Supported by grants from the Department of Defense (W81XWH-09-1-0127), the NIH (2-R01-CA089569), the Elsa U. Pardee Foundation, a Marsha Rivkin Center for Ovarian Cancer Research Pilot Study Award, and generous philanthropic support from Section of Urology and Section of Research in the Department of Surgery, University of Chicago.

所有小鼠饲养，维护和安乐死根据机构动物护理和使用委员会（IACUC）的指导方针和芝加哥动物资源中心的大学的监督下。 用于实验研究1.准备动物<ol><li>使动物能够适应新的住房和环境，运输和处理潜在的生理影响中恢复过来。 注:下面的技术适用于老鼠的所有商用株。动物用于实验的数目取决于研究设计，并应与从统计学家协商进行。 </li></ol> 2.识别及腹部脂肪仓库隔离。 <ol><li>安乐死通过CO 2过量，并确认死亡辅助物理方法动物。 </li><li>由于收获腹腔脂肪库构成的内部器官摘除（二级方法），MA科中线切口接近腹股沟乳头通过腹膜壁以暴露内部器官（图1A）。 </li><li>对于网膜脂肪（OM），通过腹腔钳（ 图1C）延伸脾脏露出网膜/胰腺复杂。通过仔细修剪所有组织连接释放胰腺和脾大网膜。确保任何剩余的胰腺组织被切除4。 </li><li>对于性腺发（GF），使用镊子解除周围的卵巢和消费税由切割组织连接 （图1A上右击 ）4性腺脂肪。 </li><li>对于子宫发（UF），使用镊子解除周围子宫角和消费税由切割组织连接子宫脂肪（ 图1A右下 ）4。 </li><li>为肠系膜脂肪（MF），切小肠和幽门之间的交界处。使用镊子双手紧紧握住的自由端小肠，慢慢地"剥离"，使其脱离肠系膜脂肪。使用解剖剪刀肠系膜根部释放肠系膜脂肪（ 图1A左下 ）4。 </li><li>为Splenoportal脂肪（SF），利用镊子以暴露组织的连接脾脏胰腺的种脐的薄脂肪酸带抬起脾脏的前端。首先从胰腺释放它，然后小心地从脾解剖它切除splenoportal脂肪（ 图1B）4。 </li></ol> 3.乳斑鉴定使用姬姆萨染色<ol><li>消费网膜（参照步骤2.3）从C57BL / 6小鼠并在4℃下16小时（O / N）的5％的中性缓冲的福尔马林固定。 </li><li>石蜡包埋固定的组织，选择一个模具适合于所述组织的大小。倾熔融石蜡从石蜡储填充模具。打开纸盒，用温暖牛肝菌，转移组织到模具中。 <ol><li>石蜡包埋产生纵向部分在仔细定向组织。将标记的组织盒在成型和用力按压到位。允许模具冷却至少30分钟。 </li></ol></li><li>切从福尔马林固定的石蜡包埋（FFPE）组织块连续切片（4微米厚）。将这一小节中的预清洗过的幻灯片作为组织的色带脱落的蜡块。 </li><li>串行部分全网膜;收集和染色三分之一的部分姬姆萨染色（参见3.6）。允许与部分风干载玻片，优选O / N在室温。干的幻灯片已经准备好固定。染色第一部分为HE染色进行比较，以姬姆萨染色切片。 </li><li>由两个连续的5分钟的孵育在二甲苯Deparaffinize幻灯片。由两个连续的温育在下面再水合幻灯片:100％的乙醇，95％乙醇，最后点击わ之三。 <ol><li>采取适当的预防措施，以避免干燥的幻灯片。执行所有的步骤在室温。 </li></ol></li><li>完全浸没在含有5％吉姆萨溶液科普林缸的幻灯片（w / v的在自来水中制备）和下室温温育4分钟。在冲洗自来水幻灯片60秒，风干，并使用安装介质盖玻片安装。 </li><li>图片使用整个幻灯片扫描仪和过程与制造商的软件染色切片。在量化使用ImageJ软件4姬姆萨染色网膜部分乳斑量。 </li></ol> 4.传播卵巢癌细胞，并准备在体内和体外研究<ol><li>预暖15毫升细胞生长培养基至37℃热浴中的。由标记准备了适当大小的组织培养瓶（例如，75厘米2表面积）与细胞系，通道号，日期以及谁制备的冷冻原液人的姓名，以及日和人谁是解冻/电镀细胞。 注:在转移的研究，例如详细的信息是至关重要的，因为冷冻下来，传代次数的日期会影响转移表型。 </li><li>使用生物安全罩，吸量管10毫升培养基到T-75培养瓶的无菌环境。从液氮系统中删除单元的小瓶和检查标签以确认细胞类型。通过在37℃热浴中温热只解冻一小瓶的细胞在一个时间。 </li><li>使用无菌技术，吸管1×10 6细胞解冻到培养烧瓶中。将烧瓶在孵化器，以允许细胞附着。轻轻摇动烧瓶中来回，以形成均匀的细胞悬浮液。 <ol><li>放置在烧瓶组织培养箱中，在37℃下在5％CO 2的环境维护。使细胞粘附O / N。 </li></ol></li><li>抽吸培养基，并用新鲜的预温热的生长培养基替换。