Name: monitoring tumor metastases and osteolytic lesions with bioluminescence and micro ct imaging
Uploaded: 2011-04-14T13:00:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging at JoVE.com

纵向成像用于临床前研究，遵循的一种疾病进展或衡量治疗效果。在肿瘤学，光学方法提供了严格的工具来监测肿瘤的生长，并在每个时间点在这样的研​​究提供精确的定量细胞生长的基因表达。解剖变化，可使用高分辨率像microCT技术测量，但低的X射线剂量的纵向成像，必须使用以避免生物文物。光学和microCT图像可以被注册，提供了一个功能和解剖，确保尽可能多的信息是从的动物模型中提取数据的组合。 1。细胞制备<ol><li>卡尺提供了一个荧光素酶表达的癌症细胞株在小鼠模型的临床前研究的范围。 </li><li> MDA - MB - 231 - LUC - D2H2LN是人类乳腺肿瘤细胞株，表达荧光素酶基因，可在体内的光学指标的tumorgenesis使用。创建该细胞系是从一种自发的淋巴结转移，从一个D3H1乳腺脂肪垫肿瘤起源，被称为积极的形式转移。 </li><li>作为一个无病原体的冷冻文化容易生长标准媒体没有必要为选择标记的细胞提供。 </li><li> 90％融合瓶中要验证前注入动物的荧光素酶的活动是由胰蛋白酶消化收获。 50,000个单元格，并在微孔板配药，进行连续稀释荧光素酶活性测定。 </li></ol> 2。在动物细胞腔内注射<ol><li>注射前，动物使用3％异氟醚麻醉。 </li><li> 1-3万细胞注入50ul体积的左心室。细胞悬液中含有150ug/ml的D -荧光素，以验证注射技术。 </li><li>要确认正确的腔内注入小鼠成像在IVIS系统。劳保局信号，显示整个身体的小鼠被注射正确。如果是本地化的信号不正确注射细胞在心里。 </li></ol> 3。劳保局成像技术来监测转移<ol><li>成像穿梭支持IVIS频谱和QuantumFX microCT系统的数据捕获鼠标。 </li><li>穿梭有助于保持在同一位置的成像动物，并采用基准信号自动注册合作援助。该商会是连接isofluorance蒸发器维持麻醉。 </li><li>影像在生活图像软件4.1向导可用于自动选择曝光时间，F -停止和分级，确保定量数据的收集。 </li><li>可以采集多个图像，覆盖秒或数月，根据实验性质的纵向研究比较。 </li><li>第6天细胞注射后，劳保局IVIS光谱成像显示转移到骨9小鼠。 </li></ol> 4。溶骨性病变，采用微型CT纵向监测<ol><li>小鼠表现出明确的骨转移，可见使用劳保局在量子FX微型CT系统成像。 </li><li>量子FX微型CT系统提供高质量的图像，在足够低，使真正的纵向显微CT在临床前研究的X射线剂量。 </li><li>使用这个系统，在不到一分钟，两条腿的骨退化可纵向监测。 </li><li>小鼠用3％的异氟醚麻醉，可直接从IVIS频谱系统转移。 </li><li>两个60毫米图像缝合在一起，捕捉到整个动物视图。 </li><li>一个30毫米视野是在高分辨率成像膝关节的理想选择。 </li><li>一个足够高的分辨率来衡量和量化骨转移的单次扫描只需18秒，并提供了一​​个唯一的13mGy的剂量。 </li><li>小鼠成像微型CT系统在36天至8倍，而不会影响肿瘤的生长或动物的健康。 </li><li>微型CT成像显示，骨leisions第10天，可见，进展十分迅速，相应增加劳保局信号。 </li></ol> 5。 3D合作登记和分析BLI和UCT <ol><li>一旦图像已被抓获IVIS频谱和量子FX微型CT系统的数据都可以注册成一个三维重建。 </li><li> MicroCT图像导出为DICOM文件，并加载到生活图片4.1 3D多模式工具。 </li><li>在客厅图片软件运行DLIT方案三个维度重构的光信号。 </li><li>在软件的基准工具是用来注册的微型CT图像的光学劳保局信号。 </li><li>配准数据集，可以定量分析，来监视多个参数，在疾病的进展，在这里我们的跟踪转移性肿瘤的生长，在骨的溶骨性病变的发展。 </li> &lt;/ OL&gt;

