Name: harnessing the dna dye-triggered side population phenotype to detect and purify cancer stem cells from biological samples
Uploaded: 2017-05-10T15:00:00
Description: Watch this Scientific Journal Video about Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples at JoVE.com

Maximilian Boeschはオーストリア科学基金FWFのErwinSchrödingerFellowship（助成金番号J-3807）の支援を受けています。

このプロトコルは、標準的な制度倫理審査委員会のガイドラインを完全に遵守しています。ここに示すプロトコールを用いてヒトまたは動物の組織を調査する場合、研究者はその機関または国の関連する審査委員会からの承認を得ることが義務付けられている。 注意：生物学的サンプルを安全に取り扱うための標準的な予防措置がこのプロトコルに適用されます。これ?...

<ol>
	<li>Pattabiraman, D. R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+the+cancer+stem+cells+-+what+challenges+do+they+pose?.">Tackling the cancer stem cells - what challenges do they pose?.</a> Nat Rev Drug Discov. 13 (7), 497-512 (2014).</li><li>Boesch, 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=Heterogeneity+of+Cancer+Stem+Cells:+Rationale+for+Targeting+the+Stem+Cell+Niche.">Heterogeneity of Cancer Stem Cells: Rationale for Targeting the Stem Cell Niche.</a> Biochim Biophys Acta. 1866 (2), 276-289 (2016).</li><li>Li, 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=Suppression+of+cancer+relapse+and+metastasis+by+inhibiting+cancer+stemness.">Suppression of cancer relapse and metastasis by inhibiting cancer stemness.</a> Proc Natl Acad Sci U S A. 112 (6), 1839-1844 (2015).</li><li>Greve, B., Kelsch, R., Spaniol, K., Eich, H. T., G&#246;tte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+in+cancer+stem+cell+analysis+and+separation.">Flow cytometry in cancer stem cell analysis and separation.</a> Cytometry A. 81 (4), 284-293 (2012).</li><li>Marcato, P., Dean, C. A., Giacomantonio, C. A., Lee, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+dehydrogenase:+its+role+as+a+cancer+stem+cell+marker+comes+down+to+the+specific+isoform.">Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform.</a> Cell Cycle. 10 (9), 1378-1384 (2011).</li><li>Golebiewska, A., Brons, N. H., Bjerkvig, R., Niclou, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+side+population+assay+in+stem+cell+and+cancer+stem+cell+research.">Critical appraisal of the side population assay in stem cell and cancer stem cell research.</a> Cell Stem Cell. 8 (2), 136-147 (2011).</li><li>Hirschmann-Jax, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+&amp;#34;side+population&amp;#34;+of+cells+with+high+drug+efflux+capacity+in+human+tumor+cells.">A distinct &amp;#34;side population&amp;#34; of cells with high drug efflux capacity in human tumor cells.</a> Proc Natl Acad Sci U S A. 101 (39), 14228-14233 (2004).</li><li>Zhou, 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=The+ABC+transporter+Bcrp1/ABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype.">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nat Med. 7 (9), 1028-1034 (2001).</li><li>Boesch, 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=The+side+population+of+ovarian+cancer+cells+defines+a+heterogeneous+compartment+exhibiting+stem+cell+characteristics.">The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics.</a> Oncotarget. 5 (16), 7027-7039 (2014).</li><li>Szotek, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+side+population+defines+cells+with+stem+cell-like+characteristics+and+Mullerian+Inhibiting+Substance+responsiveness.">Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.</a> Proc Natl Acad Sci U S A. 103 (30), 11154-11159 (2006).</li><li>Brown, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+benign+and+malignant+prostate+epithelial+Hoechst+33342+side+populations.">Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.</a> Prostate. 67 (13), 1384-1396 (2007).</li><li>Mathew, G., 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=ABCG2-mediated+DyeCycle+Violet+efflux+defined+side+population+in+benign+and+malignant+prostate.">ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate.</a> Cell Cycle. 8 (7), 1053-1061 (2009).</li><li>Engelmann, K., Shen, H., Finn, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF7+side+population+cells+with+characteristics+of+cancer+stem/progenitor+cells+express+the+tumor+antigen+MUC1.">MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1.</a> Cancer Res. 68 (7), 2419-2426 (2008).</li><li>Ho, M. M., Ng, A. V., Lam, S., Hung, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+in+human+lung+cancer+cell+lines+and+tumors+is+enriched+with+stem-like+cancer+cells.">Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells.</a> Cancer Res. 67 (10), 4827-4833 (2007).</li><li>Kato, 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=Endometrial+cancer+side-population+cells+show+prominent+migration+and+have+a+potential+to+differentiate+into+the+mesenchymal+cell+lineage.">Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage.</a> Am J Pathol. 