本研究では、DOD乳がんのIDEA賞W81XWH - 08 - 1から0583までおよびNIH / NCI CA141348 - 01A1（DZ）とFAMRI臨床科学者賞（DS）によって部分的にサポートされています。イメージングインフラストラクチャは、U24 CA126608とシモンズがんセンター（P30 CA142543）とNIH 1S10RR024757 - 01によって部分的にサポートされている南西部の小動物のイメージング研究プログラムによって提供されます。

すべての動物の手順は、テキサス大学南西医療センターの動物実験使用の委員会によって承認された。 （1）in vitroで HIF -1α生物発光アッセイで <ol><li>材料と方法： <ul><li>人間の転移性乳癌細胞株MDA - MB231は、新規なHIF - 1依存性レポーター遺伝子をトランスフェクション、5HRE - ODD - lucは博士原田によって生成されました。 </li><li>低酸素状態では、酸素依存性分解ドメイン（ODD） - ルシフェラーゼ融合タンパク質の発現増強は、低酸素応答要素の5つのコピー（5HRE）によって駆動されます。 ODDの存在は、正常酸素条件下で極めて低いバックグラウンドのルシフェラーゼ活性の結果ODD - Lucのタンパク質の分解を引き起こす。したがって、この小説のシステムは、リアルタイムイメージングによる腫瘍内低酸素領域を検出するために使用することができます。この5HRE - ODD - lucの発現ベクターの構築は、原田らの1,2で報告されています。 </li></ul></li><li>正常酸素または低酸素下で培養細胞： <ul><li>組換えMDA - MB231 1％グルタミン、G418および1％ペニシリン/ストレプトマイシン400μg/ mlの抗生物質を含有する10％ウシ胎児血清（FBS）- DMEM培地で細胞を維持する。 </li><li> 2つの6ウェルディッシュの各ウェルに5HRE - ODD - lucのベクターを発現体外 HIF -1αの生物発光アッセイ、プレート3 × 10 5 MDA - MB231細胞について。 </li><li>正常酸素状態（21％O 2）の下に他の料理を維持しながら細胞は、一晩インキュベーションした後、シャーレの壁を添付して、低酸素症研究のための低酸素室（ラップス-ローゼンバーグ、（株）デルマー、CA）中に一皿を転送することができます。 </li><li>ガスシリンダーで吸気ポートのチューブを接続し、0.1％O 2とチャンバーとガスチャンバーを組み立てます。 注：どちらの入口と出口ポートのクランプはこの手順の実行中に開いている必要があります。 </li><li>チャンバーはプラスチック製のクランプを閉じることによりパージし、シール室された後、ガス源を外します。 </li><li> 5％CO 2で37℃インキュベーターでチャンバーを配置。 注：チャンバーは、文化の過度の蒸発を防ぐために加湿する必要があります。チャンバー内に20 mlの滅菌水 - これは、10を配置することによって達成することができます。 </li></ul></li><li>生物発光アッセイ： <ul><li> 24時間のインキュベーション後、培地を除去し、氷冷PBS（2X）で迅速に細胞を洗浄。 </li><li>ルシフェリン100μlの（ゴールドのバイオテクノロジー、セントルイス、MO）で冷PBSを1 mlを追加。 </li><li>様々な露光時間（1、30、60、180秒）とBLの画像を（IVISスペクトラムシステム、キャリパーライフサイエンス、ホプキントン、MA）取得。 </li><li>よくリビングイメージのソフトウェアを（キャリパーライフサイエンス）を用いてそれぞれの光強度を測定する。 </li></ul></li></ol> 2。乳がんの脳転移モデルの確立<ol><li> MDA-MB231/5HRE-ODD-luc細胞の調製<ul><li> 10％FBS、1％グルタミン、1％ペニシリン/ストレプトマイシンを含むデーム培地でMDA-MB231/5HRE-ODD-luc細胞を取得し、文化。 </li><li> 2〜3日ごとに培地を交換してください。 Tripsinizeと彼らは80％コンフルエントに達したときに細胞を洗浄。 </li><li>適切な数の細胞をカウントし、4μlの体積で10 5細胞の最終濃度で25％マトリゲル（BD Biosciences社、サンノゼ、カリフォルニア州）で無血清デームの培地で再懸濁します。 </li><li>頭蓋内注射の前に氷の上に置いて細胞。 </li></ul></li><li>外科的移植<ul><li>雌のヌードマウス（BALB / cのNU / NU、国立がん研究所、ベセスダ、MD）4-6週齢では、本研究で使用されています。 </li><li>麻酔（誘導チャンバ内のO2 /イソフルラン3％、バクスターインターナショナルインク、ディアフィールド、イリノイ州、アメリカ合衆国イソフルラン）と酸素（1 DM 3 /分）で（1％）イソフルラン外科的処置3の間で動物を維持する。 </li><li>右頭頂皮はbetadineしてから、前の切開〜70％アルコールで整形処理されるべきである。 </li><li>高速ドリルを使用して、バリ冠状縫合とsaggital縫合の外側2 mmまでの頭蓋骨が1mm前方の右半球で1mmの穴。 </li><li> 4μlの細胞混合物（10 5細胞）を10μlハミルトンシリンジ（ハミルトン社、リノ、ネバダ州）を使用してを描く。 、右尾間脳に直接細胞を注入するカスタムメイドの32Gハミルトンの針を用いて硬膜の下に1.5ミリメートル。撤退前に約30秒のための場所に針をしてください。 32G細針の使用法は、組織の損傷を最小限に抑えます。 </li><li>骨のワックスとバリ穴を埋めると吸収性縫合糸で頭皮を閉じます。 </li><li> 70％アルコールで切開領域を準備します。 </li><li>ブプレノルフィン鎮痛を2日間ごとに12時間を適用します。 </li></ul></li></ol> （3）乳がんの脳転移の腫瘍低酸素動態の in vivo生物発光イメージングで <ul><li> 8のために週に一度二週間頭蓋内移植し、繰り返しの後に縦生物発光イメージング（IVISスペクトラムシステム）を開始-10週。 </li><li>一度に3匹のマウスを麻酔（イソフルラン3％/誘導チャンバ内のO 2） </li><li>詳細以前に4で説明したように、各マウスの頸部皮下に、D -ルシフェリンの溶液（ゴールドバイオ溶液80の全体積のPBSで120ミリグラム/ kg）の管理。 </li></ul> D -ルシフェリンは、非毒性であり、無傷の血液脳関門（BBB）と細胞膜3,5を貫通することが可能であることが示されている。 <ul><li>イメージングチャンバー内に3匹のマウスを置き、撮像中に（1 DM 3 /分）酸素（1％）イソフルランで維持した。 </li><li>ルシフェリンの投与後5分後には、様々な露出時間の配列（1、30、60、180秒）とBLの画像を取得する。 </li></ul>我々の観察では、ピーク発光時間は頸部3,4のルシフェリンの皮下投与後5分程度であることを示している。 <ul><li>興味のある領域で絶対的な光子計数（フォトン/ s）を使用してリビングのイメージングソフトウェア（キャリパーライフサイエンス）でデータを分析（ROI）、手動で脳のBLI信号の輪郭を描く。 </li><li>腫瘍低酸素のダイナミクスを示すために、光子カウントのプロットの時間経過の曲線。 </li><li>すぐに最後のBLIの後、後pimonidazole、低酸素マーカーと1時間を管理する、マウスを犠牲にして脳を解剖。最適な切削温度（OCT）-80の培地と凍結℃の冷凍庫で全体の頭脳を埋め込みます。その後の組織学的クレシルバイオレット染色とルシフェラーゼ、低酸素マーカーpimonidazoleに対する免疫組織化学的染色、およびHIF -1αは、撮像観測を検証するために行った。 </li></ul> 4。代表的な結果： 図1に示すように、有意に高いBLIの信号は、24時間のための低酸素室（1％O 2）中でインキュベートしたトランスフェクションした細胞の後に観察された。弱い発光が正常酸素下での制御細胞から観察された。これは、HIF -1αを過剰発現する細胞にストレスのシグナルを誘導した3 × 10 5細胞、24時間培養後の過密な細胞集団から発生する可能性があります。それにもかかわらず、 生体 BLIの結果では腫瘍の低酸素症とルシフェラーゼ発現との間の共局在を示す免疫組織化学的染色によって検証した。 露光時間の自動化された配列は、より長い露光時間で弱い信号の捕捉を容易にする一連のイメージ、および飽和CCDなく、短い時間を使って強力な信号の連続的な買収が可能になります。 <img alt="図1" src="/files/ftp_upload/3175/3175fig1.jpg" /> in vitroで HIF -1α生物発光アッセイでは図1上段：24のために低酸素室内の6ウェルディッシュ（0.1％O 2）の各ウェル中でインキュベート3 × 10 5 MDA-MB231/5HRE-ODD-luc細胞時間はメディアの前に、除去洗浄し、1mlのPBSに置換した。 100μlのルシフェリンを各ウェルに添加した直後に、BLIは、露光時間の配列（1、30、60、180秒）で買収された。強い発光は、代表的な井戸から観察された下行：。対照として、正常酸素状態（21％O 2）放射される微弱な光の下でインキュベート3 × 10 5細胞。 <img alt="図2" src="/files/ftp_upload/3175/3175fig2.jpg" /> マウス脳の右側から腫瘍低酸素のダイナミクスの in vivo生物発光イメージングで図2。）微弱な光信号は、最初の5週間MDA-MB231/5HRE-ODD-luc乳癌細胞の頭蓋内移植後に可視化した。増加した光信号は、腫瘍の低酸素症を示し、追加の6週間にわたって観察された。B）のプロットは、光信号の定量的な光子計数の時間的経過の曲線を示した。 <img alt="図3" src="/files/ftp_upload/3175/3175fig3.jpg" /> 免疫組織化学染色によって検出されたルシフェラーゼと酸素の図3 Colocaliztion。OCTで埋め込 ​​まれた乳がんMDA-MB231/5HRE-ODD-lucの転移が付いた冷凍マウスの脳には、切片いた。 10μmのセクションでは、低酸素マーカー、pimonidazoleを用いて免疫染色抗ルシフェラーゼ染色によって検出されたルシフェラーゼ発現を空間的に相関することが発見された低酸素の腫瘍内不均一性を、明らかにした。スケールバー、100μmである。

