Name: quantitation of intra-peritoneal ovarian cancer metastasis
Uploaded: 2016-07-18T14:00:00.000+00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about Quantitation of Intra-peritoneal Ovarian Cancer Metastasis at JoVE.com

This research was supported by research grants RO1CA109545 and RO1CA086984 to M.S.S. by the National Institutes of Health/National Cancer Institute and by an award from the Leo and Ann Albert Charitable Trust (to M.S.S.).

所有体内研究通过圣母院动物护理和使用委员会的大学批准，使用雌性C57 / BL6J小鼠。 1.小鼠卵巢癌细胞培养<ol><li>使ID8鼠卵巢癌细胞培养基如下:补充有4％胎牛血清（FBS），1％青霉素/链霉素，5微克/毫升胰岛素，5微克/毫升转铁1升的Dulbecco改良的Eagle培养基（DMEM）的和5毫微克/毫升亚硒酸钠。 </li><li>转导ID8鼠卵巢癌细胞13表示使用表达RFP与选择标记（杀稻瘟菌素基因）商业采购的慢病毒颗粒红色荧光蛋白（RFP）。需要注意的是绿色荧光蛋白可以使用，但提供一个较弱的荧光信号。 <ol><li>之前立即转导，培养细胞50 - 70％汇合，并添加新鲜的培养基（无青霉素/链霉素），该包括1-毫升聚凝胺（5微克/毫升的最终浓度）。 </li><li>轻轻吸管RFP-慢病毒（20微升）中的溶液，放入盘中，并在37℃培养72小时。除去培养基并用新鲜的培养基孵育24小时。通过将杀稻瘟到培养基中（5微克/毫升）开始转导的细胞的选择和监视用荧光显微镜（ 图 1）的红细胞。 </li></ol></li><li>培养的RFP标记细胞ID8介质通过接种约10 6个细胞到组织培养皿和使用光学显微镜，直到细胞单层是汇合可视每日。 </li><li>当汇合时，用胰蛋白酶（0.25％，3分钟）收获细胞，将烧瓶返回到37℃培养箱直至细胞被分离。 </li><li>中和用6ml含血清ID8培养基的胰蛋白酶。吸管上下对培养皿的底部，然后转移到15毫升锥形管中。离心机在1200转2分钟，然后使用GL吸出介质屁股吸管。 </li><li>通过在6ml轻轻重新悬浮温PBS，离心如上1.5洗涤用磷酸盐缓冲盐水（PBS）中的细胞。在PBS重悬至5×10 6个细胞/ ml的终浓度。保持温暖的细胞（37℃），直到注射。 </li></ol> 2. ID8细胞腹腔注射和体内成像<ol><li>注射每只小鼠用2毫升经腹腔（IP）注射温暖ID8-PBS液总共1000万个单元组成。可以让肿瘤细胞在体内生长约10周，每周实时成像。 </li><li>紧跟的喷射和每星期之后，称重小鼠，然后用异氟烷（流量2.5％在0.5升/分钟的 O 2流量）麻醉每只小鼠。注意:小鼠称重，而不是作为肿瘤负荷的定量测量，而是个别小鼠物理进展整个研究更一般量度。小鼠称重TS与自己以前的加权值进行比较。 <ol><li>通过在异氟烷室放置麻醉的小鼠。通过在鼻锥暴露于2.5％的流量异氟烷整个过程中鼠标定位保持头部扫描时不断的麻醉。 </li></ol></li><li>使用脱毛霜除去每个小鼠的躯干和腹部的毛，作为黑色头发会导致荧光信号的衰减。小鼠的脱毛霜的应用程序后，用温水沐浴并用RT纸巾干燥。 </li><li>而麻醉，使用小动物的光学成像系统扫描每个小鼠的整个身体。扫描所有小鼠在每个时间点的队列。使用以下两个步骤协议 （ 图2A）: <ol><li>第1步，观察荧光标记（RFP）的肿瘤细胞，多光谱执行收购收集总共使用440纳米，460纳米，4激发滤光片5张图片80纳米，520纳米和540纳米，和600纳米的发射滤光器。使用下面的采集参数:15秒的标准曝光，将2×2像素合并，为160毫米FOV和1.1 F 光阑 。 </li><li>在步骤2中，观察到整个动物，使用开放激发滤光器（白光），一个开放的发射滤光片，0.2秒用2×2像素合并的标准曝光和16厘米的FOV取得的反射率图像。注意:实际成像时间是约1分钟。 </li></ol></li></ol> 3.鼠标和解剖图像采集<ol><li>当实时成像显示腹水广泛的腹腔疾病或动物展览积累（如由腹胀），损失或体重大于20％，嗜睡或窘迫的其他迹象的增益的情况下，安乐死使用二氧化碳每个鼠标麻醉颈椎脱位。 </li><li>前方切开皮肤沿小鼠的腹侧中线，使用尖dissectioÑ​​剪刀，从大腿的顶部到肋笼的底部。仅经由皮肤层（ 图2BI）采取极端小心切割。轻轻的皮肤从腹膜组织中分离出来，是确保不要刺破腹膜壁上。 </li><li>使用尖解剖剪刀，既沿着肋笼的底部和大腿的顶部横向切割以创建皮肤的两个折片（ 图2B II，III）。 </li><li>放置在其腹侧各小鼠（腹膜腔朝下）与皮瓣拉开，并使用小动物光学成像系统扫描每个小鼠及其机关原位 （ 图3A，B）。 <ol><li>在2.4.1.-2.4.2所述使用相同的图像采集参数。 </li></ol></li><li>继续通过提取个人腹腔器官，包括肝脏，大网膜/胰腺，胃，膈肌，小肠，大肠，左，右腹膜，卵巢，kidne解剖鼠标YS，肠系膜脂肪垫。 <ol><li>注意，枚举，并在各器官记录可见的肿瘤。使用卡尺测量每个肿瘤的直径。 </li><li>指定大于2毫米的直径小于小肿瘤，在2 至5mm的介质和大于5mm 大之间。 </li></ol></li><li>放置在器官扫描模板 （ 图4），其识别为每个器官的预定位置的器官。用PBS以保持湿润的器官。 </li><li>扫描采用小动物光学成像系统（ 图3C，D）器官的每一张纸。 <ol><li>如步骤2.4中所述使用相同的图像采集参数。 </li></ol></li><li>扫描后，在10％的甲醛地方机关，以使用于组织学后续处理。 </li></ol> 4.在定量个肿瘤负荷Ë荧光图像<ol><li>为了减少每个多光谱扫描自发荧光的量，首先使用可用的小动物成像系统软件UNMIX的文件。 <ol><li>首先加载RFP:体内频谱文件，接着Autofluor:在该未混合的图像窗口体内红色文件并选择UNMIX。 </li></ol></li><li>导出的BIP文件作为16位.TIFF（无标度）的格式。 </li><li>使用ImageJ的软件（ 体外器官，然后完整的鼠标机身采用原位器官）。 <ol><li>使用文件&gt;打开 ImageJ的中打开器官扫描模板文件中的16位.tiff文件。 </li><li>在图像&gt;调整&gt;亮度/对比度 ，选择要设置 最小显示值为 0， 最大显示值 35353，然后在图像&gt;查找表 ，选择红热 。注意:各种动物模型将需要不同的亮度水平，但以保持亮度在整个给定的研究一致是重要的。 </li><li>使用写意选择工具，画的兴趣选择（ROI）各地各器官的自由形式的区域，并使用测量工具（CTRL + M）来计算各器官的表面面积;记录在电子表格中各器官的表面积 。 </li><li>周围各器官得出的投资回报率，对投资回报率内 ​​单击，然后选择复制到只包括器官。 </li><li>在注视各个器官，在图像&gt;调整&gt;阈值 ，选择图像的阈值设置为+ 1200 低阀值和+ 1700的上限程度来选择外核层Ÿ最明亮的荧光地区和检查来选择深色背景 ;选择确定 。注意:图像可能需要在ImageJ的阈滑块手动操作，使得先前亮区域被选择用于分析的步骤中，如上所示。不同疾病模型将需要不同的阈值的限制，但为了保持整个给定研究相一致的阈值水平是很重要的。 </li><li>在分析&gt;分析颗粒 ，改变尺寸（像素^ 2）以10-∞，选择显示:大纲 ，检查只选择"显示结果"，然后单击确定来衡量这两个领域，这些明亮区域的原材料集成密度，认为肿瘤。 </li><li>记录表面区域的值，并在显示的每个肿瘤的选择的原始综合密度 <s仲&gt;含脏器表面面积同一个电子表格结果表。 </li><li>在分析&gt;工具&gt;校准条 ，校准标尺添加到器官的图像。注意:在这个例子中，这是通过改变位置向右上角 ， 填充色为黑色， 标签颜色为白色 ， 标签数为5， 小数位数为0， 字体大小为12及变焦进行系数 4.0，使粗体 。 </li><li>在文件&gt;另存为 ...，保存蒙太奇为.TIFF和.JPEG。 </li><li>使用File&gt; Open打开器官的.jpeg文件，然后在插入&gt;文本框中 </strong&gt;的信息，通过创建以下各三排器官的文本框中添加器官的标签。 </li><li>使用File&gt; Open在鼠标号码顺序打开每个在ImageJ的全身式扫描的16位.TIFF文件。 </li><li>在图像&gt;调整&gt;亮度/对比度 ，选择要设置 最小显示值为 0， 最大显示值 + 35353然后在图像&gt;查找表 ，选择红热 。 </li><li>在文件&gt;另存为 ...，保存蒙太奇作为。TIFF和JPEG格式 。 </li></ol></li></ol> 5.数据分析<ol><li>添加肿瘤选择区域和原始集成密度，分别为每只小鼠的各个器官，并记录每只小鼠的总器官肿瘤区域。 </li><li>归一记录区和生INTEGRAT为每个器官的由相应器官表面积（ 见表1）除以总肿瘤面积和总原料集成密度值的大小编密度值。 </li><li>计算并绘制两个归一化的肿瘤表面积和归一化的原始集成密度值的平均值（ 图5A，B）。 </li><li>比较通过目视计数，每个器官的转移性病灶的检查时是可见的，以肉眼肿瘤获得的那些，这些的平均值。 </li></ol>

