We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

描述的所有研究方案已经由芝加哥机构审查委员会的大学（IRB）审查。知情同意每位患者的术前和研究经芝加哥IRB的大学。生物安全柜型和手套应处理为保护人体组织时，减少污染细胞的危险中。 1.隔离小学未转化的基质细胞与文化<ol><li>人体组织收集和准备。 <ol><li>获得人网膜，2厘米3，腹部手术过程中移除的样本，并立即浸入组织在RT中的磷酸盐缓冲盐水（PBS）（图2A）。一块网膜3厘米×2cm的一般得1万小学人类间皮细胞（HPMC）和20万人类纤维原细胞或正常网膜成纤维细胞（NOF）。 </li><li>降速的PBS将组织浸渍于0.5×g离心3分钟尽快后收集（&lt;2小时的PBS），并在50ml锥形管中的组织转移到新鲜的20个ml的PBS中。通过在一个直径为15厘米，无菌培养皿（图2B）手术刀切碎切碎组织成5毫米3块 。 </li></ol></li><li>原代人腹膜间皮细胞分离8 <ol><li>以分离的HPMC，转移组织糜到50ml锥形管中，并等待1分钟为固体片浮到顶部 （图2C＆D）。 HPMC和红血细胞（RBC）将存在于底部的液体。使用移液管从管的底部，并进入一个新的锥形管中除去液体。旋液向下0.5 XG的3分钟，并吸从HPMC /红细胞沉淀上清。 <ol><li>重复这个过程三次:加20毫升的PBS到组织糜，让组织上升，从一个吸管，进入HPMC / RBC管底部去除液体，降速，并取出上清。毕竟自旋完成并且将上清液吸出，将丸粒将包含HPMC和RBC。 </li></ol></li><li>板的所有单元从沉淀成75厘米2烧瓶中15充满生长培养基毫升（含10％胎牛血清[FBS]，1％的MEM维生素[93毫克/升]，1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）。 </li><li>执行二次PBS洗分离另外的间皮细胞。 <ol><li>对于二次PBS洗涤，摇动剩余的固体组织在200rpm，37℃下在20毫升PBS中10分钟，然后等待1分钟为组织浮到顶部。除去从管的底部的液体进新管中，离心，在0.5×g离心3分钟，吸出上清液。 </li><li>板上的所有的HPMC / RBC从二级PBS洗涤，并在单独的75cm 2的烧瓶在15毫升完全培养基中。 </li></ol></li><li>要排除任何ř通过体积的PBS溶液:emaining HPMC，摇动组织糜在200rpm，37℃度10分钟在20毫升1:1的0.25％胰蛋白酶的25mM EDTA中。允许组织上升，收集液体在试管的底部到一个新的50ml管中，和自旋向下在0.5 GX 3分钟。 <ol><li>吸出上清液和板块全部HPMC / RBC在一个单独的75cm 2的烧瓶在15毫升完全培养基中。 </li></ol></li><li>培养在37℃和5％CO 2在加湿环境中铺板的细胞。饲料铺板的细胞中加入15ml充满生长培养基上天3和5不除去用过的培养基。 HPMC可分裂前5-7天培养。 <ol><li>使用在所有的实验低传代HPMC（最多2代），以尽量减少去分化原来的表型和修改。最好是使用宽视场显微镜用塑料微分干涉对比功能，或相差capabilitie 4-20x物镜20倍的目标秒（在显微镜相衬滤波器）用于取细胞的所有图像，和10x物镜（）在明场来分析免疫组织化学3,3&#39;-二氨基联苯胺（DAB）染色。 </li></ol></li><li>确认HPMC是立方形，并进行免疫组化8（ 图3A，C，E）表达细胞角蛋白8和波形。 </li></ol></li><li> NOF隔离8 <ol><li>制备出10毫升胶原酶III型溶液（10倍）证券的通过组合1:1:100×青霉素 - 链霉素的1:1混合物:胶原酶III型的1500个活性单位/毫升:透明质酸酶在PBS 714单位活性/毫升。 </li><li>在10ml的10倍胶原酶III型溶液稀释于无血清培养基（DMEM，用1％的MEM维生素[93毫克/升]摇动剁碎网膜组织后保留的HPMC隔离在200rpm，37℃进行6小时， 1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）。消化的组织的外观不透明，并且可以具有一些纤维碎片（图2E）。 </li><li>离心溶液含有NOF细胞 在悬浮液中，在0.5×g离心3分钟，在室温，吸出上清液和板在75厘米2烧瓶中的沉淀在13ml充分生长培养基。 </li><li>删除旧的媒体24小时后，用15毫升新鲜完全培养基更换。 NOF可以培养分裂前1-3天。注意NOF的扩散将停止，当细胞达到汇合。 </li><li>确认原代人成纤维细胞是平的，细长的细胞和表达波形蛋白，但不通过进行免疫组化8角蛋白8（图3B，D，F）。使用低传代诺夫的实验（最好3代之前），以尽量减少去分化 与原始表型的修饰。使用宽视场显微镜用塑料DIC能力20倍的目标采取细胞的所有阶段图像，而在明场一个10倍的目标，分析免疫组化DAB染色。 </li></ol></li></ol> 2.电镀的器官文化8 <ol><li>从75厘米培养瓶中通过用10ml的PBS，接着毫升0.25％胰蛋白酶3/25毫摩尔EDTA不超过5分钟，漂洗释放NOF。中和胰蛋白酶具有完全生长介质中的至少3倍的体积。 </li><li>转移胰蛋白酶的细胞到50ml锥形管中，离心，在0.5 GX 3分钟。除去上清液，并在5毫升完全培养基带来电池备份。使用细胞计数器计数从培养烧瓶中回收细胞。 </li><li>稀释细胞在全生长培养基的适当体积到板每100微升2,000-4,000 NOF到黑色，透明底的96孔板。电镀前加入0.5微克/ 100微升鼠尾I型胶原的细胞混合物。让细胞在37℃，坐在5％CO 2在加湿环境中至少4小时，或直至细胞附着在板表面上。 </li><li>从利用在步骤2.1和2.2中描述的相同的方法培养瓶中释放的HPMC。稀释细胞在全生长培养基的合适量为板材每50μl10,000-20,000的HPMC在NOF的顶部已经镀有Ⅰ型胶原蛋白在96孔板中。让细胞在37℃，开始实验前坐在5％CO 2在加湿环境至少18小时。 </li><li>培养绿色荧光蛋白（GFP）在整个范围内生长培养基-expressing HeyA8卵巢癌细胞。 HeyA8卵巢癌细胞通过表达桡足类cGFP（PCDH-CMV-MCS-EF1）的慢病毒载体表达的荧光标记。 HeyA8卵巢癌细胞应该是在使用的当天（对数生长期）80-90％汇合。从培养板释放细胞和利用在步骤2.1-2.2为原代细胞所描述的相同的方法计数。 </li><li>允许卵巢癌细胞完全生长培养基以回收15-20分钟，在37℃下在一锥形管与盖子松松地密封。 </li></ol> 3，附着力测定8 <ol><li>恢复后，通过在0.5×g离心纺丝3分钟沉淀卵巢癌细胞，然后除去上清液和稀释细胞在无血清培养基的适当体积达到50000个细胞/100μl的的浓度。 </li><li>倒置96孔板用HPMC / NOF器官型培养以除去用过的培养基，并在无血清培养基中添加100微升癌细胞悬浮液的每个孔中。孵育板在37℃在5％CO 2在加湿环境进行30分钟至4小时。 </li><li>倒置96孔板以除去介质和非粘附细胞。