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>

<ol>
	<li>Alizadeh, A. M., Shiri, S., Farsinejad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+review:+from+bench+to+bedside.">Metastasis review: from bench to bedside.</a> Tumour Biol. 35 (9), 8483-8523 (2014).</li><li>Roudsari, L. C., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+influence+of+angiogenesis+in+in+vitro+cancer+model+systems.">Studying the influence of angiogenesis in in vitro cancer model systems.</a> Adv Drug Deliv Rev. , (2016).</li><li>Bill, R., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+EMT+in+breast+cancer+metastasis:+Correlation+or+causality.">The relevance of EMT in breast cancer metastasis: Correlation or causality.</a> FEBS Lett. 589 (14), 1577-1587 (2015).</li><li>Kimlin, L. C., Casagrande, G., Virador, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Three-Dimensional+(3D)+Models+in+Cancer+Research:+an+Update.">In Vitro Three-Dimensional (3D) Models in Cancer Research: an Update.</a> Mol Carcinog. 52 (3), 167-182 (2013).</li><li>Obenauf, A. C., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+at+a+Distance+Organ-Specific+Metastasis.">Surviving at a Distance Organ-Specific Metastasis.</a> Trends Cancer. 1 (1), 76-91 (2015).</li><li>Sykes, J. A., Fogh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+Tumor+Cells+from+Fibroblasts.">Separation of Tumor Cells from Fibroblasts.</a> Human Tumor Cells In Vitro. 1, 1-22 (1975).</li><li>Lang, S. H., Stark, M., Collins, A., Paul, A. B., Stower, M. J., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Prostate+Epithelial+Morphogenesis+in+Response+to+Stroma+and+Three-Dimensional+Matrigel+Culture.">Experimental Prostate Epithelial Morphogenesis in Response to Stroma and Three-Dimensional Matrigel Culture.</a> Cell Growth Differ. 12 (12), 631-640 (2001).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+Oncogenesis+of+MCF-10A+Mammary+Epithelial+Acini+Grown+In+Three-Dimensional+Basement+Membrane+Cultures.">Morphogenesis and Oncogenesis of MCF-10A Mammary Epithelial Acini Grown In Three-Dimensional Basement Membrane Cultures.</a> Methods. 30 (3), 256-268 (2003).</li><li>Shaw, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+invasion+assays.">Tumor cell invasion assays.</a> Methods Mol. Biol. 294, 97-105 (2005).</li><li>Nelson, C. M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Dynamic+Reciprocity:+Engineering+Three-Dimensional+Culture+Models+of+Breast+Architecture,+Function,+and+Neoplastic+Transformation.">Modeling Dynamic Reciprocity: Engineering Three-Dimensional Culture Models of Breast Architecture, Function, and Neoplastic Transformation.</a> Semin Cancer Biol. 15 (5), 342-352 (2005).</li><li>Hedlund, T. E., Duke, R. C., Miller, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Spheroid+Cultures+of+Human+Prostate+Cancer+Cell+Lines.">Three-Dimensional Spheroid Cultures of Human Prostate Cancer Cell Lines.</a> Prostate. 41 (3), 154-165 (1999).</li><li>Le, V. M., Lang, M. D., Shi, W. B., Liu, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Collagen-Based+Multicellular+Tumor+Spheroid+Model+for+Evaluation+of+the+Efficiency+of+Nanoparticle+Drug+Delivery.">A Collagen-Based Multicellular Tumor Spheroid Model for Evaluation of the Efficiency of Nanoparticle Drug Delivery.</a> Artif. Cells Nanomed Biotechnol. 15, 1-5 (2014).</li><li>Neto, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Hanging+Spherical+Drop+System+for+the+Generation+of+Cellular+Spheroids+and+High+Throughput+Combinatorial+Drug+Screening.">A Novel Hanging Spherical Drop System for the Generation of Cellular Spheroids and High Throughput Combinatorial Drug Screening.</a> Biomater Sci. 3 (4), 581-585 (2015).</li><li>Janvier, R., Sourla, A., Koutsilieris, M., Doillon, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+Fibroblasts+are+Required+for+PC-3+Human+Prostate+Cancer+Cells+to+Produce+Capillary-like+Formation+of+Endothelial+Cells+in+a+Three-dimensional+Co-culture+System.">Stromal Fibroblasts are Required for PC-3 Human Prostate Cancer Cells to Produce Capillary-like Formation of Endothelial Cells in a Three-dimensional Co-culture System.</a> Anticancer Res. 17 (3A), 1551-1557 (1997).</li><li>Doillon, C. J., Gagnon, E., Paradis, R., Koutsilieris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Culture+System+as+a+Model+for+Studying+Cancer+Cell+Invasion+Capacity+and+Anticancer+Drug+Sensitivity.">Three-dimensional Culture System as a Model for Studying Cancer Cell Invasion Capacity and Anticancer Drug Sensitivity.</a> Anticancer Res. 24 (4), 2169-2177 (2004).</li><li>Gobeil, S., Zhu, X., Doillon, C. J., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-Wide+shRNA+Screen+Identifies+GAS1+as+a+Novel+Melanoma+Metastasis+Suppressor+Gene.">A Genome-Wide shRNA Screen Identifies GAS1 as a Novel Melanoma Metastasis Suppressor Gene.</a> Genes Dev. 22 (21), 2932-2940 (2008).</li><li>Rajan, N., Habermehl, J., Cot&#233;, M. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+Of+Ready-To-Use,+Storable+And+Reconstituted+Type+I+Collagen+From+Rat+Tail+Tendon+For+Tissue+Engineering+Applications.">Preparation Of Ready-To-Use, Storable And Reconstituted Type I Collagen From Rat Tail Tendon For Tissue Engineering Applications.</a> Nat Protoc. 1 (6), 2753-2758 (2007).</li><li>Horie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Human+Lung+Cancer-associated+Fibroblasts+in+Three-dimensional+In+Vitro+Co-culture+Model.">Characterization of Human Lung Cancer-associated Fibroblasts in Three-dimensional In Vitro Co-culture Model.</a> Biochem Biophys Res Commun. 423 (1), 158-163 (2012).</li><li>Banyard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Genes+Regulating+Migration+and+Invasion+Using+a+New+Model+of+Metastatic+Prostate+Cancer.">Identification of Genes Regulating Migration and Invasion Using a New Model of Metastatic Prostate Cancer.</a> BMC Cancer. 30 (14), 387 (2014).</li><li>Palumbo, J. S., Degen, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+Tumor+Cell+Metastasis.">Fibrinogen and Tumor Cell Metastasis.</a> Haemostasis. 31, 11-15 (2001).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Stroma,+Tumor+Blood+Vessels,+and+Antiangiogenesis+Therapy.">Tumor Stroma, Tumor Blood Vessels, and Antiangiogenesis Therapy.</a> Cancer J. 21 (4), 237-243 (2015).</li><li>Luoto, K. R., Kumareswaran, R., Bristow, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Hypoxia+as+a+Driving+Force+in+Genetic+Instability.">Tumor Hypoxia as a Driving Force in Genetic Instability.</a> Genome Integr. 4 (1), 5 (2013).</li><li>Das, V., Bruzzese, F., Kone&#269;n&#253;, P., Iannelli, F., Budillon, A., Hajd&#250;ch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+In+Vitro+Tumor+Models+for+Drug+Screening.">Pathophysiologically Relevant In Vitro Tumor Models for Drug Screening.</a> Drug Discov Today. 20 (7), 848-855 (2015).</li><li>Longati, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Pancreatic+Carcinoma+Spheroids+Induce+a+Matrix-rich,+Chemoresistant+Phenotype+Offering+a+Better+Model+for+Drug+Testing.">3D Pancreatic Carcinoma Spheroids Induce a Matrix-rich, Chemoresistant Phenotype Offering a Better Model for Drug Testing.</a> BMC Cancer. 13 (95), (2013).</li><li>Tan, P. H., Chia, S. S., Toh, S. L., Goh, J. C., Nathanm, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Spatial+Configuration+of+Tumour+Cells+Confers+Resistance+to+Chemotherapy+Independent+of+Drug+Delivery.">Three-dimensional Spatial Configuration of Tumour Cells Confers Resistance to Chemotherapy Independent of Drug Delivery.</a> J Tissue Eng Regen Med. , (2013).</li><li>Koutsilieris, M., Reyes-Moreno, C., Choki, I., Sourla, A., Doillon, C., Pavlidis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+Cytotoxicity+of+Human+MCF-7+and+MDA-MB+231+Breast+Cancer+Cells+is+Altered+by+Osteoblast-Derived+Growth+Factors.">Chemotherapy Cytotoxicity of Human MCF-7 and MDA-MB 231 Breast Cancer Cells is Altered by Osteoblast-Derived Growth Factors.</a> Mol Med. 5 (2), 86-97 (1999).</li><li>Lang, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biphasic+Response+of+Cell+Invasion+to+Matrix+Stiffness+in+Three-Dimensional+Biopolymer+Networks.">Biphasic Response of Cell Invasion to Matrix Stiffness in Three-Dimensional Biopolymer Networks.</a> Acta Biomater. 13, 61-67 (2015).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 et al., ref.#14)</td>
		</tr>
		<tr>
			<td>Fibrinogen (freeze-dried)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F8630</td>
			<td>Type I-S, 65-85% protein with &#8805;75% of protein is clottable</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>EMD Chemicals Inc.</td>
			<td>605157</td>
			<td>&#160;Gibbstown, NJ; NIH units/mg dry weight&#160;</td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Previously from BD Biosciences</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma-Aldrich</td>
			<td>A6279&#160;</td>
			<td>&#160;solution at 5-10TIU/ml (Trypsin Inhibitor Unit)&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;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&#39;s modified Eagle&#39;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&#8217;s Balanced Salt Solution&#160;</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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

個々の腎細胞がんの中のタンパク質発現変動を探るために逆相タンパク質アレイ（RPPA）の使用

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

術後免疫抑制のメカニズムを探索し、新しい周術期免疫療法を評価するための手術侵襲のマウス腫瘍モデル

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三次元文化：乳細胞の悪性形質転換正常対腺房アーキテクチャを研究するためのツール

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

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 ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

ヒト乳癌組織および細胞株からマンモスフェア形成アッセイ

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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 ...