的A260 / A280比值。 </li></ol></li><li>从RNA 2微克反向转录基因。 <ol><li>制备在冰上反应混合物，并将其转移到0.2毫升试管中。 </li><li>短暂离心管，以降速的内容和消除气泡。 </li><li>加载管放入热循环，并执行反转录在下列条件:在37℃下60分钟，然后用5分钟，在95℃。一旦运行完成，保持cDNA样品在4℃下。 </li><li>存储将样品在 - 30℃下长期贮存。 </li></ol></li><li>执行为PSA和细胞质beta肌动蛋白实时PCR分析。 β肌动蛋白被用于标准化样品。在96孔PLA上执行100纳克逆转录RNA的测定德。测定一式三份的所有样本。使用以下参数进行40个循环:在50℃下，在95℃10分钟2分钟，15秒，在95℃下变性，在60℃1分钟。在循环阈值35（CT）设置最低检测限;因此，有超过35 CT值的样本被认为是检测不到的。 </li></ol> 3.维护MB49-PSA细胞的致瘤性注:长时间在体外生长导致肿瘤发生的损失。以维持致瘤性，在MB49 - PSA的细胞系是通过鼠标每2年传代至少一次。 <ol><li>细胞。 <ol><li>收获指数通过用无菌细胞刮轻轻刮他们生长的细胞。混合细胞悬液和0.4％台盼蓝溶液的等体积。算使用血球的活细胞。不植入如果细胞的20％以上都死了。 </li><li>计算活细胞的需要（1×10的量7个细胞/毫升），并将它们转移到一个15毫升离心管中。离心机在4℃下250×g离心5分钟并弃上清。重悬在DMEM空白介质细胞沉淀。重复洗涤三次植入前除去的FBS的所有痕迹。 </li><li>保持细胞悬液在冰上，直到老鼠是准备植入。 </li></ol></li><li>植入肿瘤细胞皮下的背侧的小鼠。 <ol><li>麻醉使用麻醉剂氯胺酮和美托咪定（分别为75毫克/公斤和1毫克/千克）的混合物中的4-至6周龄C57BL6 / J小鼠，以每10克体重0.1毫升腹膜内注射。 </li><li>剃须右翼暴露皮肤。 </li><li>用一对镊子，提起皮肤，将其从下面的肌肉皮下用24克针分开并注入0.1毫升的细胞悬浮液（1×10 6个细胞）。同时除去针，捏注射部位为5至10秒，以使肿瘤细胞做不会泄漏注射部位。 </li><li>在每10克体重0.1毫升恢复与一个皮下注射阿替美唑（1毫克/千克）的小鼠。 </li></ol></li><li>通过测量使用卡钳肿瘤直径监测肿瘤的生长每两天。肿瘤体积是用式V =（AB 2）/ 2，其中"a"是最长尺寸和"b"是在垂直宽度，无论是在毫米计算。 </li><li>当肿瘤体积为至少50 立方毫米（约7天），实施安乐死用CO 2鼠标。切除肿瘤肿块，在无菌条件下一双无菌镊子和剪刀和地点的DMEM。 </li><li>在6孔培养板，剁碎的肿瘤成用无菌手术刀小块。使用1毫升注射器活塞作为"杵"，进一步分解肿瘤。 </li><li>在培养箱中添加3mL的完全DMEM中并保持细胞在37℃和5％的CO 2。 </li><li>一旦细胞坚持开发平台E（一天和两天之间），通过用无菌巴斯德吸管轻轻抽吸移除介质，碎屑和非贴壁细胞。用DMEM洗两次。小心不要扰乱细胞。 </li><li>添加1毫升的DMEM和通过用细胞刮刀刮他们收获细胞。以选择并展开单个菌落，计数用每孔血球和板1细胞在96孔培养板中的细胞。培养的细胞在DMEM 200微克/毫升潮霉素在37℃和5％的CO 2。 </li><li>更改媒体及抗生素，每4 - 5天。监测细胞直到它们在约80-90％汇合。重板上，通过移液在24孔培养板中的细胞，以去除从井中的细胞。 </li><li>一旦在24孔培养板中的细胞汇合时，通过用细胞刮刀刮撞出它们，并在6孔培养板中板它们。 </li><li>屏幕PSA分泌的不同克隆，如在步骤1中说明超低温保存最高PSA密克隆离子，并用它为将来小鼠实验。 </li></ol> 4.植入肿瘤注意:每个小鼠在50的DMEM微升在膀胱空白介质植入1×10 5 MB49 - PSA的细胞。由于在导管的死空间，始终准备额外体积（至少100微升每只小鼠额外）。另一种方法是如由卡斯曼等描述使用空气填充的注射器。 5，而不是填充注射器。 <ol><li>细胞。 <ol><li>植入前一天，当细胞是大约80％汇合时，通道中的MB49 - PSA肿瘤细胞在完全DMEM培养基（补充有10％FBS，2毫摩尔/升L-谷氨酰胺，0.05毫克/毫升青霉素 - 链霉素，和200在37℃微克/毫升潮霉素B）和5％的CO 2以1:1的分流比:2。 </li><li>上的程序的当天，通过用无菌细胞刮轻轻刮他们收获细胞。混合细胞悬浮液等体积和0.4％台盼蓝溶液。算使用血球的活细胞。不植入如果细胞的20％以上都死了。 </li><li>计算活细胞的需要（2×10 6个细胞/ mL）的量，并将其转移到一个15毫升离心管中。离心机在4℃下250×g离心5分钟并弃上清。重悬在DMEM空白介质细胞沉淀。重复洗涤三次植入前除去的FBS的所有痕迹。 </li><li>保持细胞悬液在冰上，直到老鼠是准备植入。 </li></ol></li><li>允许4-至6周大的雌性C57BL / 6J小鼠的程序前适应环境一周。所有动物工作必须在生物安全柜中进行，以保持无菌条件。 <ol><li>称量每只小鼠，并在每10克体重0.1毫升注射麻醉（75毫克/千克氯胺酮和1mg / kg的美托咪定）腹膜内。 Bodyweights应17和22 g的。通过观察没有respo确认麻醉NSE后脚趾捏。 </li><li>对于识别，使用标记耳冲耳。 </li><li>腹腔注射哈特曼的溶液或复方乳酸钠以每10克体重每1毫升0.1 -水化2小时。 </li><li>应用无菌眼药膏双眼用无菌棉灯泡，因为眨眼反射被麻醉下丢失，眼睛干燥。必要时重新申请。 </li><li>躺在上的热包（由与空气接触被激活）以维持体温的顶部纸巾仰卧位的小鼠和向下胶带的后腿。 </li></ol></li><li>用手指，施加轻柔的压力，下腹部区并收集从膀胱尿到1.5毫升离心管中。此示例作为尿PSA的基础值。 </li><li>撤回无菌多聚-L-赖氨酸（PLL），到1毫升的注射器，并附加一个24号静脉导管，与针管心针除去。使用润滑剂涂抹到导管和插件的尖端使用镊子引导导尿ERT通过尿道导管进入膀胱。感觉到阻力时停止。注意:研究人员应该与他们的需要使用润滑凝胶与麻醉剂膀胱灌注和使用后的程序镇痛药的兽医人员讨论。 </li><li>灌输锁相环50μL的缓慢，在10微升每20秒的速率，以避免膀胱输尿管回流18。留在膀胱导管20分钟用塞子防止出流。 </li><li> 20分钟后，取出导管，并通过在下腹区域轻轻按压腾出的任何内容的膀胱。使用空1毫升的注射器，刷新任何剩余的内容出了导管。 </li><li>混合MB49-PSA细胞彻底吹打，并将它们退回到1毫升注射器。连接导管和应用润滑剂。灌输1×10 5个细胞的50微升以10微升每20秒的速度缓慢。更换制动器上导管。给对照组小鼠生理盐水50μL。 </li><li> 1小时后，取出导管和腾中的任何内容膀胱。后肢上取下胶带，然后将老鼠在他们的腹侧面。恢复通过皮下注射以每10g体重0.1mL的逆转药物（1mg / kg的阿替美唑）的小鼠。 </li><li>直到蠕动观察监视老鼠。返回小鼠它们的笼子。 </li><li>在肿瘤检测协议（第5,7或8），安乐死使用的 CO 2的小鼠的完成。验尸肿瘤检测第6节中描述。 </li></ol> 5.监测肿瘤生长与ELISA 注:肿瘤的存在和生长的小鼠尿液中测量PSA分泌监控。研究人员应与监测动物健康和福祉肿瘤植入后和人性化的端点的兽医人员进行咨询。 <ol><li>有两种方法从小鼠收集尿液。 <ol><li>通过将单个小鼠个体中的代谢笼设计为从粪便物质中分离尿收集尿液过夜。如果给定了不具有冷藏，添加蛋白酶抑制剂（100微升）到尿液收集管，以防止粘合剂的降解过夜。然而，可用的代谢笼的数目限制尿液收集的该方法的效用。 </li><li>其中，它是逻辑上不能使用代谢笼（如在ABSL2间），通过使用手指施加的下腹部区域温和的压力，同时在步骤4.2.1中描述的小鼠麻醉收集点尿。治疗灌输之前这通常完成。根据我们的经验，尿液至少150微升，可采集麻醉后1小时。 </li></ol></li><li>立即置于冰上收集的尿液。血尿可以是从肿瘤植入后的第二周可见。高度溶血的样本可能影响ELISA分析。因此，尿必须放置在冰上，收集之后快速地处理。 </li><li>离心尿管在6700×g离心5分钟，在4℃以除去细胞或碎片。 </li><li>正常化在小鼠之间尿量，等分试样的差尿到新的管中，它们在-30℃下储存直到使用测定试剂盒7进行的分析利用ELISA试剂盒7肌酸酐PSA和。尽管小鼠不产生PSA，有使用ELISA结合检测非特异性的低水平。对于被认为阳性样品中，阈值必须在值比正常小鼠+3标准偏差（SD）15更大来设置。 </li></ol> 6.检测肿瘤存在下用实时PCR <ol><li>在终止时，解剖小鼠的腹腔，并定位膀胱。切除膀胱，并把它变成一个离心管。立即冻结在液氮中。 </li><li>通过在RNA抽均质化组织中提取的RNA行动试剂。 </li><li>为粘合剂进行逆转录和实时PCR分析中，如在第2如上所述。 </li></ol> 7.监测肿瘤生长与荧光成像<ol><li>标号1×10 7 MB49 - PSA细胞与近红外荧光染料19。 </li><li>重板和收获18日4，7，11，14，标记的细胞，和21，以检查是否细胞保留活性和荧光，流式细胞仪20。 </li><li>植入小鼠膀胱标记MB49-PSA细胞，在第4节如上所述。 </li><li>监测使用荧光成像系统，每两天的肿瘤生长。 </li><li>上成像的天，称重，并使用麻醉剂氯胺酮和美托咪定（分别为75毫克/公斤和1毫克/千克）的混合物中，在每10克体重0.1毫升腹膜内注射麻醉小鼠。 </li><li>剃腹部区域以暴露皮肤。 </li><li>放置小鼠在成像室，并获得使用设置有成像系统的软件气囊的图像。 </li><li>恢复通过皮下注射以每10g体重0.1mL的逆转药物（1mg / kg的阿替美唑）的小鼠。 </li></ol> 8.监测肿瘤生长与高频超声成像<ol><li>植入肿瘤的小鼠，在第4节如上所述。 </li><li>每隔两天，使用超声成像监测肿瘤的生长。 </li><li>上成像的天，称重，并使用麻醉剂氯胺酮和美托咪定（分别为75毫克/公斤和1毫克/千克）的混合物中，在每10克体重0.1毫升腹膜内注射麻醉小鼠。 </li><li>剃腹部区域以暴露皮肤。 </li><li>灌输100微升无菌0.9％盐水的成使用24号静脉导管膀胱。生理盐水将扩张膀胱和超声成像过程中提高知名度。 </li><li>放置超声平台上的鼠标和应用传导凝胶为L奥尔腹部。 </li><li>降低手持式探头对皮肤和成像平台21上获得的膀胱B模式图像。 </li><li>在每10克体重0.1毫升恢复通过皮下注入逆转药物（1mg / kg的阿替美唑）的小鼠。 </li></ol>

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The authors have nothing to disclose.

在该协议中最关键的步骤是:1）成功地保持了细胞系的致瘤性; 2）确保小鼠肿瘤细胞植入前测PSA的分泌; 3）产生用于植入的单细胞悬浮液，从而减少肿瘤大小的变化;和4）灌输细胞以缓慢的速度，以防止膀胱输尿管回流，导致在肾肿瘤细胞植入。 体外延长传代后，MB49 / MB49 - PSA细胞可失去其致瘤性，这表现在它们不能在小鼠中产生肿瘤。这可能是一个混杂因素，以是否小鼠治疗后固化。在没有肿瘤，它是不可能区分由于免疫原性的肿瘤或从小鼠没有肿瘤自发性固化小鼠通过疗法治愈的小鼠，因为在所有的细胞系是无法形成肿瘤。对付这个的一种方式是通过产生苏重新通道小鼠细胞B-皮肤肿瘤。如果该重新传代过于频繁进行，会产生过于侵略性的肿瘤。一种细的平衡是需要的确定，当细胞需要被重新传代。一般地，前一大系列的实验中，细胞系恢复，然后在小鼠中皮下植入，以确认其形成肿瘤的能力。的肿瘤数周监控，以确保不存在自发治愈。然而，如果观察到自发治愈，应该执行则在步骤3中的协议。同样，MB49 - PSA细胞产生的PSA的能力应始终被用于肿瘤植入细胞之前确认。 尿的PSA可用于监视使用MB49 - PSA细胞建立膀胱肿瘤的生长。然而，负的或"正常的"尿的PSA值并不一定意味着不存在肿瘤细胞，但只有小于1×10 5个细胞存在。尿PSA是大约4当日则计算到第7天，这似乎没有肿瘤的小鼠，根据正常小鼠+3 SD的值，可以从实验中移除。一些这些小鼠可在稍后的日期开发肿瘤。无肿瘤去除小鼠确保肿瘤的大小开始任何治疗之前非常相似。这减少了对治疗的反应，由于肿瘤大小的变化。肿瘤大小的变化的一个主要原因是植入前细胞的聚集，所以应该努力以植入前重新悬浮细胞。 然而，当使用小鼠的小号码，可能有必要将所有的小鼠在研究中，即使肿瘤不是由天4可测量 - 7.在第11天监测PSA，它仍可能进行分类这些小鼠为有过小肿瘤，而且可以相对于被执行的初始肿瘤大小（基于可测量尿的PSA的天4的存在- 7 与第11天）终点分析。是能够量化肿瘤的优点用不同的PSA值是所有的小鼠可以被包括在研究中，以及对治疗的反应可基于相对于原发肿瘤大小他们的PSA /肌酐值来确定。另一个值得关注的一点是膀胱输尿管反流。这可能会导致肿瘤发展的肾脏。减少待从100μL的灌输到50微升的体积并进行滴眼慢慢减少发生这种情况的可能性。 尿基测定法有关的一个问题是降低了尿液收集观察到原位肿瘤在膀胱生长。而尿可以容易地从小鼠中的最初几周植入后收集，用时间，尿液收集可能不足以为PSA和肌酸酐测量的体积。所需的尿的最小体积是110微升，但更多的是更好的。因此，PSA读数可能不适用于在稍后的时间点，所有小鼠。另外，在尿液中的红血细胞的裂解给出虚假高读数在PSA ELISA。非尿基试验可以克服这个问题。不幸的是，虽然荧光成像是使用简单，我们发现，小鼠饲料有红色荧光为好。这给予了很高的背景资料，其中膀胱不能轻易看到。可能的是使用绿色荧光染料或蛋白质可以克服这个问题，如由理发师等人所述。 10对于这两个荧光和超声波，小鼠需要腹面上被剃光。 超声图像似乎是在定位肿瘤PSA基因表达好。然而，超声成像的缺点是，囊必须在成像期间被鼓起以获得良好的肿瘤可视化。而图像清晰时，膀胱肿瘤是大的，这是很难从没有肿瘤分化小肿瘤的小鼠。已经报道了超声波可检测的肿瘤的直径22大约为1.5毫米，但在第4天植入后，肿瘤比这要小很多。因此，该方法可能不是良好的检测上天肿瘤4. 表1中的3种不同的肿瘤的监测系统进行比较;所述PSA ELISA是最简单的使用，并且不需要的小动物的成像系统，这可能无法在所有的动物设施。 给定的PSA基因表达的实时PCR分析的更高的灵敏度，它可以在免疫组织化学的地方用于确认是否在实验结束小鼠治愈。免疫组化，0.2的肿瘤- 0.3 mm的植入后的15到第4天检测。但是，这是不能被用于监测植入肿瘤终点分析法。在这项研究中，磁共振成像，没有被评估，但它已被报道能第10天植入8后，以确定肿瘤的存在。通过修改细胞以表达绿色荧光蛋白（GFP），它我S可能通过第7天，以确认肿瘤植入早期尿粘合剂的组合，以检测肿瘤植入和实时PCR分析在终止提供不同的治疗组间肿瘤植入和治疗效果的确认。 我们的协议的缺点是主要的ELISA测定法无法确认肿瘤的存在时本细胞的数目少于10 5个细胞。从而更灵敏的分析方法需要开发以及更快测定，以减少所需的PSA检测的时间。 ELISA需要几个小时才能完成。一种快速方法，如它是基于所述贾菲方法23和大约需要5分钟来执行，将大大简化肿瘤生长的监测肌酸酐测定。为此，被评价为检测的PSA活性的基于酶的方法，但它没有成功。使用的胶体金以及基于纳米颗粒分析系统可以减少进行测定中所用的时间次增加灵敏度24。在这方面，基于电极的传感器和使用纳米颗粒的电化学基于传感器已被开发以测定人PSA 25-27和如果成本合理可以适用于动物模型。该测定系统也可以通过使用不同的蛋白质分泌系统，例如其中最佳的条件下进行体外测定，而不是取决于哪个是依赖于组织氧合体内成像GFP或萤光素酶的改善。 而在小鼠中化学致癌与相似的人类癌症中的突变28方面产生肿瘤，它们需要更长的时间，以产生与不具有可比性的动物之间的大小，需要更大的小鼠其增加实验的成本的数字。当靶向剂识别人的蛋白质进行评价，人异种移植物的皮下植入裸鼠或重症联合免疫缺陷（SCID）小鼠是唯一的选择。虽然这些模型提供抗体识别的概念验证，缺乏一个完整的免疫系统的阻碍免疫激活的评估。一种人源化小鼠模型使用人类异种移植29膀胱癌新的发展，确实提供了一些见解免疫，但细胞被植入皮下实际上它并不概括人类疾病的环境。这在技术上是难以产生在免疫缺陷小鼠原位肿瘤。因此，尽管有其局限性同基因原位模型仍是评估和治疗的发展膀胱癌的一个很好的模型，因为它提供了正确的组织位置使控制暴露疗法模拟临床情况，并评估宿主免疫细胞的影响的能力癌症治疗过程中。这一切都与肿瘤快速发展的小鼠，并监测在具有成本效益的模型的肿瘤生长的结果的能力相结合。

该方法的目标是产生鼠正位膀胱肿瘤，并尽可能准确地监测植入的肿瘤，所以没有肿瘤植入的小鼠都没有想到在终点分析已经固化。总体上，所示的方法将减少大量小鼠实验分析的需要，并确保在确定治疗结果更高的精度。 对于癌症的原位模型的发展是重要的，因为植入肿瘤细胞皮下不概括的临床疾病的环境或启用的治疗策略的开发。膀胱的结构允许膀胱癌治疗的滴注直接进入以最小的全身效应膀胱。因此，概括这种环境的动物模型，例如原位模型，是很重要的，以评估新疗法。从任何实验装置得出的结论是dependen在该模型的局限性吨。 几种技术已经开发了用于在小鼠中生产原位膀胱肿瘤。这些依赖于损伤膀胱的糖胺聚糖层，使肿瘤细胞被植入。所使用的技术包括电烙，这导致在膀胱壁损伤的单点，在膀胱1,2-导致肿瘤发展在一个地点。然而，使用电灼肿瘤植入的成功率是取决于运营商从10变 - 90％，并且它包括危险，即膀胱壁将被删截，从而导致肿瘤在腹腔显影。化学烧灼是用硝酸银，这损害了膀胱壁3进行。同样，酸已被用来损伤膀胱壁4。胰蛋白酶也被用来损伤膀胱以及5。这些方法可能会导致在一个以上的肿瘤在膀胱的发展。此外，存在严重损坏的膀胱的危险，如果化学品留在与膀胱壁接触时间过长。通过Ninalga 等人开发的方法。使用带正电荷的聚-L-赖氨酸（PLL）6分子涂覆膀胱壁;这使得带负电荷的肿瘤细胞粘到膀胱的糖胺聚糖层。这种方法通常导致在一个以上的肿瘤在膀胱显影，但肿瘤植入是在80 - 100％的4,7。从技术上讲，它也是以执行的最简单的过程。以保证该开发肿瘤即使在大小相当，它是重要的肿瘤细胞中不植入前大团块分组。 为了评估治疗功效，最好是具有相当类似大小的肿瘤对小鼠进行这项研究。因此，可以在植入后不久量化肿瘤大小良好的检测系统是重要的。几种策略有蜜蜂用于n评估肿瘤。这些包括磁共振成像（MRI）8-10，荧光11，生物发光12,13，超声14，和酶联免疫吸附试验（ELISA）15,16。而MRI和超声不需要的肿瘤细胞中的修改，有必要对敏感设备和造影剂用于MRI。的荧光 - ，luminescence-，和基于ELISA测定所需要的肿瘤细胞的修饰来表达，可以通过这些方法来检测标志物蛋白。为发光，需要用于检测荧光素酶活性的基板;因此，有一个附加的步骤，增加成本。这两个发光和荧光需要专门的设备。为了产生荧光，绿荧光蛋白（GFP）的环化，它是由分子氧催化的，是必需的。因此，GFP表达可能会因访问氧肿瘤块内的变量，使之成为一个相当可靠的指标<sup&gt; 17。鼠膀胱癌细胞系MB49的修饰分泌人类前列腺特异性抗原（PSA）的15,16作为替代标志物是另一种策略。这些标志物还提供确认肿瘤存在在实验结束时，使它们的替代免疫组织化学的替代手段。这项研究报告原位肿瘤植入的PLL方法，并提出肿瘤检测系统，即ELISA，荧光和超声成像的比较。

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:404px;">MB49-PSA cells</td>
			<td style="width:188px;">N/A</td>
			<td style="width:188px;">N/A</td>
			<td style="width:113px;">ref (Wu QH, 2004)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640 media</td>
			<td>HyClone</td>
			<td>SH30027.01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) media</td>
			<td>Biowest</td>
			<td>L0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American, Premium</td>
			<td>Biowest</td>
			<td>S181B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30088.03&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine</td>
			<td>Biowest</td>
			<td>X0550</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin-Streptomycin</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hygromycin B</td>
			<td>Invitrogen</td>
			<td>10687-010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">free PSA (Human) ELISA kit</td>
			<td>Abnova</td>
			<td>KA0209</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TRIzol Reagent for RNA extraction&#160;</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>4374967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Mouse Actb</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Mm00607939_s1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Human KLK3</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Hs00426859_g1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">C57BL/6J female mice</td>
			<td>In Vivos</td>
			<td></td>
			<td>4-6 wk old</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia (75mg/kg Ketamine and 1mg/kg Medetomidine)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reversal drug (1mg/kg Atipamezole)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ear punch</td>
			<td>Electron Microscopy Sciences</td>
			<td>72893-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hartmann&#39;s solution or Compound sodium lactate</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ophthalmic ointment - Duratears sterile ocular lubricant ointment</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat pack - HotHands handwarmers</td>
			<td>Heatmax Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Introcan Certo IV catheter</td>
			<td>B Braun</td>
			<td>4251300</td>
			<td>24G x 3/4&#8243;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aquagel Lubricating jelly</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;Poly-L-lysine solution, 0.01%,</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Quantichrom Creatinine Assay Kit</td>
			<td>BioAssay Systems</td>
			<td>DICT-50&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluorescent dye - VivoTrack 680</td>
			<td>Perkin Elmer</td>
			<td>NEV12000&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNAlater-ICE Frozen Tissue Transition Solution</td>
			<td>Ambion</td>
			<td>4427575</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Realtime PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Software v2.3</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Metabolic Cage</td>
			<td>Tecniplast&#160;</td>
			<td>Vertical type rack for 12 cages</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSCanto I system&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSDiva software v7</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IVIS SpectrumCT in vivo imaging system&#160;</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Living Image Software v3.1</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vevo 2100 imaging system&#160;</td>
			<td>VisualSonics&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine orthotopic bladder tumor model and tumor detection system

这个协议描述膀胱肿瘤的生成中使用鼠膀胱癌细胞系MB49，其已被修饰以分泌人类前列腺特异性抗原（PSA）的雌性C57BL / 6J小鼠，对于肿瘤植入的确认的步骤。简言之，老鼠是使用注射药物麻醉，由背侧位置埋下了伏笔。尿从膀胱腾空和聚-L-赖氨酸（PLL）的50微升缓慢以10微升/ 20秒的速率用24克IV导管灌输。它是由塞住导管留在膀胱20分钟。导管被移除和PLL是通过在膀胱温和的压力腾空。这之后是鼠膀胱癌细胞系以10微升/ 20秒的速度（1×10 5个细胞/50μl）滴注。导管被塞住，以防止过早疏散。 1小时后，将小鼠恢复与逆转药物，和膀胱腾空。缓慢滴注速度是很重要的，因为它减少膀胱输尿管回流，这可能会导致肿瘤在上尿路和在肾脏发生。细胞系应该很好再悬浮，以减少细胞的聚集，因为这可能在植入后导致不均匀肿瘤大小。 这种技术诱导效率高的肿瘤。肿瘤生长是由泌尿系PSA分泌监控。 PSA标志的监测比超声波或荧光成像用于检测肿瘤的膀胱中存在的更可靠。如果不及时治疗4周 - 小鼠肿瘤约3一般达到最大大小负面影响健康。通过监测肿瘤的生长，有可能区分那些未成功地与肿瘤植入被治愈的小鼠。只有终点分析，后者可被误认为已通过疗法治愈。

从MB49细胞的PSA分泌被发现与生长培养基而变化。 MB49 - PSA是在DMEM培养基中生长，因为这导致增加的PSA的分泌（ 图1A）。为了确定所述PSA ELISA和实时PCR的灵敏度，MB49 - PSA分泌细胞的不同数目的用MB49亲代细胞混合。 PSA ELISA检测到至少1×10 5个的PSA分泌细胞/ 1×10 6个细胞（ 图1B），而实时PCR分析可以检测100的PSA分泌细胞/ 1×10 6个细胞（ 图 1C）。因此，实时PCR比ELISA更敏感，但它只能对整个膀胱进行。这就限制了它的效用，以终点分析。在多个肿瘤（ 图2A）在膀胱显影肿瘤植入结果的PLL方法。用尿液代谢笼的两个通宵集合（ 图2B </st荣&gt;，上图）和点尿样品（ 图2B，下图）可用于通过ELISA测量的PSA。对于所有的动物研究中，小鼠重量记为健康（ 图2C，上图）的量度，和尿的PSA每周（ 图2C，下图）作为肿瘤生长的度量监测。通常存在由PSA测量小鼠体重和肿瘤大小之间的良好的相关性。小鼠大的肿瘤开始减肥（ 图2C，老鼠3和5）。在图2C观察到一些小鼠可具有大的肿瘤，但仍显示一个正常的体重。因此，单独体重不是肿瘤生长的足够的措施。 PSA用作肿瘤生长（ 图3A-D）的良好替代标记。小鼠约3安乐死-尿液分析后的5天的PSA第14天膀胱横截面的图像显示肿瘤的大小和相应的第14天PSA /肌酸酐×10 6意图鼎。在肿瘤大小的变化与接收到的小鼠中的治疗。 图3，面板AD代表来自3个不同的治疗组小鼠。另一种策略细胞的PSA修改是用染料电池的标签。这种策略消除了对细胞转化和选择的需要。用荧光染料MB49 - PSA细胞标记是容易进行（ 图4A），并在体外监测表明在该信号的存活方面承诺，尽管细胞的复制（ 图4B和C）。然而， 在体内成像并不成功，因为鼠标饲料也有自然的红色荧光（ 图4D），这导致在腹部区域的非特异性荧光。在超声成像，PSA尿分泌，PSA终点分析之间进行比较。膀胱（ 图5A-H）的超声成像是好的，用于识别大的肿瘤，但它不是与小肿瘤如此成功。 <img alt="图1" src="/files/ftp_upload/55078/55078fig1.jpg" /> 图 1. MB49-PSA细胞分泌PSA和使用ELISA和实时荧光定量PCR分析检测限值的确定。 （A）的 1×10 6检测后第2天的ELISA细胞在上清液中生长在不同的生长培养基，和PSA分泌MB49 - PSA。生长培养基是:RPMI与FBS-（RPMI），RPMI有优质的FBS（RPMI（P）），RPMI一个替代的FBS（RPMI（H）），和的DMEM与FBS（DMEM）中。在DMEM培养基上生长MB49-PSA细胞有较高的PSA分泌。 （B）的 MB49 - PSA的细胞（10 6个细胞至1细胞）与亲代MB49细胞（1细胞到10 6个细胞）混合并铺板24小时。 PSA ELISA法对上清液进行，RNA从细胞中提取。最小为1×10 5 </sup&gt; PSA分泌细胞/ 1×10 6个细胞检测通过ELISA。 （℃）100的PSA分泌细胞/ 1×10 6个细胞通过实时PCR分析可检测的。 y轴值代表平均RQ（相对定量）±标准差。 RQ值是相对倍数变化，由PSA正常化对β-肌动蛋白基因（C T）值的获得，与设置为1。控制膀胱相比， <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55078/55078fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55078/55078fig2.jpg" /> 图2. 膀胱肿瘤生长和检测。 （A）肿瘤移植13天后膀胱的组织学检查。石蜡包埋膀胱切片并用苏木精（深蓝色），该站染色INS细胞核，和伊红（粉红），该污渍剩余的细胞成分。此膀胱天5和13 PSA /肌酐×106值显示为二千六百八十八分之七。该倍率为44.8X。比例尺条为1毫米。 （B）的尿通过在代谢笼中任一放置小鼠收集过夜，并从集合管上的尿转移到2毫升试管（上图）或通过在现场收集尿液从麻醉小鼠（下图）为1.5 mL管，然后将其转移到0.6毫升试管中。 （C）的肿瘤植入后，小鼠重量每周两次（上图）监测。尿的PSA测定来监测肿瘤植入和在小鼠中的生长（下图）。 y轴PSA / CREA表示归肌酐×10 6尿PSA。小鼠终止曾经有的体重超过20％的损失。注:在40日2鼠标的膀胱中检测PSA基因的表达，当它是TERMI<html>经过NAT，即使它具有低的尿的PSA。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55078/55078fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55078/55078fig3.jpg" /> 图3. PSA和膀胱肿瘤的生长。 （AD）19日（A和B）收获小鼠膀胱，一天17（C）和肿瘤植入后13天（D）的组织切片。一天4和第13天或第14天尿PSA /肌酸酐×10 6个值的每个囊的格式日显示在每一个图像的右边4/13天或14的尿的PSA /肌酐值与肿瘤大小增加。在图像AD小鼠接受不同的治疗占肿瘤大小的差异。该倍率为41.4X。比例尺条为1毫米。"https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55078/55078fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55078/55078fig4.jpg" /> 图4.配置文件的用荧光染料标记MB49-PSA细胞。 （A）染色的细胞通过流式细胞仪镀前进行分析。收获标记的细胞每隔几天来分析荧光染料的强度。 （B）在规定天后标签标记MB49 - PSA细胞的百分比在P2栅极内从（A）中 。的信号通过平均荧光强度（MFI）测得的荧光染料（C）的强度。 （D）使用的体内成像系统标记MB49 - PSA细胞移植后10天的小鼠的照片显示，在腹部区域的非特异性荧光。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55078/55078fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55078/55078fig5.jpg" /> 图5.小鼠囊超声图像。肿瘤植入和肿瘤植入后8天（B）的前小鼠气囊（A）中 。 （C）与（H）是小鼠的超声图像植入后水囊两个星期。 PSA / CREA:尿PSA归肌酐（10 6）。 PSA基因:在气囊（日志RQ）的PSA基因表达显示每个膀胱。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55078/55078fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td&gt; <td> 尿PSA </td><td> 荧光成像 </td><td> 超声成像 </td></tr><tr><td> 步骤（需要的时间） </td><td>麻醉小鼠。 收集110微升尿。 （〜1小时） 执行的PSA ELISA（总温育时间:2小时30分钟） 肌酸酐测定（温育时间:5分钟） </td><td>麻醉小鼠。 刮胡子腹部的皮毛。 进行成像。 </td><td>麻醉小鼠。 刮胡子腹部的皮毛。 导尿和灌输100μL的生理盐水注入膀胱。 涂抹导电凝胶下腹及获得超声波图像。 </td></tr><tr><td> 专用设备 </td><td> ELISA板读数器（在450nm和510nm处的波长） </td><td>成像系统</td><td>超声成像系统</td></tr><tr><td> 肿瘤细胞的变形例 </td><td> RequirED（表达PSA） </td><td>必需（荧光） </td><td>不需要</td></tr><tr><td> 优点 </td><td>可靠</td><td>简单快捷</td><td>可检测大肿瘤</td></tr><tr><td> 限制 </td><td>可能得不到足够的尿液 负值并不意味着不存在肿瘤（PSA基因表达分析增加灵敏度，但仅限于结束点分析） </td><td>需要荧光染料的精心选择，以避免过大的背景。 </td><td>无法检测小肿瘤。 一次一个鼠标。 </td></tr></tbody></table> 表1为鼠标的水囊监测肿瘤生长的比较方法。

Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

这个协议描述在雌性C57BL / 6J小鼠和肿瘤生长的监测小鼠原位膀胱肿瘤的生成。

小鼠原位膀胱肿瘤模型和肿瘤检测系统

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

a-murine-orthotopic-bladder-tumor-model-and-tumor-detection-system

Nanyang Technological University

Research

JoVE Journal

Cancer Research

14.5K 次观看.  National University of Singapore, National University Health System. 这个协议描述在雌性C57BL / 6J小鼠和肿瘤生长的监测小鼠原位膀胱肿瘤的生成。 

视频: A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

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

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

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

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

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

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

科研

癌症研究

A Mouse Model of Fatigue Induced by Peripheral Irradiation

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

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

疲劳的小鼠模型的外围照射引起

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

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

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

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

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

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

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

利用小鼠脑肿瘤球的原发染色质沉淀

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

雄激素依赖性和去势抗性前列腺癌的荧光自体小鼠模型

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.