使用多管移液器在低功耗仔细，轻轻吸管100微升PBS到每口井，并再次翻转板。重复PBS洗涤一次。 </li><li>倒置以除去PBS中，并加入100微升4％多聚甲醛至每个孔中。允许板坐20英里n至固定细胞。 20分钟后，反转板，并添加100微升PBS中。 </li><li>需要注意的是井的总荧光可以报告或细胞的数量进行量化。对于定量，一式两份使用HeyA8细胞以1百万个细胞/ ml的浓度的第一板的标准曲线。 <ol><li>通过停转1百万个细胞，除去培养基，并允许他们坐在在1ml的4％多聚甲醛20分钟使标准曲线。 </li><li>自旋细胞再次调出1毫升的PBS（重新计票的细胞，以确认100万/ ml浓度）。板50000，40000，30000，20000，万，5000，1000，和0细胞到每个孔中一式两份，并加入PBS中的50微升每孔的最终总体积。 </li></ol></li><li>底部读取荧光酶标仪（激发= 488纳米，发射= 528纳米）的培养板，比例高的井（标准曲线上50,000）。使用标准曲线计算有多少卵巢癌细胞粘附于organotyp在每个孔中（图4A-C）的集成电路培养。 </li></ol> 4.增殖试验10 <ol><li>后的卵巢癌细胞恢复（参见步骤2.5和2.6以上），并在0.5×g离心纺丝3分钟沉淀细胞，然后稀释所述细胞中的1％FBS的培养基的适当体积（DMEM有1％FBS，1％的MEM维生素[93毫克/升]，1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）来实现4000个细胞的浓度/ 150微升。 </li><li>用HPMC / NOF器官型培养倒置96孔板以除去用过的培养基，并在1％的添加150微升癌细胞悬浮液的FBS的介质到每个孔中。孵育板在37℃在5％CO 2在72小时的潮湿环境。 </li><li>底上读取天（激发= 488纳米，发射= 528纳米）的荧光读板器一旦培养板，评估相对增殖的细胞。继续测定96小时后，改变培养基在48小时（图5A-C）。要小心，不要改变介质时（反相板为首选），扰乱文化。 </li></ol> 5.侵袭试验8 <ol><li>电镀前的3D培养，在200μlPBS中添加7.5微克大鼠尾胶原I到一个24孔培养板插入件（8微米孔径）。让平板孵蛋O / N，然后小心翼翼地用吸管直接电镀HPMC / NOF器官文化到胶原蛋白涂层刀片（步骤2，上文）之前删除的PBS。 </li><li>制备卵巢癌细胞如在步骤3.1。到板的卵巢癌细胞上的刀片的器官型培养，小心使用吸管从刀片的顶部除去过量的介质。在无血清培养基中加入100μl卵巢癌细胞悬浮液的每个孔中。 </li><li>添加750微升全生长培养基到远低于插入物以诱导癌细胞vasion到器官文化。孵育板在37℃在5％CO 2在24小时的潮湿环境。 注意:卵巢癌细胞侵入器官培养将跨越所述插入并保持在插入件的底侧。 </li><li> 24小时后，用抓钳插入并简要冲洗在PBS的烧杯中，然后将插入到一个空的好。擦洗每个插入用棉签的两侧的顶部为总共1分钟。迅速将插入到含有每750微升4％多聚甲醛以及一盘。允许每个插入到传送插入到孔中填充有750微升的PBS之前坐在低聚甲醛10分钟。 </li><li>查看侵入细胞（粘附并固定到插入件的底部），用显微镜在2.5倍的放大倍率。当整个井是在视野内，调整放大倍数10倍，并采取5张图片，一个靠近中心和1中的井的每个象限，避免采取这一过于接近的边缘图像。按照同样的模式为每口井。 </li><li>使用制造商的方案来计算在每个图像单元的数量根据其协议来获得细胞每场，每孔（图6A-C）的侵入的平均数。 </li></ol>

