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.

1. Generazione di Ovcar-3 umane cellule di carcinoma ovarico stabilmente trasdotte con lentivirus contenente il Reporter Construct SOX2 Regione Regulatory <ol><li> Generare particelle lentivirali trasfettando la linea cellulare 293T-imballaggi HEK con un costrutto giornalista riconoscere una regione regolatoria SOX2 come descritto 10,21. NOTA: Il giornalista costruire inoltre contiene un dominio destabilizzazione del sistema Shield ProteoTuner avanti della proteina tdTomato fluorescenza. Shield1 lega al dominio destabilizzazione impedendo così il proteasoma a degradare la proteina fluorescente 22. </li><li> Trasdurre OVCAR-3 celle con particelle lentivirali per un periodo di tempo di 24 ore. Successivamente, rimuovere il surnatante virale e lavare le cellule con tampone fosfato salino (PBS) e coltivate in mezzo completo (RPMI supplementato con 10% FBS, 100 U / ml di penicillina, 100 mg / ml di streptomicina). </li><li> 48 ore più tardi, 10 ug / ml puroMycin sono stati aggiunti alle culture e mantenuto per 5 giorni per consentire la selezione di cellule trasdotte correttamente. </li></ol> 2. Preparazione di cellule Ordinamento e placcatura <ol><li> Aggiungi Shield1 a 1: 1.000 diluizione 24 ore prima della separazione delle cellule. Utilizzare stabilmente trasdotte Ovcar-3 celle senza trattamento Shield1 come controlli negativi (Figura 1). Aspirare i media da pallone, lavare le cellule con PBS 1x e trypsinize cellule con 0,05% tripsina-EDTA per 3 min. </li><li> Smettere di tripsina utilizzando terreno completo (vedi sopra), contare il numero di cellule, le cellule centrifugare a 300 xg a RT (15 - 25 ° C) per 5 min. </li><li> Decantare il surnatante e risospendere le cellule con cura in 0,5 - 1 ml di PBS sterile. </li><li> Utilizzare 40 micron filtro cap filtro cella per ottenere cella singola sospensione. </li><li> Regolare conta delle cellule a 5 milioni di cellule per ml. </li><li> Preparare basso attaccamento piastre ultra 96 ​​pozzetti con 100 microlitri sfere medio (MEGM completato con fattori di crescita, citochine, e supplementi,B-27, eparina di sodio; o DMEM / F12 addizionato con fattori di crescita, citochine e integratori, B-27, eparina di sodio, con o senza l&#39;aggiunta di 1% metilcellulosa, vedi anche tabella 1). Facoltativamente aggiungere antibiotici per il mezzo ad una concentrazione di 100 U / ml penicillina e 100 ug / ml di streptomicina per minimizzare il rischio di possibili contaminazioni. </li><li> Ordina RFP + e cellule RFP- in preparati piastre a 96 pozzetti dall&#39;alto, 1 cella per pozzetto (single cell-based sfere test) e di 100 cellule per bene (più saggio sfere a base di cellule), rispettivamente. Eseguire sorta su disponibile in commercio cell sorter (vedi Materiali) utilizzando la modalità singola cella, Sort installazione: 100 μ ugello, pressione della guaina 20 psi, e la resa maschera 0, maschera purezza 32, maschera di fase 16. </li><li> Valutare placcatura efficienza microscopicamente segnando pozzetti contenenti cellule (per il saggio basato su cellule singole) e contando il numero di cellule in singoli pozzetti (per il dosaggio più basato su cellule; Figura 2 </strong&gt;). </li><li> Incubare le cellule in condizioni standard in ambito di media (per la composizione vedi punto 2.6) a 37 ° C e 5% di CO 2. Supplemento giornaliero bFGF (20 ng / ml) e EGF (20 ng / ml). </li><li> Dopo una settimana, contare il numero di emergenti sfere tumorali utilizzando un microscopio standard 4X o 10X di ingrandimento e un microscopio a fluorescenza per rilevare il segnale di fluorescenza dal sistema reporter integrato. Contare sfere con un diametro superiore a 100 micron come &quot;grandi&quot; sfere e sfere di diametro 50 - 100 micron come &quot;piccole&quot; sfere (Figura 3). Essere sicuri che si contano sfere reali e non CELL cluster. NOTA: In saggi cellulari singola formazione sfera è più facile segnare microscopicamente dopo 10 (contro 7) giorni di coltura. </li><li> Calcolare la percentuale di cellule che formano sfera in RFP + e cellule rispettivamente RFP- in saggi cellulari singoli (una piastra a 96 pozzetti per ogni singolo esperimento) o più cellule-basfere sed saggi (uno anche per