<ol>
	<li>Jenkins, D. E., Hornig, Y. S., Oei, Y., Dusich, J., Purchio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Human+Breast+Cancer+Cell+Lines+that+Permit+Rapid+and+Sensitive+in+vivo+Detection+of+Mammary+Tumors+and+Multiple+Metastases+in+Immune+Deficient+Mice.">Bioluminescent Human Breast Cancer Cell Lines that Permit Rapid and Sensitive in vivo Detection of Mammary Tumors and Multiple Metastases in Immune Deficient Mice.</a> Breast Cancer Research. 7, R444-R454 (2005).</li><li>Cowey, S., Szafran, A. A., Kappes, J., Zinn, K. R., Siegal, G. P., Desmond, R. A., Kim, H., Evans, L., Hardy, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+Metastasis+to+Bone:+Evaluation+of+Bioluminescent+Imaging+and+MicroSPECT/CT+for+Detecting+Bone+Metastasis+in+Immunodeficient+Mice.">Breast Cancer Metastasis to Bone: Evaluation of Bioluminescent Imaging and MicroSPECT/CT for Detecting Bone Metastasis in Immunodeficient Mice.</a> Clinical and Experimental Metastasis. 24, 389-401 (2007).</li><li>Labrinidis, A., Hay, S., Liapis, V., Ponomarev, V., Findlay, D. M., Evdokiou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoledronic+Acid+Inhibits+Both+the+Osteolytic+and+Osteoblastic+Components+of+Osteosarcoma+Lesions+in+a+Mouse+Model.">Zoledronic Acid Inhibits Both the Osteolytic and Osteoblastic Components of Osteosarcoma Lesions in a Mouse Model.</a> Clinical Cancer Research. 15, (2009).</li><li>Kaijzel, E. L., Pluijm, G. v. a. n. d. e. r., Lowik, C. W. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Body+Optical+Imaging+in+Animal+Models+to+Assess+Cancer+Development+and+Progression.">Whole-Body Optical Imaging in Animal Models to Assess Cancer Development and Progression.</a> Clinical Cancer Research. 13, 3490-3497 (2007).</li><li>Minn, A. J., Kang, Y., Serganova, I., Gupta, G. P., Giri, D. D., Doubrovin, M., Ponomarev, V., Gerald, W. L., Blasberg, R., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Organ-Specific+Metastatic+Potential+of+Individual+Breast+Cancer+Cells+and+Primary+Tumors.">Distinct Organ-Specific Metastatic Potential of Individual Breast Cancer Cells and Primary Tumors.</a> The Journal of Clinical Investigation. 115, (2005).</li><li>Xu, H., Christensen, A., Kuo, C., Nilson, D., Lim, E., Whalen, J., Kim, J. B., Zhang, N., Singh, R., Oldfield, S., Troy, T., Rice, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-registration+of+in+vivo+Optical+Tomography+and+Micro-CT+for+Longitudinal+Studies.">Co-registration of in vivo Optical Tomography and Micro-CT for Longitudinal Studies.</a> Word Molecular Imaging Congress. , (2010).</li></ol>

作者在这篇文章中精选仪器卡尺生命科学的员工。

与microCT小动物成像已应用于监测溶骨性病变，如肿瘤的发展（2007年Cowey等人。Labrinidis等，2009），病理情况下。由于生物发光成像的荧光素酶标记的肿瘤已被广泛采用预临床，共同注册生物发光和microCT图像提供了一个更好的生物发光信号的（Kaijzel等。2007）的解剖位置的定义。荧光素酶标记的乳腺肿瘤细胞株MDA - MB - 231 - LUC - D3H2LN以前的研究表明骨和溶骨性病变的发展转移到心脏内注射（Jenkins等2005年之后。明尼苏达州等，2005）。在这项研究中，我们探索与组合的光学低剂量辐射成像microCT与全身的MDA - MB - 231 - LUC - D3H2LN转移模型的肿瘤转移和溶骨性活动的纵向研究。我们进行了IVIS Spectrum和量子外汇microCT系统和实现生物发光和我们新开发的生活图像4.1软件（恒等，2010）的CT联合登记顺序成像。 IVIS与生物发光成像提供了非侵入性检测具有高灵敏度的肿瘤细胞，使肿瘤在​​早期发现。与量子外汇成像可以准确的三维重建，骨病变和详细的解剖信息。量子FX系统提供低的辐射剂量成像，从而使病情恶化的纵向监测。骨病变和肿瘤转移的相关性被描绘的共同登记的形象，和纵向的多模跟踪病变的发展优势，清楚地表明。

monitoring tumor metastases and osteolytic lesions with bioluminescence and micro ct imaging

以下腔内交付的MDA - MB - 231 - LUC - D3H2LN细胞NU / nu小鼠，在注射的动物全身转移。生物发光成像使用IVIS频谱监察交付程序，包括DLIT重建测量肿瘤的信号和它的位置后，肿瘤细胞的分布和发展。 转移病灶骨组织的发展引发的溶骨性活动和病变胫骨和股骨评估纵向采用微型CT。成像是使用快速成像和X射线剂量低一个量子FX微型CT系统。允许多种成像会是一个累积的X射线剂量远远低于LD50低的辐射剂量。鼠标成像穿梭设备是使用顺序的形象IVIS Spectrum和量子FX小鼠实现准确的动物在生物发光和CT图像定位。光学和CT数据集共3 dimentions注册使用的生活图像4.1软件。这种多模式的方法，使肿瘤的生长和发展与溶骨性活动，同时密切监测。

Watch this Scientific Journal Video about Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging at JoVE.com

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

骨转移的实验小鼠模型，建立乳腺肿瘤细胞荧光素酶表达心内交货。纵向与生物发光和微型CT成像监测肿瘤的发展，并导致溶骨性病变。

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

monitoring-tumor-metastases-osteolytic-lesions-with-bioluminescence

Universidad San Sebastian

Research

JoVE Journal

Medicine

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视频: Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

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 ...

科研

医学

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 ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

癌症研究

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 ...

Models of Bone Metastasis

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

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

骨转移模型

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

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

使用生物发光监测小鼠乳腺癌生长和转移集落形成

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

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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

在体内的双基体生物光学成像

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

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

骨转移模型

研究使用正交乳腺癌模型的三阴性乳腺癌

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

使用生物发光监测小鼠乳腺癌生长和转移集落形成

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

副本

摘要

探索更多视频

此视频中的章节

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

在体内的双基体生物光学成像

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

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

骨转移模型

研究使用正交乳腺癌模型的三阴性乳腺癌

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

使用生物发光监测小鼠乳腺癌生长和转移集落形成

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移