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Metastasis. 5, 181-197 (1987).</li><li>Kanemoto, T., Martin, G. R., Hamilton, T. C., Fridman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synthetic+peptides+and+protease+inhibitors+on+the+interaction+of+a+human+ovarian+cancer+cell+line+(NIH:OVCAR-3)+with+a+reconstituted+basement+membrane+(matrigel).">Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (matrigel).</a> Invasion Metastasis. 11, 84-92 (1991).</li><li>Rieppi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesothelial+cells+induce+the+motility+of+human+ovarian+carcinoma+cells.">Mesothelial cells induce the motility of human ovarian carcinoma cells.</a> Int. J. Cancer. 80, 303-307 (1999).</li><li>Barbolina, M. V., Adley, B. P., Ariztia, E. V., Liu, Y., Stack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+membrane+type+1+matrix+metalloproteinase+activity+in+ovarian+carcinoma+cells+via+collagen-induced+EGR1+expression.">Microenvironmental regulation of membrane type 1 matrix metalloproteinase activity in ovarian carcinoma cells via collagen-induced EGR1 expression.</a> J. Biol. Chem. 282, 4924-4931 (2007).</li><li>Burleson, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+carcinoma+ascites+spheroids+adhere+to+extracellular+matrix+components+and+mesothelial+cell+monolayers.">Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.</a> Gynecol. Oncol. 93, 170-181 (2004).</li><li>Suzuki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMOCC-1,+a+human+monoclonal+antibody+that+inhibits+adhesion+of+ovarian+cancer+cells+to+human+mesothelial+cells.">HMOCC-1, a human monoclonal antibody that inhibits adhesion of ovarian cancer cells to human mesothelial cells.</a> Gynecol. Oncol. 95, 290-298 (2004).</li><li>Kishikawa, T., 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=Two+distinct+pattern+of+peritoneal+involvement+shown+by+in+vitro+and+in+vivo+ovarian+cancer+dissemination+models.">Two distinct pattern of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models.</a> Invas. Metast. 15, 11-21 (1995).</li><li>Casey, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+in+vitro+assay+to+measure+the+invasion+of+ovarian+carcinoma+cells+through+mesothelial+cell+monolayers.">Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.</a> Clin. Exp. Metastasis. 20, 343-356 (2003).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+human+ovarian+tumor+cells+with+human+mesothelial+cells+grown+on+extracellular+matrix.">Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix.</a> Exp. Cell Res. 160, 499-513 (1985).</li><li>White, E. A., Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+modeling+of+ovarian+cancer.">Three-Dimensional modeling of ovarian cancer.</a> Adv. Drug Deliv. Rev. 79-80, 184-192 (2014).</li><li>Kenny, H. 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=Quantitative+high+throughput+screening+using+a+primary+human+three-dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.</a> Nature Comm. , (2015).</li><li>Sawada, K., 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=c-Met+overexpression+is+a+prognostic+factor+in+ovarian+cancer+and+an+effective+target+for+inhibition+of+peritoneal+dissemination+and+invasion.">c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion.</a> Cancer Res. 67, 1670-1680 (2007).</li><li>Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP-2+functions+as+an+early+response+protein+in+ovarian+cancer+metastasis.">MMP-2 functions as an early response protein in ovarian cancer metastasis.</a> Cell cycle. 8, 683-688 (2009).</li><li>Sawada, K., 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=Loss+of+E-cadherin+promotes+ovarian+cancer+metastasis+via+alpha+5-integrin,+which+is+a+therapeutic+target.">Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target.</a> Cancer Res. 68, 2329-2339 (2008).</li><li>Mitra, A. K., 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=Ligand-independent+activation+of+c-Met+by+fibronectin+and+&#945;(5)&#946;(1)-integrin+regulates+ovarian+cancer+invasion+and+metastasis.">Ligand-independent activation of c-Met by fibronectin and &#945;(5)&#946;(1)-integrin regulates ovarian cancer invasion and metastasis.</a> Oncogene. 30, 1566-1576 (2011).</li></ol>