将烧瓶在孵化器和所有流细胞的生长。使用倒置显微镜监测细胞通过视觉检查日生长每2-3天。在达到70-80％汇合使用标准细胞培养技术来传代细胞。 注意:对于实验分析收获细胞在70％汇合，以确保细胞活跃增殖。 </li><li>吸出培养基，并冲洗，用10ml无菌磷酸盐缓冲盐水（PBS）中。吸出PBS中，添加0.25％胰蛋白酶/ EDTA（2至3ml为75厘米2表面积）孵育5分钟，在37ºC。目视确认该细胞脱离并添加生长培养基等体积的，并轻轻吸取细胞上下以获得单细胞悬浮液。 注意:该生长培养基含有血清，因为它失活胰蛋白酶，这一点至关重要。准备至少2至4倍，比所需的给定的实验的数目更多的细胞，以补偿细胞的过程中的后续步骤的损失</li><li>确定活细胞中的百分比通过台盼蓝染色法悬架使用血细胞计数或自动细胞计数。 注意:在这里，只使用细胞制备物，其中细胞≥96％是可行的。 </li><li>从烧瓶中转移细胞悬液至50ml锥形管中。颗粒细胞离心5分钟在1100 XG，在4℃。 </li><li>吸出上清，重悬在PBS中的细胞沉淀以每毫升2×10 6个细胞的浓度。 </li></ol>细胞5.腹腔注射注:在我们的实验中，小鼠在我们的屏障设施内的层流或生物安全柜处理，以限制病原体暴露的风险，为了清楚地演示了这种技术，在影随行的程序进行了认可实验室进行动物的工作。这个特定的技术不需要对动物是在麻醉下。根据批准的方案，执行此techniqUE在活的动物。用小鼠品系适合本研究。例如:使用免疫功能低下，无胸腺裸鼠人类卵巢癌细胞（SKOV3ip.1和HeyA8）定植的研究;使用免疫C57BL / 6小鼠为小鼠卵巢癌细胞（ID8）定植研究。 <ol><li>负载500微升单细胞悬液到注射器的，并把在无菌加盖地下25针。这减少了在注射前细胞剪切。 </li><li>拿起鼠标向上的脖子颈背和保持利用手掌和食指的尾巴和固定环，小指（当鼠标被限制用左手）之间的左后腿。 注意:为了避免创伤腹部器官，抑制小鼠舒服，所以它不能在注射过程中移动。 </li><li>想象横过腹部的线只是在膝盖上方，并定位在动物的右侧的点和靠近中线。进入点是颅并略微内侧的最后乳头。 注意:插入于鼠标的右侧针避免盲肠和减少穿刺肠11的风险。 </li><li>针在小鼠腹部的下部外侧区插入到约0.5厘米的深度。向后拉柱塞，以确认针没有渗透血管或其他腹膜器官。 <ol><li>如果任何流体被吸出丢弃注射器（和样品）11。一个绿色的棕色或黄色的分泌物指示针渗透进入肠道或膀胱，分别。 </li></ol></li><li>用缓慢，稳定的压力注入样品。拔出针头，并返回鼠标笼子里。处置锐器容器之前，不要再戴上注射。 </li><li>通过CO 2窒息和重要器官切除在特定时间点牺牲小鼠注射后。 </li></ol> 6.银河现货殖民体内 <ol><li> Quantita使用流式细胞仪癌细胞本地化乳白色斑点和灰<ol><li>从小鼠分离腹膜脂肪库（如步骤2.3中所述 - 2.7）与注射荧光在冰冷的PBS标记癌细胞和立即的地方组织。 <ol><li>对于这个特殊的技术，重量匹配所有脂肪脂肪组织，以大网膜。转移的组织以分离5毫升试管含有1.5无血清DMEM毫升和0.1％（重量/体积）牛血清白蛋白。来自三个独立的小鼠收获的池组织中，以确保足够的屈服细胞用于流式细胞术。 </li></ol></li><li>用手术剪，并添加剁碎的组织1.5含0.4％（重量/体积）胶原酶一孵育组织悬浮液在37℃下进行30分钟，旋转混合无血清DMEM洗涤。 <ol><li>作为可选步骤，通过素炼进一步离解组织。在这种情况下，组织的胶原酶悬浮液转移到一个microstomacher袋，塑炼对低10分钟，旋转方向5分钟后的袋子。 </li></ol></li><li>通过尼龙网过滤器（60微米孔径）过滤样品，以除去较大的碎屑。通过离心，在250×g离心收集细胞进行5分钟，在4℃下，并丢弃上清液部分。 </li><li>重悬细胞沉淀在100微升PBS加上900微升ACK裂解缓冲液并在室温下孵育1分钟。离心细胞，在250×g离心5分钟，弃上清。 <ol><li>重悬在250微升冰冷的PBS将细胞沉淀。通过一个60微米孔径尼龙网过滤所述样本以确保单细胞悬浮液。冲洗过滤用250μl冰冷的PBS中。 </li></ol></li><li>定量荧光标记细胞的群体中使用流式细胞仪配备有561-nm的黄绿色激光和一个585纳米/ 15纳米的带通滤波器的数量。设置用于基于亲本细胞和/或适当的阴性对照4的分析tdTomato阳性细胞的栅极。 </li></ol&gt; </li><li>微观和宏观转移定量<ol><li>在所需的实验终点（多个），隔离和立即将组织在适当的固定的介质。固定较大的样品，如完好性腺，子宫和肠系膜脂肪库中的10％中性缓冲的福尔马林48小时，在4℃。固定在5％的中性缓冲的福尔马林（子宫和肠系膜脂肪的网膜和splenoportal脂肪，以及组织当量）更小的样品，在4℃下进行2-16小时。 <ol><li>或者，捕捉冷冻组织通过将它们放置在一冷冻介质诸如OCT和落入液氮的适当的量。 </li></ol></li><li>转印固定组织以70％的乙醇，并储存于4℃直至在石蜡包埋。在3.2和3.3描述嵌入和工艺组织。 <ol><li>使用H＆E染色部分，以评估组织为微观转移的存在或不存在（即，簇50个细胞）。确认吨他微观转移由次级方法如IHC染色角蛋白8/18的存在，以检测卵巢癌的细胞。 </li></ol></li></ol></li></ol> 7.乳斑定植离体 <ol><li>建立基本的离体网膜器官培养条件<ol><li>放置每个网膜到单个培养板插入并置于内含有500微升含有20％FCS的DME / F12培养基的12孔组织培养板的一个孔中。在一个5％CO 2的环境中进行24至48小时和/或另外的时间点保持器官培养，在37℃。每个条件（例如，媒体类型/时间点）使用三个独立的网膜（一式三份样品）。 </li><li>为了确认组织的实验端点的完整性，修复和处理样品的步骤3中所述计数与健康有关H＆E部分坏死的脂肪细胞可以评估组织的完整性。最少〜120细胞5生物样品需要配制本10活组织的百分比。 </li><li>为了测量组织的功能，代替网膜（N = 3）在一个24孔板含有500微升的DME / F12培养基中，用20％的FCS的分开的孔。允许网膜在一个5％CO 2的环境中放置24小时调节所述介质在37℃，然后取出250μl的用于将IL-6 ELISA测定10。 </li></ol></li><li>鼠标网膜与SKOV3ip.1-GFP细胞的体外共培养<ol><li>生长并如第4和重悬以每毫升2×10 6个细胞的浓度描述制备荧光标记的细胞。 </li><li>申请〜6微升组织粘合剂向培养插入物的膜，并允许它在空气中干燥。用无菌水洗涤两次洗膜以除去任何多余的粘合剂。风干在层流罩膜。 </li><li>仔细切除网膜并将其连接到涂覆粘合剂的MEMB使用RANE无菌镊子。允许组织粘附于膜1分钟添加介质之前。加入500微升细胞悬浮液在每个培养插管各网膜的顶部。用2.5毫升的DME / F-12 10填充围绕Transwell小室的面积。 </li><li>孵育网膜与细胞悬浮液6小时，在37℃在5％CO 2的环境中。小心地取出并用的网膜约10毫升的PBS。使用适当的荧光成像系统10可视化的荧光癌细胞灶。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+pathogenesis+of+epithelial+ovarian+cancer:+a+proposed+unifying+theory.">The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory.</a> The American Journal Of Surgical Pathology. 34 (3), 433-443 (2010).</li><li>Crum, C. P., 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=The+distal+fallopian+tube:+a+new+model+for+pelvic+serous+carcinogenesis.">The distal fallopian tube: a new model for pelvic serous carcinogenesis.</a> Current Opinion In Obstetrics, &amp; Gynecology. 19 (1), 3-9 (2007).</li><li>Crum, C. P., 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=Lessons+from+BRCA:+The+Tubal+Fimbria+Emerges+as+an+Origin+for+Pelvic+Serous+Cancer.">Lessons from BRCA: The Tubal Fimbria Emerges as an Origin for Pelvic Serous Cancer.</a> Clinical Medicine, &amp; Research. 5 (1), 35-44 (2007).</li><li>Foulkes, W. D., Narod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+risk+and+family+history.">Ovarian cancer risk and family history.</a> The Lancet. 349, 878 (1997).