176 (1), 381-392 (2010).</li><li>Bleau, A. 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=PTEN/PI3K/Akt+pathway+regulates+the+side+population+phenotype+and+ABCG2+activity+in+glioma+tumor+stem-like+cells.">PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells.</a> Cell Stem Cell. 4 (3), 226-235 (2009).</li><li>Harris, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+are+enriched+in+the+side+population+cells+in+a+mouse+model+of+glioma.">Cancer stem cells are enriched in the side population cells in a mouse model of glioma.</a> Cancer Res. 68 (24), 10051-10059 (2008).</li><li>Murase, 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=Side+population+cells+have+the+characteristics+of+cancer+stem-like+cells/cancer-initiating+cells+in+bone+sarcomas.">Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas.</a> Br J Cancer. 101 (8), 1425-1432 (2009).</li><li>Boesch, M., Wolf, D., Sopper, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Stem+Cell+Detection+Using+the+DyeCycle-Triggered+Side+Population+Phenotype.">Optimized Stem Cell Detection Using the DyeCycle-Triggered Side Population Phenotype.</a> Stem Cells Int. 2016, 1652389 (2016).</li><li>Golebiewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+in+human+glioblastoma+is+non-tumorigenic+and+characterizes+brain+endothelial+cells.">Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells.</a> Brain. 136 (PT 5), 1462-1475 (2013).</li><li>Boesch, 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=Drug+transporter-mediated+protection+of+cancer+stem+cells+from+ionophore+antibiotics.">Drug transporter-mediated protection of cancer stem cells from ionophore antibiotics.</a> Stem Cells Transl Med. 4 (9), 1028-1032 (2015).</li><li>Fukunaga-Kalabis, 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=Tenascin-C+promotes+melanoma+progression+by+maintaining+the+ABCB5-positive+side+population.">Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population.</a> Oncogene. 29 (46), 6115-6124 (2010).</li><li>Dean, M., Fojo, T., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+stem+cells+and+drug+resistance.">Tumour stem cells and drug resistance.</a> Nat Rev Cancer. 5 (4), 275-284 (2005).</li><li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183 (4), 1797-1806 (1996).</li><li>Telford, W. G., Bradford, J., Godfrey, W., Robey, R. W., Bates, 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=Side+population+analysis+using+a+violet-excited+cell-permeable+DNA+binding+dye.">Side population analysis using a violet-excited cell-permeable DNA binding dye.</a> Stem Cells. 25 (4), 1029-1036 (2007).</li><li>Boesch, 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=High+prevalence+of+side+population+in+human+cancer+cell+lines.">High prevalence of side population in human cancer cell lines.</a> Oncoscience. 3 (3-4), 85-87 (2016).</li><li>Ali, M. Y., Anand, S. V., Tangella, K., Ramkumar, D., Saif, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Primary+Human+Colon+Tumor+Cells+from+Surgical+Tissues+and+Culturing+Them+Directly+on+Soft+Elastic+Substrates+for+Traction+Cytometry.">Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J Vis Exp. (100), e52532 (2015).</li><li>Azari, 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=Isolation+and+expansion+of+human+glioblastoma+multiforme+tumor+cells+using+the+neurosphere+assay.">Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay.</a> J Vis Exp. (56), e3633 (2011).</li><li>Hasselbach, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+high+grade+glioma+cell+culture+from+surgical+specimens+for+use+in+clinically+relevant+animal+models+and+3D+immunochemistry.">Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.</a> J Vis Exp. (83), e51088 (2014).</li><li>Boesch, 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=DyeCycle+Violet+used+for+side+population+detection+is+a+substrate+of+P-glycoprotein.">DyeCycle Violet used for side population detection is a substrate of P-glycoprotein.</a> Cytometry A. 81 (6), 517-522 (2012).</li><li>Patrawala, 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=Side+population+is+enriched+in+tumorigenic,+stem-like+cancer+cells,+whereas+ABCG2++and+ABCG2-+cancer+cells+are+similarly+tumorigenic.">Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic.</a> Cancer Res. 65 (14), 6207-6219 (2005).</li><li>Guha, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+promotes+apoptosis+resistance+of+breast+cancer+cells+and+enrichment+of+side+population+cells+with+cancer+stem+cell-like+properties+via+a+signaling+cascade+involving+galectin-3,+alpha9+nicotinic+acetylcholine+receptor+and+STAT3.">Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, alpha9 nicotinic acetylcholine receptor and STAT3.</a> Breast Cancer Res Treat. 145 (1), 5-22 (2014).</li></ol>