The authors have nothing to disclose.

成立腹膜微环境的一个器官模型来评估两者的细胞和先天在卵巢癌传播微环境的组件的单独和集体功能（多个）。提供了用于镀敷和自定义三维器官型培养来研究卵巢癌的细胞粘附，增殖和侵袭的具体协议。原代人网膜间皮细胞和成纤维细胞从患者分离并使用在早期通道保持正常形态和患者的变化。在这种模式下，正常成纤维细胞网膜和ECM（通常胶原I型）的分层与伯人腹膜间皮细胞单层。该模型组织学模仿腹膜衬里，使我们重新审视和卵巢癌转移的重要事件包括卵巢癌的细胞黏附，增殖和侵袭8,27。 此前，有限的3D模型有已成立调查与微环境13,14,21-25卵巢癌的互动，而这种3D器官模型是第一个重建腹腔间皮衬里施工。人类网膜从年龄和健康状况不同患者采集的。组织的健康直接影响皮细胞和成纤维细胞的分离技术。相比于组织是比较橙色与目前大小叶（&gt; 5毫米）的健康组织会出现颜色较浅非常小的小叶（1-3毫米）。多数的HPMC在初始PBS中脱落的组织从手术运送到实验室。的健康组织，然而，更有弹性的HPMC是除去，并附加PBS洗涤和1:1的PBS:胰蛋白酶消化可以导致HPMC和NOF细胞的混合培养。在健康组织中，一些HPMC能存活6小时胶原酶消化和消化O / N在1×胶原酶溶液全生长介质建议获得纯NOF文化。注意网膜必须在除去立即放入PBS或没有的HPMC将从组织回收。此外，如果将组织在RT或冰箱内储存在PBS中的时间（&gt; 2小时）的延长期间，可行的HPMC分离的数目显著减少。 采用1:5的比例成纤维细胞间皮细胞的镀敷以模仿成纤维细胞的比率间皮细胞在体内8。要注意的是分离间皮细胞的大小从病人不同而不同病人，这可能需要调整到镀比率是重要的。的一些患者的间皮细胞要小得多;在这些情况下，我们板间皮细胞（20,000）的两倍。此外，原代细胞，总是使用在早期（1-2）通路保留形态。间皮细胞可以是立方形或纺锤形，和立方形间皮细胞变得更为细长因为它们传代，或与来自卵巢癌的细胞或治疗与TGFβ10更多地暴露于条件培养基。 卵巢癌细胞可能附着并穿过间质间皮细胞更有效地侵入比从相同或不​​同的患者的上皮间皮细胞。通常情况下，我们成立了器官培养18小时启动功能检测卵巢癌细胞之前。所述间皮细胞和成纤维细胞再进行培养在一起，更多的时间，他们必须分泌不同的蛋白质，包括ECM蛋白，这可能会影响卵巢癌细胞的行为。另一个重要因素要考虑到当执行这些测定是与增加附着力，侵袭和/或增殖，包括整合酶，蛋白酶，和生长因子相关蛋白的差动增殖率和表达水平，在各癌细胞株或初级人癌症细胞类型。需要在各功能测定与器官培养物使用癌细胞时间和数目对于各癌细胞株或原发性癌细胞的类型进行优化。例如，在所描述的粘附试验，卵巢癌细胞可能是在此期间侵入培养。 显然，这种器官模型不包含在正常腹膜微环境的所有部件，如免疫，内皮，和脂肪细胞。此外，该模型依赖于辅助和主人体组织的可用性，作为主要的细胞只有前三个通道内使用。然而，该模型有很多优点中它的使用人原代细胞和已被证明在转移中发挥重要作用多种细胞类型的掺入。该模型已被用于后，调查在各类型的细胞基因和蛋白调控（即，癌细胞，正常间皮或成纤维细胞）共培养。特别是，c-Met的28，MMP-2 9,29，E-钙粘蛋白30，β1整合素10，β3整合素16，α5整合素10,31，和纤连蛋白10在早期转移的角色具有被探索。此外，该模型有扩大，以包括更多的细胞类型，如脂肪细胞6的掺入的潜力。该模型已被修改和小型化以用于高通量筛选，以确定卵巢癌的细胞粘附/侵袭的小分子抑制剂以腹膜微环境27。这种模式的未来应用将包括免疫细胞进入测定法，包括巨噬细胞的机理研究和高通量筛选的掺入。综上所述，器官细胞培养物提供了有用的模型来评价人腹膜微环境和卵巢癌的细胞之间的相互作用，与果阿升阐明的传播和疾病进展的重要分子机制，并确定潜在的治疗目标。

