本研究では、AACRヘンリー·シェパード膀胱がん研究助成金（09から60-30-LI）によって運営されている。

動物を対象とする手順は、カリフォルニア大学サンフランシスコ校の動物実験及び利用委員会（IACUC）によって承認されています。 1。細胞調製<ol><li>ヒト膀胱癌細胞株KU-7-luc2-GFP（キャリパーライフサイエンス、ホプキントン、MA）は、10％ウシ胎児血清、50μg/ mlのゲンタマイシンを含むRPMI-1640培地で栽培されています。 </li><li>培養は37℃で維持され、メディアと5％CO 2は、2〜3日ごとに変わります。 </li><li>細胞は、トリプシン消化により回収し、血球計算板を用いてカウントした。 50μlの2×10 6細胞の単一細胞懸濁液は、リン酸塩で調製した緩衝生理食塩水と氷上で保存する。 </li></ol> 2。同所性膀胱腫瘍モデル<ol><li> 8月10日週齢雌ヌード（NU / NU）マウス（シモンセン研究所Gilory、CA）を使用した。 </li><li>提供するために加温パッド上に滅菌ドレープを配置SA外科的プラットフォームを提供します。無菌の潤滑ゼリー、スタイレット針、硝酸銀溶液、および滅菌水で24-guageカテーテルが用意されています。 </li><li>一般的な麻酔薬は、吸入イソフルラン（誘導のために3％、メンテナンスのために1-2％）とノーズコーンを介して維持したままマウスで誘導される。無菌眼軟膏は、動物の目に適用され、熱パッドは体温を維持するために使用されています。 </li><li>仰臥位でマウスを置きます。 </li><li>穏やかに安定化のための鉗子で、動物の外陰部を把握する。 </li><li>削除針スタイレット24-guage静脈カテーテルを使用して、マウスの尿道にカテーテルを挿入されています。十分な潤滑を使用する必要があります。マウスの仰臥位で、尿道は外陰部のひだと膣の前方にすぐに後方に位置しています。 45度の角度は、最初は恥骨の下にある尿道をカニューレを挿入し、合格するために取られます。より浅い角度は、膀胱に尿道を通過する取得されます。穏やかな骨盤の圧力尿道がしばしば助けることができるまっすぐにしてください。注意これは尿道や膀胱穿孔につながることができますので、抵抗に対してあまりにもハードにプッシュしないように注意する必要があります。カテーテル内に見られる尿は、膀胱内腔の位置を確認します。 </li><li>スタイレット針を用いてカテーテルや膀胱内腔から尿を吸引します。スタイレット針を所定の位置にカテーテルを残し、後続のすべての願望と注射のために使用されます。これは、カテーテル内のデッドスペースをなくすことで正確な量の注入を可能にし、それが繰り返されるカテーテル挿入が不要になります。 </li><li>膀胱尿路上皮のグリコサミノグリカン層を損なうために、0.5 M硝酸銀の10μLを注入し、10秒間滞留を許可されている。これは、残りの尿と白色沈殿物を形成することがあります。 </li><li>スタイレット針を用いて沈殿物、膀胱内容を吸引して廃棄します。 </li><li> 100μLの滅菌水を注入して膀胱を洗浄します。水を吸引して廃棄します。 REPE4回洗浄の合計滅菌水で洗浄3回。 </li><li>カテーテル抜去の準備のために尿道を閉塞するために尿道口の周りに4から0絹のネクタイを配置します。 </li><li>注入する前にスタイレット針を用いて50μLで2×10 6 KU-7-luc2-GFP細胞を用意しました。 </li><li>同時に絹のネクタイで閉塞尿道を残して、カテーテルとスタイレット針を削除します。 </li><li>絹のネクタイが削除され、動物が起こされる前に、マウスが2時間全身麻酔下で維持されます。 </li></ol> 3。超音波検査<ol><li>全身麻酔は、ノーズコーンを介して気化したイソフルラン維持で誘導されています。 </li><li> RMV-704 40 MHzのプローブを用いたin vivoで VisualSonics VEVO 770高解像度マイクロイメージングシステム（VisualSonics株式会社、トロント、オンタリオ、カナダ）を用いた。 </li><li>超音波プラットフォーム上で仰臥位にマウスを置きます。 </li><li>動物の後ろ足を確保するテープで超音波プローブの操作中に過剰な運動を避けることができます。 </li><li>マウスの骨盤に超音波ゲルを適用します。 </li><li>横または縦の画像を得るために、マウスの骨盤の上にRMV-704（40 MHz）の超音波プローブを下げます。 </li><li>腫瘍が見つかった場合、画像は、腫瘍の大きさの測定のために保存することができます。 </li></ol> 4。生物発光イメージング<ol><li> 200μLの腹腔内注射（150 mg / kg）のルシフェリン基質を実行します。 </li><li>イソフルラン気化して全身麻酔を誘導し、ノーズコーンを介して維持されます。 </li><li>生物発光イメージャー内に仰臥位にマウスを置きます。 </li><li>ルシフェリン投与5分後には、1秒当たりの光子の動物から生物発光を測定します。 </li></ol> 5。 Sarnaの準備<ol><li> 50から100マイクロモルのスケールでのRNAオリゴヌクレオチドを​​合成する。 </li><li>私は、相補的な一本鎖オリゴヌクレオチドを​​アニールNTO二重saRNAs。 </li><li> [1,3]ジオキソラン（DLinKC2-DMA）、disteroylphosphatidylコリン、コレステロール、PEG-DMG - 脂質ナノ粒子（LNP） - Sarnaのは、イオン性脂質1,2 - dilinoleyl -4 - （ - ジメチルアミノエチル2）で用意されて前述のように自発的ベシクル形成の定式化プロシージャを使用して1。 </li></ol> 6。配合Sarnaのの膀胱内投与<ol><li>同所性膀胱腫瘍モデルを確立した後、マウスは脂質ナノ粒子（LNPs）（ 表1）で定式化膀胱内小活性化RNA（Sarnaの）で処理されています。 </li><li>一般的な麻酔薬が確立し、維持イソフルラン気化して、無菌眼科用潤滑剤が適用されています。 </li><li>仰臥位で動物を配置します。 </li><li>前述のように尿道カテーテルを実行します。 </li><li>尿道とカテーテルの周囲に4から0絹のネクタイを配置します。 </li><li>カテーテルからすべての尿を吸引除去し、空のシリンジとスタイレット針を用いて膀胱。 </li><li> 50μL（3 mg / kg）の総線量のために膀胱内にLNP-Sarnaのを注入します。同時に絹のネクタイで閉塞尿道を残して、カテーテルとスタイレット針を削除します。 </li><li>マウスは2時間閉塞尿道麻酔のままになります。 </li><li>膀胱LNP-Sarnaのによる治療は、腫瘍移植後4日目に開始されました。マウスは、14回、合計3日ごとに、連続的に処理した。 </li></ol> 7。代表的な結果 KU-7-luc2-GFP細胞に由来する膀胱腫瘍は、ルシフェラーゼの生物発光イメージング（ 図1AおよびC）と膀胱の超音波（ 図1BおよびD）で監視することができます。腫瘍は、しばしば細胞接種後3-5日以内に検出されています。我々は、マウスの90％以上の成功した膀胱腫瘍の移植を見つけました。膀胱dsRNAは治療が続いて起こるように、腫瘍の進行は、wiを監視することができますこれらのモダリティ番目。動物が犠牲にされた後、腫瘍の成長はまた、GFPの蛍光を用いて確認することができます。一般に、 図の1C及び1Dに視覚的に見られるように膀胱腫瘍の平均超音波の寸法との相関生物を意味する。動物を屠殺し、腫瘍はGFPによって確認された後、しかし、我々は、生物発光が超音波測定はよりも実行可能な腫瘍の存在とより密接に相関した。処置したマウスにおける残留瘢痕組織は、しばしば超音波による可視化であったが、ライブの腫瘍と区別することができませんでしたので、これはSarnaの（ようにコントロールするのではなく）で処理したマウスの間で特に明らかであった。 <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="図1" /> 図1：膀胱腫瘍の腫瘍増殖動態。 （A）生物発光イメージングは​​、それらの位置と成長を確認するために膀胱腫瘍内のルシフェラーゼ活性を測定します。 （B） 生体内膀胱ultrasoun でD 40MHzのプローブを使用すると、その寸法を正確に測定することができる腫瘍の画像を生成します。 （C）グラフは、生物発光と超音波の次元で腫瘍の漸進的増加を示す。 6匹の動物から- （標準偏差+ / - ）の測定値は平均値を表しています。 <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU international. 100, 1377-1384 (2007).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), e2523 (2011).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

