MBは、MRCの交わりから助成金によってサポートされています。 KPとAHMはウェセックス医学研究および癌研究イギリス/ RCS（イングランド）（C28503/A10013）からの助成金によってサポートされています。我々はサウサンプトン組織化学研究ユニットの大学の支援に感謝しています。

結腸外植片からの初代線維芽細胞培養の1。設立<ol><li>外科手術室から直接、正常なヒト結腸粘膜組織のサンプルを得るし、PBS 7-10中に一時停止し、100単位/ mlペニシリン、100μg/ mlのストレプトマイシン、および0.25μg/ mlのファンギゾンを補った。 </li><li>研究室では、10cmの組織培養皿の中央に試料を置き、PBS /ペンストレプトマイシン/ファンギゾンで3回洗浄する。最後の洗浄の後に吸引しないでください。 </li><li>滅菌ピンセットやメスで、（約2mm）組織を小片にカットし、12ウェルプレート中のメスの刃を使用して描画十字の中心にそれぞれの作品を配置します。それが接着することができるように組織の上そっとメスを揺する。 </li><li> 750μlの培養中の検体は、一次線維芽細胞増殖培地submeべき（DMEM、20％ウシ胎児血清（FCS）、100 U / mlペニシリン、100μg/ mlのストレプトマイシン、および292μg/ mlのL-グルタミンを補充）フロートするためにそれを可能にすることなく、組織をRGE。 2日ごとに再給紙を除き、次の5日間にわたりプレートを邪魔しないようにします。 </li><li>次の5日間に線維芽細胞が徐々に組織の端から外に成長を開始します。約7日後に、これはより見えるようになります。後約3〜4週間、内外の成長に十分な線維芽細胞が存在する場合、各ウェルに750μlのトリプシンを追加します。細胞を、T-25組織培養フラスコに、プール、転移（5〜10分かかることがあります）取り外された後。 </li></ol> 2。器官の準備線維芽細胞を含浸させ、器官ゲルの調製： <ol><li> 10％FCSおよび292μg/ mlのL-グルタミンを補充したDMEM中の器官当たり5×10 5個の細胞を含む線維芽細胞懸濁液を調製する。 </li><li>以下の比率で、氷上器官ゲルを構成している。 <ul><li>ラットテールコラーゲンの7ボリューム：マトリゲルミックス（1:1） </li><li>フィルタリングさ10倍のDMEM 1ボリューム</li><li> FCSの1の体積</li><li> 5×10 5個の線維芽細胞を含む線維芽細胞懸濁液1容量 9ゲル（ゲルあたり1ミリリットル）のために10ミリリットルを用意し、気泡を避けるために、穏やかに混合。 </li></ul></li><li> 24ウェルプレートの各ウェルに1 mlを添加し、37℃で1時間インキュベート ゲルが設定できるように加湿した雰囲気の中でCと°。 1時間後、ゲルの上に1ミリリットルのDMEM（10％FCS）/供給過剰を追加し、一晩培養器に戻る。 </li><li>次の日、ゲルおよび5×10 5のCRC上皮細胞を含む培地のプレート1ミリリットルの上から吸引媒体。 </li></ol>ゲルコーティングされたナイロンシートの製造： <ol start="5"><li>事前に1.5センチメートル×1.5センチメートルを測定無菌、オートクレーブナイロンシートを準備します。 </li><li>滅菌ピンセットを用いて、10cmの培養皿（通常は4ナイロンシートを皿ごとに収容することができる）ナイロンシートの必要な数を配置する。 </li><li>次の順序で氷上ゲルを構成する（250μlの総ボリュームナイロンシートごとに必要となる）。 <ul><li>ラットテールコラーゲンの7容積</li><li>フィルタリングさ10倍のDMEM 1ボリューム</li><li> FCSの1の体積</li><li> 1体積のDMEM、10％FCS /グラット</li></ul></li><li>溶液が黄色の場合は、溶液がピンク色に変わるまで50μlのアリコートで0.1MのNaOHを加えることによって中和する。 </li><li>各ナイロンシートに、この溶液の250μlを加え、37℃でインキュベート °Cゲルが設定できるように30分間、5％CO 2の加湿雰囲気中。 </li><li> PBS中の1％グルタルアルデヒド溶液を構成しています。ゲルがセットされた後、各10cmの培養皿に10ミリリットルを加え、1時間4℃でインキュベートする。 </li><li>ウォッシュナイロンシートはPBSおよびDMEM中で1X（10％FCS）/グルタミン酸で3倍、4℃で、DMEM / Gluの中で一晩インキュベート</li></ol>侵入用スチールグリッド上に器官ゲルを上げる。 <ol start="12"><li>三脚の形成にステンレス鋼板を折り曲げて足場グリッドを事前に準備する。使用前にオートクレーブ。</li><li> 6ウェルプレートの各ウェルにスチールグリッドを配置し、エタノールで滅菌ピンセットを使用してください。 </li><li>各グリッド上に、コラーゲン側最上でゲルコーティングされたナイロンシートを置きます。 </li><li>慎重にエタノールで滅菌スパチュラを用いて上げたナイロンシートを24ウェルプレートからの器官のゲルを移す。 </li><li>ナイロンシートが媒体に接触しているが、水没しないまで、10％FCSを補充したDMEM、10％FCS /ウェルで供給過剰を埋める。媒体は器官ゲルに接触していないことを確認してください。 </li><li> 2日毎に培地を交換し、5％CO 2の加湿雰囲気中、37℃で14日間インキュベートする。 </li></ol> 3。器官固定<ol><li>井戸からナイロンシートを含む全器官を取り出して、ラップフィルム上に置きます。 </li><li>器官ときれいな使い捨てメスを用いてナイロンシートを二等分し、室温で24時間、ホルムアルデヒド中で両方の半分を修正。 </li><li> 70％のイーサンとホルムアルデヒドを交換してくださいオール24時間後と前に、パラフィンに包埋切片、および染色を一晩のままにしておきます。 </li></ol>侵略的マージンの4。レーザーマイクロダイセクション<ol><li>膜上10μmの厚さにセクションでは、スライドをマウントした。 </li><li> 1分間のキシレンを適用することにより脱パラフィン切片、次いでキシレンを除去し、さらに1分間、75％エタノールで固定する。 </li><li> 1分間0.125％クレシルバイオレット溶液でエタノールと汚れを取り除きます。クレバイオレットは、上皮細胞が強調表示され、間質から簡単に区別することを可能にする。 </li><li>クレシルバイオレットを除去し、100％エタノールでリンスする前にさらに1分間100％エタノールを加える。 30分間空気乾燥スライドを可能にします。 </li><li>選択したプラットフォーム（ 例えばライカASシステム）を用いたレーザーマイクロダイセクションを実施しています。 </li><li>顕微鏡ステージ上に下向き顕微解剖するために染色部をガラススライドを置きます。 </li><li>回収カセットに0.5ミリリットルマイクロチューブをマウントし、50μlを加える顕微解剖組織収集先となるキャップの細胞溶解緩衝液（ 例えば、DNA、RNAまたはタンパク質溶解緩衝液）。 </li><li>直視下、関心のある組織を同定するためにジョイスティックを使用して、ソフトウェア·インタフェースを使用して、腫瘍の浸潤先端部での顕微解剖するセルを強調表示する、染色された有機部に注釈を付ける。 </li><li>強調表示されたセクションを切り出し、マイクロチューブのキャップ内に顕微解剖した組織を推進する必要があり、どちらも、発射するレーザーを指示します。 </li><li>一旦十分な材料が収集され、収集カセットを取り出すマイクロ遠心チューブを閉じ、チューブの底に溶解緩衝液を描画するために穏やかに回す。 </li><li>マイクロダイセクションが完了するまで氷上にサンプルを置きます。適切な方法による処理サンプルは、目的の分析物を抽出する。 </li></ol>

