这项工作的支持，在某种程度上，由国家卫生部以R01 CA131990（矿渣和RRM）研究院。活细胞成像显微成像和流式细胞仪的资源支持核心部分，由NIH中心授予P30 CA22453 Karmanos癌症研究所，美国韦恩州立大学和国家儿童健康与发展研究院围产期研究科韦恩州立大学。

1。准备DQ-基板<ol><li>允许冻干DQ-基板预热至室温，然后再打开小瓶，准备去离子水1毫克/毫升DQ的基板库存的解决方案，在4°C到50μL分装和存储鸿沟</li></ol> 这可能是必要的，鼓动〜5分钟，热到50°C，以促进分散的超声波水浴DQ-基板 。 <ol start="2"><li>在冰上解冻一夜之间在4的RBM℃; RBM，应处理在所有时间上冰。 </li><li>使10X磷酸盐缓冲液（PBS），溶解80克氯化钠（1.37男），2Ğ氯化钾（0.027男），14.4Ğ的Na 2 HPO 4（1米），2.4克KH 2 PO 4（0.02 mol） 800毫升的超纯水。 PBS缓冲液的pH值调整至7.4与1.0 M盐酸。带来体积为1升，过滤，消毒。 </li><li>准备胶原冰我的解决方案：冰鲜的胶原蛋白，我冰鲜10X PBS 1 8个部分。调整pH值混合7.2-7.6使用无菌0.1 M氢氧化钠。检查pH值pH值条，并用无菌水调整最终体积的10个部分。 </li><li> DQ的I型胶原I型胶原溶液稀释至终浓度为25微克/毫升和2.4毫克/毫升，分别在prechilled管准备DQ的I型胶原：胶原蛋白，我矩阵。 </li><li>准备DQ - Ⅳ型胶原：稀释DQ胶原四在RBM（Cultrex或基底膜）在prechilled管，到终浓度为25微克/毫升和12-15毫克/毫升，分别由重组基底膜（RBM）矩阵。上混合使用轻柔的移液，以避免创建气泡的冰。 </li></ol> 2。准备MAME的共同培养参见图1为一个共同培养的示意图。 <ol><li>将两个长方形塑料盖玻片（70％的酒精消毒10分钟和风干）培养皿在35毫米或35毫米μ盘使用IbiTreat。方向在这里盖玻片和μ-菜肴，这是我们使用STU直立显微镜死亡。倒置显微镜的研究，我们只用μ-菜肴。 </li><li>混合我与DQ胶原纤维母细胞所需的数字：I型胶原基质。我们在图2和图3所示的长期共同培养，使用500的成纤维细胞在10μL媒体DQ - I型胶原加60μL：I型胶原基质。 </li><li>使用100微升移液器，仔细移液器和传播70μL的成纤维细胞：DQ的I型胶原：胶原蛋白在每个盖玻片的整个表面，并留在37°C不饱和湿度培养箱中CO 2为30分钟，以巩固我矩阵。 </li><li>转让35毫米盖玻片以37°Ç孵化含5％CO 2为10分钟，以平衡的菜肴。 </li><li>从孵化器和离开，直到他们来到室温罩下的35毫米的菜肴。 </li><li>加入60μl的DQ - Ⅳ型胶原：凝固的DQ的I型胶原上RBM矩阵：嵌入式成纤维细​​胞I型胶原基质。用枪头，CArefully传播DQ的IV型胶原：RBM矩阵均匀，避免刮伤的成纤维细胞：DQ的I型胶原：I型胶原基质。 </li><li>转让35毫米含5％CO 2为10分钟，以巩固盖玻片以37°Ç孵化器的菜肴。而在DQ-IV型胶原：RBM矩阵固化，trypsinize计数上皮细胞。 </li><li> 50微升到盖玻片的上皮细胞/肿瘤细胞悬液涂有DQ的IV型胶原：RBM矩阵。地方35毫米的菜肴，含到37℃孵化器的盖玻片。允许附加到DQ的IV型胶原：RBM基质细胞的40％至60分钟。我们在图2和图3所示的长期共同培养，使用2500上皮细胞或肿瘤细胞。 </li><li>加入2毫升含有2％RBM文化中期每35毫米的菜。如果做任何药物治疗，药物应在这个时候加入。 </li><li>孵育前成像所需的时间。对于长期的文化，媒体应改为每3-4天。可维持细胞在正常培养基。 MAME的乳腺癌细胞株共同培养，我们用上皮细胞基底中等（MEBM-PRF）辅以MEGM SingleQuots的，。 </li></ol> 3。执行现场MAME的共同培养​​4D（三维+时间）成像，共聚焦显微镜<ol><li>为原则的证明，说明研究，MAME的共同培养，用荧光的细胞追踪染料标记，根据我们公布的程序1。用PBS洗涤后，共培养培养与5微米MEGM中等CellTracker的橙在37℃孵化45分钟，用PBS洗再次孵育30分钟的预热MEGMmedium 5％CO 2培养箱在37℃前成像。 </li><li>图片MAME的共同培养​​生活在低倍率蔡司LSM 510 META激光共聚焦显微镜（10X，水浸泡镜片）。 </li><li>在整个结构的整个深度间隔的光学部分被捕获。对于我们的研究中，我们捕捉10光学切片微米的间隔。 </li><li>使用光学切片重建三维图像。我们使用Volocity软件生成三维重建。 </li><li>三维重建的电影，这样可以可视化的结构，细胞：细胞间的相互作用，并与周围的矩阵互动。我们与QuikTime Player 7的电影。 </li></ol> 4。代表结果我们开发的MAME的共同培养​​是一个听话的活细胞在乳腺肿瘤和其微环境，包括各种细胞成分之间的相互作用的实时成像的实验模型。在这里，我们展示的MAME的模型概括细胞的能力，按照时间的推移这些相互作​​用在乳腺癌的进展和活细胞成像的细胞间的相互作用。我们说明到图像1 MCF10.DCIS人类乳腺原位癌细胞株和人乳腺癌癌症相关的成纤维细胞线，WS-12TI之间的相互作用的能力，超过文化周期间超过三。 3，第16和23天，共培养了成像。图2中的三个时间点显示，光学部分通过的共同培养​​的整个体积重建在3D和有代表性的例子。图2中的红色荧光代表MCF10.DCIS和WS-12TI细胞。要区分两种类型的细胞和长期的共同培养​​，可以用荧光蛋白转染或转导单个细胞类型之前建立文化。 MAME的一个MCF10.DCIS和WS-12TI差异识别的荧光蛋白标记的细胞共培养如图3所示。 DQ-胶原在这种情况下，通过整合基板，到MAME的共同培养​​，我们可以评估和量化了侵入型过渡到在水解时间的变化。我们已经建立了量化荧光裂解产品的方法，结果从DQ胶原降解2,3。我们正常化降解产物的每一个细胞的基础上，整个3D体积MAME的共同培养​​细胞核标记和计数。此外，我们还可以本地化的降解细胞外和细胞内的车厢和量化总，细胞内和细胞外降解产品正如我们前面所述2。在图2和3的绿色荧光代表裂解两个DQ胶原的基板产品。在这个时候，差异标记的胶原蛋白是无法使用，将使我们能够区分降解IV型胶原和I型胶原的降解。 在MAME的共同培养​​的相互作用是动态的，可以随时间而改变。为了获得时间和动态信息，相同的视野可以通过整个标本体积超过一段时间从几分钟到几周反复成像和光学部分，因此，在2,4-D收集数据。在图2中，我们说明，而超过23天发生的戏剧性的变化：细胞数量的变化，细胞迁移和细胞间的相互作用，结构，体积，结构，形状，结构偏离球形，侵入副产物和蛋白质。此外，我们也表现出货量的整体变化，由于MAME的共同培养​​有辱其周围的细胞外基质，超过这个时间段。在3天图2A所示，体积11万3微米。相比之下，MAME的共同培养，在16日和23日成交量为46,250微米3，图2B和C所示<img alt="图1" src="/files/ftp_upload/3661/3661fig1.jpg" /> 图1。 MAME的共同培养。示意图塑料盖玻片涂有I型胶原，含有DQ的I型胶原和成纤维细胞（洋红色）。第二层的重组基底膜（RBM）DQ-COL拉根四是增加了。肿瘤细胞（红色）被镀上顶部和2％的培养基中RBM是覆盖与每一个媒体的变化（白点）。绿色代表DQ胶原I和IV荧光裂解产物。 <img alt="图2" src="/files/ftp_upload/3661/3661fig2.jpg" /> 图2。 MAME的MCF10.DCIS人乳腺癌细胞和WS-12TI人类乳腺成纤维细胞共培养 。被嵌入的成纤维细胞胶原蛋白的I / DQ-胶原含有DQ - Ⅳ型胶原RBM我和覆盖。原位癌细胞，然后接种于含有2％RBM媒体RBM和覆盖​​。这里说明多年来共培养23天，细胞增殖，DQ - Ⅳ型胶原和我（绿色）和胶原基质的降解量减少的共同培养​​。细胞定位与原位 CellTracker橙色标签前成像（红色）。同一现场共培养超过23天的观察，拍摄的图像由C3，16和23天的共培养onfocal显微镜。 Volocity软件所产生的光学切片的三维重建。放大倍率，10X。 <img alt="图3" src="/files/ftp_upload/3661/3661fig3.jpg" /> 图3。 MAME的16一天MCF10.DCIS人乳腺癌细胞和WS-12TI人类乳腺成纤维细胞共同培养，快递RFP（红色）和YFP（白），分别 。共培养，建立和分析，如图2。这里显示两种细胞类型，然而，被差异标记，使他们可以从彼此区分。这个数字在高倍率的图像相比，图2，说明DQ的IV型胶原蛋白水解原位癌细胞弥漫性蛋白质和DQ胶原我在附近的成纤维细胞等领域的表面。放大，20X。

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我们什么都没有透露。

作为证明，MAME的共同培养​​，可用于活细胞成像在乳腺肿瘤和其微环境，包括各种细胞成分之间的相互作用的实时。在我们的实验室正在进行的研究中，我们已经使用MAME的共同培养​​，以确定过渡，从原位癌到浸润性导管癌的蛋白水解途径，以及相关的蛋白水解途径和其他途径参与这种过渡，如趋化因子/细胞因子/生长因子途径之间的相互作用。我们进一步表明MAME的模型可以用来作为筛选?...

