我们非常感谢安德鲁·霍兰德博士，萨拉·戴维斯，李舒曼，蒂姆·詹金斯博士祥盛徐的专家协助。我们承认安·肯尼迪博士的支持。 BCB支持的放射生物学培训津贴C5T32CA009677的。 JFD宝来惠康成就奖，医学科学家（1006792）的支持。 JLB支持的超类（5 R25 CA140116-03）资助。我们要感谢史蒂夫哈恩博士的鼓励和支持，使我们的研究可能。我们还要感谢大学的宾夕法尼亚州纳米生物界面中心（NBIC）博士和丹尼斯迪彻的的鼓励和有益的意见。我们认识到，小动物成像设备（SAIF）在美国宾夕法尼亚大学的MRI和光/生物发光核心设施的使用。这些技术开发项目的一部分，由美国国立卫生研究院（RC1 CA145075和K08 NS076548支持01）。

A.术前肿瘤细胞制备<ol><li>混合厚膜U251多形性胶质母细胞瘤细胞稳定表达萤火虫荧光素酶基因的慢病毒表达载体（pGreenFire，系统生物科学）。 </li><li>这些细胞被生长在10ml完整的Dulbecco的修改的Eagle培养基（DMEM）中，其中包括的DMEM补充10％小牛血清，1％青霉素 - 链霉素，1％非必需氨基酸在一个的T75组织培养烧瓶中孵育5％CO 2和37℃下</li><li>执行标准细胞培养开始，用洗涤的细胞用磷酸盐缓冲盐水（PBS），其次是胰酶消化。 </li><li>淬灭与完全DMEM的胰蛋白酶，将溶液转移到一个50毫升的锥形烧瓶中，并用Coulter计数器确定细胞浓度。 </li><li>在约3000转每分钟的3分钟，离心收集的细胞在完全DMEM。 </li><li>离心后，吸f的媒体，只留下一个在底部的50毫升锥形瓶中的细胞沉淀。 </li><li>工作迅速，以防止干燥的细胞，重新悬浮细胞在一定体积的新鲜完全DMEM以达到所需浓度的50,000个细胞/μl，并转移至2毫升无菌小瓶置于冰上。 </li><li>在低设置〜20分钟，以防止细胞粘附，细胞在冰上进行简单的振荡。细胞可以安全地用于颅内植入了两个小时后，放置在冰上。 </li></ol> B.原位异种移植植入<ol><li>称取裸体裸鼠：NCR鼠标（女/女），建立一个基线，胚胎植入前的重量。我们通常选择6-10周龄小鼠17克和24克的重量之间。 </li><li> 1小时，在植入前，预先用5毫克/公斤的非甾体类抗炎药美洛昔康经皮下注射作为手术后的疼痛和炎症的治疗药物鼠标。 </li><li> Anesthetize的动物腹膜内注射氯胺酮/赛拉嗪的混合物中，在140毫克/千克和10毫克/公斤分别的剂量。 </li><li>确认鼠标评估动物的自发的回应掐脚趾有足够的麻醉。如果动物的脚趾掐响应，延迟的过程，，直到麻醉才能生效，或考虑注入额外的麻醉（原量的25％）。 </li><li>准备的麻醉鼠标进行定位的Stoelting数字只是鼠标立体平台（Stoelting公司）挂钩鼠标的门牙牙齿的咬酒吧的吻部限制器和紧缩的鼻钳过的吻，同时确保，鼠标的头部是上一个水平的飞机。 </li><li>转移内敛鼠标Stoelting立体定向平台，调整耳杆的前端，使该动物的定位在耳道的尾端。一旦动物的颅到尾部的定位已经adjus，TED，确保咬棒的立体框架。鼠标应该搁在一个公司塑料加热板（哈佛大学设备），用胶带固定Stoelting平台，提供反馈控制温度调节在手术过程中。 </li><li>调整必要时耳杆的高度，然后推进到耳道的尾部的耳杆，把它们固定，使得鼠标的头部是在一个水平面上，并固定在手指触摸。仔细监督的耳棒放置后的呼吸窘迫的迹象。松开并重新定位，如果动物是在遇险。 </li><li>插入的润滑尖的一个直肠温度探头连接到的TCAT-2DF温度控制器（哈佛大学设备），以监控动物的温度，并以提供反馈控制的加热板定位下鼠标，以保持动物的体温在36°Ç在的程序。 </li><li>眼药膏对眼睛preveNT干燥。 </li><li>应用聚维酮碘溶液的顶部，鼠标的头部切口部位进行消毒，照顾，以避免眼睛。 </li><li>执行一个脚趾头掐确认鼠标是无意识的，提供额外的氯胺酮/甲苯​​噻嗪的初始剂量（&lt;25％），如果需要少量的。 </li><li>清洁的0.45毫米毛刺钻头（Stoelting）连接到使用酒精垫的的钻头（Foredom微钻头），然后消毒钻头，持续15秒，在一个玻璃珠灭菌器（种子发芽500，CellPoint科学），只有在钻头进入浸入珠灭菌器。 </li><li>使用无菌解剖刀，使一个0.75厘米的切口在中间头皮从尾端的眼睛的水平延伸的纵向。确认前囟的可视化。 </li><li>附加的钻头架的Stoelting平台放置一个的钻头（Foredom微钻头）与0.45毫米毛刺钻头（Stoelting）在钻头架，它固定在适当位置。 </li><li>将钻头完全在T他囟，然后零出的x，y，z坐标的数字立体显示。的钻头的前端移动到一个位置后2毫米和1.5 mm横向囟在右大脑半球和动物的颅骨与Foredom钻头钻入，刺入只有骨。可使用的无菌棉签轻轻缩回的切口的边缘，以方便的头骨的可视化。 </li><li>取出钻头和钻架的立体平台。 </li><li>将Nanomite注射器注射泵（哈佛大学设备）的立体平台。 </li><li>从冰中取出的细胞，或者轻轻涡小瓶含有肿瘤细胞短暂脉冲或轻轻一抖小瓶重新悬浮细胞。 7微升的细胞悬液通过30号“长平锥针连接到10μl注射器（Hamilton注射器）。避免大气泡的注射器，确保在最初的6μl的液体有没有气泡WH脑出血将鼠标注入的总体积。在其它的1微升的小气泡是不关心。避免重复拉伸起坐如果可能的话，以尽量减少损坏细胞的细胞悬浮液。 </li><li>定位到注射器喷射器加载的注射器。使用的醇垫，除去任何的细胞悬浮液的流体出现在针尖与癌症细胞，这可能会导致肿瘤生长在颅外空间的切口处，以防止污染。 </li><li>设置该喷射泵提供6.0微升0.5微升/分钟的速率。确保经常收集在注射器中的空气气泡没有注入到动物中，残留在注射器中，在植入后的细胞悬浮液的1微升。注入气泡，可能会导致致命的空气栓塞。 </li><li>推进注射器针头插入保持针垂直（90度）到所述颅骨钻孔。一旦针已经走过的头骨，零出coordin阿泰上的立体定向的数字显示和然后慢慢推进针尖的超过4分钟的一段时间，直到它到达一个深度为2.5毫米。针穿过后海马皮层和部分。的x，y，z坐标用于立体定向植入的选择生成的大脑半球内的横向定位的肿瘤，同时避免损伤关键大脑结构如丘脑和接近的心室，从而减少的可能性播种与肿瘤细胞的脑脊液可能会引起不良的椎管内肿瘤。选择一个相对于前囟门后的位置，以减少的可能性大，局部晚期肿瘤，溃疡进入轨道。暂停针深度2分钟，然后启动的细胞的植入。 </li><li>在植入过程中，干燥的头骨，多次与一个显微外科海绵矛，删除任何肿瘤的液体，可能已经回流的钻孔植入。避免扰乱针显微海绵。删除此液减少的在颅外空间肿瘤生长的可能性。 </li><li>注射完成后，在大脑中留下针约2分钟，然后慢慢地撤回针超过3-4分钟期间。 </li><li>松开耳朵酒吧和吻部限制器和删除鼠标从立体定向设备。 </li><li>关闭在颅骨钻孔，用无菌骨蜡沉积钻孔的无菌棉签的木端之间来回擦蜡。继续施加骨蜡，直到被完全密封孔和骨蜡密封钻孔是与相邻的头骨平齐。 </li><li>重新接近边缘的切口，用无菌棉签和应用兽医组织胶封住伤口，照顾动物的眼睛，以避免胶水曝光。 </li><li>最后，将鼠标放在一个加热垫设置为37°。动物C，直到恢复意识。的动物转移到原来的笼子，一旦鼠标警报和反应。 </li></ol> C.生物发光成像（BLI）来监测肿瘤生长及对治疗的反应简要说明遵循的生物发光成像。 <ol><li>麻醉小鼠，在一个腔室中，用2％异氟烷和氧先前与肿瘤植入。 </li><li>虽然小鼠麻醉，皮下或腹腔内注入它们的D-荧光素的钾盐在PBS中稀释至浓度为50毫克/毫升，用60微升。 </li><li>打开的流量麻醉中的生物发光成像扫描仪的鼻锥，并迅速转移的小鼠的扫描仪放置鼻的鼻锥。 </li><li>放置黑色的老鼠之间的分隔限制到相邻的肿瘤出血肿瘤生物发光信号的信号强度高与低得多的信号。 </li><li>运用活体成像软件，频繁序列性风险的D-荧光素注射后的总持续时间长达30分钟。我们看好的曝光时间设置为“自动”限制的可能性下或过度曝光的图像。没有一个单一的曝光应该是5分钟。 </li><li>在适当的时间间隔执行重复生物发光成像。串行每周成像是一个合理的选择许多纵向的实验测试对治疗的反应。 </li><li>收购后的图像，使用活体成像软件程序来分析肿瘤的生物发光成像会议期间获得的每幅图像中绘制每个肿瘤周围地区的利益（投资回报率）。第二个较小的感兴趣区域的应用到各小鼠的下侧面在每个图像中感兴趣的纠正肿瘤的生物发光信号的基于背景的生物发光信号强度的程度上为背景区域。我们更喜欢使用“Radiance”号设置，而不是“计数”作为输出生物发光信号的值。 </li><li>结果到Excel中导出的投资回报率，并确定每个鼠标的最大背景调整后的生物发光信号。 </li><li>重复生物发光成像，作为连续监测肿瘤的生长。在我们的实验中，我们执行每周BLI。我们更喜欢确定，BLI的最大信号，对于一个给定的成像会话以频繁曝光超过5 - 30分钟后，注射D-荧光素。 </li></ol> D.代表性的成果这种立体定向的植入技术相关联的一个成功的肿瘤率90-100％，并与围手术期死亡率低，通常小于5％。意想不到的副作用的风险也低，使用这种技术，包括作为播种的脊髓从注入脑室，或颅外肿瘤生长的肿瘤细胞，无论从播种的切口与肿瘤细胞或毛刺孔封闭不足等并发症一llowing颅内肿瘤扩大开幕的头骨。 移植瘤的体外分析表明预期缺氧，血管内皮生长因子的表达增加，坏死。稳定表达由我们的GBM细胞线的绿色荧光蛋白（GFP）的荧光显微镜揭示浸润这些异种移植物的性质。 图1示出了一个典型的成功GBM细胞鼠标进入脑立体定向植入的结果。这是一个T2加权脑部核磁共振成像扫描的一个9.4特斯拉磁体植入后21日内这里描述的技术进行小鼠脑。 图1显示右侧大脑半球肿瘤中（轮廓的粉红色）测量单次对焦19毫米3定位于的植入位点的精确坐标。 图2示出了生物发光成像的结果，使用技术部<html> iques这里描述为一组10只小鼠用均匀分层最大的生物发光信号强度的基础上，接受全脑照射（4戈瑞×4每天分数）或没有治疗的立体定向移植瘤。在该实验中，生物发光成像示出了放射治疗抑制移植瘤的增殖，产生在所检测到的生物发光信号中没有增加，而信号基本上增加模拟照射控制肿瘤，由于未选中的癌细胞增殖。 图1（视频）。冠状MRI节的小鼠脑含U251多形性胶质母细胞瘤的肿瘤体积与等高线（粉红色）。在9.4特斯拉的扫描仪进行扫描，使用一个自旋回波T2加权协议。 <a href="http://www.jove.com/files/ftp_upload/4089/4089movie1.m4v" target="_blank">点击这里观看电影</a> 。 再2“SRC =”/ files/ftp_upload/4089/4089fig2.jpg“/&gt; 图2。立体定位植入的多形性胶质母细胞瘤治疗的10只小鼠，无论是外照射放射治疗16 Gy的4份或不治疗。对小鼠进行成像的生物发光成像治疗前和治疗开始后，每周。该图给出相对倍的变化电流最大BLI值到治疗前的最大BLI值的比率被定义为作为倍的变化中位数计算的生物发光。

