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 sterile water. Prepare the culture medium (CM) for stem cells.
		<ol>
			<li>To prepare CM for CSC (CM-CSC), combine the following: Dulbecco&#39;s modified Eagle medium (DMEM):F12 (1:3, v:v), 5% of fetal calf serum (FCS), 0.04 mg/L of hydrocortisone, 100 U/ml of penicillin, 0.1 g/L of streptomycin, and 20 &#181;g/L of Epidermal Growth Factor (EGFR).</li>
		</ol>
		</li>
		<li>Under a laminar flow hood, trypsinize cells. Remove media, wash with sterile phosphate buffered saline (PBS) and add 1 ml of trypsin-EDTA (0.5 g/L) for a 75 cm&#178; flask. Incubate 3 min at 37 &#176;C. Stop the reaction by adding culture medium used for the parental cell line. Count the number of cells using a cell counter.
		<ol>
			<li>To prepare CM for parental cell line (CM-P), supplement DMEM with 10% FCS, 0.4 mg/L of hydrocortisone, 100 U/ml of penicillin and 0.1 g/L of streptomycin).</li>
		</ol>
		</li>
		<li>Dilute the cell suspension obtained in the step 1.1.2 to obtain 107 cells/ml in CM-P. Split the cell suspension in the 2 tubes prepared: put 100 &#181;l (106 cells) in the tube &#34;Hoechst and Verapamil&#34; and 4 ml (4 x 107 cells) in the tube &#34;Hoechst&#34;.</li>
		<li>In the sample &#34;Hoechst and Verapamil&#34;, add 10 &#181;l of 5 mM Verapamil hydrochloride solution (final concentration: 0.5 mM) and mix gently. Add 5 &#181;l of 1 g/L Hoechst solution (final concentration: 0.1 g/L). In the sample &#34;Hoechst&#34;, add 5 &#181;l of 1 g/L Hoechst solution per 106 cells (200 &#181;l total). Henceforth, keep samples protected from direct light exposure using aluminum foil.</li>
		<li>Incubate all tubes in a water bath at 37 &#176;C for 1 hr 30 min. Mix gently every 15 min to prevent cells from settling down.</li>
		<li>Centrifuge tubes at 250 x g for 5 min at 4 &#176;C. Remove supernatant and resuspend each pellet with 2 mL of 1x PBS.</li>
		<li>Centrifuge tubes at 250 x g for 5 min at 4 &#176;C. Remove supernatant. Resuspend &#34;Hoechst and Verapamil&#34; pellet in 500 &#181;l of 5 mg/L propidium iodide (PI) diluted in PBS buffer and &#34;Hoechst&#34; pellet in 4 ml of 5 mg/L PI diluted in PBS buffer.</li>
		<li>Transfer sample solutions through a 70 &#181;m cell strainer to remove aggregates and collect single cells in tubes for sample uptake on the flow cytometer. Keep samples on ice and protect from direct light exposure using aluminum foil.</li>
	</ol>
	</li>
	<li>Isolation of the Side Population Excluding Hoechst dye by Cell Sorting.
	<ol>
		<li>Perform Hoechst negative cell separation on a flow cytometry sorter with the following parameters: UV laser (355 nm), 2 detectors on the UV laser path with blue Hoechst (450/50 BP) and red Hoechst (610/20 BP) filters, and 2 collectors.
		<ol>
			<li>First, analyze sample &#34;Hoechst&#34; that serves as a positive control for staining and flow cytometer set-up.</li>
			<li>Using the cytometer software, on the &#34;Global Worksheet&#34; window, click on &#34;Dot Plot&#34; (fifth top right picture) and create a chart on the global worksheet. On ordinate, with a right click, select FSC-A (forward scatter) and on abscissa, SSC-A (side scatter) (Figure 1A). In the same way, create a SSC-W versus SCC-H dot plot. On this second dot plot, to create a P1 region, click &#34;Polygon gate&#34; (fourteenth top right picture) (Figure 1B)33. 
			Note: The P1 region will encompass single cells and discriminate doublets.</li>
			<li>Optional: Exclude PI positive cells by creating a FSC-A versus PI gated on P1. On this dot plot, select the PI negative population (P2) to exclude PI positive dead cells.</li>
			<li>Using the cytometer software, on the &#34;Global Worksheet&#34; window, click on &#34;Dot Plot&#34; and create a chart on the global worksheet. On ordinate, with a right click, select blue Hoechst-A and on abscissa, red Hoechst-A. With a right click on the population, select &#34;Show population&#34; and P1. On this dot plot, create a region (P2) to select the negative Hoechst dye side population (SP) cells that appears as a side arm on the left of the main population of cells (Figure 1C).</li>
		</ol>
		</li>
		<li>Analyze the sample &#34;Hoechst&#34; and collect 10,000 events. To ensure that the gate P2, which represents the SP population, is well positioned, analyze the sample &#34;Hoechst and Verapamil&#34; (10,000 events) to observe the disappearance of SP population (Figure 1D).</li>
