This study was supported by a grant from the Baden-W&#252;rttemberg Stiftung (Adult Stem Cells Program II) awarded to C.L. We thank Dr. Martina Konantz for critical input and review of the manuscript. We thank Emmanuel Traunecker and Toni Krebs from the DBM FACS Facility (University Hospital Basel) for assistance with FACS sorting.

OVCAR-3 insan yumurtalık karsinoma hücrelerinde 1. Üretim Stabil Lentivirüsler Sox2 düzenleyici bölge, haberci yapı İçeren kalıt <ol><li> Tarif 10,21 bir Sox2 düzenleyici bölgesini tanıyan bir raportör yapısı ile HEK 293T paketleme hücre hattı transfekte edilmesiyle lentiviral parçacıkları oluşturur. Not: raportör yapısının ayrıca ileride tdTomato floresan protein ProteoTuner kalkan sisteminin dengesizleşmesine alanı içerir. Shield1 böylece floresan proteini 22 aşağılamak için proteasom engelleyen istikrarsızlaştırma etki bağlanır. </li><li> 24 saatlik bir süre boyunca, lentiviral parçacıklarla OVCAR-3 hücreleri nakletmek. Daha sonra, viral süpernatant kaldırmak ve tam ortamda fosfat tamponlu tuz (PBS) ve kültürlenmiş hücreleri yıkayın (RPMI, 100 U / ml penisilin / ml streptomisin, 100 ug,% 10 FBS ile takviye edilmiş). </li><li> 48 saat sonra, 10 ug / ml puromycin kültürlere ilave edildi ve uygun bir şekilde kalıt aktarımlı hücrelerin seçimine izin vermek için 5 gün için muhafaza edilmiştir. </li></ol> Hücre Sorting ve Kaplama 2. Hazırlık <ol><li> 1 at Shield1 ekleyin: hücre sıralama öncesinde 1,000 seyreltme 24 saat. Negatif kontrol olarak Shield1 tedavisi (Şekil 1) bir stably transduse OVCAR-3 hücreleri kullanır. Şişeden aspire ortam, 3 dakika boyunca% 0.05 tripsin-EDTA ile hücreler 1 x hücrelerin PBS ile yıkayın ve trypsinize. </li><li> 5 dakika - (25 ° C 15) oda sıcaklığında 300 xg&#39;de hücre sayıları, santrifüj hücreleri saymak, (yukarıya bakınız) tam orta kullanarak tripsin durdurun. </li><li> Durusu süpernatant ve tekrar süspansiyon hücreleri dikkatli bir 0,5-1 ml steril PBS. </li><li> Tek bir hücre süspansiyonu elde etmek için 40 mikron hücre süzgeç kapağı filtresi kullanın. </li><li> Ml başına 5000000 hücrelere hücre sayımı ayarlayın. </li><li> , MEGM büyüme faktörleri, sitokinler ve ek takviye (100 ul ortam ultra düşük bağlanma, 96 gözlü levhalar küreler hazırlanmasıB-27, heparin-sodyum; ya da büyüme faktörleri, sitokinler ve ek B-27, ya da% 1 metilselüloz eklenmeden heparin-sodyum ile desteklenmiş DMEM / F12), aynı zamanda Tablo 1 e bakınız. İsteğe bağlı olarak mümkün kontaminasyon riskini en aza indirmek için, 100 U / ml penisilin ve 100 ug / ml streptomisin bir konsantrasyonda ortam antibiyotik ekleyin. </li><li> Sıralama TTT + ve yukarıda hazırlanan 96 oyuklu plakalara RFP- hücreleri, oyuk başına 1 hücre (tek hücre bazlı küreler deneyi), sırasıyla oyuk başına 100 hücre (çoklu hücre bazlı küreler deneyi). 100 μ meme, kılıf basıncı 20 psi ve verim maskesi 0, saflık maskesi 32, faz maskesi 16: Tek hücreli modu, kurulum sırala kullanarak (Malzeme) sıralama ticari olarak temin hücre sıralayıcı gerçekleştirin. </li><li> Mikroskopik olarak (tek bir hücre bazlı deney için) hücreler içeren kuyu puanlama ile ve çoklu hücre bazlı tahlilde (bireysel çukurlarındaki hücre saymak suretiyle kaplama verimlilik değerlendirmek; Şekil 2, </strong&gt;). </li><li> Orta alanda standart koşullar altında kuluçkaya bırakılır, 37 ° C&#39;de ve% 5 CO2 (bileşim için adım 2.6). Günlük bFGF (20 ng / ml) ve EGF (20 ng / ml), Supplement. </li><li> Bir