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2. 
The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16. 
Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

<table><tbody><tr><td>&lt;strong&gt;1. Isolation and culture of primary cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>SH3001304</td><td></td></tr><tr><td>Single-edged razor blades</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>15 cm culture dishes</td><td>BD Biosciences</td><td>353025</td><td></td></tr><tr><td>Glass flask</td><td>?</td><td>?</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000044_3616914956</td><td></td></tr><tr><td>DMEM with L-Glutamine</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning</td><td>25-020-Cl</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Shaker&amp;nbsp;</td><td>Thermo-Fisher</td><td>MaxQ 4450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Incubator</td><td>Thermo-Fisher</td><td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td><td></td></tr><tr><td>Trypsin EDTA, 1x (0.25%)</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Hyaluronidase</td><td>Worthington Biochemical</td><td>LS002592</td><td></td></tr><tr><td>T-75 Flasks</td><td>BD Biosciences</td><td>353136</td><td></td></tr><tr><td>T-175 Flasks</td><td>BD Biosciences</td><td>353112</td><td></td></tr><tr><td>Pipet tips</td><td>Rainin</td><td>P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Pipet tips</td><td>Corning</td><td>Filtered tips P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&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/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. Plating 3D culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Counter</td><td>Invitrogen</td><td>Countess</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Invitrogen</td><td>C10313</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Collagen Type I (Rat Tail)</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>96 well plate, clear bottom, black</td><td>BD Biosciences</td><td>353219</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&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/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Adhesion assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Multichannel pipet</td><td>Eppendorf</td><td>Xplorer 300</td><td></td></tr><tr><td>Paraformaldehyde solution 4% in PBS</td><td>Santa Cruz Biotechnology</td><td>sc-281692</td><td></td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>Minimax</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&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/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;5. Invasion assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Inserts (8um, 24-well)</td><td>BD Biosciences</td><td>353097</td><td></td></tr><tr><td>Cotton swabs</td><td>Q-tips</td><td>cotton swabs</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axiovert 200m</td><td></td></tr><tr><td>Cell Profiler</td><td>public domain</td><td></td><td></td></tr><tr><td>24 well plate</td><td>BD Biosciences</td><td>353047</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&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/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;6. Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Integrin &amp;alpha;V&amp;beta;3 Antibody, clone LM609</td><td>EMD Millipore</td><td>MAB1976</td><td></td></tr><tr><td>Beta 1&amp;nbsp;</td><td>Oncosynergy</td><td>OS2966</td><td></td></tr><tr><td>Alpha 5 [CD49e]</td><td>ID Pharmingen</td><td>555615</td><td></td></tr><tr><td>Beta 4 [CD104]</td><td>EMD Millipore</td><td>MAB 2058</td><td></td></tr></tbody></table>

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.