		<li>Collect the SP Hoechst dye negative cells in a 15 ml tube containing 1 ml of CM-CSC prepared in 1.1.1.1.</li>
		<li>At the end of the cell sorting, centrifuge the cell suspension at 250 x g for 5 min, remove the supernatant and resuspend the pellet with 1 ml of CM-CSC. Count the number of sorted cells using a cell counter and transfer appropriate number of cells to a culture flask (see Table 1). Add CM-CSC and incubate at 37 &#176;C and 5% CO2 atmosphere.</li>
		<li>Maintain cells in culture under the same conditions for a maximum of 2 passages until there is a minimum of 5&#160;x 107 cells (see step 3, &#34;Cell culture method&#34;).</li>
	</ol>
	</li>
</ol>
2. Selection of the CD44high/ALDHhigh Sub-population in the Side Population Sorted
<ol>
	<li>Staining 50 million of SP Cells with the ALDH Detection Kit and CD44 Antibody.
	<ol>
		<li>Prepare seven 15 ml sterile tubes labeled as follows: &#34;Unstained&#34; (Tube a), &#34;CD44-APC&#34; (Tube b), &#34;IgG1-APC&#34; (Tube c), &#34;ALDH&#34; (Tube d), &#34;ALDH and DEAB&#34; (Tube e), &#34;ALDH and CD44-APC&#34; (Tube f), &#34;ALDH, DEAB and CD44-APC&#34; (Tube g). Keep all tubes and reagents at 4 &#176;C during the staining.</li>
		<li>Prepare buffer A with 4.5 ml of the buffer 1 (contained in the kit) and 45 &#181;l of CD44-APC (Allophycocyanin) antibody (dilution 1:100). Prepare buffer B with 100 &#181;l of buffer 1 and 1 &#181;l of IgG1-APC antibody (dilution 1:100). Prepare buffer C with 4 ml of buffer 1 and 20 &#181;l of the reagent of the kit.</li>
		<li>Prepare a cell suspension of the previously sorted cells (step 1.2.5) at 107 cells /ml. Add 100 &#181;L (106 cells) of the cell suspension to tubes a, b and c, and 4 ml (4 x 107 cells) to tube f. For the moment do not put cells in tubes d, e and g.</li>
		<li>Centrifuge the tubes containing the cells at 250 x g for 5 min. Remove the supernatant and resuspend the pellets in tubes a, b and c in 100 &#181;l of buffer 1. Add 5 &#181;l of diethylaminobenzaldehyde (DEAB), an ALDH inhibitor to tubes e and g.
		<ol>
			<li>Resuspend cells in tube f with 4 ml reagent C and immediately transfer 100 &#181;l into tubes d, e and g. Incubate all tubes in a water bath at 37 &#176;C for 30 min and protect from direct light exposure using aluminum foil. Mix the cell suspension gently by vortexing after 15 min to prevent cells from settling.</li>
		</ol>
		</li>
		<li>From this moment, keep the tubes on ice and protected from direct light exposure using aluminum foil. Centrifuge all tubes at 250 x g for 5 min at 4 &#176;C and remove the supernatant.</li>
		<li>Resuspend tube f in 4 ml and tubes b and g with 100 &#181;l of buffer A. Resuspend tube c with 100 &#181;l of buffer B. To the other tubes, add 100 &#181;l of buffer 1. Incubate 10 min at 4 &#176;C.</li>
		<li>Centrifuge all tubes at 250 x g for 5 min at 4 &#176;C and remove the supernatant. Rinse once with 1 ml of buffer 1 (4 ml for tube f) and centrifuge again at 250 x g for 5 min at 4 &#176;C. Remove the supernatant. Re-suspend tube f pellet in 4 ml and all other tubes in 1 ml of buffer 1.</li>
		<li>Transfer the sample solutions through 70 &#181;m cell strainers to remove aggregates and collect single cells in appropriate tubes for sample uptake on flow cytometer.</li>
	</ol>
	</li>
	<li>Isolation of the CD44high/ALDH high Cell Population by Fluorescence Activated Cell Sorting.
	<ol>
		<li>Perform CD44high/ALDH high cell sorting on a flow cytometry sorter with the following parameters: Blue laser (488 nm) and red laser (633 nm), 1 detector on the blue laser path with a FITC filter (530/30), 1 detector on the red laser path with an APC filter (660/20) and 2 collectors.</li>
		<li>Using the cytometer software, click on &#34;Dot Plot&#34; (the fifth top right picture) to create a FSC-A versus SSC-A dot plot as described in section 1.2.1.2, to check cell morphology and select a population composed of single cells with a SSC-W versus SSC-H dot plot.</li>
		<li>Using the cytometer software, on the &#34;Global Worksheet&#34; window, click on &#34;Dot Plot&#34; and create a chart on the global worksheet. On ordinate, with a right click, select APC-A and on abscissa, FITC-A in order to select the double stained population. Create a gate using tubes d and e to select ALDHhigh cells (Figure 2A). Note: Positive cells disappear in tube e treated with DEAB (Figure 2B).
		<ol>
