Establishment, Authentication, And Characterization Of A Novel Well-differentiated Human Laryngeal Squamous Cell Carcinoma Cell Line, FD-LSC-1

Part I. Establishment of a novel well-differentiated human laryngeal squamous cell carcinoma cell line, FD-LSC-1This study aims to establish a novel laryngeal squamous cell carcinoma (LSCC) cell line of Chinese origin through primary culture of resected LSCC specimens to facilitate the basic and preclinical research of pathogenesis and biological characteristics of LSCC in current Chinese population.In this study, we attempted 40 specimens from 40 male (46-68 years old) patients diagnosed as LSCC by pathology and received surgical treatment in our hospital between June 2011 and September 2011. Among which 29 specimens derived from the epiglottis,8 specimens from the vocal cords, and 3 specimens from the metastatic lymph nodes. The collected specimens were washed, sterilized, and dissociated mechanically and enzymatically by collagenase before primary culture. The malignant laryngeal epithelia growing out of the seeded tissue fragments were purified, assessed for purity by flow cytometer and immunofluorescence, stored in liquid nitrogen at regular intervals, screened for mycoplasma infection, and resuscitated for cell viability assay. Paired normal laryngeal epithelia (PNLE) of the same patient cultured in the same condition served as a control.We found that the primary laryngeal cancer cells were difficult to survive and propagate in vitro. The major dilemmas were contamination caused by bacteria and fungus and cancer cells that did not stick to the flask. Most of the cancer cells stopped proliferating within 3 passages. Luckily, we successfully established a novel LSCC cell line, designated FD-LSC-1, from the well-differentiated epiglottic neoplasm of an untreated 68-year-old Chinese male (Stage IVa, T4aN2M0). The subject had no family history of laryngeal cancer, but did have a 40-year history of smoking and a 30-year history of alcohol use. This cell line was maintained for more than 100 passages in vitro, with the purity of 100% and without infection of mycoplasma. In addition, the FD-LSC-1 maintained a vigorous proliferation capacity after resuscitation. The PNLE were successfully subcultured till passage 8.These results suggest that the novel human LSCC cell line, FD-LSC-1, has been established successfully and immortalized naturally in vitro; that the primary laryngeal cancer cells are difficult to survive in vitro and to be established as a permanent cell line, which may be caused by the anatomic features of laryngeal neoplasms; that it is a difficult task to establish a novel LSCC cell line and one can only occasionally succeed after dozens of attempts.Part â…¡. Authentication of the novel well-differentiated human laryngeal squamous cell carcinoma cell line, FD-LSC-1This study aims to authenticate the identity of the newly established FD-LSC-1 cell line as a qualified human LSCC cell line, which comprises the authentication of human origin, the authentication of epithelial origin, and the authentication of malignant characteristics.In this study, the authoritative short tandem repeat (STR) profiling test was used by the American Type Culture Collection (ATCC) to authenticate whether the FD-LSC-1 cell line was of human origin and whether it was contaminated by any lines within the American ATCC database and the European Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) database. Seventeen STR loci plus the gender-determining locus, Amelogenin, were investigated. The phase-contrast microscope was used to observe the morphology of the cultured FD-LSC-1 cells and PNLE. Immunofluorescence was used to examine the expression of pan-cytokeratin (pan-CK) and vimentin. Transmission electron microscopy (TEM) was used to examine the epithelial ultrastructural features. The malignant characteristics of FD-LSC-1 cell line was finally authenticated, including:Giemsa staining for morphological observation, TEM for ultrastructural features examination, cytogenetic karyotypic analysis, flow cytometry for DNA content and cell cycle analysis, soft agar for colony formation capacity, and heterotransplantation in immunodeficient mice for tumorigenicity.We found that the FD-LSC-1 cell line was authenticated to be of human origin by