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乳がんの脳転移は、IV期では乳癌患者の30％で発生します。それは、高い罹患率と死亡率と関連しており、13ヶ月6の生存期間の中央値を持っています。その頭蓋の発生および進行だけでなく、病態生理学的プロファイルの我々の理解を容易にするために、この臨床的に壊滅的な疾患を模倣するために適切な動物モデルを持っている必要があります。ここで、我々は、直接マウスの脳に?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> D -ルシフェリン</td><td>ゴールドバイオテクノロジー</td><td> L - 123 </td><td> in vivo試験のために、80μlの総体積のPBSの120 mg / kgの</td></tr><tr><td>イソフルレン</td><td>バクスターインターナショナルインク</td><td> 1001936060 </td><td></td></tr><tr><td>マトリゲル</td><td> BDバイオサイエンス</td><td> 354234 </td><td></td></tr><tr><td>ハミルトンシリンジ</td><td>ハミルトン社</td><td> 1701 </td><td></td></tr><tr><td> 32Gハミルトン針</td><td>ハミルトン社</td><td> 7803〜04 </td><td></td></tr><tr><td>低酸素室</td><td>ラップス - ローゼンバーグ、（株） </td><td> MIC - 101 </td><td></td></tr><tr><td>生物発光イメージングシステム</td><td>キャリパーライフサイエンス</td><td> IVISスペクトラムシステム</td><td></td></tr><tr><td> G418 </td><td>フィッシャーサイエンティフィック</td><td> SV3006901 </td><td></td></tr></tbody></table>

