我们感谢J. Cappellani和J.邱的技术支持。这项工作是支持的资金由美国国家癌症研究所（U01 CA141451），斯塔尔癌症协会，苏珊科曼为治愈，长岛第2天城抗击乳腺癌，曼哈塞特妇女的抗乳腺癌联盟ME，和从国会导演的乳腺癌研究计划的博士后奖学金，，美国ESNESN是收件人的Leslie C.快速和威廉·伦道夫·赫斯特基金会奖学金从沃森生物科学学院。 HAA得到了资金从研究理事会挪威（160698/V40 151882），和东南地区卫生局（2007060）。

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作者有没有利益冲突的披露。按照IACUC批准的协议，所有的动物进行了实验。

肿瘤在体内的全身治疗的反应，可以显着不同的，因为在体外癌细胞的微环境影响的急性反应和复发5。诉说在体内的耐药途径的最大障碍之一是肿瘤细胞和微环境之间的相互作用的复杂性。特别是，因为已经开发固体肿瘤癌症和基质细胞，扰动的一种成分，如抗肿瘤治疗的过程中发生的细胞死亡之间的平衡，可以导致对组织的组织的戏剧性的效果。 活体成像技术提供了允许直接在现场，麻醉小鼠的肿瘤的肿瘤细胞 - 基质相互作用的可视化与抗癌药物在治疗过程中。我们使用旋转盘共聚焦显微镜，它有一个相对有限的穿透深度（见17 </sup&gt;的），但我们的外科手术和标记技术可用于与其它类型的显微镜。从这些实验中获得的影可以用来跟踪过程，包括的荧光强度的变化（ 例如，由于标记人口或诱导细胞死亡的渗透），细胞运动，注射剂的分布（ 例如，跟踪血管渗漏），和合作本地化的荧光信号5,12,17,20。 我们经常在图像小鼠超过6小时，超过18小时，并进行成像。这需要保持不断在麻醉状态下的小鼠，这些长的时间周期。通过适当的麻醉，超过90％的动物生存6个小时，约80％存活超过18小时。有关如何进行长期的麻醉先前已公布的19。在我们的经验中，有五个因素是至关重要的：避免低温，避免脱水，保持的生理血液中氧饱和度水平，U唱加湿，异氟醚的载气，并保持动物在麻醉的最低水平，在它们不显示疼痛的迹象。为了保持体温，我们养老鼠在加热毯（38°C）。为了避免脱水，在整个成像过程中，我们注入少量的生理盐水IP。保持生理血液的氧饱和度水平，我们开始与载气中的（而不是常用的100％）的21％的氧气。这使我们吸入的氧含量增加，如果鼠标 - 小时为一个实验 - 显示血氧饱和度下降。根据我们的经验，增加吸入氧气水平的时间（如30-40％）几乎总是稳定的动物，让几个小时的额外的影像。麻醉的最佳水平，为大多数小鼠与1-1.5％异氟烷在60-65/min和脉冲率400-450/min的呼吸率和结果取得。在我们的成像实验中，我们主要使用转基因小鼠模型（MMTV-PyMT和C3 [1]-标签），其中肿瘤是多焦点，允许在不同的阶段的肿瘤进展的单个成像会话期间在多个的x，y位置的平行的多个病灶成像。这减小关切鼠标鼠标相对于实验旨在识别阶段依赖性的治疗反应的变化，并减少所需的成像的动物的数目。 通过渐进的病理组织学阶段，可以在活体成像识别的MMTV-PyMT模型的（和其他自发肿瘤模型）去，虽然可以做的活体成像过程中的肿瘤性病变的病理分期是不同的，不太详细，比组织切片5,17。相比之下，移植的模型往往反映晚期肿瘤最好的，因为晚期肿瘤的癌细胞通常是孤立的。因此，这种模型更适合于研究肿瘤间质晚期肿瘤的相互作用在不同阶段之间的相互作用大于分歧。 我们的例子形象核结构所发生的变化后诱导细胞死亡的治疗。在抗癌治疗的情况下，该标记策略，而不是可能可用于在原位的图像细胞分裂，阐明，例如，细胞的增殖和休眠如何的影响的微环境。在未来，线粒体成分的荧光标记可能有可能图像线粒体细胞凋亡的细胞死亡过程中所发生的变化。 在这个协议中，我们还如何图像治疗后癌细胞的免疫细胞间的相互作用，但其他微环境的组成部分，也可以是可视化和成像的例子。现有的转基因记者基质成分包括那些为成纤维细胞（ 如 ，FSP1-EGFP 17，21αSMA-RFP，COL1A1-EGFP 21）或细胞的脉管（ 如 Tie2的-GFP 22; VEGF-GFP的23）。 活体成像允许发生在体内以下化疗给药的相互作用和过程的实时可视化。目前的技术，我们已经取得了重大的进展，在了解髓细胞如何影响治疗的反应;然而，由于肿瘤微环境是如此复杂，重大的进步仍然需要开始钻研机械地环境介导的耐药性的过程。仍然需要的最重要的进步之一是更好的标记物的识别，特定细胞群。更好的标记物的重要性，是很好的说明有两个最突出的乳房肿瘤微环境，成纤维细胞和免疫细胞的基质细胞组件。 α-平滑肌肌动蛋白（αSMA）是经常被用来识别成纤维细胞，但该蛋白也表达的血管平滑肌细胞和肌上皮细胞24。因此，尽管存在αSMA-GFP记者小鼠，在活体成像实验确定特定的细胞类型，以下是具有挑战性的。同样，一个单一的荧光标记物，如EGFP表达的c-FMS子下，往往会识别多种免疫细胞亚群的12,17。因此，虽然理想情况下，人会喜欢使用一个单独的标记来识别特定的细胞类型使用这种方法的基本生物学的复杂性构成了重大挑战。 对这个问题的部分解决方案之一是使用注射型标签，以进一步区别细胞亚群的表达相同的荧光报道。例如，荧光葡聚糖由巨噬细胞，并可以用来标记所标记的c-fms的-EGFP和FSP1-EGFP转基因报告小鼠的巨噬细胞的亚群<sup&gt; 17。抗体共轭荧光探针也被用来识别特定亚群（ 例如 GR1-阳性人口的骨髓细胞标记的C-FMS-EGFP记者17）。 不同的游标卡尺测量，它提供的小机械信息如何或为何肿瘤给药治疗，或组织学系列在不同的时间点，需要大样本的动物组织中收获来自肿瘤生成的响应曲线，我们的技术提供了直接的，可量化的信息上的动态的细胞死亡，并在小同伙的基质细胞与癌细胞上的相互作用。因此，这里报告的使用过程的优点是它提供了对药物的反应的上下文中，一个完整的肿瘤微环境中的实时的动态信息。这些信息可以到底层的进程，推动对治疗的反应导致了重要的见解&lt;/ SUP&gt;。