the ATCC and did not match any cell lines within the ATCC and DSMZ database. Morphological observation revealed that this cell line grew in a cobblestone-like pattern with vacuoles in the cytoplasm, different from the HEp-2 cells. Positive staining of pan-CK and negative staining of vimentin were detected. The epithelial ultrastructural features of desmosomes in the intercellular connections and tonofilaments in the cytoplasm were observed. Several malignant characteristics were authenticated, including:numerous mitotic figures, a large nuclear to cytoplasmic ratio, prominent large nuclei, and occasional multinucleated giant cells detected by Giemsa staining; many mitochondria, ribosomes, and rough endoplasmic reticula were observed by TEM; both numerical and structural aberrations were detected by karyotypic analysis, including a modal chromosome number of 68 and gains, deletions, and translocations of chromosomes; polyploidy of DNA content was determined, with decreased fraction of cells in G0/G1 phase, increased fraction of cells in S-phase, and similar percentage of cells in G2/M phase, compared with HeLa cells; no colonies formed in the soft agar; successful formation of xenografted tumors in the immunodeficient mice, with the pathology being well-differentiated LSCC.These results suggest that after being authenticated of human origin, epithelium origin, and malignant characteristics, the newly established FD-LSC-1 cell line accords with the biological features of squamous cell carcinoma and could be authenticated as a qualified well-differentiated LSCC cell line of human origin, without contamination of any established cell lines.Part III. Characterization of the novel well-differentiated human laryngeal squamous cell carcinoma cell line, FD-LSC-1This study aims to detailedly characterize the authenticated FD-LSC-1 cell line, including:population doubling time, migration and invasion potential, sensitivity to chemoradiation, expression of Notch1, EGFR (epidermal growth factor receptor), CK(cytokeratin)5, and Ki67 proteins, mutation of TP53 gene, infection of HPV (human papillomavirus), expression of head and neck squamous cell carcinoma (FTNSCC) biomarkers, and verification of most frequently reported dysregulated HNSCC genes and miRNAs.In this study, the CCK-8(cell counting kit-8) assay was used to measure the population doubling time of the FD-LSC-1 cell line, the transwell assay was used to assess the migration and invasion potential compared with the Hela cell line, X radiation and cisplatin (DDP) were used to examine the sensitivity to chemoradiation compared with the Hela cell line, Western blot was used to evaluate the protein levels of Notchl, EGFR, CK5, and Ki67, DNA sequencing was used to screen the mutation of TP53 tumor suppressor gene, PCR (polymerase chain reaction) was used to detect the HPV infection, immunostaining was used to label the HNSCC biomarkers, SYBR Green quantitative real-time PCR (qRT-PCR) and TaqMan qRT-PCR were used to verify the reportedly dysregulated HNSCC genes and miRNAs.We found that the in vitro doubling time of the FD-LSC-1 cell line was some 24 h. The FD-LSC-1 cell line had higher migration and invasion potentials but lower resistance to radiation and cisplatin compared with the HeLa cells. Activated signalings of Notch1 and EGFR and elevated protein levels of CK5 and Ki67 were detected. Missense and nonsense mutations of TP53 gene in exon 5 and 8 were detected. PCR detection revealed that the FD-LSC-1 cell line was negative for HPV infection. Positive staining of important HNSCC biomarkers, such as ADAM 10 and MLH1, was detected by immunohistochemistry and immunofluorescence. Fourteen genes including TERT were found to be significantly upregulated and another fourteen genes including KRT4 were found to be downregulated in the FD-LSC-1 cells by SYBR Green qRT-PCR. Nine miRNAs including miRNA-21 were found to be significantly upregulated and ten including miRNA-138 downregulated in the FD-LSC-1 cell line by TaqMan qRT-PCR.These results suggest that after being characterized in nine biological features, the human well-differentiated LSCC cell line, FD-LSC-1, has been established, authenticated, and well-characterized, which may lay the foundation of LSCC research in pathogenesis and tumor biology for the Chinese population.