この作品は、ジャクソン州立大学と米国防、アイデア賞 11-1-0205 部環境衛生 RCMI センターを通じて健康の国民の協会 (NIMHD G12MD007581) によって支えられました。

注: このプロトコルは、McArdleらに説明します。11します。図 1のタイムラインを提供します。
1. 培養プレートの準備
<ol><li>ガラス底の疎水性を下げるための 15 分の 1 M 塩酸 (HCl) 1 mL と 10 mm のガラス底を含む 35 mm の培養皿を洗ってください。</li>
	<li>リン酸バッファー生理食塩水 (PBS) すべての HCl を取り除くために 1 mL で 2 回培養プレートを洗います。</li>
	<li>文化の 70% エタノール 1 mL を 2 回洗浄します。</li>
	<li>1 ml の細胞特定の培地の培養プレートをリンス (ここでは、使用 α-MEM 10% 熱を添加した不活性 FBS と 1% 非必須アミノ酸)。 注:次の日まで加湿器では 37 ° C で CO2インキュベーターで培養液を含む処理培養プレートを格納できます。</li>
	<li>35 mm ディッシュの扱われたガラス底に細胞 (MSCs と MCF 7 共培養) を 100% の confluency に成長します。35,000 MSC 細胞および 75,000 24 h MCF7 細胞の共培養します。</li>
</ol>2. コラーゲン ゲルの調製
<ol><li>接触時にコラーゲンの重合を防ぐために冷凍庫にコラーゲン ・ ゲルをピペッティング用マイクロ ピペットのヒントを格納します。 注:これは、最も重要なステップです。</li>
	<li>ボリュームを決定するラット尾コラーゲンのため私はコラーゲン株式の濃度に基づきます。この試金のためのコラーゲン ・ ゲルの 100 μ L を準備します。 注:私は在庫ありラット尾コラーゲンの高濃度が適しています。それに応じてコラーゲンを希釈に使用するダルベッコ変更イーグル培地 x 10 のボリュームを決定します。</li>
	<li>私 (3.8 μ g/mL 在庫)、10 x ダルベッコ変更されたワシの媒体および 1 x 細胞培養液 2% 牛胎児血清と 2.4 mg/mL のコラーゲンの最終的な集中に適切な走化性因子を添加した氷ラット尾コラーゲンを結合します。</li>
	<li>コラーゲン ・ ゲルを中和するために重炭酸ナトリウム溶液の混合物の 4.5% に追加します。 注:重炭酸ナトリウム溶液を追加のボリュームは他の試薬の正確な pH によって異なります。中性の溶液を確保するか、培地の pH 指示薬を使用して pH のストリップとの混合物をテストすることをお勧めします。</li>
	<li>ガラス底培養皿の中で細胞をコラーゲン混合物の 80 μ L を横たわっていた。</li>
	<li>加湿器では 37 ° C で CO2インキュベーターで 30 分間重合コラーゲン ゲルを許可します。</li>
	<li>減らされた血清 (2%) と細胞用培地 2 mL でコラーゲン ・ ゲルをカバーし、24、48、72 時間に加湿器で CO2インキュベーターで 37 ° C でセルを孵化させなさい。 注意:プレートは、ガラス底から切り離す、コラーゲン ・ ゲルを防ぐために慎重に取り扱う必要があります。</li>
</ol>3. コラーゲンの準備の例は、3.8 μ g/mL のラット尾コラーゲン在庫を使用してゲルします。
<ol><li>氷上では、ラット尾コラーゲンの 114.5 μ L、DMEM、x 10 の 16.6 μ L と、走化性因子を含む培地の 33.1 μ L を組み合わせます。</li>
	<li>重炭酸ナトリウム 14.0 μ L でコラーゲン混合物を中和します。</li>
	<li>手順 2.5 のように続けます。</li>
</ol>4. 細胞のイメージング
<ol><li>そっとお皿からメディアを削除します。</li>
	<li>1 ml の PBS のゲルを軽くすすいでください。</li>
	<li>15 分 4% パラホルムアルデヒドの ml の 1 セルを修正します。</li>
	<li>1 mL の PBS で 2 回細胞を洗浄します。</li>
	<li>DAPI 溶液 (100 ng/mL) 室温で 20 分間と細胞を染色します。</li>
	<li>共焦点の顕微鏡を使用して、別の場所での撮影 400 μ m z スタック 16 μ m ステップの各位置のセルをイメージします。顕微鏡の使用により、白色光、アーク励起源を用いた共焦点イメージング ユニットをスキャン ディスクがあります。0.75 の開口で利用されている 20 倍レンズを使用します。</li>
	<li>画像を再構築します。<ol>
<li>与えられたゲル (約 25 画像) の画像を開き、イメージ → スタック → 画像のスタックをクリックして画像のスタックを作成します。ゲルの底に対応した最初のイメージ (z = 0 μ m)、2 番目のイメージは、z 方向の最初のステップに対応した (z = 16 μ m)、z 方向の 2 番目のステップに対応した 3 番目のイメージ (z = 32 μ m)。それぞれのイメージの深さに核を評価し、核はその特定のセルの侵入深さが鮮明で焦点に深さを指定します。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li><li>Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Wolf, K., 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, 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=Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho/ROCK+signalling+and+extracellular+proteolysis.">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li><li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <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+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro.">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198 (2008).</li><li>Staton, C. A., Reed, M. W., Brown, 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=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li><li>Poujade, 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=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li><li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-induced+cancer+cell+fusion:+a+mechanism+of+breast+cancer+metastasis.">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li><li>McArdle, T. J., Ogle, B. M., Noubissi, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types.">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li><li>Noubissi, F. K., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Cell+Fusion:+Mechanisms+Slowly+Unravel.">Cancer Cell Fusion: Mechanisms Slowly Unravel.</a> Int J Mol Sci. 17, (2016).</li><li>Meyvantsson, I., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+models+in+microfluidic+systems.">Cell culture models in microfluidic systems.</a> Annu Rev Anal Chem. 1, 423-449 (2008).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <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+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods In Enzymol. 406, 625-643 (2006).</li><li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+memory+and+dosing+influence+stem+cell+fate.">Mechanical memory and dosing influence stem cell fate.</a> Nat Materials. 13, 645-652 (2014).</li></ol>