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本文是关于多式联运临床前成像的特殊问题，布鲁克BIOSPIN赞助的一部分。

相反，使用必须在免疫缺陷小鼠中进行人卵巢癌细胞的研究，上述的协议利用免疫C57 / BL6小鼠和同基因鼠卵巢癌细胞。而这使得在肿瘤进展和转移的免疫浸润的潜在作用的评估，黑头发的腹部表面上的存在使得成像不太敏感。脱毛的使用以除去成像之前毛发增强图像采集，但费时，特别是用于需​​要纵向成像实验。无毛&#39;裸"鼠可以在这个协议中使用并且不要求基于脱毛-脱毛。此外，裸鼠缺乏...

卵巢上皮癌（EOC）是的妇科恶性肿瘤死亡的最常见原因，在美国，2015年估计21290新诊断病例，估计14,180人死亡1。妇女的绝大多数（&gt; 75％）被诊断出患有晚期疾病（III期或IV）特征的弥漫性腹腔转移，预后差。在下面的一线化疗腹腔疾病复发也很常见，并且表示死亡率2,3的一大原因。 EOC发生转移通过同时涉及从原发肿瘤到邻近腹膜器官以及通过解离直接延伸或从原发肿瘤表面为单细胞或多细胞聚集体的细胞的脱落的唯一机制。细胞脱落进入腹膜腔，其中它们抵抗脱离诱导的凋亡4。腹腔腹水的积累是常见的，如脱落的肿瘤细胞阻塞腹膜淋巴引流和Tumors产生改变血管通透性的生长因子。棚肿瘤细胞的一部分附着于腹腔器官和结构，包括肠，肝，大网膜和肠系膜的表面，于是他们锚和增殖，产生多种广泛传播继发性病变3,5。血行转移少见。因此，临床管理通常由细胞减灭术，包括"最佳减瘤"，其定义是切除所有可见肿瘤的（不管多么小）。完整细胞减少与在整体存活6,7-一个显著增加相关，并且与识别和切除病变&lt;0.5厘米挑战相关联。 小动物模型在改善疾病进展的了解，以及在预后生物标志物和新的化疗或联合治疗方法的检测鉴定证明在卵巢癌的研究工具。作为主要的卵巢癌的发病率和转移部位是腹腔，EOC转移的原位模型​​涉及到腹腔内疾病的分析和表征。虽然已经有在能力图像的肿瘤细胞的最新改进，甚至在单细胞水平，也还存在着在量化EOC的转移性肿瘤负荷显著困难。这些挑战出现因数量，大小和转移灶的解剖位置。此外，存在一个需要标签癌细胞从正常的宿主细胞区分开来。以前的研究已经利用基于抗体的标签协议或肿瘤细胞与荧光素酶8,9的转染。癌细胞的直接荧光标记首次由千岛和同事在1997年10日报道。荧光标记并不需要加入外源基片，并提供精致的肿瘤细胞的特异性，提供了更有效的手段来追踪癌症转移11,12 </suP&gt;。 这里我们描述了使用由红色荧光蛋白（RFP）一同基因原位异种移植模型的转移性疾病的定量分析的光学成像方法-tagged鼠ID8卵巢癌细胞13和免疫能力的C57 / BL6小鼠。我们证明相对肿瘤负担定量的新方法在体内和体外成像结合切除组织自发荧光的。这种方法有在设计用于评估的特定的遗传，后生或微环境改变和/或治疗方法对卵巢癌的器官特异性转移的效果的研究潜在效用。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Corning</td><td>10-014-CM</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10437-028</td><td></td></tr><tr><td>penicillin/streptomycin</td><td></td><td></td><td></td></tr><tr><td>Insulin-transferrin-sodium selenite media supplement</td><td>Sigma</td><td>I-1884</td><td></td></tr><tr><td>Bruker Xtreme small animal imaging system</td><td>Bruker Corp.</td><td></td><td></td></tr><tr><td>Bruker Multispectral software</td><td>Bruker Corp</td><td></td><td></td></tr><tr><td>lentiviral particles with Red fluorescent protein</td><td>GenTarget, Inc.</td><td>LVP023</td><td></td></tr><tr><td>trypsin for cell culture</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>depilatory cream (such as Nair Hair Remover Lotion)</td><td>purchases from drugstore&amp;nbsp;</td><td>n/a</td><td></td></tr><tr><td>ImageJ software&amp;nbsp;</td><td>http://imagej.nih.gov/ij/&amp;nbsp;</td><td></td><td>free download</td></tr><tr><td>dissecting tools (forceps)</td><td>Roboz Surgical Instrument</td><td>&amp;nbsp;RS 5130</td><td></td></tr><tr><td>dissecting tools (Scissors)</td><td>Roboz Surgical Instrument</td><td>RS 5910</td><td></td></tr></tbody></table>

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.

卵巢癌的转移机构的特征在于包括不同尺寸，包括多个小（&lt;2毫米）病变的众多病变高度漫腹膜内转移。因此，利用RFP-标记的肿瘤细胞（ 图1）和光学成像提供了另一种方法来手工计数和损伤尺寸的测量。肿瘤负荷随时间的发展可以通过每周称重小鼠和腹围的测量来评估腹水的潜在存在来确定。 体内光学成像提供了一种互补的方法来评估肿瘤负荷（ <...

Watch this Scientific Journal Video about Quantitation of Intra-peritoneal Ovarian Cancer Metastasis at JoVE.com

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

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 ...

quantitation-of-intra-peritoneal-ovarian-cancer-metastasis

Research

JoVE Journal

Medicine

10.8K 次观看.  University of Notre Dame. 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.

视频: Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

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

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

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 ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

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 ...

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

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

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 ...

腹膜内卵巢癌转移的定量

摘要

探索更多视频

此视频中的章节

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

在实时的间皮细胞卵巢癌球体附件和入侵评估

小鼠模型的非侵入性成像检测和监测卵巢癌复发

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

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

腹膜内卵巢癌转移的定量

摘要

探索更多视频

此视频中的章节

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

在实时的间皮细胞卵巢癌球体附件和入侵评估

小鼠模型的非侵入性成像检测和监测卵巢癌复发

基于人网膜的卵巢癌腹膜转移离 体 模型

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基于人网膜的卵巢癌腹膜转移离体模型