器官型培养物组装通过首先混合主人成纤维细胞与I型胶原蛋白，然后与间皮细胞的5倍的数量覆盖该培养。将培养物温育至少18小时，加入前卵巢癌细胞，研究粘附，浸润或增殖。每个测定重复与多个（N = 3-5），从不同的患者和许多井获得3D培养在每种条件下进​​行了测试的粘合（N = 5），增殖（N = 5）和侵入测定（n = 3时） 。卵巢癌细胞附着在三维器官型培养4小时（图4）之后，增殖的培养72小时（6倍HeyA8细胞倍增时间）（图5）后，和24小时（图6）之后入侵。 HeyA8卵巢癌细胞的粘附，增殖和侵袭是整合素依赖性的（ 图4-6）。具体而言，RGD多肽和阻塞antibodies〜α5整合素，β1整合素和素 αvβ3整合素抑制卵巢癌细胞附着在体培养，而RAD肽，对照抗体和β4整合素抗体没有（ 图4）10。在增殖实验中，RGD肽和封闭抗体，以α5 -和β1整合素的器官培养抑制卵巢癌细胞增殖，而RAD肽，对照抗体素 αvβ3整合素抗体和β4整合素抗体没有（ 图5）。在侵袭测定中，RGD肽和阻断抗体对α5 -和β1整合素抑制，而素 αvβ3整合素抗体通过三维器官型培养增强卵巢癌细胞侵袭。在RAD肽，对照抗体和β4整合素抗体不影响侵袭（图6）。很少，这些测定将未解释的（即，α5整合素阻断抗体时相比对照抗体不会抑制粘附，增殖或侵袭）。以前，我们已经发现某些器官型培养将在介质或PBS洗涤的过程中检测改变洗去。此外，如果在卵巢癌细胞死亡或没有回收的足够长的时间从胰蛋白酶消化，以重新表达整联，所述测定法将不起作用。因此，它始终是重要的是有一个阳性和阴性对照在每个试验所以器官型培养和卵巢癌细胞测定的质量进行评估。如这里所示的，RGD肽，α5整合素，β1整合素阻断抗体所有抑制卵巢癌的细胞粘附，侵入和增殖的器官型培养和可作为阳性CON组trols在未来的试验。同样，β4整合素阻断抗体不影响卵巢癌细胞粘附，侵入和增殖的器官型培养，并且可以用作阴性对照在将来测定。 <img alt="图1" src="/files/ftp_upload/53541/53541fig1.jpg" /> 图1.组织学人网膜转移。苏木精和（A）的正常的人网膜（MC，间皮细胞，纤维蛋白原，纤维母细胞，阿迪，脂肪细胞）和（B）的卵巢癌网膜转移（蛋氨酸，卵巢癌转移）伊红染色。酒吧面板=长度为100微米。卵巢癌转移模仿早期卵巢癌细胞转移到人类的大网膜表面的三维器官模型（C）示意图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53541/53541fig1large.jpg" target="_blank">请点击此处查看版本更大</a>锡安这个数字。 <img alt="图2" src="/files/ftp_upload/53541/53541fig2.jpg" /> 图2.分离初级人网膜细胞。（A）片网膜被收集在手术在PBS中并运送至实验室。将组织沉淀在0.5 xgx 3分钟，并转移至新鲜的PBS。（B）中的片网膜被转移到10厘米的培养皿，用PBS洗涤，并且手术刀用于切割网膜小块（0.5厘米3 ）。 （CD） 的组织片段是使用25毫升血清吸管收集并转移到50ml锥形管中的HPMC隔离。 PBS被从管中取出和HPMC是从PBS沉淀0.5 XG在37℃。 （MC，间皮细胞，红细胞，红血细胞，纤维蛋白原，成纤维细胞）。（E）NOF从remaini分离<html>孵化与透明质酸酶和胶原酶III型后纳克网膜组织。该图显示了一个6至12小时消化后组织。 NOF由纺纱0.5 XG在室温沉淀。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53541/53541fig2large.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/53541/53541fig3.jpg" /> HPMC和NOF图3.表征从网膜组织分离后 ，分离自人组织网膜。（A）的间皮细胞（HPMC） 和 （B）的成纤维细胞（NOF）的相衬图像。人类HPMC染色（C），细胞角蛋白8 和 （E）波形。人类NOF染色（F）波形，但没染色的（D），细胞角蛋白8条= 100微米，适用于所有的面板。ftp_upload / 53541 / 53541fig3large.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/53541/53541fig4.jpg" /> 图4.卵巢癌细胞粘附于器官型培养是整合素依赖性的。用50000荧光标记的卵巢癌细胞，HeyA8细胞进行（A）的粘合试验中，如在协议部分中描述。在每个孔中的总荧光报道。对照抗体是鼠IgG。每个条形代表平均值+/-标准误差均值。代表（B）的相位差和卵巢癌的细胞粘附到器官型培养的（C）的荧光图像。酒吧= 100微米，同时适用于板。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53541/53541fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a><html><img alt="图5" src="/files/ftp_upload/53541/53541fig5.jpg" /> 在器官型培养图5.卵巢癌细胞增殖是整合素依赖性的。用4000荧光标记的卵巢癌细胞，HeyA8细胞进行（A）中的增殖测定，如在协议部分中描述。报道的细胞的总数。对照抗体是鼠IgG。每个条形代表平均值+/-标准误差均值。合并后的相衬和对器官型培养卵巢癌细胞增殖在（B）的荧光图像24和​​（C）的96小时。酒吧= 100微米，同时适用于板。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53541/53541fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> tp_upload / 53541 / 53541fig6.jpg"/&gt; 图6.卵巢癌侵袭通过器官型培养是整合素依赖性的。用40000荧光标记的卵巢癌细胞，HeyA8细胞进行（A）中的侵袭实验，如在协议部分中描述。报道侵入细胞的数量（通过三维培养移动到插入件的底部）。对照抗体是鼠IgG。每个条形代表平均值+/-标准误差均值。 （B）的相衬和（C）的卵巢癌的细胞通过器官型培养侵入的荧光图像。酒吧= 100微米，适用于所有的面板。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53541/53541fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

