ハロルドバーマス、ブライアン c. ルイス、腫瘍形成の各、ダニー黄、シャロン パン、ミーガン ・ ウォン、Manasi m. Godbole を感謝いたします。Y.C.N.D. をサポートするには、DOD グラント W81XWH-16-1-0619 と NIH グラント 1R01CA204916。

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R., Sambrook, J., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2012).</li><li>Du, Y. C., Lewis, B. C., Hanahan, D., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+tumor+progression+factors+by+somatic+gene+transfer+into+a+mouse+model:+Bcl-xL+promotes+islet+tumor+cell+invasion.">Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion.</a> PLoS Biol. 5 (10), 2255-2269 (2007).</li><li>Du, Y. C., Chou, C. K., Klimstra, D. S., Varmus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+for+hyaluronan-mediated+motility+isoform+B+promotes+liver+metastasis+in+a+mouse+model+of+multistep+tumorigenesis+and+a+tail+vein+assay+for+metastasis.">Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis.</a> Proc Natl Acad Sci USA. 108 (40), 16753-16758 (2011).</li><li>Guernet, A., Grumolato, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+editing+of+the+genome+for+cancer+modeling.">CRISPR/Cas9 editing of the genome for cancer modeling.</a> Methods. , (2017).</li></ol>

著者が明らかに何もありません。

本研究ではマウスの強力なモデル、リップ-タグについて述べるRIP-tva鳥レトロ ウイルスの同定と転移の要因の解析を介して体細胞遺伝子配達を達成するために。ただし、リップ-タグ;RIP tvaマウス膵神経内分泌腫瘍を発症、このマウス モデルにおける転移因子も他の種類のがんの転移を促進するかもしれません。
我々 のアプローチの導入したがって散発的な人間の腫瘍の開発をより忠実に模倣時間制御方法で膵 β 細胞の前癌病変に体細胞の遺伝的変化を具体的には利点があります。このアプローチは、開発中の興味の遺伝子の異所性発現のため従来のトランスジェニック モデルでしばしば観察された正常組織形成の任意の潜在的な摂動を回避できます。さらに、トランスジェニック マウスを生成するよりも興味のある遺伝子を運ぶ鳥 retroviral ベクトルを生成するはるかに高速です。RCASBP から派生した鳥レトロウイルスベクターは tva を表現する細胞in vitroとin vivoに Cdna (≤2.5 kb)、Shrna、Mirna、他の Rna を配信できます。感染症 (および複数の感染) の効率、標的細胞の増殖率と細胞のアクセシビリティによって決まります。力価 &#62; 1 x 108感染単位 ml体内感染に必要です。効率的なウイルス配信達成できる生体外で誘導される細胞増殖とウイルスに感染するすべてのセルの反復暴露の可能性のためです。
マウスの生存率の正確な心臓内注射法が欠かせません。まず、ウイルスの懸濁液を描画する前にインスリン注射器で空気のスペースを 50 μ l 添加は心臓パルスを見て重要です。第二に、血の赤い脈が表示されたら、注射器の位置を修正して、注射器で血の明るい赤のパルスを見て表示されるたびにゆっくりと 10-20 μ l ウイルス懸濁液を提供することが重要です。血の明るい赤パルス停止 10-20 μ l ウイルス懸濁液の出産後、少し針を再配置、役立ちます。第三に、最後のプッシュはウイルスの懸濁液を提供するプランジャーのや血パルスを見る前に後を胸腔からすぐに取り消す必要がある針と注射部位に適用される穏やかな圧力は内部の出血を減らすを助けます。最後になりましたが、別のマウスではインスリン注射器を再使用しないでください。
将来のアプリケーションは、このRIP の-タグです。RIP tvaマウス モデル他のトランスジェニック、ノックイン、ノックアウト マウス モデルと組み合わせて使用できます。さらに、このリップ-タグを組み合わせることを思い描いてください。RIP tva単一の点突然変異、削除、逆位や転座22などゲノムを生成する CRISPR Cas9 ゲノム編集ツールでマウス モデル。

