著者らは、共焦点とタイムラプス顕微鏡に関する技術的助言のための彼の優れた技術支援のためのハリッドGhadban、アンドレアスWodarzとステファンHeermannに感謝します。著者のいずれかのための特別の利害関係はありません。この作品は、のDresにより、Forschergruppe 942のプロジェクト2（FOR942 BI 703/3-1）にドイツの研究評議会（DFG）によって資金を供給される。ベイヤー財団（バーデンWürttembergischerKrebspreis、ドイツ）と医学部の研究プログラム、ゲオルク·アウグスト大学ゲッティンゲン、ドイツ。

この新しいモデルは、以前に公開された器官型海馬脳スライスアプローチ9月12日を適応したものです。修正および追加が癌細胞の脳組織の相互作用を最適化し、再現性を保証するために導入した。調査は見直され、地元の倫理委員会によって承認された。動物は、大学医学部ゲッティンゲンでの動物のケアのためのガイドラインに従って、慎重に処理した。器官型脳切片の共培養は、2つのステップに分けることができる。第一段階は、器官型脳切片の調製である。ステップ二つの共培養モデルにおける腫瘍細胞調製物および堆積を含む。 1。器官脳スライス<ol><li> 0.2 mMグルタミン、100 U / mlペニシリン、100 mg / mlのストレプトマイシン、およびグルコースの4.5 mg / mlのを補足した最小必須培地（MEM）、からなる解剖媒体を準備します。 </li><li>生後間の任意のマウス系統からマウスを刎ねる6と8（P6-8）。 </li><li>無菌状態で頭蓋骨から急速に脳を取り出して、氷のように冷たい解剖媒体に転送することができます。 </li><li>前頭極と全脳セクションから小脳を除去します。 </li><li>クライオのりと5％のアガロースでステージに脳を固定して安定させる。 </li><li>ビブラトームを使用して水平方向に350ミクロンの厚さに脳切片をスライス。 </li><li>種や年齢に応じて、単一のマウスの脳から五時五十六全脳スライスを収集します。 </li><li> 50％MEMからなる培地を調製し、25％ハンクス平衡塩溶液（HBSS）、25％正常ウマ血清（NHS）、0.2 mMグルタミン、100 U / mlペニシリン、100 mg / mlのストレプトマイシン（シグマ社、ミュンヘン、ドイツ）、および4.5 mg / mlのグルコース。 </li><li>下部ウェルに培養培地の1ミリリットルを6ウェルプレートに0.4ミクロンポリカーボネートトランスウェル膜インサート上の各器官の脳スライスを置く。 </li><li>一晩humidifieにおける培養器官型脳切片37℃のインキュベーター中で5％のCO 2とdの雰囲気。 </li></ol> 2。スライス共培養モデル<ol><li> GFP-トランスフェクトされた腫瘍細胞または他の細胞を15％RPMI培地および85％のECMゲルからなる20μlのゲルマトリックス（ 例えば MCF-7-GFP細胞）、10 5を埋め込 ​​む。 </li><li>器官型脳切片の皮質領域に直接隣接無菌金属スペーサ（3.8ミリメートル径）に、MCF-7-ゲルマトリックスミックスを置き、2時間インキュベート。 </li><li>スペーサーを外し、3D腫瘍スフェロイドが24〜96時間のための器官のスライスと共培養することができます。 </li><li>一日おきに培養液を変更します。 </li></ol> 3。器官型脳スライス共培養アストロサイトやミクログリアの免疫蛍光染色<ol><li> 4℃で8時間、4％パラホルムアルデヒドで器官型脳切片共培養を修正しました。 </li><li> 5分間のPBST（0.5％トリトンX-100を含むPBS）でスライス共培養を洗う。 </li><li> Sをブロック1時間室温でPBST中正常ヤギ血清（1:20）でamples。 </li><li>ヤギ抗マウスTRITC（PBST中1:100）染色、続いて4℃で36時間、抗グリア線維性酸性タンパク質モノクローナル抗体（GFAP、PBST中で1:200）を用いて脳切片の共培養をインキュベートすることにより星状細胞を染色室温で1時間。 </li><li> 5分間PBSTで3回のサンプルを洗浄します。 </li><li>室温で1時間、ILB 4 -アレクサフルーア647（PBST中1:100）とミクログリア細胞を染色する。 </li><li>室温で3分間DAPIで脳切片共培養（1:1,000）を対比染色。 </li><li>マウントとDAKO蛍光封入剤で脳切片共培養をカバースリップ。 </li><li>第+ + +≥2/3浸潤した細胞（の; - ; + &lt;1/3、2/3 + + = 1/3細胞の0 =なし、次のスコアリングシステムに基づいて、腫瘍浸潤の程度を評価する腫瘍プラグとスライスとの間の接触部の長さを測定し、測定fractio）浸潤細胞で検出可能な連絡先のN。 </li></ol> 4。グリアと腫瘍細胞との間の相互作用のライブイメージング<ol><li>ライカの下で実験を行うには、10倍の倍率のレンズとライカDFC 350 FX CCDカメラでDMI 6000B顕微鏡を反転。 </li></ol>

