作者は彼らの役に立つ議論や技術支援のための産婦人科のメンバーと癌生物学に感謝します。この研究は、科学の振興（JSPS）費補助金科学研究費（; H.梶山へ15K15604）のために日本協会によってサポートされていました。

治療プロトコルは名古屋大学で採択された動物実験のためのガイドラインに従います。 細胞懸濁液の調製<ol><li>ヒト卵巣明細胞癌細胞株。 <ol><li> （100単位/ ml、ストレプトマイシン：0.1mg / mlのペニシリン）、10％ウシ胎児血清（FBS）及びペニシリン/ストレプトマイシンを含むRPMI-1640培地中でES-2細胞12を保持します。 5％CO 2の加湿インキュベーター中で37℃で培養細胞。 </li><li> 0.05％トリプシン/ 0.02％EDTA溶液（トリプシン/ EDTA）で対数増殖期の細胞を収集します。 <ol><li> （ - 25cm 2のあたり4ミリリットル、2+ の Ca 2+ / Mgを含まないPBS 3）まず、次いでPBSで細胞を1回洗浄し、細胞培養容器から古いメディアを削除します。 </li><li>次に、トリプシン/ EDTAの25cm 2のあたり1ミリリットルを追加します。トリプシン/ EDTAで細胞単層をカバーするために細胞培養容器を回転させます。トリプシン/ EDTAを除去し、廃棄した後、細胞を返します5分または細胞が分離されるまで - 3のためのインキュベーターに培養容器。 </li><li>細胞は、細胞培養容器の表面から剥離して浮いていることを確認するために顕微鏡下で細胞を調べます。 </li><li>トリプシン（25cm 2のあたり5 ml）を不活性化し、細胞を再懸濁するために、増殖培地を含有する新鮮な血清を加えます。 15ミリリットルコニカルチューブに再懸濁した細胞を転送します。 </li><li> 5分間、300×gで遠心分離します。 4℃または室温での遠心分離のいずれかを実行します。慎重に上清を除去し、（25cm 2のあたり5 ml）を成長培地で細胞を再懸濁します。 </li></ol></li><li> （トリパンブルー排除によって、例えば ）細胞カウントを2×10 8細胞/ mlの密度で増殖培地中に再懸濁します。氷の上に懸濁した細胞を置きます。 </li></ol></li><li>凍結した細胞外マトリックス（ECM）を解凍氷浴中でヒドロゲルアリコートをベース。 </li><li> 1（最後のセルC：1の比率でECMベースのヒドロゲルのアリコートに細胞懸濁液を追加oncentration：1×10 5細胞/培地-ECMベースのヒドロゲルのμl）を、徹底的に混ぜます。使用するまで氷浴中で調製した細胞懸濁液を置きます。 </li></ol>細胞懸濁液2.同所接種注：手術は、専用のスイートを必要としません。手術は別個であり、最小限の活性を有する部屋の領域で実験室のベンチトップ上で実行することができます。層流フードを使用してもよいです。無菌の手術器具を使用する必要があり、鋏は、皮膚切開を作製するために使用すべきではありません。 <ol><li>この同所接種モデルのための8週齢で雌性ヌードマウス（BALB / C NU / NU）を使用します。 </li><li> （ - 4％2）まず、イソフルラン吸入を用いてマウスを麻酔。