这项研究是支持部分由国防部乳腺癌IDEA奖W81XWH - 08 - 1 - 0583和NIH / NCI CA141348 - 01A1（DZ）和FAMRI临床科学家奖（DS）。成像的基础设施是由西南部分小动物成像研究支持计划（U24 CA126608）和西蒙斯癌症中心（P30 CA142543）和NIH 1S10RR024757 - 01。

机构动物护理和使用委员会的得克萨斯大学西南医学中心的所有动物的程序获得批准。 在体外 HIF -1α的生物发光检测1 。 <ol><li>材料和方法： <ul><li>一种新型的HIF - 1依赖记者基因转染的人类转移性乳腺癌细胞株MDA - MB231，5HRE多 - 吕克博士原田生成。 </li><li>在缺氧条件下，氧依赖性降解域（ODD）荧光素酶融合蛋白的表达增强是由5份缺氧反应元件（5HRE）。奇的存在，导致奇 - 吕克在极低的背景荧光素酶活性在常氧条件下产生的蛋白质的降解。因此，这种新颖的系统可用于检测实时成像在肿瘤缺氧地区。有报道原田等1,2本5HRE多- LUC表达载体的建设。 </li></ul></li><li>在常氧或缺氧培养细胞： <ul><li>重组的MDA - MB231细胞保持在10％胎牛血清（FBS），DMEM培养基中含有1％谷氨酰胺，400微克/毫升的G418抗生素和1％青霉素/链霉素。 </li><li> 在体外 HIF -1α的生物发光检测，板3 × 10 5 MDA - MB231细胞表达在每两个六以及菜以及5HRE多- LUC载体。 </li><li>允许附加孵育过夜后的碟壁细胞，然后转移到缺氧的研究低氧室（比卢普斯罗森堡，公司德尔马，加利福尼亚州）一盘，而保持在常氧条件下（21％O 2）下的其他盘。 </li><li>重组与气瓶连接的进气口管与0.1％O 2的和气体的腔室。 注：在此过程中，必须打开进气口和出气口夹具。 </li><li>断开气源后室已被清除和关闭塑料夹具密封腔。 </li><li>将用5％的CO 2在37℃培养箱中培养室。 注：商会必须加湿，以防止水分蒸发过快文化。这可以通过配售10 - 20毫升无菌水在会议厅内。 </li></ul></li><li>生物发光检测： <ul><li>孵育24小时后，取出培养基，用冰冷的PBS（2X）的细胞迅速。 </li><li>添加1毫升冷PBS 100μL荧光素（金生物技术，圣路易斯，密苏里州）。 </li><li>获得不同的曝光时间（1，30，60，180秒）BL成像（IVIS频谱系统，卡尺生命科学，霍普金顿马）。 </li><li>测量光的强度在每口井使用的生活图像软件（卡尺生命科学版）。 </li></ul></li></ol> 2。乳腺癌脑转移瘤模型的建立<ol><li> MDA-MB231/5HRE-ODD-luc细胞的制备<ul><li>检索和文化DEME培养基中含有10％FBS，1％谷氨酰胺和1％青霉素/链霉素MDA-MB231/5HRE-ODD-luc细胞。 </li><li>更换介质每2-3天。 Tripsinize和清洗细胞，当他们达到80％汇合。 </li><li>计数适当数量的细胞，重悬在无血清DEME介质中，他们与4μL 体积的10 5细胞终浓度25％的基质胶（BD公司，加利福尼亚州圣何塞）。 </li><li>细胞颅内注射前置于冰上。 </li></ul></li><li>手术植入<ul><li>在4-6周龄雌性裸鼠（BALB / C NU / NU;，马里兰州贝塞斯达国立癌症研究所）在这项研究中使用。 </li><li>麻醉（3％异氟醚/感应室中的氧气，异氟醚从巴克斯特国际公司，迪尔菲尔德，IL，USA），并保持与异氟醚（1％），氧（1 DM 3 / 分 ）在手术过程3中的动物。 </li><li>右顶叶皮肤应prepped优碘，然后前70％的酒精切口。 </li><li>使用高速钻，毛刺冠状缝和矢状缝合横向到2毫米的头骨，1毫米前右半球在1毫米的孔。 </li><li>绘制4μL细胞的混合物（10 5细胞）使用10μL注射器汉密尔顿（汉密尔顿公司，内华达州里诺市）。