癌の臨床管理の進歩は、CSCを標的とする治療様式の開発にも依存する。生物学的サンプルからのCSCの確実な単離を可能にする方法は、適切な標的の同定を促進し、したがって、この努力において非常に重要である。 SP分析は、ABCの薬物トランスポーターを発現するCSC集団を同定するための確立された、そして貴重な技術であり、DCVの実施は、標準的なフローサイトメトリー機器へのその適用?...

原発性癌治療の有効性は、この疾患の遺伝的および分子的理解の魅力的進歩、治療用抗体および小分子阻害剤などの標的薬物の出現のために、過去10年間で実質的に改善されている。対照的に、転移性および再発性の疾患は依然として典型的に治癒不可能であり、罹患率および死亡率はこれらの臨床的状況において依然として高いままである。 CSCは、腫瘍内の別個の亜集団を表し、クローン原性/腫瘍原性、多剤耐性および非対称細胞分裂1,2などの標準的な幹細胞特性を付与される。したがって、CSCは、転移性の進行および腫瘍の異種性を促進するだけでなく、患者の再発の素因となる治療中も持続する。したがって、治療的CSC除去は、疾患の再発を予防し、がんの長期治癒を可能にする重要な医学的必要性である3 </sup&gt;。 脆弱性の特定およびCSCを根絶する戦略の解明は、その後の発現プロファイリングおよび/または機能検査のための生物学的サンプルからの精製を可能にする方法に大きく依存する。次に、そのような方法は、これらの細胞に特異的な表面、細胞内または機能的マーカーに依存する。 CSC特異的表面マーカーには、CD44、CD133、CD24およびCD90が含まれるが、これらに限定されず、乳癌および大腸癌を含む様々な腫瘍実体におけるCSC集団を同定するために使用されてきた4 。別のマーカーであるアルデヒドデヒドロゲナーゼ（ALDH）は、細胞内局在を示し、機能的に検出することができ、その酵素的変換により光を生じるそれぞれの基質を提供することができる。この試験を用いて、CSC集団は多様な腫瘍実体においても同定されている5 。一般にSP分析と呼ばれ、ここではm<html> ABC薬物輸送体による活性色素流出を利用して、蛍光dim型幹様細胞の小さな集団を同定する6,7,8。これを達成するために、所与の試料を、受動拡散によってすべての細胞に入り、結合のために核およびミトコンドリアDNAを標的とする親油性DNA結合フルオロフォアの存在下でインキュベートする。 ABC薬物輸送体発現を欠いている非CSCsは染料を蓄積し、明るい蛍光をもたらし、CSCsは蛍光を減少させる染料を能動的に押し出す。薬物輸送体活性の薬理学的阻害は、SP表現型を排除し機能的に確認し、制御目的に使用すべきである。 SP特性を示すCSC集団が、とりわけ、卵巣癌9,10 、前立腺癌11 、&gt; 12、乳癌13 、肺癌14 、子宮内膜癌15 、神経膠腫16,17および骨肉腫18 。重要なことには、特定の腫瘍細胞識別戦略（ある種の宿主細胞集団もSP特性を示すことができる）のような追加の課題があるにもかかわらず、SPアッセイは癌細胞株および原発腫瘍組織の両方に適合する19,20 。 2つの最も確立されたSPを与える薬物輸送体は、ABCB1 / P-糖タンパク質/ MDR1 / CD243およびABCG2 / Bcrp1 / CD338である。しかし、他の薬物トランスポーターもSP表現型の分子決定因子（ 例えば 、ABCB5）であり得る22 。 ABCB1ABCG2の活性は、fumitremorgin C（FTC）6,19を用いて特異的に排除することができるが、ベラパミルで効率的にブロックすることができる。 SP分析の特別な強さは、他の染色（ 例えば 、表面マーカーおよびALDH）と組み合わせることができ、生細胞の回収を可能にし、下流の機能検査と互換性があることである。さらに、SPC検出は、CSC集団の中でABC薬物輸送体の高い保存率のために広く適用可能である（9,23）。 もともと、SP検出は、Hoechst 33342をトリガー色素24として用いて行われている。この染料は優れた解像度を達成するが、紫外線レーザー励起を必要とする。したがって、その適用性は当然、ハイエンドフローサイトメトリー機器に限定される。 DyeCycle Violet（DCV） 25の登場により、新しいAvenuこの方法を紫外線レーザー源を欠く標準的なフローサイトメトリー機器（DCV-SP細胞を分解するのに紫色レーザー源で十分）に応用することができました。重要なことに、Hoechst 33342およびDCVは共通のポンプ特異性を共有しており、どちらの色素も同じ細胞集団を同定する必要があることを示しています。 ここでは、DCVベースのSP分析の詳細な実験プロトコルを提供し、独立したラボで素早く簡単に再生することができます。我々は、この有用な細胞生物学的方法の最適化と標準化に貢献すべきであるCSC研究者のための資料としてこの論文を認識する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vybrant DyeCycle Violet</td>
