We thank Thibault Andrieu and Sebastien Dussurgey from the Flow Cytometry Platform of UFR BioSciences Gerland-Lyon-Sud (UMS3444/US8) for their advice and help during our sorting. This work was achieved within the scientific framework of ETOILE and Labex-PRIMES (ANR-11LABX-0063).

All animal procedures were performed according to local guidelines on animal care. All the details of this study were approved by the CECCAPP, a French ethics committee.
1. Selection of a Side Population (SP) by the Hoechst Dye Efflux Assay
<ol>
	<li>Staining 50 million cells with Hoechst 33342 dye.
	<ol>
		<li>Prepare two 15 ml sterile tubes with a conical bottom: one tube labeled &#34;Hoechst&#34; and one labeled &#34;Hoechst and Verapamil&#34;. Prepare 10 ml of 5 mM Verapamil hydrochloride solution in...

<ol>
	<li>Baumann, M., Krause, M., Hill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+role+of+cancer+stem+cells+in+radioresistance.">Exploring the role of cancer stem cells in radioresistance.</a> Nat Rev Cancer. 8 (7), 545-554 (2008).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proc Natl Acad Sci USA. 100 (7), 3983-3988 (2003).</li><li>Singh, S. 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=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432 (7015), 396-401 (2004).</li><li>Collins, A. T., Berry, P. A., Hyde, C., Stower, M. J., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+prostate+cancer+stem+cells.">Prospective identification of tumorigenic prostate cancer stem cells.</a> Cancer Res. 65 (23), 10946-10951 (2005).</li><li>Eramo, 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=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15 (3), 504-514 (2008).</li><li>Dalerba, 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=Phenotypic+characterization+of+human+colorectal+cancer+stem+cells.">Phenotypic characterization of human colorectal cancer stem cells.</a> Proc Natl Acad Sci USA. 104 (24), 10158-10163 (2007).</li><li>Hermann, P. 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=Distinct+populations+of+cancer+stem+cells+determine+tumor+growth+and+metastatic+activity+in+human+pancreatic+cancer.">Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.</a> Cell Stem Cell. 1 (3), 313-323 (2007).</li><li>Yang, Z. 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=Significance+of+CD90+cancer+stem+cells+in+human+liver+cancer.">Significance of CD90 cancer stem cells in human liver cancer.</a> Cancer Cell. 13 (2), 153-166 (2008).</li><li>Fang, 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=A+tumorigenic+subpopulation+with+stem+cell+properties+in+melanomas.">A tumorigenic subpopulation with stem cell properties in melanomas.</a> Cancer Res. 65 (20), 9328-9337 (2005).</li><li>Prince, M. E., 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+a+subpopulation+of+cells+with+cancer+stem+cell+properties+in+head+and+neck+squamous+cell+carcinoma.">Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.</a> Proc Natl Acad Sci USA. 104 (3), 973-978 (2007).</li><li>Clarke, 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=Cancer+stem+cells+--+Perspectives+on+current+status+and+future+directions:+AACR+Workshop+on+cancer+stem+cells.">Cancer stem cells -- Perspectives on current status and future directions: AACR Workshop on cancer stem cells.</a> Cancer Res. 66 (19), 9339-9344 (2006).</li><li>Soltanian, S., Matin, M. M. <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+and+cancer+therapy.">Cancer stem cells and cancer therapy.</a> Tumor Biol. 32 (3), 425-440 (2011).</li><li>Molyneux, 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=BRCA1+basal-like+breast+cancers+originate+from+luminal+epithelial+progenitors+and+not+from+basal+stem+cells.">BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells.</a> Cell Stem Cell. 7 (3), 403-417 (2010).</li><li>Vermeulen, 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=Single-cell+cloning+of+colon+cancer+stem+cells+reveals+a+multi-lineage+differentiation+capacity.">Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.</a> Proc Natl Acad Sci USA. 105 (36), 13427-13432 (2008).</li><li>Ratajczak, M. Z. <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+--+Normal+stem+cells+'Jedi'+that+went+over+to+the+'dark+side.'.">Cancer stem cells -- Normal stem cells 'Jedi' that went over to the 'dark side.'.</a> Folia Histochem Cytobiol. 43 (4), 175-181 (2005).