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 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>
</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

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

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 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.

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

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.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

Discovering Middle Ear Anatomy by Transcanal Endoscopic Ear Surgery: A Dissection Manual

The middle ear is located in the center of the temporal bone and bears a highly complex anatomy. The recently introduced exclusively endoscopic transcanal approach to the middle ear is a minimally invasive technique sparing the bone and mucosa of the mastoid bone, since the middle ear is accessed through the external auditory canal. This emerging method has several advantages over the traditional (microscopic) approaches to the middle ear such as the panoramic wide-angle views of the anatomy, the possibility to approach and magnify tiny structures, and the possibility of looking around the corner using angled endoscopes.
The cadaveric dissection method presented here consists of an overview on the technical requirements and a precise description of a step-by-step protocol to discover the anatomy of the middle ear. Each step and anatomical structure is carefully described in order to provide a comprehensive guide to endoscopic ear anatomy. In our opinion, this is particularly important to any novice in endoscopic ear surgery as it provides thorough anatomical knowledge and may improve surgical skills.

The aim of this article is to describe the methodology of exclusively endoscopic cadaveric middle ear dissection. Moreover, we aim to provide a comprehensive guide to endoscopic middle ear anatomy.

The middle ear is located in the center of the temporal bone and bears a highly complex anatomy. The recently introduced exclusively endoscopic transcanal ...

Porcine As a Training Module for Head and Neck Microvascular Reconstruction

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5&#215; loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes ...