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

縦画像は、疾患の進行に従うか、または治療の効果を測定するための前臨床試験で使用されています。腫瘍学では、光学的方法は、腫瘍の成長を監視し、そのような研究では各時点での細胞増殖や遺伝子発現の正確な定量を実現するために厳格なツールを提供しています。解剖学的変化がmicroCTのような高解像度の技法を用いて測定することができますが、長手方向の画像を低X線量は、生物学的なアーチファクトを回避するために使用されている必要があります。光とmicroCT画像は最大の情報が動物モデルから抽出されることを確実に機能し、解剖学的データの組み合わせを提供するために共同で登録することができます。 1。細胞の準備<ol><li> Caliperは、マウスモデルにおける前臨床研究のためのルシフェラーゼ発現する癌細胞株の範囲を提供します。 </li><li> MDA - MB - 231 - lucを- D2H2LNは、in vivoでtumorgenesisの光学的指標として使用できるルシフェラーゼ遺伝子を発現するヒト乳癌細胞株です。この細胞株は、D3H1乳腺脂肪体の腫瘍由来の自発的なリンパ節転移から作成され、積極的に転移を形成することが知られている。 </li><li>細胞は、容易に選択マーカーを必要とせず、標準のメディアで育つ無菌凍結培養物として提供されています。 </li><li>注射の前に動物にルシフェラーゼ活性を確認するには90％コンフルエントフラスコをトリプシン処理によって回収される。ルシフェラーゼ活性をマイクロタイタープレートに50,000細胞を調剤し、連続希釈を行うことによって測定されます。 </li></ol> 2。動物の細胞の心臓内注射<ol><li>注射の前に、動物は3％イソフルランを使用して麻酔する。 </li><li> 1から3000000細胞は、左心室への50ULボリュームに注入されています。細胞懸濁液は、注入のテクニックを検証するために150ug/ml D -ルシフェリンが含まれています。 </li><li>正しい心腔内注射のマウスを確認するには、IVISシステムで撮像する。体全体にBLIの信号を示すマウスが正しく注入されています。信号がローカライズされている場合、細胞は中心部に正しく注入されていない。 </li></ol> 3。 BLIイメージングは​​、転移を監視する<ol><li>イメージングシャトルは、IVISスペクトラムとQuantumFX microCTシステムの両方でデータのキャプチャにマウスをサポートしています。 </li><li>シャトルは、イメージングのための同じ位置で動物を維持するのに役立ちますし、共同登録、自動で支援するために基準信号を内蔵しています。チャンバーは、麻酔を維持するためにisofluoranceの気化器に接続されています。 </li><li>リビングイメージのソフトウェア4.1の​​イメージングウィザードが自動的に露出時間、絞りとビニングを選択し、定量的なデータの収集を確保するために使用することができます。 </li><li>複数の画像を取得し、実験の性質に応じて数秒または数か月をカバーする長期的な研究に比較することができます。 </li><li>細胞の6日目注射後は、IVISスペクトラムとBLIの画像は、9つのマウスのうち7人で骨に転移を示しています。 </li></ol> 4。マイクロCTを用いた骨溶解性病変の長手方向のモニタリング<ol><li> BLIを使用して目に見える明らかな骨転移を示したマウスはQuantum FXマイクロCTシステムで撮像した。 </li><li>量子FXマイクロCTシステムは、前臨床試験で真の長手方向のマイクロCTを有効にするのに十分な低X線量で高品質画像を提供します。 </li><li>両下肢の骨の劣化は、長手方向に分足らずでこのシステムを使用して監視できます。 </li><li>マウスは、イソフルラン3％の麻酔とIVISスペクトラムのシステムから直接転送することができます。 </li><li>二つの60ミリメートルのイメージは、全体の動物のビューをキャプチャするために一緒にステッチされています。 </li><li> 30ミリメートルFOVは、高分解能のイメージング膝関節に最適です。 </li><li>骨転移を測定し、定量化するために十分に高い解像度を持つ単一のスキャンがわずか18秒かかり、唯一の13mGyの線量を提供します。 </li><li>マウスは、動物の腫瘍の成長や健康に影響を与えることなく、マイクロCTシステムで36日間で8回まで画像化した。 </li><li>マイクロCTイメージングは​​、骨のleisionsは、BLIの信号の増加に対応する、10日目に表示されていましたし、非常に急速に進んだことを示した。 </li></ol> 5。 3D BLIとUCTの共同登録と分析<ol><li>一度イメージが三次元再構成に同時登録できるIVISのスペクトルと量子FXマイクロCTシステムのデータの両方でキャプチャされています。 </li><li> MicroCT画像はDICO​​Mファイルとしてエクスポートして、リビングイメージ4.1 3Dマルチモダリティのツールにロードされます。 </li><li>リビングイメージソフトウェアで光信号を再構成する立体的にDLITプログラムを実行します。 </li><li>ソフトウェアの基準ツールは、マイクロCT画像への光BLIの信号を共同登録するために使用されます。 </li><li> Coregisteredデータセットは、ここで我々は骨の溶骨性病変の開発と一緒に転移性腫瘍の成長を追跡している疾患の進行中に複数のパラメータを監視するために定量的に分析することができます。 </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>