</li><li>Bao, 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=Glioma+stem+cells+promote+radioresistance+by+preferential+activation+of+the+DNA+damage+response.">Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.</a> Nature. 444 (7120), 756-760 (2006).</li><li>Liu, 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=Analysis+of+gene+expression+and+chemoresistance+of+CD133++cancer+stem+cells+in+glioblastoma.">Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.</a> Mol Cancer. 5, 67 (2006).</li><li>Moncharmont, 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=Targeting+a+cornerstone+of+radiation+resistance:+Cancer+stem+cell.">Targeting a cornerstone of radiation resistance: Cancer stem cell.</a> Cancer Lett. 322 (2), 139-147 (2012).</li><li>Bertrand, 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=Targeting+Head+and+Neck+Cancer+Stem+Cells+to+Overcome+Resistance+to+Photon+and+Carbon+Ion+Radiation.">Targeting Head and Neck Cancer Stem Cells to Overcome Resistance to Photon and Carbon Ion Radiation.</a> Stem Cell Rev. 10 (1), 114-126 (2013).</li><li>Das, B., Tsuchida, R., Malkin, D., Koren, G., Baruchel, S., Yeger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+tumor+stemness+by+increasing+the+invasive+and+tumorigenic+side+population+fraction.">Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side population fraction.</a> Stem Cells. 26 (7), 1818-1830 (2008).</li><li>Diehn, 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=Association+of+reactive+oxygen+species+levels+and+radioresistance+in+cancer+stem+cells.">Association of reactive oxygen species levels and radioresistance in cancer stem cells.</a> Nature. 458 (7239), 780-783 (2009).</li><li>Chen, Z. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+stem+cell+concept+in+progression+of+head+and+neck+cancer.">The cancer stem cell concept in progression of head and neck cancer.</a> J Oncol. 2009, 894064 (2009).</li><li>Zhang, P., Zhang, Y., Mao, L., Zhang, Z., Chen, W. <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+oral+squamous+cell+carcinoma+possesses+tumor+stem+cell+phenotypes.">Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes.</a> Cancer Lett. 277 (2), 227-234 (2009).</li><li>Zhang, Q., 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+subpopulation+of+CD133(+)+cancer+stem-like+cells+characterized+in+human+oral+squamous+cell+carcinoma+confer+resistance+to+chemotherapy.">A subpopulation of CD133(+) cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy.</a> Cancer Lett. 289 (2), 151-160 (2010).</li><li>Sun, S., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Head+neck+squamous+cell+carcinoma+c-Met&#8314;+cells+display+cancer+stem+cell+properties+and+are+responsible+for+cisplatin-resistance+and+metastasis.">Head neck squamous cell carcinoma c-Met&#8314; cells display cancer stem cell properties and are responsible for cisplatin-resistance and metastasis.</a> Int J Cancer. 129 (10), 2337-2348 (2011).</li><li>Chen, Y. 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=Aldehyde+dehydrogenase+1+is+a+putative+marker+for+cancer+stem+cells+in+head+and+neck+squamous+cancer.">Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer.</a> Biochem Biophys Res Commun. 385 (3), 307-313 (2009).</li><li>Lim, Y. 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=Cancer+stem+cell+traits+in+squamospheres+derived+from+primary+head+and+neck+squamous+cell+carcinomas.">Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas.</a> Oral Oncol. 47 (2), 83-91 (2011).</li><li>Yu, Q., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface-localized+matrix+metalloproteinase-9+proteolytically+activates+TGF-beta+and+promotes+tumor+invasion+and+angiogenesis.">Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis.</a> Genes Dev. 14 (2), 163-176 (2000).</li><li>Krishnamurthy, 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=Endothelial+cell-initiated+signaling+promotes+the+survival+and+self-renewal+of+cancer+stem+cells.">Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells.</a> Cancer Res. 70 (23), 9969-9978 (2010).</li><li>Chikamatsu, K., Takahashi, G., Sakakura, K., Ferrone, S., Masuyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoregulatory+properties+of+CD44++cancer+stem-like+cells+in+squamous+cell+carcinoma+of+the+head+and+neck.">Immunoregulatory properties of CD44+ cancer stem-like cells in squamous cell carcinoma of the head and neck.</a> Head Neck. 33 (2), 208-215 (2011).