</li><li>Antoniou, 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=Average+Risks+of+Breast+and+Ovarian+Cancer+Associated+with+BRCA1+or+BRCA2+Mutations+Detected+in+Case+Series+Unselected+for+Family+History:+A+Combined+Analysis+of+22+Studies.">Average Risks of Breast and Ovarian Cancer Associated with BRCA1 or BRCA2 Mutations Detected in Case Series Unselected for Family History: A Combined Analysis of 22 Studies.</a> The American Journal of Human Genetics. 72 (5), 1117-1130 (2003).</li><li>King, M. -. C., Marks, J. H., Mandell, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+York+Breast+Cancer+Study+Group+Breast+and+ovarian+cancer+risks+due+to+inherited+mutations+in+BRCA1+and+BRCA2.">New York Breast Cancer Study Group Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2.</a> Science. 302 (5645), 643-646 (2003).</li><li>Kj&#230;r Kr&#252;ger, S., Gayther, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+and+genetic+susceptibility+in+relation+to+the+BRCA1+and+BRCA2+genes.+Occurrence,+clinical+importance+and+intervention.">Ovarian cancer and genetic susceptibility in relation to the BRCA1 and BRCA2 genes. Occurrence, clinical importance and intervention.</a> Acta Obstetricia et Gynecologica Scandinavica. 85 (1), 93-105 (2006).</li><li>Easton, D. F., Ford, D., Bishop, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+and+ovarian+cancer+incidence+in+BRCA1-mutation+carriers.+Breast+Cancer+Linkage+Consortium.">Breast and ovarian cancer incidence in BRCA1-mutation carriers. Breast Cancer Linkage Consortium.</a> American Journal of Human Genetics. 56 (1), 265-271 (1995).</li><li>Rebbeck, T. R., 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=Prophylactic+Oophorectomy+in+Carriers+of+BRCA1or+BRCA2Mutations.">Prophylactic Oophorectomy in Carriers of BRCA1or BRCA2Mutations.</a> New England Journal of Medicine. 346 (21), 1616-1622 (2002).</li><li>Olivier, R. I., 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=Clinical+outcome+of+prophylactic+oophorectomy+in+BRCA1/BRCA2+mutation+carriers+and+events+during+follow-up.">Clinical outcome of prophylactic oophorectomy in BRCA1/BRCA2 mutation carriers and events during follow-up.</a> British Journal Of Cancer. 90 (8), 1492-1497 (2004).</li></ol>