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我々は、開示することは何もありません。

脳転移の以前の組織学的研究は、特にアストロサイトやミクログリア13の居住グリア細胞の迅速かつ劇的な変化を明らかにした。癌細胞によるこれらの変化及び相互作用を研究するために、この新規な共培養システムが適している。他の研究分野は、すでに器官海馬脳スライスと長年の経験を持っています。一つの利点は、器官型海馬脳スライス培養は、長期実験に適して、週に生存可能かつ効果日間保存されていることである。 1991年の器官型海馬スライスシステムのStoppiniの導入以来、それは広く変性疾患の研究のために使用されてきた。このように、このイノベート共培養システムは、腫瘍生物学9,11内のアプリケーションの範囲が十分に確立されたアプローチの変更を表しています。修正は、腫瘍の浸潤AFのグレードを評価するために、私たちに再現可能なモデルを提供界面法で成長terwards、培養物は、理想的には、三次元構造を必要とする実験のために適している。いくつかの技術が他の細胞と器官型海馬切片を共培養するために使用されている。これらは、マクロファージ細胞や器官型脳切片14と、海馬領域15から2異なるスライスの直接共培養の間に間接的なシステムを含む。神経膠腫凝集体は、脳切片16と共培養されてきた。これらのモデルは、脳切片における細胞および分子事象を分析するために使用することができるが、腫瘍細胞、ミクログリアおよび脳実質との間の直接的な生理学的な接触を可能にしない。さらに、この方法は、骨髄由来の末梢単球/マクロファージの汚染なしミクログリアの観察を可能にする。 CCR2およびCX3CR1のトランスジェニックマウスモデルの使用は、それが単球に侵入常駐ミクログリアを区別することは困難であるという事実に重要な改善である彼らの同様の特性17,18,19に基づく。癌細胞の脳内注入は、腫瘍の進行を調査できますが、それは囲まマクロファージ様細胞が脳常駐ミクログリア集団から、または骨髄由来末梢単球/マクロファージ17から発信かどうかについて多くを伝えることはできません。 Kettenmannのチームは、マイクロマニピュレータ20を脳スライスに神経膠腫細胞を接種することに関与器官脳切片モデルを導入しました。しかし、原発性悪性神経膠腫、転移性がんから多くの点で異なっている。まず、悪性神経膠腫は、間葉系由来のもので、腫瘍と脳組織との間の境界線なしの場合と同様に、単一の細胞に侵入/神経系の外に転移して、移行されません。これとは対照的に、浸潤性増殖は、典型的な病理学的特徴であり、そのような癌は通常、移行/コホートとして侵入する。グリア細胞つつ、第2、がんは、多くの場合、脳内の上皮構造を再構築しよう（擬似）カプセルによって脳組織から腫瘍を分離してみてください。生物学的および形態学的特徴を考慮し、悪性神経膠腫および癌腫の転移は実際に比較することはできません。これらの理由から、我々は、我々は修正された脳切片に隣接する癌細胞プラグを注入しかし共存しない共培養システムを開発した。さらに、本発明者らは、小膠細胞が腫瘍プラグを入力して、容易に、明視野顕微鏡法によって検出され、共焦点顕微鏡によって後で確認することができたことを意味し、腫瘍プラグの境界でミクログリア及び星状細胞の蓄積を観察した。癌細胞は、生体内の状況で実際に、脳は21の転移を有する剖検例の63％に浸透ゾーンを見つけBaumertらによって作ら観測に匹敵する脳切片の中に侵入する。 がないため血液灌流のために、この文化の中で唯一の常在マクロファージ/ミクログリアがあります。また、理由のM従って、T細胞を同理事と、アロ反応性は存在しない、ヒト癌細胞であっても免疫応答性マウス又はラットの脳切片（NMRI、B6、又はWistar系）を用いて共培養を使用することができる。これはヌードマウスモデルの代替となりうる。四時五十六のスライスが各マウスから得ることができるので、動物の数が有意に減少し、既存の注射モデルに比べて、必要とされる。また、動物は、転移性疾患を長期間受けない、それらは繰り返し22手術手順を受けない。 この共培養系では、我々は癌細胞によるミクログリアの活性化および癌細胞浸潤を促進する能力を実証した。さらに、これは初めてである、我々の知る限り、ミクログリアが活発癌細胞23を輸送することが見出された。 すべてのこれらの利点にもかかわらず、共培養システムは、確かに、また限界がある。それはまだ試験管内で前植民地化への転移のステップが欠落してモデル化する。なぜなら灌流の不足するのではなく、血管外漏出を勉強することはできません。したがって、溢出を研究する別の方法は、血液脳関門22,24を模倣するインビボ注入モデルまたは修飾Boydenチャンバーシステム外マトリックスと、HUVECおよびアストロサイトのいずれかを使用することである。脳切片の共培養法は、まだ多くの利点とそのような常在細胞の役割に焦点を当て、特に植民地化の分析などのアプリケーションのさまざまな可能性を有する信頼性と再現性のモデルです。 （このような免疫組織化学、共焦点顕微鏡およびタイムラプス顕微鏡のような）他の確立された技術との組み合わせは、直接的な細胞間相互作用の研究をサポートしています。それは簡単な代替及び他の技術の補完であり、転移性微小環境によって課されるようにキューと効果の調査へのアクセスを提供しています。