</li><li>Chen, Y. W., 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=Cucurbitacin+I+suppressed+stem-like+property+and+enhanced+radiation-induced+apoptosis+in+head+and+neck+squamous+carcinoma--derived+CD44(+)ALDH1(+)+cells.">Cucurbitacin I suppressed stem-like property and enhanced radiation-induced apoptosis in head and neck squamous carcinoma--derived CD44(+)ALDH1(+) cells.</a> Mol Cancer Ther. 9 (11), 2879-2892 (2010).</li><li>Clay, M. 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=Single-marker+identification+of+head+and+neck+squamous+cell+carcinoma+cancer+stem+cells+with+aldehyde+dehydrogenase.">Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase.</a> Head Neck. 32 (9), 1195-1201 (2010).</li><li>Meinelt, E., 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> Technical Bulletin: Standardizing Application Setup Across Multiple Flow Cytometers Using BD FACSDiva Version 6 Software. , (2012).</li><li>Zhou, L., Wei, X., Cheng, L., Tian, J., Jiang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133,+one+of+the+markers+of+cancer+stem+cells+in+Hep-2+cell+line.">CD133, one of the markers of cancer stem cells in Hep-2 cell line.</a> Laryngoscope. 117 (3), 455-460 (2007).</li><li>Fukusumi, 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=CD10+as+a+novel+marker+of+therapeutic+resistance+and+cancer+stem+cells+in+head+and+neck+squamous+cell+carcinoma.">CD10 as a novel marker of therapeutic resistance and cancer stem cells in head and neck squamous cell carcinoma.</a> Br J Cancer. 111 (3), 506-514 (2014).</li><li>Shen, C., Xiang, M., Nie, C., Hu, H., Ma, Y., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+as+a+molecular+marker+to+screen+cancer+stem+cells+in+hypopharyngeal+cancer.">CD44 as a molecular marker to screen cancer stem cells in hypopharyngeal cancer.</a> Acta Otolaryngol. 133 (11), 1219-1226 (2013).</li><li>Kanojia, 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=Proteomic+profiling+of+cancer+stem+cells+derived+from+primary+tumors+of+HER2/Neu+transgenic+mice.">Proteomic profiling of cancer stem cells derived from primary tumors of HER2/Neu transgenic mice.</a> Proteomics. 12 (22), 3407-3415 (2012).</li><li>Higgins, D. 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The authors have nothing to disclose.

This protocol describes a reliable method for the successful isolation of CSCs from a specific cell line that is applicable to other HNSCC cell lines. Isolated head and neck CSCs are then suitable for further molecular characterization in vitro and functional validation by transplantation in immunodeficient mice19. However, some modifications can be tested depending on the side population or the CD44high/ALDHhigh percentages present in the parental cell line. For example, if the ...

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy worldwide and despite progress in current treatments, patients with advanced disease have a poor prognosis. The overall 5 year survival rate of the patient is around 30% despite the combination of therapeutic approaches including surgery, chemo-radiotherapy and targeted-therapies. Recent studies attribute local recurrence and distant metastasis to the survival of cancer stem-like cells (CSCs) following anticancer therapies1. There is accumulating evidence supporting the existence of cells presenting stem cells properties (undifferentiated status, self-renewal and differentiation capacities, and telomerase activity) in various solid tumors including breast, brain, prostate, lung, colon, pancreas, liver and skin2-10. However, the origin of CSCs remains unclear11,12. They may result from the malignant transformation of normal stem cells3,13 or dedifferentiation of tumor cells that acquire CSCs-like features14,15. Therefore, understanding distinctive pathways relating to CSCs will provide insight into early diagnosis and treatment of resistant HNSCC.
It has been proposed that CSCs also possess resistant phenotypes that evade standard chemotherapy and radiotherapy, resulting in tumor relapse compared to the bulk of tumor cells16-19 and are localized into hypoxic niches20. Numerous factors have been proposed to explain these resistances of CSCs, such as propensity to quiescence, enhanced DNA repair, up-regulated cell cycle control mechanisms, and free-radical scavenging21. Moreover, several oncogenic molecular pathways may be specifically activated in CSCs17. In order to improve knowledge of CSCs for further targeted-therapies, we need reliable methods for the identification and isolation of CSCs, owing to the heterogeneity of stem cell-related markers in various types of cancers22.