The authors have nothing to disclose.

发展疗法靶向播散细胞需要一个机械理解转移性定植，在腹膜疾病发展的关键的第一步。为了解决这些问题，我们报告，可以用来辨别网膜独特的组织构成和架构如何促进卵巢癌转移定植的方法。我们的方法的显着特征是:1）专注于在殖民化过程中的早期事件;乳白色斑和含缺陷的脂肪库2）比较; 3）可重复技术的可用性，以定量的组织乳白色斑量; 体内互补和体外4）可用...

不像大多数实体瘤，从高档浆液性卵巢癌（HGSC）转移限于腹膜腔1。因此，有效的腹腔疗法可能控制或消除HGSC。目前，标准的治疗方法是手术细胞减灭术联合化疗1-3。不幸的是，绝大多数患者体验并死于疾病复发的并发症。这些令人沮丧的统计数据显示，需要转移的殖民认识的提高，这个过程由癌细胞本地化，利用和扩散宿主组织内，形成转移灶2。 网膜是HGSC转移4-7的一个优选的早期站点。不像其他的腹膜脂肪，网膜脂肪组织含有称为乳斑，其含有B，T和NK细胞和巨噬细胞，这在腹膜homeos发挥关键作用不寻常的免疫结构TASIS 8,9。除了 ​​他们的生理功能，我们发现，乳斑发挥卵巢癌转移定植4了积极的作用。在实验性转移测定法，SKOV3ip.1，CAOV3，和HeyA8（人）和ID8（鼠; C57BL / 6）的卵巢癌细胞迅速家乳斑，这表明细胞正在朝着分泌趋化因子。有趣的是，癌细胞不定居腹膜脂肪缺乏乳斑（ 即性腺和子宫脂肪）4。 为了鉴定调节乳斑定植机制，我们优化异种移植模型中，使过转移性定植4的时间过程的细胞和分子事件的询问。这里所描述的方法的具体优点是它强调组织结构和功能，使用户能够验证假设的完全融合在体内和 m的体外模型etastatic殖民4,10。通过在腹膜脂肪库其中或者含有或缺乏乳斑比较癌细胞本地化和生长，研究者可以测试脂肪细胞和细胞的乳斑的倒伏和卵巢癌的细胞中生理相关组织进行性生长中的相对贡献（多个）。

<table><tbody><tr><td>Dissection Tools</td><td>Fine Science Tools</td><td>NA</td><td></td></tr><tr><td>Geimsa</td><td>Fluka/Sigma Aldrich</td><td>48900</td><td></td></tr><tr><td>5% Formalin</td><td>Sigma</td><td>HT501320</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td>Without calcium and Magnesium</td></tr><tr><td>26 gauge needle</td><td>BD</td><td>329652</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Collagenase</td><td>Worthington</td><td>LS004196</td><td></td></tr><tr><td>Stomacher</td><td>Seward Labsystems</td><td>Stomacher 80 Biomaster</td><td></td></tr><tr><td>Microstomacher bag</td><td>Stomacher Lab Systems</td><td>BA6040/Micro</td><td></td></tr><tr><td>ACK Lysis Buffer</td><td>Gibco</td><td>A10492-01</td><td></td></tr><tr><td>Millicell culture plate insert</td><td>Millipore</td><td>PICM01250</td><td></td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td></td></tr></tbody></table>

in vivo and ex vivo approaches to study ovarian cancer metastatic colonization of milky spot structures in peritoneal adipose