ほとんどの癌は、体細胞変異1から発生します。従来の遺伝子組み換えマウス モデル (ジェム) は、腫瘍2に特定の遺伝子変異の貢献に重要な洞察力を提供しています。ただし、彼らはいくつかの制限があります。これらのモデルの主な欠点は、組織内の一部のセルだけが遺伝の変化を取得する人間の腫瘍の形成の散発的性質を複製しないこと。トランスジェニック、ノックアウト マウスの突然変異生殖開発に影響を与える可能性があります。また、これらマウス モデルの生成は高価で時間がかかるです。
転移は、がんの分野で重要な問題です。転移のモデリングはジェムで難しくなっています。自然転移は、マウスではまれです。浸透度は可変して転移3のジェムで長い待ち時間です。転移の翼列の初期の手順が排除されますので、実験的転移モデルはマウスの循環にセルの直接注入を採用しています。
Bitransgenic マウス モデル、リップ-タグ マウス モデルにおける転移因子を勉強の上記の制限の一部を克服するために開発しました。RIP tva4。戦略は、高い同期腫瘍進行マウス モデル、 RIP タグ5、および受容体サブグループ A トリ白血病ウイルス、tva 6、7の使用を組み合わせることに基づいています。このリップ-タグ;RIP tvaマウス モデルにより、単一の bitransgenic マウス緊張体細胞染色体導入する遺伝子。SV40 T 抗原 p53 および Rb の腫瘍抑制機能を抑制、マウスは過形成、血管新生、腺腫、浸潤癌などの段階で人間の腫瘍と同様の方法で膵神経内分泌腫瘍を開発します。このRIP タグモデルは、がん、膵神経内分泌腫瘍に限定されないの認刻極印の私達の理解に非常に有益されています。また、前臨床試験8で使用されています。
体細胞遺伝子伝リップ-タグに鳥レトロ ウイルス intracardiacally の注入のためのプロトコルを提案します。RIP tvaマウス。RCASBP 由来鳥レトロ ウイルスを感染ターゲットの積極的に増殖の細胞が必要です。したがって、我々 はリップ-タグを選んだRIP tva 7 週齢、膵島の約 50% に過形成を開発するときにマウス。高力価ウイルスの左心室心腔内注射は、10-20 4の感染効率を達成するために必要です。この配信方法は、ウイルス膵島に到達する前に循環内のウイルスの粒子の重要な希釈を低減します。
このアプローチを使用すると、以前 Bcl-xl がその抗アポトーシス機能4,9の独立したがんの転移を促進することを確認しました。Bcl xLリップ-タグに発癌性個体中すべての膵 β 細胞での遺伝子を介して発現していたときこの抗アポトーシス独立転移機能は観察されなかったRIP Bcl xLモデル10をマウスします。したがって、マウス モデルは、ユニークな機会を識別し、特徴付ける腫瘍形成の後の段階で表現される場合に遺伝子の機能を提供しています。小島の 2-4%リップ-タグで腫瘍に発展するのでRIP tvaウイルス感染とすべての前癌病変なし bitransgenic マウスは、RCASBP 由来の鳥のレトロ ウイルスに感染している、癌の自然経過の選択的優位性を与える要因のみを識別できます。特に、転移要因最も簡単に認識されますこのメソッドによって膵リンパ節や他の臓器への転移はリップ-タグで通常発生しませんのでRIP tvaマウス。

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">RCASBP-Y DV plasmid</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:119px;">11478</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RCAS-RNAi plasmid</td>
			<td style="width:123px;">Addgene</td>
			<td style="width:119px;">15182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:123px;">Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine, 100x</td>
			<td style="width:123px;">Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin solution, 100x</td>
			<td style="width:123px;">Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">PBS-/-, 1X</td>
			<td style="width:123px;">Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfect</td>
			<td>Qiagen</td>
			<td>301305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyallomer centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">0.45 mm Nalgene 
			Syringe Filters with PES Membrane</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">194-2545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Insulin Syringes&#160;</td>
			<td style="width:123px;">BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">synaptophysin</td>
			<td style="width:123px;">Vector Laboratories</td>
			<td>VP-S284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">VECTASTAIN Elite ABC HRP Kit (Peroxidase, Rabbit IgG)&#160;</td>
			<td style="width:123px;">Vector Laboratories</td>
			<td>PK-6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AmpliTaq DNA Polymerase with Buffer II</td>
			<td>Life Technologies</td>
			<td>N8080153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyTaq DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21106</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification and characterization of metastatic factors by gene transfer into the novel rip-tag; rip-tva murine model

転移がんは、固形腫瘍患者の死亡の 90% を占めています。がん転移のドライバーを理解して新たな治療標的を識別するために緊急の必要性があります。転移する癌から進行を駆動する分子のイベントを調査して、bitransgenic マウス モデル、リップ-タグを開発します。RIP tva。このマウス モデルでラットのインスリン プロモーター (RIP) は、膵 β 細胞における SV40 T 抗原 (タグ) およびサブグループ (tva) トリ白血病ウイルスの受容体の発現を駆動します。マウスは、過形成、血管新生、腺腫、浸潤癌などの段階で人間の腫瘍に類似している明確に定義された段階で 100% の浸透度を持った膵神経内分泌腫瘍を開発します。リップ-タグ;RIP tvaマウスは、転移性疾患を発症しない、転移を促進する遺伝の変化を簡単に識別することができます。体細胞遺伝子の tva 表現に、前癌病変は膵 β を増殖目的遺伝子変異をかくまっている鳥でレトロ ウイルスの心腔内注射によって実現されます。力価 &#62; 1 x 108感染単位 ml は生体内で感染症の適切なと見なされます。さらに、鳥のレトロ ウイルスリップ-タグで腫瘍由来細胞株に感染します。RIP tva高効率を持つマウス。細胞は、転移性の要因を明らかにも使用できます。ここで我々 はこのマウス モデルを利用し細胞の腫瘍の転移で候補者の遺伝子の機能を評価する方法を示します。