			<td>Thermo Fisher Scientific</td>
			<td>V35003</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td>Dissolve in ethanol</td>
		</tr>
		<tr>
			<td>Fumitremorgin C</td>
			<td>Sigma-Aldrich</td>
			<td>F9054</td>
			<td>Dissolve in DMSO</td>
		</tr>
		<tr>
			<td>7-AAD (or propidium iodide)</td>
			<td>BioLegend</td>
			<td>420403</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Standard cell culture reagents (e.g., medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, etc.)</td>
			<td>Different suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample/cells of interest (e.g., human or murine cancer cell line, human or murine tumor tissue)</td>
			<td>Different suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometric instrument (analyzer or cell sorter)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Violet laser source required</td>
		</tr>
		<tr>
			<td>FACS tubes (round-bottom)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Tubes with cap recommended</td>
		</tr>
		<tr>
			<td>70 &#181;m cut-off strainers</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Optional but recommended</td>
		</tr>
		<tr>
			<td>Digestion enzyme mix (e.g., collagenase/dispase/Dnase)</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Only relevant for tissue dissociation</td>
		</tr>
		<tr>
			<td>Flurochrome-conjugated antibodies against surface markers of interest</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>ALDEFLUOR Kit</td>
			<td>STEMCELL Technologies</td>
			<td>01700</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

harnessing the dna dye-triggered side population phenotype to detect and purify cancer stem cells from biological samples

