The authors thank Gr&#225;inne Tierney and Silvia Bellissimo for their editorial assistance.

プロトコルは、IRSTヒューマン研究倫理委員会のガイドラインに従います。 注：この手順では、UCF DNAの完全性の分析を実行するために尿試料からDNAを単離しました。 LNCaPおよびMRC細胞株は、標準を構築するために使用しました。このような特定の癌遺伝子のためのDNAの抽出、DNAの定量（コントロール遺伝子について分光光度計、リアルタイムPCR、STOX1）、及びリアルタイムPCRのような技術は、（ 図1）を行いました。 1.尿収集および処理<ol><li>清潔で乾燥し、プラスチックカップでクリーンキャッチ最初の朝の尿サンプルを入手します。尿サンプルの少なくとも50ミリリットルを収集します。 </li><li> 3時間の最大4℃で尿を維持し、同じ温度で実験室に送信します。 </li><li> 2つの50 mLコニカル底ポリプロピレンチューブに研究室や転送に到着するとすぐにそれを2回反転させることにより、各サンプルを混ぜます。 </li><li>チューブを遠心4℃または室温で10分間、850×gで。 </li><li>慎重に細胞ペレットの上の上清のうちの少なくとも2ミリリットルを残して、2つの5 mLのチューブに、尿上清の上部10ミリリットルを転送します。 注：上清の上部を転送するには、細胞または細胞破片による汚染のリスクを低減します。上部と下部との間には明確な描写はありません。 </li><li>ペレットを捨て、すぐに使用するまで-80℃上清を凍結します。 </li></ol>尿上清および細胞株から2 DNA単離注：市販のキットを用い、製造業者の指示に従って細胞系（膀胱癌のための前立腺癌またはMRCために、例えば、LNCAP）からDNAを単離します。尿上清からのDNAの単離は、以下のように修正された、商業的プロトコルを使用して行われるべきです。 <ol><li>室温で尿上清の1つのアリコートを解凍します。&lt;/李&gt; <li>明確な5-mLのチューブに尿サンプルと転送尿上清1mLのを渦と混ぜます。 -80℃で残尿をフリーズします。 </li><li>サンプルに直接プロテイナーゼkの100μLを加えます。 </li><li>サンプルにAL緩衝液の1 mLを加え、ピペッティングによりよく混ぜます。 </li><li>チューブを閉じ、15分間56℃でサンプルをインキュベートします。 </li><li>インキュベーションの間、各サンプルごとに1カラムを作製し、製造業者の説明書（100％エタノールの指示された量を加える）に従って、洗浄バッファー、AW1およびAW2を準備します。 </li><li>室温に戻し、サンプルを持参し、無水エタノール1mLを追加。ピペッティングにより完全に混和します。 </li><li>列にステップ2.7で得られた混合物の650μLを加え、1分間6000×gでそれを遠心します。 </li><li>フロースルーを含むチューブを捨て、新しい、クリーンなコレクションチューブにカラムを配置します。サンプル混合物のすべてが（5回）に使用されるまで繰り返し、2.8と2.9の手順。 </li><li> 500＆を追加＃181;カラムの縁を濡らさないようにバッファAW1のL。 1分間6000×gでそれを遠心。 </li><li>フロースルーを含むチューブを捨て、新しい、クリーンなコレクションチューブと交換してください。 </li><li>カラムの縁を濡らさないようにバッファAW2の500μLを追加します。 3分間フルスピード（2万XG）でそれを遠心。 </li><li>フロースルーを含むチューブを捨て、新しい、クリーンなコレクションチューブと交換してください。 </li><li>任意の残留洗浄バッファーを除去するための手順を繰り返し2.12。 </li><li>きれいな1.5 mLチューブにカラムを置きます。溶出バッファーAEの50μLを加え、カラムを濡らすバッファことを確認するために、7分を待ちます。 </li><li> 1分間8000×gでそれを遠心。 </li><li>ミニカラムにステップ2.15から溶離液をピペットとDNAの最大の回収率を確保するために、1分間最大速度でそれを遠心します。 </li></ol> 3. DNA定量化および希釈<ol><li>分光光度計を使用して、ボットからのDNAの定量化を行いますH細胞株および尿上清試料。製造元の指示に従って、ベンチトップ分析計で試料を2μLを使用してください。 </li><li> 1ng /μLの濃度を取得し、DNAの完全性の分析まで-20℃でDNAを保存するためにUCF DNAサンプルを希釈します。 注：DNA量をリアルタイムPCR（少なくとも100 ngの）を続行するのに十分でない場合、新たなDNAの単離処理を行います。 </li><li> 0.001 0.01、0.1、1、0.5、2 NG /μL、100μL（ST1、ST2、ST3、ST4、ST5の体積でそれぞれ、および：異なる濃度の6水準を得るために、細胞株サンプルからDNAを希釈ST6）。 DNAの完全性の分析まで-20℃で細胞株DNA標準を保管してください。 </li></ol> 4. DNA完全性試験 - PCR <ol><li>プライマー（濃度はアッセイの種類によって異なります）、グリーンスーパーミックス、細胞株のDNA標準、および氷上でUCF DNA希釈した試料を解凍します。 </li><li>プレートFのストリップチューブを準備または72ウェルロータディスク。小分けした各標準および希釈サンプルおよび陰性対照のためのRNaseフリー水10μLの重複で10μL。 </li><li>各サンプルについて、各プライマーの1μL（濃度は表1に示されている）、グリーンスーパーミックスの12.5μL、およびRNaseフリー水6.5μLのミックスを調製します。 2複製のための6の標準、2反復のサンプル数、2複製、および2エキストラサンプルの陰性対照：ミックスを調製する場合、サンプルの次の番号を使用します。 </li><li>各ウェルにミックスのアリコート15μL。ピペット又はチューブをスピンしないでください。 </li><li> 表1に示したPCR条件でプロトコルを起動します。 注：29×2サンプル+ 6×2標準+ネガティブコントロール×2 = 72（UCF DNA値）：UCF DNA値の場合、29のDNAサンプルを72ウェルのロータディスクを使用して、各リアルタイム実験のために処理しました。 注：UCF DNAインテグリティ解析のためのプロトコルもすることができます（他のデバイスを使用する場合、ROX色素を添加する必要があるかもしれない）他のリアルタイムPCR装置を用いて行きました。 </li></ol> 5. DNA完全性試験 - データ分析と解釈注：以前に7-9（ 図2）に記載のように各サンプルのUCF DNA値は、各個々のPCR遺伝子の評価のための標準曲線の構築を用いて、標準曲線の補間を使用してリアルタイム機器検出システムソフトウェアによって得ました。 <ol><li>複製を評価します。サンプルはCtは破棄され、第二の実験で再評価されなければならない≥1違いで複製します。 </li><li>各試料についての中央値のCtを計算し、36≤Ct値とサンプルを検討してください。 </li><li>融解分析を使用して、PCR産物の特異性を評価します。 </li><li>標準曲線との補間によって各アンプリコンの濃度を評価します。各アンプリコンのための濃度値（ngの/μL）を得ました。</li>
</ol>

