Name: utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells
Uploaded: 2015-05-20T14:00:00.000+00:00
Duration: 11 min 6 s
Description: Watch this Scientific Journal Video about Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells at JoVE.com

We are thankful to Dr. Richard Cerione, Dr. Marc Antonyak, and Kelly Sullivan, Cornell University, for providing technical advice, and to Dr. Gerlinde Van de Walle, Cornell University, for sharing their Olympus CKX41 inverted microscope.

3％の2-ヒドロキシエチルアガロースの調製<ol><li>清浄な乾燥した100mlのガラス瓶に、2-ヒドロキシエチル0.9gのアガロースを添加（アガロースVII）を蒸留水30mlに続きます。 </li><li>電子レンジ15秒間混合し、穏やかに渦巻きます。アガロース粉末が完全に溶解するまで、この手順を少なくとも3回繰り返します。 </li><li> 15分間この溶液を含むボトルをオートクレーブ。 </li><li>アガロース溶液は、さらに、使用前に室温まで冷却します。この溶液を室温で保管してください。 </li></ol>底層の調製：0.6％アガロースゲル<ol><li>取り扱いの際にピペットで固化からアガロースを防止するために、37℃のインキュベーターで事前に暖かい、いくつかの5ミリリットルと10ミリリットルピペット。 </li><li>部分的にボトルの蓋や電子レンジで15秒のための前作られた3％の2-ヒドロキシエチルアガロース溶液を緩めます。そして、静かに別の15秒のためのソリューション、電子レンジが渦巻きます。注意：agaroを旋回するときは注意してくださいSEソリューションソリューションは、空気にさらされると波及することができたときに上昇するためです。 </li><li>ボトル内の残留固体ゲル、さらに数秒間マイクロ波が存在する場合。 </li><li>途中で固化からアガロース溶液を防止するために、次のステップの間に45℃の水浴中でアガロース溶液の入ったボトルを保管してください。 </li><li> 37℃の水浴中で暖かいMCF10DCIS媒体。注：MCF10DCISメディアはDMEM / F12、5％ウマ血清、5％のペニシリンストレプトマイシンから成ります。 </li><li>転送滅菌50mlコニカルチューブに予め温めたピペットを用いて、3％アガロース溶液3ml。 </li><li>すぐに暖かいMCF10DCISメディアの12ミリリットルを加え、穏やかにメディアとアガロースを混合するためにコニカルチューブを反転。それは後にコロニー計数を妨害するように気泡を形成しないようにしてください。 </li><li>ゆっくりと気泡を形成することなく、6ウェル培養プレートの各ウェルにこの混合物を2mlを加えます。 </li><li> 6ウェル培養プレートのhorizo​​ntaをインキュベートLLY 1時間4℃で平らな面には、混合物が固化することを可能にします。 </li><li>混合物が固化した後、30分間37℃のインキュベーターにプレートを配置します。底層が使用できるようになりました。 </li></ol>細胞を含有する層の調製：0.3％アガロースゲル<ol><li>トリプシン処理MCF10DCIS細胞および4×10 4 / mlの細胞濃度になるように、それらを希釈します。 </li><li>予め温めたピペットを用いて、3％アガロースの2ミリリットルを取り、滅菌50mlコニカルチューブに移します。 </li><li>すぐにコニカルチューブにMCF10DCISメディアの8ミリリットルを加え、穏やかにメディアとアガロースを混合することが反転します。任意の気泡を形成することは避けてください。 </li><li> MCF10DCIS細胞（4×10 4 / ml）を2ミリリットルを取り、BB-CLA（0μM（DMSO）または1μM）で処理します。 </li><li> 1：1希釈、0.6％アガロースで細胞を混ぜます。 </li><li>細胞 - アガロース混合物の1ミリリットルを取り、静かに6ウェル培養pの底層の上に追加します後期（2×10 4細胞/ ml）。 </li><li>上層が固化することを可能にするために、少なくとも15分間、4℃で平坦な面に水平に6ウェル培養プレートに置き。 </li><li>混合物が固化した後、給電層を追加する前の週の37℃のインキュベーターにプレートを配置します。 </li></ol>フィーダー層の4製造：0.3％アガロースゲル<ol><li>電子レンジ15秒あらかじめ用意された3％の2-ヒドロキシエチルアガロース溶液。静かに別の15秒のためのソリューション、電子レンジが渦巻きます。 </li><li> 45℃の水浴中でアガロース溶液のボトルを平衡化します。 </li><li> 37℃の水浴中でMCF10DCIS媒体を温めます。 </li><li> 50mlコニカルチューブに暖かいMCF10DCISメディアの9ミリリットルで3％アガロース溶液1mlを混合し、穏やかにメディアとアガロースを混合するために反転します。気泡を形成することは避けてください。 </li><li> BB-CLA（0μM（DMSO）または1μM）との混合物を扱います。 </li><li>静か用なし（この混合物の1ミリリットルを追加明は、底部とソフト層を含む6ウェル培養プレートの各ウェルに）泡。 </li><li>混合物を固化することを可能にするために、少なくとも15分間、4℃で平坦な面に水平に6ウェル培養プレートに置き。 </li><li>フィーダー層が固化した後、37℃のインキュベーターにプレートを配置します。 </li><li>コロニー形成が観察されるまで、新しいメディアで細胞を補充するために、既存のフィーダー層の上に、0.3％アガロース/中/処理溶液の1ミリリットルを重ねることによって、この給紙手順を毎週繰り返します。注：柔らかくフィーダー層で寒天は非常に柔らかく、従って、フィーダー層からの追加の栄養素が容易に細胞に到達するために、細胞を含有する層中に拡散しています。 </li></ol> 5.データ収集<ol><li>軟寒天における細胞成長の2.5週間後、光学顕微鏡を用いて、各ウェル中のコロニーの数を数えます。定量化を容易にするために、透明性の上にグリッドを印刷しにグリッドを添付6ウェルプレートは、細胞がカウント中にある場所を特定するのに役立ちます。 （各コロニーの直径によって定量化されるように）、コロニーサイズが異なりますので、採点されるコロニーを判断するための基準コロニーサイズを事前に定義。例えば、70ミクロン以下のデータ分析が大きいのコロニーの大きさがあります。 </li><li>さらにコロニー形成を防止し、将来のカウントのために4℃でサンプルを保管してください。乾燥からゲルを防ぐためにパラフィルムで6ウェル培養プレートをシール。 </li></ol>