			<li>On the same chart, create a second gate using tubes b and c in order to select CD44high cells (Figure 2C and 2D). Positive cells disappear in the tube containing IgG1-APC. Analyze tubes f and g and create a third gate that includes CD44high/ALDHhigh cells (Figure 2E and 2F). 
			Note: If positive cells are present in the tube containing IgG1-APC (Figure 2D), the interaction between cells and the APC-stained antibody is not specific.</li>
		</ol>
		</li>
		<li>Collect CD44high/ALDHhigh cells into a 15 ml tube containing 1 ml of CM-CSC. Collect also CD44low/ALDHlow into a 15 ml tube containing 1 ml of CM19. Note: Prepare CM-CSC as described in step 1.1.1.1.</li>
		<li>Centrifuge the cell suspension at 250 x g for 5 min, remove the supernatant and resuspend the pellet with 1 ml of CM-CSC. Count the number of sorted cells.</li>
	</ol>
	</li>
</ol>
3. Cell Culture Method
<ol>
	<li>After the double-sorting of the cells as described above, plate the sorted cells into an appropriate culture flask (Table 1) with CM-CSC at 37 &#176;C with 5 % CO2. After 18-24 hr, check if the cells have adhered and change the culture medium. Change the culture medium every 3 days until the expanding colonies are greater than 50% confluent.</li>
	<li>Use the appropriate volume of trypsin 0.5 g/L - EDTA (Table 1) to trypsinize cells from the culture flask. Incubate cells 3 to 5 min at 37 &#176;C. Add the appropriate volume of CM-CSC (Table 1) to stop the action of trypsin.</li>
	<li>Count the number of cells obtained and plate 4 x 105 cells in a 175 cm&#178; culture flask with CM-CSC. Incubate at 37 &#176;C and 5 % CO2. At this concentration, cells with a doubling time of 24 hr will be 70% confluent in 7 days.</li>
	<li>Use CSCs for in vitro or in vivo experiments before they have undergone 3 passages.</li>
</ol>
4. Confirmation of Tumor Potential and CSC Characteristics
<ol>
	<li>Tumor Sphere Formation to Confirm the tumor Potential of the CD44high/ALDHhigh Cells.
	<ol>
		<li>Trypsinize cells as described in 3.2. Add 1 x 106 cells to a 15 ml tube and centrifuge at 250 x g for 5 min. Remove the supernatant and re-suspend the pellet in a&#160;DMEM:F12 (3:1) medium FCS free, 20 ng/ml of rhEGF, 4 mg/L of heparin and 1x B27. Incubate cells in a 6 well low anchorage plates culture flask at 37 &#176;C and 5% CO2. 
		Note: Use DMEM:F12 (3:1) medium containing 5% of FCS, 20 ng/ml of rhEGF, 4 mg/L of heparin and 1x B27 if cell do not grow without FCS.</li>
		<li>Observe the tumor sphere formation with an optical microscope from 4 to 10 days after seeding (Figure 3A). 
		Note: Tumor sphere diameter should be more than 35 &#181;m. CD44high/ALDHhigh cells will show a faster and more enhanced tumor sphere formation in terms of number and size compared to CD44low/ALDHlow.</li>
	</ol>
	</li>
	<li>Evaluation of In Vivo Tumorigenicity After Subcutaneous Injection of CD44high/ALDHhigh Cells in NOD-SCID Mice.
	<ol>
		<li>After sorting, re-suspend cells in PBS at three different concentrations (104 cells/ml, 105 cells/ml, and 106 cells/ml). Inject subcutaneously 100 &#181;l of 104 cells/ml (103 cells) in the right flank region of 6 mice. Do the same for 105 cells/ml (104 cells), and 106 cells/ml (105 cells). 
		Note: Lower dilution than 103 cells can be tested.</li>
		<li>Inject the same concentration of CD44low/ALDHlow cells in the left flank region. Monitor injected mice for up to 10 weeks to see tumor progression19.</li>
	</ol>
	</li>
</ol>

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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. 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></ol>

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 100x</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&amp;nbsp;</td><td>CKX31</td><td></td></tr><tr><td>SQ20B cell line</td><td>Gift from the John Little&amp;rsquo;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></table>

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 ...

isolation-characterization-head-neck-squamous-cell-carcinoma

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.5K Views.  Université de Lyon. 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).

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

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Cancer Research

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

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

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

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

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.