<ol>
	<li>Francis, G., Stein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Cell-Free+Tumour+DNA+in+the+Management+of+Cancer.">Circulating Cell-Free Tumour DNA in the Management of Cancer.</a> Int.J.Mol.Sci. 16 (6), 14122-14142 (2015).</li><li>Bryzgunova, O. E., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Nucleic+Acids+in+Urine:+Sources,+Structure,+Diagnostic+Potential.">Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential.</a> Acta Naturae. 7 (3), 48-54 (2015).</li><li>Szarvas, T., 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=Deletion+analysis+of+tumor+and+urinary+DNA+to+detect+bladder+cancer:+urine+supernatant+versus+urine+sediment.">Deletion analysis of tumor and urinary DNA to detect bladder cancer: urine supernatant versus urine sediment.</a> Oncol.Rep. 18 (2), 405-409 (2007).</li><li>Togneri, F. 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=Genomic+complexity+of+urothelial+bladder+cancer+revealed+in+urinary+cfDNA.">Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA.</a> Eur.J.Hum.Genet. , (2016).</li><li>Zonta, E., Nizard, P., Taly, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+DNA+Integrity,+Applications+for+Cancer+Research.">Assessment of DNA Integrity, Applications for Cancer Research.</a> Adv.Clin.Chem. 70, 197-246 (2015).</li><li>Hao, T. B., 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=Circulating+cell-free+DNA+in+serum+as+a+biomarker+for+diagnosis+and+prognostic+prediction+of+colorectal+cancer.">Circulating cell-free DNA in serum as a biomarker for diagnosis and prognostic prediction of colorectal cancer.</a> Br.J.Cancer. 111 (8), 1482-1489 (2014).</li><li>Salvi, 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=Urine+Cell-Free+DNA+Integrity+Analysis+for+Early+Detection+of+Prostate+Cancer+Patients.">Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients.</a> Dis.Markers. 2015, 574120 (2015).</li><li>Casadio, V., 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=Urine+cell-free+DNA+integrity+as+a+marker+for+early+prostate+cancer+diagnosis:+a+pilot+study.">Urine cell-free DNA integrity as a marker for early prostate cancer diagnosis: a pilot study.</a> Biomed.Res.Int. 2013, 270457 (2013).</li><li>Casadio, V., 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=Urine+cell-free+DNA+integrity+as+a+marker+for+early+bladder+cancer+diagnosis:+preliminary+data.">Urine cell-free DNA integrity as a marker for early bladder cancer diagnosis: preliminary data.</a> Urol.Oncol. 31 (8), 1744-1750 (2013).</li><li>Salvi, 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=Circulating+cell-free+AR+and+CYP17A1+copy+number+variations+may+associate+with+outcome+of+metastatic+castration-resistant+prostate+cancer+patients+treated+with+abiraterone.">Circulating cell-free AR and CYP17A1 copy number variations may associate with outcome of metastatic castration-resistant prostate cancer patients treated with abiraterone.</a> Br.J.Cancer. 112 (10), 1717-1724 (2015).</li><li>Ishkanian, A. 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=High-resolution+array+CGH+identifies+novel+regions+of+genomic+alteration+in+intermediate-risk+prostate+cancer.">High-resolution array CGH identifies novel regions of genomic alteration in intermediate-risk prostate cancer.</a> Prostate. 69 (10), 1091-1100 (2009).</li><li>Oxley, J. D., Winkler, M. H., Gillatt, D. A., Peat, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Her-2/neu+oncogene+amplification+in+clinically+localised+prostate+cancer.">Her-2/neu oncogene amplification in clinically localised prostate cancer.</a> J.Clin.Patho. 55 (2), 118-120 (2002).</li><li>Nord, H., 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=Focal+amplifications+are+associated+with+high+grade+and+recurrences+in+stage+Ta+bladder+carcinoma.">Focal amplifications are associated with high grade and recurrences in stage Ta bladder carcinoma.</a> Int.J.Cancer. 126 (6), 1390-1402 (2010).</li><li>Tabach, Y., 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=Amplification+of+the+20q+chromosomal+arm+occurs+early+in+tumorigenic+transformation+and+may+initiate+cancer.">Amplification of the 20q chromosomal arm occurs early in tumorigenic transformation and may initiate cancer.</a> PLoS One. 6 (1), e14632 (2011).</li></ol>