しっかりとつま先のピンチの際に撤退の欠如を検証することによって麻酔深度を確認してください。マウスを麻酔した後、必要に応じて乾燥を防ぐために、目に当たり障りのない無菌眼軟膏を適用します。 </li><li> operatに麻酔マウスを置きます段腹側を下る。アルコールとヨウ素系またはクロルヘキシジンベースのスクラブの両方で手術領域を数回駆除。 </li><li>垂直方向に右肋骨弓と大腿骨の間の背骨に近い〜2 cmの切開を行います。切開部位の周りに滅菌外科用ドレープを使用してください。開口部を維持するために、クランプで切開し、皮膚の表面を修正しました。 注：リトラクターは、代わりのクランプを使用することができます。 </li><li>次に、腹腔を開くには、最初の切開下壁側腹膜で第2の切開（約1cm）を作ります。切開腹部の表面を固定します。腹腔から卵巣を除去するために、クランプで同側卵巣と卵管を囲む脂肪組織をクリップ。慎重にガーゼ上の卵巣と卵管を配置します。その後、解剖顕微鏡下でマウスの腹側を下に置きます。 </li><li>注入直前（1/2 ccで、29 G）は、インスリン注射器で細胞懸濁液を置きます。慎重に準備された細胞懸濁液を（注入1- 卵巣に2μl）を。いくつかの秒間、ECMベースのハイドロゲルのゲル化を確実にするために卵巣内針を保持します。ゲル化した後、注入された腫瘍細胞が卵巣の内部に残っています。 </li><li>慎重に体内に卵巣を返します。滅菌医療絹を有する第二の切開を縫合し、次に創傷クリップで皮膚を密封します。 </li><li>手術後、マウスは、最初の24時間の期間（ 例えば 、メロキシカム、筋肉内（IM）又は皮下（SC）、0.2ミリグラム/ kg）の鎮痛を投与されます。 </li></ol> 3.手術後の処置<ol><li>それは胸骨横臥位を維持するのに十分な意識を取り戻したまでマウスを暖かく保つためにホットパックできれいに紙の上に別々のケージに各マウスを置きます。その後、飼育および監視するためのケージに動物を返します。外科的治療後の7日 - 3毎日マウスを監視します。 </li><li>術後のマウスのモニタリング<ol><li>痛みの兆候の場合には、analgeを投与毎日SICS（ 例えば 、メロキシカム、筋肉内または皮下、0.2ミリグラム/キログラム）。 </li><li>マウスは感染症（ 例えば 、発赤、腫脹、放電）の兆候がある場合は、抗感染剤（ 例えば 、ペニシリン、イム10万IU / kg）を毎日注射を投与します。 </li></ol></li><li> 14日手術後の治療後 - 10皮膚閉鎖材料を削除します。 </li></ol>腫瘍および転移の4分析注： - 接種後3週間で腫瘍が注入された卵巣2に形成されます。 <ol><li>さらなる分析14,16-20のために（ 例えば 、腫瘍、転移、臓器）13-15二酸化炭素によりマウスを犠牲にしてから、サンプルを収集。 注： インビボイメージングシステムは、利用可能である場合には生物発光（ 例えば 、ルシフェラーゼ）、または蛍光タンパク質（ 例えば 、GFP）を産生する酵素を有する細胞は、このプロトコルで使用することができます。この修正されたプロトコルは、有用な情報を提供します原発腫瘍からの癌細胞の広がりのダイナミクス。 </li></ol>