著者は、この記事で紹介楽器を作るキャリパーライフサイエンスの従業員です。

microCTと小動物イメージングは​​、腫瘍の開発（。。; Labrinidisら、2009 Coweyら2007）などの病理学的条件下での監視溶骨性病変に適用されている。ルシフェラーゼ標識した腫瘍の生物発光イメージングを広く事前に臨床的に採用されているように、生物発光とmicroCT画像の共同登録は生物発光信号（Kaijzelら、2007）の解剖学的位置のより良い定義を提供する。ルシフェラーゼというラベルの乳房の腫瘍細胞株MDA - MB - 231 -リュック- D3H2LNと以前の研究では、イントラ心臓注射後の骨と溶骨性病変の発生に転移を示した（ジェンキンスら、2005;。ミンら、2005）。本研究では、組合せ、光と全身MDA - MB - 231 - lucを- D3H2LN転移モデルと低放射線線量microCTイメージングによる腫瘍転移と溶骨性活動の縦断的研究を検討した。我々は新しく開発されたリビングイメージ4.1ソフトウェア（恒ら、2010）とIVISスペクトラムと量子FX microCTシステムと達成生物発光とCTの共同登録を連続イメージングを行った。 IVISによる生物発光イメージングは​​腫瘍を早期に検出できるように、高感度を持つ腫瘍細胞の非侵襲的な検出を提供します。量子FXとイメージングは​​、骨病変および詳細な解剖学的情報の正確な三次元再構成を可能にします。量子FXシステムは、疾患の進行の長手方向の監視を可能にする低放射線量イメージングを、提供しています。骨病変および腫瘍転移の相関は、共同登録画像に描かれ、そして病変開発のマルチモダリティの追跡の利点は、縦方向に明確に実証された。

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

nu / nuマウスにMDA - MB - 231 - lucを- D3H2LN細胞の以下の心臓内の配達、注入された動物に開発された全身転移。 IVISスペクトラムを用いた生物発光イメージングは​​腫瘍の信号とその位置を測定するためにDLIT再建を含む配信手順に従って腫瘍細胞の分布と発展を監視するために採用した。 脛骨と大腿骨に溶骨性活動および病変のトリガー骨組織への転移病変の開発は、マイクロCTを用いて縦方向に評価した。イメージング、高速イメージングと低X線量で量子FXマイクロCTシステムを用いて行った。低放射線量では、複数のイメージングセッションがはるかにLD50以下累積X線量で実行することができます。マウスのイメージングシャトルのデバイスは、順次画像にIVISスペクトラムと量子FXの両方を持つマウスは、生​​物発光とCT画像の両方で正確な動物の位置決めを達成するために使用されました。光学とCTデータセットは、リビングイメージ4.1ソフトウェアを使用して、3 dimentionsで共同登録されていた。このマルチモードのアプローチは、溶骨性活動と同時に、腫瘍の成長と発展の綿密なモニタリングが可能になります。