权尾椎间脑，硬脑膜下1.5毫米，使用一个特制的32G汉密尔顿针直接注入到细胞。在保持前撤出约30小号针。一个32G的细针的使用，最大限度地减少组织损伤。 </li><li>用骨蜡填充的毛刺孔和关闭与可吸收缝合线头皮。 </li><li>准备用70％酒精的切口地区。 </li><li>应用丁丙诺啡镇痛两天，每12小时。 </li></ul></li></ol> （3） 在体内的生物发光成像在乳腺癌脑转移的肿瘤缺氧动态<ul><li> 8颅内植入和每周一次的重复启动纵向生物发光成像（IVIS频谱系统）两个星期后-10周。 </li><li>一次麻醉三个小鼠（3％异氟醚/ O 2的感应室） </li><li>管理解决方案的D -荧光素（120毫克/公斤，在80μL总体积的PBS，黄金生物技术），皮下注射，每只小鼠的颈部区域， 详细介绍了此前4。 </li></ul> D -荧光素是无毒的，并已被证明是能够穿透完整的血脑屏障（BBB）和细胞膜 3,5 。 <ul><li> 3小鼠放置在成像室，并保持与异氟醚（1％），氧（1 DM 3 /分）在成像。 </li><li>荧光素注射后5分钟，获得“基本法”的成像阵列与一个不同的曝光时间（1，30，60，180秒）。 </li></ul>我们的观察表明，峰值光发射时间约5分钟后皮下注射的荧光素， 在颈部3,4。 <ul><li>分析与生活成像软件（卡尺生命科学版）数据使用绝对光子计数（光子/秒），在感兴趣的区域（ROI），手工绘制的轮廓的大脑劳保局信号。 </li><li>剧情时间当然光子计数曲线，表明肿瘤缺氧动态。 </li><li>后立即去年劳保局，管理pimonidazole，缺氧标记和1小时后，牺牲小鼠，解剖的大脑。嵌入最佳切削温度（OCT）的中期和冻结在-80 ° C冰箱的整个大脑。随后的病理甲酚紫染色法和对荧光素酶，缺氧标记pimonidazole免疫组织化学染色，与HIF -1α进行验证成像观测。 </li></ul> 4。代表性的成果： 如图1所示，劳保局信号显著较高的转染细胞在缺氧（1％O 2）室24小时孵育后观察。常氧条件下的对照组细胞观察到微弱的光发射。这可能会导致拥挤的细胞群从3 × 10 5细胞应力信号，从而诱发细胞过度表达HIF -1α后24小时文化。然而， 在体内劳保局结果进行了验证通过免疫组织化学染色显示肿瘤的缺氧和荧光素酶表达的共定位。 自动化阵列的曝光时间，使连续收购的一系列图像，这有利于用较长的曝光时间捕捉微弱信号和强有力的信号，使用更短的时间内没有饱和的CCD。 <img alt="图1" src="/files/ftp_upload/3175/3175fig1.jpg" /> 图1体外 HIF -1α的生物发光检测的顶行：3 × 10 5 24 MDA-MB231/5HRE-ODD-luc以及在缺氧室6以及盘（0.1％O 2）在每个培养细胞中期小时前取出，洗净，用1 ml PBS代替。立即加入到每孔100μL荧光素后，劳保局收购阵列的曝光时间（1，30，60，180秒）。强发光代表井底部行：作为对照，3 × 10 5细胞培养下发出微弱的光常氧（21％O 2）。 <img alt="图2" src="/files/ftp_upload/3175/3175fig2.jpg" /> A）从鼠脑右侧的微弱的光信号，在体内的肿瘤乏氧动力学生物发光成像图 2。颅内植入第一个可视化的5个星期后的MDA-MB231/5HRE-ODD-luc乳腺癌细胞。观察到的光信号增加额外的6个星期，表明增加肿瘤缺氧B）的情节表明定量光信号的光子计数的时间过程曲线。 <img alt="图3" src="/files/ftp_upload/3175/3175fig3.jpg" /> 图3免疫组织化学染色检测到的荧光素酶和缺氧 Colocaliztion。轴承在OCT包埋的乳腺癌MDA-MB231/5HRE-ODD-luc的转移一个冷冻小鼠大脑切片。一个10微米的部分免疫染色标记，pimonidazole缺氧，发现瘤内的异质性缺氧，该产品被发现与抗荧光素酶染色检测的荧光素酶的表达空间的相关性。比例尺，100微米。