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著者らは、開示することは何もありません。

動物モデルは、癌細胞に対する腫瘍発生、進行、転移、及び薬効のメカニズムを分析するために不可欠です。ヒト卵巣癌細胞を有するマウスはまた、動物モデルとして使用されてきました。ここでは、同所のサイトで卵巣腫瘍細胞の投与のための低侵襲性の手順を説明します。この手順を使用して、背側脇腹から後腹膜アプローチを通じて卵巣への接種は、他の一般的に使用される技術上の重要な利点があります。まず、同所接種は、人間13,21における腫瘍環境を反映し、より効率的かつ正確な結果を提供することができます。第二に、背側脇腹からこの後腹膜アプローチはわずかな解剖必要がある（&lt;1〜2センチメートルを）ので、腹膜のアプローチに比べて迅速な方法です。したがって、この後腹膜アプローチは、マウスのための低侵襲性です。第三に、マウスの卵巣は非常に小さく、そしてレアを大量に受け入れることがとてもできません紳士。 ;シングルショットとして- （2μL/インジェクション1）この発表の手順では、高度に濃縮された細胞懸濁液（1×10 5細胞/μl）を、低接種ボリュームを使用します従って、それは、卵巣への損傷を最小限に抑えます。また、卵巣から接種された細胞の漏出を回避することができます。さらなる研究が必要不可欠であるが、説明のネズミ同所接種は、将来のEOCの治療のための新たな治療法の開発を支援するための有用なアプローチかもしれません。 このプロトコルで2つの重要なポイントがあります。まず、細胞が対数増殖期に使用されています。第二に、ECMベースのヒドロゲルで懸濁された細胞を使用するまで氷上に置きます。 ECMベースのヒドロゲルは温めるために開始すると、ゲル化プロセスが開始されます。 ECMベースのヒドロゲルのゲル化を回避するために、ECMベースのヒドロゲル及び機器（ 例えば 、チューブ、シリンジ、及びニードル）で再懸濁した細胞を使用するまで氷上に維持されます。この手法の1つの制限はあります。マウス卵巣の接種材料を大量に受け入れることができないので、細胞懸濁液の注入量は、3未満μLです。 このプロトコルは、いくつかの点で変更することができます。 インビボイメージングシステムが利用可能である場合、生物発光（ 例えば 、ルシフェラーゼ）、または蛍光タンパク質（ 例えば 、GFP）を産生する酵素を有する細胞は、このプロトコルで使用することができます。この修正されたプロトコルは、原発腫瘍からの癌細胞の広がりのダイナミクスのために有用な情報を提供します。また、このプロトコルは、マウスの卵巣を接種するためにES-2細胞を使用しています。 ES-2細胞は、明細胞癌から誘導され、この細胞株は、ヌードマウスにおける積極的な成長を示します。他の細胞株（ 例えば 、漿液性癌）も、ES-2細胞の代わりに、このプロトコルを使用することができます。

上皮性卵巣癌（EOC）は、婦人科悪性腫瘍の1の中で癌関連死亡率の最も高い割合を占めています。 EOCは、主にその後半症状に、予後不良と関連している、と多くの場合、複数の腹腔内disseminationsおよび遠隔転移2-4と関連しています。腹膜播種は、多段階プロセスです。まず、腫瘍細胞は原発巣から離れ、そして腹腔内に移動します。腫瘍細胞が腹膜中皮に付着すると、彼らは中皮5,6を介して組織に侵入し始めます。より良い腫瘍生物学（ 例えば 、癌の進行と治療反応）を理解するために、マウスモデルは、豊富な情報を提供しています。ヒト癌細胞を有する異種移植片は、広く、静脈内に、腹腔内に、ヒト癌細胞を皮下に接種されたマウスモデルに使用され、同所れます。同所gを有する動物モデルrafted腫瘍は、より効率的かつ正確heterotopicallyグラフトされた腫瘍を有する動物モデルと比較して、ヒトにおける腫瘍環境を反映した結果を生成することができます。そのため、多くのヒト腫瘍については、同所移植モデルは7月11日に設立されました。 ここでは、腹側接種に比べて侵襲性が制限されている背側脇腹から後腹膜アプローチを通じて人EOC細胞の同所接種を説明します。この技術はEOCについての有用な情報、腹腔内播種の特にメカニズムの多様性を提供することができます。

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Matrigel matrix&#160;</td>
			<td style="width:151px;">BD</td>
			<td style="width:163px;">354234</td>
			<td style="width:163px;">ECM-based hydrogel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin syringe</td>
			<td>TERUMO</td>
			<td>SS-05M2913</td>
			<td>1/2cc, 29Gx1/2&#34;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Suturing needle with suture</td>
			<td></td>
			<td></td>
			<td>11mm, 3/8, Nylon6-0</td>
		</tr>
		<tr height="20">
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murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with ovarian carcinoma cells

それは腹膜播種を示しているので、上皮性卵巣癌（EOC）は予後不良と関連しています。予後を改善するために、腹膜播種を制御することが重要です。しかし、腫瘍細胞が原発巣から切り離し、中皮に付着するかまだ不明です。適切な動物モデルの確立は、in vivoでの腹膜播種のメカニズムの理解を得るために必要とされます。現在の研究では、腹膜や遠隔臓器などの転移の発生にマウスの卵巣表面にEOC細胞の局所注射からプロセスをご紹介します。生後8週目の雌ヌードマウス（BALB / C NU / NU）を使用しました。ビューの顕微鏡視野下では、EOC細胞（中の細胞外マトリックス（ECM）の1×10 5細胞/μlのヒドロゲル/片側性卵巣/マウスをベース）背側脇腹から後腹膜アプローチにより、マウスの卵巣に注入しました。この提案された方法は、ルですマウスのためのssの侵襲的手技とは、卵巣へのダメージを最小限に抑えることができます。ここでは、EOCの元と転移性腫瘍形成の開発における方法論の手順を説明します。