がんは幹細胞に起因する疾患であり、これらの細胞の根絶が主要な治療目標となっている。 Cancer Stem Cells（CSCs）の脆弱性を解読し、適切な分子標的を特定することは、細胞系やex vivo腫瘍組織などの異種試料での特異的差別を可能にする方法に依存しています。フローサイトメトリー/ FACSは、単一細胞レベルで生物学的サンプルをマルチパラメトリックに解剖するための強力な技術であり、下流の分析のために生きた細胞を回収するための選択方法である。 CD44およびCD133のような表面マーカーならびにアルデヒドデヒドロゲナーゼ酵素活性の検出は、FACSによる腫瘍サンプルからのCSCを定義および分類するためにしばしば用いられてきた。ここで方法論的詳細に示されている相補的アプローチは、一般に側方集団（SP）と呼ばれる蛍光dim細胞の明確な集団を同定するABC薬物トランスポーターによる機能的色素押出を利用する。 SPがん細胞は、標準的な幹細胞の特徴を示し、色素押し出し薬物輸送体（最も頻繁にはABCB1 / P-糖タンパク質/ MDR1 / CD243およびABCG2 / Bcrp1 / CD338）を阻害する物質を使用して廃止され、機能的に確認され得る。さらに、SPアッセイは、表面抗原の染色、アルデヒドデヒドロゲナーゼ検出、および死細胞識別（ 例えば 、7-AADまたはヨウ化プロピジウム（PI））などの他のフローサイトメトリー評価と適合する。したがって、我々は、表現型パラメータではなく機能的に基づいて、CSC同定、単離およびサブキャラクタリゼーションを機械的に行うための価値ある、広く適用可能な方法を記載する。もともとHoechst 33342をトリガー色素として使用していましたが、ここではバイオレットレーザー光源を備えたフローサイトメーターで分解可能な、より新しいViolet色素に基づくSP表現型に焦点を当てます。

A2780細胞の代表的なSP分析であり、これは、以前に細胞の1％未満を占めるSPを保持することが示されたヒト卵巣癌細胞株である9 。細胞を、10％（ v / v ）FBS、2mM L-グルタミンおよび1×ペニシリン/ストレプトマイシンを補充したRPMI1640中で37℃で培養し、0.05％トリプシン-EDTAでの処理を用いて85％コンフルエンシーで採取した。細胞をPBSで洗浄し?...

Watch this Scientific Journal Video about Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples at JoVE.com

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

生物学的試料からの幹細胞集団の特徴付けおよび単離を可能にする方法は、癌およびそれを超える幹細胞標的化治療の進歩にとって重要である。ここでは、色素誘発性の側方集団表現型を用いた癌幹細胞単離のための詳細なプロトコールを提供する。

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 ...