hafta sonra, entegre muhabiri sisteminden floresan sinyal tespit etmek 4X veya 10X büyütme ve floresan mikroskop ile standart mikroskop kullanılarak tümör küreler ortaya çıkan numaralarını saymak. &quot;Küçük&quot; küreler olarak 100 mikron (Şekil 3) - çapı 50 ile &quot;büyük&quot; küreler olarak 100 mikron aşan bir çapa sahip küreler ve küreler Kont. Gerçek küreler saymak ve kümeler hücre değil emin olun. NOT: Tek hücre bazlı deneylerde küre oluşumu kültür 10 (7 karşı) gün sonra mikroskopik olarak puan daha kolaydır. </li><li> TTT + küre-oluşturucu hücre ve tek bir hücre bazlı deneylerde, sırasıyla RFP- hücreleri (her bir deney için, bir 96-yuvalı plaka) ve çok hücre-ba oranını hesaplamakShaw ve arkadaşları tarafından sunulan sed küreler deneyler (her bir deney için, bir oyuk). 16 Not: küre oluşturan hücrelerin (%) = (küre sayısı) / (tohumlanmıştır hücre sayısı) oranı 100 x </li></ol> Kürelerin 3. Seri Pasajlanması <ol><li> Oda sıcaklığında 10 dakika boyunca 300 x g&#39;de uygun bir steril tüp ve santrifüj her bir oyuğa içeriğini yerleştirin. Çok hücre tabanlı küreler tahliller için, birlikte iyi biri küreler toplamak. PBS ve uzun santrifüj 2 dakika ile 5 kez - KUYU 3 yıkayın. Tek hücre tabanlı küreler tahliller için, bireysel küreler toplamak. Nedeniyle hücrelerin düşük sayıda, santrifüjleme ve yıkama adımları için 1.5 ml tüpler kullanmaktadır. </li><li> % 0.05 tripsin-EDTA, 200 ul süpernatan ve tekrar süspansiyon pelet çıkarın. </li><li> Optimal hücre ayrılmasını sağlamak amacıyla, yumuşak bir çalkalayıcı içinde 5 dakika için 37 ° C &#39;de bir hücre süspansiyonu inkübe edilir. Alt hücre ölümü oranı cep hattı için trypsinization zaman Optimize: hiçbir büyük olursaküreler yavaşça 100 ul pipet kullanarak görünür çiğnemek olduğunu. Durumunda büyük küreler, hala mevcut başka bir 3 dakika tripsin ile kuluçkaya ve daha sonra toz haline getirme adıma geçin. Tek durumunda hücre tabanlı deneyler trypsinization adımı sırasında canlı hücrelerin optimum hücre verimi için zaman optimize etmek emin olun. </li><li> Tripsin inaktive tek hücre tahlilleri, ilave bir 2 dakika boyunca santrifüj halinde, 10 dakika boyunca 300 x g&#39;de 500 ul komple ortam ve santrifüj ekleyin. </li><li> Küreler orta dikkatle süpernatant ve tekrar süspansiyon hücreleri çıkarın. </li><li> Bir tek hücre süspansiyonu elde etmek için 40 mikron hücre süzgeç kapağı filtresi kullanın. </li><li> TTT + ve çoklu hücre-bassed deneylerde RFP- hücrelerin kullanılması durumunda, analiz akış sitometresi aracılığı ile her bir göze daha sonra süspansiyon içinde flüoresan hücrelerin yüzdesini değerlendirmek. </li><li> Yukarıda ayrıntılı olarak açıklandığı gibi hazırlanan tek hücre seri şarjı tahlilleri, tohum oyuk başına 1 hücre, bir ultra düşük bağlanma, 96 oyuklu bir plakaya için. Tek bir kişi alandan yaklaşık 20 ayrı kuyu tohum. Çoklu hücre bazlı primer kürelerin şarjı tahlilleri için, tohum hücreleri yeni bir 96 oyuklu bir plakaya birinci küre bir kuyudan elde edilen ve mikroskopi ile hücre sayıları ertesi gün sayısı. </li><li> Adım 2.11 tarif edilen formüle göre ikincil küreler tahlillerinde küre oluşturan hücrelerin oranını değerlendirmek. </li></ol> 4. Sonuç Analizi <ol><li> Bağımsız üçlü olarak yapılan deneylerden elde edilen sonuçları analiz ve iki taraflı Student t-testi istatistiksel analiz için normal dağılıma değerleri ve aksi Mann-Whitney-Testleri analiz için kullanabilirsiniz. </li></ol>

