UM-SCC-104: a new human papillomavirus-16-positive cancer stem cell-containing head and neck squamous cell carcinoma cell line

Abstract

Background: Few human papillomavirus (HPV)(+) head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established University of Michigan-squamous cell carcinoma-104 (UM-SCC-104), a new HPV(+) HNSCC cell line from a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSCs).

Methods: Tumor cells were tested for biomarker expression by immunohistology, and the presence of HPV was assessed by several methods.

Results: UM-SCC-104 has a unique genotype, contains HPV-16, and expresses E6/E7. Inoculation of aldehyde dehydrogenase (ALDH)(+) and ALDH(-) cells in an immunocompromised mouse resulted in tumor growth from the ALDH(+) cells after 6 weeks that recapitulated the histology of the primary, whereas ALDH(-) cells did not produce tumors.

Conclusion: UM-SCC-104, a new HPV-16, CSC-containing HNSCC cell line will aid in studying recurrent HPV(+) tumors. The aggressive nature of this tumor is consistent with high uniform expression of epidermal growth factor receptor (EGFR) and a functionally significant proportion of ALDH(+) CSCs.

Fig 1

PCR-MassARRAY using MALDI-TOF mass spectroscopy to detect and type human papillomavirus. Diagram depicting three-step process of HPV detection by competitive multiplex PCR amplification using 15 hrHPV type-specific primer pairs for HPV E6 & hGAPDH. Amplification of HPVE6 occurs in episomal and/or integrated viral DNA as well as each of the synthetic competitors.

Fig 2

Establishment of UMSCC-104 from a recurrent oral cavity squamous cell carcinoma. A, Outgrowths of squamous cells from explanted tissue placed in culture flasks. B, UM-SCC-104 at passage 8 growing in separate colonies which is its typical phenotype. C, Primary tissue stained with hematoxylin and eosin demonstrating infiltrating nests of tumor cells with abundant pink (keratinized) cytoplasm. Histology also showed intercellular bridges and focal squamous whorls typical of squamous cell carcinoma. D, Tumor from xenograft derived from cells expressing high ALDH expression. Similar to the primary tissue section, morphology consists of invasive poorly differentiated squamous cells.

Fig 3

Identification of cancer stem cells from primary tissue. A, Flow cytometry analysis in which uptake of an ALDH substrate (BAAA) is inhibited with DEAB. This inhibition serves as a baseline for fluorescence activity. B, When BAAA is incubated with the cells without the inhibitor, an ALDH high population stands out (2.32%). Cells were collected from both the ALDH high population (P5) as well as the ALDH low population (P4). C, 5,000 cells were implanted in the flank of a NOD/SCID mouse. Cells collected in the ALDH high population grew tumor and cells from the ALDH low population did not produce tumor. D, Flank tumor of mouse derived from injection of ALDH high cells.

Fig 4

Immunohistologic staining of the patient’s resected anterior floor of mouth tumor (Primary Tumor), UM-SCC-104 cancer stem cell initiated murine xenograft tumor (CSC Derived Tumor), and UM-SCC-104 cells grown on glass slides (UM-SCC-104). Sections of the primary tumor tissue, the CSC derived tumor and the cell line all exhibited strong and diffuse nuclear and cytoplasmic staining for p16^INK4a. Cell membrane staining was also strong and diffuse for EGFR in all three tumor cell samples. Approximately 10% of the resected tumor cells over-expressed p53, while staining was negative for RB, Bcl-2 and cyclin D. There was 10% focal staining of RB and cyclin D and 5% focal staining for p53, while no expression of Bcl-2 was seen on mouse tissue sections. UM-SCC-104 cultured on chamber slides uniformly over-expressed p16^INK4a, EGFR and Rb. Focal staining of cyclin D1 (20%), Bcl-2 (20%) and p53 (30%) was presentin the cell line.

Fig 5

In Situ Hybridization for high risk HPV and p16^INK4a immunohistochemistry staining of the patient’s tumor tissue. A, In situ hybridization (blue stain) demonstrating rare punctate signals (arrows) within the nucleus indicative of integrated HPV DNA. B, p16^INK4a immunohistochemistry demonstrating dark nuclear and diffuse cytoplasmic staining on an adjacent tumor tissue section to that used for in situ hybridization.

Fig 6

MassARRAY analysis of HPV 16 DNA from UM-SCC-104 and the floor of mouth tumor tissue. In each case two peak areas representing the extension products of the target allele from the tumor cells and the corresponding competitive template allele separated by MALDI-TOF Mass Spectroscopy by the 80 Dalton mass difference resulting from the single base difference between wild-type HPV16 and the HPV16 competitor.

Fig. 7

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) detection of HPV16 E6 and E7 oncogenes in cDNA prepared from the UM-SCC-104 cell line, the patient’s anterior floor of mouth frozen tumor tissue and the HPV-positive control Caski and UM-SCC-47 cell lines. No bands were seen in HPV-negative UM-SCC-38 cell line. HPV16 E6 primer sets amplify the full length E6 (476 bp) and the alternately-spliced E6* variant (297 bp) in UM-SCC-47, Caski and UM-SCC-104. With the cDNA from the floor of mouth tumor tissue the E6 bands were too faint to show in the photograph, but the E7 amplicon was more abundant and easily visible.