リップ-タグの生体内および生体外での感染率RIP tva RCASBP ベースのウイルスによる腫瘍細胞は ~ 20% 〜 80% とそれぞれ20。RIP-タグ;RIP tvaマウス モデル、各マウス 400 の膵島の約 4% に腫瘍20; 開発自然したがってウイルスによって遺伝子の潜在的な効果の組織学的表現型分析の各マウスにおける腫瘍細胞の十分な量があります。このシステムを使用して、転移 Bcl-xl の新規核関数は識別された9だった。RIP の-タグです。RIP tva RCASBP-Bcl xL感染マウス発生率が高い癌のコントロール ウイルス感染マウスよりも RCASBP-ALPP (対 74% 96%) を出展しました。さらに、RCASBP-Bcl-xlの 47%-リップ-タグを感染RIP tvaマウス コントロール マウス20に転移は見つかりませんでした、16 週齢 (図 3 a)、安楽死させたとき膵臓のリンパ節転移を開発しました。
さらに、スクリーニングがん遺伝子リップ-タグのライブラリRIP tvaマウス膵リンパ節および肝21 (図 3 b 、 3 C) への転移を促進する最初の遺伝子を識別し、。この遺伝子は、ヒアルロン酸を介した運動アイソ フォーム B (RHAMMB) タンパク質の受容体をエンコードし、EGFR シグナル伝達21がアクティブになります。我々 は、その特定の肝臓転移は、N134 腫瘍細胞が当初21受信者免疫不全マウスの肺毛細血管床を通る循環実験的転移の尾静脈アッセイで締めくくっていることが実証。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55890/55890fig1.jpg"> 
図 1: RCASBP(A)、代替服務研究委-X、および代替服務研究委 Y 構造の概略図。クローニング サイトは青、太字のフォントで表示されます。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55890/55890fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55890/55890fig2.jpg"> 
図 2: 心腔内注入を配置します。解剖学的ランドマークは、(白で記載) 胸骨上水平の破線で表示されます。マウスの皮膚に胸骨切痕と胸膜のランドマークとなると針は半ばに胸骨から 1 mm を挿入され、わずかに左胸骨の (解剖学的)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55890/55890fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55890/55890fig3.jpg"> 
図 3: 免疫染色による転移性膵 β 細胞します。写真は、代表的なシナプトフィジン膵リンパ節 (A、B) または (C) 肝臓転移性膵神経内分泌腫瘍の染色を見せます。RIP の-タグです。RIP tvaマウスが 7 週齢で示された RCASBP レトロ ウイルスに感染しているし、16 週齢で安楽死させた。スケールバー = 50 μ m. 元の倍率 = 20 X。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55890/55890fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model at JoVE.com

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

リップ-タグを利用した体細胞遺伝子の転送システムを実証するためのプロトコルを提案します。RIP tvaマウス モデル転移遺伝子の機能を研究します。鳥のレトロ ウイルスは、成体マウスの膵 β 細胞の前癌性、非侵襲的病変への遺伝子導入を確保するため intracardiacally が配信されます。

同定と新規リップ-タグ; に遺伝子導入による転移因子の解析RIP tvaマウスモデル

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

identification-characterization-metastatic-factors-gene-transfer-into

Nanyang Technological University

Research

JoVE Journal

Cancer Research

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

8.7K ビュー.  Weill Cornell Medicine. リップ-タグを利用した体細胞遺伝子の転送システムを実証するためのプロトコルを提案します。RIP tvaマウス モデル転移遺伝子の機能を研究します。鳥のレトロ ウイルスは、成体マウスの膵 β 細胞の前癌性、非侵襲的病変への遺伝子導入を確保するため intracardiacally が配信されます。

ビデオ: Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

肝臓における大腸転移の高度な動物モデル：イメージング技術および転移性クローンのプロパティ

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

がん研究

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

定量的および前臨床治療の縦評価のための転移性腎細胞癌の同系マウスモデル

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

周辺照射による疲労のマウスモデル

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌の細胞外マトリックスと舌扁平上皮癌生体外の再構成の作製

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

アンドロゲン依存性去勢抵抗性前立腺癌の生物発光と蛍光の同所性同種同系マウスモデル

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

ウイルスの遺伝子改変マウスの前立腺に CRISPR ガイドの配信

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

濃縮と確立された皮下マウス腫瘍における腫瘍免疫と免疫のない微小のキャラクタリゼーション

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

培養 3 D 回転楕円体モデルでのマウス前立腺の葉の解剖組織学的特性及び

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉腫に対する三次元骨細胞外マトリックス モデル

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

虚血多灌流損傷の設定における転移性肝腫瘍のマウスモデル

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...