高档浆液性卵巢癌（HGSC），广泛腹膜转移的原因，仍然有一个非常预后较差;妇女只有不到30％是活确诊5年后。大网膜是HGSC转移形成一个首选站点。尽管这样的微环境的临床重要性，网膜脂肪组织卵巢癌进展的贡献仍充分研究。网膜脂肪是不寻常的，因为它含有一种被称为乳斑，这是由B，T，以及NK细胞，巨噬细胞和祖细胞周围的脉管的稠密巢结构。乳斑起到网膜的生理功能，这需要进行腹膜稳态中起关键作用。我们已经表明，乳斑也促进腹膜脂肪的卵巢癌的转移性定植，在腹膜转移的发展中的关键步骤。在这里，我们描述的方法我们开发评估和量化每个乳斑itoneal脂肪和学习他们的体内和体外大网膜组织的卵巢癌细胞转移的殖民功能的贡献。这些方法推广到更多的小鼠模型和细胞系，从而使卵巢癌转移形成的研究，从细胞的初始定位，以乳白色斑结构的广泛腹膜转移的发展。

腹膜脂肪库的鉴定和组织学检查 总解剖解剖可以识别四种的腹部脂肪（ 图1A）的五个主要来源。从顶部中心顺时针方向分别是:大网膜（OM;概述）坐落在胃和脾脏，性腺脂肪（GF）围绕左侧卵巢（OV），子宫脂肪（UF）附着于子宫角（嗯）和肠系膜（MY）附接到小肠（SI）。卵巢（OV），子宫角（嗯），和小肠（SI）表示为参考点。该splenoportal脂肪（SP）?...

Watch this Scientific Journal Video about In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose at JoVE.com

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

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In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

Cancer

SCIENTISTS IN ACTION

13.0K 次观看.  The University of Chicago. We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

视频: In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

科研

医学

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

建立卵巢癌-网膜相互作用的 3D 离体模型

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

作者聚焦:用于研究癌症-脂肪微环境相互作用的高级离体模型 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

基于人网膜的卵巢癌腹膜转移离 体 模型

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

癌症研究

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Harvesting Peritoneal Fat Depots: A Technique to Study Ovarian Cancer Colonization of Peritoneal Adipose Tissues in Mouse Models

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">In Vivo</a><a href="https://www.jove.com/t/52721">&#160;and&#160;Ex Vivo&#160;Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we use a euthanized female mouse model bearing ovarian cancer. We then describe a protocol to identify and extract fat depots or adipose tissues associated with the five primary peritoneal organs to study metastatic cancer cell colonization.

收获腹膜脂肪库:一种研究小鼠模型中腹膜脂肪组织卵巢癌定植的技术

Source: Krishnan, V. et al. In Vivo&#160;and&#160;Ex Vivo&#160;Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Animal model studies

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we perform an intraperitoneal injection of ovarian cancer cell suspension in a mouse model to study the colonization of ovarian cancer cells in peritoneal adipose tissues and their subsequent metastasis.&#160;

腹腔注射细胞:一种将癌细胞输送到小鼠模型腹膜脂肪组织中的方法

Source: Krishnan, V. et al. Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. J. Vis. Exp. 
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在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

摘要

探索更多视频

此视频中的章节

前溶瘤病毒的活组织的体内感染

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

在实时的间皮细胞卵巢癌球体附件和入侵评估

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

腹膜内卵巢癌转移的定量

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

收获腹膜脂肪库:一种研究小鼠模型中腹膜脂肪组织卵巢癌定植的技术

腹腔注射细胞:一种将癌细胞输送到小鼠模型腹膜脂肪组织中的方法

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

摘要

探索更多视频

此视频中的章节

前溶瘤病毒的活组织的体内感染

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

在实时的间皮细胞卵巢癌球体附件和入侵评估

基于人网膜的卵巢癌腹膜转移离 体 模型

基于人网膜的卵巢癌腹膜转移离 体 模型

基于人网膜的卵巢癌腹膜转移离 体 模型

腹膜内卵巢癌转移的定量

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

收获腹膜脂肪库:一种研究小鼠模型中腹膜脂肪组织卵巢癌定植的技术

腹腔注射细胞:一种将癌细胞输送到小鼠模型腹膜脂肪组织中的方法

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型