In HNSCC, stem-like tumor-initiating cells have been isolated from primary patient tumors by sorting cells expressing different CSC biomarkers (such as drug efflux transporters expression23, CD44high, CD24low CD133high, c-Met+ phenotype10,24,25, or ALDHhigh activity26) or cultivating primary patient tumor to form squamospheres that have CSC properties. Nevertheless, the number of squamospheres decreases dramatically after two passages, thus giving a small sample size for further characterization studies27. Therefore, in vitro assays starting from well-established cell lines is an easier solution to design experiments in order to improve knowledge of CSCs.
The aim of this article is to propose a method to isolate CSCs from HNSCC cell lines using multiparametric flow cytometric analysis and cell sorting. The expression of CD44 in correlation with several CSCs properties including ALDH activity, Side Population (SP) phenotype, spheroid formation ability and tumorigenicity are used to isolate and characterize this sub-population of CSCs. CD44, a cell-surface glycoprotein, is involved in cell adhesion and migration. CD44 is highly expressed in many solid tumors CSCs28, including in head and neck cancer models29-31. Moreover, CD44high cells can generate in vivo a heterogeneous tumor whereas CD44low cells cannot10. The SP assay is based on the differential potential of cells to efflux the Hoechst dye22 via the ATP-binding cassette (ABC) family of transporter proteins overexpressed within the CSC membrane. This assay includes the use of ABC transporter inhibitors such as verapamil in control samples. Aldehyde dehydrogenase (ALDH) is an intracellular enzyme that is involved in converting retinol to retinoic acid during early stem cell differentiation25,26. Cells that exhibit high ALDH activity show stem-like cell behavior in HNSCC26 and a very few number of ALDHhigh cells are able to generate tumors in vivo26,32.
The combination of these markers and properties was successfully used by Bertrand et al. to study the resistance in vitro and in vivo of these CSCs to photon and carbon ion radiation19. Their results clearly showed that the combination of various cell markers and properties are more selective for useful studies on HNSCC CSCs populations than single-marker approaches.

<table>
	<tbody>
		<tr>
			<td>Fetal Calf Serum Gold</td>
			<td>GE Healthcare</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone water soluble</td>
			<td>Sigma-Aldrich</td>
			<td>H0396-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100 X</td>
			<td>Dominique Dutscher</td>
			<td>L0022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>61965-026</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 Nut Mix (1X) + GlutaMAX-I</td>
			<td>Gibco</td>
			<td>31765-027</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Promega</td>
			<td>G5021</td>
			<td>The solution must be prepared just before use because it is very unstable</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>StemcellTM Technologies</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma-Aldrich</td>
			<td>14533</td>
			<td>Corrosive, acute toxicity (oral, dermal, inhalation) category 4</td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>V-4629</td>
			<td>Acute toxicity (oral, dermal, inhalation) category 3</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td>Acute toxicity (oral, dermal, inhalation) category 4</td>
		</tr>
		<tr>
			<td>ALDEFLUOR Kit</td>
			<td>Stem Cell</td>
			<td>1700</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44-APC, human antibody</td>
			<td>Miltenyi Biotech</td>
			<td>130-095-177</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG1-APC, human antibody</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-189</td>
			<td></td>
		</tr>
		<tr>
			<td>Z1 coulter particle</td>
			<td>Beckman Coulter</td>
			<td>6605698</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus&#160;</td>
			<td>CKX31</td>
			<td></td>
		</tr>
		<tr>
			<td>SQ20B cell line</td>
			<td>Gift from the John Little&#8217;s Laboratory</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>FaDu cell line</td>
			<td>ATCC</td>
			<td>HTB-43</td>
			<td></td>
		</tr>
		<tr>
			<td>Low anchorage plates</td>
			<td>Thermo Fischer Scientific</td>
			<td>145383</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva software v8.0.1</td>
			<td>BD Biosciences</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
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isolation and characterization of a head and neck squamous cell carcinoma subpopulation having stem cell characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

The isolation of CSCs from HNSCC cell lines required two successive sorting because of the very low percentage of CSCs in the parental cell line. The first sorting was based on the ability of CSCs to exclude the Hoechst dye due to drug efflux transporters. This resulted in acquisition of 1-5% of the total cell population sorted (Figure 1). During the Hoechst dye negative cell sorting, check the size and granulation of sorted cells by looking at the FSC-A versus SSC-A dot ...