ogni singolo esperimento) presentato dalla Shaw et al. 16 NOTA: Percentuale di cellule che formano sfera (%) = (numero di sfere) / (numero di cellule seminate) x 100 </li></ol> 3. Passaging seriale di sfere <ol><li> Posizionare il contenuto di ciascun pozzetto in un idoneo tubo sterile e centrifugare a 300 xg per 10 min a RT. Per il multi-cellulari sfere saggi, raccogliere insieme le sfere di un bene. Lavare il ben 3 - 5 volte con PBS e centrifugare 2 min più. Per i singoli cellulari-sfere saggi, raccogliere le singole sfere. A causa del basso numero di cellule, utilizzare 1,5 ml tubi per centrifugazione e lavaggio passi. </li><li> Rimuovere il surnatante e risospendere pellet in 200 ml di 0,05% tripsina-EDTA. </li><li> Per realizzare la separazione cellulare ottimale, incubare la sospensione cellulare a 37 ° C per 5 minuti in un agitatore morbido. Ottimizzare tripsinizzazione tempo per la vostra linea cellulare a basso tasso di morte cellulare: se no grandesfere è visibile triturare delicatamente con un puntale 100 microlitri. In caso di grandi sfere sono ancora presenti, incubare con tripsina altri 3 minuti e poi procedere al passo triturazione. In caso di singolo saggi cellulari assicurarsi di ottimizzare i tempi per la resa ottimale delle cellule delle cellule viventi durante la fase di tripsinizzazione. </li><li> Per inattivare la tripsina, aggiungere 500 microlitri mezzo completo e centrifugare a 300 xg per 10 min, in caso di saggi cellulari singoli, centrifugare per altri 2 minuti. </li><li> Rimuovere accuratamente le cellule surnatante e risospendere in sfere di media. </li><li> Utilizzare un filtro tappo filtro cella 40 micron per ottenere una cella singola sospensione. </li><li> In caso di utilizzo RFP + e le cellule RFP- in saggi di multi-cellulari bassed, valutare la percentuale di cellule fluorescenti in ogni pozzetto dopo risospensione tramite citofluorimetro analisi. </li><li> Per i saggi replating serie di singole cellule, di semi 1 cella per bene in un nuovo minimo attaccamento piastra a 96 pozzetti ultra preparati come sopra. Da una sfera individuale, seminare circa 20 singoli pozzi. Per i saggi replating di multi-sfere primarie a base di cellule, le cellule di semi ottenuti da un pozzetto di sfere primarie in una nuova piastra a 96 pozzetti e contano i numeri cellulari giorno successivo al microscopio. </li><li> Valutare la proporzione di cellule che formano sfera in sfere secondarie test utilizzando la formula descritta al punto 2.11. </li></ol> Analisi 4. Risultato <ol><li> Analizzare i risultati di esperimenti condotti in triplicato indipendenti e utilizzare Test t su due lati per analizzare i valori normalmente distribuiti e altrimenti Mann-Whitney-test per l&#39;analisi statistica. </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.

Sfere culture sono un metodo ampiamente utilizzato per saggiare il potenziale delle cellule staminali del cancro e arricchire per le cellule staminali, come in una vasta gamma di cellule tumorali umane 15,25,26. In queste condizioni di coltura, le cellule tumorali che non hanno la capacità di auto-rinnovamento sono tenuti a differenziare e infine sottoposti a morte cellulare. Anche se possono inizialmente formare gruppi di cellule o persino sfere tumorali soprattutto a dosaggi primarie, non sono in grado di ...

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.

In sfere convenzionali saggi, quasi il 40% delle RFP + OVCAR-3 celle contro il 20% delle cellule RFP- ha dato luogo a una sfera singolo tumore nel primario test sfere (Figura 4A). Inoltre, sfere formate da RFP + cellule erano di dimensioni maggiori di quelle formate da cellule RFP-. Quando placcato in saggi cellulari singoli, cellule RFP + anche formati più sfere di cellule RFP-, confermando i risultati di cui sopra. Tuttavia, c&#39;è stata una tendenza verso un minor nume...

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.

La valutazione delle proprietà sulle cellule staminali in cellule di carcinoma ovarico umano Utilizzo multi e 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 ...

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

Research

JoVE Journal

Medicine

9.5K Views.  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.