<ol>
	<li>Pardal, R., Clarke, M. F., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+the+principles+of+stem-cell+biology+to+cancer.">Applying the principles of stem-cell biology to cancer.</a> Nat Rev Cancer. 3 (12), 895-902 (2003).</li><li>Reya, T., Morrison, S. J., Clarke, M. F., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+cancer,+and+cancer+stem+cells.">Stem cells, cancer, and cancer stem cells.</a> Nature. 414 (6859), 105-111 (2001).</li><li>Ahmed, N., Abubaker, K., Findlay, J. K. <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+stem+cells:+Molecular+concepts+and+relevance+as+therapeutic+targets.">Ovarian cancer stem cells: Molecular concepts and relevance as therapeutic targets.</a> Mol Aspects Med. 39, 110-125 (2014).</li><li>Silva, I. 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=Aldehyde+dehydrogenase+in+combination+with+CD133+defines+angiogenic+ovarian+cancer+stem+cells+that+portend+poor+patient+survival.">Aldehyde dehydrogenase in combination with CD133 defines angiogenic ovarian cancer stem cells that portend poor patient survival.</a> Cancer Res. 71 (11), 3991-4001 (2011).</li><li>Kryczek, I., 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=Expression+of+aldehyde+dehydrogenase+and+CD133+defines+ovarian+cancer+stem+cells.">Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells.</a> Int J Cancer. 130 (1), 29-39 (2012).</li><li>Stewart, J. 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=Phenotypic+heterogeneity+and+instability+of+human+ovarian+tumor-initiating+cells.">Phenotypic heterogeneity and instability of human ovarian tumor-initiating cells.</a> Proc Natl Acad Sci U S A. 108 (16), 6468-6473 (2011).</li><li>Ginestier, 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=ALDH1+is+a+marker+of+normal+and+malignant+human+mammary+stem+cells+and+a+predictor+of+poor+clinical+outcome.">ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.</a> Cell Stem Cell. 1 (5), 555-567 (2007).</li><li>Chang, 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=ALDH1+expression+correlates+with+favorable+prognosis+in+ovarian+cancers.">ALDH1 expression correlates with favorable prognosis in ovarian cancers.</a> Mod Pathol. 22 (6), 817-823 (2009).</li><li>Cannistra, S. 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=CD44+variant+expression+is+a+common+feature+of+epithelial+ovarian+cancer:+lack+of+association+with+standard+prognostic+factors.">CD44 variant expression is a common feature of epithelial ovarian cancer: lack of association with standard prognostic factors.</a> J Clin Oncol. 13 (8), 1912-1921 (1995).</li><li>Bareiss, P. 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=SOX2+expression+associates+with+stem+cell+state+in+human+ovarian+carcinoma.">SOX2 expression associates with stem cell state in human ovarian carcinoma.</a> Cancer Res. 73 (17), 5544-5555 (2013).</li><li>Pham, D. 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=SOX2+expression+and+prognostic+significance+in+ovarian+carcinoma.">SOX2 expression and prognostic significance in ovarian carcinoma.</a> Int J Gynecol Pathol. 32 (4), 358-367 (2013).</li><li>Lengerke, 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=Expression+of+the+embryonic+stem+cell+marker+SOX2+in+early-stage+breast+carcinoma.">Expression of the embryonic stem cell marker SOX2 in early-stage breast carcinoma.</a> BMC Cancer. 11, 42 (2011).</li><li>Lapidot, 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=A+cell+initiating+human+acute+myeloid+leukaemia+after+transplantation+into+SCID+mice.">A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.</a> Nature. 367 (6464), 645-648 (1994).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255 (5052), 1707-1710 (1992).</li><li>Dontu, 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes Dev. 17 (10), 1253-1270 (2003).</li><li>Shaw, F. 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=A+detailed+mammosphere+assay+protocol+for+the+quantification+of+breast+stem+cell+activity.">A detailed mammosphere assay protocol for the quantification of breast stem cell activity.</a> J Mammary Gland Biol Neoplasia. 17 (2), 111-117 (2012).</li><li>Leis, O., 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=Sox2+expression+in+breast+tumours+and+activation+in+breast+cancer+stem+cells.">Sox2 expression in breast tumours and activation in breast cancer stem cells.</a> Oncogene. 31 (11), 1354-1365 (2012).</li><li>Higgins, D. 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=Brain+tumor+stem+cell+multipotency+correlates+with+nanog+expression+and+extent+of+passaging+in+human+glioblastoma+xenografts.">Brain tumor stem cell multipotency correlates with nanog expression and extent of passaging in human glioblastoma xenografts.</a> Oncotarget. 4 (5), 792-801 (2013).</li><li>Wang, Y. J., Bailey, J. M., Rovira, M., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphere-forming+assays+for+assessment+of+benign+and+malignant+pancreatic+stem+cells.">Sphere-forming assays for assessment of benign and malignant pancreatic stem cells.