Name: three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation
Uploaded: 2016-08-18T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

注：いいえ倫理考慮動物およびヒトの癌細胞​​が購入または親切に私たちに提供されたので。 1.コラーゲンプラグ（擬似原発腫瘍）の作成<ol><li>コラーゲン分散液を準備します。以前に17を報告した 、または購入として私は、ラットの尾の腱（RTT）から型コラーゲンを抽出し、滅菌することができるのいずれか。 （; 5 2分の実行高速設定）均一な混合のために凍結乾燥したRTTコラーゲン（0.02 N酢酸で3.25から3.50 mg / mlで）ブレンダーを使用して分散させます。 </li><li>ハーベスト（トリプシン-EDTA、通常）とは、血球計数器を使用して生細胞をカウントするためのトリパンブルー排除を使用します。所望の細胞密度（プラグあたり5×10 4細胞）に調整します。 </li><li>無菌条件下で別々に全ての溶液（NaOHを、ウシ胎児血清、DMEM 5倍、 炭酸水素ナトリウム）（ 表1）を準備し、氷上で冷やしておきます。注：種々の溶液の添加の順序は、細胞内の浸透圧または酸性ショックを防止することが重要です。 </li><li>可能な限り迅速に最終コラーゲン溶液（5ミリリットル）に細胞分散（1.25×10 6細胞）を実行します。気泡を避けながら（上下にピペッティングすることによって）十分に混合し、その後すぐに96ウェルプレートの各ウェルにすぐに使用できる溶液200μlを分配します。穏やかに気泡を除去するために、ウェルの内部に均一に溶液を広げるために、細胞培養フードの作業領域表面上のマルチウェルプレートに衝突します。 </li><li> （このステップは96ウェルプレート当たり約15〜20分かかります）すべてのウェルを充填した後、インキュベーターに格納します。 </li><li>一晩に2時間から37℃でプレートをインキュベートします。コラーゲンゲル化（ すなわち 、原線維）は30分以内に起こります。一晩のインキュベーションを行うために培養し（100μl/ウェル）、培地を追加します。 </li></ol>フィブリンゲルの2第一層<ol><li>フィブリノーゲン溶液の調製。 注：フィブリンゲルの同じバッチは、理想的には、より再現resulのために使用されるべきですフィブリンゲル形成などのTSは、商業的な凍結乾燥フィブリノーゲンの異なるバッチ間で変化してもよいです。 <ol><li>常に新たに調製したフィブリノゲン溶液を使用します。水和物結晶の形成を回避するために、バイアルを開く前に室温に凍結乾燥したフィブリノーゲンを持参してください。 </li><li>徐々に（最低限必要な最終容量の15％超過を調製考える3 mg / mlでの作業濃度での Ca 2+ / Mgの2+ハンクス平衡塩溶液（HBSSに）予め温めた（37℃）でフィブリノゲンを溶解： 例えば 、5ミリリットルソリューションの5.75ミリリットルで17.25 mg）を。 <ol><li>フィブリノゲン断片を可溶化するために最初に予め温めたHBSSの滴下を追加します。ビーカー内のへらでより大きなフラグメントを打破します。混合を容易にするために、随時ビーカーを攪拌します。手順中の攪拌プレートを使用しないでください。サスペンションの上下にピペッティングすることによって、残りの粉末を溶かします。 </li></ol></li><li> steriながら生ぬるいフィブリノゲン溶液を保ちます0.22μmのフィルターを通すことにより、溶液をlizing。注：HBSSが十分に暖かいではないか、フィブリノゲンが完全に溶解していない場合は、解決策は、フィルターを詰まらせることがあります。該当する場合は、フィブリノーゲン濃度を低下させ、ひいてはフィブリン血餅剛性もよく、一度か二度フィルターを交換してください。 </li><li>別の方法として、その最終的な音量を調整しながら、すぐに使用できるフィブリノーゲン溶液に、目的とする細胞（ 例えば 、内皮細胞）をサスペンド。 </li></ol></li><li>トロンビン溶液を準備します。 <ol><li> ddH 2 O（50 NIH単位/ ml）の原液を調製し、その後、0.22μmのフィルターを使用して滅菌します。 </li><li>フィブリンゲルを生成するために0.0075（V / V）：フィブリノーゲン/トロンビン比≥1使用。 </li></ol></li><li>フィブリンゲルを生成します。 <ol><li>無菌フィブリノゲンを維持し、すべての次のステップの間、氷上でストック溶液をトロンビン。フィブリンゲルは、24ウェルプレート中に形成させました。 </li><li>速やかにオーバーレイ番目フィブリノゲン溶液（200μL/ウェル）で各ウェルのE面の気泡の形成を回避しつつ。一度に6ウエルを処理します。 </li><li>フィブリノゲン溶液は、完全に、ウェルの表面を覆って、45°の角度でプレートを傾け、ウェルの中心にトロンビンをドロップすることにより、第1のウェルにトロンビン溶液の1.5μlを添加し、その後緩やかにするために、水平プレートを旋回したら1-2秒。 <ol><li>層流フード（5〜10分）、ゲル化/凝固プロセスが完了するまで下に安定した位置にプレートを残す（NB：重合プロセスは、インキュベーターにプレートを輸送することにより、 例えば 、邪魔してはいけません）。 </li></ol></li><li>最初の6ウェルを重合した後、全てのウェルが処理されるまで、次の6ウェルについて同じ配列（ すなわち 3前の手順）を繰り返します。 </li></ol></li></ol>フィブリンゲルとに挟まれたコラーゲンプラグの3第2層<ol><li>オプションA：（すぐにコラーゲンプラグを使用）。 <ol><li>フィブリンゲルの第1層は微妙にプレートを傾けることによって、すべてのウェル中で重合していることを確認します。コラーゲンプラグの移動を容易にする（フィブリンゲルを含む）を24ウェルプレートで並んでコラーゲンゲルプラグの側面を含む96ウェルプレートを置き。 </li><li>コラーゲンプラグを含むプレートの各ウェルにHBSSの1滴を追加します。 </li><li>薄い（ハンドルとして使用）、注射器に取り付けられた針または（ビデオを見る）マイク​​ロスプーンを使用してウェルから各コラーゲンプラグを取り外します。コラーゲンプラグがウェルにセンタリングされ、その無菌性が良好に維持されていることを確認しながら、1または2つのマイクロスプーンを使用して第1のフィブリンゲル層の上に、各コラーゲンプラグを転送します。 </li><li>フィブリノゲン溶液の第二の層と、以前に形成されたフィブリンゲルをオーバーレイ（300μL/ウェル）、2.3で説明したようにトロンビンを導入し、最小限の1を維持：一度に0.0075比と6ウェルのシーケンスを&lt;/李&gt; </ol></li><li>オプションB（成長因子低減基底膜（GFRBM）の薄層でコラーゲンプラグをコーティング）。 <ol><li>事前にすべての準備ソリューションや楽器を冷却し、GFRBMの凍結アリコートを解凍中に過度の加熱速度に非常に敏感であるため、取り扱い時に（ 例えば 、ピペット、チップ、試験管）4℃または氷上で保管してください（メーカーの指示に従ってください） 。 </li><li>プレートのウェルから除去した後、純粋なGRFBM溶液100μlを含む氷上で1.5 mlの遠心管中で2分間、各コラーゲンプラグを浸します。 </li><li>前述したように、それは、よく中心で確保しつつ、第1のフィブリン層の上にそれぞれコーティングされたプラグを転送します。 GRFBMがゲルを形成することができるように5分間37℃で、プラグを含むプレートをインキュベート。ステップ3.1.4でのように、第2のフィブリン層を追加します。 </li></ol></li></ol> 4.細胞培養培地条件<ol><li> 4（培養液で各ウェルを充填00μl）を。培地および補充物は、細胞株および実験条件に基づいて選択されます。 </li><li> 100カリクレイン阻害剤単位（KIU）/ mlの最終濃度で培養培地へのアプロチニン、抗線維素溶解剤を追加します。 注：試験した細胞株に使用される条件の下で細胞培養インキュベーターでプレートを保管してください。 </li><li>一日おきに新鮮な培地で培養液を補充するか、実験的なスケジュールに従って、およびアプロチニンを追加します。新鮮な培地を追加する前に、わずかに（30-35°の角度で）プレートを傾け、慎重に一定の観察下に馴化培地を吸引しながら、ウェルの側面にピペットを傾け。 </li></ol>

