私たちは、博士に感謝しています。ダイアンパルミエリおよびNCIのパトリシアSteegの私たちに、MDA-MB231Br細胞を提供するため。私たちは、技術的、合議サポートのために博士はラルフメイソン氏ジェイソンReneauさんとラモナロペスに感謝します。この作品は、DOD IDEA賞W81XWH-08-1から0583までとW81XWH-12-1から0317によって部分的にサポートされていました。 MRI実験は、NIH BTRP＃のP41-RR02584施設、高度なイメージング研究センターで行われた、超音波ガイド下心臓内注射は1S10RR02564801下VisualsonicsのVevoの770を用いて行った。

全ての動物手順は、テキサス大学南西医療センターの施設内動物管理使用委員会によって承認された。 1。 MDA-MB231/Br-GFP細胞の調製<ol><li> 10％FBS、1％グルタミンおよび1％ペニシリン/ストレプトマイシンを含むDMEM培地（親切に博士パルミエおよびSteegの、NCIによって提供される）MDA-MB231/Br-GFP細胞を取り出し、培養物。 </li><li>細胞と培地の色の状態を観察し、2〜3日ごとに培地を交換してください。 </li><li>トリプシン処理およびステップ1.3.1-1.3.5に概説されるように80％のコンフルエンスに達したときに細胞を回収する。 <ol><li>完全に古い培地を除去し、穏やかに細胞を洗浄するために5ミリリットルのPBS（1X）を追加します。皿からPBSを削除します。 </li><li>皿に1.5ミリリットルのトリプシンを追加します。すべての細胞をトリプシンで覆われていることを確認するために静かにお皿を傾けてください。バックセルインキュベーターに料理を入れて、1分間37℃で保管してください。 </li><li>インキュベーターからお皿を取り出し、観察する細胞を確保するために、光学顕微鏡下で細胞が皿から外します。 </li><li>トリプシンの効果を停止する培地3ミリリットルを追加します。遠心管中の細胞の混合物を回収します。 5分間2000rpmで混合を遠心分離する。 </li><li>慎重に培地を除去し、均一になるまで5ミリリットルの無血清培地中で細胞を再懸濁。 </li></ol></li><li>適切な数の細胞をカウントし、100μl量の1.75×10 5細胞の最終濃度で無血清DMEM培地で再懸濁します。 <ol><li> 50μlの細胞混合物を取り、50μlのトリパンブルーを追加。混合した後、10μlの混合物を取り、慎重に細胞計数スライドに追加します。正確な細胞数の推定を確実にするために複数のサンプルを使用してください。 </li><li>細胞計数スライドを1つずつ終了し、細胞計数器で固執すると、自動的にカウントを開始します。すべてのカウント結果の平均を取ると、総細胞数を計算します。 </li><li>再び細胞を遠心分離し、それらを再懸濁ボリュームμL1.75×10 5 cells/100の最終濃度になるように無血清培地の適切なボリューム。 </li></ol></li><li>前心臓内注射に氷上で場所細胞。 </li></ol> 2。超音波画像誘導心臓内注射<ol><li> 6〜8週齢の間の雌ヌードマウス（BALB / C NU / NU）を使用します。 </li><li>イメージング·システムにログインします。トランスデューサー704（40 MHz）を初期化します。 </li><li>新たな研究を開始し、情報を入力します。 </li><li>麻酔（誘導室中の3％isoflurane/100％のO 2）とノーズコーンを介して全体の手順の間に、100％O 2（1 dm 3の/分）にイソフルラン（2％）を有する動物を維持する。まだ引っ込め反射はつま先のピンチで観察されていない場合に麻酔が確認された。 </li><li> 37℃に撮影台の温度を設定する仰臥位での加熱された撮影台に、麻酔したマウスをテープで固定します。 </li><li>イムの前​​に、37℃での超音波ゲルを保つ高齢化。マウスの胸部にゲルを適用します。 </li><li>ホルダーにトランスデューサを取り付けます。希望の撮像深度に達するまでトランスデューサを下げます。左心室は、ランドマークとして上行大動脈（ 図1A）で識別されるまで、ステージを移動します。左心室のクリアな視界が可視化される段階をロックします。 </li><li> 22 Gの針を1ミリリットルのシリンジに細胞混合物100μlを描画します。マウント注射器の注射器を配置し、固定します。 </li><li>マウスの胸に向けて前進注射器を移動して、慎重に針の先端が（マウスに入る前に）撮像視野に入るまでなるので、横に移動します。 </li><li>左心室ダウン目指して針の高さと角度を調整します。 </li><li>超音波イメージングの指導の下で左心室に速やかに皮膚や筋肉層を通ってインターコース空間に注射針を貫通している。 左ventriに針の挿入が成功の指標CLEは、注射器に新鮮な動脈血（暗赤色の静脈血とは対照的にピンク色）の還流である</li><li> （ 図1B）を徐々に細胞混合物を注入する。 </li><li>完了すると、針を撤回変換器を持ち上げ、湿らせたガーゼで超音波ゲルをきれいにし、テープを取り外します。 </li><li>予熱したパッドできれいなケージを準備します。 </li><li>パッドの上に動物を置き、完全に回復するまで動物を観察します。 </li><li>動物の行動2日間、24時間毎に監視します。 </li><li>一般的な条件や実験マウスにおける合併症の神経学的徴候が日常監視されています。 </li></ol> 3。頭蓋内腫瘍発生のMRIモニタリング<ol><li>脳転移の頭蓋内開発を監視するために9.4テスラの磁石を使用してください。 </li><li> 2週間腫瘍移植後のMRIを開始し、最長3週間週1回繰り返します。 </li><li> 3％のイソフルランを動物に落ち着いたし、遺伝子の下でそれらを維持するRAL麻酔（1.5％イソフルラン）。 </li><li>監視および実験を通して、動物の体温および呼吸を一定に保つ。 </li><li>高解像度マルチスライス（1ミリメートルと14スライス厚の隙間）のT 1 -とT 2強調冠状後頭蓋窩に前頭葉までの領域をカバーする画像は、次のパラメータを使用して取得されたT 1強調画像を。複数のスライス（SEMS）エコースピン、TR / TE = 400 msec/20ミリ、マトリックス：X 256 256、平面解像度のFOV 20×20ミリメートル、：X 78 78ミクロン2。 T 2強調画像：複数のスライスエコー高速スピン（FSEMS）シーケンス、TR / TE = 2500 msec/48ミリ、8エコートレイン、マトリックス：256×256、FOV 20×20ミリメートル、平面解像度：78 X 78ミクロン28,9。 腫瘍病変は、T 2強調画像上の正常な脳組織よりも明るく表示されます。 </li><li>腫瘍の大きさを手動enhanのを概説することによってT 2 -重み付け画像について決定されるボランティア8によって書き込まMATLABプログラムを用いて、各画像上での質量の一部をcing。 所与の腫瘍直径の大部分はスライス厚さ（1mm）よりも小さい、腫瘍の大きさは、平面領域ではなく、容積として提示される。 </li></ol> 4。 H＆E染色は、転移の確認<ol><li>担癌脳を解剖、すぐに最後のMRスキャン後のマウスを犠牲にして、OCT媒体でそれを埋め込み、-80℃で凍結させ</li><li>セクションクライオスタットで厚さ10μmの冠状脳標本のシリーズ。 </li><li>脳切片にH＆E染色を行う。 </li></ol>