</a> Methods Mol Biol. 980, 281-290 (2013).</li><li>Li, Y. F., Xiao, B., Tu, S. F., Wang, Y. Y., Zhang, X. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+and+identification+of+colon+cancer+stem+cell-derived+spheres+from+the+Colo205+cell+line.">Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line.</a> Braz J Med Biol Res. 45 (3), 197-204 (2012).</li><li>Wu, F., 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+two+novel+phenotypically+distinct+breast+cancer+cell+subsets+based+on+Sox2+transcription+activity.">Identification of two novel phenotypically distinct breast cancer cell subsets based on Sox2 transcription activity.</a> Cell Signal. 24 (11), 1989-1998 (2012).</li><li>Banaszynski, L. A., Chen, L. C., Maynard-Smith, L. A., Ooi, A. G., Wandless, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+reversible,+and+tunable+method+to+regulate+protein+function+in+living+cells+using+synthetic+small+molecules.">A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.</a> Cell. 126 (5), 995-1004 (2006).</li><li>Kawase, Y., Yanagi, Y., Takato, T., Fujimoto, M., Okochi, H. <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+multipotent+adult+stem+cells+from+the+skin:+transforming+growth+factor-beta+(TGF-beta)+facilitates+cell+growth.">Characterization of multipotent adult stem cells from the skin: transforming growth factor-beta (TGF-beta) facilitates cell growth.</a> Exp Cell Res. 295 (1), 194-203 (2004).</li><li>Walia, 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=Loss+of+breast+epithelial+marker+hCLCA2+promotes+epithelial-to-mesenchymal+transition+and+indicates+higher+risk+of+metastasis.">Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis.</a> Oncogene. 31 (17), 2237-2246 (2012).</li><li>Leung, E. 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=Non-small+cell+lung+cancer+cells+expressing+CD44+are+enriched+for+stem+cell-like+properties.">Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties.</a> PLoS One. 5 (11), e14062 (2010).</li><li>Bertolini, 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=Highly+tumorigenic+lung+cancer+CD133++cells+display+stem-like+features+and+are+spared+by+cisplatin+treatment.">Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment.</a> Proc Natl Acad Sci U S A. 106 (38), 16281-16286 (2009).</li><li>Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. <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-like+cells+can+be+isolated+with+drug+selection+in+human+ovarian+cancer+cell+line+SKOV3.">Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3.</a> Acta Biochim Biophys Sin (Shanghai). 42 (9), 593-602 (2010).</li><li>Wang, H., Zhang, Y., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+and+breast+cancer+spheres+are+similar+in+transcriptomic+features+and+sensitive+to+fenretinide).">Ovarian and breast cancer spheres are similar in transcriptomic features and sensitive to fenretinide).</a> Biomed Res Int. 2013, 510905 (2013).</li><li>Du, Y. R., 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=Effects+and+mechanisms+of+anti-CD44+monoclonal+antibody+A3D8+on+proliferation+and+apoptosis+of+sphere-forming+cells+with+stemness+from+human+ovarian+cancer.">Effects and mechanisms of anti-CD44 monoclonal antibody A3D8 on proliferation and apoptosis of sphere-forming cells with stemness from human ovarian cancer.</a> Int J Gynecol Cancer. 23 (8), 1367-1375 (2013).</li><li>Xiang, 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=Interleukin-17+produced+by+tumor+microenvironment+promotes+self-renewal+of+CD133+cancer+stem-like+cells+in+ovarian+cancer.">Interleukin-17 produced by tumor microenvironment promotes self-renewal of CD133 cancer stem-like cells in ovarian cancer.</a> Oncogene. , (2013).</li><li>Liu, T., Cheng, W., Lai, D., Huang, Y., Guo, L. <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+primary+ovarian+cancer+cells+in+different+culture+systems.">Characterization of primary ovarian cancer cells in different culture systems.</a> Oncol Rep. 23 (5), 1277-1284 (2010).</li><li>Soritau, O., 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=Enhanced+chemoresistance+and+tumor+sphere+formation+as+a+laboratory+model+for+peritoneal+micrometastasis+in+epithelial+ovarian+cancer.">Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.</a> Rom J Morphol Embryol. 51 (2), 259-264 (2010).</li><li>Shi, M. F., 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+cancer+stem+cell-like+cells+from+human+epithelial+ovarian+carcinoma+cell+line.">Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line.</a> Cell Mol Life Sci. 67 (22), 3915-3925 (2010).</li><li>Zhang, 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=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68 (11), 4311-4320 (2008).</li><li>Liang, 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=The+hypoxic+microenvironment+upgrades+stem-like+properties+of+ovarian+cancer+cells.">The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells.</a> BMC Cancer. 12, 201 (2012).</li></ol>