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Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

The overall goal of this procedure is to isolate cancer stem cells from head and neck squamous cell carcinoma or HNSCC cell lines. This method can help answer key questions in the cancer treatment field about how to use chemotherapy to overcome tumor radioresistence. The main advantage of this technique is that it uses a combination of markers to achieve a high specificity in cancer stem cells from HNSCC cell lines.Demonstrating the procedure will be by Prescillia Battiston-Montagne our technician and Marion Gilormini, a PhD from my lab. To select a side population of cancer stem-like cells by Hoechst dye efflux assay first label one conical tube Hoechst and one Hoechst and Verapamil. Next, wash an HNSCC cell culture with sterile PBS followed by the addition of one milliliter of Tripsyn EDTA.After three minutes at 37 degrees Celsius stop the reaction with fresh cell culture medium and count the cells. Dilute the culture to one times 10 to the seventh cells per milliliter and complete parental cell line medium. Then add 100 microliters to the Hoechst and Verapamil tube and four milliliters of cells to the Hoechst tube.Next, gently mix 10 microliters of five millimolar Verapamil hydrochloride solution into the Hoechst and Verapamil sample followed by five microliters of Hoechst per one times 10 to the sixth cells to both tubes. Then cover the samples with aluminum foil and place them at 37 degrees Celsius. After 90 minutes with gentle mixing every 15 minutes centrifuge the tubes and resuspend the pellets in two milliliters of PBS.After a center centrifugation, resuspend the Hoechst and Verapamil pellet in 500 microliters of propidium iodide in PBS and the Hoechst pellet in four milliliters of the same. Now filter the samples through 70-micron cell strainers into FACS tubes to remove any aggregates and place the samples on ice protected from light. Next, load the Hoechst sample onto the flow cytometer and open the Global Worksheet window in the flow cytometer software.Click on Dot Plot to create a graph on the Global Worksheet and right click to select the forward and side scatter parameters. Create a side scatter wide by a side scatter height dot plot in the same way and then click polygon gate to create a P1 region in the second graph to select the single cells and to discriminate the doublets. Next, create a red Hoechst area by a blue Hoechst area dot plot and right click on the cells to highlight the P1 population.Then create a P2 region to select the negative Hoechst dye side population of cells that appears as a side arm to the left of the main population of cells and collect 10, 000 of the Hoechst sample events. To confirm that the P2 gate representing the side population is positioned correctly, collect 10, 000 Hoechst and Verapamil sample events as well. The side population should disappear.Then sort the side population of Hoechst dye negative cells into a 15-milliliter conical tube containing one milliliter of complete cancer stem-like cell medium. At the end of the sort, spin down the cells and resuspend the side population pellet in one milliliter of fresh complete cancer stem-like cell medium. Then count the cells and seed them at the appropriate number into a new cell culture flask.To select the CD44-high/ALDH-high subset from the sorted side population cells, first label seven sterile 15-milliliter conical tubes as indicated. Next, dilute the sorted cells to one times 10 to the seven cells per milliliter in stem cell medium and aliquot 100 microliters of cells to the unstained CD44-APC and IGG1-APC tubes and four milliliters of cells to the ALDH and CD44-APC tube, all on ice. Spin down the cells and resuspend the unstained CD44-APC and IGG1-APC pellets in 100 microliters of buffer one from the ALDEFLUOR kit.Then add five microliters of the ALDH inhibitor DEAB to the ALDH and DEAB and ALDH DEAB and CD44-APC tubes. Resuspend the cells in the ALDH and CD44-APC tube in four milliliters of reagent C containing four milliliters of buffer one and 20 microliters of the reagent from the kit and immediately transfer 100 microliters of these cells into the ALDH, ALDH and DEAB, and ALDH DEAB and CD44-APC tubes. Then place all of the tubes in a 37-degrees Celsius water bath for 30 minutes protected from light mixing the samples after 15 minutes by gentle vortexing.At the end of the incubation, centrifuge all of the samples and discard the supernatants. Then