<ol>
	<li>Schouten, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+brain+metastases-translation+to+new+therapies.">The biology of brain metastases-translation to new therapies.</a> Nat. Rev. Clin. Oncol. , (2011).</li><li>Lockman, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+blood-tumor+barrier+permeability+determines+drug+efficacy+in+experimental+brain+metastases+of+breast+cancer.">Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer.</a> Clin. 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利害の対立が宣言されていません。

本研究では、この研究におけるすべての動物は、脳転移を発症しない動物の死亡が観察されないように、超音波画像誘導左心室注入は、正確さを保証することを実証した。画像誘導注射の美しさは最終的に左心室に皮膚の針貫通のパスとは従って、厳密な解剖学的ランドマークを必要とする手動の手順とは区別され、画像の下に監視し、調整することができるということである。 <p class="j...

脳転移は、成人において最も一般的な頭蓋内悪性腫瘍である。予後はさらに積極的な治療と4〜6ヶ月の生存期間の中央値で、非常に悪い。乳がんは脳転移1〜3の高い罹患率との三大原発性癌の一つです。いくつかの脳指向性乳癌株は、心臓内又は頚動脈内注射後4脳転移を発症することが可能である。左心室への腫瘍細胞の直接注入は、肺毛細血管床を迂回し、従って、内臓転移を最小限に抑えながら、脳転移の形成の発生率を増加させることができる。 MDA-MB231Brラインは、げっ歯類モデル5,6の脳転移を開発するために最も広く使用されているヒト乳癌株の一つである。 他の多くの研究4,7と同様に、我々は我々の以前の研究で、心臓内注射の手動手順を実行した。しかしながら、50％しか成功率は、手動で得られた前臨床試験が失敗した場合、注入し、マウスの割合を繰り返し侵襲的処置で死亡した。ここでは、無胸腺マウスの左心室に脳探索乳癌細胞の注射を確保するために画像誘導の手順の使用を導入している。縦方向の高解像度のMRIは、脳転移の頭蓋開発に追従するために適用される。

<table border="0" cellpadding="0" cellspacing="0" width="783">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">DMEM</td>
			<td style="width:136px;">HyClone</td>
			<td style="width:87px;">SH30022.01</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Trypsin</td>
			<td style="width:136px;">Mediatech</td>
			<td style="width:87px;">25-051-C1</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Automatic cell counter</td>
			<td style="width:136px;">Biorad</td>
			<td style="width:87px;">TC10</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
			<td style="width:136px;">Baxter International Inc.</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
			<td></td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
			<td style="width:87px;"> </td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Sakura Finetek USA</td>
			<td style="width:87px;">4583</td>
			<td style="width:147px;"> </td>
		</tr>
	</tbody>
</table>

ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

乳癌脳転移は、ステージIVで乳癌患者の30％で生じる、高い死亡率と関連している。生存期間の中央値は6ヶ月です。それは、臨床シナリオにおける転移細胞の広がり血行動態を模倣するために適切な動物モデルを有することが重要である。ここでは、無胸腺ヌードマウスの左心室に脳熱帯乳癌細胞の正確な注入を案内する小動物超音波イメージングの使用を導入している。縦方向のMRIは、脳転移の頭蓋内の開始と成長性を評価するために使用されます。超音波ガイド下心臓内注入は、正確な注入とここに高い成功率だけでなく、確実にだけでなく、大幅に我々の以前の手作業に比べて、死亡率を減少させた。 インビボ高解像度MRIは310程度と小さいように、高信号多発性病変の可視化を可能にする3週間のT 2強調画像上の直径で注射後。 FollowはアップMRIは頭蓋内腫瘍の増殖および脳全体にわたって分配する転移の数の増加を明らかにする。

MRI（平面解像度78ミクロン）の高空間分解能で、高信号病変は、直径が310ミクロン（ 図2）のように小さいとして同定することができる。本研究では転移は非常に小さく、壊死や浮腫の開発が最小限であることから、T 2強調画像上の高信号病変は真に腫瘍塊を表していた。 長手MRI研究は、腫瘍成長のインビボで非侵襲的評価を可能にする。 <str...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

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

乳癌脳転移マウスモデルをMDA-MB231/Br-GFP細胞の超音波画像誘導の心臓内注射で確立される。多焦点頭蓋内転移の発生は、高解像度の9.4 T MRIを用いて長手方向にモニタされた。

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 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.6K ビュー.  University of Texas Southwestern Medical Center. 乳癌脳転移マウスモデルをMDA-MB231/Br-GFP細胞の超音波画像誘導の心臓内注射で確立される。多焦点頭蓋内転移の発生は、高解像度の9.4 T MRIを用いて長手方向にモニタされた。 

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

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

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

ラットモデルでの経頭蓋集束超音波を用いた血液脳関門のMRI誘導破壊

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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 ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

縦二光子イメージングに続いてマウスの急性脳外傷

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝転移の前臨床マウスモデル

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

腫瘍増殖を研究するためにマウスの乳房脂肪パッド中に乳癌細胞の同所注入。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.