<ol>
	<li>Hamburger, A. W., Salmon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+bioassay+of+human+tumor+stem+cells.">Primary bioassay of human tumor stem cells.</a> Science. 197, 461-463 (1977).</li><li>Wang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signaling+regulating+anchorage-independent+growth+of+cancer+cells.">Molecular signaling regulating anchorage-independent growth of cancer cells.</a> Mt Sinai J Med. 71 (6), 361-367 (2004).</li><li>Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., Pruijn, 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=PAD,+a+growing+family+of+citrullinating+enzymes:+genes,+features+and+involvement+in+disease.">PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.</a> Bioessays. 25 (11), 1106-1118 (2003).</li><li>Horibata, S., Coonrod, S. A., Cherrington, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+peptidylarginine+deiminase+enzymes+in+disease+and+female+reproduction.">Role for peptidylarginine deiminase enzymes in disease and female reproduction.</a> J Reprod Dev. 58 (3), 274-282 (2012).</li><li>Mohanan, S., Cherrington, B. D., Horibata, S., McElwee, J. L., Thompson, P. R., Coonrod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+peptidylarginine+deiminase+enzymes+and+protein+citrullination+in+cancer+pathogenesis.">Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis.</a> Biochem Res Int. , 895343 (2012).</li><li>McElwee, J. 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=Identification+of+PADI2+as+a+potential+breast+cancer+biomarker+and+therapeutic+target.">Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.</a> BMC Cancer. 12, 500 (2012).</li><li>Knight, J. S., 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=Peptidylarginine+deiminase+inhibition+disrupts+NET+formation+and+protects+against+kidney,+skin+and+vascular+disease+in+lupus-prone+MRL/lpr+mice.">Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice.</a> Ann Rheum Dis. , 1-8 (2014).</li><li>Miller, F. R., Santner, S. J., Tait, L., Dawson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF10DCIS.com+xenograft+model+of+human+comedo+ductal+carcinoma+in+situ.">MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ.</a> J Natl cancer Inst. 92, 1185-1186 (2000).</li><li>Fan, D., Morgan, L. R., Schneider, C., Blank, H., Fan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+evaluation+of+human+tumor+chemosensitivity+in+the+soft-agar+assay+and+its+clinical+correlations.">Cooperative evaluation of human tumor chemosensitivity in the soft-agar assay and its clinical correlations.</a> J Cancer Res Clin Oncol. 109, 23-28 (2000).</li><li>Hamburger, A. W., White, C. P., Dunn, F. E., Citron, M. L., Hummel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+human+tumor+colony+growth+in+soft+agar+by+serum.">Modulation of human tumor colony growth in soft agar by serum.</a> Int J Cell Cloning. 1 (4), 216-229 (1983).</li><li>Anderson, S. N., Towne, D. L., Burns, D. J., Warrior, U. <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+soft+agar+assay+for+identification+of+anticancer+compound.">A high-throughput soft agar assay for identification of anticancer compound.</a> J Biomol Screen. 12, 938-945 (2007).</li></ol>