The authors declare no competing financial interests.

UCF DNAの完全性の分析は、尿中のDNAの完全性を評価するための新しい、非侵襲的方法です。最近、膀胱9および前立腺癌7,8の早期診断のために提案されました。 UCF DNA完全性テストの利点と欠点の数は、一緒に将来の見通しで、ここで説明されています。 アプローチの主な利点は、それが安価で、非侵襲的な方法と分子生物学的手法の基本的な知識を必要?...

無細胞DNAは、アポトーシスまたは壊死の機構によって、血液および細胞死に起因する尿中で検出することができます。血中の無細胞DNAは、広く様々な疾患における診断および予後目的、特に癌1のために研究されています。ただし、以下は、尿、無細胞（UCF）DNAの役割について知られています。 UCF DNAは、糸球体濾過システムを介して、または、この体液2（ 例えば、尿路上皮細胞または前立腺細胞）との接触に直接来て、細胞から通過する血液に由来し得ます。バイオマーカーの供給源としてUCF DNAの使用は、主に尿路細胞3,4から直接UCF DNAの割合が高いと、腎、膀胱、前立腺癌の早期診断のために検討されています。 リトルは、UCF DNA単離とそれを特徴付けるための最良の方法について知られています。腫瘍細胞は正常細胞よりも長いDNA断片を放出するという仮説評価与えられました無細胞DNAの完全性は、血液循環5にDNAの起源を解明する試みにおいて研究されてきました。いくつかの研究は、血液中の無細胞DNAの完全性は、癌6多くの種類のための良好な診断テストを表し、同じ仮説が尿7-9に関連して提案されていることを実証しました。 本稿では、膀胱および前立腺癌の検出への適用の可能性とともに、UCF DNAインテグリティ解析のための新しい方法を説明します。具体的には、UCF DNAの完全性は、より長い250bpのが前立腺癌および膀胱癌を含む固形腫瘍において増大したDNAコピー数を有することが知られている4つの領域において試験したよりフラグメント：C-MYC（8q24.21）、HER2を （17q12.1 ）、BCAS1（20q13.2）、およびAR（Xq12）10-14。特定の癌遺伝子ではなく、ランダムな配列は、癌患者の無細胞画分でそれらを発見する確率を高めるために選択しました。の主な利点の一つこの方法は、柔軟であり、他の領域は、腫瘍の種類や特性に基づいて選択することができるということです。