place all of the tubes on ice and resuspend the ALDH and CD44-APC pellet in four milliliters of buffer A, a CD44-APC and ALDH DEAB and CD44-APC pellets in 100 microliters of the same. Resuspend the IGG1-APC pellet in 100 microliters of buffer B and the unstained ALDH and ALDH and DEAB tubes in 100 microliters of buffer one.After 10 minutes, spin down all of the samples again followed by a wash in 1 milliliter of buffer one for all of the samples except the ALDH and CD44-APC sample, which is washed in four milliliters of buffer one. After the second centrifugation, resuspend all of the pellets in one milliliter of fresh buffer one except for the ALDH and CD44-APC sample, which is resuspended in four milliliters of buffer one. Now load the ALDH and CD44-APC tube onto the flow cytometer and create an APC by fitsy dot plot to visualize the double stained population.Next, load the ALDH tube to create a gate for ALDH-high cells. The positive cells should disappear when the ALDH and DEAB tube is loaded. In the same graph, create a second gate using the CD44-APC and IGG1-APC tubes to select the CD44-high cells.The positive cells will disappear when the IGG1-APC tube is loaded. Then load the ALDH and CD44-APC and ALDH DEAB and CD44-APC tubes to create a third gate that includes the CD44-high/ALDH-high cells. Use double-staining ALDH-CD44 tube to position the last gate on the double-positive cells.Then sort the CD44-high/ALDH-high cells into a 15-milliliter conical tube containing one milliliter of complete cancer stem-like cell medium collecting the CD44-low/ALDH-low cells in one milliliter of complete cell culture medium as desired. After the second sort, plate the CD44-high/ALDH-high cells into an appropriate cell culture flask with complete cancer stem-like cell medium in the cell culture incubator for 18 to 24 hours. When the cells have attached to the bottom of the flask, change the culture medium and return the flask to the incubator.To confirm the tumor potential of the CD44-high/ALDH-high cells, trypsinize the cancer stem-like monolayer as demonstrated to obtain a single-cell suspension and transfer one times 10 to the six cells to a 15-milliliter conical tube. After spinning down the cells, resuspend the pellet in serum-low medium supplemented with recombinant human epidermal growth factor heparin and B27 and incubate the cells in a low-anchorage petri dish in the cell culture incubator until tumor spheres are observed by optical microscopy. When the sorting is performed for the first time on a new cell line, it is necessary to confirm the tumor potential of the cell line as evidenced by its ability to form tumor spheres in serum-free medium in vitro.Moreover, QPCR should demonstrate a high expression of beta-catenin and other stem-like cell markers by the CD44-high/ALDH-high cells. The CD44-high/ALDH-high cells should also be able to form tumors in situ when injected in low quantities compared to CD44-low/ALDH-low cells. Once mastered, each cell sorting should be able to be completed in five hours if they are performed properly.While attempting this procedure it is important to remember to protect the fluorochrome-labeled samples from direct light exposure. Following this procedure, other methods like invasion migration assays can be performed to answer additional questions about the involvement of cancer stem cells in metastatic process. After its development, this technique paved the way for researchers in the field of cancer to explore the mechanisms of tumor escape in HNSCC cancer.After watching this video you should have a good understanding of how to isolate cancer stem cells from HNSCC cell lines using multiparametric, flow cytometric analysis, and cell sorting. Don't forget that working with tumor cells and intercalating DNA agents can be extremely hazardous and that precautions such as wearing a lab coat and gloves should always be taken performing this procedure.

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8.4K visualizações.  Université de Lyon. The overall goal of this procedure is to isolate cancer stem cells from head and neck squamous cell carcinoma or HNSCC cell lines. This method can help answer key questions in the cancer treatment field about how to use chemotherapy to overcome tumor radioresistence. The main advantage of this technique is that it uses a combination of markers to achieve a high specificity in cancer stem cells from HNSCC cell lines.Demonstrating the procedure will be by Prescillia Battiston-Montagne ou...