We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

倫理ステートメント すべての動物実験は、ニューヨーク市立大学ハンターカレッジの施設内動物管理使用委員会（IACUC）によって承認されました。 注：8 BALB / cマウスは、この実験手順で使用しました。五BALB / cマウスは、原発腫瘍を産生する5×10 6 BNL 1ME A.7R.1マウス肝細胞癌細胞を移植しました。無移植で三BALB / cマウスを対照として使用しました。 BNL 1ME A.7R.1は、マウス非腫瘍形成性肝細胞株BNL CL.2から化学変換によって得られたマウスの肝細胞癌細胞株です。 BNL 1ME A.7R.1は、アメリカン・タイプ・カルチャー・コレクション（ATCC）から入手しました。 1.手術前<ol><li>すべての生存の手術のためのマウスは健康であることを確認してください。マウスは健康であることを確実にするために、皮膚のテンティングによって脱水の兆候を検査します。疾患又はdのいずれかの証拠でマウスを使用しないでくださいこれらの実験でistress。 </li><li>彼らは低体重ではないであることを確認するために、マウスを計量。重量は少なくとも16〜20グラムであることを確認してください。 </li><li>マウスあたりの移植のための5×10 6 BNL 1ME A.7R.1マウスHCC細胞を準備します。 <ol><li> BNL 1ME A.7R.1マウスHCC細胞の70％コンフルエント細胞培養皿 - 10ミリリットルの60％から培地を吸引。 5ミリリットルPBSで2回洗浄します。皿にトリプシンの2 mlを加え、5分間空気および5％CO 2の加湿インキュベーター中、37℃でインキュベートします。 </li><li>その後、ピペット10μlを10倍の倍率で顕微鏡下で細胞計数のためのマニュアル血球計数器に細胞をトリプシン処理。 4大隅の正方形で見つかったセルの合計数をカウントし、細胞の総数によって希釈係数を掛けます。そして、カウント正方形の数で割ると、細胞濃度（1ml当たり細胞）のために10 6を掛け。 </li><li>計算を使用して、無菌マウスあたり5万個の細胞を収集1.5mlの微小管。 1.5mlの微小管にラベルを付けます。 </li></ol></li><li>リン酸緩衝生理食塩水（PBS）100μl中5×10 6 BNL 1ME A.7R.1マウスHCC細胞を再懸濁します。手術で移植まで氷上でこれらの細胞を保管してください。 </li><li>このような麻酔導入と髪が汚染を避けるために、滅菌装置・材料から離れクリッピングなどの手術のためのマウスの準備のために必要な活動を行います。 </li><li>毎分1リットル（約2.1％）の一定濃度の流量で酸素と一緒にイソフルラン気化器を使用して、イソフルランの吸入によってマウスに全身麻酔を誘導します。注：システムは、自立で、精密気化器、酸素供給及びガス/麻酔掃気のために必要な呼吸回路にリンクされているチャコールキャニスタを含んでいます。 </li><li>麻酔は、手術中に痛みを受けてから動物を防ぐために十分であることを確認してください。つま先のピンチを実行し、非responために動物を観察siveness麻酔を確認しました。 </li><li>その時、ノーズコーンにバルブライン、シールボックスに、オープンを切り替えます。 </li><li>すぐにマウスを麻酔されると、乾燥やダメージから予防措置として角膜に眼軟膏の少量を適用します。 </li><li>その後、適切に（メンテナンス麻酔のために）配置され、接続されたノーズコーンと、準備領域に動物を移動します。この準備エリアで脱毛することにより手術のための外科的切開部位を準備します。 </li><li>手術の面積は、他のすべての活動の自由位置にあることを確認してください。 </li><li>作業面は、手術前に頑丈かつ適切に消毒、汚れていないとすっきりであることを確認してください。リネンまたはそれ以上の外科手術用トレイの滅菌ピースを置きます。 </li><li>術野が手術を行う者のみに利用可能であることを確認してください。外科的な部屋は、適切なdisinfeで洗浄し、適切なクリーナーで掃引し、噴霧されていることを確認ctant。 </li><li>空気の流れは、サイトの汚染につながる塵を広げることができるようにそれはまた、ドアや窓から離れて配置されていることを確認してください。 </li><li>滅菌器具で生存手術を行います。どちらのメーカーの推奨またはオートクレーブに応じて消毒剤で楽器を浸します。使用するための準備ができている基本的な無菌の消耗品を使用してください。 </li><li>無菌区域の汚染を防ぐために機能に応じて個別の楽器。 </li><li>動物の肝臓にアクセスするために切開部位の周囲に約1インチ（正中切開）を剃ります。手術部位から離れた動物から毛を削除します。 </li><li>シェービング後、せっけん消毒剤（ベタジン外科的スクラブ）の領域をスクラブし、滅菌PBS溶液で余分な消毒剤を洗い流します。約5分を持続全体スクラブプロセスで、この数回を実行します。削除し、使用する手袋を廃棄しました。 </li><li>その後、このようなsurgicなどの防護服に変更アルガウン、スクラブトップ、およびフェイスマスク。次に、手術用手袋の上に置く前に、適切なスクラブ液で手を消毒します。注意：これは脱落し、潜在的な汚染物質と手術部位の汚染を防止するために必要なステップです。 </li></ol> 2.手術<ol><li>麻酔の全期間を通じて暖かさのための外部熱源を提供します。麻酔は、体温調節の乱れの原因となるため、動物は、いくつかの低体温症が発生する可能性があります。従って、体温を維持します。 </li><li>手術を通して、その背中の上にマウスを置きます。 </li><li>サージカルテープの作品で手術テーブルにフォア手足、後肢およびマウスの尾を修正。 </li><li> xyphisternumにすぐに劣る直線切開を行います。 </li><li>肝臓を露出させ、滅菌自己保持手術用開創器を使用してください。 </li><li>シリンジはゆっくりと慎重に、20G針を有する5×10 6 BNL 1ME A.7R.1を含む100μlのPBSを注入肝臓の右葉にマウスHCC細胞。 </li><li>針を除去した後、原因肝臓の高い血管分布に任意の潜在的な出血を止めるために、少なくとも1分間滅菌綿棒を適用します。 </li><li>外科用クリップを用いて皮膚の閉鎖に続いて吸収性縫合糸で皮下組織を閉じます。その後、ベータダインスクラブで再び傷をきれいにします。注意：これは、手術創の閉鎖を保証します。 </li><li>皮下に生理食塩水を100μlを与えます。 </li></ol> 3.手術後<ol><li>すぐに手術後、マウス条件の世話人を通知するために、手術後のIDカードできれいにケージにマウスを転送します。食料と水を提供します。注：最初の24時間の期間後に手術のためのウェットフードと水を単独で収容された場合は、当社の経験では、ちょうど手術を受けたマウスは最高の運賃になります。 </li><li> 38ºCを超えない熱を発生する熱ランプで動物を暖かく保ちます。 Uマウスが完全に回復するまで、温度を測定する温度計をSE。 </li><li>ハウスの快適性を確保するために、紙の寝具と好ましくはケージ内の動物。 </li><li>最初の回復後、不快感、出血、痛みや苦痛の徴候のために動物を監視します。痛みを和らげるためにブプレノルフィン（0.05 -0.1ミリグラム/キログラム）を与えます。密接に約2時間の活動、行動、外観だけでなく、水や食料の消費量を監視します。歩行した場合、マウスを定期的に通常の住宅のためのバックエリアに動物を移動し、24時間後に手術のために6-12時間間隔で監視し続けます。必要に応じて重量チェックを実行します。 </li><li>しかし、動物が療養問題を抱えている場合、適切な支持療法の行動を取ります。マウスは回復できない場合は、人道的に、CO 2窒息と頸椎脱臼を経由して、マウスを安楽死させます。安楽死のために承認された機関のガイドラインに従ってください。 </li><li>手術後、通常observabあるHCCの臨床的証拠をチェックしますルボディコンディションスコアの大幅な削減など。注：この時点では、原発腫瘍は、おそらく肝臓で開発しており、転移性預金は、おそらく肺に開発しました。 </li><li>この時点で、人道的な安楽死によって実験に関与し（実験及びコントロール）すべてのマウスを生け贄に捧げます。二酸化炭素窒息にマウスを件名：非常にゆっくりと5〜10分かけてチャンバー内に二酸化炭素を放出します。これは、呼吸停止の原因となります。 </li></ol>