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The authors have no disclosure.

重要な技術脚注として、ギャップは、中枢および末梢ゲルとの間の界面に存在しないことが必須です。それ以外の場合は、フィブリンゲルに侵入/移行する細胞の能力を低下させる可能性があります。トロンビンは、適切に希釈されていない場合、コラーゲンとフィブリンゲルとの間の空間は、培養の最初の24時間の間に形成してもよいです。試験した細胞株は、それによって両方のゲルの間?...

癌細胞の浸潤/遊走およびその後の転移の確立の基本的および生物医学的特性を調査することは熱心な研究1,2の主題です。転移は、がんの究極の段階であり、その臨床管理はとらえどころのないままになります。細胞および分子レベルでの転移のより良い理解は、より効率的な治療法3の開発を可能にします。 転移性細胞のいくつかの特性は、それらの幹細胞性と移行し、内と原発腫瘍5から侵入する遷移状態（ 例えば 、類上皮間葉移行）を取得する可能性を含む試験管 4 で検討することができます。それは事実上の血液/リンパ循環の寄与を除外しかし、浸潤/転移過程のin vitro評価は、課題でした。コラーゲンゲルに腫瘍断片を埋め込む器官培養物previou有しますずる癌の攻撃性をモニターするために使用されて。腫瘍の複雑さ（ 例えば 、非癌性細胞の存在）を保持しているが、腫瘍断片を、制限された培地の拡散にさらされている、サンプリング変化、および間質細胞6の異常増殖です。別の方法は、三次元（3D）細胞環境を模倣する細胞外マトリックス（ECM）の構成要素内の癌細胞を成長させることにあります。コラーゲンゲルおよび/または基底膜由来のマトリックス中の乳癌細胞株の増殖は、3次元細胞培養物の最もよく特徴づけ実施例の中です。特定の三次元細胞培養環境を使用して、標準的な条件下で増殖させた乳癌細胞について観察された無秩序なアセンブリは、乳腺腺房と管状構造7-10の自発的形成を逆転させることができます。また、腺癌細胞由来の多腫瘍スフェロイドの形成は、（別の技術を使用して集まっ例えば、滴、浮遊スフェロイド、寒天埋め込 ​​み）を吊りは現在最も一般的に使用される三次元細胞培養アッセイ11-13を構成しています。しかし、このアッセイは、スフェロイドを形成することができる癌細胞株の制限されたセットによってこれらの条件で細胞を研究するために利用可能な短い期間によって制限されます。 この視覚化技術では、我々は、本明細書中に関心の癌細胞は、代わりに基底膜由来のマトリックスで被覆することができる擬似原発腫瘍のインビトロ形成を可能にするために、コラーゲンゲル中に包埋された洗練された三次元細胞培養アッセイ法を導入します。一旦形成されると、擬似原発腫瘍はその後、癌細胞は、2つの行列の区画との間の界面を横断することを可能にする無細胞マトリックス（ここでフィブリンゲル）に挟まれている（ 図1参照）。興味深いことに、攻撃的な癌細胞と共に擬似原発腫瘍に由来する二次性腫瘍のような構造は、で表示されフィブリンゲル。このような三次元培養系は、例えば、抗癌剤、遺伝子発現および細胞-細胞および/ ​​または細胞-ECM相互作用14-16、調査に必要な柔軟性を提供します。 <img alt="図1" src="/files/ftp_upload/54322/54322fig1.jpg" /> 図1： メソッドの概要癌研究のためのモデルとして、3D細胞培養系を生成するための方法の概略要約<a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.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>

<table><tbody><tr><td>Freeze-dried collagen</td><td>Sigma-Aldrich</td><td>C7661</td><td>from rat tail tendon (soluble dispersion) or home-made (see Rajan &lt;i&gt;et al&lt;/i&gt;., ref.#14)</td></tr><tr><td>Fibrinogen (freeze-dried)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F8630</td><td>Type I-S, 65 - 85% protein with &amp;ge; 75% of protein is clottable</td></tr><tr><td>Thrombin</td><td>EMD Chemicals Inc.</td><td>605157</td><td>&amp;nbsp;Gibbstown, NJ; NIH units/mg dry weight&amp;nbsp;</td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</td><td>Corning</td><td>356234</td><td>Previously from BD Biosciences</td></tr><tr><td>Aprotinin</td><td>Sigma-Aldrich</td><td>A6279&amp;nbsp;</td><td>&amp;nbsp;solution at 5 - 10T IU/ml (Trypsin Inhibitor Unit)&amp;nbsp;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Micro-spoons</td><td>Fisher Scientific</td><td>2140115</td><td>Fisherbrand Handi-Hold Microspatula</td></tr><tr><td>96 well plate, round base</td><td>Sarstedt</td><td>3925500</td><td></td></tr><tr><td>24 well plate</td><td>Sarstedt</td><td>3922</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s Medium</td><td>Sigma Chemical, Co.</td><td>D5546</td><td>DMEM</td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>CAA15-701</td><td>FBS, Canadian origin.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma Chemical, Co.</td><td>T4049</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>Sigma Chemical, Co.</td><td>H8264</td><td>HBSS</td></tr></tbody></table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

