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. 2007 Jan 16;104(3):973-8.
doi: 10.1073/pnas.0610117104. Epub 2007 Jan 8.

Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma

M E Prince  1 , R Sivanandan, A Kaczorowski, G T Wolf, M J Kaplan, P Dalerba, I L Weissman, M F Clarke, L E Ailles
Affiliations

Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma

M E Prince et al. Proc Natl Acad Sci U S A. .

Abstract

Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD44(+) cancer cells, which typically comprise <10% of the cells in a HNSCC tumor, but not the CD44(-) cancer cells, gave rise to new tumors in vivo. Immunohistochemistry revealed that the CD44(+) cancer cells have a primitive cellular morphology and costain with the basal cell marker Cytokeratin 5/14, whereas the CD44(-) cancer cells resemble differentiated squamous epithelium and express the differentiation marker Involucrin. The tumors that arose from purified CD44(+) cells reproduced the original tumor heterogeneity and could be serially passaged, thus demonstrating the two defining properties of stem cells: ability to self-renew and to differentiate. Furthermore, the tumorigenic CD44(+) cells differentially express the BMI1 gene, at both the RNA and protein levels. By immunohistochemical analysis, the CD44(+) cells in the tumor express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. BMI1 has been demonstrated to play a role in self-renewal in other stem cell types and to be involved in tumorigenesis. Taken together, these data demonstrate that cells within the CD44(+) population of human HNSCC possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation and form unique histological microdomains that may aid in cancer diagnosis.

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Conflict of interest statement

Conflict of interest statement: I.L.W. was a member of the scientific advisory board of Amgen and owns significant Amgen stock; and I.L.W. cofounded and consulted for Systemix, is a cofounder and director of Stem Cells, Inc., and recently cofounded Cellerant, Inc.

Figures

Fig. 1.
Fig. 1.
Isolation of tumorigenic cells. Flow cytometry was used to isolate subpopulations of cells based on their CD44 expression. Dead cells (7AAD+ or PI+) and Lin+ cells were eliminated from all analyses. The percentage of CD44+ vs. CD44− is shown.
Fig. 2.
Fig. 2.
Tumor morphology. (A) Representative tumor in a mouse at the CD44+ injection site. (B) Histology of the tumor resulting from a CD44+ injection site. (C) Histology from the corresponding primary tumor.
Fig. 3.
Fig. 3.
Phenotypic diversity in tumors arising from CD44+ Lin− cells. CD44 staining pattern of live cancer cells from primary unpassaged tumor, tumor resulting from the implantation of CD44+Lin− cells from the primary tumor (first passage), and tumor resulting from the implantation of CD44+Lin− cells from the first passage tumor (second passage).
Fig. 4.
Fig. 4.
Immunohistochemical analysis of a well differentiated HNSCC. Serial sections were stained with antibodies to CD44 (A), Cytokeratin 5/14 (B), Involucrin (C and E), or an isotype control (D). A horseradish peroxidase-conjugated secondary antibody was used for detection. CD44+ regions were CK5/14+ and Involucrin− (white arrows), whereas CD44− regions were CK5/14− and Involucrin+ (black arrows).
Fig. 5.
Fig. 5.
Quantitative RT-PCR and immunofluorescence for BMI1. (A) CD44+Lin− and CD44−Lin− cells from three HNSCC samples were double-sorted, and purified RNA was assayed for the presence of BMI1 transcripts by quantitative RT-PCR. Human embryonic stem cells (ES) were used as a positive control for PCR, and transcript levels were normalized to expression of β-actin. All three samples were primary patient samples. BMI1 was expressed in the CD44+Lin− population but was undetectable in the CD44−Lin− population in samples 1 and 2 and 4-fold lower in the CD44−Lin− population in sample 3. (B and C) A well differentiated HNSCC was costained for CD44 (green) and Bmi1 (red). (B) The CD44/BMI1 overlay. (C) The BMI1/Hoechst 33342 overlay, to highlight nuclei in blue. BMI1 protein was seen in the nuclei of the majority of CD44+ cells and was rare in the CD44− region of the tumor.
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