The overall goal of this procedure is to detect putative cancer stem cell populations in biological samples based on dye extrusion capacity of the side population cells. This method addresses a key question in cancer stem cell research regarding reliable means for detection and purification of cancer stem cells from biological samples. The main advantage of this technique is that it deploys functional markers and therefore providing better resolution.In addition, expression of these ABC drug transporters is highly conserved among cancer stem cells and therefore we can use this technique for many different cancer entities. The implications of this technique extends toward the discovery of anti-cancer stem cell therapeutics because it facilitates downstream analysis such as gene expression profiling and target validation in highly purified cancer cells. So this method can provide insight into the characteristics of cancer stem cells.We can also use this method to characterize as a systems comprising ABC drug transporter expressing populations such as the bone marrow. Demonstrating the procedure will be Elisabeth Hoflehner, a technician from my laboratory. To begin, culture the desired human cancer cell line at 37 degrees celsius in 10 to 30 milliliters of RPMI 1640 medium supplemented with 10%FBS, two millimeter L-glutamine, and antibiotics.When cells reach approximately 70 to 90%confluence, remove the medium and add two milliliters of 0.05%trypsin-EDTA to detach the cells from the culture dish surface. Then, wash the cells with 10 milliliters of culture medium, transfer the suspension to a clean 15 milliliter tube, and spin it at 250 times G at four degrees celsius for five minutes. Once the cells are pelleted, discard the suprenatent and resuspend the pellet in one milliliter of culture medium.Mix a small aliquot of the cell suspension with trypan blue solution and count the cells using a hemocytometer. To prepare test samples, suspend the cells in fresh culture medium to obtain a cell suspension of one time ten to the six cells per milliliter. After adjusting the cell density, transfer three milliliters of the sample to a 15 milliliter round bottom tube labeled as test.Then, add 10 micromolar of violet dye to the sample and gently vortex the suspension. Next, prepare inhibition controls by transferring 500 microliters of the dye containing cell suspension to two flow cytometry tubes labeled as control. Add 50 micromolar of Verapamil to the first.And 20 micromolar of Fumitremorgin C to the second tube. And mix the solutions by gently pipetting. Cover all tubes with caps and incubate them at 37 degrees celsius in the dark for 90 minutes.Gently agitate the samples every 15 minutes. Once the staining is completed, wash the inhibition controls in three to four milliliters of ice cold PBS. And prepare aliquots of the test sample for subsequent costaining with antibodies.Wash this aliquots as well and filter all samples through 70 micron cut off strainers to remove potential cell aggregates. And then centrifuge the suspensions at 250 times G at four degrees celsius for five minutes. After resuspending the resulting pellet in 100 microliters of PBS, maintain the cells on ice protected from light.Next, add an appropriate panel of flurochrome conjugated antibodies to the cells prestained with violet dye. Vortex the suspensions and incubate them at four degrees celsius in the dark for 20 to 30 minutes. After incubation, wash the cells in three to four milliliters of ice cold PBS, filter the sample if desired.And then centrifuge at 250 times G at four degrees celsius for five minutes. Discard the supernatent and resuspend the pelleted cells in 100 microliters of PBS. Finally, add 7-AAD to the cells prestained with violet dye.And incubate the samples for five to 10 minutes in the dark. To analyze the cells using the flow cytometer, gate an appropriate cell population on a FSC SSC dot plot. Then, using different FSC signals, exclude from the selected population all cell aggregates.Exclude cells revealing positive 7-AAD staining to obtain the population of viable cells. To create a dot plot for blue and red dye emission, display the blue fluorescent channel on the y-axis and the red fluorescent channel on the x-axis. Switch both channels to the linear mode and adjust the detector voltage accordingly so that the side population is located in the lower left part of the plot.Then, gate the side population. And stop the acquisition. Next, load the inhibition controls and acquire the data.Finally, create a dot plot displaying the red florescence of violet dye on the x-axis and an appropriate florescence channel on the y-axis plotted in logarithmic mode. Load the sample then adjust the detector voltages accordingly and identify the faction of the side population cells that express the investigated surface. Then, record the data.Presented here are representative results of the side population analysis carried out in human ovarian cancer cells. The side population distinguished by the analysis of blue and red-violet emission was identified in the left lower quadrant of the dot plot and constituted 0.70%of the whole analyzed population. The later use of efflux inhibitors resulted in the disappearance of the florescence signal observed for the previously identified cells confirming their side population phenotype.Finally, the human ovarian cells were also characterized in terms of ABCG2 expression. ABCG2 positive staining was observed in 0.67%of cells corresponding to the identified side population. Once mastered, this procedure can be done in approximately two to three hours if performed properly.When attempting this procedure, it is important to remember that the side population phenotype strictly depends on an optimal dye concentration to cell density ratio. Hydration of both variables is therefore highly recommended prior to the experiment. Following this procedure, other methods like gene expression profiling can be performed in order to further characterize side population cells hence to identify novel cancer stem cell specific targets for future drug development.After its development, this technique paved the way for researchers in the field of stem cell biology to explore the characteristics of tissue stem cells and cancer stem cells, both in mice and men. After watching this video, you should have a good understanding on how to perform violet dye triggered side population SA and how to detect putative cancer stem cells in biological samples in your own lab. Don't forget that working with biological samples can be extremely hazardous and that precautions such as the use of PPE and biological safety cabinets should be taken when working with this procedure.

Research

JoVE Journal

Cancer Research

Cell-Free DNA Integrity Analysis in Urine Samples

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 ...

がん研究

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

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. ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

李 Fraumeni 症候群患者由来の誘導多能性幹細胞を用いた骨肉腫をモデリング

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

婦人科腫瘍および乳がん腫瘍から癌幹細胞球体を取得する

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Analysis of Side Population in Solid Tumor Cell Lines

固形腫瘍細胞株における側集団の解析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

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

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 ...

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

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 ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

薬物耐性表現型を調節する因子を同定するためのプールされたshRNAライブラリースクリーニング

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples

粗生物学的サンプル中のトポイソメラーゼ1活性のシンプルで高速なローリングサークル増幅ベースの検出

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, ...