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco, Darmstadt,Germany</td>
			<td>24020</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco, Darmstadt , Germany</td>
			<td>32360</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>PAA Laboratories Inc., C&#246;lbe, Germany</td>
			<td>E15-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (PBS)</td>
			<td>Pan Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal horse serum (NHS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>10091148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>B. Braum, Melsungen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine-Penicillin-Streptomycin solution</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica SM 2000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane</td>
			<td>BD Falcon, Heidelberg,Germany</td>
			<td>353090</td>
			<td>transwell membrane insert (0.4 &#956;m pore size)</td>
		</tr>
		<tr>
			<td>ECM gel</td>
			<td>Trevigen, R&#38;D, Wiesbaden-Nordenstadt, Germany</td>
			<td>3432-005-01</td>
			<td>Basement membrane extract</td>
		</tr>
		<tr>
			<td>Metallic spacer</td>
			<td>Kig GmbG, Kirkel, Germany</td>
			<td>DIN 433</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-lapse microscope and camera</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>DMI 6000B microscope and a DFC 350 FX CCD camera</td>
			<td></td>
		</tr>
		<tr>
			<td>Anatomy microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>Stemi SV11</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1.04005.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG, F(ab&#39;)2- TRITC</td>
			<td>Santa Cruz, Heidelberg, Germany</td>
			<td>SC3796</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 647 conjugate</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>132450</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6&#39;-diamidino-2-phenylindole dihyfrochloride)</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent mounting medium</td>
			<td>DAKO, Glostrup, Denmark</td>
			<td>S3023</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture system with an organotypic brain slice and 3d spheroid of carcinoma cells