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The authors declare that they have no competing financial interests.

この記事では、この方法の詳細な説明は、（ 図2）ことが最近OgunwobiとHCC転移の同所性同系マウスモデルの確立に成功の同僚によって報告された与えられている3。この手順のために、腫瘍の取り込み率は、一般的に高いです。我々は以前の100％3の腫瘍取り込み率を観察しました。しかし、テイク率は研究者の能力に応じて可変とすることができます。トレーニン?...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table><tbody><tr><td>Micro Dissecting Tweezer&amp;nbsp;</td><td>Roboz</td><td>(RS-5040)</td><td>Tip 0.20 x 0.12 mm</td></tr><tr><td>Graefe Micro Dissecting Forceps, serrated curved tip</td><td>Roboz</td><td>(RS-5111)</td><td>1 X 2 teeth, curved tip width 0.6 mm</td></tr><tr><td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6501)</td><td>Blunt 3 X 3 prongs, depth 4 mm, spread 25 mm</td></tr><tr><td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6503)</td><td>Blunt, 3 X 3 prongs, depth 4 mm, spread 19 mm</td></tr><tr><td>Jameson Caliper (Measures tumors)</td><td>Roboz</td><td>(RS-6466)</td><td>80 mm/3 inch scale, chrome plated&amp;nbsp;</td></tr><tr><td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td><td>Roboz</td><td>(RS-5852)</td><td>23 mm blades, length 4 inches, flat shanks and large rings</td></tr><tr><td>Scalpel with blades, for delicate dissecting procedures</td><td>Roboz</td><td>(RS-9861-36)</td><td></td></tr><tr><td>Scalpel handle&amp;nbsp;</td><td>Roboz</td><td>(RS-9884)</td><td>Solid</td></tr><tr><td>Littauer Stitch Sissors</td><td>Roboz</td><td>(RS-7074)</td><td>Length 4.5 inches</td></tr><tr><td>Brown needle holder, for easy suture tying</td><td>Roboz</td><td>(RS-7960)</td><td>Convex jaw, fine serrations</td></tr><tr><td>Reflex 7MM wound clips with reflex 7 clip applier</td><td>Roboz</td><td>(RS-9262)</td><td>safe, secure alternative method of wound closure</td></tr><tr><td>Instrument tray and lid</td><td>Roboz</td><td>RT-1350S</td><td></td></tr><tr><td>Mini-clipper with detachable blade</td><td>Roboz</td><td>(RS-5903)</td><td></td></tr><tr><td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td><td>Roboz</td><td>Ds-400, Ds-401, DS-501</td><td>For fast decontamination of micro-dissecting instruments.&amp;nbsp;&amp;nbsp; Instruments decontaminate within 15 sec.&amp;nbsp;</td></tr><tr><td>Microinjection needles</td><td>VWR</td><td>BD305125</td><td>25G Needle</td></tr></tbody></table>

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.

Balb / cマウスの肝臓は、5×10 6 BNL 1ME A.7R.1マウスHCC細胞を移植しました。 HCC開発の臨床的証拠は63日、手術後に観察しました。これにより、マウスを人道的に安楽死させました。剖検を安楽死させたマウスで行いました。肺と肝臓を切除し、慎重な肉眼的検査は肉眼で見える腫瘍を同定するために実施しました。 1マウスでは、表在性腫瘍は腹壁（ 図1A）にはいくつかのフ...

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

癌の転移拡散は、癌関連死の主要な原因です。私たちは、固形臓器腫瘍で転移のメカニズムを研究するための「患者様」同所同系マウスモデル系を確立するための私たちの生存の手術方法の詳細な説明を提供します。

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 ...

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12.2K ビュー.  Hunter College of the City University of New York. 癌の転移拡散は、癌関連死の主要な原因です。私たちは、固形臓器腫瘍で転移のメカニズムを研究するための「患者様」同所同系マウスモデル系を確立するための私たちの生存の手術方法の詳細な説明を提供します。 

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

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 Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

乳癌の肝転移を研究するためのポータル静脈注射モデル

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

大腸癌の同所性マウスモデル

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

生物学

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

肝炎症と線維症の設定に起因する肝細胞癌の腫瘍性肝細胞誘発性オルコトピックマウスモデル

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

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 ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

免疫不全マウスの皮下および肝内ヒト肝細胞癌異種移植片の生成

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

肝細胞癌の同所性モデルマウスにおける非侵襲的PET/MRイメージング

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

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, ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.