<ol>
	<li>Nystrom, M. L., Thomas, G. J., Stone, M., Mackenzie, I. C., Hart, I. R., Marshall, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J Pathol. 205 (4), 468-475 (2005).</li><li>Zhang, L., 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=miR-153+supports+colorectal+cancer+progression+via+pleiotropic+effects+that+enhance+invasion+and+chemotherapeutic+resistance.">miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance.</a> Cancer Res. , (2013).</li><li>Bullock, M. D., 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=Pleiotropic+actions+of+miR-21+highlight+the+critical+role+of+deregulated+stromal+microRNAs+during+colorectal+cancer+progression.">Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.</a> Cell Death Dis. 20 (4), (2013).</li><li>Jenei, V., Nystrom, M. L., Thomas, 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=Measuring+invasion+in+an+organotypic+model.">Measuring invasion in an organotypic model.</a> Methods Mol Biol. 769, 223-232 (2011).</li><li>De Wever, O., Demetter, P., Mareel, M., Bracke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+myofibroblasts+are+drivers+of+invasive+cancer+growth.">Stromal myofibroblasts are drivers of invasive cancer growth.</a> Int J Cancer. 123 (10), 2229-2238 (2008).</li><li>Fries, K. 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=Evidence+of+fibroblast+heterogeneity+and+the+role+of+fibroblast+subpopulations+in+fibrosis.">Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.</a> Clin Immunol Immunopathol. 72 (3), 283-292 (1994).</li><li>Moutasim, K. A., 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=Betel-derived+alkaloid+up-regulates+keratinocyte+alphavbeta6+integrin+expression+and+promotes+oral+submucous+fibrosis.">Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.</a> J Pathol. 223 (3), 366-377 (2011).</li><li>Winter, J., Jung, S., Keller, S., Gregory, R. I., Diederichs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+roads+to+maturity:+microRNA+biogenesis+pathways+and+their+regulation.">Many roads to maturity: microRNA biogenesis pathways and their regulation.</a> Nat Cell Biol. 11 (3), 228-234 (2009).</li><li>Li, 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=Comparison+of+miRNA+expression+patterns+using+total+RNA+extracted+from+matched+samples+of+formalin-fixed+paraffin-embedded+(FFPE)+cells+and+snap+frozen+cells.">Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.</a> BMC Biotechnol. 7 (36), (2007).</li><li>Szafranska, A. E., 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=Accurate+molecular+characterization+of+formalin-fixed,+paraffin-embedded+tissues+by+microRNA+expression+profiling.">Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.</a> J Mol Diagn. 10 (5), 415-423 (2008).</li></ol>