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没有利益冲突的声明。

在本文中所描述的小鼠肿瘤细胞立体定向植入的方法，可重复生成肿瘤的浸润和快速增长的模式，临床多形性胶质母细胞瘤2，6-8，合理地概括。此技术特别适合均匀地不同的可比尺寸和生物学性质，并在特定的解剖位置的重现性肿瘤的治疗组中是可取的分层小鼠的实验。立体定向植入肿瘤细胞的技术，我们描述应该是容易实现的，大多数的翻译研究实验室7,9-11。

<table border="1"><tbody><tr><td> 描述 </td><td> 提供者 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>数字只需鼠标立体定位仪</td><td> Stoelting </td><td> 51730D </td><td>对小鼠植入的立体平台</td></tr><tr><td>氯胺酮/甲苯​​噻嗪</td><td></td><td></td><td>注射麻醉</td></tr><tr><td> Puralube兽医软膏（眼） </td><td> Amazon.com </td><td></td><td>为了防止干燥鼠标的眼睛</td></tr><tr><td>钻套的立体平台</td><td> Stoelting </td><td> 51681 </td><td></td></tr><tr><td>微电机电钻</td><td> Stoelting </td><td> 51449 </td><td>对于通过颅骨钻孔</td></tr><tr><td> 0.45毫米硬质合金钻头</td><td> Stoelting </td><td> 514551 </td><td></td></tr><tr><td>无菌棉拭子</td><td> Fisher Scientific则</td><td> 23-400-100 </td><td></td></tr><tr><td>玻璃珠干燥灭菌器（种子发芽500） </td><td>布伦特里科学</td><td> GER-5287 </td><td>金属手术器械消毒</td></tr><tr><td>鼠标直肠探头</td><td>布伦特里科学</td><td> RET-3-ISO </td><td>适合的温度控制器</td></tr><tr><td>温度控制器（TCAT-2DF） </td><td>哈佛设备</td><td> 727561 </td><td>维持动物的体温在手术过程中的温度控制器</td></tr><tr><td>小加热板</td><td>哈佛设备</td><td> 727617 </td><td>在手术过程中使用温度控制器的保暖鼠标。加热板适合在鼠标下的立体平台。 </td></tr><tr><td>一次性手术刀</td><td> BD巴德 - 帕克</td><td> 2015-11 </td><td>第10刀</td></tr><tr><td> 10微升注射器</td><td>汉密尔顿</td><td> 7635-01 </td><td>对于注射肿瘤细胞</td></tr><tr><td> 30号针，1“长平</td><td>汉密尔顿</td><td>各个</td><td> 10μl注射器必须兼容</td></tr><tr><td> Nanomite可编程注射泵</td><td>哈佛设备</td><td> 704507 </td><td>数字立体定位装置的电动注射器注射器</td></tr><tr><td>纤维素的无菌手术矛海绵</td><td> UltraCell的</td><td> 40410 </td><td>要干外科领域</td></tr><tr><td>骨蜡</td><td>爱惜</td><td> W31 </td><td>以密封钻孔</td></tr><tr><td> Tissumend II合成可吸收组织粘合剂</td><td>兽药产品实验室</td><td> 3002931 </td><td>以密封该切口</td></tr><tr><td>热水泵变暖垫</td><td> Gaymaŕ </td><td> TP-650 </td><td>温暖的小鼠在手术后期间</td></tr><tr><td> IVIS Lumina的II </td><td>卡尺生命科学</td><td></td><td>生物发光成像仪</td></tr><tr><td> D-荧光素钾盐</td><td>金生物技术</td><td> LUCK-1 </td><td>荧光素生物发光成像</td></tr></tbody></table>

stereotactic intracranial implantation and in vivo bioluminescent imaging of tumor xenografts in a mouse model system of glioblastoma multiforme

胶质母细胞瘤（GBM）是一种高品位的原发性脑肿瘤在人类中位生存期仅为14.6个月，尽管标准的三模态的治疗包括手术切除，手术后的放射治疗和替莫唑胺化疗1。显然需要新的治疗方法，以提高患者生存率和生活质量。 GBM的动物模型的，概括人类疾病还允许串行成像技术监测肿瘤的生长和治疗反应，将有助于发展更有效的治疗策略。在本文中，我们描述了我们的技术，精确的立体定向植入生物成像GBM肿瘤细胞的裸鼠移植瘤，概括主要临床特征的GBM 2的大脑。这种方法产生的肿瘤是可重复和精确的解剖位置，同时允许在体内生物发光成像的连续监测内颅移植瘤的生长和治疗3-5。这种方法也很好的耐受性低的围手术期发病率和死亡率的动物。

Watch this Scientific Journal Video about Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme at JoVE.com

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

我们描述一个综合性的方法，精确，立体的人多形性胶质母细胞瘤细胞植入到裸鼠的大脑和随后的串行<em在体内</em&gt;成像技术来监测治疗所造成的异种移植物的生长和响应。

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 ...

stereotactic-intracranial-implantation-vivo-bioluminescent-imaging

Research

JoVE Journal

Medicine

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

25.4K 次观看.  University of Pennsylvania. 我们描述一个综合性的方法，精确，立体的人多形性胶质母细胞瘤细胞植入到裸鼠的大脑和随后的串行

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

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

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 ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

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 Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

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 ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...