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

これはよく腫瘍低酸素症は悪性化を促進し、否定的に治療反応に影響を与える重要な役割を果たしていることが認識されている。 その場でについて少し知識がいるため、これらの根深い同所性腫瘍で、それを監視する効率的な手段の欠如の悪性脳腫瘍の頭蓋内開発中の腫瘍低酸素は、生体内で、ある。ルシフェラーゼ遺伝子を発現する生細胞により放出される光の検出に基づいて、生物発光イメージング（BLI）は、急速に臨床動物試験において、治療に応答して、腫瘍の成長または腫瘍サイズの変化を評価するために、特に、癌研究のために採用されています。また、プロモーター配列の制御下にレポーター遺伝子を発現させることにより、特定の遺伝子発現は、BLIによる非侵襲的に監視することができます。低酸素ストレス下では、シグナリング応答は、種々の遺伝子の転写を駆動するために主に低酸素誘導因子-1α（HIF -1α）を介して媒介されています。したがって、我々は、安定的にヒト乳癌MDA - MB231細胞（MDA-MB231/5HRE-ODD-luc）にトランスフェクションされたHIF -1αレポーター構築物、5HRE - ODD -ルシフェラーゼを、使用している。 体外 HIF -1α生物発光アッセイでによって実行される正常酸素状態の細胞（21％O 2）コントロールとして機能しながら、BLIの前に24時間のための低酸素室（0.1％O 2）にトランスフェクション細胞をインキュベートする。低酸素下細胞で観察された有意に高い光子フラックスは、正常酸素状態のものと比較して、そのプロモーターに増加HIF -1αの結合（HREの要素）を、示唆している。細胞は、乳がんの脳転移モデルを確立するためにマウスの脳に直接注入される。腫瘍低酸素動態の in vivo生物発光イメージングでは移植後2週齢を開始し、週に一度繰り返されます。 BLIは増加頭蓋内腫瘍の低酸素を示す、腫瘍が進行するように脳からの光信号を増加させる明らかにする。組織学的および免疫組織化学的研究は、in vivoイメージングの結果で確認するために使用されています。ここで、我々は、頭蓋内腫瘍の低酸素症を監視するために、in vivoで生物発光イメージングでのヌードマウスおよびアプリケーションにおける乳がんの脳転移の外科的確立をin vitroで HIF -1α生物発光アッセイでのアプローチを紹介します。

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

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

低酸素誘導因子-1αの活性の生物発光イメージングは​​、乳がんの脳転移のマウスモデルにおける頭蓋内腫瘍の低酸素症の開発を監視するために適用されます。

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 ...

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

Research

JoVE Journal

Medicine

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

19.5K ビュー.  University of Texas Southwestern Medical Center. 低酸素誘導因子-1αの活性の生物発光イメージングは​​、乳がんの脳転移のマウスモデルにおける頭蓋内腫瘍の低酸素症の開発を監視するために適用されます。

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

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

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

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

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

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

医学

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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

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

同所性マウス脳腫瘍モデルのコンビネーション放射線治療

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

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

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

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

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

定位頭蓋内注入と<emインビボ&gt;</em多形性膠芽腫のマウスモデル系における腫瘍異種移植片の&gt;生物発光イメージング

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

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

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

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

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 ...

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.

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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

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

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

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

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

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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

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

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

縦MRIが続く乳がんの脳転移のマウスモデルを開発するために、超音波画像誘導心臓内注射

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

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

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

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

乳癌の新規トランスジェニックマウスモデル：アデノ-Creを持つ乳管上皮の標的とすることにより、転移性乳癌の開始

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

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

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

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

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

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

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