単層での哺乳動物細胞の培養は、広く様々な生理学的および分子プロセスを研究するために使用されます。しかし、成長細胞を研究するために、このアプローチは、多くの場合、不要なアーティファクトを生成します。したがって、多くの場合、細胞外マトリックス成 ​​分を用いた3次元（3D）環境内での細胞培養は、によりインビボ組織または臓器において天然にその近い類似に興味深い代替として登場しました。我々は、フィブリンゲルからなる疑似一次macrospherical腫瘍および（ii）の周辺の無細胞区画としてコラーゲンゲル作用に埋め込 ​​まれた癌細胞を含む中心コンパートメント、二つの区画、すなわち（i）を用いて、三次元細胞培養システムを開発しましたすなわち、中心部に使用されるものとは異なる細胞外マトリックス成 ​​分は、ここで癌細胞が遊走する（浸潤フロント）および/ ​​または第二またはサテライト腫瘍を表す微小球腫瘍を形成することができます。末梢コンパートメント内の衛星腫瘍の形成であります著しく、この3D培養系は、ユニークなネイティブの腫瘍細胞の既知の攻撃性または転移性の起源、と相関。この細胞培養アプローチは、癌細胞の浸潤性および運動性、細胞 - 細胞外マトリックス相互作用と抗がん剤の特性を評価する方法としてを評価するために考慮されるかもしれません。

前述したように、この三次元細胞培養アッセイの興味深い特徴は、癌細胞は、隣接するフィブリンゲルにコラーゲンプラグからの移行だけでなく、二次性腫瘍（ 例えば 、衛星腫瘍様構造）を確立することができないだけです。これは、直接、特に長い作動距離コンデンサ（ 図2）を用いて、ゲルの厚さを通って低及び高倍率で倒立位相差顕微鏡を?...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

癌細胞の浸潤や衛星腫瘍形成を探検するための三次元培養アッセイ

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

Research

JoVE Journal

Medicine

10.3K ビュー.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

ビデオ: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF -β誘発性浸潤を測定する回転楕円体アッセイ

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

医学

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

侵略を勉強し、膀胱癌の治療を評価するための 3次元細胞培養システム

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

がん研究

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

3D培養におけるがん細胞浸潤とT細胞毒性のモニタリング

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

腫瘍間質相互作用のための三次元共培養モデル

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

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 ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌細胞のスキャフォールドフリー3D培養におけるヘテロ多細胞間質細胞による浸潤の促進

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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 ...

3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

Source: Cvetkovi&#263;, D., et al. <a href="https://www.jove.com/v/51341/quantification-breast-cancer-cell-invasiveness-using-three">Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model.</a> J. Vis. Exp. (2014).
This video provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment.

3D培養基底膜アッセイ:3D基底膜タンパク質マトリックス上でがん細胞を培養し、細胞浸潤を研究する

Source: Cvetkovi&#263;, D., et al. Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model. J. Vis. Exp. (2014).
This video ...

JoVE Encyclopedia of Experiments

乳がん

試験管研究

3-Dimensional Culture of Lung Carcinoma Cells: A Method To Study Cell-Matrix Interactions

Source: Chen, S. et. al. <a href="https://www.jove.com/v/60644/multidimensional-coculture-system-to-model-lung-squamous-carcinoma">Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression</a>. J. Vis. Exp. (2020).
This video describes the technique of 3-D culture of lung carcinoma cells to study the cell-matrix interactions. In the protocol, we will perform the 3D culture of TUM622 lung carcinoma cells to explore its potential of forming organoids in vitro.

肺癌細胞の3次元培養:細胞-マトリックス相互作用を研究する方法

Source: Chen, S. et. al. Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression. J. Vis. Exp. (2020).
This video describes the ...