该协议是在实验室博士理查德海恩斯（MIT）的初步测试和优化。提供经费的NYSTEM IDEA奖（LX）和露丝属Kirschstein国家研究服务奖（LX）。

1。样品制备<ol><li>种植细胞〜70％在其​​特定的生长因子或胎牛血清媒体的融合（如MC - 1细胞，含10％胎牛血清的DMEM）。从板吸媒体，轻轻地用1 × PBS（8克/ L的NaCl，0.2 g / L的氯化钾，1.15克/升的Na 2 HPO 4 0.7 H 2 O，0.2 g / L的KH 2 PO几次4，pH值7.3）。 </li><li>吸PBS和添加2毫升versene 0.05％胰蛋白酶（0.014克/ L的酚红和0.2 g / L的EDTA钠在1 × PBS，pH值7.2）。轻轻摇动板的板，以便从细胞脱落。在显微镜下观察细胞。它通常需要2-5分钟细胞分离。 </li><li>添加媒体充裕量，含胎牛血清（或大豆胰蛋白酶抑制剂）淬火胰蛋白酶的活性，并收集在50毫升猎鹰管细胞。使用hemacytometer计数细胞。加载到一个干净的hemacytometer细胞悬液10μL。每毫升细胞数等于平均每五个平方乘以​​10 4＃细胞。 </li><li>离心机细胞在1000转速（或〜200 XG）在一个3分钟的台式离心机。 </li><li>小心地取出，而不会干扰细胞沉淀的媒体。在适当的音量汉克斯平衡缓冲液（HBSS）重悬细胞终浓度达到〜5 × 10 6细胞/ ml。 </li><li>通过猎鹰70微米的细胞滤网过滤细胞排除大量的细胞聚集。吸管直接放置到过滤器通过一个标记5ML猎鹰文化管的尖端。快速到文化管悬浮液通过过滤弹出。 </li><li>计数细胞，再使用hemacytometer稀释到终浓度为2.5 × 10 6 /毫升。保持细胞在冰上。 </li><li>确定细胞的可行性。台盼蓝混合一些细胞和测量使用hemacytometer总细胞的细胞死亡（蓝色）的百分比。细胞活力，应在注射之前，≥90％。 </li></ol> 2。静脉注射<ol><li>更换手套。轻轻地抢一个免疫缺陷小鼠裸照，NOD SCID，或核供应国集团，杰克逊实验室的尾巴和拉成的鼠标限位，其反对的狭缝和它的尾巴坚持小开口，在后面的限位回， 。 </li><li>慢慢地沿着缝隙向内滑动环和锁定到位，一旦环捕获鼠标口。鼠标不应该能够自由移动，但应该有正常的呼吸率。 </li><li>主要尾静脉。四个主要血管在鼠尾。尾部的背，腹两侧的血管动脉。静脉尾部的侧面。 </li><li>绘制成1ml注射器超过200μL的准备细胞。将30 G1 / 2英寸的针头和推任何可能存在的气泡。注射器中的细胞最终体积为200μL（即5 × 10 5细胞总数）。 </li><li>尾静脉注入细胞。 <ol class="lalpha"><li>从尾部的末端开始，所以，如果第一次试验失败，尾巴更近端的地区可以使用了第二次尝试。 </li><li>用70％的乙醇尾巴。直拉尾巴。按住尾部用拇指尖和支持注射用食指点。 </li><li>针插入静脉注入细胞。确保在注射针头和注射器静脉平行，否则针戳透过血管壁，并注入到相邻的尾巴组织细胞。 </li><li>注射后撤回针。血液应该从注射部位充沛，如果成功注入了。按一块干净的纸巾或注射部位的棉签，促进凝血，触诊尾部向上推入静脉进入流通领域的任何残留的样品。 </li><li>从限位释放鼠标并返回它的笼子。在实验室笔记本记录注射过程中（例如，有多少试验了注入细胞，以及如何被注射多细胞）。 </li><li>确定注射后的细胞的可行性，如样品制备第9步。这一步提供了保证整个注射过程中，细胞停留活着。在注射实验结束，细胞活力下降，但应在80％以上。 </li><li> （可选）旋转剩下的细胞，用PBS漂洗一次颗粒。未来的分析（例如，Western印迹，以确认基因表达或击倒）冻结，在-80℃的颗粒。 </li></ol></li><li>通常一两个月后，老鼠将被解剖和转移的位置是非常确定。龙是为转移的主站点，因为它包含的细胞后，他们遇到的第一个毛细血管床进入流通领域。 </li><li>冲洗后用PBS，每个肺（或含有转移的其他组织）叶（图1）在解剖显微镜下观察。 1肺转移更容易被检测肺部固定在福尔马林一夜之间检测转移的肺癌双方的数量，四叶肺转移的数字相加为肺转移的总数。以来的转移将apppear作为对比相邻的黑褐色肺组织发白点。 </li></ol> 3。培养细胞肺转移<ol><li>从小鼠体内分离出肺转移。每个人都应该单独培养。 </li><li>每个转移是由一个70微米的细胞过滤器上的针帽（无菌）剁碎。 </li><li>冲洗细胞过滤器中并收集细胞，在培养皿中通过过滤器通过与几个MLS。 </li><li>在37 ° C孵育至少4天无干扰的细胞。 </li><li>菜用PBS洗去血液或组织碎片。 </li><li>添加新鲜培养基。在开始的时候，癌症细胞和成纤维细胞生长板，但渐渐地，成纤维细胞就会死亡和被癌细胞所取代。如果癌细胞进行任何的耐药基因，选择相应的抗生素的细胞。 </li><li>评估使用对人类特有的蛋白抗体的免疫源性细胞纯度。我们使用抗人波形蛋白抗体（NCL - VIM - V9，Novocastra）。我们推导出的细胞中含有通常超过99％的人类细胞，甚至在选药的情况下。新产生的细胞可以再次被注入到免疫缺陷小鼠，以测试他们的转移能力，如上所述。 </li></ol> 4。代表性的成果<ol><li>在这个实验结束时，高度转移性的癌细胞株，通常会引起许多肺转移（图1），1，而很少肺转移，将来自不良的转移性癌细胞株。 </li><li>细胞衍生的使用这种方法通常会引起更多的转移比亲行，当他们再次测试，使用这种检测。 1-3 </li></ol><img src="/files/ftp_upload/1942/1942fig1.jpg" alt="图1" /> 图1。后尾静脉注射癌细胞的小鼠肺代表的图像。免疫缺陷小鼠的尾静脉注入5 × 10 5转移性人黑色素瘤细胞株，丹参细胞，4 。两个月后，肺部孤立和分散之间的正常肺组织转移被发现。