<ol>
	<li>Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+hypoxia-response+enhancers+and+an+oxygen-dependent+proteolytic+motif+enables+real-time+imaging+of+absolute+HIF-1+activity+in+tumor+xenografts.">The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.</a> Biochem Biophys Res Commun. 360, 791-796 (2007).</li><li>Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+HIF-1+imaging+for+determining+optimal+timing+of+combining+bevacizumab+and+radiotherapy.">Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy.</a> Int J Radiat Oncol Biol Phys. 75, 463-467 (2009).</li><li>Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, e8051-e8051 (2009).</li><li>Contero, A., Richer, E., Gondim, A., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+quantitative+bioluminescence+imaging+for+assessing+tumor+burden.">High-throughput quantitative bioluminescence imaging for assessing tumor burden.</a> Methods Mol Biol. 574, 37-45 (2009).</li><li>Zhao, D., Richer, E., Antich, P. P., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antivascular+effects+of+combretastatin+A4+phosphate+in+breast+cancer+xenograft+assessed+using+dynamic+bioluminescence+imaging+and+confirmed+by+MRI.">Antivascular effects of combretastatin A4 phosphate in breast cancer xenograft assessed using dynamic bioluminescence imaging and confirmed by MRI.</a> FASEB J. 22, 2245-2451 (2008).</li><li>Chang, E. L., Lo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+central+nervous+system+metastases+from+breast+cancer.">Diagnosis and management of central nervous system metastases from breast cancer.</a> Oncologist. 8, 398-410 (2003).</li><li>Palmieri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vorinostat+inhibits+brain+metastatic+colonization+in+a+model+of+triple-negative+breast+cancer+and+induces+DNA+double-strand+breaks.">Vorinostat inhibits brain metastatic colonization in a model of triple-negative breast cancer and induces DNA double-strand breaks.</a> Clin Cancer Res. 15, 6148-6157 (2009).</li><li>Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodality+imaging+of+hypoxia+in+preclinical+settings.">Multimodality imaging of hypoxia in preclinical settings.</a> Q J Nucl Med Mol Imaging. 54, 259-280 (2010).</li><li>Tatum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Moeller, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+effects+of+HIF-1+blockade+on+tumor+radiosensitivity.">Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity.</a> Cancer Cell. 8, 99-110 (2005).</li><li>Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+and+distinct+functions+of+hypoxia+in+primary+tumor+growth+and+organotropic+metastasis+of+breast+cancer.">In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer.</a> Cancer Res. 70, 3905-3914 (2010).</li><li>Zhao, D., Chang, C. H., Kim, J. G., Liu, H., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Near-Infrared+Spectroscopy+and+Magnetic+Resonance+Imaging+Monitoring+of+Tumor+Response+to+Combretastatin+A-4-Phosphate+Correlated+With+Therapeutic+Outcome.">In vivo Near-Infrared Spectroscopy and Magnetic Resonance Imaging Monitoring of Tumor Response to Combretastatin A-4-Phosphate Correlated With Therapeutic Outcome.</a> Int J Radiat Oncol Biol Phys. 80, 574-581 (2011).</li></ol>