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">QIAamp DNA Mini Kit 
			&#160;</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">51304</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">iQ SYBR&#160;Green Supermix, 100 x 50 &#181;l rxns, 2.5 ml (2 x 1.25 ml)</td>
			<td style="width:216px;">Biorad</td>
			<td style="width:119px;">1708880</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">IDT custom DNA oligos&#160;</td>
			<td style="width:216px;">IDT&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">HPLC purification, 100nMole DNA oligo</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">NanoDrop 1000 Spectrophotometer</td>
			<td style="width:216px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Other spectrophotometric methods could also be used to quantify DNA</td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:277px;">Rotor-Gene 6000</td>
			<td style="width:216px;">Corbett</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Another Real Time PCR instrument could also be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">microcentrifuge</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">one centrifuge for 50 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">incubator</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">&#160;-80&#176;C freezer</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">-20&#176;C freezer</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">10 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">20 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">200 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1000 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">pipette tips (10;20;200;1000)</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1,5 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">50ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">15 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Rotor-Disc 72 Rotor</td>
			<td style="width:216px;">Corbett</td>
			<td>&#160;9018899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Strip Tubes and Caps, 0.1 ml (250)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">981103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Collection Tubes (2 ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Buffer AL (264 ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Proteinase K (10ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19133</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free dna integrity analysis in urine samples

血漿または血清中の無細胞循環DNAの存在は、広く多くの種類の癌のためのバイオマーカーの適切な供給源であることが示されているが、いくつかの研究は、尿、無細胞（UCF）DNAの潜在的な使用に焦点を当ててきました。正常なアポトーシス細胞が高度に細分化DNAを作製し、その癌細胞はより長いDNAを解放仮説から出発し、UCF DNAの完全性の潜在的な役割は、前立腺や膀胱癌および健康な個体の患者とを区別することが可能な早期診断マーカーとして評価しました。 C-MYC、BCAS1、HER2、およびAR：UCF DNAの完全性の分析は、250塩基対よりも長い4系統の4定量的リアルタイムPCRのもとに提案されています。しばしば、膀胱および前立腺癌において増加したDNAのコピー数を有する配列は、分析のために選択したが、この方法は柔軟性があり、そしてこれらの遺伝子は、INTEの他の遺伝子で置換することができ残り。バイオマーカーの供給源としてUCF DNAの潜在的な有用性は、すでにこのようにUCF DNAの特性化に関するさらなる研究のための道を開く、泌尿器科悪性腫瘍のために実証されています。 UCF DNAの完全性試験は、非侵襲的、迅速、かつ実行が容易で、分析を行うために必要な尿のわずか数ミリリットルであるという利点を有します。

全遊離DNA濃度は、1.51の間および138 ngの/μLの範囲を示す、分析したすべてのサンプルについて、分光光度法により定量可能でした。五対照試料は、データの再現性のために使用した二つの独立したリアルタイムの実験は、C-MYC、HER2、BCAS1、AR、及びSTOX1について実施しました。変動（CV）の係数は、2つの独立した実験間の再現性?...

Watch this Scientific Journal Video about Cell-Free DNA Integrity Analysis in Urine Samples at JoVE.com

Cell-Free DNA Integrity Analysis in Urine Samples

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

尿サンプル中の無細胞DNAインテグリティ解析

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

cell-free-dna-integrity-analysis-in-urine-samples

Research

JoVE Journal

Cancer Research

13.7K ビュー.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

ビデオ: Cell-Free DNA Integrity Analysis in Urine Samples

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP&#160;cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP&#160;assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

生物学的サンプルからの癌幹細胞の検出と精製のためのDNA色素誘発側母集団表現型の活用

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

がん研究

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

コメットアッセイを用いた DNA 損傷の in Vitro評価

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

タッチ捺印細胞診を用いた臨床病理標本から質の高い腫瘍 DNA を取得する簡易法

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

アンチ PD L1 抗体は生検で非小細胞肺癌患者からの細胞のサンプル集中 22 3 を使用して

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

便DNA完全性検出による大腸癌リスクと罹患率の評価

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

構造DNA改変解析と患者由来異種移植片を用いたがんの標的治療の試験

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

大腸癌細胞の3次元スフェロイド培養におけるプロバイオティクスの細胞遊離上清を用いた細胞死の評価

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

消化器癌におけるDNAメチル化のゲノムワイド解析

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

がん患者の血漿サンプルにおける無細胞DNAの検出

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.