<ol>
	<li>Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+an+animal+melanoma+model+correlate+with+aggressiveness+of+human+melanoma+metastases.">Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases.</a> Mol Cancer Res. 6 (5), 760-760 (2008).</li><li>Clark, E. A., Golub, T. R., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406 (6795), 532-532 (2000).</li><li>Xu, L., Begum, S., Hearn, J. D., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPR56,+an+atypical+G+protein-coupled+receptor,+binds+tissue+transglutaminase,+TG2,+and+inhibits+melanoma+tumor+growth+and+metastasis.">GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.</a> Proc Natl Acad Sci U S A. 103 (24), 9023-9023 (2006).</li><li>Kozlowski, J. M., Hart, I. R., Fidler, I. J., Hanna, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+melanoma+line+heterogeneous+with+respect+to+metastatic+capacity+in+athymic+nude+mice.">A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.</a> J Natl Cancer Inst. 72 (4), 913-913 (1984).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multigenic+program+mediating+breast+cancer+metastasis+to+bone.">A multigenic program mediating breast cancer metastasis to bone.</a> Cancer Cell. 3 (6), 537-537 (2003).</li></ol>

转移是导致死亡的癌症患者的原因。它包括四个主要步骤：从他们的主要位点，其进入流通领域的项目（血管内），从退出流通（外渗），并在远处器官的生存和成长的癌细胞脱落。在人类的转移被认为是一个缓慢的过程，往往表现经过多年的潜伏期。要研究及其进展及时，在免疫缺陷小鼠建立了相对快速的实验检测上述4自成立以来，已有效地用于隔离不同转移能力的细胞，确定基因的?...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10313-021</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30088.03</td>
<td>&nbsp;</td>
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<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175-095</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S9888</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>6838-04</td>
<td>&nbsp;</td>
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<td>Na2 HPO4.7H2O</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
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<td>&nbsp;</td>
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<td>Mallinckrodt Chemicals</td>
<td>7100-12</td>
<td>&nbsp;</td>
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<td>Phenol red</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P3532</td>
<td>&nbsp;</td>
<tr>
<td>EDTA-Na</td>
<td>&nbsp;</td>
<td>EMD Biosciences</td>
<td>4010</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin 2.5%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Ultrapure</td>
<td>200-CSGP</td>
<td>&nbsp;</td>
<tr>
<td>50 ml Falcon Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89039-656</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge Tubes</td>
<td>&nbsp;</td>
<td>Axygen Scientific</td>
<td>MCT-175-C</td>
<td>&nbsp;</td>
<tr>
<td>Falcon 70&#181;m Cell Strainer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
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<td>5ml Falcon Culture Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60818-576</td>
<td>&nbsp;</td>
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<td>Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>15170-208</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>329650</td>
<td>&nbsp;</td>
<tr>
<td>30 &frac12; gauge needle</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>Mouse restrainer</td>
<td>&nbsp;</td>
<td>Plas Labs Inc.</td>
<td>551-BSRR</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scissors</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-940</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-902</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ1500</td>
<td>&nbsp;</td>
<tr>
<td>Benchtop Centrifuge</td>
<td>&nbsp;</td>
<td>Thermoscientific</td>
<td>75003491</td>
<td>&nbsp;</td>		</tbody>
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experimental metastasis assay

转移是导致死亡的癌症患者的原因。要了解转移的机制，一个实验性转移的检测使用免疫缺陷小鼠的建立。本文描绘在这个实验中涉及的程序，包括样品制备，静脉注射，培养细胞和肺转移。简言之，人体的癌细胞数量预先确定的准备<em&gt;在体外</em&gt;，并直接进入流通​​通过免疫缺陷小鼠尾静脉注射。一个少数细胞生存在流通领域的动荡，并成长为在内部器官，如肺，转移。小鼠注射一定时期后解剖。转移的组织分布在解剖显微镜下决定。计算转移的数量在一个特定的组织，它直接注入癌细胞的转移能力相关。出现转移，分离和培养<em&gt;在体外</em&gt;细胞系，这往往比亲行增强转移能力时，再注射到免疫缺陷小鼠。这些高转移的衍生工具，成为有用的工具，用于确定基因或分子途径，规范转移进展。

Watch this Scientific Journal Video about Experimental Metastasis Assay at JoVE.com

Experimental Metastasis Assay

本文介绍的，是用来确定人类癌症细胞系的转移潜能的一个实验性转移检测的程序。

实验转移的含量

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Research

JoVE Journal

Medicine

20.2K 次观看.  University of Rochester Medical Center. 本文介绍的，是用来确定人类癌症细胞系的转移潜能的一个实验性转移检测的程序。

视频: Experimental Metastasis Assay

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 ...

科研

医学

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

人体实验肺炎球菌运输

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

"以病人为像"肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

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 ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...