癌の脳転移を有する患者は予後不良である。しかし、転移部位の処理はほとんど、特に、居住者（間質）細胞の役割を調べた。原発癌の研究でも、予後1,2に、転移に対する微小環境の影響を示しています。特に腫瘍関連マクロファージ（TAM）のサポート移動、浸潤や増殖3。興味深いことに、転移の主要な標的部位は、CNSにおける肝臓やミクログリアにおけるクッパー細胞などの組織特異的マクロファージを保有。また、転移部位も、星状細胞のような他の組織特異的細胞を有する。最近では、アストロサイト、癌細胞4,5の増殖および持続性を促進することが示された。したがって、これらの組織特異的な細胞型の機能は、脳転移6,7の過程で非常に重要であると思われる。 これらの観​​察にもかかわらず、しかし、これまで生体内で/直接明視野顕微鏡で、特に脳転移形成の際にグリアの反応を可視化するために利用可能なin vitroモデルには適しているのはありません。癌細胞の生体ライブイメージングにおける最近は彼らの脳の植民地化の動作8を示 ​​した。しかし、この方法は非常に手間がかかりコストがかかり、技術的に複雑である。また、動物実験のこれらの種類は、小さな直列に制限されている実質的な（ガラス板の注入により、腫瘍細胞の注射、繰り返し麻酔と長期固定の）動物のためのストレスが付属しています。常在細胞との相互作用はまだ示されていないのに対し、また、in vivoイメージングは、これまでに、癌細胞の可視化に限定される。最後に、免疫担当動物内ヒト癌細胞の調査は8不可能です。 これらの理由から、我々は、共培養システムを確立しconsiマトリゲル（3Dセル球）に埋め込まれた器官型マウス脳スライスや上皮細胞のスティング。 3D癌細胞球体は、隣接する脳組織の浸潤を調べるために、脳切片縁部に直接隣接して配置した。これは蛍光であっても、明視野顕微鏡法によってグリア細胞と癌細胞との間の形態学的変化との相互作用を視覚化することを可能にします。共培養実験の後、脳組織または3Dスフェロイドは、さらに、分子分析（ 例えば、定量RT-PCR、IHC、または免疫ブロット）のため、ならびに共焦点顕微鏡による調査のために回収し、使用することができる。この方法は、脳切片に対して有害な影響なし日間生体脳組織内のイベントを監視するために適用することができる。モデルはまた、ドナー組織からの細胞による選択的抑制、住民細胞の置換は、与えられた遺伝子型の明確な影響を判断することができます。最後に、共培養モデルが実行可能な選択肢である目標と薬理操作をテストするときに、生体内へのアプローチ。