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

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. ...

modeling-early-steps-ovarian-cancer-dissemination-an-organotypic

Nanyang Technological University

Research

JoVE Journal

Medicine

26.5K 次观看.  University of Chicago. 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. 

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

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

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 ...

医学

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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 ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

生物工程

Modeling Ovarian Cancer Communication for Imaging Mass Spectrometry Analysis: A Method to Identify Small Molecule Chemical Communicators in Metastatic Ovarian Tumor Development

Source: Zink, K. E. et. al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/59490">Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry.</a> J. Vis. Exp. (2019)
This protocol describes a technique for sample preparation by co-culturing ovarian explants with the fallopian tube cancer cells. These samples are visualized using imaging mass spectroscopy, or IMS,&#160; to detect small molecule exchange between the cells and tissues leading to tumor development.

用于成像质谱分析的卵巢癌通讯建模:一种在转移性卵巢肿瘤发展中识别小分子化学通讯器的方法

Source: Zink, K. E. et. al. Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry. J. Vis. Exp. (2019)
This ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Assays & techniques

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

Source: Koya, Y. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/54353">Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells.</a> J. Vis. Exp. (2016)
This video describes the protocol for creating a murine experimental model to study tumor development and peritoneal metastasis. This ovarian cancer model is generated by the orthotopic inoculation of human epithelial ovarian cancer cells into the ovaries of a female mouse.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

摘要

探索更多视频

此视频中的章节

原发肿瘤发展小鼠实验模型和腹膜转移

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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

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造型卵巢癌传播在人类腹腔的器官文化的早期步骤

摘要

探索更多视频

此视频中的章节

原发肿瘤发展小鼠实验模型和腹膜转移

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

异型三维

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

模拟卵巢癌多细胞球体行为动态三维微型腹膜

用于成像质谱分析的卵巢癌通讯建模:一种在转移性卵巢肿瘤发展中识别小分子化学通讯器的方法

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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