实体肿瘤包括两大隔间：癌细胞和基质成分（蜂窝和非蜂窝）协助维持一个适当的环境为肿瘤的生长1。这些间质的的的选民中发挥着重要的的角色，，在的开发，“的进展，和”转移中的许多类型的癌症，其中包括那些的的乳房2-5的的的中 。的的间质的环境也地影响对治疗的反应2,4,5。因此，确定癌细胞如何响应常规和新疗法的原封未动的微环境的上下文内，重要的是我们进一步认识癌症生物学和改善目前的治疗策略。此外的是，对于理解的生物学的肿瘤复发的，定义了如何癌症的细胞-间质的相互作用的会，随着投药的治疗而改变是至关重要的的的的。 器官型的的合作培养系统已被有用的，用于研究肿瘤 - 基质的的相互作用的<s最多&gt; 6，但它是明显的的，，目前可用的的方法不能成功地扼要重述的完好无损的的肿瘤微环境在体外培养，置于特别是相对于到的免疫细胞的血管功能和征聘。事实上，癌症细胞的反应，顷在体外观察到的的治疗，的客人也常常显着不同的从那些在体内 ，在那里的微环境是完整的5的中观察到的。因此，， 在了上的的治疗反应正研究肿瘤-基质的的的的相互作用和他们的的的的影响的体内实验模型为中，可能会更好地反映临床相关的的机制，7。 研究到在体内的抗肿瘤治疗的响应的主要手段，已被通过来自处理过的动物或患者，的的派生的组织的的肿瘤的大小和的组织学评估的测量的。 ，则即使，在显微镜和荧光记者，中的的进步不会明显缩小超过的过去的的两个几十年来已经启用了的的间-和细胞内的的流程的可视化，在高分辨率下在活的的，麻醉后的的的动物（的活体成像）8 JOURNAL。这些的活体显微镜技术已被证明到是尤其是宝贵的的用于在空间上上和时间上地解剖所述在体内动力学的肿瘤-基质的蜂窝式的和子-蜂窝式的的的决议的的相互作用在8,9的 。 由的活体成像的揭开了的被的生产工艺进行过包括的的抗-肿瘤的T细胞的的细胞毒作用10,11，T细胞的系和髓系的的服务小区12的之间的相互作用，的动态变化的“在胶原蛋白的的组成和”组织以下食疗的治疗13日 ，巨噬细胞-而引发相关癌症的细胞的的血管内的和转移“的变化14日 ，和血管通透性的15。 有是三个主要的的的的为的活体成像的要求。这些措施包括的组织中的适当的准备和曝光为成像，的的的组织的组件的的兴趣时在荧光灯的标签中，和一个已配对的显微术-摄影相机S变体系能够采集图像16。的用于，以解决这些的要求的的最常见的的战略包括：1）异位准备工作（ 例如 ，。，的耳朵或眼睛简直轨道的的接种的），永久的的成像的窗户，的或形象化的的准备工作（ 例如 ，。，背鳍的皮肤瓣）; 2）转基因荧光记者，的，并荧光血管注射剂到组织成分贴上标签;，和3）与的光电倍增管或押记偶联型的移动设备（CCD）相机，分别为9配对的多光子或共聚焦显微镜系统。 ，我们使用一个外科手术准备的的的皮肤的皮瓣模型，以暴露的腹股沟的的乳腺腺为表达的编码荧光记者，的转殖基因的的的麻醉动物的，的成像技术在。的画像顷得到的过了一段中的12小时到第40小时，使用一个增强型CCD（ICCD）的摄像头与一个四-激光纺丝磁盘共聚焦显微镜观察系统17配对。在以这种方式中的影像学已经允许我们，以研究这样的的的的进程，，作为在的体内的药物分布，阶段-依赖的的的CH中emotherapeutic的的反应，的化疗-诱导的细胞死亡，系和髓系的细胞的行为5开始的类型。 我们提供了一个的的活体的成像的癌症细胞的反应，的，以的的抗-癌的疗法和在乳房癌症的小鼠模型中的中的肿瘤 - 基质的的相互作用施治，的的协议，用于。此协议可以用于来跟踪的两种癌症细胞和间质组件的行为和死亡的与内容广泛且具有的各种各样的转基因的和可注射的的的荧光标记在转基因和器官移植两种模型中为期限最长可达到第40小时在一个单一的的成像届会议的。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> MMTV-PyMT小鼠</td><td>杰克逊实验室</td><td> 2374 </td><td></td></tr><tr><td> C3（1）标记的小鼠</td><td>杰克逊实验室</td><td> 13591 </td><td></td></tr><tr><td> ACTB ECFP小鼠</td><td>杰克逊实验室</td><td> 3773 </td><td></td></tr><tr><td> ACTB-H2B-EGFP小鼠</td><td>杰克逊实验室</td><td> 5418 </td><td></td></tr><tr><td> 1×PBS </td><td>内部</td><td></td><td></td></tr><tr><td> 2 MD葡聚糖，荧光素共轭的</td><td> Invitrogen公司</td><td> D-7137 </td><td>重组2 O（4毫克/毫升，储存在-20°C）蒸馏水稀释至1 mg / ml的1×PBS </td></tr><tr><td> 10 kD的葡聚糖的Alexa Fluor 647-共轭</td><td&gt; Invitrogen公司</td><td> D-22914 </td><td>重组2 O（4毫克/毫升，储存在-20°C）蒸馏水稀释至1 mg / ml的1×PBS </td></tr><tr><td>盐酸阿霉素</td><td> Sigma-Aldrich公司</td><td> 44583 </td><td>重组在卫生署2 O和存储在4°C，在1×PBS稀释</td></tr><tr><td> Isothesia（异氟醚） </td><td>巴特勒动物保健供应</td><td> 029450 </td><td> 250毫升</td></tr><tr><td>碘化丙啶</td><td> Invitrogen公司</td><td> P3566 </td><td> 1毫克/毫升，稀1:15 1×PBS中</td></tr><tr><td>氮</td><td></td><td></td><td></td></tr><tr><td>氧</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> 18G×1½“定期锥针</td><td> BD </td><td> 305196 </td><td></td></tr><tr><td> μManager </td><td>淡水河谷实验室，加州大学旧金山分校</td><td> <a href="www.micro-manager.org" target="_blank">www.micro-manager.org</a> </td><td>开放源码软件</td></tr><tr><td>酒精棉签</td><td> BD </td><td> 326895 </td><td> 70％的异丙醇拭子</td></tr><tr><td>麻醉系统</td><td>分子影像产品，公司</td><td></td><td></td></tr><tr><td>盖玻片</td><td>康宁</td><td> 2940-245 </td><td>第1½，24x50毫米; 70％的异丙醇擦拭消毒</td></tr><tr><td> Curity纱布（无菌） </td><td>肯德尔</td><td> 6939 </td><td></td></tr><tr><td>显微镜幻灯片</td><td>康宁</td><td> 2948-75x25 </td><td>预清洗，如果没有预先清洗，消毒用70％的异丙醇湿巾</td></tr><tr><td>硬化精剪</td><td>精细科学工具</td><td> 14090-11 </td><td>不锈钢，锋利的尖，直头，26毫米的切削刃，11厘米长2对; </td></tr><tr><td>电热毯</td><td> Gaymar产业</td><td></td><td></td></tr><tr><td>热珠灭菌器</td><td>精细科学工具</td><td> 18000-45 </td><td>在使用前打开约30分钟;消毒工具，在&gt; 200℃下进行30秒</td></tr><tr><td> Imaris </td><td>位面</td><td> <a href="www.bitplane.com" target="_blank">www.bitplane.com</a> </td><td></td></tr><tr><td> KRAZY胶</td><td> Elmer的产品</td><td> KG484 </td><td></td></tr><tr><td>实验室带（½“） </td><td></td><td></td><td></td></tr><tr><td>实验室磁带（1“） </td><td></td><td></td><td></td></tr><tr><td>盖保丽龙航运冷却器</td><td></td><td></td><td>这将被用作手术平台</td></tr><tr><td>微解剖钳</td><td> Roboz </td><td> RS-5153 </td><td> 1x2齿，轻微的曲线，0.8毫米刀头宽，4“长度</td></tr><tr><td>微解剖钳</td><td> Roboz </td><td> RS-5135 </td><td>锯齿状的，轻微的曲线，0.8毫米的刀尖宽度4“长度</td></tr><tr><td>显微镜</td><td></td><td></td><td>纺纱盘共聚焦，XYZ压电的阶段，epifluourescence capablility 17,25; </td></tr><tr><td>显微镜载物台插入</td><td>应用科学仪器</td><td></td><td>自定义制作，有两个圆形成像端口</td></tr><tr><td> MouseOx血氧饱和度，软件和传感器</td><td> STARR生命科学</td><td> <a href="www.starrlifesciences.com" target="_blank">www.starrlifescience</a>s.com </td><td></td></tr><tr><td>干的Nalgene超级砂劳越实验室soakers </td><td> Thermo Scientific的</td><td> 74218-00 </td><td>排队手术的平台，切成四分</td></tr><tr><td>喷雾器</td><td>索尔特实验室</td><td> 8901 </td><td>加湿气体，防止刺激肺组织</td></tr><tr><td>滑尖一次性结核菌素注射器（1ml）中</td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> Surflo翅输液器</td><td>泰尔茂</td><td> SV-23BLK </td><td> 23G x¾“超薄针，12”管</td></tr><tr><td>聚维酮碘喷雾</td><td> Purdue Pharma公司</td><td> BASP3H </td><td></td></tr></tbody></table>