最初に、すべての使用癌細胞（ヒト：MCF-7およびマウス：410.4）は器官型マウス脳スライスに侵入した。浸潤は、種に依存しない方法で広範囲の用途を示しているので、種に依存しない（ 図1A）であった。さらに、ミクログリアだけでなく、これまで生体内および患者サンプル13で説明したように界面に蓄積アストロサイト。長期間にわたるタイムラプスイメージングは​​、スライスの実行可能性を明らかにしただけでなく、細胞間相互作用を観察するための良いプラットフォームを提供するだけでなく。次に、癌細胞はまた、脳切片（ 図1B1-B2）に侵入し、つつ、時間経過実験により、小グリア細胞は、3Dセル球体を入力して記録した。また、我々は、以前はそれにより癌の浸潤を補助するために、依然として謎の機構によって癌細胞を輸送するミクログリアの能力を示した。免疫蛍光標識技術を使用して、我々侵入プロセスの間、これらの細胞間の緊密な相互作用を示唆し、脳組織および腫瘍細胞プラグ（ 図1C-D）の両方において、腫瘍細胞および間質細胞の同時局在（ 例えば、ミクログリアおよびアストロサイト）に記載。異なる種の遺伝子型および株からの細胞を研究するための用途の広い範囲を示す-マウスの脳切片は、ヒト（ 図1C1-C3）またはマウス（ 図1D1-D3）癌細胞のいずれかで培養した場合に匹敵する結果が得られた。 <img alt="図1" src="/files/ftp_upload/50881/50881fig1.jpg" fo:content-width="4.75in" fo:src="/files/ftp_upload/50881/50881fig1highres.jpg" /> 図1。マウスとヒト癌細胞の3次元回転楕円体と脳切片共培養モデル。マウス乳癌器官全脳スライス共培養中の癌細胞の浸潤のA）の定量化細胞株410.4またはヒト乳癌細胞株MCF-7。データは、n≥38と、各グループ内の脳切片に細胞浸潤の程度の割合を表す。これら二つのグループ（クラスカルウォリス検定）の間に有意差は認められなかった。日2（B1）と5日目（B2）から、MCF-7-GFP細胞と代表画像との器官型全脳スライス共培養のB）タイムラプス画像がされました示す。CD）共焦点顕微鏡画像は、癌腫、アストロサイトとミクログリアのコホートを示した。二重星状細胞の染色（抗GFAP-TRITC、赤）とミクロ​​グリア350ミクロンの厚さの器官型脳切片およびGFP-トランスフェクトされた腫瘍細胞プラグ（緑色）と全体の共培養（ILB4-アレクサフルーア647、紫）：3D- MCF-7-GFP（C1-C3）および3D-410.4-GFP（D1-D3）。白いダッシュは脳切片と腫瘍の浸潤前線の端を示した。スケールバーは50μmで表す。ミクログリア、アストロサイトAをNDの腫瘍の共局在（C1およびD1）、アストロサイト-の腫瘍共局在（C2およびD2）とスライス共培養におけるミクログリア腫瘍共局在（C3とD3）。 <a href="https://www.jove.com/files/ftp_upload/50881/50881fig1highres.jpg" target="_blank">拡大画像を表示するには、ここをクリックしてください</a>

Watch this Scientific Journal Video about Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells at JoVE.com

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

癌細胞との器官型脳切片の共培養は、脳組織の癌細胞浸潤の過程で蛍光だけでなく、明視野（ビデオ）顕微鏡によって形態学的変化を可視化することができます。このモデル系は、細胞の交換や補充的なアプローチを可能にし、操作及び分析を幅広く提供しています。

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

coculture-system-with-an-organotypic-brain-slice-3d-spheroid

Research

JoVE Journal

Medicine

20.6K ビュー.  University of Göttingen. 癌細胞との器官型脳切片の共培養は、脳組織の癌細胞浸潤の過程で蛍光だけでなく、明視野（ビデオ）顕微鏡によって形態学的変化を可視化することができます。このモデル系は、細胞の交換や補充的なアプローチを可能にし、操作及び分析を幅広く提供しています。 

ビデオ: Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6Jマウスにおける間質細胞と癌細胞の乳腺移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

医学

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 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

リアルタイムで中皮細胞の卵巣癌スフェロイドアタッチメントおよび浸潤の評価

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一次多発性骨髄腫細胞の薬剤感受性の特徴付けのための器官のハイスループットシステム

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

腫瘍微小環境と標的薬物療法研究のための脳の腫瘍/器官スライス共培養系

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

3Dの設定で侵略を評価するために、癌細胞スフェロイドアッセイ

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

腫瘍細胞移動の研究および抗浸潤性薬物の患者特異的効果の研究のためのヒト神経膠芽腫の器官型スライス培養モデル

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

対称Bihemispheric死後脳人間に健康と病理学的脳条件を研究するためにカッティング

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.