没有利益冲突的声明。

乳腺癌脑转移发生在IV期乳腺癌患者的30％。这是与高发病率和死亡率，并有一个中位生存期13个月6。是需要有合适的动物模型，模仿这种破坏性疾病，在临床上为了便于我们了解其颅内的启动和进展，以及病理生理型材。在这里，我们已经开发注入人类乳腺癌细胞，直接进入鼠脑，原位乳腺癌脑转移模型。我们以前的经验表明，一个放射学可视化（MRI）的颅内病变植入约2周后出现。这个...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> D -荧光素</td><td>黄金生物技术</td><td> L - 123 </td><td> 120毫克/公斤的PBS在体内研究的总体积80μL </td></tr><tr><td>异氟醚</td><td>巴克斯特国际公司</td><td> 1001936060 </td><td></td></tr><tr><td>基质胶</td><td> BD公司</td><td> 354234 </td><td></td></tr><tr><td>汉密尔顿注射器</td><td>汉密尔顿公司</td><td> 1701 </td><td></td></tr><tr><td> 32G汉密尔顿针</td><td>汉密尔顿公司</td><td> 7803-04 </td><td></td></tr><tr><td>低氧室</td><td>比卢普斯罗森堡，公司</td><td> MIC - 101 </td><td></td></tr><tr><td>生物发光成像系统</td><td>卡尺生命科学</td><td> IVIS光谱系统</td><td></td></tr><tr><td> G418 </td><td> Fisher Scientific则</td><td> SV3006901 </td><td></td></tr></tbody></table>

in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model

众所周知，肿瘤的缺氧起着促进恶性进展和治疗反应产生负面影响重要作用。有一点知识在原地，在体内 ，在缺氧的发展，因为缺乏有效的手段来监测这些深层次的原位肿瘤恶性脑肿瘤的颅内肿瘤，。生物发光成像（劳保局），表达的荧光素酶基因的活细胞所发出的光检测的基础上，已迅速采用为癌症研究，特别是评估肿瘤的生长或应对，在临床前动物研究治疗肿瘤的大小变化。此外，通过一个控制下的一个子序列的记者基因表达，特定的基因表达是可以被监测的非侵入性劳保局。缺氧压力下，主要通过缺氧诱导因子-1α（HIF -1α）介导的信号反应，以驱动各种基因的转录。因此，我们必须使用一个记者HIF -1α建设，5HRE多- LUC，稳定转染人乳腺癌MDA - MB231 细胞在体外HIF -1α的生物发光检测（MDA-MB231/5HRE-ODD-luc）是由孵化在低氧室，24小时前劳保局（0.1％O 2）转染的细胞，而细胞在常氧（21％O 2）作为一个控制。显着较高的光子通量细胞缺氧条件下的观察表明其启动的增加HIF -1α结合（HRE元素），比在常氧。细胞是直接注射到老鼠大脑建立一个乳腺癌脑转移瘤模型，在体内的肿瘤乏氧动力学生物发光成像。植入后2周开始，并反复每周一次。劳保局揭示增加脑肿瘤进展的光信号，表明增加了颅内肿瘤缺氧。组织学及免疫组织化学研究，以确认在体内成像结果。在这里，我们将介绍在体外 HIF -1α的生物发光检测，乳腺癌裸鼠和应用，在体内的生物发光成像技术来监测颅内肿瘤缺氧的脑转移瘤的手术建立的方法。

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

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

缺氧诱导因子-1α活动的生物发光成像是用于监测在乳腺癌脑转移瘤小鼠模型的颅内肿瘤缺氧发展。

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 ...

in-vivo-bioluminescence-imaging-tumor-hypoxia-dynamics-breast-cancer

Research

JoVE Journal

Medicine

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

19.5K 次观看.  University of Texas Southwestern Medical Center. 缺氧诱导因子-1α活动的生物发光成像是用于监测在乳腺癌脑转移瘤小鼠模型的颅内肿瘤缺氧发展。

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

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 ...

科研

医学

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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 ...

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 ...

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 ...

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 ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

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 ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...