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Medicine

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

Published: May 11th, 2016

DOI:

10.3791/53958

1UCBL1, UMR/CNRS 5822, Laboratoire de Radiobiologie Cellulaire Moléculaire, Université de Lyon, 2Hospices-Civils-de-Lyon, Centre Hospitalier Lyon-Sud

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 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 "side population" (SP). As the SP cells represent a low percentage (<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 β-catenin.

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....

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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

  1. Staining 50 million cells with Hoechst 33342 dye.
    1. Prepare two 15 ml sterile tubes with a conical bottom: one tube labeled "Hoechst" and one labeled "Hoechst and Verapamil". Prepare 10 ml of 5 mM Verapamil hydrochloride solution in.......

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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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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 .......

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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).

....

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

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