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?...

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><tbody><tr><td>MB49-PSA cells</td><td>N/A</td><td>N/A</td><td>ref (Wu QH, 2004)</td></tr><tr><td>RPMI 1640 media</td><td>HyClone</td><td>SH30027.01&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM) media</td><td>Biowest</td><td>L0102</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) South American</td><td>Biowest</td><td>S1810</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) South American, Premium</td><td>Biowest</td><td>S181B</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>HyClone</td><td>SH30088.03&amp;nbsp;</td><td></td></tr><tr><td>L-glutamine</td><td>Biowest</td><td>X0550</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Biowest</td><td>L0022</td><td></td></tr><tr><td>Hygromycin B</td><td>Invitrogen</td><td>10687-010</td><td></td></tr><tr><td>free PSA (Human) ELISA kit</td><td>Abnova</td><td>KA0209</td><td></td></tr><tr><td>TRIzol Reagent for RNA extraction&amp;nbsp;</td><td>Ambion</td><td>15596026</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td><td>Applied Biosystems</td><td>4374967</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystems</td><td>4304437</td><td></td></tr><tr><td>TaqMan Gene Expression Assay &amp;ndash; Mouse Actb</td><td>Applied Biosystems</td><td>4331182</td><td>Mm00607939_s1</td></tr><tr><td>TaqMan Gene Expression Assay &amp;ndash; Human KLK3</td><td>Applied Biosystems</td><td>4331182</td><td>Hs00426859_g1</td></tr><tr><td>C57BL/6J female mice</td><td>In Vivos</td><td></td><td>4 - 6 wk old</td></tr><tr><td>Anesthesia (75 mg/kg Ketamine and 1 mg/kg Medetomidine)</td><td>Local pharmacy</td><td></td><td></td></tr><tr><td>Reversal drug (1 mg/kg Atipamezole)</td><td>Local pharmacy</td><td></td><td></td></tr><tr><td>Ear punch</td><td>Electron Microscopy Sciences</td><td>72893-01</td><td></td></tr><tr><td>Hartmann&#039;s solution or Compound sodium lactate</td><td>B Braun</td><td></td><td></td></tr><tr><td>Ophthalmic ointment - Duratears sterile ocular lubricant ointment</td><td>Alcon</td><td></td><td></td></tr><tr><td>Heat pack - HotHands handwarmers</td><td>Heatmax Inc</td><td></td><td></td></tr><tr><td>Introcan Certo IV catheter</td><td>B Braun</td><td>4251300</td><td>24 G x 3/4&amp;Prime;</td></tr><tr><td>Aquagel Lubricating jelly</td><td>Local pharmacy</td><td></td><td></td></tr><tr><td>Poly-L-lysine solution, 0.01%,</td><td>Sigma</td><td>P4707</td><td></td></tr><tr><td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>4693159001</td><td></td></tr><tr><td>Quantichrom Creatinine Assay Kit</td><td>BioAssay Systems</td><td>DICT-50&amp;nbsp;</td><td></td></tr><tr><td>Fluorescent dye - VivoTrack 680</td><td>Perkin Elmer</td><td>NEV12000&amp;nbsp;</td><td></td></tr><tr><td>RNAlater-ICE Frozen Tissue Transition Solution</td><td>Ambion</td><td>4427575</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment and Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>7500 Realtime PCR System</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>7500 Software v2.3</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Metabolic Cage</td><td>Tecniplast&amp;nbsp;</td><td>Vertical type rack for 12 cages</td><td></td></tr><tr><td>BD FACSCanto I system&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva software v7</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>IVIS SpectrumCT &lt;em&gt;in vivo&lt;/em&gt; imaging system&amp;nbsp;</td><td>Caliper Life Sciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>Living Image Software v3.1</td><td>Caliper Life Sciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>Vevo 2100 imaging system&amp;nbsp;</td><td>VisualSonics&amp;nbsp;</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</...

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

Research

JoVE Journal

Cancer Research

14.6K 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

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

医学

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

癌地致小鼠膀胱肿瘤的磁共振成像评价

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

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

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

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

小鼠膀胱肿瘤器官的培养、操作和正交移植

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

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Bladder Tumor Organoid Model: A Method for Orthotopic Transplantation of Bladder Organoids into Recipient Murine Bladder

Source: Kim, Y. et al. <a href="https://www.jove.com/video/60469" target="_blank">Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids.</a>&#160;J. Vis. Exp.&#160;(2020)
In this video, we describe the orthotopic transplantation of cultured bladder tumor organoids into the murine bladder to study the role of the organ environment on developing tumors.

膀胱肿瘤类器官模型:一种将膀胱类器官原位移植到受体小鼠膀胱的方法

Source: Kim, Y. et al. Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids.&#160;J. Vis. Exp.&#160;(2020)
In this ...

JoVE Encyclopedia of Experiments

Urinary Tract Cancer

In vivo studies

Modeling Orthotopic Bladder Tumor in Mouse Model: A Procedure for Intravesical Administration of Cancer Cells into Murine Bladder

Source: Tham, S. M. et al. <a href="https://www.jove.com/video/55078" target="_blank">A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.</a>&#160;J. Vis. Exp.&#160;(2017)
In this video, we describe the intravesical procedure of delivering cancer cells into the bladder of a mouse model. This procedure helps generate an orthotopic bladder tumor model that can be used to develop therapeutic strategies.

小鼠模型中原位膀胱肿瘤建模:将癌细胞膀胱内给药到小鼠膀胱中的程序

Source: Tham, S. M. et al. A Murine Orthotopic Bladder Tumor Model and Tumor Detection System.&#160;J. Vis. Exp.&#160;(2017)
In this video, we describe ...