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

骨転移の実験的マウスモデルは、ルシフェラーゼを発現する乳腺腫瘍細胞の心臓内の送達後に設立されました。開発腫瘍と溶骨性病変の結果は、生物発光とマイクロCT画像を縦方向にモニターした。

生物発光とマイクロCTイメージングで腫瘍転移と溶骨性病変の監視

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

Research

JoVE Journal

Medicine

24.5K ビュー.  Caliper Life Sciences.  中文 

ビデオ: 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.

マウスモデルにおける乳がんの脳転移の腫瘍の低酸素動態の in vivo生物発光イメージングに

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

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.

生体デュアル基質生物発光イメージングにおける

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 マウスの多発性骨髄腫, 骨髄異種のマルチ モーダルな発光とポジトロニック放出断層レントゲン写真撮影/計算断層イメージング

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

がん研究

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.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

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.

骨転移モデル

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.

交感性乳癌モデルを用いたトリプルネガティブ乳癌の研究

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.

尾静脈転移アッセイとリアルタイムインビボイメージングの併用による乳癌転移性コロニー形成と肺の負担の定量化

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.

固形腫瘍移植による骨原発性骨腫瘍と骨転移のモデリング

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.

生物発光を用いたマウスにおける乳がん増殖および転移性コロニー形成のモニタリング

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>同所性注射された乳房腫瘍細胞の自発的な肺転移を測定するためのin vivoイメージング

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.

生物発光とマイクロCTイメージングで腫瘍転移と溶骨性病変の監視

文字起こし

要約

さらに動画を探す

この動画の章

マウスモデルにおける乳がんの脳転移の腫瘍の低酸素動態の in vivo生物発光イメージングに

生体デュアル基質生物発光イメージングにおける

NOG マウスの多発性骨髄腫, 骨髄異種のマルチモーダルな発光とポジトロニック放出断層レントゲン写真撮影/計算断層イメージング

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

骨転移モデル

交感性乳癌モデルを用いたトリプルネガティブ乳癌の研究

尾静脈転移アッセイとリアルタイムインビボイメージングの併用による乳癌転移性コロニー形成と肺の負担の定量化

固形腫瘍移植による骨原発性骨腫瘍と骨転移のモデリング

生物発光を用いたマウスにおける乳がん増殖および転移性コロニー形成のモニタリング

インビボ同所注射された乳房腫瘍細胞の自然肺転移を測定するためのイメージング

生物発光とマイクロCTイメージングで腫瘍転移と溶骨性病変の監視

文字起こし

要約

さらに動画を探す

この動画の章

マウスモデルにおける乳がんの脳転移の腫瘍の低酸素動態の in vivo生物発光イメージングに

生体デュアル基質生物発光イメージングにおける

NOG マウスの多発性骨髄腫, 骨髄異種のマルチ モーダルな発光とポジトロニック放出断層レントゲン写真撮影/計算断層イメージング

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

骨転移モデル

交感性乳癌モデルを用いたトリプルネガティブ乳癌の研究

尾静脈転移アッセイとリアルタイムインビボイメージングの併用による乳癌転移性コロニー形成と肺の負担の定量化

固形腫瘍移植による骨原発性骨腫瘍と骨転移のモデリング

生物発光を用いたマウスにおける乳がん増殖および転移性コロニー形成のモニタリング

インビボ 同所注射された乳房腫瘍細胞の自然肺転移を測定するためのイメージング

NOG マウスの多発性骨髄腫, 骨髄異種のマルチモーダルな発光とポジトロニック放出断層レントゲン写真撮影/計算断層イメージング

インビボ同所注射された乳房腫瘍細胞の自然肺転移を測定するためのイメージング