<html>六ヌードマウスは、このプロトコルに記載のように、それらの卵巣は、ヒト明細胞癌細胞株ES-2を接種していました。 図1Aに示すように、2週間後、6匹のマウスの5は、ES-2細胞を接種した卵巣の腫瘍を有していたが、反対側の卵巣には腫瘍（コントロールとして使用）を接種せずに存在しませんでした。また、5匹のマウスの2は、腹水を持つ複数の腹膜disseminationsを示しました。細胞は、肝臓（ 図1B）に転移します。接種部位での腫瘍塊とし、制御側の両方の卵巣は、解剖切片、ヘマトキシリン-エオシン（HE）で染色し、顕微鏡（ 図2）で分析しました。卵巣癌細胞の大多数はなく、反対側の、接種卵巣で観察されました。細胞形態は、親腫瘍と同じでした。 <img alt="&lt;/ htmlの">「図1」SRC = &quot;/ファイル/ ftp_upload / 54353 / 54353fig1.jpg&quot; /&gt; 図1： ヒト明細胞癌細胞のES-2と同所接種による腫瘍形成。 AとBは、BALB / cのNU / NUマウスはES-2細胞を同所接種を受けました。 2週間後、マウスを剖検のために屠殺しました。 （矢印によって示されるように）Bは、細胞は肝臓に転移します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54353/54353fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54353/54353fig2.jpg" /> 図2： ヒト明細胞癌細胞のES-2と同所接種と卵巣のヘマトキシリン-エオシン染色。 A及びB、ES-2細胞の接種と卵巣、ヘマトキシリン-エオシン（HE）で染色しました。同様の染色は、明細胞癌に比べて見られましたヒト卵巣インチCとD、コントロールとして、接種せずに卵巣のHE染色。 スケールバーは100μm（A、C）、20ミクロン（B、D）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54353/54353fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells at JoVE.com

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

卵巣癌の腹膜播種の多段階プロセスを明確にするために、現在の研究では、これらの腫瘍細胞と同所接種を介して元の腫瘍の開発と腹膜転移のマウス実験モデルを提示します。

オリジナル腫瘍の開発のマウス実験モデルと腹膜転移<em&gt;経由で</em卵巣癌細胞で&gt;同所接種

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

murine-experimental-model-original-tumor-development-peritoneal

Nanyang Technological University

Research

JoVE Journal

Cancer Research

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

10.4K ビュー.  Bell Research Center for Reproductive Health and Cancer. 卵巣癌の腹膜播種の多段階プロセスを明確にするために、現在の研究では、これらの腫瘍細胞と同所接種を介して元の腫瘍の開発と腹膜転移のマウス実験モデルを提示します。 

ビデオ: Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

腫瘍転移に対する低酸素のテストの効果のためのインビボモデル

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

がん研究

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

マウス同所性膀胱腫瘍モデルおよび腫瘍検出システム

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

無細胞性と細胞の肺転移モデル

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

腫瘍免疫プロファイル用のマウスおよび治療応答評価における扁平上皮癌細胞の粘膜接種

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

ヒト尿路上皮細胞癌および大腸癌腫瘍増殖および自発性転移に対する患者由来の眼科医異所性Xenograft移植片モデル

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

下流の分子分析と転移性電位評価のための循環腫瘍細胞のマイクロマニピュレーション

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

肺扁平上皮癌進行をモデル化する多次元共培養システム

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

腫瘍細胞の横尾静脈注入後の肺転移の病理学的解析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

固形腫瘍移植による骨原発性骨腫瘍と骨転移のモデリング

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...