live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer

肿瘤微环境中起着举足轻重的作用，在肿瘤的发生，发展，转移和抗癌疗法的反应。三维共培养系统经常被用来阐明肿瘤 - 基质的相互作用，包括它们的作用，药物的反应。然而，许多完整的微环境中发生在体内的相互作用不能被完全复制在这些体外设置。因此，直接可视化这些进程在实时了解肿瘤反应疗法和识别肿瘤细胞之间的相互作用和基质，可以影响这些反应已成为一个重要的工具。在这里，我们提供了一种方法，使用旋转盘的活，麻醉小鼠的共聚焦显微镜直接观察药物分布，癌症细胞的反应和在给药后的全身性治疗乳腺癌模型中的肿瘤 - 基质相互作用的变化。我们为labeli描述程序ng的不同肿瘤组件，用于观察治疗反应治疗的动物，和用于暴露长达40小时的用于成像的肿瘤组织的外科手术。此协议从所获得的结果的时间推移的电影，在其中可以使用图像分析软件评价药物浸润，肿瘤细胞死亡和基质细胞迁移等过程。

Watch this Scientific Journal Video about Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer at JoVE.com

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

我们描述了一种方法，成像响应于抗肿瘤治疗的<em在体内</em在单细胞分辨率。

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 ...

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Research

JoVE Journal

Medicine

24.6K 次观看.  Watson School of Biological Sciences. 我们描述了一种方法，成像响应于抗肿瘤治疗的

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

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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 ...

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 ...