LCLは、カリフォルニア大学サンフランシスコ校が提出したとアルナイラム·ファーマシューティカルズにライセンスされていますSarnaのに関連した特許出願を保留中の名前の発明者である。

我々は、転写活性化にp21のCIP1/WAF1（P21）のプロモーターを標的とLNP策定Sarnaの腫瘍の膀胱内治療に続いてマウス同所性異種移植膀胱腫瘍モデルの確立のためのプロトコルを提示します。2、3の結果、腫瘍ができる生物発光イメージングと膀胱の超音波で確認し、監視されます。同所性モデルは、より密接に内因性の膀胱癌を近似している尿や尿路上皮の環境を提供することに?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> KU-7-lucを-GFP細胞</td><td>キャリパー</td><td> 128091 </td><td></td></tr><tr><td>胸腺ヌードマウス（NU / NU） </td><td>シモンセン研究所</td><td> 6から7週齢</td><td> SIM：（NCR）NU / NU fisol </td></tr><tr><td>超音波装置</td><td> Visualsonics </td><td> VEVO 770 </td><td> RMV-704プローブ</td></tr><tr><td>生物発光ユニット</td><td>キャリパー</td><td> IVISスペクトラム</td><td></td></tr><tr><td>エクセルsafeletカテーテル</td><td>フィッシャー</td><td> 14-841-21 </td><td> 24G X¾ &quot; </td></tr><tr><td>硝酸銀</td><td>シグマ</td><td> S8157 </td><td></td></tr><tr><td>ハミルトンシリンジ</td><td>ハミルトンの共同mpany </td><td> 80301 </td><td></td></tr><tr><td> LNP-Sarnaの</td><td>アルナイラム医薬品</td><td></td><td></td></tr></tbody></table> 表1特異的試薬および装置。

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

私たちは、同所移植マウス膀胱腫瘍モデルでは膀胱内に配達小さな活性化RNA（Sarnaの）と膀胱癌を治療するための新たな手法を提案する。マウスモデルは、吸入全身麻酔下に尿道カテーテル法によって確立されます。化学熱傷は、その後膀胱グリコサミノグリカン層を破壊するために膀胱内の硝酸銀溶液を用いた膀胱粘膜に導入され、細胞が付着することを可能にします。滅菌水で数回洗浄後、ヒト膀胱癌KU-7-luc2-GFP細胞は2時間に住むために膀胱にカテーテルを通して注入されています。膀胱腫瘍のその後の成長は 、in vivo膀胱超音波および生物発光イメージングによって確認され、監視されています。腫瘍はその後脂質ナノ粒子（LNPs）に策定Sarnaので膀胱内に処理されます。腫瘍の成長は、超音波検査および生物発光で監視されています。この手順のすべてのステップは、添付のビデオで実証されています。

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

超音波検査および生物発光イメージングによる膀胱内に配達Sarnaのとモニタリング腫瘍成長の抗腫瘍効果を評価するために同所性膀胱腫瘍モデルを確立します。

同所性膀胱腫瘍モデルと膀胱内Sarnaの治療の評価

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

an-orthotopic-bladder-tumor-model-evaluation-intravesical-sarna

Research

JoVE Journal

Medicine

14.6K ビュー.  University of California, San Francisco. 超音波検査および生物発光イメージングによる膀胱内に配達Sarnaのとモニタリング腫瘍成長の抗腫瘍効果を評価するために同所性膀胱腫瘍モデルを確立します。

ビデオ: An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

マウス膀胱癌の同所性モデル

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

医学

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 ...

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 ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

甲状腺未分化癌の同所性マウスモデル

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

遺伝子送達研究のための同所膀胱癌モデル

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

ヒト前立腺癌転移の同所マウスモデル

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

マウス同所性膀胱異種移植片の最小侵襲性の確立

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

皮下腫瘍モデルにおける腫瘍浸潤白血球サブセットの評価

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

の有効性の評価<em&gt; H。ピロリ</em同所性マウスモデルにおける膀胱癌の治療のための治療ツールとして&gt;タンパク質HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

肝細胞癌転移の「患者様」同所同系マウスモデル

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...