Establishment and genetic characterization of six unique tumor cell lines as preclinical models for sinonasal squamous cell carcinoma

Abstract

Sinonasal squamous cell carcinomas (SCC) are rare tumors, etiologically related to occupational exposure to wood and leather dust. In spite of surgical and radiotherapeutic advances, the 5 year survival is still 30-50%. Therefore, alternative treatment options are needed. We report the establishment and characterization of six unique human sinonasal SCC cell lines, named SCCNC1, 2, 4, 5, 6 and 7. In vitro growth and invasion characteristics were evaluated and genetic profiles were compared to those of the original primary tumors. The population doubling times ranged from 21 to 34 hours. Cell lines SCCNC2 and 7 were highly invasive in matrigel. Five cell lines carried a high number of copy number alterations, including amplifications and homozygous deletions, while one showed only three abnormalities. Sequence analysis revealed three cell lines with TP53 mutation and none with KRAS or BRAF. Overexpression of p53 was observed in five, and of EGFR in four cell lines. None of the cell lines showed strong immunopositivity of p16 or presence of human papilloma virus. In conclusion, we have created six new cell lines that are clinically and genetically representative of sinonasal SCC and that will be a useful tool for the preclinical testing of new therapeutic agents.

Figure 1. Photomicrographs of the in vitro growing cell lines (left column) and representative H&E stained paraffin sections of the corresponding primary tumors (right column).

Figure 2. Proliferation and invasion in matrigel.

(A) In the exponential growth phase, the cell lines showed population doubling times between 21 and 34 hours. After passaging, SCCNC7 and SCCNC5 settled and continued proliferating quickly, whereas the other cell lines were slower in adhering and resuming proliferation. (B and C) Cell lines SCCNC2 and SCCNC7 showed the strongest capacity to invade with 439 and 153 cells in the matrix after 24 hours, respectively. All experiments were done in triplicate.

Figure 3. Microarray CGH profiles of cell lines at passages between 6 and 10 (left column) and their corresponding primary tumors (right column).

All data points are expressed as log2-ratios, ordered continuously from left to right as chromosome 1 up to chromosome X (here numbered as 23). In cell lines SCCNC1,2,4,6 and 7 many chromosomes show copy number alteration, including homozygous deletion and high level amplification. The majority of the aberrations are also present in the primary tumor. Cell line SCCNC5 harboured only three copy number changes and these were not detected in the primary tumor.

Figure 4. P53 expression analysis by immunofluorescence on the in vitro growing cell lines (left column) and by immunohistochemistry on the corresponding primary tumors (right column), showing positivity in all cell lines except SCCNC4.

Figure 5. P16 (left column) and EGFR (right column) expression analysis by immunohistochemistry on the primary tumors.

SCCNC4 shows a weak p16 immunostaining. EGFR overexpression is observed in SCCNC1, 4, 5 and 7.