The authors have nothing to disclose.

Küreler kültürleri kanser kök hücre potansiyeli tahlil, insan tümör hücrelerinin 15,25,26 geniş bir kök-benzeri hücreler için zenginleştirmek için yaygın olarak kullanılan bir yöntemdir. Bu kültür koşulları altında, kendini yenileme yeteneğinden yoksun kanser hücreleri ayırt etmek ve en sonunda hücre ölümü beklenmektedir. Başlangıçta hücre kümeleri veya özellikle ilköğretim tayinlerde bile tümör küre oluşturabilir rağmen, onlar yüzünden kendini yenileyen özellikler...

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.
A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable6. Consistently, in ovarian cancer, other than for example in breast carcinoma7, expression of ALDH1 associates with favorable outcome8 and expression of the proposed stem cell marker CD44 variant has no prognostic value9. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells10 and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.
By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice10,13.
An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells14. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast17, brain18, pancreas19 and colon20 tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models10. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells10. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo10 suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.
In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

<table border="0" cellpadding="0" cellspacing="0" width="636">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:189px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-Attachment-plate&#160;</td>
			<td>Corning</td>
			<td style="width:119px;">3474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGM</td>
			<td>Lonza</td>
			<td>CC-3151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>Lonza</td>
			<td style="width:119px;">CC-4136</td>
			<td>SingleQuots&#8482; Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrocortison</td>
			<td>Lonza</td>
			<td style="width:119px;">CC-4136</td>
			<td>SingleQuots&#8482; Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF</td>
			<td>Lonza</td>
			<td style="width:119px;">CC-4136</td>
			<td>SingleQuots&#8482; Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGF</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>end concentration: 20 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>end concentration: 20 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Invitrogen/ Gibco</td>
			<td>17504-044</td>
			<td>end concentration: 1x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin-Natrium-25000 IE</td>
			<td>Ratiopharm</td>
			<td style="width:119px;">N68542.02</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FCS&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Gibco</td>
			<td style="width:119px;">21875-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s PBS (1x)</td>
			<td>Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shield1&#160;</td>
			<td>Clontech</td>
			<td style="width:119px;">632189</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td>Gibco</td>
			<td style="width:119px;">21041-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12 (powder)</td>
			<td>Gibco</td>
			<td style="width:119px;">42400-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl cellulose</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">M0387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td>applichem</td>
			<td style="width:119px;">A2856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cell sorter&#160;</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>Aria III cell sorter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS analyser</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>accuri c6 flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:112px;">Olympus&#160;</td>
			<td style="width:119px;"></td>
			<td>IX50 Osiris</td>
		</tr>
	</tbody>
</table>

evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

Geleneksel küreler deneylerinde, RFP + OVCAR-3 hücrelerinin genel RFP- hücrelerin% 20, yaklaşık% 40, birincil küreler deneyi (Şekil 4A) &#39;de tek bir tümör alanına yol açmıştır. Ayrıca, RFP + hücreleri tarafından oluşturulan küreleri RFP- hücrelerden oluşan daha büyük boyutta olmuştur. Tek bir hücre bazlı deneylerde kaplama olduğunda, TTT + hücreleri aynı zamanda, yukarıda sonuçları teyit RFP- hücrelerden daha fazla küreler oluşturulur. B...

Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Çoklu ve tek hücre bazlı küreler tahliller kullanılarak, insan yumurtalık karsinoma hücrelerinde Kök Hücre Özelliklerinin Değerlendirilmesi

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

evaluation-stem-cell-properties-human-ovarian-carcinoma-cells-using

Research

JoVE Journal

Medicine

9.5K Görüntüleme.  University Hospital Basel. In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.