著者らは、利害の相反を開示していない。

ここでは、特に上皮細胞および間質細胞の3次元共培養構築物における転移性進行の最も初期の段階で癌関連間質に侵入する能力を獲得した腫瘍細胞を分離し、特性化する方法について説明します。 ex vivoでのヒト結腸繊維芽細胞を含有する合成基質と並置CRC上皮細胞からなる共培養モデルは3次元でCRC浸潤を研究するために使用した。 インビボ条件の...

器官型共培養物は、必須の細胞外マトリックス成 ​​分1〜3を含むコラーゲンゲルにおける悪性上皮細胞および間質細胞を並置することによって3次元での生体内腫瘍微小環境を再構築するのを助ける組織工学モデルである。主に腫瘍浸潤を測定する方法として考えられ、organotypics インビボ動物モデルへの依存を低減し、そのような浸潤細胞が単分散状態2-4に人為的に強制されたトランスウェルアッセイのような他のインビトロ技術の欠点を回避する。線維芽細胞などのこれらのモデルにおける間質細胞の封入は、悪性の浸潤および転移3-4の調節における腫瘍微小環境の重要な役割を反映し、しかし、間質の表現型の異質性は、例えば、ある器官の生理的関連性を最大にするためにモデル、臓器の特定、解剖学的に正確なex vivoで可能な限り3,6線維芽細胞が含まれるべきである。 Organotypicsは、腫瘍細胞および間質細胞の間の相互作用を研究するための汎用プラットフォームであり、ますます化学的阻害剤および標的化遺伝子改変7,8の腫瘍浸潤に及ぼす影響を調べるために新規な方法で使用されている。浸潤性腫瘍マージンを研究することは、特にエキサイティングな見通しである。器官型モデルでは、確立された結腸直腸癌（CRC）細胞株は、典型的には、井戸重層上皮層を生成し、断面において、細胞外マトリックス（ECM）を浸潤する能力を獲得した細胞が容易に識別可能である。 インビボでの腫瘍および腫瘍関連間質細胞の確立マイクロ地形異質、レーザーマイクロダイセクションを使用して、これらの細胞を抽出し、分離して、それらを研究に照らして、大腸癌転移の起源に関する重要な生物学的な洞察を明らかにすることができるとどのようにそれがより効率的になることがあり対象とした。私N以下の方法論、組織サンプルを寄付し、すべての患者が書面によるインフォームドコンセントを提供し、研究は教育機関、地域研究倫理委員会によって承認された。