The authors have nothing to disclose.

軟寒天におけるコロニー形成の速度は、細胞型9によって異なり。したがって、細胞の数を最適化し、それに応じて調整されるべきで開始します。提案さ始動範囲は6ウェルプレートを使用してあたり4×10 2〜1×10 5細胞の間です。また、コロニーの大きさは、各細胞の増殖速度に応じて変化します。そのため、コロニーの大きさのために事前に定義されたカットオフは、下流定?...

Both non-transformed (normal) and transformed cells can readily proliferate in a 2D monolayer culture. This form of adherent cell growth is quite dissimilar from that which occurs in vivo where, in the absence of mitogenic stimulation, cells do not often rapidly divide within their microenvironment. The soft agar assay on the other hand is distinct from 2D culture systems because it quantifies tumorigenicity by measuring a cell&#8217;s ability to proliferate and form colonies in suspension within a semi-solid agarose gel1. In this setting, non-transformed cells are unable to rapidly propagate in the absence of anchorage to the extracellular matrix (ECM) and undergo apoptosis, a process known as anoikis. In contrast, cells that have undergone malignant transformation lose their anchorage dependence due to activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and Rac/Cdc42/PAK. Therefore, these cells are able to grow and form colonies within the semi-solid soft agar matrix2.
A common use of the soft agar assay is to test whether specific compounds, such as PADI inhibitors, are able to suppress tumor growth in vitro. In general, colony count or colony sizes are quantitative read-outs from the assay that can be compared between control and treatment groups to assess differences in cellular tumorigenicity. Therefore, if one finds that colony formation is inversely correlated with increasing drug concentration, then a conclusion could be drawn that the drug is an effective inhibitor of tumorigenicity in vitro. On the other hand, if the drug does not affect colony formation, the drug is either not at the appropriate dosage or it is not an effective tumorigenic inhibitor. Aside from using a soft agar assay to test the anti-tumor effect of a drug, this assay can also be used to probe the relationship between a specific gene and tumorigenesis. For example, the effect of suppressing PADI2 expression on tumorigenicity can be addressed by PADI2-specific siRNA treatment.
PADIs are calcium-dependent enzymes that post-translationally modify proteins by converting positively charged arginine residues into neutrally charged citrulline in a process known as citrullination or deimination3-5. We have recently found that peptidylarginine deiminase 2 (PADI2) may function as a novel breast cancer biomarker and that PADI inhibitors represent candidate therapies for early stage breast cancers6. For example, we have previously demonstrated that a &#8220;pan-PADI&#8221; inhibitor, Cl-amidine, suppresses the proliferation of breast cancer cells using 2D monolayers and that the inhibitor suppressed the growth of 3D tumor spheroids6. In this report, we extend these studies, and highlight the utility of the soft agar assay, by testing the efficacy of a new PADI inhibitor, BB-CLA, in suppressing the growth of MCF10DCIS breast cancer colonies7. We note that we used MCF10DCIS cells for this experiment because they are oncogenic derivatives of non-transformed human MCF10A cells and because they contain high steady state levels of PADI2 protein8. We hypothesize that PADI2 enzymatic activity plays a key role in the tumorigenicity of this cell line and that BB-CLA-mediated inhibition of PADI2 activity will suppress cancer progression.

<table><tbody><tr><td>Zeiss Axiopot</td><td>Carl Zeiss Microscopy</td><td>1021859251</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>DMEM/F-12</td><td>Lonza BioWhittaker</td><td>12-719F</td><td></td></tr><tr><td>HyClone Donor Equine Serum</td><td>Fisher Scientific</td><td>SH30074.03</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>2-Hydroxyethylagarose: Type VII, low gelling temperature</td><td>Sigma-Aldrich</td><td>39346-81-1</td><td></td></tr></tbody></table>

utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

軟寒天コロニー形成アッセイは、癌細胞の腫瘍形成能を記録幅広い用途に使用することができます。この手法の主な利点は、半固体マトリックスが選択的足場に依存しない方法で増殖できる細胞の増殖に有利に働くということです。この特性は、主に癌細胞によってではなく、正常細胞によって示されます。我々は、主に薬剤による腫瘍増殖阻害の有効性を試験するために、この技術を使用?...

Watch this Scientific Journal Video about Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells at JoVE.com

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

乳癌細胞における腫瘍形成の阻害剤を同定するための軟寒天コロニー形成アッセイの活用

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

utilization-soft-agar-colony-formation-assay-to-identify-inhibitors

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

28.5K ビュー.  Cornell University. Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro. 

ビデオ: Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

NFκB活性および乳癌幹細胞を阻害するアスピリン、フマル酸プロドラッグの合成とキャラクタリゼーション

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

がん研究

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

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

腫瘍性細胞行動の尺度としてのインビトロ侵血アッセイにおけるマウス乳腺腫瘍細胞の使用

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

遺伝学

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

低から中程度のスループットで培養哺乳動物細胞の細胞増殖と生存に影響を与える化合物を同定する戦略

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

生化学

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

ヒト乳腺上皮細胞における発癌性転換のインビトロ評価

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

アン<em&gt;インビトロ</em&gt;骨髄中のエストロゲン感受性乳癌の休眠モデル：分子機構の研究と仮説生成のためのツール

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

軟寒天コロニー形成アッセイ

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

生物学

Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

Source: Horibata A. et. al., <a href="https://jove.unicartagenaproxy.elogim.com/v/52727/utilization-soft-agar-colony-formation-assay-to-identify-inhibitors"> Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.</a> J. Vis. Exp. (2015).
This video describes a soft agar colony formation assay to test the effects of a PADI enzyme inhibitor and BB-Cl-amidine on breast cancer tumorigenicity in vitro. This assay also enables the quantitative assessment of cell transformation potential within the context of genetic perturbations.

軟寒天コロニー形成アッセイ:癌細胞増殖に対する新規化合物の影響をテストする方法

Source: Horibata A. et. al.,  Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells. J. Vis. ...

JoVE Encyclopedia of Experiments

乳がん

試験管研究

Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

Source: Kesarwani, M., et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/58194">Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence.</a> J. Vis. Exp. (2019).
This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

メチルセルロースベースのCFUアッセイ:白血病細胞のコロニー形成に対する抗癌化合物の影響を決定する方法

Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).
This video describes the ...