著者は、競合する関心が存在しないことを宣言しています。

ここでは、様々 な細胞などの微小環境に依存したり、まれにあるセルに便利です簡単な反転垂直浸潤能の測定法について述べる.この 3 D 浸潤アッセイの細胞は彼らの元の微小環境の維持し、走化性因子を含むコラーゲン ゲルで覆われています。セルは移行し、コラーゲンに侵入します。このアッセイで細胞を染色し、ゲル内で簡単に監視できます。シンプルさとこのアッセイの再現性は、3 ...

細胞運動は細胞の正常発達的および生理学的プロセスです。ただし、パターンと移行し、侵入する細胞の能力の変化は、炎症性疾患、がん転移1,2,3などの病態に関連付けられます。転移がんで最も悪い理解の側面といえます。転移、がん細胞は細胞外マトリックス (ECM) が低下する、ローカル組織および intravasate に侵入する必要があります。循環では、がん細胞は、遠くのサイトに発信する、extravasate、二次性腫瘍を形成します。したがって、能力 ECM を低下させる細胞の移行して侵入するが、転移能に関連します。異なるアプローチは、分析、細胞の移行と侵略の機能を定量化して開発されています。最もよく使われる方法には、創傷治癒の試金、微速度顕微鏡観察、ボイデン室アッセイが含まれます。創傷治癒の試金4と時間経過顕微鏡検査、細胞遊走に予備的な洞察力を与えるだろう簡単で便利な試金。ただし、細胞が生体内で存在する 3 D 環境を反映して 2 D の試金はどちらも。研究では、細胞が 2 D 15,6と比較して 3 D 環境に異なる反応することを示しています。さらに、創傷治癒の試金は再現し、量的な結果7,8,9をもたらす困難です。創傷治癒のアッセイの 3 D の変更、セルの除外ゾーンの試金はもっと生理学的に関連するアッセイが細胞融合イベント10,11 から生じるハイブリッド細胞など数の少ないセルに適していません.
ボイデン室アッセイは、細胞研究関連微小を提供する 3 次元試金です。ただし、セルの元の微小環境から削除して移行し、走化性因子含む培地に向かって下方に侵入・ ボイデン ・ アッセイ系の上部室に預けられるが必要です。この 3 D のアプローチは細胞の微小環境やで限定数10、11,12に脆弱性の移行および侵略の機能の分析に適していません。細胞の遊走・浸潤を分析する新しいアプローチは、マイクロ流路勾配商工会議所アッセイ13です。適した、移行および侵略の研究の信頼できる、この試金はより高価な典型的な実験室の技術的に困難な場合が。ここで、微小環境に敏感な細胞を含む多くの細胞の種類と互換性があるおよび/または数の制限は、単純な反転垂直浸潤アッセイについて述べる。この試金はフーパーらによって提案されたプロトコルから適応されました。14します。 この分析では、セルのまま彼らの元の環境での移行と侵略が走化性因子の11を含むコラーゲン ・ ゲル内上方に移動ことを監視します。この試金は再現はされて最適化し、35 mm ディッシュで検証します。それは、異なるセル文化のサイズ、異なるマトリックス付着、および異なる塩基の強さなどさまざまな分析条件に適応できます。この試金は、創薬プロセスの初期スクリーニングのための in vitroプラットフォームとして役立つことができます。

<table><tbody><tr><td>MatTek P35G-1.5-10C</td><td>MatTek Corporation, Ashland, MA</td><td>P35G-1.5-10-C</td><td>mattek glass bottom dishes</td></tr><tr><td>Rat tail collagen I</td><td>Corning, Corning, NY, USA</td><td>354236</td><td>Collagen gel</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>H1758</td><td>HCl</td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>10010023</td><td>PBS</td></tr><tr><td>10X Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>D2429</td><td>10X DMEM</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Confocal Fluorescent microscope</td><td>Olympus America, Center Valley, PA, USA</td><td>Olympus IX81</td><td>Confocal&amp;nbsp; microscope</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td><td>SH30070.03</td><td>FBS</td></tr><tr><td>Ti:Sapphire multi-photon laser scanning microscope</td><td>Prarie Technologies, Sioux Falls, SD, USA</td><td>40x NA 0.8 objective</td><td>MPLSM</td></tr><tr><td>Imaris software</td><td>Bitplane Inc. Zurich, CH</td><td>Imaris 7.5.2</td><td>Imaris software</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>D9542</td><td>DAPI</td></tr><tr><td>Paraformaldehyde</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>50980487</td><td>PFA</td></tr><tr><td>&amp;alpha;-minimum essential medium</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>M0894</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC; Manassas, VA, USA</td><td>HBT-22</td><td></td></tr><tr><td>MSC</td><td>Trivedi and Hematti, 2007</td><td></td><td></td></tr><tr><td>20X lens UPLAPO</td><td>Olympus America, Center Valley, PA, USA</td><td>36-064</td><td>Olympus UPLSAPO 20X Objective</td></tr></tbody></table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

3 D 浸潤アッセイを開発しているが、その微小環境の整合性に影響を与えずに細胞を研究課題が残っています。ボイデン室必要セルが元の文化場所から避難して、新しい環境に移動するなど、従来の 3次元の試金します。だけでなく、これが、微小環境に固有な細胞プロセスを混乱させる、それは多くの場合細胞の損失の結果します。まれ、かの微小環境に非常に敏感な細胞を扱うとき、これらの問題は特に挑戦的です。ここでは、両方の問題を回避する 3 D 浸潤能の測定法の開発について述べる.この分析では、小規模でも細胞をめっきし、走化性因子を含む ECM 行列をセルの上に置いた。これはセル変位を必要としない、上向きマトリックスへの侵入セルをできます。このアッセイでは、細胞の形態と同様、細胞浸潤は、コラーゲン ・ ゲル内で評価できます。この試金を使用して、我々 は稀で、敏感な細胞の侵襲的な容量を特徴付ける乳癌細胞 MCF7 と間葉系/多能性の融合から生じるハイブリッド細胞幹/間質細胞 (MSCs)。

ガラス下部およびラット尾コラーゲン入り 35 mm 培養皿を用いて開発し、生体外で3 D 侵略を最適化する私はプロトコルを試金。我々 は癌細胞 MCF7 と MSC 胸を示したこのアッセイを使用して細胞が 0.04 ± 1.8 μ m、μ m ± 76.0 41.3 平均距離にコラーゲン ・ ゲルに 48 時間後それぞれ侵入しとき IGF1 (18.6 ng/mL) は、走化性因子 (図 2) として使用され?...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

このプロトコルでは、細胞の微小環境を維持しながら三次元の設定のセルの移行と侵略の機能を定量化に使用できる反転垂直浸潤能の測定法について説明します。この試金は稀および/または敏感な細胞に適しています。

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

moving-upwards-simple-flexible-vitro-three-dimensional-invasion-assay

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Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

6.0K ビュー.  University of Minnesota - Twin Cities. このプロトコルでは、細胞の微小環境を維持しながら三次元の設定のセルの移行と侵略の機能を定量化に使用できる反転垂直浸潤能の測定法について説明します。この試金は稀および/または敏感な細胞に適しています。

ビデオ: Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

モデリングとイメージング、3次元の集団細胞浸潤

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

三次元スフェロイド浸潤アッセイにおける完全ヒト腫瘍ベースマトリクス

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

がん研究

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

アン試験管内でフルオロブロックの腫瘍の浸潤アッセイ

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

生物学

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

移行, 侵略, と不死化妊娠初期絨毛細胞の増殖を評価する生体外の試金

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

発生生物学

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

変更された<em&gt;インビトロ</em媒介する癌細胞浸潤におけるホルモン、サイトカインおよび/または成長因子の潜在的な役割を決定するために&gt;浸潤アッセイ

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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.

3D培養物へのIn vitro細胞浸潤を解析するための代替戦略

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

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 ...

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

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TGF -β誘発性浸潤を測定する回転楕円体アッセイ

モデリングとイメージング、3次元の集団細胞浸潤

三次元スフェロイド浸潤アッセイにおける完全ヒト腫瘍ベースマトリクス

アン試験管内でフルオロブロックの腫瘍の浸潤アッセイ

移行, 侵略, と不死化妊娠初期絨毛細胞の増殖を評価する生体外の試金

変更された

3D培養物へのIn vitro細胞浸潤を解析するための代替戦略

3D培養物へのIn vitro細胞浸潤を解析するための代替戦略

3D培養物へのIn vitro細胞浸潤を解析するための代替戦略

三次元（3D）腫瘍スフェロイド浸潤アッセイ