<table border="1"><tbody><tr><td> 试剂/设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>重组基底膜（Cultrex的） </td><td> trevigen </td><td> 3445-005-01 </td><td>媲美基底膜（BD公司） </td></tr><tr><td> I型胶原</td><td>凝聚力实验室</td><td> 5005-B的</td><td></td></tr><tr><td> DQ-基板 （I型胶原，Ⅳ） </td><td> Invitrogen公司</td><td> DQ-COL-D12060 DQ-COL四D12052 </td><td></td></tr><tr><td> 22毫米的塑料盖玻片</td><td>尔科技有限公司</td><td> 12-547 </td><td>减少了一半，70％的乙醇和空气干燥，使用前10分钟离开。 </td></tr><tr><td>细胞培养基</td><td>龙沙</td><td> MEBM-PRF CC-3153 MEGM的CC-4136 </td><td>无酚红</td></tr><tr><td> ibiTreatμ-碟</td><td> ibidi </td><td> 80136 </td><td></td></tr><tr><td> volocity软件</td><td>在Perkin-Elmer </td><td>版本5.5 </td><td>其他品牌的成像软件，可以用来产生三维重建和电影。 </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

我们已经开发出三维共培养模型，我们长期MAME的（M ammary 1 rchitecture 和 m icroenvironmenténgineering），用于活细胞成像中实时细胞：细胞间的相互作用。我们的总体目标是发展架构，以原位癌的乳腺病变的研究进展侵入型的模型，概括。具体来说，我们开发的模型来分析预恶性乳腺上皮细胞变异和肿瘤微环境，提高或降低原位癌乳房上皮细胞的浸润性导管癌的进展已牵连的其他类型的细胞之间的相互作用。最新研究其他细胞类型的肌上皮细胞，成纤维细胞，巨噬细胞和血液和淋巴微血管内皮细胞。除了MAME的车型，其目的是概括乳房内的细胞相互作用过程中癌症的发展，我们已经制定了前列腺癌的进展同类机型。 在这里，我们说明建立3D共培养与使用的活细胞成像和功能的蛋白质检测，遵循共同培养的乳腺导管原位癌 （DCIS）的细胞和成纤维细胞，随着时间的推移，侵入型的过渡，在程序这种情况下超过二十三天在文化。 MAME的共同培养​​由多个层组成。成纤维细胞中嵌入底层的I型胶原。放在一层重组基底膜（RBM），其中原位癌细胞接种。最后的2％RBM包括顶层和补充每一个媒体的变化。图像与侵入型的进展相关的蛋白质，我们用染料荧光淬火（DQ）的基质蛋白（DQ胶原我与I型胶原和DQ的IV型胶原混合层与中间层的RB混合M）和观察使用共聚焦显微镜的活文化。光学部分被捕获，处理和Volocity可视化软件的三维重建。在MAME的共同培养​​23天的过程中，原位癌细胞增殖和凝聚到大的侵入结构。成纤维细胞迁移，并成为纳入到这些入侵结构。 DCIS的结构表面的胶原蛋白的荧光蛋白片段协会发现，细胞内，也分散在周围的矩阵。目标蛋白，趋化因子/细胞因子和蛋白激酶途径或修改的共同培养​​的细胞组成，可以减少侵袭，MAME的模型可以用来作为新的治疗方法的临床屏幕的药物。

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

我们已经制定了活细胞成像在乳腺肿瘤细胞和其他细胞之间的相互作用，在自己的微实时三维共培养模型，侵入型的影响进展。这些模型可以作为目标旁诱导蛋白，趋化因子/细胞因子和蛋白激酶途径侵袭牵连的药物临床前屏幕。

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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14.3K 次观看.  Wayne State University. 我们已经制定了活细胞成像在乳腺肿瘤细胞和其他细胞之间的相互作用，在自己的微实时三维共培养模型，侵入型的影响进展。这些模型可以作为目标旁诱导蛋白，趋化因子/细胞因子和蛋白激酶途径侵袭牵连的药物临床前屏幕。

视频: MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

原位Xenografting人类荧光素酶标记的恶性外周神经鞘瘤细胞在体内测试候选治疗药物

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

科研

医学

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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 ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

对小鼠淋巴瘤模型监测肿瘤进展和治疗效果生物发光基于肿瘤定量方法

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

体外肺转移及微环境的实时成像

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...