<table><tbody><tr><td>Colorectal cancer cell lines - example shown SW480</td><td>ATCC</td><td>ATCC CCL-228</td><td></td></tr><tr><td>Collagen</td><td>BD Biosciences</td><td>354265</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354234</td><td></td></tr><tr><td>Nylon membrane</td><td>Merck Millipore</td><td>VVLP01300</td><td></td></tr><tr><td>Metal grid</td><td>The Mesh Company</td><td>WSS20-A4</td><td>themeshcompany.com</td></tr><tr><td>Laser microdissection platform</td><td>Leica Microsystems</td><td>Leica AS LMD</td><td></td></tr><tr><td>Membrane mounted slides</td><td>Molecular devices</td><td></td><td></td></tr><tr><td>Cresyl Violet</td><td>Merck Millipore</td><td>1052350025</td><td></td></tr></tbody></table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

大腸がんに侵入（CRC）細胞は、姉妹細胞から自由になる間質に浸透し、細胞外マトリックス（ECM）を改造する能力を獲得している。細胞のこの表現型が異なるグループの生物学を特徴づけることは、実質的に転移カスケード時に初期事象の我々の理解を向上させることができます。 腫瘍浸潤、悪性上皮と癌関連間質間の双方向の相互作用によって促進さ動的なプロセスである。腫瘍間質界面での細胞特異的応答を調べるために、我々は器官共培養し、レーザーマイクロダイセクション技術を組み合わせている。 線維芽細胞などのキーの間質成分が、がん上皮細胞と共培養3 - dimentioanallyである、器官のモデルは、近pHの検討が浸潤や癌間質の相互作用を可能にする、高度に操作可能な実験的なツールですysiological条件。 レーザーマイクロダイセクション（LMD）は、ミクロンレベルの精度で腫瘍組織内の様々な階層の外科解剖と抽出を伴う手法である。 ゲノム、トランスクリプトームおよびエピジェネティックなプロファイリングでこれらの技術を組み合わせることで、我々は、腫瘍細胞に侵入し、間質組織、周囲の分子特性をよく理解できるようにする、そうすることで、潜在的に、CRCにおける薬剤開発のための新規バイオマーカーと機会を明らかにすることを目指しています。

我々は、CRC細胞株及び間質細胞の複数の組み合わせに対して、上記の方法を適用した。ここで紹介する一つの例は、一次ヒトex vivoで結腸線維芽細胞と上皮SW480 CRC細胞株である。 3次元共培養物は、浸潤性腫瘍前線におけるセル間の差次的に発現されるmiRNAの比較のために顕微鏡およびレーザーキャプチャーマイクロダイセクション（ 図2）に供した（ 図1）は ?...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

合成からのレーザーマイクロダイセクション細胞や間質、組織工学大腸癌モデルの分子プロファイリングは、浸潤性前面に腫瘍細胞および癌関連の間質細胞との間の界面に特徴的な生物学を特徴付け、分析する新規かつ操作可能なアプローチを表しています。

大腸癌細胞の3次元モデルにおける侵襲性腫瘍微小環境の分子プロファイリングと<em&gt;生体外</em&gt;線維芽細胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

molecular-profiling-invasive-tumor-microenvironment-3-dimensional

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

11.0K ビュー.  University of Southampton School of Medicine. 合成からのレーザーマイクロダイセクション細胞や間質、組織工学大腸癌モデルの分子プロファイリングは、浸潤性前面に腫瘍細胞および癌関連の間質細胞との間の界面に特徴的な生物学を特徴付け、分析する新規かつ操作可能なアプローチを表しています。 

ビデオ: Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

腫瘍の4DライブセルイメージングのためのMAMEモデル：微小環境の相互作用に影響を与える悪性進行

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

医学

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

がん関連線維芽細胞分化とインビトロ研究における侵入のためのより生理学的システムとしての3Dスフェロイドモデル

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

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 ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

大腸癌幹細胞を研究するスフェロイドの3次元モデル

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

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 ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

3D多細胞肺腫瘍スフェロイドから分離された癌関連線維芽細胞におけるミトコンドリアの健康評価

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

の調製と分析<em&gt;インビトロ</em&gt;三次元乳癌サロゲート

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

生物工学

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 ...

がん生物学における3Dモデル:ヘテロスフェロイドの浸潤ダイナミクス

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

著者スポットライト:頭頸部がんの進行過程におけるがんと免疫細胞との間のクロストークの解読

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

大腸癌細胞の3次元モデルにおける侵襲性腫瘍微小環境の分子プロファイリングと

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